Nanotechnology Research Methods for Food and Bioproducts

Nanotechnology Research Methods for Foods and Bioproducts Padua_ffirs.indd i 1/23/2012 10:29:14 PM Nanotechnology Re...

Author: Graciela Wild Padua PhD | Qin Wang PhD

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Nanotechnology Research Methods for Foods and Bioproducts

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Nanotechnology Research Methods for Foods and Bioproducts Edited by

Graciela W. Padua Department of Food Science and Human Nutrition University of Illinois Urbana, Illinois USA

Qin Wang Department of Nutrition & Food Science University of Maryland College Park, Maryland USA

A John Wiley & Sons, Ltd., Publication

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This edition first published 2012 © 2012 by John Wiley & Sons, Inc. Wiley-Blackwell is an imprint of John Wiley & Sons, formed by the merger of Wiley’s global Scientific, Technical and Medical business with Blackwell Publishing. Editorial Offices: 2121 State Avenue, Ames, Iowa 50014-8300, USA The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK 9600 Garsington Road, Oxford, OX4 2DQ, UK For details of our global editorial offices, for customer services and for information about how to apply for permission to reuse the copyright material in this book please see our website at www.wiley.com/ wiley-blackwell. Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Blackwell Publishing, provided that the base fee is paid directly to the Copyright Clearance Center, 222 Rosewood Drive, Danvers, MA 01923. For those organizations that have been granted a photocopy license by CCC, a separate system of payments has been arranged. The fee codes for users of the Transactional Reporting Service are ISBN-13: 978-0-8138-1731-6/2012. Designations used by companies to distinguish their products are often claimed as trademarks. All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. The publisher is not associated with any product or vendor mentioned in this book. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold on the understanding that the publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required, the services of a competent professional should be sought. Library of Congress Cataloging-in-Publication Data Nanotechnology research methods for foods and bioproducts / edited by Graciela W. Padua, Qin Wang. p. cm. Includes bibliographical references and index. ISBN 978-0-8138-1731-6 (hardcover : alk. paper) 1. Food–Biotechnology. 2. Nanotechnology. I. Padua, Graciela W. II. Wang, Qin, 1973TP248.65.F66N36 2012 664–dc23 2011039277 A catalogue record for this book is available from the British Library. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books. Set in 10/12pt Times by SPi Publisher Services, Pondicherry, India 1

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Contents

Foreword Contributors

1 Introduction Graciela W. Padua References

1

2 Material components for nanostructures Graciela W. Padua and Panadda Nonthanum

5

2.1 Introduction 2.2 Self-assembly 2.3 Proteins and peptides 2.3.1 Amyloidogenic proteins 2.3.2 Collagen 2.3.3 Gelatin 2.3.4 Caseins 2.3.5 Wheat gluten 2.3.6 Zein 2.3.7 Eggshell membranes 2.3.8 Bovine serum albumin 2.3.9 Enzymes 2.4 Carbohydrates 2.4.1 Cyclodextrins 2.4.2 Cellulose whiskers 2.5 Protein–polysaccharides 2.6 Liquid crystals 2.7 Inorganic materials References 3 Self-assembled nanostructures Qin Wang and Boce Zhang 3.1 Introduction 3.2 Self-assembly 3.2.1 Introduction 3.2.2 Micelles 3.2.3 Fibers 3.2.4 Tubes

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3

5 6 8 8 9 9 10 10 10 10 11 11 11 11 12 13 14 14 15 19 19 20 20 20 21 23

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Contents

3.3 Layer-by-layer assembly 3.3.1 Introduction 3.3.2 Nanofilms on planar surfaces from LbL 3.3.3 Nanocoatings from LbL 3.3.4 Hollow nanocapsules from LbL 3.4 Nanoemulsions 3.4.1 Introduction 3.4.2 High-energy nanoemulsification methods 3.4.3 Low-energy nanoemulsification methods 3.4.4 Nanoparticles generated from different nanoemulsions and their applications References 4 Nanocomposites Graciela W. Padua, Panadda Nonthanum and Amit Arora 4.1 4.2 4.3 4.4 4.5

33 34 41

Introduction Polymer nanocomposites Nanocomposite formation Structure characterization Biobased nanocomposites 4.5.1 Starch nanocomposites 4.5.2 Pectin nanocomposites 4.5.3 Cellulose nanocomposites 4.5.4 Polylactic acid nanocomposites 4.5.5 Protein nanocomposites 4.6 Conclusion References

41 42 43 44 45 46 46 47 47 48 50 50

5 Nanotechnology-enabled delivery systems for food functionalization and fortification Rashmi Tiwari and Paul Takhistov

55

5.1 Introduction: functional foods 5.2 Food matrix and food micro-structure 5.3 Target compounds: nutraceuticals 5.3.1 Solubility and bioavailability of nutraceuticals 5.3.2 Interaction of nutraceuticals with food matrix 5.4 Delivery systems 5.4.1 Overcoming biological barriers 5.4.2 Nano-scale delivery systems 5.4.3 Types/design principles 5.4.4 Modes of action 5.5 Examples of nanoscale delivery systems for food functionalization 5.5.1 Liposomes 5.5.2 Nano-cochleates 5.5.3 Hydrogels-based nanoparticles 5.5.4 Micellar systems

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55 56 58 60 61 64 64 65 67 69 72 72 74 75 75

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Contents

5.5.5 Dendrimers 5.5.6 Polymeric nanoparticles 5.5.7 Nanoemulsions 5.5.8 Lipid nanoparticles 5.5.9 Nanocrystalline particles 5.6 Conclusions References 6 Scanning electron microscopy Yi Wang and Vania Petrova 6.1 Background 6.1.1 Introduction to the scanning electron microscope 6.1.2 Why electrons? 6.1.3 Electron–target interaction 6.1.4 Secondary electrons (SEs) 6.1.5 Backscattered electrons (BSEs) 6.1.6 Characteristic X-rays 6.1.7 Overview of the SEM 6.1.8 Electron sources 6.1.9 Lenses and apertures 6.1.10 Electron beam scanning 6.1.11 Lens aberrations 6.1.12 Vacuum 6.1.13 Conductive coatings 6.1.14 Environmental SEMs (ESEMs) 6.2 Applications 6.2.1 Zein microstructures 6.2.2 Controlled magnifications 6.2.3 Nanoparticles 6.3 Limitations 6.3.1 Radiation damage 6.3.2 Contamination 6.3.3 Charging References 7 Transmission electron microscopy Changhui Lei 7.1 Background 7.2 Instrumentations and applications 7.2.1 Interactions between incident beam and specimen 7.2.2 Conventional TEM 7.2.3 Scanning TEM 7.2.4 Analytical electron microscopy 7.3 Sample preparations 7.4 Limitations References

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77 78 80 81 83 85 85 103 103 103 104 104 105 106 107 107 108 109 109 110 111 111 111 111 112 115 117 119 120 122 124 126 127 127 128 129 130 136 139 142 143 143

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viii

8

9

Contents

Dynamic light scattering Leilei Yin

145

8.1 8.2 8.3 8.4

The principle of dynamic light scattering Photon correlation spectroscopy DLS apparatus DLS data analysis 8.4.1 Multiple-decay methods 8.4.2 Regularization methods 8.4.3 Maximum-entropy method 8.4.4 Cumulant method References

145 151 152 156 158 158 159 159 160

X-ray diffraction Yi Wang and Phillip H. Geil

163

9.1

163 163 165 165 168

Background 9.1.1 Introduction 9.1.2 Classical X-ray setup 9.1.3 X-ray sources 9.1.4 X-ray detectors 9.1.5 Wide-angle X-ray scattering and small-angle X-ray scattering 9.2 Applications 9.2.1 Example: X-ray characterization of zein–fatty acid films 9.2.2 Temperature-controlled WAXS References 10

11

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169 169 170 176 179

Quartz crystal microbalance with dissipation Boce Zhang and Qin Wang

181

10.1 Background and principles 10.2 Instrumentation and data analysis 10.2.1 Sensors 10.2.2 Data analysis 10.3 Applications 10.4 Advantages References

181 183 183 184 185 190 192

Focused ion beams Yi Wang

195

11.1 Background 11.1.1 Introduction to the focused ion beam system 11.1.2 Overview of the FIB 11.1.3 Ion beam production 11.1.4 Ion–target interaction 11.1.5 Basic functions of the FIB system 11.1.6 SEM and SIM

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Contents

11.1.7 SEM and FIB combined system 11.1.8 3D nanotomography with application of real-time imaging during FIB milling 11.1.9 3D nanostructure fabrication by FIB 11.2 Applications 11.2.1 Polymers 11.2.2 Biological products 11.2.3 Example: self-assembled protein structures 11.3 Limitations References 12

ix

201 201 202 202 202 203 203 207 214

X-ray computerized microtomography Leilei Yin

215

12.1 12.2 12.3 12.4 12.5 12.6

215 215 217 220 226 228 229 230 230 231 232

Introduction X-ray generation X-ray images X-ray micro-CT systems Data reconstructions Artifacts in micro-CT images 12.6.1 Ring artifacts 12.6.2 Center errors 12.6.3 Beam-hardening artifacts 12.6.4 Phase-contrast artifacts 12.7 A couple of issues in X-ray micro-CT practice 12.7.1 The spatial resolution, and associated issues of contrast and field of view 12.7.2 Localized imaging and sample-size reduction References Index

232 232 233 235

A color plate section falls between pages 194 and 195

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Foreword Ample Opportunities for Nanotechnology in Foods and Bio-based Products to Benefit Society

Food, like clean water and fresh air, is essentially important to human living. However, the current global agricultural production and food security are facing serious longterm sustainability challenges, exacerbated by population growth and climate changes. It has been estimated by authority that more than 1 billion people experienced food insecurity in 2011 – a daunting image of one out of every seven people suffering hunger and malnutrition. Yet the world population is expected to increase from 7 billion today to more than 9 billion by 2050. The Food and Agriculture Organization (FAO) of the United Nations predicts that food production will need to increase by 70% over the next four decades to meet the anticipated demand. Given the fact that new land resources for cultivation are extremely limited, the majority of the production increase will have to come from technological innovations and new approaches. Nanotechnology has been actively pursued for about 10 years to enhance our capabilities to address the grand challenges facing agriculture and food systems, and its momentum is continuing. Nanoscale science, engineering, and technology, often simply referred to as nanotechnology, emerged around the year 2000 as a new distinctive frontier for scientific research and development in broad fields including physics, chemistry, biology, engineering, materials sciences, social sciences, and in almost all industry sectors from semiconductors and electronics, energy, space, medicine and pharmaceutics, food and nutrition, agriculture and forestry, to natural resources and the environment, and many others. The recognition of novel properties of matter at the nanoscale and the newly developed capabilities to precisely study and manipulate such properties was necessary, but not sufficient, to champion for a national R&D initiative. Visionary scholars and government leaders articulated a long-term vision for the transformative potential of nanotechnology R&D to benefit society, and thus ignited the establishment of national nanotechnology initiatives first in the USA, followed by major research powerhouse countries, and now by most countries in the world. Substantial investments in nanotechnology R&D by governments and the private sector have sustained steady advancement of new solutions and products over the last decade. The US National Nanotechnology Initiative (NNI) has contributed a cumulative $14 billion, including about $1.8 billion in 2011, in nanotechnology R&D. Many nanotechnology breakthroughs have begun to impact the marketplace. The current value of nanotechnology-enabled products in the USA is estimated at about $91 billion. Current trends suggest the market impact of nanotechnology-enabled products will achieve $3 trillion worldwide by 2020. Nanotechnology has been touted to have the potential to revolutionize agriculture and food in the 21st century. Numerous exploratory research projects and publications have clearly shown ample evidence of this in a broad range of critical challenges

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Foreword

and opportunities facing agriculture and food systems. Innovative ideas have emerged to develop nanotechnology-enabled solutions for global food security through improving productivity and quality, adaptation and mitigation of agricultural production systems to climate changes, improving nutritional quality of foods, enhancing food safety and biosecurity through better detection of pathogens and contaminants as well as novel intervention technologies, and development of biobased products and bioenergy alternatives. Some examples include novel uses and high value-added products of nano-biomaterials of agricultural origins for food and non-food applications, nanoscale-based sensing mechanisms and devices for reliable early detection of diseases and monitoring of physiological biomarkers for optimal agricultural production, precision agriculture technologies including ones to efficiently manage applications of agricultural chemicals and water resources, and water quality improvements. Persistent investment and support will bring these pioneer work and many other creative ideas to fruition in the near future. Responsible development and deployment of nanotechnology is critically important to nanotechnology R&D not only because it will impact the ultimate success in propelling economic growth and job creation, but also the environment, human health and consumer safety. The food science community has a long tradition of ensuring food safety. Agriculture production and allied industries are fully aware of the importance of safety and the environmental implications of agricultural chemical applications. Investigations in risk assessment and characterization of nanosized materials and their uses in agricultural production and foods have been, and will continue to be, a high priority in nanotechnology R&D. Analytical instrumentation, test standards and experimental protocols, both in vitro and in vivo, will be further developed and used. This book is among the first covering the intersection of foods and nanotechnology. It is unique in presenting two interrelated but also independent sections, namely materials and analytical techniques, in one combined volume to give the reader a convenient access of references. The material section deals with common food components, nanostructure formation, processes and mechanisms, macromolecular and supramolecular structures and functionality, food and nutrition applications, and nanocomposites in food packaging. The analytical section details seven instrumentations that are among the most important characterization tools in nanoscale science research and technology development. The reader will find rich information detailed by experts in the fields of food science and nanotechnology from some of the most prominent research institutes in the USA. Researchers and students may be inspired and empowered to eagerly engage in addressing the key challenges in securing the supply and availability of food to all, improving human health and wellbeing through better foods, and developing high value-added bioproducts of agricultural origin. The potential of nanotechnology to significantly advance technical solutions for sustainability, vulnerability and human health can be clearly envisioned. These chapters may crystallize new visions and innovative approaches to advance food science and technology in the future. The book editors, Drs Padua and Wang, being two active practitioners and front runners in this field, have indeed made a valuable contribution to the professionals of food and biomaterials nanotechnology, and broadly, to food and agricultural sciences. Hongda Chen USDA National Institute of Food and Agriculture US National Nanotechnology Initiative

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Contributors

Amit Arora Department of Paper Science and Engineering, University of Wisconsin – Stevens Point, WI

Paul Takhistov Department of Food Science, Rutgers, The State University of New Jersey, New Brunswick, NJ

Phillip H. Geil Department of Materials Science and Engineering, University of Illinois, Urbana-Champaign, IL

Rashmi Tiwari Department of Food Science, Rutgers, The State University of New Jersey, New Brunswick, NJ

Changhui Lei Materials Research Laboratory, University of Illinois, UrbanaChampaign, IL

Qin Wang Department of Nutrition and Food Science, University of Maryland, College Park, MD

Panadda Nonthanum Department of Food Science and Human Nutrition, University of Illinois, Urbana-Champaign, IL

Yi Wang Department of Food Science and Human Nutrition, University of Illinois, Urbana-Champaign, IL

Graciela W. Padua Department of Food Science and Human Nutrition, University of Illinois, Urbana-Champaign, IL

Leilei Yin Beckman Institute, University of Illinois, Urbana-Champaign, IL

Vania Petrova Center for Microanalysis of Materials, Frederick Seitz Materials Research Laboratory, Urbana, IL

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Boce Zhang Department of Nutrition and Food Science, University of Maryland, College Park, MD

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Electron gun

Beam column

Anode Electron beam

Spray aperture First condenser lens

Scan coils

Second condenser lens

Magnification control

Computer system for signal processing

Condenser aperture Objective lens

Stigmator SE detector

Final aperture BSE detector

X-ray detector

Scan generator

Amplifier

Sample stage High vacuum pump

Multichannel analyser

Plate 6.1 A schematic diagram of an SEM system. A thermionic electron gun is shown as an example. Details of electron guns are given in Fig. 6.4.

Nanotechnology Research Methods for Foods and Bioproducts, First Edition. Edited by Graciela W. Padua and Qin Wang. © 2012 John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc.

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Crystal oscillation

Crystal oscillation

Crystal oscillation

Clean surface

Energy does not dissipate – ΔD = 0

Rigid film

Energy dissipates slowly – ΔD > 0, small

Soft film

Energy dissipates rapidly – ΔD > 0, large

Plate 10.1 Energy dissipation changes when adhering to rigid or soft materials.

Top view

Bottom view Coating layer Quartz crystal

Side view Coating layer Gold Quartz Gold

Contact electrodes

diam. 14mm

Contact electrodes

5MHz Plate 10.2 QCM-D sensor overview.

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PNA-DNA hybridization DNA-DNA hybridization DNA 0.4 × 10

–6

ΔD

PNA

DNA

DNA

0

–5

–10

–15

–20

Δf (Hz) Plate 10.3 Comparison of two antibodies on an antigen-covered sensor. Reprinted from Specificity of DNA Hybridisation on a Functionalised Lipid Bilayer (Q-Sense application note), with permission from Q-sense. Available on www.q-sense.com.

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(a) NaCl 8,5

0

8

7,5

–5

7

A

6,5 B

B

–15

6

5,5 A

–20

5

B

4,5

A

–25

4

A

3,5

B

3

2,5

–30

f-1 f-3 f-5 D-1 D-3 D-5

–35 –40

A 0

Dissipation (1E-6)

Frequency (Hz)

–10

2

1,5 1

0,5 0

10 20 30 40 50 60 70 80 90 100 110 120 Time (min)

(b) 8,0 7,0

Sauerbrey Model

Thickness [nm]

6,0 5,0 4,0 3,0 2,0 1,0 0,0 A1st layer

A 2nd layer

A 3rd layer

A 4th layer

A 5th layer

NaCl

Water

Plate 10.4 (a) Frequency and dissipation responses of a real-time polyelectrolyte multilayer formation. (The large buffer step is generated by different solution properties when changing from water to NaCl and back.) (b) Thickness data after each of five polymer A adsorptions, NaCl and water. Reprinted from Real Time Thickness Monitoring of Polyelectrolyte Multilayer Formation in Situ (Q-Sense application note), with permission from Q-sense. Available on www.q-sense.com.

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ΔD (10–6)

Δf (Hz) Exposure to Mefp–1

0

Rinsing Exposure to Nal O4

Δfn = 1 16 14

Δfn = 3 /3

–20

12

Δfn = 5 /5

–40

10

ΔDn = 1

8

ΔDn = 3

–60

6 4

–80

2

ΔDn = 5

0

–100 0

20

40 Time (min)

60

80 Release of water

Exposure to NaIO4 After rinsing Collapsed sturcture Plate 10.5 Monitoring of thickness and hydration changes in an adhered protein layer. Reprinted from Structural change of Adsorbed Protein Layer (Q-Sense application note), with permission from Q-sense. Available on www.q-sense.com.

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PNA binding

DNAfc and DNAmm

Buffer rinsing

Δf (Hz)

0 –5 Mismatch PNA-DNAmem

–10 –15

biotin-PNA

Fully complementary PNA-DNAfc

–20 0

1000

2000

3000

4000

Time (s)

Fully complementary PNA-DNAfc

2

ΔD (10–6)

1.5 biotin-PNA 1

Mismatch PNA-DNAmem

0.5 0 0

1000

2000

3000

4000

Time (s)

biotin-PNA

DNAmin Rinse

Functionalized lipid bilayer Rinse

Reversable binding

Irreversable binding

DNAfc Plate 10.6 Binding of single-strand biotin-PNA to mismatched DNA (DNAmm) and fully complementary DNA (DNAfc). Reprinted from Specificity of DNA Hybridisation on a Functionalised Lipid Bilayer (Q-Sense application note), with permission from Q-sense. Available on www.q-sense.com.

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Δmass (ng/cm2) 1400 IgG

1. IgG (positive control) on gold 1200 2. Polyurethane urea (PUUR) on 1000 gold

Anti-C3c

3. Polystyrene (PS) on gold

800

4. Hydrophobic self-assembled mono-layer (SAM) on gold

600

5. Titanium (Ti)

400

6. Heat-inactivated sera(i.sera) (negative control) on gold

200

PUUR

PS SAM Ti i. sera

0 0

5

10

15

20

25

30

Time (min) Plate 10.7 Binding of anti-C3c to various substrate surfaces. Reprinted from Protein Adsorption as Biocompatability Evaluation Method for Implant Surfaces (Q-Sense application note), with permission from Q-sense. Available on www.q-sense.com.

(a)

Δm (ng/cm2)

1000

Δ mSPR Δ mQCM

800

Step 2

600

Step 3

400 200

Step 1

0 0

4

8

12

16

20

Time (min)

(b)

2.5 ΔDQCM

ΔD (10–6)

2

Step 2

1.5 1 0.5 0

Step 3 Step 1 0

4

8

12

16

20

Time (min)

Step 1

Step 2

Release water

Step 3

Plate 10.8 QCM-D and SPR data during the formation of a lipid bilayer. Reprinted from Lipid Bilayer Formation; A Comparison Between QCM-D, SPR and AFM (Q-Sense application note), with permission from Q-sense. Available on www.q-sense.com.

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Ion source Suppressor Extractor Spray aperture First lens Computer system Upper octopole for signal processing Variable aperture

Ion column

Blanking deflector

Vacuum chamber

Blanking aperture Deflection octopole Second lens Ion beam Ion detector Gas injection system

Secondary electron detector Sample stage Plate 11.1

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Schematic diagram of an FIB system.

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1

Introduction

Graciela W. Padua

Nanoscale-sized particles are not new – they exist naturally. However, our ability to visualize, understand and control matter at the nanoscale is new. Recent recognition of the impact of nanoscale materials on the overall structure and functionality of foods and biological tissues is driving new interest in their study. This new body of knowledge, along with the methodology used to create it, is the subject matter of nanoscale science and technology for food and biological materials. Novel structures and new functionalities are expected to be the product of this new knowledge. It is increasingly recognized that many of the structure-building elements in foods are the result of self-assembly of nanosized molecules into particles or at interfaces. Thus, the ability to control the assembly of biomacromolecules in a matrix spanning several length scales (the size of a large protein molecule is ∼5–10 nm) will become an integral part of food product design. The next wave of food innovation will require a shift of focus from macroscopic properties to those at the nanoscale. Future development of food products will require an understanding of the relations between nano- and higher length scale structures and their impact on functionality, including physicochemical, nutritional and sensorial. Examples of structures being examined at the nanoscale are liposomes, micelles, nanotubes, hydrogels, dendrimers and nanocomposites. Such structures are used or proposed to be used for enhancement of nutritional value of foods, improving flavor profiles, preserving freshness, improving packaging and preventing disease. Nanotechnology has high potential in food science and technology. Major impacts are foreseen in nutrition, food quality, food packaging and food safety assurance. ●

Nutrition: Controlled delivery of bioactive compounds by micro- and nanoencapsulation is foreseen to yield significant benefits in nutrition and wellness. A goal is the development of more effective delivery systems, able to deliver bioactive compounds directly to the appropriate sites, maintain their concentration at

Nanotechnology Research Methods for Foods and Bioproducts, First Edition. Edited by Graciela W. Padua and Qin Wang. © 2012 John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc.

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2

●

●

●

Nanotechnology Research Methods for Foods and Bioproducts

suitable levels for long periods of time, and prevent their premature degradation. Nanostructured materials, such as liposomes, micelles and nanospheres, could be used to develop high-performance delivery vehicles for biologically active substances, such as nutraceuticals. Food quality: Nanostructured systems for the design of novel food matrices are being studied. Another area of development is the use of nanostructured carriers for enhanced delivery of flavors in foods. Food packaging: Nanocomposites for improving properties of packaging materials are in development. The addition of natural nanosized materials can render plastics lighter, stronger, more heat-resistant, with improved oxygen, carbon dioxide, moisture and volatile barrier properties. Such materials could enhance considerably the shelf-life and safety of packaged foods. With the emphasis on sustainability, nanocomposite technology may be applied to the development of biopolymers as viable packaging materials. Food safety assurance: Nanotechnology is helping design antigen-detecting biosensors to facilitate early identification of pathogen contamination.

The development of food materials through nanoscience involves understanding of the precise assembly and ordering of structures at a molecular scale that subsequently control the organization and integration of structures over several length scales. Food scientists and technologists will find themselves increasingly engaged in nanoscience and nanotechnology.1 This book covers nanoscale materials and structures (Part One), where the properties of food materials and biological components are described, self-assembly is explained, and the formation and applications of nanocomposites and nanocolloids are reviewed. Food nanotechnology is an expanding field. This expansion is based on the advent of new technologies for nanostructure characterization, visualization and construction. Indeed, nanotechnology is possible due to various techniques and instruments for detection and imaging that have only recently become available to researchers and engineers. They are expected to provide insights into meso- and nanostructural changes in food and biological systems and their relationship with their macroscopic properties.2,3 This book introduces the reader to a selection of the most widely used techniques in food and bioproducts nanotechnology. It is intended as a quick reference and a guide in the selection of research tools. The focus is on state-of-the-art equipment; thus, it contains a description of the tool kit of a nanotechnologist. The book will provide concise explanations for the technical basis of the methods being described, will highlight research opportunities and will point out methods’ pitfalls and limitations. Part Two covers nanostructure characterization techniques, starting with scanning electron microscopy (SEM), then transmission electron microscopy (TEM), dynamic light scattering(DLS), X-ray diffraction (XRD), Quartz crystal microbalance with dissipation (QCM-D) and focused ion beam, through to micro-computer tomography. This book is addressed to workers new in the field of nanotechnology. It is meant to inform students in formal and informal settings, new researchers and product development teams in the expanding field of food and bioproducts nanotechnology.

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Introduction

3

The rapid implementation of nanotechnology concepts in industry and academia creates the need for information on instruments and methods among researchers and product development teams. Also, the advent of new structures had led to regulatory reexamination of materials involved. The selection of appropriate characterization instruments and methods is critical to this endeavor.

References 1. Sanguansri, P. and Augustin, M.A. (2006) Nanoscale materials development: a food industry perspective. Trends Food Sci Technol 17, 547–556. 2. Hermansson, A., Langton, M. and Lorén, N. (2000) New approaches to characterizing food microstructures. MRS Bull 25, 30–36. 3. van der Linden, E., Sagis, L. and Venema, P. (2003) Rheo-optics and food systems. Current Opinion in Colloid & Interface Science 8, 349–358.

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2

Material components for nanostructures

Graciela W. Padua and Panadda Nonthanum

Abstract: Nanosized structures are commonly found in foods and biological products. They may be natural formations, such as casein micelles, or the result of processing, such as nanosized fat globules in homogenized milk. Nanotechnology is not involved in their formation; however, it is involved in their characterization and analysis. Nanoscience is involved in the formation of novel products, for example whey protein nanotubes, which are prepared by a combination of enzymatic and chemical treatments. The induced formation of nanostructures by provision of the correct environmental conditions involves nanoscale science. This chapter presents some of the most frequently used materials in nanoscale developments for foods and bioproducts. Keywords: nanostructure; micelle; liposome; self-assembly; supramolecular; length-scale; amyloidogenic protein; cyclodextrin; amphiphile; montmorillonite

2.1

Introduction

Food products naturally contain nanosize ingredients. Globular proteins may vary between 10 and several hundred nanometers in diameter. Milk naturally contains nanostructures, such as casein micelles. Many polysaccharides are ribbon-shaped polymers that are less than 1 nm thick. Also, nanostructures may be produced during routine food processing operations, such as homogenization. When milk is homogenized, fat globules are reduced to about 100 nm in size. The natural or fortuitous formation of such structures does not involve nanotechnology; however, their characterization does. Novel nanotubes from whey proteins are formed by combinations of enzymatic, chemical and physical treatments. The induced formation of nanostructures by providing the right environmental conditions involves nanoscale science. This chapter will present some of the most frequently used materials involved in food and bioproducts nanoscale developments. Nanotechnology Research Methods for Foods and Bioproducts, First Edition. Edited by Graciela W. Padua and Qin Wang. © 2012 John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc.

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Nanoscale-size foods include micelles, liposomes and nanoemulsions. Micelles are spherical structures 5–100 nm diameter. They form spontaneously when surfactants are dissolved in water. Micelles are able to encapsulate nonpolar molecules such as lipids, flavors, antimicrobials, antioxidants and vitamins. Ingredients which are not normally water soluble can be solubilized using micelles. Such systems are referred to as microemulsions. Micelles have been used in the pharmaceutical industry for a long time but have only recently attracted the interest of the food industry. Liposomes are spherical, polymolecular aggregates with a bilayer shell configuration. They vary in size from 20 nm to several hundred micrometers. Liposomes are formed by polar lipids, which are frequently found in nature, for example phospholipids from soy and eggs. Like micelles, liposomes can encapsulate a broad spectrum of functional ingredients. The difference however is that liposomes can encapsulate both water-soluble and fat-soluble ingredients. Liposomes are used to encapsulate sensitive proteins so that they retain their function irrespective of adverse environmental conditions, such as unfavorable pH. The shelf-life of milk products, for example, can be extended using liposomes. Nanoemulsions are fine, oil-in-water emulsions with a mean droplet size of 50–200 nm. They do not scatter visible light and are hence transparent. Due to their small particle size, nanoemulsions remain stable for long periods. The bioavailability of lipophilic substances can be increased considerably by means of nanoemulsions. Nanoemulsions have been in use for some time in parenteral nutrition. They also show unique textural properties, even at low oil concentration, and have the consistency of a viscous cream, which makes them interesting for the development of reduced fat products. Inorganic materials such as SiO2 can be prepared as colloids, where particles are nanosized. However, the particles tend to form aggregates larger than 100 nm. Silicon dioxide is not a new product; it has been used in the food industry for many years. Its structure and the fineness of its particles have not been altered.1

2.2

Self-assembly

The self-assembly of biomolecules has gained much attention from scientists in various fields of interest. It is a new realization that self-assembly is ubiquitous in the natural world. Both inorganic and organic molecules self-organize into complex structures.2 The well-defined structures of such supramolecular assemblies turn out to be prerequisites for their biological function. Examples are hemoglobin, phospholipid membranes, actin microtubules, membrane channels, collagen and casein micelles. Inspired by nature, nanoscale scientists and nanotechnologists attempt to synthesize functional structures via a “bottom-up” approach.3 Linear assemblies such as rod- and tube-like structures are of particular interest, since they have unique properties potentially useful in several applications.4 It is important to make a distinction between self-assembly and aggregation. Aggregation is a self-association reaction, usually irreversible, that leads to the formation of amorphous, flocculent aggregates which are heterogeneous in structure and biologically inactive.5 Both noncovalent and covalent interactions may be involved. Self-assembly is a special kind of aggregation that occurs towards a state

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of minimum free energy via noncovalent intermolecular forces, such as hydrogen bonds, electrostatic interactions, hydrophobic interactions and van der Waals forces. Although those interactions are small in magnitude, the large number of interactions in the final assembly is significant.6 The large entropic cost of ordering is only slightly offset by the favorable enthalpy gained from those weak interactions. The homogeneously ordered structure formed is in thermodynamic equilibrium, determined by conditions such as temperature, pressure, pH and the chemical potential of the molecules involved.7 Self-assembly relies on balancing the forces of attraction and repulsion between the molecular building blocks that form supramolecular structures. The forces between the building blocks can be influenced by a number of factors. By changing the temperature, concentration, pH and ionic strength of the system, mechanical force (pressure, shear, extension and ultrasound) or electric and magnetic field strengths, a large variety of ordered structures may be obtained. The final structure will depend on how chemical, physical and processing conditions are applied to influence the balance of intramolecular and intermolecular forces between the components in the system. Examples of self-assembled nanostructures in foods include the casein micelle, the structures formed in protein–polysaccharide coacervates, and liposomes. Ordered structures result from the coexistence of long-range repulsion forces (e.g. thermodynamic incompatibility, phase separation, excluded volume and columbic repulsion) and short-range attraction (e.g. hydrogen bonds and electric dipole interaction). Macromolecules of defined shape self-assemble due to the effects of excluded volume interactions. Block copolymers consist of binary polymer structures that self-assemble via phase-separation effects.8 The nature of the structures formed depends on the size of the polymer, its shape, the composition of the solution and its bulk phase, and environmental stress. Assembling a spatially defined supramolecular structure requires bi- or multifaceted building blocks.9 Bifaceted moieties or amphiphiles are widely found in nature. Amphiphilic molecules primarily use their hydrophobic sides to develop molecular associations and hydrophilic faces to interact with water. In this context, proteins are unrivalled in their potential to become building blocks. Protein folding, which is dominated by hydrophobic effects, is critical for molecular self-assembly. Protein folds are the result of sequence patterns in the primary structure, where hydrophobic (H) and polar (P) effects are due to the chemistries of amino acids. For example, alternating H and P residues produces β-strands, and H residues spaced sequentially three and four residues apart give α-helical structures. Proteins develop specific assemblies by using subtle differences in the chemistries of hydrophobic amino acids. Thus, protein geometry, which invariably has an impact on the ability of proteins to self-assemble and the characteristics of resulting assemblies, is defined at the lowest levels of structural hierarchy. In this notation, a-helical motifs probably offer the richest architectural flexibility. Among synthetic nanostructures, fibrous assemblies stand out as examples of hierarchical design. The study of fibrous assemblies has dominated the field of peptide self-assembly over the last decade. For example, peptide nanofibers are morphologically and scale-wise similar to the native extracellular matrix (ECM), which is a fibrous product of self-assembled collagen. ECM is an indispensable part of tissue growth and development and mimicking it synthetically may have a notable impact

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on regenerative medicine. The technological potential of nanostructures assembled from peptides is considerable in a diversity of fields.

2.3 2.3.1

Proteins and peptides Amyloidogenic proteins

Amyloidogenic proteins are polypeptides capable of self-assembly into β-sheet linear aggregates;10 such structures were first described in association with several diseases, but are now known to be of more general significance. Indeed, it has become apparent that many proteins that do not possess structural similarities or sequence homology with disease-related proteins are also capable of undergoing fibrillar self-assembly. Hierarchical self-assembly has been shown to be a powerful tool for the creation of nanostructured materials. Polypeptide fibrillar self-assembly may follow a two-step process. In the first step, protein molecules are assembled into elongated fibrils under conditions where the formation of intermolecular interactions is favored over intramolecular ones. The resulting nanofibrils are highly stable and rigid. These densely hydrogen-bonded structures can be formed from a range of different peptides and proteins. In the second step, the fibrils are cast into thin films. During the casting process, the fibrils align in the film plane and in the presence of plasticizers stack with nematic order, resulting in materials that have a hierarchy of length scales: nanometer ordering within the fibrils and micrometerscale ordering in the stacking of the fibrils. Such films have many similarities with films produced from monomeric proteins for applications in edible packaging materials. However, the two-step assembly process results in a well-defined hierarchy of length scales. Using this method, films with lateral dimensions up to the centimeter scale can be routinely fabricated. Film characterization by X-ray diffraction (XRD) reveals that they possess a high level of order at the nano- and micrometer scales. Insight into the structure of the material can be obtained from examining the characteristic distances and orientations of the two major length scales present. The individual β-strands composing the nanofibrils are separated by 4.8 Å along the fibril direction. Sheets, composed of laterally associated strands in parallel arrays, have a characteristic distance of 12 Å in the direction perpendicular to the strand repeat. When plasticizing molecules such as polyethylene glycol (PEG) are added to the suspension of nanostructures before casting, further ordering is observed and the fibrils adopt a nematic order in the solid phase. Liquid-crystalline behavior of elongated biomolecular assemblies in solution is well known and the presence of a plasticizer could enable this orientational order to be preserved in the solid phase through interfibril lubrication, competition for interfibril interactions, or by enhancing the tendency for orientational ordering as a result of depletion interactions. Owing to the combination of accurate self-assembly and chemical versatility with regard to possibilities for surface functionalization, such protein-based nanostructured films represent an attractive path towards new multifunctional materials built from the bottom up. Nanometer-sized tubular structures may be formed by self-assembly of partially hydrolysed α-lactalbumin.11 Hydrolysis is needed to disassemble α-lactalbumin into

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peptides prone to self-assembly. At neutral pH and in presence of an appropriate cation, such building blocks self-assemble to form micrometer-long tubes with a diameter of only 20 nm. These long, straight nanotubes withstand heating and mechanical deformation. The α-lactalbumin nanotubes show that it is possible to create interesting nanostructures based on food proteins. Because of their linearity, nanometer-sized cavity and controlled disassembly, the α-lactalbumin nanotubes have potential to be used as novel ingredients with specific functionality.2

2.3.2

Collagen

Collagen is the most abundant protein in the body of invertebrates and is the principal structural element of the ECM of connective tissue. The protein self-assembles into a triple helix which contains the amino acid glycine at about every third position. The production of novel materials from collagen has attracted interest from a wide variety of biomedical applications, especially tissue engineering. In terms of food applications, collagen is the starting material for gelatin and mats spun from collagen may thus have interesting properties in clarification or gelation applications. Collagen, along with other proteins including gelatin, casein, zein and eggshell membrane protein, has been made into nanofibers by electrospinning. This is a process where a high voltage is used to create an electrically charged jet of polymer solution or melt, which dries or solidifies into a fine fiber in the micro- or nanoscale.12 Collagen nanofibers with a size range of 100 ± 40 nm and with few bead defects were fabricated at 8.3 wt% from type I calf-skin collagen, while a less uniform fiber matrix was obtained from type I collagen of human placenta (fiber sizes ranged from 100 to 730 nm). The type III collagen from human placenta was electrospinnable, forming defect-free nanofibers at concentrations of 4 wt%, yielding fibers of 250 ± 150 nm. Blending type I and type III collagen led to fibers with average diameters of 390 ± 290 nm.13

2.3.3

Gelatin

Gelatin is derived from collagen by acidic (gelatin A) or alkaline (gelatin B) extraction and thermal denaturation. Like collagen, gelatin or blends of gelatin and synthetic biodegradable polymers have become attractive in the field of tissue engineering due to their excellent biocompatibility, biodegradability, low cost and wide availability. Gelatin, while soluble in water, cannot be electrospun from aqueous solutions due to the extensive hydrogen bonding – which results in gel formation in the capillary of the syringe at room temperature – the low volatility of water and the excessively high surface tension. Li and coworkers14 succeeded in electrospinning 15 wt% gelatin in 10 wt% ethanol solution. Smooth and homogeneous fibers with an average diameter of 212 nm were produced. Blending of 4.5 wt% gelatin with hyaluronic acid at ratios ranging from 10 : 1 to 10 : 3 in an aqueous environment resulted in smooth, uniform fibers. Fiber diameters increased from 200 to 400 nm with increasing hyaluronic acid concentration or gelatin concentration. Crosslinking of these gelatin fibers in 80% (v/v) ethanol solution rendered the gelatin and gelatin– hyaluronic acid composite fibers water resistant.

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2.3.4

Caseins

Caseins are a group of flexible milk proteins of molecular weight ranging from 19 to 25 kDa. More than 55% of the amino acids that make up casein molecules contain polar side groups. Thus, there is an extensive network of inter- and intramolecular hydrogen bonds among casein molecules, which are believed to be responsible for their poor electrospinnability.

2.3.5

Wheat gluten

Wheat gluten is a polydisperse plant protein that consists of a higher-molecularweight protein, glutenin, and a lower-molecular-weight protein, gliadin. It is extensively used in the food industry to strengthen dough networks and to manufacture texturized protein products such as meat analogues. Its viscoelastic properties stem from a combination of disrupting and reforming disulfide bonds and chain entanglement, leading to a structure that is similar to that of a crosslinked melt. An acetic acid-extracted wheat protein fraction produced fibers by electrospinning. Circular, flat, and ribbon-like fibers with a broad range of diameters (25 nm–5 μm) were produced. The number of ribbons increased with increasing concentrations of high-molecular-weight polymers in gluten.

2.3.6

Zein

Zein is the major storage protein of corn. It accounts for roughly 50 wt% of corn endosperm proteins. Zein is not a single protein but rather a mixture of proteins of different molecular weights and solubilities. Zein fractions are classified according to their solubility as type α, β, γ or δ. Zein has been used in industry for the manufacture of fibers, buttons and binders, among other things. One of its best known uses is as a coating material for food or tablets. Ribbon-like but bead-defect-free fibers were obtained by electrospinning of 30–50 wt% zein in 70 wt% aqueous ethanol with fiber diameter increasing from 1 to 6 μm as the polymer concentration was increased. The occurrence of ribbons instead of cylindrical fibers was explained by the rapid evaporation of ethanol, which first caused a skin formation around the fibers and then a collapse of the outer skin due to the subsequent evaporation of the residual solvent. Another condition yielding fibrous structures with almost no bead defects was 30–40 wt% zein in 80–90 wt% ethanol. Glacial acetic acid, on the other hand, produced ribbon-like fibers with 27–30 wt% zein solutions. Fibers had a narrower diameter distribution, ranging from 1 to 5.6 μm.

2.3.7

Eggshell membranes

Eggshell membranes consist of a macroporous interwoven protein fiber network. It is crucial in the formation of eggs and the development of the chicken embryo. The membrane consists of a complex mixture of various proteins such as collagen type I, V and X, as well as osteopontin and sialoprotein (two phosphoproteins). This membrane is a byproduct that is typically discarded during the manufacturing of

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pasteurized egg yolk and white. Its utilization has been limited due to the presence of high concentrations of heavy metals. Nevertheless, the presence of polycationic functional groups, its biological origin and its composition have generated interest in its use for tissue engineering and as an enzyme immobilization platform. However, eggshell membrane proteins are stabilized by an extensive network of disulfide bonds, preventing direct electrospinning.

2.3.8

Bovine serum albumin

Bovine serum albumin (BSA), while not being electrospinnable, has attracted interest from researchers working on the production of nanofibers due to the fact that it has been widely used in the pharmaceutical and biomedical industries as a model protein for the encapsulation and delivery of drugs. Like the previously described approaches, combination with other polymers such as poly(ethylene glycol), poly(vinyl alcohol) and dextran resulted in the formation of fibers that allowed for a controlled release of the protein. Other studies involved the use of BSA as a carrier for functional bioactive compounds such as the human nerve growth factor.

2.3.9

Enzymes

Enzymes are biological catalysts, essential to the control of most chemical reactions occurring in living cells. Difficulties for their industrial-scale utilization have been attributed to a loss of activity in environments different from those in which they normally function. Only a limited number of studies on enzyme electrospinning have been published, despite the great appeal that such structures could hold as mini reactors. In most cases, these bioactive compounds were attached to prefabricated nanofibers through adsorption and subsequent covalent linkage of the enzyme to the surface of fibers. Examples of enzymes attached to surfaces include lipase and catalase. Sawicka and coauthors15 described the successful electrospinning of poly(vinyl pyrrolidone) with urease. The authors reported that enzyme activity was retained after processing in the high electrical field, producing nanofibers of 7–100 nm intersected by spherical urease aggregates of 10–800 nm. The large surface area of the nanofibers greatly improved the reaction rate with the substrate and thus reduced the response time. Composite fibers containing 1.3 wt% chymotrypsin had an average fiber diameter of 815 ± 190 nm. Interestingly, the relative bioactivity of the enzyme was better retained in nanofibers over the course of two weeks when compared with the bioactivity of unencapsulated chymotrypsin, which rapidly decreased to below its detection limit within one day.

2.4 2.4.1

Carbohydrates Cyclodextrins

Cyclodextrins (CDs) are cyclic oligosaccharides in which 6 (α), 7 (β), 8 (γ) or 9 (δ) glucose monomers16 are linked through α(1–4) glycosidic bonds with the formation of a hollow truncated conical structure. They are crystalline, homogeneous, nonhygroscopic and nontoxic substances formed during enzymatic decomposition

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of amylase. CDs contain a relatively hydrophobic internal cavity, which can include various inorganic and organic molecules, and a hydrophilic surface, which has primary and secondary hydroxyl groups. The diameter of the CD cavity increases in proportion to the number of glucose units – 5.7, 7.8 and 9.5 Å for α, β and γCD, respectively – but the depth remains the same: 7.9 Å. The volume of the cavity is 174, 262 and 472 Å for α, β and γCD, respectively.17 CDs are used in the production of nanoscale materials to improve morphology and size distribution. In the food industry, CDs are used for the prevention of losses during storage due to lipid oxidation, decomposition by light, thermal decomposition and volatility. The conversion of volatile substances from gas or liquid state into solid powders through complexation with CD is of a great practical value. Thus, the CO2–αCD complex can be used as baking powder and ethanol–βCD complexes can be used for the preservation of products. With the use of CD, fragrant or medicinal substances are extracted from plants, and compounds with a bitter taste, such as phenylalanine in protein hydrolyzate, are removed. The bitter taste of grapefruit juice is removed by adding 0.3% of βCD. Soybeans are exempt from the typical grassy smell and astringent taste when mixed with CD. 80–90% of cholesterol can be removed from eggs, pork or cheese through the formation of inclusion complexes (ICs) with CD. The unpleasant taste of coffee or tea instant drinks, remaining after extraction or boiling, or caused by an inappropriate raw material, can be removed by adding CD. The most important property of CD for both practical application and scientific research is the ability to selectively form ICs with other molecules, ions and radicals.18 The formation of ICs is based on the interactions of a noncovalent nature, electrostatic, van der Waals and steric effects. The driving forces of the formation of ICs are van der Waals (or hydrophobic) interactions between hydrophobic “guest” molecules and the CD cavity, and hydrogen bonds between polar functional groups of “guest” molecules and CD hydroxyl groups. One of the promising new areas of application of CDs is the production of nanoscale materials. Magnetite nanoparticles were prepared in the presence of βCD by the formation of ICs between metal oxide and the CD.19 In this case, βCD was used simultaneously as a stabilizer of and finisher for nanoparticles, “selecting” the particles with a size less than 2 nm. The synthesis of magnetite nanoparticles covered with a coating of βCD molecules for use in medical diagnosis and therapy was reported by Racuciu and coworkers.20 Nanoparticles suspended in a pH-neutral medium are obtained by chemical deposition of iron (II) and (III) salts in the presence of βCD. CD supramolecular complexes, also known as molecular necklaces, consist of linear molecular chains on which ring molecules in particular CDs are “threaded”. Molecular necklaces can be formed in the process of self-organization by spontaneous threading of CD molecules on long polymer molecules such as PEGs. The use of CDs opens up a wide range of possibilities for the surface modification of nanoparticles, particularly for the phase transfer between aqueous and organic media.

2.4.2

Cellulose whiskers

Micro- and nanocrystals of cellulose can be obtained by acid hydrolysis of cellulose fibers. Nanocellulose crystal dimensions are typically 100–300 nm length and 3–10 nm width. These fiber-like crystals, often referred to as nanowhiskers, display

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an elastic modulus of 120–150 GPa and are readily obtained from renewable biosources such as bacteria, wood, cotton and sessile sea creatures called tunicates.21 Cellulose whiskers, and their use as a reinforcing material in composites, are a relatively new field within nanotechnology and have generated considerable interest within the biopolymer community. The incorporation of small amounts of highstiffness, high-aspect-ratio nanometer-sized fillers into polymers is a design approach for the creation of new materials with tailored mechanical properties. Because of their strongly interacting surface hydroxyl groups, cellulose nanowhiskers have a significant tendency for self-association, which is an advantage in the formation of load-bearing percolating architectures within the host polymer matrix. The effective reinforcement of polymers observed for this class of materials is attributed to the formation of rigid nanowhisker networks in which stress transfer is facilitated by hydrogen bonding between nanowhiskers.22 Cellulose nanowhiskers ranging in size from 10 to 20 nm in width, with an aspect ratio of 20–100, were added at low concentrations (2–10% (w/w)) to starch gels and films as reinforcing agents. Significant changes in mechanical properties, especially maximum load and tensile strength, were obtained for fibrils derived from several cellulosic sources, including cotton, softwood, and bacterial cellulose. For extruded starch plastics, the addition of cotton-derived microfibrils at 10.3% (w/w) concentration increased Young’s modulus by fivefold relative to a control sample with no cellulose reinforcement. However, addition of microfibrils does not always change mechanical properties in a predictable direction. Whereas the tensile strength and modulus of extruded thermoplastic starch and cast latex films increased with addition of microfibrils, these parameters decreased when microfibrils were added to a starch– pectin blend, implying that complex interactions are involved in the application of these reinforcing agents.23 Cellulose whiskers were used to reinforce soy protein isolate (SPI) plastics. Cellulose whiskers with an average length of 1.2 mm and diameter of 90 nm were prepared from cotton linter pulp by hydrolysis with sulfuric acid. The effects of whisker content on the morphology and properties of glycerol-plasticized SPI composites were investigated by scanning electron microscopy, dynamic mechanical thermal analysis, differential scanning calorimetry, ultraviolet-visible spectroscopy and tensile testing. Strong interactions between whiskers and SPI matrix were observed upon addition of whiskers at 0–30 wt%. Cellulose whiskers reinforced the composites, while preserving their biodegradability.24

2.5

Protein–polysaccharides

Protein–polysaccharide nanostructures are used as structuring agents in food colloids such as whey protein–gum arabic and sodium caseinate–gum arabic. Novel protein– polysaccharide aggregates may be formed by using static high-pressure technology, for example, to dissociate and reassemble native casein micelles in the presence of interacting hydrocolloids such as low-methoxyl pectin or i-carrageenan. In model emulsion studies, rheological and stability properties can be attributed to the presence of associative interfacial interactions between protein and polysaccharide ingredients. Examples include whey protein–carboxy methylcellulose, sodium caseinate–low-methoxyl

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pectin, sodium caseinate–high-methoxyl pectin, β-lactoglobulin–high-methoxyl pectin, canola protein–k-carrageenan, whey protein–xanthan gum and sodium caseinate– gellan gum.25

2.6

Liquid crystals

Amphiphilic lipids such as monoglycerides, phospholipids and glycolipids selfassemble spontaneously in water to form various well-ordered nanostructures: a fluid isotropic micellar phase (L2), a lamellar phase (Lα), an inverted hexagonal phase (H2) and a reversed bicontinuous cubic phase (V2). The type of phase formed by a specific system may be predicted utilizing the critical packing parameter (CPP = ν/(lc a0), where lc is the effective length of an amphiphile chain, a0 is the effective amphiphile headgroup area and ν is the average volume occupied by the amphiphile chain). Molecules with a CPP less than one will preferentially form normal phases, while those with a CPP greater than one – that is, molecules with an effective reverse wedge shape – will preferentially form inverse phases. Reversed bicontinuous cubic phases consist of a single continuous curved lipid bilayer forming a complex network with 3D cubic symmetry, which separates two continuous but nonintersecting water channels. It is generally believed that the morphology of bicontinuous cubic phases can be described as infinite periodic minimal surfaces (IPMS), where the minimal surface is defined by the lipid bilayer center. Minimal surfaces have zero mean curvature, meaning that they are, at all points, as convex as they are concave. To date, three types of IPMS have been observed in amphiphile–water systems, namely the gyroid (G), diamond (D) and primitive (P) surfaces, which correspond to space groups Ia3d (G), Pn3m (D) and Im3m (P).26 Polar lipids self-assemble in aqueous environments into lamellar, cubic and hexagonal (HII) liquid crystalline phases. These phases provide important functional properties due to their molecular organization. Cubic phases are the most complex of the lipid–water liquid crystals. Structure determination was first done by nuclear magnetic resonance (NMR) diffusion measurements, combined with an XRD analysis. Electron microscopy studies indicate that each cubic particle is a single crystal. Particles of the bicontinuous cubic phase appear to be the most important with regard to application possibilities, involving encapsulation of enzymes with stabilization of their native structures. Future developments include the incorporation of enzymes in foods. Such nanoparticles may function as micro reactors and be utilized, for example, for inactivation of oxygen radicals or flavor production and release during the storage of food products.27

2.7

Inorganic materials

Numerous processes have been developed to impart oxygen barrier properties to transparent plastic films used in packaging applications.28 Silicon oxide (SiOx) barrier films are particularly useful due to their low oxygen transmission rate (OTR), high transparency and microwaveability.29

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SiOx coatings are produced using plasma-enhanced chemical vapor deposition. Despite being a relatively mature technology, SiOx coatings suffer from a variety of problems, including cracking and poor adhesion to plastic substrates. Polymer multilayer (PML) is a more recent technology that uses conventional sputtering to build an alternating polymer/ceramic film. The thickness of each layer is an order of magnitude thicker than that deposited using layer-by-layer (LbL) deposition. Adding clay to polymers is a third technique used to reduce OTR. Montmorillonite (MMT) layered silicates are ceramic platelets with a high aspect ratio (typically more than 100 nm in diameter and 1 nm thick). This high aspect ratio creates a tortuous path for gas molecules moving through the polymer matrix, leading to a large diffusion length that retards permeation. LbL assembly using polymers and nanoclay is reported as a means to produce thin films with barrier properties that are unrivaled by the technologies described above. LbL assembly is an aqueous coating technology whereby films are produced through alternate exposure of a charged (or polar) substrate to water-based solutions (or mixtures) containing charged (or polar) ingredients. Each pair of complementary layers is referred to as a bilayer, which is typically 1–100 nm thick depending on chemistry, molecular weight, temperature, counter ion, ionic strength and pH. Thin films of negatively charged sodium MMT clay and cationic polyacrylamide were grown on a polyethylene terephthalate film using LbL assembly. After 30 clay–polymer layers were deposited, with a thickness of 571 nm, the resulting transparent film had an OTR below the detection limit of commercial instrumentation (0.005 cc/m2/day/atm). This unique combination of materials and processing can reduce the OTR of polyethylene terephthalate (PET) by four orders of magnitude without diminishing its transparency or flexibility. This technology suffers from moisture sensitivity, but lamination with a water-barrier film allows the low OTR to be maintained at 23°C and 95% relative humidity (RH). Films made by LbL processes provide transparency (>93% throughout the visible light spectrum) and flexibility, making them good candidates for foil replacement in a variety of packaging applications. This high barrier behavior is believed to be due to a brick wall nanostructure present within the thin film, which produces an extensive tortuous path for a diffusing oxygen molecule. The combination of oxygen barrier and transparency displayed by LbL composites makes them ideal candidates for food packaging.

References 1. Greßler, S., Gazsó, A., Simkó, M., Nentwich, M. and Fiedeler, U. (2010) Nanoparticles and nanostructured materials in the food industry. Nano Trust Dossiers 004en, 1–5. 2. Graveland-Bikker, J.F. and de Kruif, C. G. (2006) Unique milk protein based nanotubes: food and nanotechnology meet. Trends Food Sci Technol 17, 196–203. 3. Drexler, K.E. (1981) Molecular engineering: an approach to the development of general capabilities for molecular manipulation. Proc Natl Acad Sci USA 78, 5275–5278. 4. Bittner, A.M. (2005) Biomolecular rods and tubes in nanotechnology. Naturwissenschaften 92, 51–64. 5. Kentsis, A. and Borden, K.L.B. (2004) Physical mechanisms and biological significance of supramolecular protein self-assembly. Curr Protein Peptide Sci 5, 125–134.

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6. Rajagopal, K. and Schneider, J.P. (2004) Self-assembling peptides and proteins for nanotechnological applications. Curr Opin Struct Biol 14, 480–486. 7. Oosawa, F. and Asakura, S. (1975) Thermodynamics of the Polymerization of Protein, London: Academic Press. 8. Förster, S. and Konrad, M. (2003) From self-organizing polymers to nano- and biomaterials. J Mater Chem 13, 2671–2688. 9. Ryadnov, M. (2007) Peptide alpha-helices for synthetic nanostructures. Biochem Soc Trans 35, 487–491. 10. Knowles, T.P.J., Oppenheim, T.W., Buell, A.K., Chirgadze, D.Y. and Welland, M.E. (2010) Nanostructured films from hierarchical self-assembly of amyloidogenic proteins. Nat Nanotechnol 5, 204–207. 11. Graveland-Bikker, J.F., Ipsen, R., Otte, J. and de Kruif, C.G. (2004) Influence of calcium on the self-assembly of partially hydrolyzed alpha-lactalbumin. Langmuir 20, 6841–6846. 12. Kriegel, C., Arrechi, A., Kit, K., McClements, D.J. and Weiss, J. (2008) Fabrication, functionalization, and application of electrospun biopolymer nanofibers. Crit Rev Food Sci Nutr 48, 775–797. 13. Matthews, J.A., Wnek, G.E., Simpson, D.G. and Bowlin, G.L. (2002) Electrospinning of collagen nanofibers. Biomacromolecules 3, 232–238. 14. Li, J., He, A., Zheng, J. and Han, C.C. (2006) Gelatin and gelatin-hyaluronic acid nanofibrous membranes produced by electrospinning of their aqueous solutions. Biomacromolecules 7, 2243–2247. 15. Sawicka, K., Gouma, P. and Simon, S. (2005) Electrospun biocomposite nanofibers for urea biosensing. Sens Actuators B 108, 585–588. 16. Chernykh, E.V. and Brichkin, S.B. (2010) Supramolecular complexes based on cyclodextrins. High Energy Chem 44, 83–100. 17. Nepogodiev, S.A. and Stoddart, J.F. (1998) Cyclodextrin-based catenanes and rotaxanes. Chem Rev 98, 1959–1976. 18. Dodziuk, H. (2006) Cyclodextrins and their Complexes: Chemistry, Analytical Methods, Applications, Weinheim: Wiley-VCH. 19. Bocanegra-Diaz, A., Mohallem, N.D.S. and Sinisterra, R.D. (2003) Preparation of a ferrofluid using cyclodextrin and magnetite. J Braz Chem Soc 14, 936–941. 20. Racuciu, M., Creanga, D., Badescu, V. and Airinei, A. (2007) Synthesis and physical characterization of magnetic nano-particles functionalized with beta-cyclodextrin. J Optoelectron Adv Mater 9, 1530–1533. 21. Goetz, L., Mathew, A., Oksman, K., Gatenholm, P. and Ragauskas, A.J. (2009) A novel nanocomposite film prepared from crosslinked cellulosic whiskers. Carbohydr Polym 75, 85–89. 22. Capadona, J.R., Shanmuganathan, K., Triftschuh, S., Seidel, S., Rowan, S.J. and Weder, C. (2009) Polymer nanocomposites with nanowhiskers isolated from microcrystalline cellulose. Biomacromolecules 10, 712–716. 23. Orts, W.J., Shey, J., Imam, S.H., Glenn, G.M., Guttman, M.E. and Revol, J. (2005) Application of cellulose microfibrils in polymer nanocomposites. J Polym Environ 13, 301–306. 24. Wang, Y., Cao, X. and Zhang, L. (2006) Effects of cellulose whiskers on properties of soy protein thermoplastics. Macromol Biosci 6, 524–531. 25. Dickinson, E. (2009) Hydrocolloids as emulsifiers and emulsion stabilizers. Food Hydrocoll 23, 1473–1482. 26. Kaasgaard, T. and Drummond, C.J. (2006) Ordered 2-D and 3-D nanostructured amphiphile self-assembly materials stable in excess solvent. Phys Chem Chem Phys 8, 4957–4975.

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27. Larsson, K. (2009) Lyotropic liquid crystals and their dispersions relevant in foods. Curr Opin Colloid Interface Sci 14, 16–20. 28. Jang, W., Rawson, I. and Grunlan, J.C. (2008) Layer-by-layer assembly of thin film oxygen barrier. Thin Solid Films 516, 4819–4825. 29. Bieder, A., Gruniger, A. and von Rohr, P.R. (2005) Deposition of SiOx diffusion barriers on flexible packaging materials by PECVD. Surf Coat Technol 200, 928–931.

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3

Self-assembled nanostructures

Qin Wang and Boce Zhang

Abstract: Self-assembly is also known as self-association and self-organization. It has been considered as the most powerful strategy to create novel technologies and nanostructured materials, including spheres, layers, fibers and tubes, which have been generated and extensively studied in the agriculture, food and pharmaceutical areas. A wellestablished consensus is that a morphological difference can have substantial influence on a material’s physical, chemical and biological properties. Therefore, in this chapter, we explain mechanism and preparation methods according to the morphological category of the nanostructure. In the section on self-assembly, we focus on formation and fabrication technology in the production of micelles, fibers and tubes from food biopolymers. Because of the exclusively broad applications of layer-by-layer assembly and nanoemulsion, these two concepts are introduced individually. Keywords: self-assembly; layer-by-layer assembly; nanoemulsion; fabrication

3.1

Introduction

Nanostructures from food biopolymers, including spheres, layers, fibers and tubes, have been generated and studied in the areas of agriculture, food and pharmaceuticals. These structures can be produced by a series of fabrication methods, including self-assembly, layer-by-layer (LbL) construction and nanoemulsion templates. Each of the three methods has unique advantages and disadvantages and will be discussed in detail in this chapter. The nanostructures are generated from a group of polymers, both synthetic and natural. However, this chapter will emphasize nanostructures derived from food polymers, including but not limited to polylactic acid (PLA), chitosan, milk proteins (i.e. casein and whey protein), alginate, gelatin, carrageenan, zein and soy proteins. Furthermore, a comparison between the nanostructures of these natural polymers and of synthetic ones will be illustrated. The examples of nanostructures will be introduced and discussed in terms of their physicochemical Nanotechnology Research Methods for Foods and Bioproducts, First Edition. Edited by Graciela W. Padua and Qin Wang. © 2012 John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc.

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properties and their potential applications in different areas. Mechanisms of formation of different types of nanostructures using the presented methods will also be included, to better explain the theory part of nanostructure formation.

3.2 3.2.1

Self-assembly Introduction

Self-assembly is also known as self-association and self-organization. It has been considered the most powerful strategy to create novel technologies and nanostructured materials.1,2 Self-assembly often happens “when certain types of components under appropriate environmental and preparation conditions undergo assembly, leading to well-defined structures to minimize the free energy of the system”.3 However, selfassembly can also result in a reduction of entropy in the system, which is thermodynamically unfavorable. Therefore, there must be some forces balancing the reduction of entropy. These forces are mainly weak interactions, including hydrophobic interactions, electrostatic interactions, hydrogen bonds, π–π interactions and van der Waals forces.3 According to McClements,3 the self-assembly phenomenon can be divided into spontaneous self-assembly, directed self-assembly and undirected self-assembly. These are distinct in their requirements for certain conditions (e.g. pH, temperature, order of mixing or ionic strength), with spontaneous self-assembly having the least requirements. Examples of self-assembly structures of micelles, tubes and fibers will be discussed in detail in this section, with an attempt to compare their formation mechanisms as well as their physicochemical properties.

3.2.2

Micelles

Micelle structures can be self-assembled from polysaccharides, proteins, peptides and copolymers. The primary driving force is the reduction of systematic free energy, which is mainly contributed by weak interactions, again including hydrophobic interactions, hydrogen bonds, π–π, electrostatic interactions and van der Waals forces.3 Numerous studies have reported the self-assembly of peptides or copolymers into micelle structures.4–9 In early studies, peptides were designed to mimic traditional surfactants. These surfactant-like peptides could spontaneously self-assemble into well-ordered nanostructures.9 Copolymers can be roughly divided into several groups based on their subunit arrangements, such as alternating copolymers, periodic copolymers, block copolymers, graft copolymers and so on. However, based on the specificity of biopolymers, alternating copolymers and periodic copolymers were relatively rare, so only block copolymers and graft copolymers were reported as building blocks of micelles. Several techniques were used in preparing surfactant-like peptide molecules, such as mimicking natural lipids, modifying hydrophilic heads and modifying hydrophobic tails. Yang et al.10 referred natural lipids as a guideline in their study. A class of oligopeptides was prepared to mimic natural lipids. Each peptide consisted of a charged or polar head as a hydrophilic group, and a tail of hydrophobic amino acid residues. These peptides self-assembled into micelles, membranes and even tubes.

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The study also revealed that the critical aggregate concentration (CAC) was closely related to the amino acid sequence, such that CAC decreases with the addition of a hydrophobic tail.10 Santoso4 reported a modification method which altered the monodispersity of the self-assembled nanostructures. In this method, a string of glycine tail was covalently bound to the hydrophilic head of aspartic acid. The reduction in the length of the glycine tail from 10 residues to 4 residues resulted in a significant increase in monodispersity. The monodisperse micelles also showed potential application as a scaffold material for constructing diverse nanodevices and as a delivery vessel for encapsulating rudimentary enzymes.4 Zhang et al.11 also studied how the charge of the hydrophilic head might affect self-assembly behaviors and applications of the surfactant-like peptides.5,6 The cationic hydrophilic head consisted of one or two lysine or histidine residues, whereas the anionic head was designed with acidic residues (i.e. aspartic acid). Both cationic and anionic peptides were likely to form nanotubes, nanovesicles and a 3D network. However, the application was distinct because cationic micelles were capable of encapsulating and delivering small waterinsoluble or large biological molecules, including negatively charged nucleic acids.6 Another category of self-assembled micelles was obtained from copolymers. Several studies focused on block copolymers and graft copolymers of both polypeptides and polysaccharides. Carlsen12 developed an amphiphilic block copolymer based on polypeptides. In the study, the polypeptide-based copolymer was proposed to be controllable over intra–intermolecular interactions, and consisted of stable and modifiable secondary and tertiary structures. Peng et al.7 investigated self-assembled micelles from chitosan-derived copolymer. In their studies, chitosan was first modified to N-phthaloyl-carboxymethylchitosan. The modified chitosan molecule self-assembled into vesicles or micelles under different conditions in water and N,N-dimethylformamide solution. Prepared structures had a diameter of 80–240 nm, which was a potential carrier for bioactive compounds. A recent finding applied chitooligosaccharides as a backbone, which was grafted by polycaprolactones as branches.8 Then the graft copolymer selfassembled into giant vesicles in a water–dioxane mixture. Gao also claimed the vesicles as a carrier for semiconductor materials, including quantum dots and metal clusters.8

3.2.3

Fibers

Since the development of the concept of supramolecular chemistry and weak interaction, many self-assembled supramolecular polymers have been created, such as self-assembly monolayers, liquid crystals, self-assembly colloids and so on. The conceptual and technological advances have enabled the possibility of using molecular self-assembly to create nanofibers. Nanofibers can self-assemble from various components, such as polypeptides and polysaccharides. According to the physicochemical properties of building-block components, two techniques (i.e. electrospinning and hydrogel formation) are usually involved in making nanofibers. 3.2.3.1 Electrospinning Electrospinning is a mature technique, patented early in textile manufacturing. It applies a high voltage to draw a fine electrically charged fiber from a polymer solution. The process is believed to be noninvasive and does not involve severe chemical

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Syringe High-voltage power supply

Spinneret

V Polymer threads Fiber Collector

Fig. 3.1

Schematic view of the electrospinning process.

reactions or high temperatures in producing fiber threads. A schematic view of the electrospinning technique is shown in Fig. 3.1. Nowadays, electrospun nanofibers have many applications, such as in tissue engineering, artificial organ implant materials, drug delivery, wound dressing and so on. However, there are certain limitations or requirements on the polymer materials used to produce electrospun materials. First, the polymer threads are generated due to the high voltage (usually 5–50 kV) between the spinneret and the collector. The electrical power can only draw the polymer solution into threads when the polymer itself carries a charge. However, in the case of most biopolymers, such as most polysaccharides and protein on its isoelectric point (PI), the molecules are normally uncharged. Therefore, this limitation narrows the application of the electrospinning technique to a wide selection of biopolymer materials. Changes from either chemical modification or experimental conditions are necessary for uncharged biopolymers. For instance, Wu13 applied starch acetate in formic acid/water solution to produce electrospun fibers. The fibers had a diameter of 50 ± 5 μm, which showed a low initial burst and constant drug release rate in the resultant experiment. Other studies adopted chitosan,14–16 which was the only natural polysaccharide to carry a positive charge in acidic solution. In these studies, thin and homogenous chitosan fibers with diameters around 100 nm were reported. In Geng’s study,16 the formation of fine fibers was attributed to the reduction of surface tension due to the high electric field (5 kV/cm) applied in the electrospinning device. Second, for most protein-based materials, structure is critical in maintaining functionality. The electrospinning process doesn’t involve chemical reactions or high temperatures, which can damage proteins’ primary, secondary and tertiary structures. However, high voltage plus the aforementioned chemical modification and environmental change may induce protein denaturation. Therefore, when severe environmental conditions (e.g. severe pH or organic solvent) are applied, bioactive

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protein molecules are no longer suitable for electrospinning. As a result, only inert storage proteins (i.e. albumin and zein) have been studied in producing electrospun protein fibers. Bovine serum albumin (BSA) was electrospun into proteinous fiber, which was then combined with fluorescein isothiocyanate.17 The fluorescent fiber was designed as a pH sensor. Yao et al.18 studied a combination of zein and poly-L-lactide acid (PLLA) nanofibers produced by electrospinning. The electrospun fibers showed a diameter of around 100 nm, which showed strong tensile strength and large elongation. 3.2.3.2 Hydrogel formation Because not all biopolymers are capable of electrospinning, hydrogel formation was used as an alternative method of producing biopolymer nanofibers. In this method, most biopolymer materials are oligomeric peptides. A 16-residue β-sheet peptide can self-assemble into a network of interwoven nanofibers in salt solution.11 The nanofibers have a diameter of 10–20 nm, which can lead to hydrogel fabrication for potential application of 3D cell cultures and tissue engineering.19 Silva et al.20 designed a peptide amphiphilic molecule which presented bioactive epitopes. The peptide was then proven to be capable of self-assembly into a 3D cell culture network with calcium, and promoted the rapid formation of human neuron from a neural progenitor cell.20 Other studies21,22 also investigated pH and salt-triggered peptide selfassembly. Gao et al.23 concluded that all these peptidic nanofibers resulted in the formation of hydrogels (e.g. hydrogelation). They also mentioned that hydrogelation was an obvious macroscopic change in materials’ texture and appearance, so this visual feature enabled an initial assay for identifying nanofiber self-assembly. This rapid examination method was proposed to accelerate the exploration of a large variety of biopolymers and of the formation of molecular nanofibers and hydrogels.23

3.2.4

Tubes

A nanotube is a nanometer-scale tubelike structure, and was reported by many studies as a potential bioactive carrier. The earliest nanotube was a carbon nanotube, which had a similar structure to fullerene, known as C60. Carbon nanotubes have been studied as a potential drug delivery vessel,24,25 but their toxicity has become a major concern.26 Therefore, research interest has turned to biocompatible materials, such as biomaterials. Most of the biomaterial nanotubes are made from peptides and proteins. There is no study regarding the self-assembly of nanotubes from oligosaccharide or polysaccharide. This absence of a study in carbohydrate is due to the structural specificity of protein and the mechanism of protein nanotube self-assembly, as the formation of peptide nanotubes is due to the hydrogen bond between “the oxygen atom of the precipitating carbonyl group and the hydrogen atom of the amino group”.27 Several studies have investigated molecular dynamics simulations of protein nanotube formation through computational validation methods.27–29 Tarek proposed three possible schemes for the self-assembly of peptide nanotubes:27 (i) a cyclic peptide self-assembles in one dimension, along the tube axis; (ii) a unit cell is made of an asymmetric unit that is repeated in two dimensions, both along the tube axis and along its wrapping axis; and (iii) a unit cell is repeated as above, but the

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nanotube comprises several asymmetric units, where each such unit consists of two self-assembling domains. Both oligomeric peptides and proteins have been examined in previous studies. Several structural similarities were shared between those building-block components. Tarek27 prepared a cyclic peptide with eight amino acids – cyclo[(L-Trp-D-Leu)3-LGln-D-Leu] – as subunits. The repeating unit consisted of an even number of alternating L and D α-amino acids. The alternated L/D chirality of the amino acids resulted in a unique structure in which all lateral chains were oriented toward the outside of the cyclic peptides. The self-assembly nanotube was obtained by stacking subunits through hydrogen bonds.27,30 Other studies involved β-helical protein in the formation of protein nanotubes.31,32 Jenkins33 indicated that β-helical protein usually contained a repeating helical strand-loop motif, where each repeating unit provided one or more parallel β-sheet(s). The protein primary structure was critical to these secondary and tertiary structures, such as asparagine (or glutamine) ladders, hydrophobic residues (e.g. Valine, Isoleucine, Leucine) and stacking of aromatic amino acids (phenylalanine, tyrosine and histidine) and aliphatic proline rings.30 Further, the left-handed β-helical structure was preferable for protein nanotubes, because the helical structure was symmetrical and regular, and stabilized by a network of aforementioned interactions.30 Such protein nanotubes have been reported using capsid proteins,34 tubulin,35,36 actin,37–39 amyloid proteins40 and so on. These protein nanotubes were all reported as efficient delivery vessels. Alongside self-assembled peptide or protein nanotubes, an enzyme-instructed protein nanotube was also reported in making α-lactalbumin nanotube.41 The process started with a partial hydrolysis of the milk protein by alcalase, an alkaline protease extracted from Bacillus licheniformis. After calcium was introduced into the system, hydrolyzed milk protein self-assembled into a nanotube structure. The characterization then indicated the size of the protein nanotube, which had a 19.9 nm eternal diameter and an 8.7 nm internal diameter.41

3.3 3.3.1

Layer-by-layer assembly Introduction

LbL assembly is a coating technology used to build up alternating multilayers of polyelectrolytes through electrostatic deposition. The principle behind this technique is the utilization of electrostatic attraction and complex formation between negatively charged polyions and positively charged polycations to form the thin layers. Surfacecharge inversion during each adsorption step limits each layer’s thickness and prepares the surface for the subsequent adsorption of the oppositely charged polyelectrolyte. Besides the electrostatic interaction, the driving force behind multilayer film formation is the gain in entropy due to the release of counterions, very similar to what is observed in the formation of polyelectrolyte complexes.42–44 It has been established that films can contain more than 1000 polyelectrolyte multilayers (PEMs). The thickness of formed polyelectrolye layers is highly dependent on polyelectrolyte coating solutions, including but not limited to pH,45 ionic strength,46 molecular weight,47 chemistry,48 counter ion49 and temperature.50 Each pair of complementary layers is referred to as a

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bilayer, which is typically 1–100 nm thick.51 It is claimed in many publications that the architecture of the resulting film can be designed and controlled with nanometer precision to meet different requirements such as thickness, mechanical properties, biocompatibility, controlled permeability, targeting and optical or magnetic properties.52–54 The functional properties of PEMs through LbL deposition are similar to those of thin films prepared from other methods (e.g. Langmuir–Blodgett, selfassembled monolayer, casting method). A variety of important functions have been identified, including encapsulation of drugs and nutraceuticals, control of permeability (i.e. moisture and gas), incorporation of antioxidant and antimicrobial agents, and smart delivery systems with controlled or triggered release mechanisms. For foodindustry applications, prevention of texture degradation and encapsulation of colors and flavors have also been proposed for LbL thin films.3 Moreover, the universal character of the method does not impose any restriction on the type of polyelectrolyte. To date, more than 50 different charged macromolecules, including synthetic and natural polymers, have been tested and used for LbL assembly.53 For synthetic polymers, poly(allylamine hydrochloride) (PAH) is typically used as the polycation, and poly(styrenesulfonate) (PSS) as the polyanion. For natural polymers, chitosan, gelatin and poly-L-lysine (PLL) are commonly used as polycations, and a group of polysaccharides (e.g. alginate, dextran sulfate (DexS), carboxymethylcellulose (CMC), carrageenan, pectin, polyglutamic acid (PGA) and so on) as polyanions. The LbL assembly technique has been used to build multifunctional thin films on planar surfaces. It has also been applied in particulate systems to fabricate solid and hollow capsules with core materials having various shapes and sizes ranging from micro- to macroscopic. In this section, we will discuss the approaches of building thin films on either flat surfaces or core materials separately, and compare them in terms of the formation procedures and factors that have effects on the thin film and capsule constructions. In addition, their applications in medical and food areas are included, aiming to claim LbL assembly’s high potential in different research studies.

3.3.2

Nanofilms on planar surfaces from LbL

LbL thin films on planar surfaces can be produced through alternate exposure of a charged or polar substrate to water-based solutions (or mixtures) containing oppositely charged (or polar) ingredients, as shown schematically in Fig. 3.2. LbL films on flat surfaces are being studied for a wide variety of applications in the fields of biomedicine, electronics, sensors and optical devices, including nanopatterning,55,56 nanobioreactors,57,58 electronic devices,59–61 polymer catalytic electrodes,62 field-effect transistors,63 antireflection coatings64 and humidity sensors.65 Two comprehensive review papers have been published recently on the biomedical applications of PEM films, by Boudou54 and Ai.52 In these reviews, the authors claim that many biomedical devices are made of nonbiocompatible materials (e.g. synthetic polymers) and need to be coated with a biocompatible layer in order to achieve improved biocompatibility, reduced immunological response and controlled-release properties. Typically, in LbL assembly linear polyion solutions with concentrations of 0.1–3.0 mg/ml were used, and the deposition time for monolayer formation was

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LbL

Substrate Fig. 3.2

Substrate

The self-assembly of LbL thin film on a planar substrate.

10–15 minutes. Thin-film properties (e.g. film thickness, mechanical properties and stability) are greatly influenced by the ionic strength, pH and concentration of the polyelectrolyte solutions, and the molecular weight of the polyelectrolytes. For the LbL process, the pH of the polyelectrolyte solutions should be selected to maintain a high degree of polyion ionization. At low pH, the negative charge of the polyanions is often achieved by pendant sulfonate with pKa = 1 or carbonate groups with pKa = 4∼5. At basic pH, phosphate-buffered saline (PBS) is often used to maintain the charge of many polyanions and polycations. The thickness of each layer in the LbL film can be finely controlled by changing the ionic strength of the solution, which in turn induces polymer coil formation. A thicker film is usually derived from a higher-ionic-strength solution.56 For example, Lvov66 demonstrated that alternating adsorption of PSS and poly(dimethyldiallylammonium chloride) from solutions with ionic strengths 0.01 and 1 M NaCl resulted in a bilayer growth step variation from 1.6 to 6 nm. The thickness of the film was also found to be increased by the molecular weight of chitosan and hyaluronan (HA). However, this effect was only attributed to the difference in film growth onset and not to actual differences in mass deposited per layer.28 On the other hand, the physical properties of a PEM, including the tensile strength, Young’s modulus and visceoelastic property could be characterized using a series of methods (e.g. atomic force microscopy (AFM), quartz crystal microbalance (QCM) and surface plasmon resonance (SPR)). The stiffness of a PEM can be modulated from a few kPa to several GPa, depending on both intrinsic factors (e.g. structure of polyelectrolyte) and extrinsic factors (e.g. pH, ionic strength and fillers). It was found that PEMs built with ι-carrageenan as polyanions and PAH as the polycation were three times stiffer than films made of λ-carrageenan and PAH. The mechanical property can be reinforced by adding fillers into polyelectrolyte polymers. For example, nanoparticles such as montmorrillonite,67 carbon nanotubes68 and metal oxides have been added to PEMs. By evaluating their tensile properties, it was found that both tensile strength and Young’s modulus increased by up to two orders of magnitude compared to the pure polyelectrolye.69 Mixing soft polyelectrolyte with stiff polyelectrolyte is another strategy to change the mechanical properties of thin films.70–73 The stability of LbL-formed polyion films varies with their hydrophobicity. Hydrophilic films could usually last longer (more than 1 month) than plasma-treated polymer surfaces (several days), since the latter lost their hydrophilic surface property after a few days.74 The hydrophobic films, which are insoluble in water and many organic solvents, can be stable for up to several months.75

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Core Fig. 3.3

Scheme of LbL formation over solid cores.

Another factor which has tremendous effect on thin-film properties is the growth mode of the polyelectrolyte. Films formed from a linear growth mode (often synthetic polymers, such as PSS/PAH) have a stratified structure, each polyelectrolyte layer interpenetrating only its neighboring layers. They often form thiner and stiffer films than films grown by the exponential mode. However, thicker films, and thus less dipping cycles, can be achieved by using both synthetic polymers (e.g. polyacrylic acid) and natural polymers (polyaminoacids/polysaccarides, e.g. PLL/alginate) that grow exponentially. Either film roughness or polyelectrolyte diffusion in and out of the film was found to be at the origin of the growth.46,76 Traditionally, thin film construction is realized by spin coating, solution casting, thermal deposition or the Langmuir–Blodgett technique. A primary advantage of the LbL self-assembly technique over other techniques is its ability to coat the targeted surface with an ordered structure and nanometer thickness on supports of various shapes and sizes.

3.3.3

Nanocoatings from LbL

Potentially, the multifunctional nanocoatings could be formed through LbL deposition technology. Figure 3.3 illustrates the formation procedure for building coatings on solid core materials by the LbL technique. The laminated coatings can be applied on both micro- and macroscopic objects. Modification of nanoparticle surfaces with polyelectrolyte LbL shells allows for modulation of nanoparticle cell uptake rate and ratio, providing a template for their modification with tumor-targeting agents, increasing nanoparticle colloidal stability and controlling loading/releasing characteristics.52,77–80 Polyphenol-loaded gelatin nanoparticles with diameters of 200–300 nm have been coated by polyelectrolytes, including PSS/PHA, PGA/PLL and DexS/protamine sulfate, through the LbL assembly process.81 Lipid droplets at sizes ranging from several nanometers to micrometers have also been coated by laminated nanocoatings as a stabilizing layer.3 The nanocoatings are made of β-lactoglobulin, pectin and other food polymers. Moreover, biobased nanocomposties with high biodegradability and controlled permeability or barrier properties have been generated through the LbL process.82 Highly deacetylated chitosan and eucalyptus wood cellulose nanowhiskers are used as a polycation and a polyion, respectively. Using the same concept, macroscopic objects, such as fruits (e.g. apples) and vegetables (e.g. tomatoes), are proposed to be coated by the thin coatings through LbL assembly. However, this exploration is still in its infant stage. Different functional agents, including antimicrobials, antioxidants and antibrownings, can be incorporated into the coatings to achieve the desired multifunctional properties. Vargas et al.83 studied the formation of thin coatings using the LbL technique on planar hydrogel

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(agar-pectin, agar-carrageenan) surfaces, which were used to mimic fresh-cut vegetable and fruit surfaces. They found the anionic pectin and carrageenan molecules migrated from hydrogel surfaces to interact with the droplets of a cationic protein over time, making them more negative and aggregate. Therefore, pH becomes very important, since the droplets only stick to the hydrogel surfaces when the droplets are positively charged (pH < pI). Recently, it was found that laminated coatings from chitosan/eugenol can be formed on fruit and vegetable surfaces and provide protection against microbial growth.3

3.3.4

Hollow nanocapsules from LbL

PEM in the form of hollow capsules can be fabricated on various colloid templates, such as polymeric particles, organic or inorganic crystals and blood cells.84–86 Stepwise, a charged particle is first placed in a dilute solution of polyelectrolyte having opposite charges. The electrostatic driving force makes the coating possible and subsequently changes the particles’ apparent charge. Any excess polyelectrolyte is then removed by centrifugation and washing several times. After cleaning, the deposition is carried out again with a polyelectrolyte solution of opposite charge, and the process can be repeated as many times as needed. Finally, the cores are dissolved to obtain hollow nano- to microcapsules. In order to fabricate intact capsules consisting only of coating materials, the process of core dissolution should result in 100% elimination of the core, with little effect on the PEM. However, this is complicated from a chemical point of view, since the molecular weight of the core materials is typically hundreds of times greater than that of the polymers forming the shell. It then becomes an issue to completely remove the core after the shell is formed. There are three groups of core materials. For the first, synthetic organic cores, popular examples are melamine formaldehyde and polystyrene. These can be removed by dissolving in low-pH water and the organic solvent tetrahydrofuran, respectively. Polylactic acid or polylactic-co-glycolic acid, as an example of a natural polymer, can be dissolved in an acetone and N-methyl-2-pyrrolidinone mixture. The challenge of using organic core materials is that they are hard to remove even after several washing steps and they can interact with coating shells.87 In some cases, the capsule stays intact during the dissolution process only if it is assembled with less than 8–10 polyelectrolyte layers.87,88 The second group is the inorganic small molecules and ions. These have the advantage of an absence of osmotic stress upon dissolution; therefore, the complete removal of core is relatively easy to realize. As a consequence, these capsules can comprise a high number of layers and have a lower permeability than large moleculetemplated capsules.89,90 The last group, biological core cells (e.g. erythrocyte cells), can be used as a template to form hollow PEMs by the LbL process. The removal of the cell cores can be achieved by an oxidation with sodium hypochlorite solution. However, the treatment with NaOCl could change the chemical composition of the capsules dramatically.86 Permeability is an important factor for hollow-capsule applications in diverse areas mainly related to exploitation of systems with controlled- and sustained-release properties. It is usually decided by layer thickness, porosity, pH, ionic strength and

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the structure of the electrolyte multilayers, as well as by chemistry, charge and the size of the permeable compound.53 Studies on capsules of the typical PEM, PSS/PAH, revealed that it is semipermeable. It is permeable to low-molecular-weight compounds but impermeable to polymers with molecular weights larger than 4 kDa.91 The semipermeability of the PEM microcapsules is of special interest for controlled drug release. Recently, several studies have been devoted to PSS/PAH microcapsules with respect to the permeation properties.84,92 Fluorescent dyes such as pyrene and fluorescein microparticles were used as model drugs and coated with various numbers of PSS/PAH multilayers. By adding ethanol or changing the pH value of the dispersion solution, drug release from these microcapsules was realized, and the thickness dependence of the release profiles was quantified. Because the coating materials are available in a wide range, from both synthetic and natural origins, there are a number of possibilities to be explored in this area. Researchers from different research areas focus their attentions on different aspects of LbL. For instance, food scientists are interested in obtaining films with great mechanical properties, and in the biodegradability of films constructed from natural (even edible) polyelectrolytes. Cell biologists wish to use LbL in building implantable biomaterials and in tissue engineering; though they still need ultrathin films for hosting cells and tissues, more emphasis is placed on biocompatibility and cytotoxicity. Therefore, depending on the application, the functions and properties of PEMs from LbL deposition need to be checked and utilized accordingly. In summary, since the pioneering work by Decher and coworkers, who first introduced the LbL assembly of the PEMs about 15 years ago,93,94 this technology has gained attention as an attractive method for a broad range of applications, as illustrated above. LbL assembly is of great interest because of its simplicity, the control over coating thickness and composition that can be obtained at the nanometerlength scale, and the fact it allows us to bypass the use of harsh solvents. The downside of this technology is the long dutation, with multiple deposition steps, as well as the tedious particle cleaning required after each deposition to remove the excess nonadsorbed polyelectrolytes. However, this concern can be extenuated by changing the deposition method from dipping to spray deposition. The latter method was found to be effective even under conditions for which dipping failed to produce homogeneous films, when an extremely short contact time was required.95 Moreover, the rinsing step involved in the dipping method could be skipped, thus making it possible to speed up the whole deposition process. Another concern is that the working concentration is very low, in order to prevent polyelectrolyte-induced particle flocculation, leading to a low overall production rate.

3.4 3.4.1

Nanoemulsions Introduction

An emulsion is defined as a system made up of two immiscible phases (e.g. water and oil). One is the continuous phase, the other is the dispersed phase, usually in the form of small liquid droplets. Two basic emulsions are oil-in-water (o/w) and water-in-oil (w/o).

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Some food examples of basic emulsions include salad dressing (o/w), milk (o/w) and chocolate (w/o). Complex emulsion systems have been generated and studied, including multiple emulsions (e.g. w/o/w), solid lipid particles (SLP-o/w), multilayer emulsions (M-o/w) and filled hydrogel particles (o/w/w). Depending on the size of the dispersed phase, an emulsion can be classified as a conventional emulsion, microemulsion or nanoemulsion. Conventional emulsions and microemulsions usually have high interfacial energy and are thermodynamically unstable due to the free energy of emulsion formation being greater than zero. Surfactants at a weight ratio of 10–30%, with different hydrophilic–lipophilic balance (HLB) values (e.g. triglyceride, lecithin and polysorbate), are added to lower the interfacial tension and make the emulsions stable to some extent. Over the past few decades, the concept of nanoemulsions has been introduced to the emulsion family; they are claimed to have the ability to stabilize at a reduced amount of emulsifiers (2–8%) compared to that required for the same formulations of conventional and microemulsions.96 Nanoemulsions are nano-sized emulsions, typically exhibiting diameters of up to 500 nm. Nanoemulsions are also frequently known as submicron emulsions, fine-dispersed emulsions, miniemulsions, ultra-fine emulsions and so on, but all are characterized by a great stability in suspension due to their very small size, essentially the consequence of significant steric stabilization between droplets.97 Nanoemulsions are usually generated by dispersion (or high-energy) emulsification methods (e.g. high-pressure homogenization).98 However, condensation (or lowenergy) methods, such as the phase-inversion temperature (PIT) method, can also be used to produce nanoemulsions.97 The formation of such nanoscale droplets by the former method is governed by directly controllable processing parameters such as the amount of energy, amount of surfactant and nature of the components. When the latter technique is applied, nanoemulsion formation is ruled by intrinsic physicochemical properties and the behavior of the systems. Nanoemulsions have been used and proposed for many applications in the food and pharmaceutical industries, including encapsulation and delivery systems for drugs and bioactive compounds.

3.4.2

High-energy nanoemulsification methods

High-energy emulsification methods involve mechanical breakdown of emulsion into nanometric size, while generating huge interfacial areas using high-shear stirring, high-pressure homogenization96 or ultrasound homogenization.99 There are two steps in the process: drop creation, the deformation and disruption of macrometric initial droplets; and surfactant adsorption at the interfaces, to ensure steric stabilization. Three groups of devices – rotor/stator mixer, ultrasound generator and high-pressure homogenizer (e.g. microfluidizer) – are used to produce nanoemulsions in both industry and research labs. The latter two have been reported to be more efficient than the first for providing good dispersion with uniform nano-sized droplets.100 Ultrasonic emulsification is widely used in the lab. Two mechanisms have been proposed to produce nanoemulsions. In the first, the application of an acoustic field produces interfacial waves, which become unstable, eventually resulting in the eruption of the oil phase into the water medium in the form of droplets.101 In the

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second, the application of low-frequency ultrasound generates acoustic cavitation, namely the formation and subsequent collapse of microbubbles by the pressure fluctuations of a sound wave. The collapse of each bubble causes extreme levels of highly localised turbulence. The turbulent microimplosions act as a very effective method of breaking up primary droplets of dispersed oil into droplets of submicron size.102 The ultrasonic emulsification (400 W, 24 kHz) has been used to produce food nanoemulsions containing water, flaxseed oil as a bioactive compound, and Tween 20 as a surfactant.99 Results showed that emulsions with a mean droplet size as low as 135 nm were achieved, and were comparable to emulsions prepared with a microfluidizer operated at 100 MPa. The authors suggest that in order to achieve a commercial outcome, significant additional work is required to optimize equipment design, especially in minimizing the ultrasonic “hot zone” and preventing contamination of samples from transducer-tip erosion. In summary, the ultrasonic homogenization method has the advantage of low instrumentation and equipment contamination, but suffers from the fact that it is hard to scale up for industrial applications. On the other hand, high-pressure homogenization is the most widely used emulsifying method for preparing nanoemulsions in the food industry, due to its ability to control the droplet size and give a large choice of compositions.96 Microfluidizer (Manton–Gaulin device) is used to generate particle–particle collisions through the microfluidic channel architectures, rather than a straight shear field, to cause particle size reduction. It is designed to force macroemulsions to pass through narrow gaps by imposing high pressures. The fluid accelerates in the microchannels of the microfluidizer to a high velocity of 300 m/s, resulting in the generation of nanoscaled emulsion droplets.103 One example applied high-pressure homogenization (1500 bar) to produce food nanoemulsions containing water, canola oil and a surfactant in order to form encapsulation systems for bioactive compounds such as epigallocatechin gallate (EGCG) and curcumin.96 Thus-formed nanoemulsions were proven to be able to improve the pH stability of EGCG and enhance the solubility of curcumin. Moreover, the anti-inflammation activity of nanoemulsified curcumin against 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced edema of mouse ears has been found to be increased by 85% compared to control, possibly due to its nanoscaled size. Another example confirmed the possibility of using high-pressure homogenization to produce stable o/w nanoemulsions, using β-lactoglobulin and ι-carrageenan as stabilizing agents.104 For all high-energy emulsification methods, the nature and amount of surfactants are important parameters and need to be carefully examined. The addition of encapsulating molecules and polymeric matrix, however, appears not to influence the emulsification process. In brief, high-energy nanoemulsification methods are of practical interest in generating nano-sized particles, since the formulation parameters are directly controllable.

3.4.3

Low-energy nanoemulsification methods

The low-energy counterpart of the nanoemulsification method involves diverting the intrinsic physicochemical properties of the surfactants, cosurfactants and formulated excipients.99 It is claimed to have many advantages over the aforementioned high-energy

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methods, including lower energy input, better protection of drug degradation and denaturation, better preserving activity during processing and easier scale-up. Two types of method, namely spontaneous nanoemulsification (constant temperature)105–109 and PIT (constant composition),110–116 are proposed in the literature and developed intensively. The spontaneous emulsification method uses the rapid diffusion of water-soluble solvent, solubilized first in an organic phase, then moving towards the aqueous one when the two phases are mixed. The source of energy is believed to be the interfacial turbulence, closely related to the surface tension gradient induced by the diffusion of solutes between the two phases. The process is known as condensation and spontaneously increases entropy and thus decreases the Gibbs free energy of the system. The process only happens under specific conditions, so finding the right condition is necessary and is often guided by following the diffusion pathway within the phase diagram. Moreover, the equilibrium phase diagram needs to be carefully studied and the phases analyzed and characterized.97 This method has been applied to form nanoemulsions of both nonionic and ionic surfactants. The attraction of formulating o/w nanoemulsion systems lies in their ability to incorporate hydrophobic drugs and polyphenolic compounds into the oil phase, thereby enhancing their solubility and bioavailability.117 One article found that when a hydrophobic drug (Ramipril) was formulated into a nanoemulsion through a spontaneous process, the in vivo bioavailability of the drug increased 230 and 540% compared to that of conventional capsule form and the drug suspension, respectively.118 The nanoemulsion was made of Sefsol 218 (oil), Tween 80 (surfactant), Carbitol (cosurfactant) and standard buffer solution at pH 5 (aqueous phase). Another article reported that nanoemulsions were prepared by adding potassium hydroxide solutions to the ionic surfactant, composed of hexadecane–oleic acid–C12E10 mixtures at constant temperature (25°C), with a final water concentration of 80%.119 The authors found that the smallest droplet size could be obtained along the emulsification path and the equilibrium could be achieved near the nanoemulsion region, with all the oil dissolved in a phase, in this case a cubic liquid crystalline phase. It has also been found that the most probable breakdown mechanism of the nanoemulsions formed is Ostwald ripening.119 The PIT method uses the specific properties of nonionic surfactants (e.g. polyethoxylated surfactants) to modify their partitioning coefficient as a function of the temperature, and leads to the creation of biocontinuous phases when the temperature is close to the PIT. The surfactants will break up to generate an o/w type of nanoemulsion. The phase inversion of the surfactants is temperature-dependent, simply because their affinities for water and oil are governed by temperature. When the composition of emulsion is fixed, the phase will be changed from one type of emulsion (e.g. o/w) to its opposite (e.g. w/o) by rapid cooling or by a sudden dilution in water or oil. PIT is an organic, solvent-free and low-energy method and allows easy industrial scaleup. The influence of the extrinsic factors such as ionic strength, pH, temperature and composition parameters (surfactant amount and type, and water-to-oil ratio) on the formulation and consequence of formation of nano-sized emulsion droplets has been reported. Using the PIT method, nanoemulsions were prepared using a poly(oxyethylene) nonionic surfactant and a polymerizable acrylic monomer (lauryl acrylate) as the oil phase. Inversion of the emulsion, followed by rapid cooling, resulted in emulsions with an average droplet size as low as 25 nm.120 In one study,

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a group of surfactants (AOT, Brij 30 and HCO 040), concentrations of surfactants (2–10%) and types of fatty acid (lauric acid, myristic acid, palmitic acid and stearic acid) were screened using a PIT emulsification method.121 The authors found that sodium bis(2-ethylhexyl) sulfosuccinate (AOT) (6% (w/w))-stabilized nanoemulsions made with lauric acid at 0.85% (by weight) oil-to-water ratio has the best stability. The HLB temperature and droplet size for this emulsion are 38°C and 230 nm, respectively. Moreover, ultraviolet-absorbing flavornoid extracts (i.e. Cinnamomum japonicum Sieb and Sophora japonica L) from herbal plants were incorporated into the nanoemulsion. In vitro skin permeation experimental results indicated that the most effective flavornoid extract concentrations were around 2%.121 In general, the low-energy spontaneous emulsification method and solvent-free PIT method appear relatively adaptable and easy to handle. They can be used to generate nanoparticles at a low energy cost, free from the toxicity of organic solvent, and with a relatively low amount of surfactant (at 5% (w/w)). However, the literature mainly reports formulation of nanoemulsions based on high-energy methods. Therefore, development and adaptation of the low-energy emulsification methods in the nanoemulsion field need more effort in the future.

3.4.4

Nanoparticles generated from different nanoemulsions and their applications

Different types of nanoparticle (e.g. polymeric nanospheres, nanocapsules and solid lipid nanoparticles) can be obtained from nanoemulsions when they serve as templates. Nanoparticles are defined as solid colloidal particles with sizes ranging from 10 to 1000 nm. They are built from macromolecules or molecular assemblies. The fundamental advantage of nanoparticles compared with other colloidal systems (e.g. liposomes, hydrogels and microemulsions) is their great kinetic stability and rigid morphology. Two groups of nanoparticles will be discussed in this section: nanospheres, which have a homogeneous structure in the whole particle, and nanocapsules, which exhibit a typical core-shell structure. For synthetic polymers, nanospheres usually form through an in situ polymerization process,97 while for natural polymers (e.g. food biopolymers), they are formed from preformed macromolecules. Two steps are followed: the first is to dissolve or disperse the macromolecules in the solvent phase (mainly organic solvent), while the second is to remove the solvent from the formulation by evaporation or diffusion shock and therefore cause polymer precipitation within an organic phase template. There are lots of examples in the literature of the use of this method to generate nanospheres for drug delivery systems, controlled-release formulations, and recently for encapsulating bioactive compounds (e.g. polyphenol and omega-3 oils). PLA– polyethylene glycol (PEG) nanoparticles were constructed from a double emulsion (w/o/w) by two-step sonication followed by evaporation of the solvent (ethyl acetate/ methylene chloride) in order to encapsulate a plasmid DNA.122 Chitosan nanospheres were prepared by the double-emulsion method and used as encapsulation carriers for hydrophobic and hydrophilic drugs (Gantrez, carbopol, polycarbophill and salicylic acid) aimed at achieving a controlled-release property in oral care products.17,123

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The same evaporation method was applied to generate chitosan-modified poly(DLlactic-co-glycolic acid) (PLGA) nanospheres, which were investigated for their cell toxicity and uptake properties.124 Chitosan–PLGA nanospheres demonstrated low cytotoxicity, similarly to non-PLGA nanospheres. Cellular uptake of PLGA nanospheres increased with decreasing diameter to the submicron level and with chitosan-mediated surface modification. Novel core-shell hydroxyapatite/chitosan nanocapsules were synthesized in a multiple emulsion (w/o/w).125 The emulsion was made of diammonium phosphate solution as an inner aqueous phase, cyclohexane as an oil phase and calcium nitrate solution and chitosan solution as an outer aqueous phase. The forming mechanism of core-shell spheres and the influence of temperature on the morphology of the nanospheres were investigated. The diameter of the resulting core-shell nanospheres was 100–200 nm and the thickness of the chitosan shell was about 10 nm. The study concluded that at different reaction temperatures the morphologies of the products would be changed.

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54. Boudou, T., Crouzier, T., Ren, K., Blin, G. and Picart, C. (2010) Multiple functionalities of polyelectrolyte multilayer films: new biomedical applications. Adv Mater 22, 441–467. 55. Hua, F., Cui, T. and Lvov, Y. (2002) Lithographic approach to pattern self-assembled nanoparticle multilayers. Langmuir 18, 67123. 56. Ai, H., Lvov, Y.M., Mills, D.K., Alexander, J.S. and Jones, S.A. (2003) Coating and selective deposition of nanofilm on silicone rubber for endothelial cell adhesion and growth. Cell Biochem Biophys 38, 103–114. 57. Lvov, Y. and Caruso, F. (2001) Biocolloids with ordered urease multilayer shells as enzymatic reactors. Anal Chem 73, 4212–4217. 58. Fang, M., Grant, P.S., McShane, M., Sukhorukov, G., Golub, V. and Lvov, Y. (2002) Magnetic bio/nanoreactor with multilayer shells of glucose oxidase and inorganic nanoparticles. Langmuir 18, 6338–6344. 59. Gao, M., Richter, B. and Kirstein, S. (1997) White-light electroluminescent from selfassembled Q-CdS/PPV multilayer structures. Adv Mater 9, 802–805. 60. Lee, J.K., Mattoussi, H., Yoo, D., Wu, A. and Rubner, M. (1997) Thin film light emitting heterostructures: from conjugated polymers to ruthenium complexes to inorganic nanocrystallites. Polymer Prep 38, 351–352. 61. Ho, P., Kim, J. and Burroughes, J.H. (2000) Molecular-scale interface engineering for polymer light-emitting diodes. Nature 404, 481–484. 62. Farhat, T.R. and Hammond, P.T. (2006) Engineering ionic and electronic conductivity in polymer catalytic electrodes using the layer-by-layer technique. Chem Mater 18, 41. 63. Cui, T., Liu, Y. and Zhu, M. (2005) Field-effect transistors with layer-by-layer selfassembled nanoparticle thin films as channel and gate dielectric. Appl Phys Lett 87, 183105. 64. Hiller, J.A., Mendelsohn, J.D. and Rubner, M.F. (2002) Reversibly erasable nanoporous anti-reflection coatings from polyelectrolyte multilayers. Nature Mater 1, 59–63. 65. Nohria, R., Khillan, R.K., Su, Y., Dikshit, R., Lvov, Y. and Varahramyan, K. (2006) Humidity sensor based on ultrathin polyaniline film deposited using layer-by-layer nano-assembly. Sens Actuators B Chem 114, 218–222. 66. Lvov, Y. (2000) Electrostatic layer-by-layer assembly of protein and polyions. In: Lvov, Y. and Möhwald, H.M. (eds) Protein Architecture: Interfacial Molecular Assembly and Immobilization Biotechnology, New York: Dekker, pp. 125–167. 67. Tang, Z., Kotov, N.A., Magonov, S. and Ozturk, B. (2003) Nanoscale artificial nacre. Nat Mater 2, 413. 68. Gheith, M.K., Sinani, V.A., Wicksted, J.P., Matts, R.L. and Kotov, N.A. (2005) Singlewalled carbon nanotube polyelectrolyte multilayers and freestanding films as a biocompatible platform for neuroprosthetic implants. Adv Mater 17, 2663. 69. Srivastava, S. and Kotov, N.A. Composite layer-by-layer (LBL) assembly with inorganic nanoparticles and nanowires. Acc Chem Res 41, 1831. 70. Hubsch, E., Ball, V., Senger, B., Decher, G., Voegel, J.-C. and Schaaf, P. (2004) Controlling the growth regime of polyelectrolyte multilayer films: changing from exponential to linear growth by adjusting the composition of polyelectrolyte mixtures. Langmuir 20, 1980. 71. Cho, J., Quinn, J.F. and Caruso, F. (2004) Fabrication of polyelectrolyte multilayer films comprising nanoblended layers. J Am Chem Soc 126, 2270. 72. Ball, V., Bernsmann, F., Betscha, C., Maechling, C., Kauffmann, S., Senger, B., Voegel, J.C., Schaaf, P. and Benkirane-Jessel, N. (2009) Polyelectrolyte multilayer films built from poly(l-lysine) and a two-component anionic polysaccharide blend. Langmuir 25, 3593. 73. Salomaki, M. and Kankare, J. (2009) Influence of synthetic polyelectrolytes on the growth and properties of hyaluronan-chitosen multilayers. Biomacromolecules 10, 294.

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74. Yoo, D., Shiratori, S. and Rubner, M. (1998) Controlling bilayer composition and surface wettability of sequentially adsorbed multilayers of weak polyelectrolytes. Macromolecules 31, 4309–4318. 75. Decher, G., Lvov, Y. and Schmitt, J. (1998) Proof of multilayer structural organization of poly-cation/polyanion self-assembled films. Thin Solid Films 244, 772–777. 76. Picart, C., Mutterer, J., Richert, L., Luo, Y., Prestwich, G.D., Schaaf, P., Voegel, J.-C. and Lavalle, P. (2002) Molecular basis for the explanation of the exponential growth of polyelectrolyte multilayers. Proc Natl Acad Sci 99, 12531. 77. Qiu, X., Leporatti, S., Donath, E. and Möhwald, H. (2001) Studies on the drug release properties of polysaccharide multilayers encapsulated Ibuprofen microparticles. Langmuir 17, 5375–5380. 78. Ai, H., Pink, J.J., Shuai, X., Boothman, D.A. and Gao, J. (2005) Interactions between self-assembled polyelectrolyte shells and tumor cells. J Biomed Mater Res 73A, 303–312. 79. Zahr, A.S. and Pishko, M.V. (2007) Encapsulation of paclitaxel in macromolecular nanoshells. Biomacromolecules 8, 2004–2010. 80. Zhou, J., Moya, S., Ma, L., Gao, C. and Shen, J. (2009) Polyelectrolyte coated PLGA nanoparticles: templation and release behavior. Macromol Biosci 4, 326–335. 81. Shutava, T., Balkundi, S., Vangala, P., Steffan, J., Bigelow, R., Cardelli, J., O’Neal, D. and Lvov, Y. (2009) Layer-by-layer-coated gelatin nanoparticles as a vehicle for delivery of natural polyphenols. ACS Nano 3(7), 1877–1885. 82. de Mesquita, J.P., Donnici, C.L. and Pereira, F.V. (2010) Biobased nanocomposites from layer-by-layer assembly of cellulose nanowhiskers with chitosan. Biomacromolecules 11, 473–480. 83. Vargas, M., Weiss, J. and McClements, D.J. (2007) Adsorption of protein-coated lipid droplets to mixed biopolymer hydrogel surfaces: role of biopolymer diffusion. Langmuir 23, 13059–13065. 84. Caruso, F., Yang, W., Trau, D. and Renneberg, R. (2000) Microencapsulation of uncharged low molecular weight organic materials by polyelectrolyte multilayer selfassembly. Langmuir 16, 8932. 85. Leporatti, S., Voigt, A., Mitlöhner, R., Sukhorukov, G.B., Donath, E. and Möhwald, H. (2000) Scanning force microscopy investigation of polyelectrolyte nano- and microcapsule wall texture. Langmuir 16, 4059–4063. 86. Moya, S., Dahne, L., Voigt, A., Leporatti, S., Donath, E. and Mohwald, H. (2001) Polyelectrolyte multilayer capsules templated on biological cells: core oxidation influences layer chemistry. Colloids Surf A Physicochem Eng Asp 183, 27. 87. Gao, C.Y., Moya, S., Lichtenfeld, H., Casoli, A., Fiedler, H. and Donath, E. (2001) The decomposition process of melamine formaldehyde cores: the key step in the fabrication of ultrathin polyelectrolyte multilayer capsules. Macromol Mater Eng 286, 355–361. 88. Gao, C.Y., Leporatti, S., Moya, S., Donath, E. and Mohwald, H. (2001) Stability and mechanical properties of polyelectrolyte capsules obtained by stepwise assembly of poly(styrenesulfonate sodium salt) and poly(diallyldimethyl ammonium) chloride onto melamine resin particles. Langmuir 17, 3491. 89. Antipov, A.A., Sukhorukov, G.B., Leporatti, S., Radtchenko, I.L., Donath, E. and Mohwald, H. (2002) Colloids Surf A Physicochem Eng Asp 198, 535. 90. Antipov, A.A., Shchukin, D., Fedutik, Y., Petrov, A.I., Sukhorukov, G.B. and Mohwald, H. (2003) Colloids Surf A Physicochem Eng Asp 224, 175. 91. Sukhorukov, G.B., Brumen, M., Donath, E. and Möhwald, H. (1999) Hollow polyelectrolyte shells: exclusion of polymers and donnan equilibrium. J Phys Chem B 103, 6434. 92. Antipov, A.A., Sukhorukov, G.B., Donath, E. and Möhwald, H. (2001) Sustained release properties of polyelectrolyte multilayer capsules. J Phys Chem B 105, 2281.

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93. Decher, G., Hong, J.D. and Schmitt, J. (1992) Buildup of ultrathin multilayer films by a self-assembly process. 3. Consecutively alternating adsorption of anionic and cationic polyelectrolytes on charged surfaces. Thin Solid Films 210, 831–835. 94. Lvov, Y., Decher, G., Haas, H., Möhwald, H. and Kalachev, A. (1994) X-ray analysis of ultrathin polymer-films self-assembled onto substrates. Physica B 198, 89–91. 95. Lzquierdo, A., Ono, S.S., Voegel, J.C., Schaaf, P. and Decher, G. (2005) Dipping versus spraying: exploring the deposition conditions for speeding up layer-by-layer assembly. Langmuir 21, 7558. 96. Wang, X., Wang, Y.-W. and Huang, Q.R. (2009) Enhancing stability and oral bioavailability of polyphenols using nanoemulsions. In: Huang, Q.R., Given, P. and Qian, M. (eds) Micro/Nano-Encapsulation of Active Food Ingredients, ACS Symposium Series 1007, pp. 198–213. 97. Anton, N., Benoit, J. and Saulnier, P. (2008) Design and production of nanoparticles formulated from nano-emulsion templates: a review. Journal of Controlled Release 128(3), 185–199. 98. Huang, Q.R., Yu, H. and Ru, X.M. (2010) Bioavailability and delivery of nutraceuticals using nanotechnology. J of Food Sci 79, R50–R57. 99. Kentish, S., Wooster, T.J., Ashokkumar, A., Balachandran, S., Mawson, R. and Simons, L. (2008) The use of ultrasonics for nanoemulsion preparation. Innovative Food Sci & Emerging Tech 9, 170–175. 100. Abismail, B., Canselier, J.P., Wilhelm, A.M., Delmas, H. and Gourdon, C. (1999) Emulsification by ultrasound: drop size distribution and stability. Ultrason Sonochem 6, 75–83. 101. Li, M.K. and Fogler, H.S. (1978) Acoustic emulsification. Part 1. The instability of the oil-water interface to form the initial droplets. Journal of Fluid Mechanics 88, 499–511. 102. Li, M.K. and Fogler, H.S. (1978) Acoustic emulsification. Part 2. Break-up of the larger primary oil droplets in a water medium. Journal of Fluid Mechanics 88, 513–528. 103. Asua, J.M. (2002) Miniemulsion polymerization. Prog Polym Sci 27, 1283–1346. 104. Ru, Q.M., Cho, Y.H. and Huang, Q.R. (2009) Biopolymer-stabilized emulsions on the basis of interactions between β-lactogloblulin and ι-carrageenan. Frontiers of Chemical Engineering in China 3, 399–406. 105. Davies, J.T. and Haydon, D.A. (1957) An investigation of droplet oscillation during mass transfer. II. A dynamical investigation of oscillating spherical droplets. Proc 2nd Int Congr Surf Act London 1, 417. 106. Groves, M.J. (1978) Video disposable diaper having an emulsion concentrate. Chem Ind 12, 417–423. 107. Rubin, E. and Radke, C.J. (1980) Dynamic interfacial tension minima in finite systems. Chem Eng Sci 35, 1129–1138. 108. Miller, C.A. (1988) Spontaneous emulsification produced by diffusion: a review. Colloids Surf 29, 89–102. 109. Pouton, C.W. (1997) Formulation of self-emulsifying drug delivery systems. Adv Drug Deliv Rev 25, 47–58. 110. Shinoda, K. and Saito, H. (1968) The effect of temperature on the phase equilibria and the types of dispersions of the ternary system composed of water, cyclohexane, and nonionic surfactant. J Colloid Interface Sci 26, 70–74. 111. Shinoda, K. and Saito, H. (1969) The stability of o/w type emulsions as a function of temperature and the hlb of emulsifiers: the emulsification by pit-method. J Colloid Interface Sci 30, 258–263. 112. Forster, T., Schambil, F. and von Rybinski, W. (1992) Production of fine disperse and long-term stable oil-in-water emulsions by the phase inversion temperature method. J Disp Sci Technol 13, 183–193.

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113. Forster, T., Schambil, F. and Tesmann, H. (1990) Emulsification by the phase inversion temperature method: the role of self-bodying agents and the influence of oil polarity. Int J Cosmet Sci 12, 217–227. 114. Sing, A.J., Graciaa, A., Lachaise, J., Brochette, P. and Salagers, J.L. (1999) Interactions and coalescence of nano-droplets in translucent o/w emulsions. Colloids Surf A 15, 231–239. 115. Izquierdo, P., Esquena, J., Tdros, T.F., Dederen, J.C., Feng, J., García-Delma, M.J., Azemar, N. and Solans, C. (2004) Phase behavior and nano-emulsion formation by the phase inversion temperature method. Langmuir 20, 6594–6598. 116. Solans, C., Izquierdo, P., Nolla, J., Azemar, N. and Garcia-Celma, M.J. (2005) Nanoemulsions. Curr Opin Colloid Interface Sci 10, 102–110. 117. Lawrence, M.J. and Rees, G.D. (2000) Microemulsion-based media as novel drug delivery systems. Adv Drug Deliv Rev 45, 89–121. 118. Shafiq, S., Shakeel, F., Talegaonkar, S., Ahmad, F.J., Khar, R.K. and Ali, M. (2007) Development and bioavailability assessment of ramipril nanoemulsion formulation. European Journal of Pharmaceutics and Biopharmaceutics 66, 227–243. 119. Solè, A., Maestro, C.M., Pey, C., González, C., Solans, J.M. and Gutiérrez, J. (2006) Nano-emulsions preparation by low energy methods in an ionic surfactant system. Colloids and Surfaces A: Physicochem Eng Aspects 288, 138–143. 120. Spernath, L., Regev, O., Levi-Kalisman, Y. and Magdassi, S. (2009) Phase transitions in o/w lauryl acrylate emulsions during phase inversion, studied by light microscopy and cryo-TEM. Colloids and Surfaces A: Physicochem Eng Aspects 332, 19–25. 121. Ling, I.M., Li, W.H. and Wang, L.H. (2009) In vitro skin permeation efficiency study on natural flavornoid extracts cncorporated into nano-emulsions. Asian Journal of Chem 21, 6237–6246. 122. Perez, C., Sanchez, A., Putnam, D., Ting, D., Langer, R. and Alonso, M.J. (2001) Poly(lactic acid)-poly (ethylene glycol) nanoparticles as new carriers for the delivery of plasmid dna. J Control Release 75, 211–224. 123. Taghizadeh, S.M. and Javan, R.S. (2010) Preparation and investigation of chitosan nanoparticles including salicylic acid as a model for an oral drug delivery system. E-POLYMERS, Article Number 36, March 25. 124. Tahara, K., Sakai, T., Yamamoto, H., Takeuchi, H., Hirashima, N. and Kawashima, Y. (2009) Improved cellular uptake of chitosan-modified PLGA nanospheres by A549 cells. International J of Pharmaceutics 382, 198–204. 125. Li, X.N., Chen, X.M., Li, S.P. and Peng, Z.M. (2010) Synthesis and characterization of core-shell hydroxyapatite/chitosan biocomposite nanospheres. J of Wuhan Univ of Techn-Mater Sci 25(2), 252–256.

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4

Nanocomposites

Graciela W. Padua, Panadda Nonthanum and Amit Arora

Abstract: Nanocomposites represent a new strategy in improving the mechanical strength, thermal stability and gas-barrier properties of polymers. In food packaging, nanocomposites are better able to withstand the stress of thermal food processing, transportation and storage. Fibers, platelets and particles have been used for decades to form polymer composites with enhanced properties. In nanocomposites, fillers have at least one dimension smaller than 100 nm. The most promising nanoscale-size fillers are layered silicate clays. In food packaging, a major emphasis is placed on the development of high barrier properties against oxygen, carbon dioxide, flavor compounds and water vapor. The nanoscale plate morphology of clays promotes gas-barrier properties. Decreasing watervapor permeability is critical to the development of biopolymers as sustainable packaging materials. Nanocomposites may advance the utilization of biopolymers in food packaging. Several examples are cited. Keywords: nanocomposite; food packaging; barrier property; nanoclay; biopolymer

4.1

Introduction

Over the past few decades, polymers have replaced conventional materials (metals, ceramics, paper) in packaging applications due to their functionality, light weight, ease of processing and low cost. The use of synthetic polymers is ubiquitous in food packaging, where they provide mechanical, chemical and microbial protection from the environment and allow product display. The polymers most frequently used in food packaging are polyethylene, polypropylene, polystyrene, polyvinyl chloride (PVC) and polyethylene terephthalate (PET).1,2 High-density polyethylene is used in applications such as milk bottles and bags. Low-density polyethylene is used for trays and general-purpose containers. Polypropylene has excellent chemical resistance, it is strong, and it has the lowest density among the plastics used in packaging. It has a high melting point, making it Nanotechnology Research Methods for Foods and Bioproducts, First Edition. Edited by Graciela W. Padua and Qin Wang. © 2012 John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc.

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ideal for hot-filling operations. It is employed in film and microwavable containers.3 PET is clear, tough and has good gas- and moisture-barrier properties. Plastic soft drinks and mineral water bottles are generally made of PET. It has good resistance to heat, mineral oils, solvents and acids.4 However, despite their enormous versatility, a limiting property of polymeric materials in food packaging is their inherent permeability to gases and vapors, including oxygen, carbon dioxide and organic vapors. Biopolymers are notorious for their high water-vapor permeability. This has boosted interest in developing new strategies to enhance barrier properties. Polymers are often added with fillers to form composites with enhanced barrier properties. Composites typically consist of a polymer matrix or continuous phase and a discontinuous phase or filler.5 Fibers, platelets and particles have been used for decades to form polymer composites with enhanced mechanical and thermal properties. A recent breakthrough in composite materials was the advancement of nanotechnology. Nanocomposites are materials in which the filler has at least one dimension smaller than 100 nm. Mechanical, thermal and barrier properties of nanocomposites are often markedly different from those of their component materials. Polymer nanocomposites promise a new crop of stronger, more heat resistant and higher-barrier materials.

4.2

Polymer nanocomposites

Nanocomposites represent a new alternative to conventional technologies for improving polymer properties. Nanocomposites exhibit increased mechanical strength, improved heat resistance and increased barrier properties compared to their neat polymers and conventional composites.6–8 A classic example is the use of nanosized MMT clay to improve mechanical and thermal properties of nylon.9 When used in food packaging, nanocomposites are better able to withstand the stress of thermal food processing, transportation and storage.6,10 Also, because of their improved mechanical properties, nanocomposites may allow downgauging, thus reducing materials usage. Particle fillers used and proposed in the literature include the nanoclays MMT and kaolinite, carbon nanotubes (CNTs), and graphene nanosheets. MMT and kaolinite clays consist of nanometer-scale platelets of magnesium aluminum silicate (Fig. 4.1). Their dimensions, 1 nm thick and 100–500 nm diameter, result in platelets of high aspect ratio.11 Clay structure is formed by hundreds of layered platelets stacked into particles or tactoids 8–10 μm in diameter. The effect of nanoclays on polymer properties stems mainly from their high surface to volume ratio, since polymer–filler interactions are governed by interfacial forces. Clay particles should be exfoliated as individual platelets and uniformly dispersed within the polymer matrix in order to take full advantage of the potential high surface area.6 Exfoliated nanoclays are effective at improving the gas-barrier properties of polymeric materials. When dispersed into polymers, they create a maze structure that presents a tortuous path to moving gases, greatly slowing their permeation rate.12 Traditional composite structures contain large quantities of filler (approximately 60% vol), but in nanocomposites, dramatic changes in properties are possible at very low loads (<2% vol). The carbon-based graphene nanoplates (GNPs) can form heat-resistant, high-barrier nanocomposites that are promising in food packaging applications. GNPs 20–60 nm in thickness and 0.5–25 μm in diameter dispersed in poly(methyl

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Fig. 4.1

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Stacks of kaolinite nanoplates.

methacrylate) (PMMA) were reported to increase the glass transition temperature of PMMA by 30°C at 1–5 wt% loading.13 GNPs were dispersed by high-speed shearing methods. Functionalized graphene sheets were reported to be remarkably effective at enhancing the fracture toughness, fracture energy, stiffness, strength and fatigue resistance of epoxy polymers at significantly lower loading fractions in comparison to nanoparticles, nanoclays and CNTs. This was attributed to their enhanced specific surface area, two-dimensional geometry and strong nanofiller-matrix adhesion.14 CNTs, another carbon-based nanofiller, have attracted considerable attention due to their intrinsic mechanical and electrical properties. Improvements in modulus and strength of 30% and 15%, respectively, have been reported for 1 wt% loading of functionalized single-walled CNTs in epoxy. However, the use of CNTs in nanocomposites to date has been limited by challenges in processing and dispersion, and their prohibitively high cost.

4.3

Nanocomposite formation

Nanoclay technology relies on the high surface area of clay platelets, in excess of 750 m2/g, and high aspect ratio (100–500). However, MMT clays come in platelet clusters with low surface exposed. Thus, processing at high shear or sonication techniques are necessary to deaggregate or exfoliate the clusters and increase the surface area exposed to the polymer.15 There are three types of polymer–clay formation, namely (i) tactoid, (ii) intercalated and (iii) exfoliated6,16 (Fig. 4.2). Tactoid structures remain clustered in a polymer when the interlayer space of the clay gallery does not expand, usually due to its poor affinity with the polymer. No true

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Fig. 4.2

Tactoid, intercalation and exfoliation.

nanocomposites are formed this way.17 Intercalated structures are obtained at moderate expansion of the clay interlayer. In this case, interlayer spaces expand slightly as polymer chains penetrate the basal spacing of clay, but the shape of the layered stack remains. This is the result of moderate affinity between polymer and clay. In the case of exfoliated structures, clay clusters lose their layered identity and are well separated into single sheets within the continuous polymer phase. This is due to a high affinity between polymer and clay. Clay aggregates must be exfoliated into single platelets and distributed homogeneously throughout the polymer phase to take full advantage of the nanoplates’ potential high surface area.6,18 Dispersion of clay layers into the polymer is affected by a mismatch between the hydrophobic/ hydrophilic character of the polymers and of the clay. Polymers are typically hydrophobic, while clays are hydrophilic. Nanoclays are often chemically modified to render their surface more hydrophobic and improve their compatibility with polymers. Fatty acids are commonly used for this purpose. Several authors have described the mechanism for silicate clay nanocomposites formation. Vaia and Giannelis19 have used a lattice-based thermodynamic model that examines the entropic and enthalpic contributions during the formation of a polymerlayered silicate nanocomposite to understand the driving forces for intercalation and exfoliation of layered silicates in long-chain polymer matrices. In their estimation, despite the entropy loss associated with polymer confinement, an entropy gain associated with layer separation balances the entropy loss of polymer intercalation. This consideration led to the conclusion that the enthalpy of mixing dominates all free-energy considerations. A study by Mackay and others20 suggested that the dispersion of nanoparticles in a polymer was a result of a favorable enthalpy of mixing due to increased molecular contacts between the polymer and the dispersed nanoparticles. This was attributed to the increased accessible area of the nanoparticle caused by dispersion. Their results indicated that nanoparticles were capable of being dispersed in polymers if the size of the nanoparticles was smaller than the radius of gyration of the polymers.

4.4

Structure characterization

Characterization is an essential part of materials research. Important aspects of nanocomposite structure characterization include particle dispersion, changes in bulk matrix and the nature of particle–polymer interfaces. Recent advances in characterization techniques, especially for the elucidation of microstructure, have

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allowed the advancement of nanotechnology. The most common techniques used to probe nanocomposite structures are scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD) – both wide angle (WAXS) and small angle (SAXS) – infrared (IR) spectroscopy and atomic force microscopy (AFM).21 Ray and Okamoto6 gave a detailed description of nanocomposite characterization techniques in their review paper. Analytical methods to probe the morphology of polypropylene–clay nanocomposites were discussed by Morgan and Gilman.22 They recommended TEM as the means to qualitatively assess the degree of dispersion of clay in polymer matrices and observe the structure of silicates. The types of structure that can be determined by TEM are polymer structure, void size and shape, filler size, shape and distribution, local crystallinity and crystal size. Changes in the polymer matrix may be assessed by polarized light microscopy with assistance from TEM and/or AFM. SEM fracture analysis continues to be the best method for assessing structure–property relationships, especially for toughness.21 The degree of intercalation, exfoliation and dispersion has been traditionally characterized by XRD.17 Three types of nanostructure can be identified by XRD. When the basal spacing of a mixture is the same as that of the clay cluster, the structure is considered a tactoid with no polymer chains inside the clay gallery (interlayer space). In intercalated structures, the d-space is increased as the interlayer space is expanded, thus the 2q position in the X-ray spectra is decreased (smaller value). Exfoliated structures show no peaks in XRD, indicating that polymer chains have penetrated the gallery and widened the interlayer space until the regular stacks of clay layers become disordered, so that X-rays cannot detect any regular structure. Exfoliation is achieved when clay stacks no longer show an XRD peak. Protein–clay nanocomposite research by Chen and Zhang23 demonstrated that MMT tactoids were delaminated into thin lamellas in soy protein. The d-spacing values increased from 1.4 nm for the MMT tactoid to a value ranging from 2 to 3 nm.

4.5

Biobased nanocomposites

Biopolymers have attracted considerable attention as sustainable replacements for conventional plastic packaging materials. Biopolymers include plant-derived materials (starch, cellulose, other polysaccharides, proteins), animal products (proteins, polysaccharides), microbial products (polyhydroxybutyrate) and polymers synthesized chemically from naturally derived monomers (polylactic acid, PLA). Most reports on the formation and properties of biopolymer films are focused on their application as edible films. Pertinent reviews are available.24–34 For packaging applications, biopolymers present relatively poor mechanical and barrier properties, which currently limits their industrial use. Especially challenging is the development of moisture-barrier properties, due to the intrinsic hydrophilic nature of biopolymers. However, it has been suggested that inherent shortcomings of biopolymer-based packaging materials may be overcome by nanocomposite technology. The forming of nanocomposites to improve mechanical, thermal and barrier properties of polymers remains a promising option. Researchers may be inspired by the various nanocomposites seen in nature. Seashells are natural nanocomposites of aragonite, a carbonate mineral, at 95 and

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1% organic biopolymer by volume. Seashells have superior mechanical strength and toughness. Bone tissue is another example of a nanocomposite in nature. Its structure may serve as a model for the development of biomimetic materials. Its building block is the mineralized collagen fibril. Bone consists of nanosize hydroxyapatite (Ca5(PO4)3OH) plates dispersed in a collagen matrix, forming an orderly layered array.35 The mineral layers impart hardness and the protein matrix lends toughness. Bone provides mechanical support to skeletal tissues and serves as a reservoir of minerals, especially calcium and phosphates.36 Engineered biopolymer-layered silicate nanocomposites are reported to have markedly improved physical properties, including higher gas-barrier properties, tensile strength and thermal stability.6,17,18,37,38 Chemically treated nanoscale silicate plates incorporated with appropriate polymers can provide effective barrier performance against water, gases and grease.39 The hyper-platy, nanodimensional thickness crystals create a tortuous path structure that resists penetration. Detailed reports on biopolymer nanocomposites have been published by several researchers.8,37,40,41

4.5.1

Starch nanocomposites

Starch has been extensively investigated as a choice material for food packaging applications due to its environmental compatibility, wide availability and low cost.42,43 Blends with synthetic polymers and the addition of inorganic materials6,40,44,45 have been proposed to improve the water resistance of starch. In recent literature, starch–clays are the most often cited biodegradable nanocomposites, investigated for various applications including food packaging.40,44–49 Significant improvements in mechanical properties were reported; both Young’s modulus and tensile strength increased with the addition of MMT clay. Cyras and others45 reported that effective diffusion coefficients for nanocomposites were lower than for starch alone. This suggested that addition of MMT reduced water uptake of starch films, possibly due to the tortuous structure formed by the exfoliated clay. Recently, a starch/ ZnO–carboxymethylcellulose (CMC) nanocomposite was prepared using ZnO nanoparticles stabilized by CMC as the filler in glycerol plasticized-pea starch.50 When the ZnO–CMC content varied from 0 to 5 wt%, tensile strength increased from 3.9 to 9.8 MPa, although elongation at break was reduced from 42.2 to 25.8%. Water vapor permeability (WVP) was reported to decrease significantly. Zhao and others38 give an overview of the current status of starch-based nanocomposite properties, processing and applications.

4.5.2

Pectin nanocomposites

Nanosized MMT (at 0.5, 1 and 2 wt%) was successfully dispersed in an aqueous pectin solution (5 wt%) using a high-pressure fluidizer in acidic conditions. High shear induced orientation of nanoclay platelets. Instead of full exfoliation, stacks of 10–15 nm-thick swelled nanoclay layers were formed during high-shear fluidization. Anionic pectin interacted with the cationic edges of the nanoclay stacks, bonding the stacks into a laterally oriented film and preventing nanoclay agglomeration. Such

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hybrid films showed significantly improved barrier properties against oxygen, slightly decreased water vapor transmission, and grease resistance.51

4.5.3

Cellulose nanocomposites

Biopolymer nanocomposites from fruit and vegetable purees and cellulose nanofibers (CNFs) have recently been studied as film-forming edible materials. CNFs were added to improve the tensile properties, WVP and glass transition temperature of mango puree films.17 Tensile strength increased (4.09 to 8.76 MPa) with increase in CNF concentration from 0 to 36%. It was proposed that the formation of a fibril network within the matrix was effective at increasing tensile strength and Young’s modulus, especially at higher concentrations of CNF. The addition of CNF was also effective in improving the water vapor barrier of the films (2.66 to 1.67 g·mm/kPa·h·m2). The WVP was significantly decreased when CNF was incorporated at loadings of at least 10% (10 g/100 g). Cellulose films in themselves are poor water vapor barriers due to the inherent hydrophilic nature of polysaccharides. Burdock52 found that the cellulose derivative hydroxypropyl methylcellulose (HPMC) was a promising material for edible coatings or packaging films. De Moura and others53 proposed nanocomposites using chitosan (CS) as the nanofiller in HPMC to improve mechanical and film barrier properties. Different concentrations of CS nanoparticles were incorporated in HPMC to evaluate changes in mechanical properties, WVP and oxygen permeability. Incorporation of CS nanoparticles in the films improved tensile strength (30.7 to 66.9 MPa) and film barrier properties. SEM analysis revealed that CS nanoparticles tended to fill porous spaces in the HPMC matrix improving film tensile properties and WVP. Oxygen permeability was reduced significantly by incorporating CS–poly(methacrylic acid) (PMAA) in HPMC matrix (182 to 142 cm3·μm/mm2·d·kPa). Further reduction in permeability was observed with reduction in nanoparticles size (142.3 cm3·μm/ mm2·d·kPa at 110 nm and 110 cm3·μm/mm2·d·kPa at 59 nm). Cellulose nanowhiskers, with lengths ranging from 25 to 50 nm and cross-sections around 5 nm, were prepared by acid hydrolysis of highly purified cellulose microfibers (50–100 μm length, 10–20 μm cross-section) and used to reinforce carrageenan matrices at 1–5 wt%. Cellulose nanowhiskers dispersed well in the matrix, especially at low filler content. However, TEM and WVP data suggested that at nanofiller loadings above 3 wt%, cellulose nanowhiskers (CNWs) agglomerated, possibly due to hydrogen bonding-induced self-association. A WVP drop of 70% was seen at around 3 wt% load of CNWs. The permeability drop was attributed to a strong reduction in water uptake rather than a diffusion-driven tortuosity effect. On the other hand, the addition of the parent cellulose microfibers also resulted in permeability reductions at low filler loadings, but those were smaller per filler volume compared to the CNWs.54

4.5.4

Polylactic acid nanocomposites

PLA has received attention as a sustainable, biocompatible, biodegradable material with good mechanical and optical properties. Lactic acid, the monomer of PLA, may easily be produced by fermentation of carbohydrate feedstock. Thus, PLA offers

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more disposal options and its manufacture is less environmentally burdensome than traditional petroleum-based plastics.55,56 However, the large-scale use of PLA as a packaging material is still hampered by its high cost and low performance compared to commodity polymers. The most important limitation to the application of PLA in food packaging is its low gas-barrier properties. Nanocomposite technology has the potential to improve polymer properties and expand the applications of PLA.37,55,57,58 Nanocomposites of amorphous PLA and chemically modified kaolinite were studied by Cabedo and others.59 They observed good interaction between polymer and clay, which led to an increase in oxygen-barrier properties of about 50%. The study also included the addition of plasticizers to overcome the inherent brittleness of PLA. In general, plasticizers lower the gas-barrier properties of polymers. In that study, the effect of plasticizers on the oxygen permeability of PLA was offset by the formation of the kaolinite nanocomposite. The combination of PLA and MMT layered silicate may result in a nanocomposite with barrier properties suitable for food packaging applications.57,58

4.5.5

Protein nanocomposites

The film-forming ability of various proteins has been utilized in industrial applications for a long time.60 Animal-derived proteins used in commercial applications are mainly casein, whey protein, collagen, egg white and fish myofibrillar protein.38 Plant-based proteins under consideration include soybean protein, zein (corn protein) and wheat gluten.61–63 Compared with nonionic polysaccharide films, protein films have better oxygen-barrier properties and lower WVP due to their more polar nature and more linear (non-ring) structure, and lower free volume.64 However, serious concerns remain regarding their performance in food packaging, including their high modulus, high water adsorption and high gas permeability. Significant efforts have been made to improve the properties of various proteins, applying nanocomposites technology, mainly usingnano clays. Whey protein has received significant attention as an edible film and coating material. Sothornvit and Krochta65 reported the formation of whey protein transparent films that also acted as oxygen barriers. TiO2 was added to form a nanocomposite with improved antimicrobial properties. Zhou and others66 indicated the potential of whey/TiO2 nanocomposites to be used as food-grade, biodegradable packaging materials. Addition of small amounts (<1 wt%) of TiO2 nanoparticles significantly increased the tensile properties of whey protein isolate (WPI) films (1.69 to 2.38 MPa). Similar studies regarding interactions in ZnO–whey protein nanocomposites have been reported.67 Soy protein has been of great interest to researchers for its thermoplastic properties and its potential as a biodegradable plastic. However, because of its poor response to moisture and high rigidity, its biodegradability has not been exploited effectively.68 Like starch, soy protein is also blended with plasticizers to overcome brittleness. However, the use of plasticizers further decreases barrier properties. Soy protein nanocomposite films showed reduced WVP and improved elastic modulus and tensile strength compared to counterparts without fillers.23,50,69–71 Dean and Yu69 reported an increase in tensile strength (47%) of resulting films when soy protein nanocomposite

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dispersions were treated with ultrasound. Chen and Zhang23 investigated the mechanism of interaction between soy protein and MMT clay by correlating their structure and properties. The surface electrostatic interaction between the soy protein (+ charge) and the MMT layers (− charge), as well as hydrogen bonding between − NH and Si-O groups, were believed to be the major interacting mechanisms in protein–MMT systems. Such mechanisms resulted in improved mechanical strength of nanocomposites. Young’s modulus (E) increased from 180.2 to 587.6 MPa with an increase of the MMT content from 0 to 20 wt%. Tensile strength improved from 8.77 to 15.43 MPa when MMT content increased from 0 to 16%. Bionanocomposite films of soy protein isolate (SPI) and MMT were prepared using melt extrusion.72 The effects of pH, MMT content and extrusion parameters on the structure and properties of SPI–MMT bionanocomposite films were investigated by XRD, TEM and SEM. The arrangement of MMT in the soy protein matrix ranged from exfoliated at lower MMT content (5%) to intercalated at higher MMT content (15%). There was a significant improvement in the mechanical (tensile strength and per cent elongation at break) and dynamic mechanical properties (glass transition temperature and storage modulus), thermal stability and WVP of the films with the addition of MMT. The effects of clay content, homogenization parameters and pH on the mechanical and barrier properties of fish gelatin/nanoclay composite films were investigated by Bae and others.73 Unmodified sodium MMT clay in a glycerol solution was treated with ultrasound and added to fish gelatin solutions, before casting into films. The addition of 5% nanoclay (w/w) increased the tensile strength from 2.37 to 3.30 MPa. At 9% (w/w) loading, gelatin films exhibited the largest improvements in oxygen- and water-barrier properties. Oxygen permeability decreased from 402.8 to 114.4 g·m/m2·day·atm and WVP decreased from 31.2 to 8.1 ng·m/m2·s·Pa. XRD and TEM data suggested that ultrasonication resulted in exfoliation of the silicates. Zein, a relatively hydrophobic protein found in corn kernels, is known to readily form films.74–76 Zein is used in the food industry as a coating agent and has shown potential as a biodegradable polymer.75,77 However, zein products, although less water sensitive than other biopolymers, still show high WVP and low tensile strength when compared with commodity polymers. As was the case with other biopolymers, its inherent brittleness may be ameliorated by the use of plasticizers, which, on the other hand, further decrease water vapor- and gas-barrier properties. Recent work by Arora and Padua78 is concerned with the formation of zein nanocomposites using kaolin nanoclays. Kaolin-based barrier coatings give useful properties when applied to paper and paperboard. Such nanocomposites may serve to replace fluorocarbons in extruded polymer barrier coatings.79 The objective of the study was to evaluate changes in the water resistance of zein-coated paper as a result of added kaolinite. Preliminary results indicated that water vapor permeation decreased by 50%. A reduction in water absorption and oil permeation rate was also observed. Lower permeability was possibly due to the formation of a tiled nanoplate structure, kept in place by a film of zein on the paper substrate (Fig. 4.3). Nanoplate layering may have contributed to overcoming the water sensitivity of both the paper substrate and the protein film. Further research is needed to investigate the properties of zein–nanoclay films.

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Fig. 4.3

4.6

Protein–nanoclay composite.

Conclusion

This chapter illustrated the potential of nanocomposites in food packaging. The main driver is the need for better barrier properties from polymeric materials. Better barriers against the migration of oxygen, CO2, water vapor and flavor compounds would have a major impact on the shelf-life of fresh and processed foods. In the case of biopolymers, improving water- and gas-barrier properties is a critical issue. Nanocomposites technology is still in its early stages. MMT and kaolinite clays have shown good potential for improving the properties of polymeric materials. GNPs are novel highly promising carbon-based nanosized fillers. Best effects are generally observed at low loads (∼5 wt%). Good compatibility between filler and polymer is essential, hence the importance of chemically modified clays. With respect to processing, although exfoliation is recognized as a processing goal, an orderly array of platelets in the polymer matrix, which would maximize effectiveness, is still largely unachieved. Improvements in barrier properties are often reported at 50% that of the neat polymer. Thus, polymers of intrinsically better barrier properties will render even better products as nanocomposites. Further improvements could be expected from the development of more compatible filler–polymer systems, better processing technologies and a systems approach to the design of polymer–plasticizer–filler.

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5

Nanotechnology–enabled delivery systems for food functionalization and fortification

Rashmi Tiwari and Paul Takhistov

Abstract: The use of functional foods for health enhancement is of great public interest. Recognition of the powerful functionality of micronutrients has established a new group of food ingredients, called nutraceuticals, positioned between nutrition and medicine. But despite the growing interest in supplementing foods with nutraceutical ingredients such as antioxidants, phytochemicals, omega-3 fatty acids, proboitic bacteria and others, incorporating these components into existing food formulations presents many challenges due to the high concentration of bioactives required to provide specific health benefits, the disagreeable taste and aroma associated with most nutrients, chemical instability and undesirable interaction with other ingredients in the food system, and the risk of reducing bioavailability due to active inability to reach the target site to provide desirable functionality. Various drug delivery systems such as liposomes, solid lipid nanoparticles, micelles, and polymer micro/nanoparticles have shown much promise in controlled release and targeted drug delivery. Colloidal particles in the size range 10–100 nm are considered nanoparticles. Conversion of coarse particles of nutraceutical compounds to nanoparticles or nanosuspensions can be effective in handling the solubility and bioavailability challenges in the delivery of nutraceuticals. This chapter will review a variety of nanoparticles that are currently under consideration as carriers of therapeutic molecules. These include liposomes, cochelates, coacervates, hydrogels, dendrimers, polymeric micelles, nanoemulsions, and others. Keywords: functional food, nutraceutical, antioxidant, phytochemical, omega-3 fatty acid, proboitic bacteria, liposome, cochelate, coacervate, hydrogel, dendrimer, polymeric micelle, nanoemulsion

5.1

Introduction: functional foods

The application of functional foods for health enhancement is of great public interest,1–4 reflected in the rapidly expanding market for food functionalization and fortification which is expected to reach $177 billion in 2013.5 Nanotechnology Research Methods for Foods and Bioproducts, First Edition. Edited by Graciela W. Padua and Qin Wang. © 2012 John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc.

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Understanding the powerful functionality of macro and micro nutrients has established a new group of food ingredients positioned between nutrition and medicine.6 People who consume fruits and vegetables have shown lower risk of cancer and certain other chronic diseases, based on epidemiological studies.7 These benefits have been attributed to certain dietary components termed nutraceuticals. A revolution has taken place with functional foods containing nutraceuticals since food is being linked to cure for diseases.8 However, there is now a need for food grade delivery systems that provide substantial health benefits including prevention and/or treatment of chronic diseases.1,2 Such products are regarded as solutions to today’s major public health problems. New technologies need to be developed to deliver nutraceuticals, improving their stability and bioavailability. Due to the complexity of the food matrix, there is a major research question: What are the challenges we may encounter in delivering these nutraceuticals for their health benefits?

5.2

Food matrix and food micro-structure

The choice of food system used for fortification with nutraceuticals plays a critical role. Most foods are dispersed systems and are physically heterogeneous, multicomponent and multi-phasic. The structure and properties of food systems depend on composition, processing and storage conditions. Eventually, the food structure determines a range of quality aspects for food systems. In general, a food system can be represented as a matrix composed of protein, carbohydrates, fat, nutrients, colors, flavors, and food additives. Water is also an important component of food matrices. Manufactured foods represent a broad spectrum of structured matrices. For example, beer foam is a dispersion containing gas bubbles, milk is a dispersion containing fat droplets and protein aggregates, and salad dressing is an emulsion. But other manufactured foods are structurally complicated in that they contain several different structural elements of widely varying size and state, e.g. materials obtained by extrusion, dough, and bread, have very complex structures. Thus, when considering the option of delivering nutraceuticals using a nanoparticle one needs to understand how a structured food matrix interacts with nanoparticles. The choice of foods to fortify with nutraceuticals is also affected by their composition and processing conditions. In fruit juices and drinks, low pH could cause loss of vitamin A, folic acid and calcium, whereas different heat treatments could cause loss of heat labile vitamins: thiamine, folic acid and ascorbic acid. For example, a significant reduction in vitamin B1 (thiamin), B2 (riboflavin), B3 (niacin), B6, and folate has been reported under the influence of different milk processing regimes.9,10 Similar losses on heat processing have been reported for ascorbic acid in orange juice.11 In yogurt, the low pH could pose a great challenge in stabilizing nutraceuticals.12 In high protein food, calcium and magnesium could destabilize proteins.13 Ascorbic acid could involve itself in a browning reaction.14,15 Lycopene emulsion was found to be more stable in orange juice and skim milk as compared to water.16 Orange juice contains vitamin C, which may help to prevent lycopene oxidation. In the case of skim milk, proteins chelate metal ions prevent degradation of lycopene. Degradation in water was attributed to dissolved oxygen.

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Gas bubbles Plant cell and cellular walls Protein assemblies

Crystals Fat / oil droplets Starch granules

Food polymers

Powders Fibers

1 nm Fig. 5.1

10 nm

100 nm

1 μm

10 μm

100 μm

Scale of the structural elements in food matrices.

The various elements contributing to food structure include plant cells, cell walls, meat fibers, small particulate materials, powders, starch granules, protein assemblies, food polymer networks, crystals, oil-droplets, gas bubbles and colloidal particles.17 The different length scales in food microstructure are shown in Fig. 5.1. Food functionality is highly dependent on its microstructure and with nanotech even smaller particles are part of food structure.18 Today many microand nanostructures are generated based on colloidal science.19 Air bubbles are important structural elements in solid foams such as bread, whipped cream, or cappuccino.17 The physical structure of food can have physical, chemical or geometrical effects on nanoparticles. For example, milk contains protein and fat particles suspended in an aqueous medium. Properties of emulsions, creams and textured products depend on the structural interactions of various micro particles. The texture of fruits, vegetables and meats also arises from the interaction of particles (cells) within a matrix. Thus, an important question is how to design nanoparticle size, shape and surface physical and chemical properties, and also how they are affected by the food matrix. As nutraceuticals are affected by food matrix, nanoparticles offer needed protection. However, the next steps of concern are: (i) How would nanoparticles interact with food phases solid, liquid, or gas? (ii) How are their chemical, physical or geometrical properties affected by interactions with food particles and surfaces? (iii) Which different interactions – specific or non-specific, like van der Waals, steric, electrostatic – occurring between nanoparticles and food particles become of concern? The successful delivery of nutraceuticals into our bodies through a food system poses a big challenge. There are many concerns associated with nutraceuticals which

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demand a search for a delivery vehicle. There are several hurdles which need to be overcome before we start benefiting from these nutraceuticals, some of which are discussed below. These issues to a large extent answer the big question: Why do we need a delivery system for nutraceuticals? Efficient delivery systems are today’s demand to overcome the following issues associated with delivering nutraceuticals through a food system.

5.3

Target compounds: nutraceuticals

Nutraceuticals can be defined as food or food ingredients that aid in the prevention and/or treatment of diseases/disorders.20 There are numerous reports in the literature regarding health benefits of the various bioactive compounds. Recently, moderate coffee consumption has been associated with reduced risk of Type II diabetes, cancer, and liver disease.21 This effect has been attributed to the bioactive compounds in coffee. Consumption of fish oil, rich in omega-3 fatty acids, prevents atherosclerosis and is beneficial against tissue inflammation.22 The fetus and young children require omega-3 fatty acids for normal brain and nervous tissue development.3,23,24 Another example includes tea catechins, which are reported to be bioactive.25,26 But to get the real benefit from these nutraceuticals, researchers recommend consuming 10 cups of tea per day which is impractical. Edible vaccines can be created by adding antigens to food.27 Antioxidants like carotenoids may be useful in prevention of cancer.28 Vitamins play a critical role in brain functionality, e.g. vitamin C acts as a scavenger for free radicals in the brain.29 Quercetin can function as an antioxidant as it scavenges radicals, inhibits lipid oxidation and chelate metals, and could prevent cardiovascular disease.30,31 Curcumin is a potent antioxidant, anti-inflammatory and anti-Leishmania compound.32 Vitamins E and C, β-carotene and selenium act as antioxidants and help reduce oxidative stress and prevent cardiovascular disease, cancer and age-associated macular degeneration.33 LDL oxidation can be reduced by the antioxidants Vitamin E, C and β-carotene.34 Table 5.1 summarizes some of the links between nutraceuticals and their biological activity along with their chemical names, bioavailability and solubility data. The RDA and AI values mentioned in the table are mainly for traditional nutrients, as for new nutracetucials these values are yet to be established. Despite the growing interest in supplementing food with nutraceutical ingredients such as antioxidants, phytochemicals, omega-3 fatty acids, probiotic bacteria and others, incorporating these components into existing food formulations presents many challenges,35 due to the: ● ● ●

●

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high concentration of bioactives required to provide specific health benefits; disagreeable taste and aroma associated with most bioactive compounds; chemical instability and undesirable interaction with other ingredients in the food system; risk of reducing bioavailability or inability to reach its target site to provide desirable functionality.

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Table 5.1

Nutraceutical health benefits and dose levels.

Category

RDA/AI

Health benefits

Solubility

Vitamins A D E K C B1 B2

900 μg ∼7 μg 15 × 103 μg 120 μg 90 × 103 μg 1.2 × 103 μg 1.3 × 103 μg

Prevents certain cancers and skin disorders Essential for bones and teeth Antioxidants and immune booster Essential for blood clotting Antioxidant Essential in neurological function Maintains healthy eye, skin and nerve function Proper brain function Synthesis of coenzyme A Produces essential proteins Catalyst for essential metabolic reactions Necessary for genetic material for cells

Lipid Lipid Lipid Lipid Water Water Water

Oxidation of fatty acids Lipotropic agents Amino acid transport Insulin activity

Water Water Water Water

Building bones ATP processing Energy processing Electrolyte Electrolyte Essential amino acids and proteins

Water Water Water Water Water Water

Biosynthesis of thyroxin Required for hemoglobin Required for peroxidase Brain mineral Insulin regulation

Water Water Water Water Water

Neutralizes free radicals Healthy vision Healthy vision Prostate health

Lipid Lipid Lipid Lipid

Heart health Antitumor, antiinflammatory Reduced risk of heart disease

Lipid Lipid Lipid

Inhibitors of cell proliferation Strong antioxidants can treat cancer

Lipid Lipid

Mental and visual function

Lipid

B3 16 × 103 μg B5 5 × 103 μg B6 ∼1.5 × 103 μg B7 300 μg B9 400 μg B12 2.4 μg Vitamin-like compounds L-carnitine Choline ∼500 × 103 μg Inositol (B8) Taurine Minerals Macrominerals Calcium >200 × 103 μg Magnesium >200 × 103 μg Phosphorus >200 × 103 μg Potassium >200 × 103 μg Sodium >200 × 103 μg Sulfur >200 × 103 μg Trace minerals Iodine <200 mg Iron <200 mg Selenium <200 mg Manganese <200 mg Chromium <200 mg Carotenoids Beta-carotene Lutein Zeaxanthin Lycopene Flavonoids Catechins Quercetin Kaemferol Isoflavones Genistein Daidzein Fatty acids Omegas Phytoestrogens Lignans Saponins Tangeretin

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Water Water Water Water Water Water

Antioxidant Hypercholestersolemic effect Antimutagenic, antiproliferative

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Pharmaceutically active compound

Highly permeable

Soluble

Well absorbed

Low soluble

Rate of solvation limits bioavailability

Low permeable

Soluble

Fast solvation but absorption is limited by permeation rate

Low soluble

Poor bioavailability

Fig. 5.2

Intestinal absorption pattern.

5.3.1

Solubility and bioavailability of nutraceuticals

Solubility is a measure of the maximum amount of solute that will dissolve in a solvent. The intermolecular forces between solvent and solute determine solubility. Solubility is affected by temperature and pressure as they alter this balance. Solubility is also affected by complex-forming ions and common-ion effects. Solutes dissolve best in solvents of similar polarity. The rate of dissolution is an important factor to be considered for controlled drug delivery. It is affected by the compound’s crystalline properties (crystalline and amorphous) and presence of polymorphism. Partition coefficient is a measure of differential solubility of a compound in a hydrophobic vs. a hydrophilic solvent. Nutraceuticals could be water or fat soluble. This difference in solubility poses a great challenge to incorporating them in food, and greatly affects their bioavailability. Bioavailability refers to the degree and rate at which a substance is adsorbed in a living system or is made available at the site of physiological activity. Bioavailability is 100% when compounds are administered intravenously, but decreases when administered via other routes (e.g. orally) due to incomplete absorption and first pass metabolism. Absolute bioavailability is the availability of an active drug in systemic circulation after non-intravenous administration. Relative bioavailability is the bioavailability of a molecule when compared with another formulation of the same compound, or when it is administered via a different route. The biopharmaceutics classification system (BCS) guides through predicting intestinal drug absorption patterns based on solubility and intestinal permeability (see Fig. 5.2) and can be used as a guide for nutraceuticals. Bioavailability differs greatly among nutraceuticals. For example, curcumin, a potent antioxidant and anti-inflammatory,32,36 is practically insoluble in water, making it less bioavailable. The bioavailability of quercetin is 17% and is strongly affected by the attached sugar moiety.37 Bioavailability differs greatly from one polyphenol to another. The bioavailability of tea polyphenols such as EGCG is not high. The peak plasma concentration of epigallocatechin gallate EGCG (free and conjugated form) is approximately 1 μM.38,39

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The bioavailability of carotenoids is also very poor.40 Lycopene is more bioavailable in oil than water.41 In food, carotenoids are bound to proteins, further reducing their bioavailability.42–44 Iron has very low bioavailability and adding high amounts has undesirable organoleptic effects. Factors such as aw, pH, osmolarity, ionic content, presence of fat, lipid, and proteins could affect its bioavailability. There are many factors that affect bioavailability in vivo due to specific interactions with human tissues, such as: poor absorption from GIT; degradation or metabolism of drug prior to absorption; hepatic first pass effect; drug taken with or without food; and disease. Omega-3 and omega-6 fatty acids could be made available in vivo only after they are hydrolyzed from dietary fats by pancreatic enzymes.45 Flavonoids exist as glycosides and have low bioavailability as they are rapidly metabolized and have limited absorption.46–48 Similarly, resveratol has low bioavailability due to rapid metabolism and elimination.49 Another concern with nutraceuticals is that they present different molecular and physical forms, not all readily adsorbed. Polarity, molecular weight, physical state, hydrophobic and hydrophilic character50 significantly affect their bioavailability. Overcoming cellular and tissue barriers, like crossing blood brain barrier and GIT, is also a challenge for many nutraceuticals. This property causes problems in delivering these compounds in food systems as well as affecting their bioavailability or absorption in biological systems, hence demand for a delivery vehicle. Vitamin losses vary in different foods considerably during both processing and storage of the final product. Stability of vitamins is affected by a number of factors, such as temperature, moisture, oxygen, light, pH, and vitamin–vitamin interactions, among others.51 The most unstable vitamins are the B group and C. The breakdown of vitamins can be accelerated by vitamin–vitamin interactions.52 In general, minerals are resistant to processing but some are lost during cooking. Minerals are affected by pH, heat, and air, and may react with other food components including proteins and carbohydrates.51 Carotenoids (e.g. lycopene) can be easily isomerized by heat, acid or light. They can be easily oxidized due to their large number of conjugated double bonds.16, 53–55 Such reactions can cause color loss of carotenoids in foods and are major degradation mechanisms. Anthocyanins’ stability in foods is greatly affected by pH and heat.56 Catechins are unstable to pH and heat.57 They are stable in black tea and unstable in green tea. Catechin isomerizes with browning at pH > 6. Due to high degree of unsaturation omega-3 fatty acids are very susceptible to oxidation and oxidization products may be harmful.58–61 Thus, incorporating nutraceuticals into existing food formulations presents many challenges, e.g. fortification of beverages with vitamin E. Dispersed vitamin E droplets rise to the top to form a whitish ring, increasing turbidity and changing product appearance62 which is undesirable.

5.3.2

Interaction of nutraceuticals with food matrix

There are many physical and chemical factors, including heat, moisture, exposure to air or light, and acid or alkaline environment, that affect the stability of nutraceuticals. Exposure to these factors during processing, distribution and storage affect stability.

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Nano-scale carrier

Nutraceutical compound Surface properties

Format

Molecular weight Polarity Chemical nature Physical form

Composites Particulates Structure Porous Compact Nano-scale delivery system

FOOD

Food matrix environment

Food matrix structure

Food processing

Water content pH Enzymes Ionic strength Composition

Fibrous Liquid Amorphous Crystalline

Thermal Mechanical Non-thermal Fermentation

Fig. 5.3

Factors affecting delivery of nutraceuticals via nanoparticles.

The nanoparticle delivery system when added to food has to be stable against food conditions, processing, presence of other components, and food microstructure as shown in Fig. 5.3. Nanoparticles should be able to withstand various food conditions of aw, pH, ionic strength, and must be stable against the presence of enzymes, metal ions, proteins and lipids. The delivery system should also be stable against food processing methods such as baking, pasteurization, or extrusion. Stability is expected to extend beyond processing and into packaging and transportation in order to have longer shelf life. Food matrix influences nutraceuticals’ bioavailability. Some nutraceuticals show increased absorption, whereas others show decreased absorption in the presence of certain foods. A high- or low-fat meal can affect bioavailability. Nutraceutical binding to metal ions such as calcium is influenced as some of the food components, e.g. milk and yogurt, contain calcium. High fat in foods can improve the bioavailability of poorly soluble nutraceuticals via improved solubilization as well as transport to the lymphatic system, whereas a low-fat diet will decrease the bioavailability. On the other hand, high protein content in food may impede absorption of certain amino acid-based nutraceuticals by competing for absorption. Calcium and heavy metals in certain foods, such as milk and

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Table 5.2

Examples of nutraceuticals’ incompatibility in foodstuffs and the human body.

In foods ● ● ●

63

Interaction of iron with unsaturated fatty acids leads to rancidity. Metal catalyzes degradation of vitamin C. Iron accelerates vitamin C loss in fruit juices.

In vivo ● ● ● ● ●

Zinc absorption is significantly affected by iron. Calcium inhibits iron and zinc absorption. High consumption of β-carotene competes with lutein and lycopene absorption. Flavonoids bind non-heme iron, inhibiting intestinal absorption. Pantothenic acid may compete with biotin for intestinal and cellular uptake.

yogurt, may bind to nutraceuticals and form insoluble complexes. Polycyclic aromatic hydrocarbons in smoked food, the presence of enzyme inducers in certain spices, and cruciferous vegetables may affect the bioavailability of certain nutraceuticals. A lot of data are available on the effect of food on drug performance in vivo. Interaction depends on physicochemical and biopharmaceutical drug properties and food-induced physiological changes. Food can cause an increase or decrease in absorption of drugs. An excellent compilation on this topic is available from.63 Food has a positive effect on drugs with high pKa by stabilizing them in the stomach. Even with a low pKa, nutraceuticals may precipitate due to food composition.63 Poorly solubilized drugs could have increased stabilization in the presence of food. Acid labile drugs have the potential to undergo degradation in the presence of food. The effect of food on physicochemical and biopharmaceutical drug properties depends on the food’s ability to influence GIT. pH solubilization effects and lymphatic absorption, residence time and blood flow could affect drug bioavailability. For example, a food matrix with a high fat content increases bioavailability of poorly soluble drugs. High protein food can prevent absorption of amino acids drug due to competition. Metals in food may form insoluble complexes with drugs. Similar to the effect of food on drug absorption, food and food components can have a huge impact on both the physicochemical and biopharmaceutical properties of neutraceuticals. Food effects on controlled release of nutraceuticals pose unique challenges. Thus it is important to protect food ingredients from various food effects, nanoparticles could offer a solution. There are many reasons why one would like to combine multiple nutraceutical compounds in the same formulation due to their synergistic effects. For example, the combination of vitamin E, C and β-carotene has been shown to be useful in reducing low density lipoprotein oxidation and subsequent atherosclerosis. Vitamin C scavenges free radicals, Vitamin E protects against polyunsaturated fatty acids, and β-carotene provides antioxidant activity.34 However, different nutraceuticals are often incompatible with each other within a formulation or in vivo, demanding a delivery vehicle, some of which are listed in Table 5.2. Nutraceuticals’ performance could also be affected by the presence of drugs in vivo and vice versa. Some of these effects are as listed below, e.g. anti-seizure drugs may reduce the level of biotin in the blood.64 Calcium could decrease absorption of

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antibiotics (tetracycline and quinoline). Cholesterol-lowering agents can reduce absorption of fat soluble vitamins and carotenoids. Coenzyme Q10 has been reported to decrease the anticoagulation effect of drugs.65,66 In cultured breast cancer cells, curcumin has been shown to inhibit apoptosis induced by chemotherapeutic agents.67 Flavonoids can inhibit intestinal drug metabolizing enzymes. Cholesterol-lowering drugs could decrease absorption of folic acid.68 Delivery vehicles are required to shield/protect nutraceuticals for greater health benefits. These vehicles will help protect nutraceuticals from the environment, make them more bioavailable, and help their delivery to the appropriate site in order to perform the required function. Nanoparticles could function as delivery vehicles.

5.4

Delivery systems

The smallest capillaries in the human body are 5–6μm in diameter, thus the delivery vehicle to be distributed by the blood stream must be smaller.69 The dissolution rate linearly depends on surface area. Decreasing particle size from microns to nanometers increases surface area, causing an increase in the dissolution rate.70 There is a restriction on the particle size that can cross the intestinal lumen into the lymphatic system following oral delivery. Particle size is critical for controlling diffusion through the mucosal gel layer. Particle size, type and composition of polymer, particle surface charge, hydrophilic/hydrophobic balance used for microand nanoencapsulation are crucial for uptake and transport across the mucosal barrier.71 Cystic fibrosis sputum offers a size-dependent barrier to transport of nanospheres. Large nanospheres of 560 nm are completely blocked, whereas smaller particles of 120nm are moderately retarded. Thus particle size has influence in transport phenomena.72 Uptake of particles increases with decreasing particle diameter.73 Particles of 3–10 μm do not migrate through the lymphatic system whereas particles larger than 10 μm are not taken by GIT.74 There are studies proving that the particle uptake depends on the size, with smaller nanoparticles showing relatively greater efficacy of uptake than larger micro particles,75 making them a better choice of delivery vehicle.

5.4.1

Overcoming biological barriers

Nanoparticles show higher uptake intracellularly: 100nm particles showed higher uptake compared to 1μm particles in CaCo-2 cells.76 As compiled by Delie,77 uptake of nanoparticles in the GIT tract is dependent on size, nature of polymer, zeta potential, coating with lectins or adhesion factors; even the presence of nutrients could influence their uptake.77 Nanoparticle size is important for uptake into the epithelial of GIT and sizes smaller than 500nm are required. Nanoparticle surface properties and hydrophilic/hydrophobic balance also affects their uptake. The submicron size of nanoparticles has helped in improving the therapeutic and pharmacokinetic performance of drugs, thus enabling effective drug delivery by various routes.78 The nano size of these systems allows crossing biological barriers, ameliorates tissue tolerance, and improves cellular uptake and transport.79–84 Surface properties of nanoparticles could be modified as desired to achieve controlled and targeted release of drugs/nutraceuticals. Nanoparticles have offered solutions to

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many problems associated with drug delivery, and the same may be extended to the delivery of nutraceuticals. Thus there is a direct correlation between particle size and in vivo exposure. It is found that nanoparticle colloidal dispersions provide the highest exposure, suggesting dissolution-limited absorption. Due to small particle size and increased surface area, nanoparticles could overcome the narrow absorption window. These studies can be correlated with the bioavailability and absorption characteristics of the healthpromoting constituents in foods. The increase in absorption can be achieved by formation of nanoparticles without affecting the mechanism of biological systems. The performance in biological systems can be further improved by controlled release and surface modification of these nanoparticles.

5.4.2

Nano-scale delivery systems

Colloidal particles in the size range of 10 to 100nm are termed nanoparticles.85,86 Different molecules could be bound to nanoparticles by sorption, incorporation or chemical bonding. Today, nanoparticles have found their way into many applications, including clinical diagnostics assays, imaging, and drug delivery systems.87–89 Colloidal gold, iron oxide crystals and quantum dots semiconductor nanocrystals are examples of nanoparticles (size range of 1–20nm) that have diagnostic applications in biology and medicine.90 Nanoparticle classification based on dimensions, phase composites and manufacturing is compiled in Fig. 5.4. The choice of a delivery vehicle depends on the drug/nutraceutical potency, size, stability, solubility and charge. Delivery vehicles could be differentiated based on the following attributes (adapted from91), as listed below: ● ● ● ●

drug loading capacity; possibility of drug targeting; in vivo fate of carrier; acute and chronic toxicity;

Nanoparticulate system

Dimension

1-D

2-D

3-D

Tubes Fibers Platelets

Films Coatings Multilayer laminates

Particles Hollow spheres

Fig. 5.4

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Phase

Single phase

Multi phase

Crystalline amorphous particles and layers

Suspensions Emulsions Foams

Composites

Matrix composites Coated particles

Classification of nanoparticulate systems.

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Nanotechnology Research Methods for Foods and Bioproducts

scaling-up production; physical and chemical stability, including processing, post-processing, and shelflife stability; overall costs.

Nanoparticles possess a very high surface-to-volume ratio and, hence, a high dissolution rate. They have several advantages due to enhanced solubility, stability and long circulation time.92–96 Nanoparticle drug carriers help reduce drug toxicity.97 Delivery of hydrophobic compounds is a major challenge, but nanoparticle delivery systems like polymeric nanoparticles, nanocapsules, solid lipid nanoparticles, nanogels and drug nanoparticles offer solutions for their delivery. In vivo and in vitro studies have demonstrated that nanoparticles are promising carrier systems for drug targeting. Nanoparticles have shown the ability to deliver their cargo to a wide range of tissues in the body for sustained periods of time.69 They have been used to deliver hydrophilic drugs, proteins, vaccines, and biological macromolecules to cells and tissues.69,76 The uptake of nanoparticles by endothelial cells is shown to be time- and concentration-dependent.98 Delei77 has provided an excellent review compiling different studies on uptake of nano- and micro particles by the GI tract.77 These systems can be used to provide targeted (cellular/tissue) delivery of drugs, peptides, or genes. They help solubilize drugs for intravascular delivery and improve stability of therapeutic agents, especially proteins, peptides and nucleic acids against enzymatic degradation.99–101 Overcoming the blood–brain barrier is a challenge for many drugs and nanoparticles have being shown to be promising for delivering therapeutic molecules across the blood brain barrier.79,102 Nanoparticles can be formulated for targeted delivery to the lymphatic system, brain, arterial valve, lungs, liver, and spleen or made for long-term circulation.69 Nanoparticles enter the circulation; they are coated by plasma protein (opsonization).103 Opsonization renders particles recognizable by the body’s major defense system, whereas the RES specialized cells are phagocytic.104 Macrophage (Kupffer) cells of liver and spleen play a critical role in the removal of opsonized particles. Thus application of nanoparticles requires surface modification that would ensure particles were non-toxic, biocompatible and stable to RES. Particles with hydrophobic surfaces are removed from the circulation readily by phagocytic cells of the RES, but particles with hydrophilic surface (e.g. hydrogels) are shown to clear slowly.105 Initial disadvantages of nanoparticles, such as rapid uptake by the reticuloendothelial system due to phagocytosis by macrophages in the liver and spleen,85 have been overcome by steric stabilization of nanoparticles. PEGylation106 is used for covalent modification of biological macromolecules and surfaces, shielding of antigenic and immunogenic epitopes, shielding receptor mediated uptake by RES, and preventing recognition and degradation by proteolytic enzymes. PEGylation increases the size of polypeptides, thus reducing renal filtration and altering biodistribution. The nanoparticles’ surface characteristics could be modified for targeted delivery.107 The most common coatings are dextran, PEG, PEO, proloxamers and polyoxamines.108 The role of dense brushes of polymers is to inhibit opsonization and hence allow longer circulation time.109 A further strategy in avoiding RES is by reducing particle size.80,92

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Nanoparticles adhere to the surface of intestinal mucosa through bioadhesion processes, increasing bioavailability.110 Bioadhesion could occur through specific (ligands) or nonspecific (surface properties) interactions. The bioadhesive potential of a drug delivery system is affected by affinity of the material to biological support, intensity of phenomena, and relative duration of adhesive bonds and elimination rate of adhered particles.111 Modifying surface properties of PVM/MA nanoparticles with BSA and lectin has been shown to affect their adhesive property. Nanoparticles could be targeted to specific cells and locations in the body with the help of appropriate ligands. The carrier can be activated by a change in environmental pH, chemical stimuli, magnetic field, or heat. Nanoparticles have offered several solutions towards specific and targeted delivery of anticancer drugs such as paclitaxel, doxorubicin, 5-flurouracil, camptosor and topotecan, solving different issues associated with each drug.112 These include decreased solubility in aqueous solution, number of side effects, faster clearance and high hydrophobicity. Silica, alumina and titania are biocompatible. Ceramic-based nanoparticles carrying photosensitized drugs could be irradiated with light of suitable wavelength which results in effective generation of singlet oxygen which causes significant damage to tumor cells.113 Nanoparticles are very effective in controlled delivery of drugs. This characteristic property may be carried over to food systems. Production of nanoparticles has proved to be an effective tool in dealing with solubility problems in drug delivery. The oral delivery of drug particles in the form of nanoparticles has the following advantages: improved bioavailability, improved dose delivery, enhanced absorption rate. For optimal drug action, the most efficient way is to deliver the drug to the desired site of action in the body and decrease or avoid the side-effects at non-target sites. Various drug delivery systems such as liposomes,114 solid lipid nanoparticles,115 micelles,116 and polymer micro/nanoparticles117 have shown much promise in controlled release and targeted drug delivery. Among them, biocompatible and biodegradable polymer nanoparticles were preferable candidates for DDS.118 Food particles act similarly to drug particles. Thus conversion of coarse particles of nutraceutical compounds to nanoparticles or nanosuspensions can be effective in handling the solubility and bioavailability problems in the delivery of nutraceuticals.119 Thus, similar to drug delivery, there are different goals to be achieved in food nutraceuticals delivery. Nutraceuticals need to be made further bioavailable, protected from the environment (heat, moisture, and oxidation), and delivered to the appropriate site for them to perform the required function. Those factors are compiled in Table 5.3 to provide insight into the design of delivery vehicles. Efficient delivery systems are required to enhance the stability and bioavailability of nutraceuticals. Nanoparticles may be useful in delivering nutraceuticals through food systems as shown in Table 5.4.

5.4.3

Types/design principles

A wide variety of nanoparticles are currently under investigation for carriers of therapeutic molecules, including liposomes, cochelates, coacervates, hydrogels, dendrimers, polymeric micelles, carbon nanoparticles, drug nanoparticles,

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Table 5.3

Comparative challenges for delivery of nutraceuticals: in food and in vivo.

In food

In vivo

High concentration of actives required to provide specific health benefits Disagreeable taste and aroma associated with most nutrients Chemical instability and undesirable interaction with other ingredients in the food system Risk of reduction of bioavailability due to active inability to reach the target site for the desired functionality Poor solubility

Protection of the nutraceutical Control of the duration of action of the nutraceutical Maintenance of nutraceutical levels in the human body Targeting of the nutraceutical to a particular part or cells in the body Overcoming of certain cellular and tissue barriers, etc.

Table 5.4

Effects of delivery systems.

Problem

Implications

Effect of delivery system

Poor solubility, e.g. CoQ10

Delivery is difficult as the hydrophobic nature of the nutraceutical may precipitate in aqueous media

Rapid breakdown, e.g. flavanoids

Loss of activity, e.g. loss due to pH

Susceptible to oxidation, e.g. omega fatty acid

Oxidative products are harmful

Degradation, e.g. theaflavins Hydrophobic nutraceuticals, e.g. ECG and EGCG Unstable to heat, acid and light, e.g. lycopene

Loss of activity to metabolization by enzyme Difficult to incorporate in food systems

A delivery system such as liposomes provides both a hydrophilic and a hydrophobic environment, enhancing solubility A delivery system such as dendrimers can protect against premature degradation A delivery system such as coacervates can help increase thermal stability and protect against oxidation Delivery system provides protection against such degradations A delivery system such as hydrogels can be used

Loss of activity due to cis/trans isomerization

Delivery system provides protection against heat, acid, light, etc.

nanoemulsions, and other nanoparticles (e.g. magnetic), with each class offering various advantages and disadvantages.120 Nanoparticle-based delivery vehicles are widely different in structure.121 Nanoparticles also differ in makeup, morphology and size. Nutraceuticals could be attached to surfaces, encapsulated/entrapped, adsorbed/ suspended or dissolved in nanoparticle delivery systems as depicted in Fig. 5.5. Nanoparticles not only offer controlled release characteristics but also protect the encapsulated agent from degradation. Depending on particle charge, surface properties and relative hydrophobicity, nanoparticles can be designed to adsorb preferentially on specific organs or tissues.

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(a)

(b)

(c)

69

(d)

Fig. 5.5 Loading mechanisms for nanoparticulate delivery systems: (a) dissolution, dispersion, absorption; (b) physical/chemical adsorption, chemical bonding, layer-by-layer deposition; (c) coacervation, encapsulation; (d) entrapment.

5.4.4

Modes of action

The two modes of action of nanoparticle delivery systems can be broadly categorized into controlled release and targeted delivery of active compounds. As compiled by,118 release rate of nanoparticles depends on desorption of the surface bond drug, diffusion through the matrix, diffusion through the polymer wall, nanoparticle matrix erosion and combined erosion and diffusion processes. Thus release is governed by diffusion and biodegradation. 5.4.4.1

Controlled release

Controlled release over an extended duration could be highly beneficial against rapid metabolism and elimination of nutraceuticals from the body.122 With the controlled release system, the rate of administration is matched by the rate of elimination and the drug concentration is maintained within the therapeutic window. Nanoparticles offer a promising solution to the controlled release of drugs.123 Controlled release offers several advantages: ● ● ● ● ●

release at the required rate; fewer applications; reduced danger of overdose; fewer side effects; economic advantages.

In distribution control, the aim is to target the release to the precise site, whereas in temporal control delivery systems aim to deliver over an extended duration or at a specific time during treatment.124 Temporal control drug delivery systems aim to control drug release over prolonged periods of time or at a specific time during treatment. Rate of release is matched by rate of elimination, maintaining the optimum therapeutic window. Distribution control aims to target the release to a precise site of action in the body.124,125 There are several factors that affect the release rate of the entrapped drug, such as molecular weight, size, and distribution morphology and make up.126 Controlled release could be obtained through different sensing mechanisms. The physical, chemical, and biochemical means of achieving the control release are compiled in Table 5.5.

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Table 5.5

Controlled-release mechanisms.

Physical methods

Chemical methods

Biochemical methods

Osmotic pressure Hydrodynamic pressure Vapor-pressure-activated Mechanically activated Sonophoresis-activated Iontophoresis-activated Hydration-activated

pH Ion-activated Hydrolysis-activated

Enzyme-activated Small-molecule-activated

Most controlled release systems fall into one of three categories: diffusion, solvent activated, or polymer degradation.127,128 In a diffusion system, the drug is either encapsulated in a polymer membrane or suspended within a polymer matrix. Typically, water diffuses into the membrane or matrix, the drug dissolves and diffuses out of the polymer. In a membrane system, diffusion of water through the polymer is the ratedetermining step and the rate of release is constant and proportional to the concentration of drug initially present. In a matrix system, the rate of release depends upon the amount of drug present at a particular time, thus the release rate is time dependent. A solvent-activated system consists of a semi-permeable membrane containing a small laser-drilled hole. Within the membrane is a high concentration of an osmotic agent, either the drug itself or a salt that causes water to enter through the membrane. The drug is then forced out through the hole as a result of the increased pressure. The drug release proceeds at a constant rate in a solvent-activated system.129 In polymeric degradation, the drug is contained within a polymer membrane or matrix. The polymer is designed to degrade and release the drug at a specific location in the body. As the polymer degrades, the drug is freed and made available. The degradation could be caused in three different ways.129 Many degradation systems use a combination of these mechanisms: (i) Water-soluble polymers are made insoluble by cross-linking them together, and when the cross-links are broken at some point in the body, the polymer dissolves. (ii) Water-soluble polymers are made soluble by hydrolysis or ionization of side groups. (iii) Insoluble polymers are used that are cleaved into soluble monomers 5.4.4.2

Targeting delivery

Drug delivery systems alter the pharmacokinetics and biodistribution of therapeutics. Targeted and modulated release of drugs with delivery systems like nanoparticles help in disease treatment.104,130 Targeted delivery could be classified as targeting delivery to a particular organ, to a specific cell or to a structure within a cell. Targeted delivery can be achieved by either active or passive targeting. By conjugating the therapeutic agent with tissue or a cell-specific ligand, active targeting of a therapeutic agent can be achieved.131,132 Passive targeting is achieved by coupling the therapeutic agent to macromolecules that passively reach the target organ.133 Various methods have been proposed for targeting delivery based on a chemical, biological or physicochemical nature. The chemical method involves the use of

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modified forms of active drugs. These active compounds can accumulate preferentially in targeted sites and then are cleaved to release active compounds by means of enzymatic processes.134 Biological approaches to drug targeting include the use of monoclonal antibodies where a conjugate of the drug is formed by coupling it chemically to an antibody or a fragment thereof using an appropriate spacer. The conjugate can be directed towards an antigen residing on or within the target tissue. Ligands such as sugar or lectin, which can be directed to specific receptors found on cell surfaces, can be utilized for targeted delivery.103 A physical method can be used for the site-specific delivery of drugs in the form of polymeric and colloidal carriers; here, targeting can be achieved either by passive methods where a polymer conjugate or particle system is captured by a physiological uptake mechanism such as filtration or macrophages sequestration, or by active targeting by the attachment of a homing moiety such as monoclonal antibodies or ligands. Coupling with PEG or inert polymers to therapeutic molecules decreases drug clearance by the kidneys and the immune system.135 Calcium phosphate nanoparticles (80nm size) surface modified could be used to deliver entrapped DNA molecule in vivo to the liver.136 Polysaccharides could be attached to liposomes, nanoparticles and polymeric micelles for targeted delivery.137 Endothelium is an important target for drug or gene therapy. To achieve targeted delivery to endothelium, drug conjugates and immunoliposomes have being studied.98 In the liver, the asialoglycoprotein receptor could be targeted using nanoparticles attached with appropriate ligands.138,139 These nanoparticles are internalized by hepatocytes through receptor-mediated mechanisms.140 Carrier systems can be directed to bone marrow by modifying the apo-lipoprotein adsorption onto the surface.141,142 To enhance drug absorption, its intestinal residence time needs to be longer. Intestinal bioadhesion helps achieve this; lectin is one such example.143,144 Lectins are proteins able to provide specific binding to biological surfaces bearing sugar residues located at the surface of the epithelial cells, thus giving a bioadhesive nature to polymers with attached lectin.145 An example of this is the binding of lectins to nanoparticles of poly(methyl vinyl ether-co-maleic anhydride) (PVM/MA).111 Among others, bioadhesions include microbial bioadhesion and immunoglobulin. Vitamin B12 is absorbed by receptor-mediated endocytosis in the intestine after binding to intrinsic factors from the stomach and hence could be used as a bioadhesion.146 Cancer today is a major concern. Targeted delivery using degradable and nondegradable polymer nanoparticles offers treatments for cancer.109,112 Liposomes with ligands have also being targeted to be used as anticancer drugs.147 Thermallyresponsive targeted drug delivery systems have been shown to release drugs in a tumor with the help of heating by regional hyperthermia.148 Dextran-doxorubicin entrapped in chitosan nanoparticles has been shown to reduce side effects and improve therapeutic efficacy in the treatment of solid tumors.149 Nanoparticles act as vehicles to specifically deliver cancer-fighting drugs to tumors.109 Folate receptors are expressed on the surface of cancer cells and can be targeted using nanoparticles by attaching folic acid-derived antibodies.112 To achieve tumorspecific targeting, antibodies that can recognize the specific antigens on the tumor cells can be attached to the nanoparticle surface.150 The attachment can be brought about by nonspecific adsorption, specific adsorption, or by covalent linkage. Non-specific

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adsorption is achieved by incubating the nanospheres with monoclonal antibodies (Mabs). Specific adsorption is obtained by first coupling the nanoparticle to a ligand, which can specifically bind to antibodies. The Mabs are incubated with the precoated nanoparticle and bind to the spacer molecule via noncovalent linkage. Covalently bound Mabs offer the advantage of maintaining their integrity, unlike other methods.

5.5 5.5.1

Examples of nanoscale delivery systems for food functionalization Liposomes

Liposomes are phospholipids vesicles.114,151,152 The colloidal suspension consists of thermodynamically stable lipid bilayer membranes separated by water. These nanosized biodegradable lipid vesicles have aqueous space surrounded by a lipid bilayer. These hollow micro spheres are formed by self-assembly of phospholipids in water above their transition temperaturev.153–155 Liposomes can be used for targeted delivery at 50°C (transition temperature of phospholipids) where the content is released immediately. The biocompatibility of liposomes along with their amphiphilic character and smaller size make them promising delivery systems. Liposomes are classified by their size and number of bilayers as either small unilamellar vesicles (SUV) (10–100nm) or large unilamellar vesicles (LUV) (100–3000nm). If more than one bilayer is present, separated from each other by aqueous spaces, then they are referred to as multilamellar vesicles (MLV).114,156–159 These phospholipid bilayer membranes can entrap both hydrophilic and hydrophobic drugs, where lipophilic drugs can be incorporated into lipid bilayers while hydrophilic drugs are solubilized in the inner aqueous core. The performance of these vesicles is determined by size, surface charge, surface hydrophobicity and membrane fluidity.160 They have been shown to be effective in reducing systemic toxicity and preventing early degradation of encapsulated drugs.161,162 In the case of a liposome, several release mechanisms could be employed, such as pH-dependent neutralization of charged functional group, pH-dependent hydrolysis, and thiolysis, thermo sensitive, using lipids with transition temperature or integration of fusogenic components like viral protein or synthetic peptides into the liposomal membrane.163 The rapid clearance of liposomes from blood due to opsonization triggers uptake by the mononuclear phagocytic system (MPS) or the reticulo-endothelial system (RES), which is one of the major drawbacks. This has been overcome by the development of stealth liposomes, which utilize a surface coating of a hydrophilic carbohydrate or polymer. Stealth liposomes may be formed with anchored and covalently attached polymers. PEGylation offers steric stabilization, prevents adsorption of proteins responsible for phagocytotic removal and extends circulation time. The most commonly used is the lipid derivative of polyethylene glycol (PEG).164–166 They are characterized by their long circulating nature within the body as compared with the classical liposomes. Polyethyloxazoline and polymethyloxazoline have been also suggested as good alternatives to PEG.167

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The liposome surface could also be modified by introducing positive or negative charges. In attempts to increase the specificity of interaction of liposomal ingredients to the site of its action, targeting moieties (ligands) are coupled to the liposome surface. These include antibody molecules or fragments resulting in immunoliposomes and naturally occurring or synthetic ligands such as peptides, carbohydrates, glycoproteins, or receptor ligands like folate or transferrin.147 On the basis of molecular targets, vasoactive intestinal peptide receptors (VIP-R), which are over expressed in human breast cancer, VIP-grafted sterically stabilized liposomes, are synthesized.168 Sterically stabilized liposomes with an antibody attached on the surface have shown to reduce tumors in mice.169,170 Lectin binding to carbohydrates on certain cells has been used for the synthesis of lectin-liposome conjugate by covalent binding.171 The novel drug delivery system liposome-in-micro sphere which is a polymerbased and lipid-based controlled release system, integrates the benefits and avoids the downfalls of polymer and lipid-based systems.172–173 The cationic liposome binds to DNA and coats it with a cationic layer which helps in cell engulfment and transfer of genes.174 Agglomerated liposomes offer potential for aerosol drug carriers.175 Linking phospholipids by covalent bonds has been shown to increase the stability of polymerized liposomes in the GI tract.176 Iron sulphate particles have also been incorporated in polymerized liposomes, thereby making them magnetically responsive.177 The application of an external magnetic field around the mucous intestinal area is capable of retaining the liposome in the intestine and increases their delivery efficiency. Encapsulation of liposomes in polymeric matrix of micro spheres for stability is also studied.172 Hydrophilic polymers like dextran178 and hydrophobic biodegradable polymers of poly(L-lacide)- polyethylene glycol- poly(L-active) copolymers and chitosan172 as the coating have been reported. The encapsulation with hydrophobic material is done by a modified water-in-oil-water (w/o/w) double emulsion solvent extraction/evaporation process. Agglomerated liposomes with the help of cross-linking have being shown to provide controlled and modulated release of drugs.175 Linking the phospholipids by covalent bonds has been shown to increase the stability of polymerized liposomes in the GI tract. The post insertion method offers a great opportunity to prepare the targeted liposome allowing the therapy to be tailored to the needs of individual patients.147 There are several different types of liposome-based delivery systems that can be used for nutraceuticals delivery: Virosomes: Liposomal formulations that have viral envelope proteins anchored in their lipid membrane. These are effective immunogens.179 Transferosomes: A liposome consisting of phospatidylcholine and cholate with enhanced skin-penetrating properties for cutaneous applications.180 Arachaesome: A liposome prepared using archaeo bacteria membrane lipids that are reported to induce superior immune response comparable to immunization with complete Freunds adjuvant.181 Niosomes: A non-ionic surfactant vesicles with enhanced chemical stability.182,183 Proteosome: A liposome prepared by reconstitution of the bacteria’s outer membrane and composed mainly from proteins that provide high immunogeneity.184 Magnetoliposome: A magnetoliposome consists of a liposome filled with nanosized magnetic nanoparticle.185–186

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Photo sensitizer-based liposome: A liposome associated with photodynamic agent.187 PEGylated liposome: They have potential to form nano-sized micelles in aqueous environments and hence can incorporate hydrophobic drugs.188 Polyplexes/lipopolyplexes: Assemblies which form spontaneously between nucleic acid and polycations or cationic liposome and are used in transfection protocols.189 There are many advantages of liposomes as delivery systems including: (a) enabling delivery of higher drug doses; (b) targeting of specific cells, tissues or organs; (c) both hydrophobic and hydrophilic character with biocompatibility; (d) introduction of positive and negative charges to lipid provides the liposome with a surface charge; (e) LUV have high encapsulation efficiency, simple production methods and good stability over time; and (f) liposomes impart stability to water soluble materials in high water activity applications. However, some of the disadvantages190 include: (a) interaction with lipoproteins; (b) increased free radical production; (c) complete saturation of the immune system causing toxic and adverse effects; (d) the aqueous delivery form of liposomeencapsulated ingredients is a major drawback which brings concerns in terms of large-scale production, storage, shipping, and use of antimicrobials (low temperature shipping and storage could increase the cost; freeze drying could offer a solution but is an expensive process); (e) chemical and physical stability problems might lead to liposome aggregation and drug degradation during storage and compromise the performance of liposome-based drug carriers; (f) liposome can entrap up to tens of thousands of drug molecules but have trouble with larger therapeutic molecules such as protein, if small liposome diameters are desired for reasons of biodistribution; and (g) at physiological temperatures liposomes are fluid bilayer membranes which are susceptible to pH or lipase degradation destroying the encapsulated material. Currently, liposomes are one of the delivery systems applied to foods. Liposomeencapsulated enzyme concentrates have being used in the curd during cheese fermentation.191 Bromelain-loaded liposomes have being used as a meat tenderizer to improve enzyme stability.192 Free nisin adheres to fat, leading to lower accessibility to bacterial cells. Liposomes are reported to protect nisin.193 Incorporation of vitamin C in the inner core of liposomes194 has been shown to prevent degradation against copper, ascorbate oxidase and lysine. Milk fat globule membrane (MFGM) acts as a natural emulsifying agent preventing flocculation and coalescence of fat globules in milk. MFGM-derived phospholipids could be used to make liposomes using micro fluidization techniques. MFGM can form liposomes for delivering bioactive compounds.195

5.5.2

Nano-cochleates

Cochleates are stable, lipid-based vaccine carriers and delivering formulations. Cochleates are composed of phosphatidylserine (PS), cholesterol and calcium. Cochleates have different properties and are structurally distinct from liposomes and are structurally distinct from liposomes. Liposomes contain aqueous space within the compartments bounded by the lipid bilayers. Cochleates are maintained into rolled-up forms by calcium ions and are large, continuous, solid, lipid bilayer sheets with no internal

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aqueous space.196 The two positive charges on the calcium ion interacts with a negative charge on the phospholipids on the two opposing bilayers.197 Oral administration of cochleate vaccines has been shown to induce strong, longlasting circulating and mucosal antibody response and long-term immunological response. Protein, peptides and DNA can be formulated into cochleate-based vaccines.198 Cochleates can be formulated with viral surface glycoprotein useful as a vaccine delivery system. Cochleates have being shown to induce antigen-specific immune response in vivo. DNA cochleates are more potent than naked DNA. Bio Delivery Sciences International has developed nano cochleates made from soy and calcium that can carry and deliver nutrients such as vitamins, lycopenes, and omega fatty acids directly to cells.199–201

5.5.3

Hydrogels-based nanoparticles

Hydrogels are 3D hydrophilic polymeric networks. The network is formed by crosslinking polymer chains with covalent bonds, hydrogen bonding, Van der Waals interactions or physical entanglement.202 Hydrogels are composed of insoluble homopolymers or copolymers and are capable of swelling in water.203 Hydrogels have been extensively used in the development of smart drug delivery systems. This network of hydrophilic polymers can swell in water and hold a large amount of water whilst maintaining its structure.204 Hydrogels can protect drugs from hostile environments, e.g. the presence of enzymes and low pH in the stomach. They provide desirable protection for drugs, especially proteins, from the potentially harsh environment in the vicinity of the release site.205–209 Hydrogels containing such ‘sensor’ properties can undergo reversible volume phase transitions or gel–sol phase transitions upon only minute changes in the environmental condition and are called ‘intelligent’ or ‘smart’ hydrogels.202,210 The various physical (temperature, electric fields, solvent composition, light, pressure, sound and magnetic fields) and chemical (pH, ions and specific molecular recognition) stimuli can be utilized to induce various responses of the smart hydrogel systems. Hydrogels can be classified based on,206 nature of side groups as neutral or ionic; method of preparation as homopolymer or copolymer; physical structure as amorphous, semi crystalline and hydrogen bonded and based on environmental sensitivity towards temperature, pH, and ionic strength. In recent years, various hydrogels for drug delivery have been developed, such as Novels hydrogels, environmentally-responsive hydrogels connected to biosensors,204 bioadhesive,211 glucose sensitive,212 pH and thermosensitive hydrogels.149,213 Hydrogels contain components that are not GRAS and hence not suitable for food applications. Food proteins could be used to develop environmentally-sensitive hydrogels.214 Cold set β-lactoglobulin emulsion gels protect α-tocopherol under GIT conditions and that release is related to matrix degradation.215

5.5.4

Micellar systems

Polymeric micelles (<100nm size) are potential carriers for poorly soluble drugs in their inner core.216 Polymeric micelles are copolymers having diblock, multiblock structures with a hydrophilic shell and hydrophobic polymer core. Micelles are

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formed in solution as aggregates in which the component molecules (e.g. amphiphilic AB type or ABA type block copolymers, where A is hydrophobic, B is hydrophilic component) are arranged in a spheroidal structure with hydrophobic cores shielded from water. Micelles are dynamic aggregates of amphiphilic-like surfactant molecules that form spontaneously when the surfactant concentration exceeds the critical micelle concentration. However, formation of micelles is a reversible process and as the surfactant concentration falls below the critical micelle concentration by dilution or other effects, micelles spontaneously break up. The hydrophilic shell makes the micelle water-soluble while the hydrophobic core contributes to the microenvironment needed for stability. First, the hydrophobic core of micelles aid in the solubilization of hydrophobic molecules in aqueous solutions because hydrophobic molecules associate with the core microenvironment preferentially; secondly, the incorporated compounds are prevented from diffusing out due to steric effects.217 There are three classes of block copolymers depending on type of functionalization:218 (i) The first class contains one or more functional groups on the hydrophobic block, which allows cross linking of the core. (ii) In the second class, substituents are located on the hydrophilic part of the molecule and enable cross-linking of the micellar corona. (iii) The third class is surface functionalized with ligands that allow drug delivery devices to be directed to the specific site. Core cross-linked polymeric micelles are synthesized majorly by attaching a polymerizable group at the hydrophobic chain end. Surface functionalization of the micelles has been achieved by attaching aldehydes, glucose and galactose to PLA-PEG diblock copolymers through ring opening polymerization, using protected gars as the initiator.219 Drugs can be trapped physically within the hydrophobic core or can be linked covalently to component molecules of micelles. Polymeric micelles, as drug delivery devices, offer potential due to biocompatibility, low cytotoxicity, biodegradability and ability to encapsulate drugs. Membrane proteins play a crucial role in disease mechanisms and, hence, are biomarkers in disease monitoring prevention and treatment. Polymeric micelles have being shown to stabilize membrane proteins incorporated in the inner core.220 Polymeric micelles could be used as delivery systems for anti-cancer drugs, to transport plasmid DNA, antisense oligonucleotides and for delivery of diagnostic agents to specific organs in the body.221–226 Polymeric micelles can access more regions in body due to their smaller size as compared to liposomes. There are several advantages of polymeric micelles: (a) small and uniform particle size distribution; (b) the hydrophilic corona prevents interaction with blood components and the micelles’ small size prevents immune system recognition, thus there is a long circulation time in the blood stream; (c) active targeting is possible by modifying peripheral chain ends of polymers with targeting specific ligands; and (d) for the release of drugs once micelles have reached their target, degradation or stimuli-responsive micelles offer great promise. One of the major drawbacks of polymeric micelles includes thermodynamic instability.124 Physical stability is also an issue with polymeric micelles as a rapid release of incorporated drug may occur in vivo.227

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Dendrimers

Dendrimers were discovered in the early 1980s. Dendrimers are highly branched macromolecules which are organized in layers called generations that represent repeating monomer units of these macromolecules.228 The branched structures consist of three structural components: multifunctional center core; branched unit; and surface groups.229 Cavities in the core structure and folding of the branches create cages and channels, surface groups are amenable to modification and the presence of many chain ends is responsible for their high solubility, miscibility and reactivity. Dendrimer solubility is strongly influenced by the nature of surface groups. Dendrimers are highly soluble in a large number of organic solvents.230 A number of poly (D, L, lactide) -b-polyethylene glycol (PLA-PEG) have been synthesized in this way by the attachment of a methacryloyl group to the PLA chain end.231 Dendrimers offer a great potential in controlled delivery of bioactive compounds. They have been shown to have potential as delivery systems, protecting drugs within the macromolecular interior from their environment and offering increased stability. Dendrimers can achieve tissue targeting as nanoscopic container molecules due to prolonged residence time in blood circulation.229 Drug molecules can be loaded by two methods, either in the interior of the dendrimers or attached to surface groups. Dendrimers are stable structures for encapsulation and, due to modifiable end groups, allow improved water solubility and easy attachment of drug molecules. The presence of reactive functionality on the peripheral shell of dendrimers has also led to synthesis of dendrimer-conjugate with high control over loading of the active compounds and with reduced alteration of the physical and chemical properties of the dendrimers. In the case of drugs embedded in the polymer matrix, release can be obtained by physical or chemical modifications such as swelling of the polymer, diffusion or chemical erosion. When the drug is covalently bound to the polymer, the drug diffuses more slowly than the free drug and can be absorbed in specific interfaces.232,233 Dendrimers could be used to increase entrapment of drugs in liposomes, Purohit et al.234 recently studied the interaction of ampipathic cationic dendrimers with charged and neutral liposomes. Amine terminated PAMAM dendrimers showed increased encapsulation of a model acidic drug in liposomes; this was because dendrimers were entrapped by charge interaction that created pH and solubility gradients across the bilayer and led to an influx of acidic molecules into the liposome.235 Dendrosomes, the phospholipids and dendrimer composites also offer good entrapment of bioactive ingredients.234,236 Current gene therapy (by viruses) has many risks associated with it and thus have triggered the search for new synthetic DNA-based delivery systems. The high cytotoxicity and low delivery efficiency associated with linear cationic lipids and cationic oligopeptides have limited their application.237 Dendrimers have been tested for gene delivery; they were shown to form stable complexes with plasmid DNA and reported to be effective in gene delivery.236 Dendrimers were used as carriers for the antitumor drug 5-flurouracil.238 They faced issues of stability and leakage of the encapsulated ingredient due to the relatively open network and hemolytic toxicity due to –NH2 groups on the periphery. Coating dendrimers with polymers is one of the best options to increase their stability.

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A widely-used polymer for these purposes is poly (ethylene glycol) (PEG). PEGylation improves the stability and efficacy and also reduces hemolytic toxicity issues.239,240 End functional groups of PEG are functionalized by converting them to carboxylic acid derivatives and then to NHS ester.241 PEGylation of dendrimers was found suitable for delivery of the anticancer drug 5-FU. It helps increase drug loading and circulation and reduces drug release. PEGylated dendrimers showed increased drug release in vivo compared to non-PEGylated complexes.240 Encapsulation of guest molecules (GM) into a dendritic box was reported by Jansen and co-workers242 and Klajnert.243 The box could be opened by hydrolysis or photochemically. Dendritic unimolecular micelles were shown to entrap the model drug indomethacin.244 Dendrimers have several advantages: (a) ease of preparation and functionalization; (b) ability to display multiple copies of surface groups for biological recognition; and (c) drug could be loaded in their interior or attached to the surface. Dendrimers, however, have poor capacities to incorporate active compounds due to the smaller size 5–10nm.

5.5.6

Polymeric nanoparticles

Polymeric nanoparticles may be synthesized either from a monomer or from a preformed polymer.245 Depending on the method of preparation, nanoparticles, nanospheres or nanocapsules can be obtained with different properties and release characteristics for the encapsulated agent.150 Nanoparticle is a collective name for nanosphere and nanocapsule.123 Nanocapsules are vesicular systems in which the drug is confined to a cavity surrounded by a unique polymer membrane. Nanospheres have a matrixtype structure where active compounds can be adsorbed at their surface, entrapped or dissolved in matrix.92 Nanocapsules have a reservoir system in which an oily core is surrounded by a thin polymer wall, thus having a polymeric shell and an inner core where active compounds are dissolved in the core or adsorbed at the surface. Polymeric nanoparticles consist of biodegradable polymers. Most biodegradable polymers consist of synthetic polyesters like polycyanoacrylate, poly (lactic acid) (PLA), polyglycolic acid (PGA), poly (-caprolactone) (PCL), poly (alkylcyanoacrylate) (PACA).246 Apart from these, there are several natural polymers that are used for the synthesis of nanoparticles. Chitosan, gelatin, sodium alginate and other hydrophilic/biodegradable polymers come into this category.118 Polymeric particles used as delivery systems have provided peptides with protection from extensive degradation in the gastro intestinal tract.150 Lipophilic drugs can be easily incorporated in the oily core of nanocapsules or solubilized in nanospheres whereas hydrophilic compounds can be adsorbed on the surface.247 Polymeric nanoparticles are taken up by the intestinal tract.77 Encapsulation of oligonucleotides (ODN) using nanocapsules help avoid the use of positively-charged compounds to ensure ODN associations, thus avoiding the protein interaction and toxicity associated with this type of molecule.248 Antisense oligonucleotides can be protected from cellular uptake by conjugating them with polymeric nanoparticles.249–251 In order to completely shield oligonucleotides from nuclease attack, a new system based on polyisohexyl cyanoacrylate (PIHCA) nanocapsules with aqueous core was prepared by interfacial polymerization

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of isobutylcyanoacrylate in a w/o emulsion.248 For lymphatic targeting, nanocapsules coated with hydrophobic polymers tend to be easily captured by the lymphatic cells in the body.81 However, the usefulness of conventional nanoparticles is limited due to their hydrophobic nature, which leads to their uptake by the macrophages and thus not reaching the targeted site. A high curvature resulting in small size (<100nm) and/or a hydrophilic surface are required to reduce such opsonization reactions.252 Small sized nanospheres have been prepared from polyvinyl pyrrolidine (50–60nm) and chitosan (100nm).149,253 Surface modification with polymeric and biological materials have offered great solutions for specific applications or targeting desired locations in the body. Developments of so-called ‘stealth’ particles that are invisible to the macrophages have shown much promise. They use hydrophilic polymers that repel plasma proteins, polyethylene glycol (PEG), poloxamines, poloxamers and polysaccharides to coat conventional nanoparticle surfaces, which can be introduced at the surface either by adsorption of surfactants or by the use of block or branched copolymers.252,254 Use of surfactants like polysorbate 80 on PIBCA nanospheres, poloxamer 407 and poloxamine 908 on PMMA nanospheres has been the first strategy in directing the nanoparticle. However, the covalent linkage of copolymers is the preferred method as it reduces the chance of desorption upon dilution. This approach has been employed with poly (lactic acid) (PLA), polycaprolactone (PCL), and poly (cyanoacrylate) that are chemically coupled to PEG. All of these methods are reviewed by.69,109 Lectins are proteins able to provide specific binding to biological surfaces bearing sugar residues located at the surface of the epithelial cells. Thus polymers with attached lectin have a bioadhesive nature.145 An example for this is the binding of the lectins to the nanoparticle of poly(methyl vinyl ether-co-maleic anhydride) (PVM/ MA)111 and core(polyester)-shell(polysaccharides).255 Drug delivery for cancer therapy is one of the most important applications of nanoparticles. To achieve tumor-specific targeting, antibodies can be attached to the nanoparticle surface that can recognize the specific antigens on tumor cells.256 The attachment can be brought about by nonspecific adsorption, specific adsorption, or by covalent linkage.110 Non-specific adsorption is achieved by incubating the nanospheres with monoclonal antibodies. Specific adsorption is obtained by first coupling the nanoparticle to a ligand, which can specifically bind to the antibodies. The Mabs are incubated with the precoated nanoparticle and bind to the spacer molecule via noncovalent linkage. Covalently bound Mabs offer the advantage of maintaining their integrity, unlike other methods. The several advantages associated with polymeric nanoparticles include: (a) small size can penetrate through smaller capillaries and are taken up by cells; (b) use of biodegradable materials allows sustained drug release within the target site over a period of days or even weeks; (c) in comparison to solid lipid nanoparticles or nanosuspensions, polymeric nanoparticles consist of biodegradable polymers; (d) increased stability of any volatile pharmaceutical agents, and easily and cheaply fabricated in large quantities by a multitude of methods; (e) can be engineered specifically, allowing them to deliver a higher concentration of pharmaceutical agents to a desired location, thus the possible chemical modification of these polymers offers great advantages; and (f) polymeric nanoparticles have been found to be more

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stable in vivo and during storage compared to liposomes. Disadvantages include: (a) polymer-conjugated drugs carrying capacity and high drug loading influence water solubility of the product and biodistribution of the conjugate;257 and (b) could trigger polymer-related toxicity, organic solvent residue, scaling up of production process and polymer hydrolysis during storage. Nutrients or nutraceuticals can be added into the aqueous phase before the introduction of monomers of after its polymerization. They can be either incorporated into the nanospheres during the polymerization process or they can be adsorbed onto the surface of the matrix. Adsorption capacity is related to the hydrophobicity of the polymer and the surface area of the carrier. For highly nonpolar molecules, longer alkyl chains should be used to increase the affinity. Polymers could be degraded by surface erosion releasing nutrients or nutraceuticals.

5.5.7

Nanoemulsions

Nanoemulsions are true emulsions with a dispersed phase droplet 50–100nm in diameter and a continuous phase. The lipophilic cores are separated from the aqueous phase by a monomolecular layer of a surfactant material which makes them suitable for nanoencapsulation of oil-based bioactives. Nanoemulsions are submicron droplets with narrow size distribution. Emulsions have surfaces stabilized by adsorption of some surfactants or phospholipid. Generally, an emulsion consists of two immiscible liquids (usually oil and water), with one of the liquids dispersed as small spherical droplets in the other. Typically, the diameters of the droplets lie somewhere between 1 and 100μm. Emulsions can be conveniently classified according to the distribution of the oil and aqueous phases. A system that consists of oil droplets dispersed in an aqueous phase is called an oil-in-water (O/W) emulsion, whereas a system that consists of water droplets dispersed in an oil phase is called water-in-oil (W/O) emulsion. The substance that makes up the droplets in an emulsion is referred to as the dispersed phase, whereas the substance that makes up the surrounding liquid is called the continuous phase. In addition to the conventional O/W or W/O emulsions described above, it is also possible to prepare various types of multiple emulsions, e.g. oil-in-water-in-oil (O/W/O) or water-in-oil-in-water (W/O/W) emulsions.258 Emulsions are thermodynamically unfavorable systems that tend to break down over time due to a variety of physicochemical mechanisms, including gravitational separation, flocculation, coalescence and Ostwald ripening.259–261 Stabilization of nanoemulsions can be achieved through electrostatic stabilization, steric stabilization or static by solid particle at interface or by increasing viscosity of emulsion. It is possible to form emulsions that are kinetically stable for a reasonable period of time by including substances known as stabilizers, e.g. emulsifiers or texture modifiers.35 Emulsifiers are surface-active molecules that absorb to the surface and prevent the droplets from aggregating whereas texture modifiers thicken or gel the continuous phase, improving stability by retarding or preventing droplet movement. The shelf life of emulsion-based products is greatly governed by the selection of appropriate stabilizers. Emulsions offer a viable option for drug delivery.262,263 by selective uptake into lymphatic regions in the order oil-water>water-oil>aqueous solution.81 Different

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emulsions263 are listed below that could be used as delivery systems. The various emulsion systems are: oil-in-water emulsion carrier for oil soluble drugs; lipid emulsion; water-in-oil emulsion for water soluble drugs; self emulsifying drug delivery systems; lipid nanoemulsions; solid emulsions; multiple emulsions; water-in-oilin-water emulsions; and modified emulsions. Emulsions (o/w, w/o, o/w/o or w/o/w) are frequently found in the food industry and this could be a promising delivery system for nutraceuticals. The several advantages of nanoemulsions include: (a) their small size reduces gravity force and Brownian motion, which may prevent creaming or sedimentation; (b) steric stabilization prevents any flocculation or coalescence of droplets; and (c) unlike micro emulsions which require high concentration of surfactant, nanoemulsions can be prepared with less surfactant concentration. The small size and high kinetic stability make nanoemulsions suitable for delivery of active compounds in beverages. 5.5.7.1 Double emulsions Double emulsions are complex liquid dispersion systems known as emulsions of emulsions, in which droplets of one liquid are further dispersed in another liquid. The internal droplet can serve as an entrapping reservoir for active ingredients that can be released by a controlled transport mechanism. However, sizes of droplets and thermodynamic instability are major drawbacks.264,265 The compartmentalized structure of a double emulsion globule is suitable for drug delivery. Using double emulsion encapsulation helps increase water solubility. The structure of a double-emulsion globule makes it a possibility for applications in drug delivery, especially where the drug is soluble in the emulsion’s oil. Drugs with poor solubility in aqueous media can be solubilized in the interfacial layer of emulsions. Use of double emulsions for this purpose was suggested as early as the 1960s. A double emulsion can be of two general types: water-in-oil-in-water (W/O/W) or oil-in-water-in-oil (O/W/O). Specifically, W1/O/W2 emulsion consists of a continuous aqueous phase (W2) in which oil globules (O) are dispersed. For medical applications, a water-soluble therapeutic component can be solubilized within the inner W1 phase of the emulsion globule. There are several advantages to this type of delivery. For instance, a therapeutic substance solubilized within the internal phase shows extended release, which can lessen toxic effects. Variations in the type and concentrations of surfactants can allow control of the stability of and release from a double-emulsion globule, making these molecules a key component in designing a double-emulsion system for any practical application.264,266

5.5.8

Lipid nanoparticles

5.5.8.1 Solid lipid nanoparticles (SLN) Solid lipid nanoparticles comprise a high melting point triglycerides core with a phospholipid coating and can incorporate both hydrophilic and lipophilic drugs.267–271 Solid lipid nanoparticles were developed as an alternative carrier system to emulsions, liposomes, and polymeric nanoparticles as a colloidal carrier system for

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controlled drug delivery. These are nanoparticles with a matrix being composed of a solid lipid, i.e. the lipid is solid at room temperature and also at body temperature.91,272 With solid lipids, drug mobility is considerably reduced compared to liquid oil offering controlled drug release. The process used is melt emulsification: the lipid is melted and the drug dissolved in it and surfactants with GRAS status are used to avoid aggregation and to stabilize the dispersions. Solid lipid nanoparticles are known to adhere to the gut wall and release the drug where it should be absorbed. Moreover, they have absorption-promoting properties not only for lipophilic drugs but all drugs in general. Stable drug-loaded SLN with sufficient loading capacity could be formulated for controlled delivery.273 The in vivo fate of solid lipid nanoparticles depends on the administration route and interaction with biological surroundings. Cationic solid lipid nanoparticles are being studied for gene transfer.274,275 Apart from advantages such as the possibility of controlled release and drug targeting, increased drug stability, high drug payload and incorporation of lipophilic and hydrophilic drugs feasibility, SLNPs have no toxicity of the carrier and they also avoid organic solvents and have no problem with respect to large-scale production and sterilization. Some of the disadvantages include: (a) high pressure induced drug degradation; (b) lipid crystallization’ (c) drug incorporation implied localization of the drug in the solid lipid matrix; (d) coexistence of different lipid modifications and different colloidal species; (e) low drug loading capacity; (f) storage stability issues; and (g) possibility of increase in particle size and drug explosion. 5.5.8.2

Nanostructure lipid carriers (NLCs)

NLCs were introduced to overcome the potential difficulties with SLNs.276–278 These are characterized such that a certain nanostructure is given to the particle matrix by a process similar as for SLN, but in this case, the lipid matrix is prepared from a solid lipid and liquid oil.279,280 The compound to be encapsulated needs to be sufficiently lipophilic to be used in SLN or NLC applications, and where they are hydrophilic they need to be made lipophilic by conjugating them with a lipid moiety.262 5.5.8.3

Lipid drug conjugate (LDC)

The limitations of SLN can be overcome by LDC. The approach for the synthesis of LDC is the delay of dissolution and increase in stability when soluble molecules are incorporated into insoluble matrices. This also leads to increased permeation of lipophilic drug molecules through the gut wall. These conjugates are prepared by salt formation (e.g. an amino group containing molecules with fatty acid) or, alternatively, by covalent linkage (e.g. ether, ester). The conjugates are melted, dispersed in a hot surfactant solution, and homogenized at high pressure.277 The LDC has been shown to have potential applications in brain targeting of hydrophilic drugs.281 5.5.8.4

Coacervate nanoparticles

Coacervation offers unique controlled release possibilities based on mechanical stress, temperature or sustained release. They are synthesized by a phase separation process when interaction between the two biopolymers are favored in a rich solvent phase with a very small amount of biopolymers and a rich biopolymers phase forming the so-called

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coacervates.282–285 The nature of interactions is electrostatic. The charge is the most significant factor affecting coacervation, and the maximum coacervation is formed at a pH value where they carry equal and opposite charges. A very large number of hydrocolloid systems have been evaluated for coacervation.286 Coacervation is typically used to encapsulate flavor oils287 but can also be used to encapsulate fish oils132 nutrients, vitamins, preservatives and enzymes.288 Coacervates can be used to encapsulate nutraceuticals for controlled release.289 Encapsulation of nutraceuticals using coacervates enhances their chemical and thermal stability. Coacervates from gelatin-pectin were shown to have fat mimetic and flavor encapsulation ability.290 The appeal of frozen baked foods has being shown to improve upon heating by utilizing encapsulated flavor oils in complex coacervate microcapsules using gelatin and gum Arabic.291 Microcapsules of vitamin A palmitate prepared by gelatin-acacia complex coacervates convert an oily vitamin to solid powders, prevent degradation from the environment, and enhance stability.289 However, coacervation has some disadvantages: it is292 expensive, complex, and cross-linking shell material involves glutaraldehye/enzymatic cross-linkers.

5.5.9

Nanocrystalline particles

The production of nanocrystals and nanosuspensions is called nanoisation.293 Nanosuspensions solve several solubility-related problems of poorly soluble drugs.294 It consists purely of poor water soluble drugs suspended in an appropriate dispersion media. The approach towards the synthesis of nanocrystals is based on the traditional knowledge that the dissolution of any substance is positively related to its surface area. Thus the concept of nanoionization with resultant nanocrystals of 200–400nm diameter has been thought to give increased saturation solubility. Milling the substance and then stabilizing smaller particles with coating, forms nanocrystals in a size range to suit oral delivery and intravenous injection.293,295 The drug is homogenized by high pressure homogenization,294 wet milling,78 or alternative techniques such as nanocrystallization from saturated solution states or spray drying.298 The development of nanocrystals in non-aqueous media or the media with reduced water content has also been shown.280,297 The advantage of the process is that any drug or compound can be diminuted to the nanoscale and temperature-sensitive drugs can be processed by applying temperature control jackets or homogenizing at 0°C or below. Nanocrystals can also be injected intravenously as an aqueous suspension, nanosuspension. Stabilization of the nanosuspension is achieved by electrostatic stabilization (charged surfactants), or by steric stabilization (nonionic surfactants or polymers). Poorly soluble compounds show increased dissolution rate and absorption in GIT when formulated as nanosuspensions.298 Oral administration of drugs in the form of nanocrystals helps improve bioavailability, dose proportionality, reduce fed/ fasted and inter-subject variability, and enhance absorption rate.295 5.5.9.1 Colloidosomes Colloidosomes are hollow, elastic shells whose size, permeability, elasticity, strength and compatibility can be precisely controlled. They are fabricated by self-assembly of colloidal particles onto the interface of emulsion droplets.299–302

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5.5.9.2

Aquasomes (carbohydrate ceramic nanoparticles)

Aquasomes are novel delivery vehicles stabilized by carbohydrates that prevent the destructive drug carrier interaction.303,304 The nanoparticles are spherical 60–200nm particles, used for drug and antigen delivery, which are adsorbed on to the surface of these particles.306 Particle core is composed of nanocrystalline calcium phosphate or ceramic diamond and is covered by a polyhydroxyl oligomeric film.

5.5.9.3

Cubosome

Bicontinuous cubic phases consist of two separate continuous but nonintersecting hydrophilic regions divided by a lipid layer that is contorted into a periodic minimal surface with zero average curvature.306 Continuous and periodic structures result in a very high viscosity of bulk cubic phases. Compared to liposomes, they have a much higher bilayer area to particle volume ratios. Cubosome structure can be changed by modifying the environmental conditions such as pH, ionic strength, or temperature, thus achieving controlled release of carried compounds. Cubosomes may be used in the controlled release of solubilized bioactives in the food matrix as a result of their nanoporous structure (50–10nm),214 and their ability to solubilize hydrophobic, hydrophilic and amphiphilic molecules and their biodegradability and digestibility by simple enzyme action.307 The cubic phase is strongly bioadhesive, so may find application in flavor release via its mucosal deposition and delivery of effective compounds.

5.5.9.4

Hollow capsules (Nan containers)

Mechanical properties of the capsule can be manipulated by material selection from thin polymeric, oligomeric or lipid shells to organic, inorganic composites and to totally organic capsules.308 These capsules are stable against high salt concentrations. Dimensions of 100nm to 10μm, thin walls and high modularity lead to designed properties. Permeability is pH switchable by varying intermolecular interactions, solvents, changing salt concentration, and incorporating thermosensitive co-polymers.

5.5.9.5

Ceramic nanoparticles

Ceramic particles with entrapped biomolecules136,309–310 have great potential applications in drug delivery. Particles such as silica, alumina, and titanium are known for their compatibility with biological systems and can effectively protect doped molecules (enzymes) against denaturation induced by external pH and temperature.310,311 Surfaces can be modified with different functional groups.312,313 They offer many advantages, such as easy preparation to the desired size, shape and porosity. Their ultra-low size help evades RES, also these particles do not show any swelling or porosity change with change in pH.

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5.5.9.6 Polyelectrolyte Surface coating colloid particles with consecutive adsorption of oppositely charged polyelectrolyte forms films in the nm range. Polyelectrolyte multilayers contain many charged groups available for binding metal ions. Multifunctional organic and composite films are made by adsorption. Particles are coated layer-by-layer by the adsorption technique. Polyelectrolyte capsules are produced using layer-by-layer absorption of polyelectrolytes onto oppositely charged particles or layer of polyelectrolyte. By this method, microparticles with novel biofunctionality can be produced.313 5.5.9.7 Nanotubes and nanowires Nanotubes (with a diameter of 100nm and height of 100 micron) are spherical selfassembling lipid nanotubes with increased internal volume and option to functionalize the inner or external surface.314–316 Cylindrical geometry allows encapsulation (of drugs which can be covalently bound to surface or internally) followed by fabrication and modification of vehicles.

5.6

Conclusions

Nanoparticle delivery systems may have a wide range of applications in the food industry. The direct application of nanotechnology in the food industry is at its primary level and, hence, this area holds a lot of potential for future applications of sophisticated techniques. The benefits of foods as drugs or nutrition through controlled release technology are enormous but the industry is still in its infancy. In the next decade we will see remarkable progress. With increasing knowledge about controlled drug release we will be able to provide more therapeutic agents, to deliver the complex drugs that are becoming available through genetic engineering techniques, be more able to target drugs to specific cells, and we will learn to make drug release contingent on physiological stimuli. This would contribute to great savings and better efficiency.

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228. Tomalia, D.A., Naylor, A.M. and Goddard III, W.A. (1990) Starburst dendrimers: Molecular-level control of size, shape, surface chemistry, topology, and flexibility from atoms to macroscopic matter. Angewandte Chemie International Edition in English 102, 138–175. 229. Aulenta, F., Hayes, W. and Rannard, S. (2003) Dendrimers: A new class of nanoscopic containers and delivery devices. European Polymer Journal 39, 1741–1771. 230. Hawker, C.J. and Frechet, J.M.J. (1996) Comparison of linear, hyperbranched, and dendritic macromolecules. In Step-Growth Polymers for High-Performance Materials (Hedrick, J. and Labadie, J., Eds), pp 132–144, American Chemical Society, Washington D.C. 231. Liu, S. and Armes, S.P. (2001) Recent advances in the synthesis of polymeric surfactants. Current Opinion in Colloid & Interface Science 6, 249–256. 232. Kohn, J. (1993) Design, synthesis, and possible applications of pseudo-poly(amino acids). Trends in Polymer Science 17, 206–212. 233. Nathan, N., Zalipsky, S. and Kohn, J. (1994) Strategies for covalent attachment of doxorubicin to poly(PEG-Lys), a new water-soluble poly(ether urethane). Journal of Bioactive and Compatible Polymers 9, 239–251. 234. Purohit, G., Sakthivel, T. and Florence, A.T. (2001) Interaction of cationic partial dendrimers with charged and neutral liposomes. International Journal of Pharmaceutics 214, 71–76. 235. Khopade, A.J., Caruso, F., Tripathi, P., Nagaich, S., Jain, N.K. (2002) Effect of dendrimer on entrapment and release of bioactive from liposomes. International Journal of Pharmaceutics 232, 157–162. 236. Sarbolouki, M.N., Sadeghizadeh, M., Yaghoobi, M.M., Karami, A. and Lohrasbi, T. (2000) Dendrosomes: A novel family of vehicles for transfection and therapy. Journal of Chemical Technology and Biotechnology 75, 919–922. 237. Haensler, J. and Szoka, F.C. (1993) Polyamidoamine cascade polymers mediate efficient transfection of cells in culture. Bioconjugate Chemistry 4, 372–379. 238. Zhuo, R.X., Du, B. and Lu, Z.R. (1999) In vitro release of 5-fluorouracil with cyclic core dendritic polymer. Journal of Controlled Release 57, 249–257. 239. Bhadra, D., Bhadra, S.,Jain, P. and Jain, N.K. (2002) Pegnology: A review of PEGylated systems. Pharmazie 57, 5–29. 240. Bhadra, D., Bhadra, S., Jain, S. and Jain, N.K. (2003) A PEGylated dendritic nanoparticulate carrier of fluorouracil. International Journal of Pharmaceutics 257, 111–124. 241. Veronese, F.M., Caliceti, P., Pastorino, A., Schiavon, O., Sartore, L., Banci, L. and Scolaro, L.M. (1989) Preparation, physico-chemical and pharmacokinetic characterization of monomethoxypoly(ethylene glycol)-derivatized superoxide dismutase. Journal of Controlled Release 10, 145154. 242. Jansen, J., Debrabandervandenberg, E. M.M. and Meijer, E.W. (1994) Encapsulation of guest molecules into a dendritic box. Science 266, 1226–1229. 243. Klajnert, B. and Bryszewska, M. (2001) Dendrimers: Properties and applications. Acta Biochimica Polonica 48, 199–208. 244. Liu, M.J., Kono, K. and Frechet, J.M.J. (2000) Water-soluble dendritic unimolecular micelles: Their potential as drug delivery agents. Journal of Controlled Release 65, 121–131. 245. Couvreur, P., Couarraze, G., Devissaguet, J.P. and Puisieux, F. (1996) Nanoparticles: Preparation and Characterization. Marcel Dekker, New York. 246. Fattal, E., Vauthier, C., Aynie, I., Nakada, Y., Lambert, G., Malvy, C. and Couvreur, P. (1998) Biodegradable polyalkylcyanoacrylate nanoparticles for the delivery of oligonucleotides. Journal of Controlled Release 53, 137–143. 247. Barratt, B. (2000) Therapeutic applications of colloidal drug carriers. Pharmaceutical Science & Technology Today 3, 163–171.

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248. Lambert, G., Fattal, E., Pinto-Alphandary, H., Gulik, A. and Couvreur, P. (2000) Polyisobutylcyanoacrylate nanocapsules containing an aqueous core as a novel colloidal carrier for the delivery of oligonucleotides. Pharmaceutical Research 17, 707–714. 249. Godard, G., Boutorine, A.S., Saison-Behmoaras, E. and Helene, C. (1995) Antisense effects of cholesterol-oligodeoxynucleotide conjugates associated with poly (alkylcyanoacrylate) nanoparticles. European Journal of Biochemistry 232, 404–410. 250. Schwab, G., Chavany, C., Duroux, I., Goubin, G., Lebeau, J., Helene, C. and SaisonBehmoaras, T. (1994) Antisense oligonucleotides adsorbed to polyalkylcyanoacrylate nanoparticles specifically inhibit mutated Ha-ras-mediated cell proliferation and tumorigenicity in nude mice. Proceedings of the National Academy of Sciences of the United States of America 91, 10460–10464. 251. Zimmer, A. (1999) Antisense oligonucleotide delivery with polyhexylcyanoacrylate nanoparticles as carriers. Methods 18, 286–295. 252. Storm, G., Belliot, S.O., Daemen, T. and Lasic, D.D. (1995) Surface modification of nanoparticles to oppose uptake by the mononuclear phagocyte system. Advanced Drug Delivery Reviews 17, 31–48. 253. Sharma, D., Chelvi, T.P., Kaur, J., Chakravorty, K., De, T.K., Maitra, A.N. and Ralhan, R. (1996) Novel Taxol formulation: Polyvinylpyrrolidone nanoparticle-encapsulated Taxol for drug delivery in cancer therapy. Oncology Research 7–8, 281–286. 254. Stolnik, S., Illum, L. and Davis, S.S. (1995) Long circulating microparticulate drug carriers. Advanced Drug Delivery Review 16, 195–214. 255. Rodrigues, J.S., Santos-Magalhaes, N.S., Coelho, L.C.B.B., Couvreur, P., Ponchel, G. and Gref, R. (2003) Novel core(polyester)-shell(polysaccharide) nanoparticles: Protein loading and surface modification with lectins. Journal of Controlled Release 92, 103–112. 256. Leroux, J.C., Allemann, E., Jaeghere, F.D., Doelker, E. and Gurny, R. (1996) Biodegradable nanoparticles: From sustained release formulations to improved site specific drug delivery. Journal of Controlled Release 39, 339–350. 257. Duncan, R. (1992) Drug-polymer conjugates: Potential for improved chemotherapy. Anti-Cancer Drugs 3, 175–210. 258. Dickinson, E. and McClements, D.J. (1995) Advances in Food Colloids, 1st ed. Blackie Academic and Professional, Glasgow. 259. Coupland, J.N. and McClements, D.J. (2001) Droplet size determination in food emulsions: Comparison of ultrasonic and light scattering methods. Journal of Food Engineering 50, 117–120. 260. Dickinson, E. (1992) Introduction to Food Colloids. Oxford University Press, Oxford. 261. McClements, D.J. (2005) Food emulsions: Principles, Practice, and Techniques. CRC Press, Boca Raton. 262. Muller, R.H. and Keck, C.M. (2004) Challenges and solutions for the delivery of biotech drugs: A review of drug nanocrystal technology and lipid nanoparticles. Journal of Biotechnology 113, 151–170. 263. Nakano, M. (2000) Places of emulsions in drug delivery. Advance Drug Delivery Review 45, 1–4. 264. Benichou, A., Aserin, A. and Garti, N. (2004) Double emulsions stabilized with hybrids of natural polymers for entrapment and slow release of active matters. Advances in Colloid and Interface Science 108-109, 29–41. 265. Garti, N. and Benichou, A. (2001) Double Emulsions for Controlled-release Applications: Progress and Trends. Marcel Dekker, Inc. 266. Lawson, L.B. and Papadopoulos, K.D. (2004) Effects of a phospholipid cosurfactant on external coalescence in water-in-oil-in-water double-emulsion globules. Colloids and Surfaces A: Physicochem. Eng. Aspects 250, 337–342.

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267. Müller, R.H., Rühl, D., Runge, S.A., Schulze-Forster, K. and Mehnert, W. (1997a) Cytotoxicity of solid lipid nanoparticles as a function of the lipid matrix and the surfactant. Pharmaceutical Research 14, 458–462. 268. Müller, R.H., Maaßen, S., Schwarz, C. and Mehnert, W. (1997b) Solid lipid nanoparticles (SLN) as potential carriers for human use: Interaction with human granulocytes. Journal of Controlled Release 47, 261–269. 269. Gasco, M.R. (1997) Solid lipid nanospheres from warm micro-emulsions. Pharmaceutical Technology Europre 9, 56–58. 270. Heiati, H., Phillips, N.C. and Tawashi, R. (1996) Evidence for phospholipid bilayer formation in solid lipid nanoparticles formulated with phospholipid and triglyceride. Pharmaceutical Research 13, 1406–1410. 271. Mukherjee, S., Ray, S. and Thakur, R.S. (2009) Solid lipid nanoparticles: A modern formulation approach in drug delivery systems. Indian Journal of Pharmaceutical Science 71, 349–358. 272. Muller, R.H., Mehnert, W., Lucks, J.S., Schwarz, C., Zur Muhlena, A., Meyhers, H., Freitas, C. and Ruhl, D. (1995) Solid lipid nanoparticles (SLN): An alternative colloidal carrier system for controlled drug delivery. European Journal of Pharmaceutics and Biopharmaceutics 41, 62–69. 273. Schwarz, C. and Mehnert, W. (1999) Solid lipis nanoparticles for controlled drug delivery II. Drug incorporation and physicochemical characterization. Journal of Microencapsulation 16, 205–213. 274. Tabatta, K., Kneuerb, C., Sametic, M., Olbricha, C., Müllera, R.H., Lehrc, C. and Bakowsky, U. (2004) Transfection with different colloidal systems: Comparison of solid lipid nanoparticles and liposomes. Journal of Controlled Release 97, 321–332. 275. Tabatta, K., Sameti, M., Olbrich, C., Müller, R.H. and Lehr, C.M. (2004) Effect of cationic lipid and matrix lipid composition on solid lipid nanoparticle-mediated gene transfer. European Journal of Pharmaceutics and Biopharmaceutics 57, 155–162. 276. Uner, M. (2006) Preparation, characterization and physico-chemical properties of solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC): Their benefits as colloidal drug carrier systems. Pharmazie 61, 375–386. 277. Muller, R. H., Radtke, M. and Wissing, S.A. (2002) Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) in cosmetic and dermatological preparations. Advanced Drug Delivery Review 54, S131–155. 278. Radtke, M. and Muller, R.H. (2000) Comparison of structural properties of solid lipid nanoparticles (SLN) versus other lipid particles. Proceedings of International Symposium Control Release Bioactive Materials 27, 309–210. 279. Müller, R.H., Mäder, K. and Gohla, S. (2000) Solid lipid nanoparticles (SLN) for controlled drug delivery: A review of the state of the art. European Journal of Pharmaceuticas and Biopharmaceutics 50, 161–177. 280. Müller, R.H., Mäder, K., Lippacher, A. and Jenning, V. (2000) Solid-lipid (semi-solid) liquid particles and method of producing highly concentrated particles dispersions. German Patent Application, 199 45 203.2 281. Olbrich, C., Gessner, A., Kayser, O. and Müller, R.H. (2002) Lipid-drug-conjugate (LDC) nanoparticles as a novel carrier system for the hydrophilic antitrypanosomal drug diminazenediaceturate. Journal of Drug Targeting 10, 387–396. 282. Bakan, J.A. (1980) Microencapsulation using coacervation/phase separation technique. In Controlled Release Technology: Methods, Theory and Applications (Kydonieux, A.F., Ed.), pp 84–104, CRC Press, Boca Raton. 283. Burgess, J.E. and Carless, D.J. (1984) Microelectrophoretic studies of gelatin and acacia for the prediction of complex coacervation. Journal of Colloid and Interfacial Science 98, 1–8.

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284. Dervichian, D.G. (1954) A comparative study of the flocculation and coacervation of different systems. Discussion of the Faraday Society 18, 231–239. 285. Dobetti, L. and Pantaleo, V. (2002) Application of a hydrodynamic model to microencapsulation by coacervation. Journal of Microencapsulation 19, 139–151. 286. Prokop, A., Hunkeler, D., DiMaris, S., Haralsom, M.A. and Wang, T.G. (1998) Water soluble polymers for immunolisation I: Complex coacervation and cytotoxicity. Advanced Polymer Science 136, 1–51. 287. Korus, J., Tomasik, P. and Lii, C.Y. (2003) Microcapsules from starch granules. Journal of Microencapsulation 20, 47–56. 288. Dubin, P.L., Muhoberac, B.B. and Xia, J. (1998) Preparation of Enzyme-Polyelectrolyte Coacervate Complexes and their Properties. Patent number: US 5834271A. 289. Junyaprasret, M., Mitrevej, A., Sinchaipand, N., Boonme, P. and Wurster, D.E. (2001) Effect of process variables on the microencapsulation of vitamin and palmitate by gelatin acacia coacervation. Drug Delivery and Industrial Pharmacy 26, 561–566. 290. Strauss, G. and Gibson, S.M. (2004) Plant phenolics as cross-linkers of gelatin gels and gelatin-based coacervates for use as food ingredients. Food Hydrocolloids 18, 81–89. 291. Yeo, Y., Bellas, E., Firestone, W., Langer, R. and Kohane, D.S. (2005) Complex coacervates for thermally sensitive controlled release of flavor compounds. Journal of Agriculture and Food Chemistry 53, 7518–7525. 292. Gouin, S. (2004) Micro encapsulation industrial appraisal of existing technologies and trends. Trends in Food Science and Technology 15, 330–347. 293. Rabinow, B.E. (2004) Nanosuspensions in drug delivery. Nature Reviews Drug Discovery 3, 785–796. 294. Muller, R.H., Jacobs, C. and Kayser, O. (2001) Nanosuspensions as particulate drug formulations in therapy: Rationale for development and what we can expect for the future. Advanced Drug Delivery Reviews 47, 3–19. 295. Liversidge, G.G. and Cundy K.C. (1995) Particle size reduction for improvement of oral bioavailability of hydrophobic drugs. Absolute oral bioavailability of nanocrystalline danazol in beagle dogs. International Journal of Pharmaceuticals 125, 91–97. 296. Sarkari, M., Brown, J., Chen, X.X., Swinnea, S., Williams, R.O. and Johnston, K.P. (2002) Enhanced drug dissolution using evaporative precipitation into aqueous solution. International Journal of Pharmaceuticals 243, 1731. 297. Grau, M. J., Kayser, O. and Muller, R.H. (2000) Nanosuspensions of poorly soluble drugs – reproducibility of small scale production. International Journal of Pharmaceutics 196, 155–159. 298. Jia, L., Wong, H., Cerna, C. and Weitman, S.D. (2002) Effect of nanonization on absorption of 301029: Ex vivo and in vivo pharmacokinetic correlations determined by liquid chromatography/mass spectrometry. Pharmaceutical Research 19, 1091–1096. 299. Dinsmore, A.D., Hsu, M.F., Nikolaides, M.G., Marquez, M., Bausch, A.R. and Weitz, D.A (2002) Colloidosomes: Selectively permeable capsules composed of colloidal particles. Science 298, 1006–1009. 3001. Saraf, S., Rathi, R., Kaur, D. and Saraf, S. (2011) Colloidosomes: An advanced vesicular system in drug delivery. Asian Journal of Science Alert 4, 1–15. 301. Noble, P.F., Cayre, O.J., Alargova, R.G., Velev, O.D. and Paunov, V.N. (2004) Fabrication of hairy colloidosomes with shells of polymeric microrods. Journal of American Chemical Society 126, 8092–8093. 302. Shilpi, S., Jain, A., Gupta, Y. and Jain, S.K. (2007) Colloidosomes: An emerging vesicular system in drug delivery. Critical Reviews in Therapeutic Drug Carrier Systems 24, 361–391. 303. Saraf, S. (2008) Aquasomes: An overview. Pharmaceutical Information 6. See http:// www.pharmainfo.net/reviews/aquasomes-overview

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304. Shahabade, S.G., Bhosale A.V., Mutha S.S., Bhosale N.R., Khade P.H., Bhadane N.P. and Shinde S.T. (2009) An overview on nanocarrier technology: Aquasomes. Journal of Pharmacy Research 2, 1174–1177. 305. Kossovsky, N., Gelman, A., Rajguru, S., Nguyen, R., Sponsler, E., Hnatyszyn, H.J., Chow, K., Chung, A., Torres, M. and Zemanovich, J. (1996) Control of molecular polymorphisms by a structured carbohydrate/ceramic delivery vehicle – aquasomes. Journal of Control Release 39, 383–388. 3067. Spicer, P. T ( 2009) Cubosomes: Bicontinuous liquid crystalline nanoparticles. In Dekker Encyclopedia of Nanoscience and Nanotechnology (Contescu, C.I. and Putyera, K., Eds), pp 1018–1028, CRC Press, Boca Raton. 307. Borne, J., Naylander, T. and Khan, A. (2002) Effect of lipase on onoolein-based cubic phase dispersion (cubosomes) and vesicles. Journal of Physical Chemistry B 106, 10492–10500. 308. Caruso, F., Caruso, R. and Möhwald, H. (1998) Nanoengineering of inorganic and hybrid hollow spheres by colloidal templating. Science 282, 1111–1114. 309. Cherian, A.K., Rana, A.C. and Jain, S.K (2000) Self-assembled carbohydrate-stabilized ceramic nanoparticles for the parenteral delivery of insulin. Drug Development and Industrial Pharmacy 26, 459–463. 310. Jain, T.K., Roy, I., De, T.K. and Maitra, A. (1998) Nanometer silica particles encapsulating active compounds: A novel ceramic drug carrier. Journal of American Chemical Society 120, 11092–11095. 311. Lal, M., Levy, L., Kim, K.S., He, G.S., Wang, X., Min, Y.H., Pakatchi, S. and Prasad, P.N. (2000) Silica nanobubbles containing an organic dye in a multilayered organic/inorganic heterostructure with enhanced luminescence. Chemical Matters 12, 2632–2639. 312. Badley, R.D., Ford, W.T., McEnroe, F.J. and Assink, R.A. (1990) Surface modification of colloidal silica. Langmuir 6, 792–801. 312. Sukhorukov, G.B., Rogach, A.L., Zebli, B., Liedl, T., Skirtach, A.G., Köhler, K., Antipov, A.A., Gaponik, N., Susha, A.S., Winterhalter, M. and Parak, W.J. (2005) Nanoengineered polymer capsules: Tools for detection, controlled delivery and sitespecific manipulation. Small 1, 194–200. 313. Martin, C.R. and Kohli, P. (2004) The emerging field of nanotube biotechnology. Nature Review of Drug Discovery 2, 29–37. 315. Goldstein, A.S., Amory, J.K., Martin, S.M., Vernon, C., Matsumoto, A.M. and Yager, P. (2001) Testosterone delivery using glutamide based complex high axial ratio microstructures. Bioorganic and Medicinal Chemistry 9, 2819–2825. 316. Ram Rao, C.N. and Govindaraj, A (2005) Nanotubes and Nanowires. RSC Publishing.

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6

Scanning electron microscopy

Yi Wang and Vania Petrova

Abstract: Scanning electron microscopy (SEM) is a traditional electron microscopy technique widely used for structure characterization in materials science, chemistry, biology and polymer science. In this chapter, we introduce the principles of SEM operation and the set up of a typical SEM, including the electron sources, electron beam, lenses and apertures. In SEM, secondary electrons, backscattering electrons and characteristic X-rays can be collected to give structural or chemical composition information. Environmental scanning electron microscopy (ESEM), which operates without exposing volatile samples to high vacuum, is introduced for biological applications. SEM is one of the most powerful and maneuverable tools in nanotechnology. We present, as an example, the use of SEM for the characterization of protein (zein) micro- and nanostructures formed by evaporation-induced self-assembly. Finally, we introduce three limitations encountered in SEM experimentation that can impact the quality of the SEM images. These are radiation damage, contamination and charging effect. Keywords: scanning electron microscopy; SEM; structure characterization; environmental scanning electron microscopy; ESEM; nanotechnology; zein; charging; contamination

6.1 6.1.1

Background Introduction to the scanning electron microscope

The scanning electron microscope (SEM) is one of the most versatile instruments available for the examination and analysis of microstructure morphology and chemical composition. The SEM has over two orders of magnitude improvement in resolution over the light microscope, which has been of great importance to scientific research. The SEM was developed by replacing the light source with a high-energy electron beam. By taking advantage of the short wavelengths of electrons, focused by electromagnetic lenses, the SEM can achieve high-resolution imaging with a Nanotechnology Research Methods for Foods and Bioproducts, First Edition. Edited by Graciela W. Padua and Qin Wang. © 2012 John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc.

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large depth of field. The interaction between the electron beam and the specimen under study produces a variety of signals, including backscattered electrons (BSEs), secondary electrons (SEs) and characteristic X-rays, used to gain insight into the morphology, phase, chemical composition and crystallography of materials. The principles of SEM operation, including the formation of an electron beam, the position of the detector and the amplification of the signal current, were established in the 1930s. A system with 50 nm resolution was built in 1948.1 Improved performance came later, including imaging of the three-dimensional nature of surfaces and increasing resolution down to 1 nm. Although the transmission electron microscope (TEM) has an order of magnitude resolution advantage, the SEM avoids the difficulties of TEM sample preparation and offers the ability to examine the surfaces of bulk specimens to visualize their topography over a wide range of magnifications.

6.1.2

Why electrons?

The resolution of the images taken by a system is primarily determined by the wavelength of the probing radiation. The shorter the wavelength, the smaller the feature that can be observed. Thus, the shorter wavelength of electrons compared to visible light gives them an advantage in achievable resolution. Electrons can be extracted from various sources. After acceleration by an electrical potential along the evacuated column, they can form into a finely focused beam and scan over a surface of interest. When the primary electrons strike a specimen surface, a wide range of useful interactions occur, generating various charged particles and photons, which can be collected and used to form images, a diffraction pattern or a chemical spectrum. In modern SEM systems, the imaging resolution is better than 1 nm. It is mainly limited by the physics of the interactions between the electrons and the sample.

6.1.3

Electron–target interaction

SEM images depend on the acquisition of signals produced by the interaction between the electron beam and the specimen. There are generally two kinds of process involved, elastic scattering and inelastic scattering. Elastic scattering comes from the deflection of the incident electron upon hitting the specimen’s atomic nucleus or outer-shell electrons. Elastic scattering has negligible energy loss during the collision and the scattered electron has a wide-angle directional change. When the directional change is more than 90°, the electrons become BSEs, which produce a useful imaging signal. In inelastic scattering, a significant energy is transferred to a sample electron, which is set in motion as an SE and in turn can undergo scattering or leave the sample. If the SE is ejected from an inner orbital, the atom is ionized; the resulting electron hole will be filled from the outer-shell electrons. The characteristic deexcitation energy can be emitted either by an X-ray photon or by another electron, which is called an Auger electron. Spectroscopic analysis of the characteristic X-ray and the Auger electrons provides chemical information about the sample. When the finely focused electron beam strikes the specimen surface, the energetic electrons will penetrate into the sample to encounter and collide with a sample atom at some depth. The region of interaction, from which a variety of signals are produced,

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High Z Low E

High Z Low Z

High E

Low E

Fig. 6.1

Excitation volumes at different atomic numbers (Z) and beam energies (E).

is in the range of micrometers. The shape of the interaction region is a teardrop for low-atomic-number specimens and a hemisphere for specimens of high atomic number (see Fig. 6.1). The depth of the interaction volume depends on the energy of the incident beam, the incidence angle and the specimen composition. The volume and depth of the interaction region increase with an increase of beam energy or a decreasing atomic number of the specimen, because the higher the atomic number, the higher number of particles on the electron path. The various elastic and inelastic particles have different mean-free paths in the sample and thus have different zones of signal emission, as presented schematically in Fig. 6.2. For example, although SEs and Auger electrons are produced throughout the interaction volume, they have very low energies and can only escape from a thin layer near the sample surface. To analyze the sample visually, SEs or BSEs can be collected to form an image. To determine information about the sample composition, X-ray or Auger electron spectra may be recorded.

6.1.4

Secondary electrons (SEs)

SEs are the group of low-energy electrons, typically below 50 eV, which leave the sample as a result of the collision sequence generated by the primary beam. The SE emission coefficient (SEEC), the ratio of the number of SEs leaving the surface to the number of incident primary electrons, generally rises as the beam energy is lowered or at a higher angle of incidence beam. The SE yield, the number of SEs emitted per incident electron, is relatively independent of the atomic number (unlike BSEs), and

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SE, thickness = 50–500Å

Auger electron, thickness = 10Å BSE

Characteristic X-ray

Fig. 6.2

Continuum X-ray

Illustration of interaction volumes for various electron–sample interactions.

for a beam energy of 20 kV has a value of 0.1 for most elements. The exceptions are carbon, for which the SEEC is anomalously low at 0.05, and gold, which has its SEEC anomalously high at 0.2. Gold is the preferred coating material for nonconductive samples. The mean free path of SEs in many materials is approximately 1 nm. Thus, although electrons are generated in the whole excitation volume, only those originating from the near-surface region of the sample can be expected to be detected. The volume of SE production is very small compared with that of BSEs and X-rays, as shown in Fig. 6.2. Therefore, the resolution using SEs is better and is determined by the electron beam size. The small depth production of detected SEs makes them very sensitive to topography and they are the most commonly used signal for SEMs.

6.1.5

Backscattered electrons (BSEs)

In addition to SEs, BSEs can also be detected to form an image. BSEs are electrons that have gone through single or multiple scatterings and escaped from the surface with an energy greater than 50 eV. Generally, 10–50% of the beam electrons are backscattered toward their source, which represents 60–80% of the energy of the primary beam. The elastic collision between an electron and the specimen nucleus causes the electron to bounce back with wide-angle directional change. The backscattered signal is high when the electron beam strikes a high-atomic-number specimen; because of high positive charges on the nucleus of the specimen, more electrons are backscattered. The zone of production of BSEs is larger than that of SEs, because BSEs have high energy, which prevents them from being absorbed by the sample. Thus the resolution of a BSE image is lower than that of an SE image (1 nm). BSEs can produce images in the SEM with both compositional and topographical information. Relatively smooth surfaces are suitable for this imaging mode, because slight topographical irregularities cause artifacts due to shadowing.

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Electron gun

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Beam column

Anode Electron beam

Spray aperture

Computer system for signal processing

Scan coils

First condenser lens

Magnification control

Second condenser lens Condenser aperture Objective lens

Stigmator Scan generator SE detector Amplifier

Final aperture BSE detector

X-ray detector

Sample stage High vacuum pump

Multichannel analyser Fig. 6.3 A schematic diagram of an SEM system. A thermionic electron gun is shown as an example. Details of electron guns are given in Fig. 6.4. To see a color version of this figure, see Plate 6.1.

6.1.6

Characteristic X-rays

When an inner-shell electron is displaced by collision with a primary electron, an outer-shell electron will fall into the inner shell to rebalance the orbital charge and the atom will become ionized. An X-ray photon is emitted to bring the atom back to the ground state. The various shells of an atom have discrete amounts of energy, and X-ray radiation is characteristic of the atom from which the photon was released. X-ray analysis in an SEM involves the identification of radiation of a specific energy or wavelength for elemental analysis of the sample. Both qualitative and quantitative information can be obtained, with lateral resolution in the range of micrometers. The microscope can produce images in X-ray, where characteristic X-rays from a given element are detected and their position recorded or “mapped”. This allows the production of maps showing the distribution of specific elements. These maps can be recorded alongside corresponding SE or BSE images.

6.1.7

Overview of the SEM

The SEM system includes the SEM beam column, sample stage, signal detectors and signal processing system for real-time observation and image recording (see Fig. 6.3). The beam column, sample stage and detectors are placed in a vacuum chamber with the control electronics and high-voltage supplies connected. The column consists of an electron gun, lenses and apertures, coils for beam scanning, and a deflector/ acquisition system for collecting and processing of the signal information, which will be displayed as images or graphs on a monitor.

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(a)

Filament heating supply

High voltage supply Bias 0.1–30kV resistor − V +

Wehnelt cylinder Electron beam crossover Anode

(b)

Extracting voltage V0

Accelerating voltage V1

Field emission tip 1st Anode 2nd Anode Crossover

Fig. 6.4 Schematic diagrams of (a) a thermionic electron gun and (b) a field-emission electron gun (FEG).

6.1.8

Electron sources

The SEM system requires that the electron gun produces a stable electron beam with high current, small spot size, adjustable energy and small energy dispersion. There are two main types of electron source used in SEM systems: thermionic and fieldemission electron guns. The properties of the beams they produce vary considerably. In the thermionic electron gun (Fig. 6.4a), electrons are emitted from a heated filament, focused by negative electrical potential (∼500 V) applied to the Wehnelt cylinder and then accelerated by a voltage of 0.1–30 keV toward the anode. The thermionic electron gun provides a high (1 pA to 1000 nA) and stable beam current, which make it suitable for X-ray analysis. Ultra-high vacuum is not required for this type of electron source. In a field-emission electron gun (FEG) (Fig. 6.4b), a strong electric field (109 V/m) is used to extract electrons from a very sharp single-crystal tungsten tip. An FEG requires two anodes: the first regulates the field strength and emission current at the tip, and the second accelerates the electrons to the final operating voltage. The cold FEG operates at room temperature. An FEG has a much higher source brightness (current per solid angle) and lower energy dispersion than a thermionic gun, but it requires a high vacuum – as much as ∼10−8 Pa – in the chamber. The small crossover of the beam produced by an FEG allows highresolution SEM imaging.

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Scanning electron microscopy

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Lenses and apertures

The diameter of electron beam crossover produced by the electron gun is too large to form a high-resolution image. Electromagnetic lenses below the electron gun are used to demagnify the image of the crossover (10–20 μm for a thermionic gun) to the final spot size on the sample (1 nm–1 μm). For a field-emission source, the final spot can be reduced to 1 nm. A series of electromagnetic lenses is used to demagnify and focus the electron beam. Electromagnetic lenses consist of a copper wire coil inside a cylindrical soft-iron shell. The coil current generates a magnetic field with axial symmetry. The electrons experience both radial and circumferential forces and spiral towards the center of the lens, thus being focused. Increasing the field strength by increasing the current causes the focal point to move upwards, reducing the beam spot size. There are two kinds of lens in an SEM column, condenser and objective. The electrons first pass through the condenser lens, which demagnifies the crossover diameter of the electron beam produced by the electron gun to a smaller size. The condenser lens plays an important role in determining the final beam size and beam current. Usually two condenser lenses are used, paired together and controlled by a single knob. The final lens in the column is the objective lens, which focuses the electron beam on the sample surface by moving the crossover of the beam along the optical axis. The objective lens gap houses scanning coils, which cause the beam to raster the sample surface; stigmator coils, which correct the aberration of the lenses, called astigmatism; and an objective aperture, which controls the beam convergence angle. For high image resolution, the SEM sample should be close to the gap, because lens aberration, which enlarges the final probe size, increases with focal length. Spot-size reduction for optimum resolution is shared equally by lenses in the column. Reduction is achieved by the appropriate combination of lens demagnification and aperture size. Several apertures, depending on the system design, are used in the column. Apertures are used to exclude scattered electrons and to control spherical aberrations in the final lens. An aperture after the condenser lens limits the angular range of electrons that are allowed to travel through the objective lens, while a final aperture in the objective lens determines the final convergence semiangle of the electron beam.

6.1.10

Electron beam scanning

The scanning electron beam is made possible by the scan coils that are placed in the objective lens. Controlled by the scan coils, the electron beam moves along the x- or y-axis over the specimen surface and generates a point-to-point image of the scanning area with help from detectors. The intensity of each screen pixel is determined by the signal level arriving at the detector at each point. The ratio of the area rastered on the sample to that of the recording media gives the magnification. A change in the magnification is a change in the size of the area scanned.

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Fig. 6.5

6.1.11

SEM images showing astigmatism.

Lens aberrations

The electromagnetic lenses bring in aberrations that affect the focusing of the electron beam. The aberrations cause the electrons to be focused in slightly different planes and result in a beam with a finite minimum diameter, which is called the disc of least confusion, rather than an infinitely sharp point. These aberrations are the limiting factor in determining the ultimate system resolution. For spherical aberrations, electrons passing through the edge of the lens are focused more strongly than those passing through the center. The spherical aberration can be reduced by using a lens with shorter foci or by using an aperture in the condenser to stop electrons travelling to the edges of the objective lens. For chromatic aberrations, electrons, particularly with low beam energies, are focused in different planes due to the energy spread among those arriving at the lens. The smaller the energy spread of the electrons in the primary beam, the smaller the effect of the chromatic aberration. The field-emission sources, with a smaller energy spread than the thermionic sources, have the advantage of smaller chromatic aberration. The chromatic aberration can be reduced by increasing the primary beam energy or by using a smaller aperture. Smaller apertures, however, reduce the beam current. Thus there is an optimum aperture size to achieve good signal-tonoise ratios and small aberrations. Astigmatism is caused by asymmetries in the magnetic field of any contamination on the lenses or apertures. With astigmatism, there are two different focal lengths in the perpendicular direction and the overall beam diameter is larger than it should be. Astigmatism can be corrected by using stigmators, which apply a counter field in the x or y direction to reshape the beam to a circular cross-section. When working at high magnifications, it is essential to regularly check and correct for astigmatism in a routine focus–stigmate–focus to achieve the best results. Figure 6.5 provides SEM images showing astigmatism. The particles are not perfectly imaged because the figure is focused in one direction, shown as sharp edges, and not focused in the perpendicular direction, shown as blurred edges. The astigmatism can be fixed by using the stigmator to modify the foci of the electron beam on the x- or y-axis separately in order to reshape the beam to a circular cross-section.

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Scanning electron microscopy

6.1.12

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Vacuum

A high-vacuum environment is needed to avoid contamination of the electron source and scattering of gas molecules during electron travel. The high vacuum throughout the column depends on the electron source, and is in the range of 10−3 to 10−5 Pa for a thermionic electron source and 10−6 to 10−8 Pa for FEG. The specimens placed in the vacuum chamber should be vacuum-friendly, which means no volatile components are allowed. So, many biological specimens, foams, emulsions and food systems which contain water or oils cannot be placed directly in the vacuum chamber to be imaged. Preparations, including chemical fixing, dehydration in a graded alcohol series, freeze drying and critical point drying, which can remove potentially volatile substances, are required beforehand. A proper preparation procedure should not change the structural or chemical nature of the specimen to be examined.

6.1.13

Conductive coatings

The bombardment of samples by relatively high-energy electrons quickly results in a build-up of negative charge unless the sample is electrically conductive. Commonly, insulating samples are sputter-coated with a conductive material such as gold, platinum or palladium. The electrically conductive coating does not make an insulating specimen conductive, it just provides a ground plane for the electrical field. If applied too thickly, conductive coatings can obscure small features.

6.1.14

Environmental SEMs (ESEMs)

Charging and volatile samples can be examined without special preparation with an SEM that operates without exposing the sample to high vacuum. In a variablepressure or environmental SEM (ESEM), the electron gun is maintained at high vacuum, while a control amount of gas, up to 20 torr, is allowed into the sample chamber. A fine aperture separates the gun and sample chamber to prevent excessive gas from getting into the gun chamber. The interaction between the electron beam and the air molecules creates a cloud of positive ions around the electron beam. These ions will neutralize the negative charge – from electrons – collecting on the surface of a nonconductive sample. By controlling the pressure in the sample chamber, the number of gas molecules interacting with the electron beam is maintained at a level that is sufficient to prevent charging but does not deflect the beam to prevent imaging.

6.2

Applications

As one of the most powerful and maneuverable tools in nanotechnology, the SEM has played an important role in the research of nanomaterials, including nanoparticles, nanoporous structures, nanocrystalline oxides, nanostructured semiconductors and thermoelectric materials, observing their morphologies in low and high magnifications and confirming their orientations. The SEM also has its applications in biomaterials,

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Fig. 6.6

SEM image of free zein microspheres.

such as three-dimensional nanofibrous scaffolds and polymer-based nanocomposite scaffolds for bone and tissue engineering, and nanospheres for controlled delivery of bioactive factors. In addition to its main ability to offer a 2D visualization of large areas of the sample, the SEM can provide diversified qualitative and quantitative information on many physical properties, including size, morphology, surface texture and roughness. Moreover, advanced manipulation of samples during SEM experiments, coupled with high-resolution microscopy techniques like TEM, can provide key information about the morphology of the nanomaterials at the nanometer scale, as well as insights into the different reaction pathways. This section provides three selected examples to show the advantages of SEM in nanotechnology applications.

6.2.1

Zein microstructures

An SEM was used to characterize the morphology of zein self-assembled microstructures. Zein is able to self-assemble into microspheres, films, rods and fibers through evaporation-induced self-assembly (EISA) processes.2,3 SEM images of EISA samples are given in Figs 6.6, 6.7 and 6.8. Fig. 6.6 shows free microspheres formed from dilute zein solutions (3 mg/ml). The average of the microsphere diameter was 1 μm. Figure 6.7 shows closely packed spheres obtained at higher zein concentrations (10 mg/ml). Microsphere diameter reached about 3 μm. As shown in Fig. 6.8, when zein concentration in solution reached 100 mg/ml, microspheres were fused into a film. In Fig. 6.9, the zein structure shown is a composite of microspheres and microfilms. Clusters of spheres were placed on the film surface. The film had pores, through which spheres below the film layer could be seen. The formation of the spheres and films structure, which can help us understand the mechanism of the

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Scanning electron microscopy

Fig. 6.7

SEM image of closely packed zein microspheres.

Fig. 6.8

SEM image of a zein lamellae film.

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zein self-assembly, was highly interesting to researchers. Figure 6.10 shows SEM images of 100 mg/ml zein in 30% ethanol solution after EISA, and the structure shows zein microspheres containing smaller spheres. Zein was not dispersed in 30% ethanol solution. Phase separation occurred when zein was dissolved in a solution with an ethanol concentration lower than 40% or higher than 90%.4

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Fig. 6.9

Fig. 6.10

SEM image of a zein sphere–thin film combined structure.

SEM images of zein microspheres containing smaller spheres.

100 mg/ml zein, compared to 0.2–5 mg/ml zein, was more difficult to dissolve.3 The single-phase solution was made with the help of sonication to avoid phase separation. The uncommon microstructure – microspheres containing smaller spheres – is shown in Fig. 6.10, when the zein and ethanol mixtures were close to

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Scanning electron microscopy

Fig. 6.11

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SEM images of zein microfibers.

phase separation, was unlike the typical sphere, bicontinuous and lamellar phases shown in the phase diagram made by Wang and Padua.3 Figure 6.11 gives SEM images of 5 mg/ml zein in 30% ethanol solution after EISA, and shows that zein spheres connected into short fibers, which connected each other to make a network. Like Fig. 6.10, the microstructure after EISA was atypical when the zein and ethanol mixture was close to phase separation.

6.2.2

Controlled magnifications

The SEM has a resolution as low as 1 nm. Thus, it is capable of revealing microstructural details which cannot be observed by an optical microscope. The magnification in an SEM can be controlled over a broad range from 10 to 500 000×. Figure 6.12 shows images of the cross-section of a sample, made of zein and nanoclay, at two different magnifications. The nanoclay was believed to be nanoflakes with a large area-to-volume ratio. In Fig. 6.12a, with magnification 650×, the film is not smooth but shows layer separation and structural gaps. Figure 6.12b is taken at the same point as Fig. 6.12a, but at higher magnification, 11 000×. The high-magnification image shows details of the film structure. The film is made of layers of nanoclay held together by zein spheres, and zein spheres are visible on the surface of the flakes. Zein microspheres are believed to stay between every two nanoclay flakes and to be attached to both, increasing the tensile strength of the nanocomposite structure. Figure 6.13a shows an SEM image of zein microspheres with a magnification of 600×. The sample is not evenly distributed. The areas in white or grey are covered

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Fig. 6.12

SEM images of zein layers at different magnifications.

Fig. 6.13

SEM images of zein microspheres at different magnifications.

Fig. 6.14

SEM images of zein microstructures at different magnifications.

by zein, while the areas in black are not covered at all. Although the sample is not evenly distributed, the pattern of covered surface is regular, since the boundary lines of the sample pattern are always straight in the same direction. However, the microstructure is not clearly seen in Figure 6.13a. Figure 6.13b is taken at the same point, but with a magnification of 14 000×. It shows zein microspheres closely packed layer by layer. Because the sample was prepared by EISA, capillary forces made the

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Scanning electron microscopy

Fig. 6.15

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SEM images of zein microstructures at different magnifications.

spheres packed and the boundary lines straight and parallel at the end of the evaporation. Figure 6.14 shows another two SEM images of a zein microstructure, with magnifications of 50 and 2200×. In Fig. 6.14a, the zein film is in grey and the white circles are openings within it. With a much higher magnification (Fig. 6.14b), it can be seen that connected zein microspheres make clusters inside the circles. Figure 6.15 shows a sequence of SEM images of a zein microstructure. In Fig. 6.15a, with magnification 190×, it can be seen that several zein spheres are on an unsmooth zein film. There are some irregular-shaped zein particles and some small openings on the zein film. In Fig. 6.15b, when the magnification increases to 950×, it is observed that there are several layers inside the small opening on the zein film. At a higher magnification, 2300× (Fig. 6.15c), it is seen that there are microspheres between the first and second layers. Openings are visible, which may lead to further observation of a combined structure of spheres, openings and layers on the third layer.

6.2.3

Nanoparticles

SEM was applied to observe zein microspheres which were hundreds of nanometers in size. Figure 6.16 shows an SEM image of two zein nanospheres prepared by EISA. The two spheres were about 500 nm in size and are connected. The surfaces of the spheres were not smooth but had small zein particles laid on top.

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Fig. 6.16

SEM image of zein nanoparticles.

Fig. 6.17

SEM images of zein microspheres prepared by freeze drying.

Figure 6.17 shows SEM images of zein microspheres prepared by freeze drying. As a comparison, the zein spheres in Figs 6.6 and 6.17 were prepared by EISA only and by EISA with the additional step of freeze drying, respectively. The morphologies of the two samples are different. In Fig. 6.6, the spheres are dispersed into layers, while in Fig. 6.17a the spheres have kept their structure in space and been made into a cluster. The reason for this is that the freeze drying fixed the zein structure against

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Scanning electron microscopy

Fig. 6.18

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SEM images of zein microspheres obtained by centrifuge.

gravity, and the zein spheres did not go through arrangement by the capillary force, due to the convective solvent flowing right before EISA finished, so they kept their structure as in the solution. As shown in Fig. 6.17b, the zein spheres made by freeze drying were much smaller, about 800 nm in size, than those made by EISA, which were 1–2 μm. This could be explained by the self-assembly mechanism of zein during EISA: zein followed a layer-adsorption mechanism of sphere growing. The growing of the zein spheres was stopped by the application of freeze drying, so they were not as large as those that had finished the whole EISA process. Figure 6.18 shows SEM images of zein microspheres prepared by centrifuge. Figure 6.18b is a partial enlargement of Fig. 6.18a. Figure 6.18 shows that the centrifuge caused the zein spheres to be deformed and to stay closely attached to their neighbors. The size of the spheres was about 200–300 nm, smaller than those made by either freeze drying or EISA only. The reason for this is that the growth of the zein spheres prepared by centrifuge was stopped earlier than that of the spheres prepared by freeze drying, and much earlier than that of the spheres going through the whole EISA process.

6.3

Limitations

Bombarding a specimen with charged particles can result in some problems. First, the interaction between electrons and the specimen may cause damage to the specimen. Second, if the specimen is not a particularly good electrical conductor, negative charge will accumulate inside its bulk, which may affect the imaging process. Radiation damage can result from primary or secondary radiation. Primary radiation damage includes the excitation of an individual atom or group of atoms, ionization and atom displacement. Secondary radiation damage includes the emission of electrons, X-rays or light, temperature increase, electrostatic charging, bond scission, cross-linking, mass loss and the formation of a carbonaceous coating. Most radiation damage is caused by plasmon excitation and atomic ionizations, which give rise to specimen heating. In polymers and biopolymers, degradation occurs during electron beam bombardment. Scission takes place at random points along the chain and can

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Fig. 6.19

SEM images of a zein microstructure after radiation damage.

lead to the production of smaller molecules, which can diffuse to the surface and leave by sublimation. Scission is more likely to happen in molecules with large side groups, such as proteins. The ends of polymer chains are susceptible to depolymerization. Following primary ionization and the formation of side-chain free radicals and hydrogen free radicals, cross-linking may occur. Hydrogen radicals diffuse easily to abstract another hydrogen radical, forming a hydrogen molecule and leaving another polymer radical behind. Various lengths of polymer radical join up in pairs to form cross-links, leading to a more radiation-resistant specimen. This process is associated with small mass losses. Scission and cross-linking occur simultaneously, and scission may predominate.

6.3.1

Radiation damage

The interaction of the electron beam and the sample transfers energy from the beam to the sample, which might damage the sample surface if the heating is accumulated. Figure 6.19 shows the morphology of a zein sample surface with radiation damage. The zein self-assembled into small disk-like particles with diameters of 200 nm or even less. Due to high-magnification electron beam scanning, the surface of the sample was heated and broken open. It was a thin film, with the zein particles on top of or buried within it. The damage to the sample occurred when the electron beam scanned a 500 × 500 nm2 square, shown in dark at the center of Fig. 6.19. Figure 6.20 shows SEM images of a zein and acid mixture sample after EISA. The three images were taken in the same area with the same magnification, 2200×, in a time sequence 0 seconds (Fig. 6.20a), 10 seconds (Fig. 6.20b), and 20 seconds (Fig. 6.20c). The image sequence demonstrates the radiation damage on the sample surface made by the electron beam scanning. At time 0, there was a broken area at the

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Fig. 6.20

SEM images of a zein microstructure, showing radiation damage.

Fig. 6.21

SEM images of a zein microstructure after radiation damage.

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right side of the image. Cracks were formed around a zein sphere. At 10 seconds, there were more cracks all over the image area. The heat accumulation made the radiation damage worse. At 20 seconds, there were more cracks, and the cracks shown in Fig. 6.20b became wider and bigger. The radiation damage was caused by the energy transferred from the electron beam to the sample. The damage became worse as the electron beam continually scannned and focused the area.

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Fig. 6.22

SEM image of a zein microstructure after radiation damage.

The radiation damage occasionally helps in the investigation of the sample structure under the surface. Figure 6.21a shows an SEM image of a zein and acid mixture sample after EISA. The electron beam heated the surface layer of its scanning area, and the surface layer was removed by the accumulated heat. The structure under the surface layer was porous, with many small holes, while the surface layer was a solid film with small openings, much fewer in number than the pores under the surface layer, on top. In Fig. 6.21b, when the electron beam kept scanning on the sample surface, one part of the surface layer was removed by a weak explosion resulting from energy accumulation under the layer. Because the acid solution was a mixture of chemical compounds that had different interactions with zein, there were a variety of structures under the surface layer. There were small spheres on top, rods and big spheres buried inside, and films at the bottom, as shown in Fig. 6.21b. Low-concentration zein with surfactant makes very soft film. For very soft materials, the electron beam will burn the sample if it keeps scanning and focusing on it. In Fig. 6.22, the electron beam burned a sample in an area of 1.5 × 1 μm2. When investigating very soft materials with an SEM, the electron beam should be controlled at a very low strength to avoid burning.

6.3.2

Contamination

Contamination is a serious problem in electron microscopy, especially at low accelerating voltages and high magnifications, which have become necessary for biological samples under an SEM. Contamination is caused by a cross-linking of adsorbed organic molecules under electron irradiation. The main source of adsorbed organic molecules is the organic molecules in the atmosphere and comparable

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Scanning electron microscopy

Fig. 6.23

SEM images of a zein microstructure, showing contamination.

Fig. 6.24

SEM image of a zein microstructure, showing contamination.

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quantities from the vacuum – by diffusion and rotating pump oils – as well as from vacuum grease, rubber gaskets and the finger marks of the user. When the concentration of adsorbed molecules is decreased in the irradiated area by irradiation damage and cross-linking, further molecules can diffuse from unirradiated areas, which results in a continuous growth of contamination films or needles in the case of a stationary electron probe. Figure 6.23 shows SEM images with contamination. In Fig. 6.23a, a square-shaped zein particle was focused and scanned by an electron beam at a scale of 200 nm. Due to high magnification, a frame-like mark was created, and at the corner of the mark there was a particle deposited by contamination. Figure 6.23b shows a similar phenomenon, with a line at the left side and a particle deposited at the upper corner of the scanning window. Figure 6.24 shows another kind of contamination. Because of

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Fig. 6.25

SEM image of a zein microstructure, showing charging effect.

a long scanning time under the electron beam, the zein particle at the right of the image was coated in a thin layer of adsorbed molecules deposited by the beam.

6.3.3

Charging

Charging artifacts can look quite different, from a fast scan for visual observation to a slow scan for image recording. Charging is one of the barriers to taking a good image by SEM. Figure 6.25 shows a zein particle on a zein film surface. The particle appears bright due to the increased emission of SEs at the rough surface. The size of the particle was such that more electrons were absorbed than emitted, which resulted in a negative charging. This negative charge caused a distortion of the electric field. Generally, the electric field directed the SEs emitted from the sample surface, which had low positive potential, toward the SE detector, which had high positive potential on its grid. Due to the high absorption of SEs by its rough surface, the particle had negative potential. The negative-charged particle caused an electric field, which connected induced positive charges on the zein film to the negative charges on the particle. The result was a dark voltage contrast beside the particle. The darkening of the zein film started when the electron probe, which scanned the area point-by-point from top to bottom and from the left-hand to the right-hand side, first struck the particle. The dark area was extended more to the right-hand side than to the left-hand side, more obviously shown in Fig. 6.26, because the charging of the particle was decreased by conduction as the electron probe moved along each line and always approached from the left-hand side. This decrease of negative charging can also be seen as a dark area below the particle when the electron probe moved to the bottom and could not restore

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Scanning electron microscopy

Fig. 6.26

SEM image of a zein microstructure, showing charging effect.

Fig. 6.27

SEM image of a zein microstructure, showing charging effect.

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the negative charge of the particle. Figs 6.25 and 6.26 are typical examples of the charging effect. However, charging can result in quite different image artifacts depending on the specimen geometry, conductivity and electron emission. Additional SEM image showing charging effects are given in Figs 6.27 and 6.28.

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Fig. 6.28

SEM images of a zein microstructure, showing charging effects.

Figure 6.27 shows an irregularly shaped zein particle with an extremely rough surface on the zein film. In Fig. 6.27, both the radiation damage on the zein film and the charging due to the zein particle appear. Because of the high absorption of SEs by the particle, and since the thus-caused electric field connected the induced positive charges on the zein film to the negative charges on the particle, the zein film around the particle was damaged. The zein film was originally smooth, as seen in the lowerright corner of Fig. 6.27. After the radiation damage, the zein film surface was rough, with textures. Because of the charging effect, the region right below the particle on the left could not be observed. Figures 6.28a and b show the charging effects caused by large particles. Because of their large sizes, the SEM images were destroyed by the charging effects, which dominate the whole image. Because of the shape and rough surface of the particles and clusters, charging effects occurred across a larger area of the image. The most common procedure to prevent charging is sputter-coating of the specimen with a conductive layer. Sputter-coating provides a uniform coating film on rough surfaces. A disadvantage of this method is that the fine structure of the coating film caused by the grain size is visible at high resolution. In addition, specimen details can be buried by the coating film to a depth of the order of a few nanometers.

References 1. Zworykin, V.K., Hillier, J. and Snyder, R.L. (1942) A scanning electron microscope. American Society for Testing and Materials (Aug), 15–23. 2. Wang, Q., Yin, L.L. and Padua, G.W. (2008) Effect of hydrophilic and lipophilic compounds on zein microstructures. Food Biophysics 3, 174–181. 3. Wang, Y. and Padua, G.W. (2010) Formation of zein microphases in ethanol-water. Langmuir 26, 12897–12901. 4. Shukla, R. and Cheryan, M. (2001) Zein: the industrial protein from corn. Ind Crop Prod 13, 171–192.

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7

Transmission electron microscopy

Changhui Lei

Abstract: Electrons accelerated to a few hundred kilovolts will produce many signals when interacting with a thin specimen. A transmission electron microscope (TEM) can detect and analyze these signals to obtain images, diffractions and different spectra from an area of hundreds down to a few nanometers at atomic resolution. The images and diffractions can be obtained through a conventional TEM mode, where a parallel electron beam illuminates the interested area, or through a scanning TEM (STEM) mode, where a focused beam is scanned across the specimen. Two important signals used for chemical analysis are the characteristic X-ray and energy-loss electrons. STEM has good spatial resolution, which makes the microscope and its attached spectrometers a powerful instrument in investigating the structure and chemistry of nanomaterials. Keywords: transmission electron microscopy; TEM; scanning transmission electron microscopy; STEM; energy-dispersive spectrometer; EDS; electron energy-loss spectrometry; EELS; image; diffraction pattern; nanomaterial; composition analysis

7.1

Background

It is well known that accelerated electrons have wave–particle duality. Transmission electron microscopy (TEM) is a very good example of the application of this duality. The wave property enables an electromagnetic lens to focus the accelerated electrons; consequently, the accelerated electrons can act as a source to image materials. TEM applies an electron beam to interact with and transmit through an ultra-thin specimen, and forms an electronically magnified image of the specimen via electromagnetic lenses for detailed observation. There are a variety of types of imaging techniques, generally based on two main classes of microscopy technique. Conventional TEM (CTEM), the original form of TEM, illuminates a stationary beam on an electrontransparent specimen. The beam can be either parallel or convergent. When the electron beam emerges from the specimen, it carries information about the specimen Nanotechnology Research Methods for Foods and Bioproducts, First Edition. Edited by Graciela W. Padua and Qin Wang. © 2012 John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc.

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and forms an image via the objective lens of the microscope. CTEM is based on an image formation process that is conceptually analogue to the process in classical light microscopes. The resolution is determined by the wavelength of the accelerated electrons and the quality of the imaging optics behind the specimen. Another type of TEM is scanning TEM (STEM), where a focused electron probe is scanned across an electron-transparent specimen that allows the selection and detection of transmitted electrons. The signal is amplified and used to modulate the intensity of images, usually displayed on a cathode-ray tube (CRT) that is scanned synchronously with the electron probe. STEM is essentially a “mapping” technique whose resolution is mainly determined by the probe size – that is, by the formation optics of the electron probe before the specimen. The geometric magnification is determined by the ratio of the area displayed on the image unit to the area synchronously scanned by the electron probe on the specimen. The accelerated electrons, as particles, can interact with thin specimens and generate variable signals. Collecting and analyzing these signals will give the chemical information and a lot of other details about the sample, as most of the produced signals have characteristic energies that identify the elements present, which is in the scope of “analytical electron microscopy” (AEM). With the recent advance of certain novel technologies, in particular nanotechnology, the increasing importance of TEM has been obvious, as TEM techniques allow the study of the microstructure (morphology), the structure (symmetry of crystals) and the composition of a single specimen using the same instrument. The synergism of these techniques has the potential to provide detailed structural and chemical information from the same area, which is becoming important for the analysis of areas at the nanometer scale. This chapter shall present a brief introduction to a few common TEM methods and their applications.

7.2

Instrumentations and applications

A typical TEM instrument consists of the following parts: an electron gun, illumination lenses, display/recording units and vacuum systems. The electron gun is used to generate the accelerated electrons. The source used to emit electrons might be a tungsten hairpin, a Lab6 crystal or a field-emission gun (FEG), in the order of increasing brightness. Both tungsten and Lab6 are thermionic emission sources in which the electrons are obtained by heating the filament, made from a tungsten hairpin or a Lab6 crystal. The FEG source applies an electric field to a fine tip, normally a ZrO2-coated [310] W crystal, to produce “monochromatic” electrons that are very coherent or have a narrow energy spread. FEG is important for a STEM dark-field (DF) image and analytical functions. The accelerated voltages are usually at the range of 80–300 kV. Most commercial TEMs are operated at 200 kV for material investigations or 120 kV for beam-sensitive samples. The lenses used in TEMs are electromagnetic lenses; one TEM instrument may contain several sets of lenses in order to obtain the desired illuminations. Each lens might be a composite lens, which can either form images or adjust (deflect or shift) the beam path. The images can be displayed on a fluorescent viewing screen or a CRT, and are finally recorded on photographic plates or with a charge-coupled device (CCD). The electron gun, lenses and specimen have to be sealed in vacuum as the accelerated electrons

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Incident electrons ~ 200 kV Backscattered electrons (BSEs) Auger electrons

Sample

Secondary electrons (SEs) Characteristic X-rays

Transmitted beam

Elastically Inelastically scattered electrons scattered electrons Direct beam Fig. 7.1 A schematic illustration of the interaction between a beam of high-energy electrons and a thin sample.

will lose energy as a consequence of the collision with air molecules. This section presents a brief description of the setups and basic knowledge about a few common TEM techniques.

7.2.1

Interactions between incident beam and specimen

Figure 7.1 summarizes a few signals produced during the interaction between a beam of high-energy incident electrons and a thin specimen. Most electrons transmit through the specimen, neither changing direction nor losing energy, but a few may transfer a small portion of energy to the specimen. Some of the forward electrons are scattered in other directions; these scattered electrons are classified as elastically scattered and inelastically scattered electrons depending upon whether they lose energy or not. Transmitted electrons are also called coherent or incoherent when considering their wave nature. The elastically scattered electrons change directions without losing energy. The number of elastically scattered electrons strongly depends on the specimen. Most of elastically scattered electrons are coherent for a crystal sample; they are usually found at certain relatively low angels (<10°) by obeying Bragg’s equation, 2d Sinq = nl, where d is the interplanar distance of the specimen, l is the wavelength of the accelerated electrons and q is the scatter angle. These scattered electrons are called diffraction beams as well when considering their wave nature. The inelastically scattered electrons are almost incoherent; their numbers decrease rapidly with the increase of scatter angles. In addition, heavy elements cause inelastically scattered electrons at high angles more readily than light elements. The variety of TEM imaging techniques is based on the selection and application of different scattered transmitted electrons, for example the elastic scattered electrons for CTEM and the incoherent electrons for DF images of STEM. The incident electrons can transfer part of their energies to the electron clouds of atoms within the specimen by moving a few electrons to high energy levels or outer

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shells, or even knocking a few electrons out of the specimen. The minimum amounts of energy that must be transferred to eject electrons are unique to different elements. In addition, the energy loss causing the excitation of inner-shell electron clouds and possible interference between neighboring atoms reflects the bonding structure of elements in a specimen. The analysis of the energy losses of transmitted electrons gives us their chemical information as well as the possible number and distance of nearest neighbors; that is, electronic information on the atoms in the specimen. When the incident electrons create vacant inner energy levels by ejecting the inner electrons of atoms to higher energy levels, X-ray photons are emitted out of the specimen surface as the outer-shell electrons jump into the vacant inner energy levels (core levels). The energy of X-ray photons equals the energy difference between the two shells involved and is unique to each element. Hence, the X-ray photons have characteristic energies, and can act as the fingerprint to identify the elements present. The energy-loss electrons and the characteristic X-rays are two important signals used in AEM to obtain chemical information of the specimens.

7.2.2

Conventional TEM

Figure 7.2 is a schematic illustration of ray diagrams of a conventional electron microscope, according to the geometrical optics approximation. It presents two key operating modes: (a) image mode and (b) diffraction mode. Both modes are based on the same lens configuration. The condenser lens is employed to form an incident beam on the sample. Depending on the microscopy technique, the beam could be parallel or focused/convergent. The post-specimen lens is essentially a three-lens system: an objective lens, an intermediate lens and a projector lens. Each lens may be a composite lens. The objective lens first focuses the transmitted beams at its back focal plane, and then forms a magnified image at its image plane (first image). The first image is further magnified through the intermediate and projector lenses. The final image is projected on either a fluorescent viewing screen or a TV monitor, and is recorded either on negative plates or with a CCD. The objective lens is the most important, as it carries the original information about samples and determines to a large extent the resolution and the contrast of images. The resolution of the microscopes depends on the wavelength and the quality of the objective lens. The wavelength of electron waves at 200 kV is 0.00 271 nm, or 0.00 251 nm after the relativistic correction. The imperfection of an objective lens can be corrected with the stigmatism correctors in a modern microscope. Hence, the short wavelength gives much space to improve the resolution, which is around 0.2 nm for most modern commercial TEM instruments. The intermediate and projector lenses mainly provide the desired magnification. By tuning the currents of lenses or changing the combination of setups of the objective, intermediate and projection lenses, a series of magnification times ranging from 30× to 1000 k× can be realized. The objective lens provides two types of information. The first image, or the magnified image formed at the image plane of an objective lens, reveals the microstructure (morphology) of samples. The microstructure of samples will be displayed on the viewing screen if the first image of the objective lens serves as the object of the intermediate and projector lenses (Fig. 7.2a). Meanwhile, the transmitted beams scattered in different directions interfere with each other and form a so-called

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(a)

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(b) Electron source

Condenser lens condenser aperture Object Objective lens

Objective aperture

Focal plane of objective 1st image

Selector aperture

Intermediate/ projector lens

Screen final image Image

Diffraction

Fig. 7.2 Ray diagrams of two operation modes in a conventional TEM, according to the geometrical optics approximation: (a) image mode and (b) diffraction mode.

diffraction pattern at the back focal plane of the objective lens. Information at the back focal plane of an objective lens usually reveals the structural (symmetry) information of crystalline specimens. By tuning the lens currents, the back focal plane of an objective lens can coincide with the object plane of the intermediate/projector lenses; hence, the electron diffraction pattern at the back focal plane of the objective lens is magnified and displayed on the viewing screen (Fig. 7.2b). This operation is called selective area diffraction (SAD). Images and the corresponding electron diffraction patterns are equally important for a detailed study of materials; both should always be produced from the same area, with the specimen orientation unchanged. For this reason, most commercial microscopes have the ability to interswitch easily between the image mode and the diffraction mode. The diffraction patterns formed at the back focal plane of an objective lens are planar sections of the reciprocal space of specimens. Each dot on a diffraction pattern corresponds to a transmitted beam scattered by the specimen. The strong beam at the center is the directly transmitted beam that carries most of the intensity of the postspecimen beams; other beams around the center beam are called diffraction beams and usually follow the Bragg’s equation if the specimen is a crystal. The types or characters of diffraction patterns are of course related to the structures (symmetry) of

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Fig. 7.3 Examples of three types of common microstructure and the corresponding electron diffraction patterns: (a) polycrystalline Au particles, (b) a single crystal of a triangular Ag tablet and (c) amorphous TiOx powder.

specimens. Three types of diffraction pattern are routinely observed: the concentric multiple-ring from a polycrystalline sample, a dotted pattern from a single crystal, and vague rings from the amorphous material. Figure 7.3 shows examples of three types of electron diffraction pattern and the corresponding microstructures. The structure of samples can be retrieved by analyzing and indexing the diffraction patterns as well. The reciprocal lattice of an unknown crystal can be reconstructed through a series of electron diffraction patterns obtained by tilting the specimen in three dimensions. As a result, the symmetry of the crystalline specimen is derived. The electron diffraction technique is a useful complement to X-ray diffraction, especially for materials only available at small scales or in small amounts. Even fine powders usually contain many small pieces of sufficient size to produce singlecrystal electron diffraction patterns that allow the approximate determination of lattice parameters. In addition, electron diffraction patterns often reveal weak reflections due to superstructures or the imperfection of crystals, which are usually invisible in regular X-ray powder diffraction. A good example of the applications of electron diffraction techniques to the structural study of a few Li-battery samples is presented in the references by Lei et al.1,2 Three sets of apertures, which are movable metal blades with holes of different sizes, can be placed into the ray path according to the requirements of imaging techniques (Fig. 7.2). These apertures allow the electrons defined by the holes to travel through the imaging system. The condenser aperture located above the samples determines the number of incident electrons on the sample. A small condenser

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aperture helps form a small probe size and decreases the beam damage to samples, but will lower the intensity of images. The objective aperture, placed close to the back focal plane of the objective lens, is used to select a number of transmitted electron beams for imaging. The image usually called the diffraction contrast image is the map of the intensity distribution in the selected beam if a single beam is selected for imaging materials; it is called the bright-field (BF) image when a directly transmitted beam is selected or a DF image if a scattered beam is highlighted. Only the part producing the selected diffracted beam is bright in the DF image. The DF image has better image contrast but a poor intensity compared to the BF image. A detailed theory of the diffraction contrast images of thin crystals can be found in the book by Hirsch et al.3 Both BF and DF techniques are good for the study of a large area of defects in a thin crystal. The explanation of BF images of noncrystalline samples such as polymers and biological samples is simplified as the qualitative mass–thickness contrast, where the objective aperture blocks or “absorbs” more electrons scattered from heavy elements or thick areas than from light elements or thin areas; consequently, heavy elements or thick areas appear darker in BF images. The aperture at the image plane of the objective lens is called the SAD aperture and is usually used to select a specific area for structural analysis or for diffraction. SAD apertures usually define a relatively large area of several micrometers. The SAD aperture is very important when multiple phases are present within the same sample. The combination of BF, DF and SAD techniques generally provides an overview of the shape, distribution, size and possible symmetry of phases in specimens, and is suitable for the study of a large area at a relatively low magnification, usually between 10 000 and 100 000×. Figures 7.4a and b present a BF and a DF image, respectively, of the domain structure in a Li1.2(Mn0.4Co0.4)O2 sample. The inserted SAD patterns are the superimposition of several sets of diffraction patterns, suggesting the coexistence of a few small domains. The domain walls are vague in the BF image, while the DF technique reveals one of the domains. Figures 7.4c and d are the corresponding schematic illustrations of the setup of objective apertures in ray paths. If a crystal sample is tilted to a specific zone axis, and a big objective aperture is used to collect multiple beams for imaging specimens at a magnification of around 500 000×, the final image on the fluorescent viewing screen is a lattice image, usually called a high-resolution electron microscopy (HREM) image, which has become an important method in the study of complicated crystal structures and their defects at the atomic scale. Figure 7.5a is a schematic illustration of the ray diagram of the lattice image technique. The lattice image obtained this way is the array of dots that directly relate to the structural periodicity of specimens at the atomic level. Figure 7.5b is the lattice image of a multiply twinned Au particle. The dots change the stacking sequence at locations marked with dashed lines. The dot arrays at two sides of a boundary are mirror-related (that is, the boundary is a twin boundary). Generally, the dots bear no direct connection to atomic positions in the crystal. The theory behind the high-resolution image is complicated, and beyond the scope of this chapter. A simple consideration of the image formation is treated in two steps. First, electrons propagate through a very thin sample and produce a two-dimensional periodic electron distribution at the exit face, which images the projected lattice potential or the projected electron density of the specimen. This could be expressed as a twodimensional transmission function q (x,y) that consists of the amplitude and phase of

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(c)

(d) Incident beam specimen

Objective lens

Diffracted beam

Diffracted beam

Objective aperture

Transmitted beam BF image

Transmitted beam DF image

Fig. 7.4 (a) A BF and (b) a DF image of the domain structure in a Li-battery sample. Beams used for imaging are marked in insets. A small domain hardly visible in the BF image is revealed by the DF technique. The ray diagram and the positions of objective apertures are shown in (c) BF and (d) DF.

the electron beams emerging from the column at the position (x,y) of the exit surface after dynamic diffraction in the sample. In a second step, the exit wave acts as a planar distribution of point sources, which interfere and form the diffracted beams that move forward in the lens system of the microscope. The diffraction pattern can approximately be described as the Fourier transform Q (u,v) = F [q (x,y)] of the object function q (x,y). This diffraction pattern acts in turn as a source of Huyghens wavelets, which interfere with each other to form the final image after the approximately linear magnification by the optical lens. The image is, in turn, the Fourier transform ψ (x,y) = F [Q (u,v)] of the diffraction pattern, which will be directly interpretable only if the beams interfere with the correct phases. Obviously, the image depends on the beam numbers admitted by the objective aperture; the image will exhibit more details as the number of included diffractive beams increases.

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(a) Incident beam Sample

q(x,y)

Objective lens Objective aperture

First image

Q(u,v)

ψ(x,y)

Fig. 7.5 (a) Ray diagram for the formation of the lattice image technique. (b) The lattice image of a twinned Au particle.

The details of the recorded lattice image depend on many experimental factors, such as the sample thickness, the scattering ability of the atoms within the specimens, the number of diffractive beams admitted by the objective aperture, the defocus value and so on. This phenomenon is obvious even from the image of a wedge-shaped sample obtained on an FEG microscope. In practice, a limited number of diffracted beams are included within the objective aperture and used to obtain lattice images, in order to remove unwanted details or “noise”. The precise interpretation of a highresolution image, or the connection between dot contrast and the position of atoms in a specimen, requires a careful comparison between the recorded images and a

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FEG Anode & condensor lens

Scanning system

Scanning coils Objective lens

Sample Image process unit ADF detector BF detector EELS Magnetic prism

Display unit

ZLP

Spectrum process unit Fig. 7.6 Schematic illustration of the configuration of a STEM instrument integrated with an EELS spectrometer.

series of simulated images based on a structural model of specimens imaged at variable thickness and defocus values. The image simulation is carried out when the correlation between dots and the precise positions of atoms is requested. Dots in the lattice image are intuitively assumed to represent the projections of atom columns along the beam direction in many situations where the periodicity but not the position of atoms is of interest.

7.2.3

Scanning TEM

Figure 7.6 is a schematic illustration of the configuration of a STEM instrument. Similarly to CTEM, a condenser lens forms a fine convergent electron probe of about 1 nm or less. Unlike CTEM, STEM does not have to need a post-specimen lens, but requires a small and brilliant source, which is usually a FEG. The incident beam is focused on the sample and is scanned across the area of interest through a scanning coil. A convergent electron diffraction pattern is formed behind the specimen. Parts of the diffracted beams are selected by an appropriate detector placed below the specimen and the signals detected thereby are modulated and displayed on a screen. A control system enables the detector and the images on the displaying unit to be synchronous with the probe scanning across the specimen. Hence, the intensity of each point on the image corresponds to the signals from a point of the specimen. Two kinds of detector are usually used. A BF image is obtained when the detector is

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placed below the directly transmitted beam. Images obtained from the BF mode are similar to those recorded with CTEM techniques. A DF image is obtained if one or several beams outside the central beam is selected. A donut-shaped detector is usually designed to exclude the direct beam and obtain a DF image, called the annular dark field (ADF) image. The resolution of the STEM technique depends on the probe size, and the high spatial resolution is achieved by making the effective probe size as small as possible. A modern commercial STEM instrument installed with an FEG can form a probe size in the order of 1 nm diameter or less. The signal in the STEM technique is generated in a serial or time-dependent form, which makes it easy to apply online image processing or a high-spatial-resolution analysis of the chemical information of the specimens. The advantage of the high spatial resolution makes it possible to park the fine beam at a specific location in order to obtain the composition from a small area comparable to the beam size. Like the interaction occurring in the conventional TEM, electron beams that have interacted with samples will yield incoherently scattered electrons, resulting from thermal diffuse scattering and in particular Rutherford scattering, simultaneously with coherent scattering (diffraction beams). In the STEM mode, these incoherently scattered electrons can be used to image materials through the proper electron optics, and an ADF detector can be used for the capture of these electrons. The Rutherford scattering contributes to the large portion of the incoherently scattered electrons at high angles. The incoherent imaging condition occurs approximately when the ADF detector is placed at a high-collection-angle (typically >50 mrad) position, which forms a high-angle annular dark field (HAADF) image (Fig. 7.6). The number of incoherently scattered electrons that usually occur at relatively large scattering angles depends on sample thickness and the composition of the specimen. The dot intensity of a HAADF image increases positively with the average Z value of atoms in the corresponding column. Thick areas and high-mass elements will produce bright contrast in HAADF images as a consequence of the steady increment of average Z values with the thickness and heavy elements within the areas. The contrast in HAADF images is basically a mass–thickness effect. The contrast of a dot varies monotonously with the defocus in the case of an incoherent imaging condition, whereas it is a rapidly oscillating function under coherent imaging conditions such as HREM. This has important consequences. The brightness of a dot imaging a given atomic column may change from bright to dark and vice versa with the adjustment of defocus in HREM images, whereas the relative brightness of a dot remains consistently the same, or independent of defocus values, in the case of incoherent imaging. The HAADF imaging technique meets the incoherent condition to a large extent. It is possible to obtain an HAADF image at the atomic scale, provided that the proper optics, a fine and strong probe, and an ultrathin clean sample are available. It is understandable that structure retrieval is in principle possible and simpler using the HAADF technique than in HREM. HAADF images at the atomic scale can therefore be interpreted on an experiential and simple basis even for a relatively thick sample. HAADF images are sometimes called Z-contrast images. HAADF technique is well suited to the study of geometric shapes and large variations in the chemistry of samples. Figure 7.7 provides a comparison of TEM and STEM techniques through images of Pd particles on a carbon nanotube. The STEM image, Fig. 7.7c, clearly shows plenty of bright Pd nanoparticles of

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Fig. 7.7 A comparison of CTEM BF images and the corresponding STEM DF image of Pd nanoparticles coating a carbon nanotube. The multiple-beam BF images are recorded at (a) near-optimal focus and (b) defocus conditions. (c) is the HAADF image of the same area.

different sizes on a dark tube. The mass effect (ZPd = 46 and ZC = 6) on the HAADF image is obvious. Figure 7.7a is the BF image recorded near the optimal focus and reveals particles similar to those shown in the STEM image. In contrast, a few small particles are out of contrast in Fig. 7.7b, which is a slightly defocused BF image; the contrast of the tube walls turns vague as well, due to the defocus effect.

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(a) M

L

Lα K

e–

Kα1 Lβ Kα2 Kβ

0

5

10 Energy (keV)

SrK β

SrK α CuK β

CuK α

TiK α TiK β

SiK / SrL α

Intensity

OK

(b)

15

20

Fig. 7.8 (a) Schematic illustration of the principle of characteristic X-rays. (b) An EDS spectrum from an SrTiO3 film.

7.2.4

Analytical electron microscopy

The incident electrons will lose energy when they transfer sufficient energy to shell electrons to change the orbital states of atoms within a sample; this process produces several kinds of signals, of which the most commonly used in AEM are the characteristic X-rays emitted from the specimen surface and the energy-loss electrons passing through the specimen. The X-ray photons can emit from the sample surface when the outer-shell (L, M, etc.) electrons of an atom jump into the vacant inner-energy level (the core level) that is created through an ionization process, for example the ejection of an inner (K) shell by the high-energy incident electrons (Fig. 7.8a). The X-ray photons whose energies equal the energy difference between two related shells are characteristic or unique to elements, and can therefore be used as fingerprints to identify the elements in samples. One element can emit X-rays of different energies. These X-ray photons are named Kα1, Lα2, …, where the letters K, L, M, … relate to the excited shell, the Greek letters α, β, χ,… reveal the difference between the excited shell and the shell filling the vacant, and the numbers 1, 2, 3, … denote the various subshells. More details on the label rules can be found in the book by Williams.4 The X-ray

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spectrometers attached to TEM instruments usually cannot distinguish different subshells due to the limited resolution. Hence, X-ray peaks are labeled only with K, L, M and α and β except at very high energies. Two types of X-ray spectrometer are available: wavelength-dispersive spectrometers that is based on the dispersion of the wavelengths of X-rays, and energy-dispersive spectrometers (EDSs) that is based on energy analysis. The EDS is usually applied in most TEM instruments as it can produce a rapid analysis even from a small amount of material. An X-ray EDS mounted sideways on the microscope column slightly above the specimen captures the X-rays emitted from the upper surfaces of specimens within a certain solid angle from a specific takeoff direction, converts those X-rays through an X-ray transparent window into pulse signals, stores the signals in appropriate energy channels and finally displays the stored results through a computer. The spectrum consists of the characteristic X-ray lines of different energies superimposed on a continuous background of bremsstrahlung. Figure 7.8b shows a typical EDS spectrum taken from an SrTiO3 sample. Elements, Ti and Sr in this spectrum, may show families of peaks of different excited shells. Families of peaks are important and helpful in the identification of elements as one peak sometimes cannot be unambiguously indexed due to the close energies between the characteristic lines of two different elements. For instance, the peak around 1.85 keV can be identified as either Sr (Lα = 1.87 keV) or Si (Kα = 1.83 keV) due to the resolution limit, and an Sr Kα peak at 14.2 keV will help to rule out the possibility of Si. EDS is a good technique for the analysis of elements ranging from B to U, preferably for heavy elements. The peak positions are important for qualitative analysis, but the quantitative analysis requires a careful examination of both the shape and the height of peaks. The energy losses of incoherent transmitted electrons can be measured via an electron spectrometer mounted below the samples. The electrons are first selected by means of an entrance aperture, then pass through a magnetic prism to produce the spatial separation of electrons of different energies and are collected by a scintillator located at the dispersion plane of the spectrometer, and finally are read out and displayed using a computer system (Fig. 7.6). The spectrum is interpreted in terms of what causes the energy loss. Figure 7.9a presents an electron energy-loss spectrometry (EELS) spectrum recorded from an Li1.2(Mn0.4Co0.4)O2 powder. It contains three ranges, the zero loss peak (ZLP), the low-loss range (<50 eV) that corresponds to plasmon oscillations or inter- and intra band transitions, and the high-loss range (>50 eV), whose intensity is usually hundreds of times lower than ZLP. The plasmon peak reflects the interactions between beam electrons and the weakly bound outershell electrons of atoms within the specimen. The intensity of the background drops rapidly with the increase of energy at the high-loss range. The inner-shell ionization or inelastic interaction between beam electrons and inner-shell electrons is generally a high-energy process; the peaks usually called “edges” at the high-loss range of the spectrum reflect such inelastic interactions. A specific minimum-energy, or the critical ionization energy, is required to excite the inner-shell electrons or ionize the atom, which is the onset of the absorption edges on a high-loss spectrum. The critical ionization energy is characteristic of elements, which makes EELS a useful fingerprint technique for the identification of elements. The ionization edges are not simple Gaussian shapes, but have a “fine structure” that reflects chemical and structural effects or the electronic information. The possible EELS edges due to the

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(a) ZLP

High loss Low loss X500

CK

OK

plasmon peak

MnL2,3 CoL2,3

0

200

400

600

800

Energy Loss (ev)

Energy

(b)

N

4f 4d 4p 4s

N6,7 N4,5 N2,3 N1

M

3d 3p 3s

M4,5 M2,3 M1

L

2p 2s

L2,3 L1

K 1s Shell subshell

K Edge name

Fig. 7.9 (a) An EELS spectrum taken from an Li1.2(Mn0.4Co0.4)O2 sample. (b) The possible edges due to inner-shell ionization and their notations in the EELS technique.

inner-shell ionizations are schematically shown in Fig. 7.9b. As in X-rays, the edges are named after K, L, M,… shell excitations. The “dual” edges, such as L2,3 and M4,5, exist when subshell edges are not easily resolvable. The experimentally accessible energy loss is usually below 1 keV for most currently commercial EELS detectors. EELS is a technique complementary to EDS that works best from 1 to 20 keV. It is especially good for the light elements B, C, N, O and F, which are easily detected with EELS but hard to identify with EDS. EELS is perhaps best developed for these very light elements and for many 3D transition metals, where the K or L2,3 excitation edges look to be sharp and well-defined. A careful examination of the positions and details of the inner-shell edges of EELS curves will help in understanding the bonding or chemical structures of elements, which is usually impossible from a regular EDS spectrum. The relative positions of characteristic peaks on EDS and EELS spectra will identify the elements present in the specimen, whereas the peak heights approximately give the quantitative results. The user-friendly commercial software can provide both

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qualitative and semiquantitative analyses of EDS and EELS data. The integration of STEM and EELS/EDS enables compositions to be obtained from a point, a line and a small area. In addition, the integration makes it possible to image/map the spatial distributions of different elements at the nanoscale. An important feature of electron microscopy is the possibility of performing both a qualitative and a quantitative elemental analysis of the same area to provide the electron diffraction pattern and the image simultaneously in the same instrument.

7.3

Sample preparations

Sample preparations are just as important as TEM experiments. The objective of all sample preparation methods is to obtain a sufficiently large thin slice that is transparent for electrons with energies of 100 kV or more. The thickness of specimens is usually a few hundred nanometers, or less for a specimen containing heavy elements. All samples loaded on a standard TEM holder should be 3 mm in diameter or less. The preparation methods depend on the type of material and the observation methods. A universal way to prepare samples does not yet exist, but the methods are classified into three categories. If the samples you want to study are nanoparticles dispersed in a solution, you can drop the liquid on the carbon film supported on a Cu grid. The liquid will evaporate in a dry environment, leaving a distribution of the interesting materials on the support film. This method is also good for the study of small cells embedded within a solution. Many brittle materials, such as dry powders, are most easily prepared by crushing them in a clean pestle and mortal (preferably in an inert liquid). The liquid containing the particles can be ultrasonically stirred to obtain uniform dispersion and is then dropped on a carbon-coated grid for TEM study. If the materials have to be crushed dry, the agglomeration caused by the electrostatic forces will be a problem. It is sometimes possible to use the carbon film to pick up a few pieces on which some thin areas are available for direct TEM observation. If the materials for TEM study are solid and not sensitive to the beam damage, a small disk of about 3 mm diameter is first cut from the bulks, then mechanically ground down to about 100 μm thickness or less, and finally milled to electron transparency with Ar+ ions. This is a good way to obtain a sample with a large transparent area. A focused ion beam (FIB) instrument that applies a beam of focused Ga+ ions to mill samples, does not require the pregrinding procedure and is well suited to sample preparation from specific sites of bulk materials. Ultramicrotomy is a very direct way to prepare TEM samples from bulk materials that are not eligible for milling methods. It applies a special cutting instrument called an ultramicrotome to section materials down to 100 nm or even less. The ultramicrotome operates by repeatedly moving a specimen past a special knife blade that is made from glass or diamond. The knife will cut the soft material into pieces, or cause a partially controlled fracture if the material is brittle. The advantage of ultramicrotomy is that it will create a large sample of uniform thickness without altering the chemistry of the samples. The disadvantage is that it introduces a deformation structure to materials, and therefore is ineligible for defect study. Ultramicrotomy is extremely useful when you want to study tiny bulks such as fibers or particles that are

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too small to thin individually but are too large for direct observations. You can also use epoxy to capsulate the porous samples, and slice the cured samples, because such samples are not thinned mechanically. Sample preparation for many biological samples requires special training as the samples have to be preserved in a condition that is close to their living state. The procedure generally includes fixation that preserves the structures, dehydration that removes water, infiltration, which fills the samples with supporting materials for subsequent treatments, embedding of materials into epoxy, and finally sectioning of pieces for TEM study with ultramicrotomes. A simple introduction can be found in the book by Weakley.5

7.4

Limitations

The limitations of the application of TEM as a powerful technique in the study of nanomaterials are obvious, although the advantages are significant. First, it is hard to avoid beam damage, where the high-energy incident electrons affect the structure and/or the chemistry of a specimen, which is a serious issue even for many soft materials in food and agriculture engineering. A good way to lower the beam damage is to use the low-voltage microscope, which is usually operated at 120 kV or even less. The minimum dose method was developed to search the area of interest at low magnification and record images at high magnification in order to minimize the beam damage. The cryo-TEM technique, where samples are studied at cryogenic temperatures (generally liquid nitrogen temperatures), was developed for the direct observation of beam-sensitive materials in their native environments. Good experience is required to prepare samples for cryo-study. A second limitation is that TEM usually presents two-dimensional images of a thin sample. Hence, it is hard to discern the microstructure or to obtain the distribution of materials along the beam direction. Currently in-development tomography techniques or a tilting series of images may help to solve this problem, but they require a long experiment time and a stable sample under beam illuminations. Third, electron microscopy, as a poor sampling technique, usually gives a local scenario rather than a big picture. The area studied by TEM is very small; it is assumed that the information obtained from this small portion represents the whole picture of the sample. It is possible to miss local information where the sample is not observed. A cross-examination of the sample will help to reduce the possibility of such “omissions”. Finally, samples must be prepared for TEM observation. In addition, samples have to be observed in vacuum, which may not reflect their true environment. It has been a challenge to observe samples from parts of materials without changing their true structures under their original circumstances.

References 1. Lei, C.H., Wen, J.G., Sardela, M., Bareño, J., Petrov, I., Kang, S.H. and Abraham, D.P. (2009) Structural study of Li2MnO3 by electron microscopy. J Mater Sci 44, 5579–5587.

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2. Lei, C.H., Bareño, J., Wen, J.G., Petrov, I., Kang, S.H. and Abraham, D.P. (2008) Local structure and composition studies of Li1.2Ni0.2Mn0.6O2 by analytical electron microscopy. J Power Sources 178, 422–433. 3. Hirsch, P.B., Howie, A., Nicholson, R.B., Pashley, D.W. and Whelan, M.J. (1977) Electron Microscopy of Thin Crystals, 2 edn, New York: Krieger Publishing Company. 4. Williams, K.L. (1990) X-Ray Spectrometry, London: Allen and Unwin. 5. Weakley, B.S. (1972) A Beginner’s Handbook in Biological Electron Microscopy, London: Churchill Livingstone Press.

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8

Dynamic light scattering

Leilei Yin

Abstract: Dynamic light scattering(DLS) particle sizing characterizes the temporal structure of particles’ Brownian motion in liquid suspension, which carries critical information about the size of the particles. By measuring the temporal structure instead of angular distribution, DLS is able to measure particles as small as a few nanometers, much smaller than the wavelength of light used. Accordingly, the core components of DLS hardware are a high-quality photon counting device and a digital autocorrelator. Advanced algorithms developed to perform inverse Laplace transformations process the autocorrelation spectra to acquire the desired sizing information. Keywords: particle sizing; dynamic; Brownian motion; autocorrelation; inverse Laplace transform

8.1

The principle of dynamic light scattering

Light scattering has been a very important probe into the structure and dynamics of molecules and particles. When light encounters a small particle, the alternating electrical field of the light synchronizes the polarization of the electrons in the particle. The small particle then acts as a secondary source of light emission: the scattered light. The intensity, angular distribution and polarization of scattered light carry information about the particle’s size, shape and electrical character. When the particle’s size is close to or larger than the wavelength of light, the light scattered from different positions on the particle will not necessarily be the same but rather will have a fixed phase relation. The superposition of all scattered parts results in an overall scattered light with an angular distribution. The refraction index of the particle’s material brings more complicated phase and even amplitude relations into the equation. It has turned out to be so complicated that today there is only an analytical solution for spherical particles, produced by Mie and Debye in the early Nanotechnology Research Methods for Foods and Bioproducts, First Edition. Edited by Graciela W. Padua and Qin Wang. © 2012 John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc.

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20th century.1,2 This type of light scattering is referred as Mie scattering. The angular distribution of scattered light has since been used to determine the sizes of large spherical particles relative to their wavelengths. It is the theoretical base for static light scattering, as compared to dynamic light scattering (DLS), the main topic of this chapter. When a particle’s size is much smaller than the wavelength of light, the electrical field at every position is nearly the same. The whole particle becomes a “point” source of scattered light. Thus, no angular character of the scattering is related to the size of the particle anymore. Static light scattering is quite ineffective for subwavelength particle sizing. While Mie scattering has been very successful in explaining scattering by particles in gas, it alone is insufficient in dealing with particles in liquid, where strong molecule–molecule interactions are seen. The destructive superposition of correlated molecule–molecule interactions reduces the scattered intensity by more than an order. However, what failed Mie scattering was proven to be critical for sizing subwavelength particles in liquid by DLS. To understand how DLS works, it is beneficial to look into how static light scattering loses its effectiveness on subwavelength particles. As described above, the angular distribution of scattered light becomes uniform when a particle is much smaller than the wavelength of light. The intensity of light scattered by a particle can be described as: I s ∝ ( Es )2 ∝ f (nl , n p )V 2 I 0

(8.1)

where Is is the intensity of scattered light, Es is the electric field of scattered light, f(nl ,np) is a function determined by refraction indices of liquid nl and the particle np , V is the volume of the particle and I0 is the intensity of incident light. The intensity of scattered light drops significantly (by the 6th order of the diameter of the particle) with reduced particle size. And f(nl,np) helps further bring down Is when nl and np are close in value. Therefore, the scattering intensity quickly becomes too low for sizing of particles in the nanometer range. Small particles in liquid are not stationary; experiencing constant collision with the molecules in the liquid, the small particles undergo a random movement known as Brownian motion. Brownian motion was first observed on pollen suspended in water, but is more rapid and violent in smaller particles. The random motion of particles in liquid brings major changes to two aspects of the scattered light: the frequency and the phase. The frequency change is simply due to the Doppler effect caused by the motion of the particles. Leon Brillouin3,4 first studied the scattering frequency shifts of particles suspended in liquid. He predicted a pair of frequency shifts, now known as the Brillouin doublet, away from the original frequency of the incident light.

ω = ± cs k s

(8.2)

where cs is the velocity of sound in the liquid and ks is the scattering vector: ks = n .

4π

λ

sin

θ 2

(8.3)

where n is the refraction index of the liquid, l is the wavelength of light in vacuum and q is the scattering angle.

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I

Ic

IB

w 0 − c sk s Fig. 8.1

w0

w 0 + csk s

w

Brillouin doublets of light scattering by particles in liquid.

Landau and Placzek5 later explained the details of the Brillouin doublet, as well as the strong central line seen in Fig. 8.1. They concluded that the intensity ratio of the central line and Brillouin doublet is: I c CP − CV = IB CV

(8.4)

where CP is the specific heat at constant pressure and CV is the specific heat at constant volume. The width of the central line is determined by the thermal diffusivity, and the width of the Brillouin doublet depends on the sound absorption of the liquid. It had been very difficult to experimentally observe the Brillouin doublet before the implementation of laser technology, because the frequency shift is generally very small and the doublets were not completely separated from the strong central line. After the invention of the laser, a highly monochromatic light source, reliable experimental measurements of Brillouin doublets using a spectrometer or Fabry–Perot interferometer became practical for the analysis of particle size in liquid. Even with a high-quality laser and a sensitive detector, particle sizing by frequency measurement is still relatively complicated and has its limits. The reason for this is that the frequency shift is still relatively small, and the accuracy of the measurement relies heavily on the stability of the laser and the quality of the Fabry–Perot interferometer or spectrometer. Such methods have difficulty in accurately detecting a frequency shift below 106 Hz. It is rare to have a condition in which particles in liquid wander violently enough to cause so much frequency shift. The frequency method of particle sizing is not used as much as photon correlation spectroscopy, which will be examined in detail in this chapter.

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l2

l4

l3

Photo detector Fig. 8.2

Phase relation of scattered light from two particles.

The second aspect of scattered light affected by Brownian motion is the phase. As a particle changes its position, with tiny displacements in an otherwise uniform field of incident light, the phase of scattered light is also changed when it is observed at a fixed position. Even though the phase of light is hard to measure directly, the superposition, or interference, of a large amount of scattering with changing phase results in a fluctuation in intensity, which is easy to detect. Again, it is important to notice the use of laser rather than another incoherent light source in DLS because DLS’s basic principle relies on the superposition of a coherent scattered field, instead of the sum of intensity. From each of many small particles in the path of the laser beam, a scattered wave is generated, which reaches the detector at a fixed position. Because all scattered waves are generated by the same monochromatic laser beam, and because each particle is small enough to be treated as a point source, the accumulative signal at the detector is a simple superposition of the electromagnetic field (not intensity) from all the scattered waves; each particle carries a phase determined by its relative position. In the simplest case of two-particle scattering, shown in Fig. 8.2, E1 and E2 are the fields scattered by particles 1 and 2, and the phase difference between E1 and E2 at the

(l1 + l3 ) − (l2 + l4 ) .2π . The superposition of E

and E2 can be any value 1 λ between E1 − E2, the case of total destructive superposition, and E1 + E2, the case of total constructive superposition. Because of the Brownian motion, the particles are constantly changing their relative positions; therefore, the phase difference keeps changing accordingly. The principle of DLS is that the temporal character, not the intensity or angular distribution, of the “random” fluctuation of total scattered light detector is

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I

T

I

T

I

T Fig. 8.3 Demonstration of light scattering from small (top), medium (middle) and large (bottom) particles.

is affected by the particles’ sizes. In general, small particles wander in liquid relatively rapidly, and the motion of larger particles is slower. In reality, of course, there are more than just two particles in the laser beam, but the size-dependent fluctuation of sum scattering stands just as it is in the simple two-particle case. Fig. 8.3 illustrates typical scattering from particles of different sizes. The mobility of a particle undergoing Brownian motion, described by a term called the diffusion coefficient, D, is affected by the radius of the particle, R, through the well-known Stokes–Einstein equation:6,7 D = KT

6phR

(8.5)

where K is the Boltzmann constant, T is the temperature of the liquid in Kelvins and h is the viscosity of the liquid. Note that h is also a function of T. Most liquid solvents have lower viscosities at higher temperatures and vice versa, so a particle’s mobility increases greatly not only from increased temperature in the numerator, but also from decreased viscosity in the denominator of the equation. And for most of the commonly used solvents, the viscosity changes with temperature at a much higher rate than does the temperature itself. For example, the viscosity of water drops from 1.002 cP at 293 K to 0.7 975 cP at 303 K,8 a 20% drop with only a 3.4% increase in

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Kelvin-scale temperature. So temperature is perhaps the most important experimental parameter in a DLS measurement in producing an accurate result. A mathematic algorithm called autocorrelation is used to quantatively evaluate the scattered light in the time domain. The time-domain autocorrelation of a function is defined as: C (t ′ ) ≡ I s (t ) × I s (t + t ′ ) = lim

T →∞

1 2T

∫

T −T

I s (t )I s (t + t ′ ) dt

(8.6)

In plain language, the autocorrelation indicates how similar the function Is(t) is to itself after a time interval t′. If a function changes slowly in time, it will still look like itself after a short amount of time. The result of the autocorrelation is a relatively large value. If the time interval is much larger than the function’s temporal character, the function Is(t + t′) becomes almost irrelevant to Is(t′). The autocorrelation becomes smaller than the mean value of expectation C(t′ → 0). Back to the case of DLS: C(t′ → ∞) is referred as the baseline of an autocorrelation function. The scattered light seems totally irrelevant to itself after a long time interval. Any shorter time interval will yield an autocorrelation larger than the baseline; the function will seem to be more similar to itself some time ago. For a suspension of uniform-sized particles with minimum interaction between one another (sufficiently diluted), the autocorrelation

4.8

Autocorrelator output (log scale)

4.6 4.4 4.2 4 3.8 3.6 3.4

0

10

20

30

40

50

60

Channel number Fig. 8.4 The autocorrelator output of dynamic light scattering of 90 nm polystyrene spheres in water, by a NiComp ZLS 380 DLS/zeta potential particle sizer. The autocorrelator output is presented in log scale after subtracting the baseline. A total of 64 channels are used to calculate the autocorrelation function. The channel width in this measurement is 12 μs. The autocorrelation function of this near-monodisperse particle suspension is very close to a simple exponential decay curve.

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of a scattered light signal turns out to be an exponentially decayed curve in the time domain, as demonstrated in Fig. 8.4: ⎛ t′⎞ C (t ′ ) = [C (t ′ = 0) − C (t ′ → ∞)] exp ⎜ − ⎟ + C (t ′ → ∞) ⎝ t⎠

(8.7)

where t is the characteristic time constant of this decay function. It is very clear in this simplified case that C(t′ → ∞) is the baseline of the correlation function and that the maximum value occurs at C(t′ = 0). In fact, t is directly connected to diffusion coefficient D by: t = (2 Dks )−1

(8.8)

where ks is the scattering wave vector described in (8.3). This section presented the the basic principles of autocorrelation DLS particle sizing: how Brownian motion of particles in liquid induces fluctuation of light scattering, how the temporal structure of the fluctuation is determined by the particle size and how to temporally analyze the seemingly random fluctuation of the scattered light.

8.2

Photon correlation spectroscopy

The scattered light becomes extremely weak when the particles’ size is much smaller than the wavelength of light, as well as when the concentration of particles in liquid is very low. The scattered light reaching the detector soon decreases to pulses of photons instead of a continuous wave of light. The integral form of the autocorrelation function needs to adapt to the photon correlation function in order to process the pulses of scattered photon. Figure 8.5 is an illustration of the output from the photodetector. The low-level scattered light is a string of pulses, where each pulse represents

T Channel width n=4

n=5

n=3

n=6

n=2

n=5

n=4

n=1

n=5

T Fig. 8.5 Illustration of output signals from a photodetector (top) and after going through a discriminator (bottom). In the nine gate periods shown, the number of scattered photons ranges from one to six.

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a photon that has reached the photodetector. The difference in amplitude is mostly due to the response variation of the detector. The number of pulses in unit time is proportional to the intensity of scattered time. An electronic device called a discriminator is used to regulate each pulse into a digital pulse with fixed amplitude and width for further processing by the digital autocorrelator. The first step an autocorrelator takes is to choose an appropriate length of time in which to count the number of pulses received within that period of time. The chosen length of time is called the “gate period” or “channel width” for an autocorrelator. The criterion for choosing the channel width is that the intensity fluctuation ought to be clearly represented by the change in pulse numbers within the channel width. An overly long channel width will contain too many pulses, so that the fluctuation is averaged out. Assuming the random pulses of photons reaching the photodetector follow the Poisson process, the average photon number N detected within the channel width will have a standard deviation of N . The photon counts in channel widths should reflect that fluctuation in pulse number. Modern digital electronics can apply a wide range of channel widths, from tens of nanoseconds to seconds, for a wide variety of particle sizes and concentrations. Once the channel width is determined, the integral form of the autocorrelation function in (8.6) changes into a series of equations, since now we have a discrete time interval kΔt′: N −1

C ( Δt ′ ) = ∑ ni ni +1 i=0

N −1

C (2 Δt ′ ) = ∑ ni ni + 2 i=0

(8.9)

N −1

C (k Δt ′ ) = ∑ ni ni + k i=0

Each autocorrelation equation is assigned to a unit called “channel” in the instrument. For example, an autocorrelator with 64 channels will have designated units to calculate from C(Δt′) to C(64Δt′). With an appropriate Δt′, the results of C(Δt′) to C(64Δt′) represent the autocorrelation of scattered light pretty well. The same mathematics of particle sizing can be applied to the digitized autocorrelation function.

8.3 DLS apparatus A typical DLS setup consists of a laser source, sample container, photodetector, photon correlation electronics and a computer. The laser provides a stable, coherent and continuous light source. An ideal laser for DLS experiments should provide adequate power, low noise, suitable wavelength and long coherent length. The laser should be powerful enough that even low-scattering particles can scatter enough light to be detected. For strongly scattering particles, a neutral density filter can easily reduce the incident light power so that the photon detector and autocorrelator won’t be saturated by strong scattering. The laser’s wavelength should be in the range of the detector’s most sensible spectrum, but since more detectors are available than lasers,

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a detector is usually chosen to match the wavelength of the laser. The output of the laser should be as stable as possible in both power and frequency. Any noise in output power will be proportionally reflected in the scattered light, which will affect the accuracy of the correlation function. The coherent length of the laser is as important as its power stability. The detected signal is the superposition of coherent scattering from many particles in the path of the laser beam. Short coherent lengths will introduce phase noise into the total scattered light, which affects the autocorrelation function in the same way as noise in the laser’s output power. Popular lasers in DLS systems are gas and semiconductor diode lasers. The gas laser is the earliest and most well-developed laser source. In DLS instruments, lowor medium-power He–Ne lasers are used for high-scattering sample measurements. For low-scattering samples, a high-power Argon laser is a more suitable source, but it comes at a higher cost. He–Ne lasers produce a red 632.8 nm wavelength with an output power from a couple to tens of milliwatts. An argon laser works at 483 or 514 nm wavelength: greenish blue or bright green color. Typical output power starts at a couple of hundred of milliwatts. A gas laser’s emission is a highly collimated circular beam. The beam cross-section profile of the TEM00 mode is Gaussian. TEM00 mode means the wave numbers in the two transverse directions are both 0. On a plane perpendicular to the beam direction, the phase of the electromagnetic field is uniform. Thermal expansion of the gas plasma tube will cause drifts in beam properties, both power and wavelength. Some lasers incorporate fine-tuning devices to stabilize the resonant cavity; if a laser doesn’t, one should wait for its temperature to stabilize before starting DLS measurements. Recently, semiconductor diode laser devices have been able to deliver satisfactory performance and cost for DLS instrumentation. With compact size, laser diodes are capable of a few milliwatts to a few hundred milliwatts of output power in a wide selection of wavelengths. The most common emission wavelength of laser diodes is in the near-infrared (IR) and red range, but they are extending into shorter wavelengths rapidly. Now affordable green diode lasers are commercially available, with outputs up to tens of milliwatts. Without the resonant cavity formed by the mirrors at the ends of a gas laser tube, the output beam is less ideal than a gas laser beam. Some accessory devices have been implemented to improve the beam quality of diode lasers. Thermal control is necessary to stabilize output wavelength and power, as well as to prolong the life of the diode by keeping the diode junction temperature under control. Focusing optics and spatial filters help to form a collimated circular beam with the desired TEM00 mode. The sample container holds the particle suspension and places it in the optical path of the laser source and the photodetector. The container’s material ought to be chemically compatible with the liquid solvent. Commonly used sample containers in commercial DLS instruments are either square cuvettes or cylindrical tubes, as shown in Fig. 8.6. Cuvettes can be made from plastic, glass or fused quartz. The plastic cuvettes are low-cost and very often disposable, but they are incompatible with some organic solvents. Glass and quartz cuvettes are compatible with most chemicals, except for some acids and strong alkaline solutions. Fused-quartz cuvettes are less popular than glass ones because of their higher cost, but quartz offers less absorption or scattering when a short-wavelength laser is used. Low scattering by the sample holder’s material is more important in small-angle-scattering or back-scattering measurements.

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Nanotechnology Research Methods for Foods and Bioproducts Cylindrical tube

Square covet

qs qair Focused Scattered light laser beam Photodetector

Scattered light Focused laser beam

Photodetector

Fig. 8.6 Popular sample containers in scattering apparatus: (left) square cuvette and (right) cylindrical tube.

Glass cylindrical tubes are a great choice in many situations; their cylindrical shape allows both incident and scattered light to perpendicularly pass the glass wall at any scattering angle if the cylindrical tube is centered on the cross of both light paths. Using a square cuvette induces an angle correction due to the difference in refraction indices between liquid and air, unless the incident light and scattered light are perpendicular to the cuvette walls. The actual scattering angle in liquid is ⎡1 ⎤ θ s = cos −1 ⎢ cos(θ air )⎥ , where nl is the refraction index of the solvent and qait is the n ⎣ l ⎦ scattering angle in air. The sample container and its holder also provide a controlled environment for the sample during DLS measurements. It has been mentioned before that the temperature affects the DLS result greatly through direct mobility of the particles and the viscosity of the surrounding liquid. Most DLS instruments have apparatus to control and stabilize the sample temperature. The most popular device is the Peltier thermoelectric heat pump. A Peltier heat pump uses electric current to create heat flow across the junction between two materials, usually semiconductors for their high thermoelectric efficiency. Such a device can heat or cool an object by simply reversing the electric current. The sample temperature should be controlled and stabilized with sub-Kelvin accuracy at least, preferably 0.1 Kelvin accuracy. The photodetector in a DLS instrument is critical to the system’s performance. A good photodetector should be highly sensitive, high-speed and low-noise. The scattering in DLS experiments is generally low-level, even down to a single photon. The photodetector should be sensitive enough to reliably detect low-level scattered light. The response time of the photodetector should be short, so that when photons reach the detector with short time intervals, they can be counted correctly. A fast photodetector can measure a higher-scattering sample more accurately. The noise level of a photodetector is measured as dark current, which is the electric output even when no photon reaches the photodetector. The dark current should be as low as possible, since it is also processed by the autocorrelator regardless of its origin. Excessive dark current will cause problems, especially for low-scattering samples. Photomultiplier tubes (PMTs) were among the earliest-developed photodetectors, and are still widely used in DLS instruments. The basic structure of a PMT is illustrated in Fig. 8.7. It consists of a photocathode, an anode and a series of interstages called dynodes, all concealed in a vacuum housing. A transparent window allows photons to reach the photocathode while maintaining a high vacuum inside the PMT. The photocathode contains a thin

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Dynodes

Window

155

Anode

Amplifier

e e

e e e e e e

Photocathode Electrons Fig. 8.7

Simplified internal structure of a PMT.

layer of photoelectrical material, most commonly alkaline metals. Low-energy photoelectrons will be produced due to the photoelectrical effect when photons hit the photocathode. The photoelectrons leave the photocathode and are accelerated to the nearest dynode by the applied electric field. More low-energy secondary electrons (SEs) will be produced after accelerated photoelectrons collide with the first dynode. Several dynodes are then held at more and more positive potential, each in turn producing more SEs. Eventually they will produce enough electrons that they can be collected by the anode. A typical PMT multiplies the number of photoelectrons by five to seven orders. The spectral response of the PMT is very wide, depending on the photocathode material and the window material. Alkaline metals have strong photoelectrical effects, from deep ultraviolet (UV) to the long end of visible light. Changing the photocathode material to InP or InGaAs can extend the response spectrum into the mid-IR range. Glass is the most common window material. At short wavelengths, special material such as quartz is needed to reduce absorption by the window. A highvoltage power supply is necessary to place the anode and dynodes at the correct potential to produce more SEs. The high voltage varies from several hundred to several thousand volts. A higher voltage increases the gain to the number of electrons, but also increases the dark current at output. Since electrons are actually flying inside the PMT over a relatively long path, careful shielding from any external electromagnetic field is necessary to keep the PMT in normal working condition. An avalanche photodiode (APD) is a relatively new type of photodetector that combines the advantages of the photodiode and the PMT. An APD is a semiconductor device that generates electron–hole pairs when it is exposed to light and a high voltage bias is applied to the P–N junction. Its simplified structure is shown in Fig. 8.8. The bias creates an electric field similar to that between dynodes in the PMT, which can increase the energy of the generated photoelectrons to produce more SE–hole pairs at the anode. An APD is like a single-multiplying-stage PMT made by a semiconductor instead of metal plates in a vacuum tube. The result is a photodetector that is more sensitive and less noisy than a regular photodiode but still compact in size. Since it only contains one stage in which to multiply the number of electrons, the APD is not as sensitive as a PMT. The latest APDs are capable of very high-speed detection of photons up to several GHz. The spectral response of an APD is also very wide, from

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Window

P contact (anode)

P N N contact (cathode) Fig. 8.8

p region (depletion region)

P+

Simplified schematics of an APD.

the low end of visible light to near-IR for silicon APDs, and from near-IR to mid-IR for InGaAs APDs. The P–N junction of an APD can be fabricated to a very small size (tens of microns across), so it is ideal to couple such an APD detector to optical fibers. Like other semiconductor devices, the noise of an APD can be greatly reduced when the device operates at a low temperature. As a matter of fact, almost all APD module are actively cooled in applications. And because it is quite small in size, a compact Peltier heat pump (semiconductor thermoelectrical device), often integrated into the housing, can easily lower and maintain the temperature of an APD photodetector.

8.4 DLS data analysis As mentioned above, the autocorrelation function of uniform-sized particles is a single exponential decay curve. Correspondingly, a single exponential decay correlation function most likely indicates a monodisperse particle distribution. However, uniformsized particles are almost never found in any real sample. The task then is to find out how to apply our knowledge of the DLS of uniform-sized particle to a mixture of different sizes. Particle sizing from a measured autocorrelation function is a much more complicated and tricky study. It is relatively easy to calculate the autocorrelation function from a known size distribution, whether monodisperse or polydisperse, but the reverse process is much less trivial and certain. If the sample contains a mixture of particles of several sizes, one can expect there to be several decay time constants ti, or decay rates Gi = 1/ti, in the autocorrelation function: C (t ) = ∑ N i exp( −Γ i t )

(8.10)

i

where Ni is the weighting coefficient of each size. If i is sufficiently large, the sum function can be expressed with an integral: C (t ) = ∫ N (Γ )exp( −Γt )d Γ

(8.11)

This is a standard Laplace transformation. Now, if one has an autocorrelation function and wants to calculate the distribution function N(G ) of different decay rates, ideally

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an inverse Laplace transformation will simply yield the result. However, a unique solution to the inverse Laplace transformation only exists when the data are noise-free and absolute precision in calculation is achievable, which is impossible in any real autocorrelation measurement and digital algorithm. There are fluctuation and noise in the acquisition of the autocorrelation function, rounding errors in digital calculation and limited gate widths cutting off long decay components in the autocorrelation function. This is the so-called “ill condition” of the inverse Laplace transformation.9–12 With the difficulties in acquiring a full solution through the inverse Laplace transform, particle size distribution is still achievable by DLS. By following some these steps, a close representation of the actual particle size distribution can be deciphered from the autocorrelation functions. (i) Apply preliminary knowledge to the sample to greatly reduce variables in mathematical calculation. For example, assuming the sample’s size distribution is the Gaussian curve will reduce the variables to only two: mean size and deviation. (ii) Apply limits to the size distribution to be extracted from the autocorrelation functions, such as limited size range, limited number of particle sizes. As a matter of fact, in almost all commercial DLS instruments, the choice of forms and the numerical fitting algorithms are closely guarded trade secretes. The success of these instruments depends on the ability of our preliminary knowledge and assumptions to correctly analyze the acquired correlation function. Details of the algorithm are not the focus of this chapter. Knowledge of samples and understanding of data are more important to the practice of particle sizing with DLS. The standard by which to evaluate the goodness of a fit is often the test of c 2 (chi squared). c 2 is defined as: n

χ2 = ∑ i =1

[Cex (τ ) − Cthe (τ )]2

σ2

(8.12)

where Cex(t) is the measured autocorrelation function, Cthe(t) is the theoretical or expected autocorrelation function of chosen forms, n is the number of comparison points and s is a standard error (a constant can be used in its place). The smaller c 2, the better the fit. There are many popular algorithms to help computers find the most optimized parameters in forms to minimize c 2. Generally, the fewer numbers of variable parameters in the forms, the easier and better the chance optimized fitting can be achieved, given the forms chosen are indeed correctly describing the size distribution of a sample, of course. The minimized c 2 value acquired from a fitting process is often an excellent indicator of the validity of the chosen forms. A repeatable, small c 2 value first indicates the choice of correct forms, and the optimized parameters reveal the most probable size distribution of the particles. There is one very important point that needs to be noted: all particles are assumed to be spherical in DLS experiments. The theoretical background mentioned previously assumes we are dealing with spherical particles. With a nonspherical shape, the rotation of particles in liquid induces other terms into the scattered light. There have been theoretical studies of the rotational terms,13−17 but they have not yet materialized in commercial instruments. Such a level of theoretical study is beyond the scope of this chapter. In some cases, a DLS instrument can somehow give consistent and repeatable results on nonspherical particle samples. However, this does not mean the presented data should be considered an exact result. What they mean is that the DLS data of the sample are a close fit to the correlation function of spherical particles with

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the presented size distribution. Some preliminary knowledge of the sample is very important in the correct understanding and interpretation of DLS experiments. The simplest type of polydisperse sample would be a known shape of distribution with a few parameters left to adjust. But one needs a great deal of knowledge about the sample to confidently believe the known distribution is a valid form. The Gaussian form is a very popular form for samples with a well-defined mean diameter and a symmetric, bell-shaped curve of size distribution. The Gaussian form assumes there is only one peak in the size spectrum and that the deviation from center peak size should be relatively small. To fit to the Gaussian form, only two parameters need to be adjusted: mean size and deviation from mean size. It is a relatively fast and simple algorithm for modern computing. The standard Gaussian function G(x,m) is: G ( x, μ ) =

⎛ ( x − μ )2 ⎞ exp ⎜ − 2σ 2 ⎟⎠ ⎝ 2πσ 2 1

(8.13)

where m is the mean size and s is the deviation. For samples with few known distribution characters, data analysis methods have to allow the weighting function W(G ) to be adjusted with more freedom. Some valuable approaches have been developed to deal with more complicated or less understood samples in DLS measurements.

8.4.1

Multiple-decay methods

The multiple-decay methods are a natural extension of single-decay analysis of the autocorrelation function. If an autocorrelation function indeed contains two exponential components then: C (t ) = W1 exp( −Γ1t ) + W2 exp( −Γ 2 t ) + B0

(8.14)

Here are five variables in the fit to the autocorrelation function: decay rate G1 and G2, corresponding to two particle sizes; their weighting coefficients W1 and W2; and the baseline B0, which can be acquired from the far end of the autocorrelation function or else considered as a variable too. It is still practical to find optimal fit with 4∼5 variables in the double decay situation. But with even one more decay component, the number of variables rises to 6∼7, which already makes a direct approach of fitting very difficult. By gradually adding decay components and applying non-negative restrictions, researchers have developed methods to resolve more than a dozen decay components.18−22 The major limit of the multiple-decay methods is that the interpreted data are limited to a series of discrete sizes. Information between these discrete points is lost.

8.4.2

Regularization methods

Regularization methods9,10,19,20,23,24 are designed to acquire smooth distributions of W(G ) instead of the series of discrete weighting coefficients W(Gi) in multiple-decay 2 methods. A regularization parameter a is added to the evaluation of c :

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χ 2 (α ) = χ i2 + α ℑW (Γ )

2

159

(8.15)

where ℑ is an operator to W(G ). ℑW (Γ) can exist in various forms in different cases, such as ΣW(G ) or the second derivative of W(G ). A small or zero a means the 2 regular mean square minimization of ci . With a larger a, any peak-like component 2 in the distribution function W(G ) is penalized in the minimization of c (a). a is in effect a smoothing coefficient which when chosen properly leads to more distribution information between peaks in W(G ).

8.4.3

Maximum-entropy method

The maximum-entropy method was developed to solve the ill-conditioned inverse Laplace transformation, not only mathematically but also using principles from thermal dynamics. Among all possible solutions to the autocorrelation function, the one 2 with the maximum entropy ought to be the most probable. The c of fitting includes entropy as the second term: ⎛ t ⎞⎤ 1 ⎡ χ = ∑ 2 ⎢C (ti ) − ∑ W (Γ j )exp ⎜ − i ⎟ ⎥ ⎝ Γ j ⎠ ⎥⎦ i σi ⎢ ⎣

2

2

(8.16)

Various constriction conditions have been discussed in previous research by Livesey et al.,25 Nyeo et al.,26 Elster et al.27 and Provencher et al.28

8.4.4

Cumulant method

Cumulant expansion was developed by Koppel.29 The correlation function C(t), minus baseline C(∞), is expanded into a series: 1 1 1 ln[C (t ) − C (∞)] = C0 − K1t + K 2t 2 − K 3t 3 ... 2 2! 3! K1 = 〈Γ〉 K 2 = 〈(Γ − 〈Γ〉)2 〉 K 3 = 〈(Γ − 〈Γ〉)3 〉 K 4 = 〈(Γ − 〈Γ〉)4 〉 − 〈(Γ − 〈Γ〉)3 〉

(8.17)

Here it is assumed the correlation function contains several different decay rates and that 具Γ典 is the average decay rate: ∞

〈Γ〉 = ∫ ΓW (Γ )d Γ 0

(8.18)

The Cumulant expansion method is suitable for small t and relatively fast decay correlation functions. It has been proved to be easy to apply and fairly reliable. In most cases, expansion to the third order of t is adequate for analysis.

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References 1. Mie, G. (1908) Beiträge zur Optik Trüber Medien, Speziell Kolloidaler Metall-Lösungen. Ann Phys 25, 377–445. 2. Debye, P. (1909) Das Verhalten von Lichtwellen in der Nähe Eines Brennpunktes Oder Einer Brennlinie. Ann Phys 30, 755–776. 3. Brillouin, L. (1914) Diffusion de la lumiere par un corps transparent homogene. C R Seances Acad Sci 158, 1331–1334. 4. Brillouin, L. (1922) Diffusion de la lumière et des rayons X par un corps transparent homogène: influence de l’agitation thermique. Ann Phys 17, 88–122. 5. Landau, L. and Placzek, G. (1934) The structure of undisplaced scattered lines. Physik Z Sowjetunion 5, 172. 6. Einstein, A. (1906) Zur Theorie der Brownschen Bewegung. Ann d Phys 324, 371. 7. Reif, F. (1965) Fundamentals of Statistical and Thermal Physics, New York: McGrawHill. 8. Weast, R.C. (1985–1986) Handbook of Chemistry and Physics, 66 edn, Boca Raton, FL: CRC Press. 9. Provencher, S.W. (1982) A constrained regularization method for inverting data represented by linear algebraic or integral equations. Comput Phys Commun 27, 213–227. 10. Provencher, S.W. (1982) CONTIN: a general purpose constrained regularization program for inverting noisy linear algebraic and integral equations. Comput Phys Commun 27, 229–242. 11. McWhirter, J.G. and Pike, E.R. (1978) On the numerical inversion of the Laplace transform and similar Fredholm integral equations of the first kind. J Phys A: Math Gen 11, 1729–1745. 12. Honerkamp, J. and Weese, J. (1990) Tikhonovs regularization method for ill-posed problems: a comparison of different methods for the determination of the regularization parameter. Continuum Mech Thermodyn 2, 17–30. 13. Doi, M. and Edwards, S.F. (1978) Dynamics of rod-like macromolecules in concentrated solution. Part 1. J Chem Soc, Faraday Trans 2. 74, 560–570. 14. Maguire, J.F., McTague, J.P. and Rondelez, F. (1980) Rotational diffusion of sterically interacting rodlike macromolecules. Phys Rev Lett 45, 1891–1894. 15. Maguire, J.F., McTague, J.P. and Rondelez, F. (1981) Rotational diffusion of sterically interacting rodlike macromolecules. Phys Rev Lett 47, 148. 16. Highsmith, S., Wang, C.C., Zero, K., Pecora, R. and Jardetzky, O. (1982) Bending motions and internal motions in myosin rod. Biochemistry (NY) 21, 1192–1197. 17. Zero, K.M. and Pecora, R. (1982) Rotational and translational diffusion in semidilute solutions of rigid-rod macromolecules. Macromolecules 15, 87–93. 18. Cantor, D.G. and Evans, J.W. (1970) On approximation by positive sums of powers. SIAM J Appl Math 18, 380–388. 19. Provencher, S.W. (1976) An eigenfunction expansion method for the analysis of exponential decay curves. J Chem Phys 64, 2772–2777. 20. Provencher, S.W. (1976) A Fourier method for the analysis of exponential decay curves. Biophys J 16, 27–41. 21. Jakeš, J. (1988) A numerical method of fitting a multiparameter nonlinear function to experimental data in the L1 norm. Applications of Mathematics 33, 161–170. 22. Jakeš, J. and Šteˇpánek, P. (1990) Positive exponential sum method of inverting Laplace transform applied to photon-correlation spectroscopy. Czechoslovak Journal of Physics 40, 972–984.

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23. Provencher, S.W., Hendrix, J., De Maeyer, L. and Paulussen, N. (1978) Direct determination of molecular weight distributions of polystyrene in cyclohexane with photon correlation spectroscopy. J Chem Phys 69, 4273–4276. 24. Provencher, S.W. (1979) Inverse problems in polymer characterization: direct analysis of polydispersity with photon correlation spectroscopy. Die Makromolekulare Chemie 180, 201–209. 25. Livesey, A.K., Licinio, P. and Delaye, M. (1986) Maximum entropy analysis of quasielastic light scattering from colloidal dispersions. J Chem Phys 84, 5102–5107. 26. Nyeo, S.L. and Chu, B. (1989) Maximum-entropy analysis of photon correlation spectroscopy data. Macromolecules 22, 3998–4009. 27. Elster, C. and Honerkamp, J. (1991) Modified maximum entropy method and its application to creep data. Macromolecules 24, 310–314. 28. Provencher, S.W. (1992) Low-bias macroscopic analysis of polydispersity. In: Harding, S.E., Sattelle, D.B. and Bloomfield, V.A. (eds) Laser Light Scattering in Biochemistry, Cambridge: Royal Society of Chemistry, pp. 92–111. 29. Koppel, D.E. (1972) Analysis of macromolecular polydispersity in intensity correlation spectroscopy: the method of cumulants. J Chem Phys 57, 4814–4820.

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9

X-ray diffraction

Yi Wang and Phillip H. Geil

Abstract: X-ray diffraction is an analytical technique that reveals the crystallographic structure of materials, especially thin films. In this chapter, we introduce X-rays and X-ray diffraction, and we present the general set up for the collection of X-ray diffraction data. Because X-ray diffraction yields the atomic structure of a material, it is also called characteristic X-ray. We present the X-ray tube and the synchrotron radiator as the two general types of X-ray source. We introduce small-angle X-ray scattering (SAXS) and wide-angle X-ray scattering (WAXS), which belong to a family of X-ray scattering techniques used in the characterization of materials. X-ray scattering is a universal tool for exploring the structure of matter. We present the use of X-ray scattering to characterize protein composite films. SAXS and WAXS are used to determine the orientation and molecular spacing of thin films. Keywords: X-ray diffraction; small-angle X-ray scattering; SAXS; wide-angle X-ray scattering; WAXS; structure; film; zein

9.1 9.1.1

Background Introduction

9.1.1.1 X-rays X-rays are electromagnetic radiation with a short wavelength of about 0.01–10 nm, located between γ-radiation and ultraviolet (UV) rays. X-ray radiation was discovered by Roentgen in 1895, but as the nature of radiation was not yet understood, Roentgen called them X-rays.1 Von Laue’s diffraction theory, developed around 1910, began the research into using a crystal as a diffraction grating which was carried out by his assistants, Friedrich and Knipping. Their results, published in 1912, provided evidence for the existence of lattices in crystals and the wave nature of X-rays. Nanotechnology Research Methods for Foods and Bioproducts, First Edition. Edited by Graciela W. Padua and Qin Wang. © 2012 John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc.

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A heated filament under vacuum releases thermal electrons, and X-rays are produced when the electrons are accelerated towards, and eventually strike, a metal anode at a high potential.2 The deceleration of these electrons as they penetrate matter is the most important process in the generation of X-rays. If a bombarding electron should eject an electron from the K shell of an atom, the resulting vacancy will be filled by an electron falling from a higher-energy shell, the energy difference being radiated in the form of an X-ray photon. The X-rays resulting from the fall of electrons from the L or M shells into the K shell are known as Kα or Kβ radiation. Since the various shells have fixed energies for a given type of atom, these radiations also have a fixed energy, a fixed frequency and a fixed wavelength characteristic of the atom. They are known as characteristic X-rays. 9.1.1.2

X-ray scattering and X-ray diffraction

X-rays are electromagnetic waves, and thus they cause vigorous vibrations of the shell electrons of the atoms of substances through which they pass. The formation of a composite wave by superimposition of two or more waves is known as interference. The incident wave, confined initially to a single direction of propagation, will be deflected and propagate in new and different directions after meeting an obstructing object, the size of which is in the order of the wavelength of the incident wave. This latter phenomenon is known as diffraction. When electrically charged particles, such as electrons, are accelerated by the incident X-rays, secondary radiation is always emitted. Because the secondary emission is stimulated by the oscillating electric field of the incident X-rays, it is synchronous with it, and consists of X-rays with the same frequency and wavelength as the incident X-rays. This scattering, without a change in wavelength, is known as elastic scattering or Thomson scattering and is the main type of scattering involved in X-ray diffraction. X-rays also behave as particle-like photons having momentum. Momentum is transferred when these particles collide inelastically with electrons, and the radiation is converted into X-rays with lower energy. Since the energy change varies with the collision conditions, these scattered X-rays have a continuous spectrum at wavelengths greater than that of the incident X-rays. This scattering is known as inelastic scattering or Compton scattering and is of historical importance as experimental evidence for the dual wave/particle nature of X-rays. However, these scattered X-rays do not result in interpretable diffraction. In addition, some photons are dissipated, and some lose their energy by ejecting an electron from an atom, or due to the photoelectric effect; an example is X-ray fluorescence. Accelerated by the incident X-rays, free electrons and electrons in the shells of atoms can give rise to coherently scattered X-rays (i.e. with no change in wavelength). The intensity varies with the observation direction. Every type of atom has its own characteristic number of extranuclear electrons. Their distribution throughout the space surrounding the nucleus may be taken as continuous, and the amplitude of the coherent scattering due to this atomic electron cloud can be found from the Fourier transform of the electron density around the nucleus. The scattering from an atom may be conveniently regarded as a wave of amplitude originating from the position of the atomic nucleus. The amplitude of the scattered X-rays from an assembly of atoms can, therefore, be found by appropriate summation of the scattered waves

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originating from all points. The composite amplitude of the scattered waves from a discontinuous set of points may be found by a simple summation of the products of their individual scattering amplitudes and the appropriate exponential phase terms for their path differences relative to a convenient origin. X-ray diffraction yields the atomic structure of materials and is based on the elastic scattering of X-rays from the electron clouds of the individual atoms in the system. X-ray diffraction requires a source that is as monochromatic as possible. Because of its comparatively high intensity, Kα radiation is usually chosen, which means that it must be isolated from the accompanying background of continuous radiation and Kβ radiation. Since the radiation emitted from an X-ray source is actually a mixture of X-rays of various wavelengths, in order to obtain substantially monochromatic X-rays it is necessary to eliminate unwanted wavelengths as much as possible. This can be achieved by diffraction of the mixed radiation by means of a crystal and subsequent isolation of the desired characteristic radiation on the basis of its orientation. Another method utilizes the fact that the intensity of the characteristic X-rays is very high. When the beam is passed through a substance that specifically absorbs the unwanted X-rays, the relative intensity of the characteristic radiation is increased because of absorption of the radiation at all other wavelengths.

9.1.2

Classical X-ray setup

The main pieces of hardware needed for the collection of X-ray diffraction data are an X-ray source and an X-ray detector. In principle, every scattering pattern can be recorded using the classical X-ray diffraction setup. Using the ideal instrument, we could vary the scattering angle 2θ to record a scattering curve. In the detector, the scattering intensity is measured in units of counts per second.

9.1.3

X-ray sources

The X-rays are usually generated using two different methods or sources. The first is a device called an X-ray tube, in which electromagnetic waves are generated from the impacts of high-energy electrons with a metal target.2 The metal target must be continuously cooled because nearly all the kinetic energy of the accelerated electrons is converted into heat when they decelerate rapidly during the impacts. X-ray tubes are the simplest and the most commonly used sources of X-rays that are available in a laboratory of any size, and thus, are known as laboratory or conventional X-ray sources. Conventional X-ray sources usually have a low efficiency, approximately 1% or less, and their brightness is fundamentally limited by the thermal properties of the target material. X-ray tubes produce two kinds of beam, the point-focused and the line-focused beams, which are nearly identical, but have different brightnesses – the point focus is brighter than the linear one – and therefore different applications. For example, in powder diffraction, the use of the linear focus is justified by the need to maintain as many particles in the irradiated volume of the specimen as possible. The line of focus is typically 0.1–0.2 mm wide and 8–12 mm long. In single-crystal diffraction, point focus is employed, because the typical size of a specimen is as small as 0.1–1 mm.

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The high brightness of a point-focused beam enables one to achieve a high scattered intensity in a single-crystal diffraction experiment. The second method is a much more advanced source of X-ray radiation, called the synchrotron. In a synchrotron, high energy electrons are confined in a storage ring. When they move in a circular orbit, the electrons accelerate toward the center of the ring, thus emitting electromagnetic radiation. The synchrotron sources are extremely bright since thermal losses are minimized and there is no target to cool. Their brightness is only limited by the flux of electrons in the high-energy beam. Obviously, given the cost of both the construction and the maintenance of a synchrotron source, owning one would be prohibitively expensive and inefficient for a laboratory. All synchrotron sources are multiple-user facilities, which are constructed and maintained using governmental support. In general, there is no difference in principle in the diffraction phenomena produced using conventional X-ray tubes and synchrotrons. 9.1.3.1

X-ray tubes

There are two kinds of X-ray tube. First is the sealed X-ray tube. The sealed tube consists of a stationary anode coupled with a cathode, with both placed inside a metal/glass or a metal/ceramic container sealed under high vacuum. The usual power is several or tens of kilowatts. It is limited by the necessity of keeping the temperature in the anode below the melting point. The X-rays exit the tube through beryllium windows. A standard sealed tube has four Be windows located 90° apart around the circumference of the cylindrical body. One pair of opposite windows corresponds to a point-focused beam, while the second pair results in a line-focused beam. In a sealed X-ray tube, electrons are emitted by the cathode. The tungsten filament is electrically heated by a voltage of 30–60 kV.3 Because the tube is under vacuum and the cathode is at a high negative potential with respect to the metal anode, the electrons are accelerated and reach the anode at high speed. Generally, the anode is a copper plate on to which the electron beam is focused to a focal spot, normally 0.4 × 8 mm. Most of the electron energy is converted to heat, which is removed by cooling the anode, usually with water. However, a small part of the energy is emitted as X-rays in two different ways: as a smooth function of the wavelength and as sharp peak at specific wavelengths. The sharp peaks in the spectrum are due to electron transition between inner orbitals in the atoms of the anode material. The high-energy electrons reaching the anode shoot electrons out of low-lying orbitals in the anode atoms. Electrons from higher orbitals occupy the empty positions and the energy released in this process is emitted as X-ray radiation of a specific wavelength: Kα radiation if it comes from a transition from the L shell to the K shell and Kβ for a transition from the M shell to the K shell. Because of the fine structure in the L shell, Kα is split into Kα(1) and Kα(2). When copper is the anode material, the values for λ are λ(Kα(1)) = 1.54 051 Å, λ(Kα(2)) = 1.54 433 Å and λ(Kβ) = 1.39 217 Å. For emission of the characteristic lines in the spectrum, a minimum excitation voltage is required. For example, for the emission of the CuKα line, V should be at least 8 kV. If a higher voltage is applied, the intensity of the line is stronger with respect to the continuous radiation, up to about V/Vmin = 4. The intensity of the line is also

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proportional to the tube current, at least as long as the anode is not overloaded. A normal setting is V = 40 kV with a tube current of 37 mA for a 1.5 kW tube. The second kind of X-ray tube is the rotating-anode X-ray tube, which was introduced around 1960. The rotating anode enables significant improvement in the dissipation of heat. In a standard sealed X-ray tube, the heating of the anode caused by the electron beam at the focal spot limits the maximum power. Too much power would ruin the anode. This limit can be moved to a higher power loading if the anode is a rotating cylinder instead of a fixed piece of metal. In a rotating-anode X-ray source, a massive disk-shaped anode is continuously rotated at a high speed while being cooled by a stream of chilled water. By rotating the anode of the X-ray tube, the power of the incident electron beam is spread on a circular ring. Thus it is possible to increase the power of the tube to ∼15–18 kW and in some reported instances to 50–60 kW – which is up to 20 times greater when compared to a standard sealed X-ray tube – without “burning” the anode material. High-power rotating anodes are less robust than the medium-power ones. The resultant brightness of the X-ray beam increases proportionally to the input power. With a rotating-anode tube, small source widths (0.1– 0.2 mm) with a high brilliance are possible. A disadvantage is that it requires continuous pumping to keep the vacuum at the required level. 9.1.3.2

Synchrotrons

Synchrotrons are devices for circulating electrically charged particles at nearly the speed of light. The particles are injected into the storage ring directly from a linear accelerator or through a booster synchrotron. Originally these machines were designed for use in high-energy physics as particle colliders. When the particle beam changes direction, the electrons or positrons are accelerated toward the center of the ring and therefore emit electromagnetic radiation and, consequently, lose energy. This energy loss is compensated for by a radio-frequency input at each cycle. Synchrotron radiation was first directly observed in 1947 at a General Electric laboratory in the USA. The main aim of the physicists was to study colliding particles. Synchrotrons are extremely large and expensive facilities: the ring has a diameter of ten to a few hundred meters. Synchrotron radiation covers a very wide spectral range, from the infrared (IR) to hard X-rays. Since there is no target to cool, the brilliance of the X-ray beam that can be achieved in synchrotrons is 4–12 orders of magnitude higher than that from a conventional X-ray source. Tremendous energies are stored in synchrotron rings, where beams of accelerated electrons or positions move in a circular orbit, controlled by a magnetic field, at relativistic velocities. The electrons accelerated up to several GeV have a continuous spectrum, and are now used as a powerful X-ray source. This is because synchrotron radiation has excellent properties such as high intensity, very broad continuous spectral range, narrow angular collimation, small focal size, high degree of linear/circular polarization, regularly pulsed time structure, ultra-high vacuum environment and computability of properties. In addition to the extremely high brilliance of the X-ray beam, another important advantage of synchrotron radiation sources is in the distribution of the beam intensity as a function of wavelength. The high intensity, observed in a broad range of photon energies, allows for easy selection of nearly any desired wavelength.

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Nanotechnology Research Methods for Foods and Bioproducts

X-ray detectors

The X-ray detector is an integral part of any diffraction analysis system, and its major role is to measure the intensity and, sometimes, the direction of the scattered beam. The detection is based on the ability of X-rays to interact with matter and to produce certain effects or signals, for example to generate particles, waves and electrical current, which can be easily registered. In other words, each photon entering the detector generates a specific event, or better yet a series of events, that can be recognized, and from which the total photon count (intensity) can be determined. Obviously, the detector must be sensitive to X-rays, and should have an extended dynamic range and low background noise. Efficiency, linearity and proportionality are the important characteristics of X-ray detectors. The detector efficiency is how quick the detector collects X-ray photons and then converts them into a measurable signal. Detector efficiency is determined by two parameters, a fraction of the X-ray photons that pass through the detector window and a fraction of the photons that are absorbed by the detector and thus result in a series of detectable events. The product of the two fractions, which is known as the absorption or quantum efficiency, should usually be between 0.5 and 1. The linearity of the detector is critical in obtaining correct intensity measurements (photon count). The detector is considered linear when there is a linear dependence between the photon flux and the rate of signals generated by the detector per second. The proportionality of the detector determines how the size of the generated voltage pulse is related to the energy of the X-ray photon. Usually, a high detector proportionality enables one to achieve additional monochromatization of the X-ray beam in a straightforward fashion: during the registration, the signals that are too high or too low and thus correspond to photons with exceedingly high or exceedingly low energies, respectively, are simply not counted. Historically, the photographic film was the first and the oldest detector of X-rays, having been in use for many decades. Just like visible light, X-ray photons photosensitize fine particles of silver halide when the film is exposed to X-rays. Photographic film is a classical detector for X-ray radiation, but it is not used much at present because of the availability of far more sensitive image plates and area detectors. The single advantage of film over other present-day area detectors is its superior resolution, resulting from its fine grain. Image plates are used in the same manner as X-ray film but have several advantages. Image plates are made by depositing a thin layer of an inorganic storage phosphor on a flat base. X-ray photons excite electrons in the material to higher energy levels. Part of this energy is emitted very soon as normal fluorescent light in the visible-wavelength region. However, an appreciable amount of energy is retained in the material by electrons trapped in color centers. It is dissipated only slowly over a period of several days. This stored energy is released on illumination with light. In practical applications, a red laser is used to scan the plate and blue light is emitted. The red light is filtered away and the blue light is measured with a photomultiplier. With certain precautions, the light emitted is proportional to the number of photons to which each particular position of the plate was exposed. Image plates are at least 10 times more sensitive than X-ray film and their dynamic range is much wider.

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Therefore, the entire range from strong to weak reflections can be collected with one exposure on a single plate. Although photographic film and image plates are “area detectors”, the use of this term is restricted to electronic devices that detect X-ray photons on a two-dimensional surface and process the signal immediately after photon detection. They are also called position-sensitive detectors, because both the intensity of a diffracted beam and the position at which it hits the detector are determined. A basic difference with image plates is that area detectors scan through a diffraction spot every 0.1° or so, giving a three-dimensional picture of the spot.

9.1.5

Wide-angle X-ray scattering and small-angle X-ray scattering

Wide-angle X-ray scattering (WAXS) and small-angle X-ray scattering (SAXS) belong to a family of X-ray scattering techniques that are used in the characterization of materials. The scattered intensity, I(q), is a function of the magnitude q of the scattering vector. The magnitude q = 4πsin(θ)/λ, where θ is half of the angle between the incident X-ray beam and the detector measuring the scattered intensity, and λ is the wavelength of the X-rays. Scattering experiments are carried out in different angular regions. SAXS is recorded at very low scattering angles, 2θ, close to 0°,4 while WAXS concentrates on scattering angles, 2θ, larger than 5°. In WAXS, the distance from the sample to the detector is shorter than in SAXS. Since the larger the diffraction angle, the smaller the length scale probed, WAXS is used to determine crystal structure on the atomic length scale, while SAXS is used to explore microstructure on the colloidal length scale. WAXS is often used to determine the crystalline structure, orientation and crystallinity of polymers. For film samples, the diffraction pattern generated by WAXS can determine the phase composition and texture of the film, the crystallite size and the presence of film stress. SAXS can observe the typical nanostructures in semicrystalline materials and thermoplastic elastomers. SAXS patterns contain information about the shape, size and size distribution of macromolecules, characteristic distances of partially ordered materials, and pore sizes.

9.2

Applications

X-ray scattering is a universal tool in many branches of natural science for exploring and qualitatively and quantitatively analyzing the structure of matter, such as the crystalline structure and physical properties of materials and thin films. X-ray scattering techniques are based on observing the scattered intensity of an X-ray beam hitting a sample as a function of incident and scattered angle, polarization and wavelength or energy. When X-rays are directed in solids, they will scatter in predictable patterns based upon the internal structure of the solid. A crystalline solid consists of regularly spaced atoms that can be described by imaginary planes. The distance between these planes is called the d-spacing. Every crystalline solid will have a unique pattern of d-spacing, which is a “fingerprint” for that solid. In fact, solids with the same chemical composition but different phases can be identified by their patterns of d-spacing.

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SAXS is used to determine the micro- or nanoscale structure of ordered systems. The materials can be solid or liquid and can contain solid, liquid or gaseous domains of the same or different materials in any combination. The method is accurate, nondestructive and usually requires only a minimum of sample preparation. Applications are very broad and include colloids of all types, metals, cement, oil, plastics, proteins, foods and pharmaceuticals, and can be found in research as well as in quality control. In the case of biological macromolecules, such as proteins, the advantage of SAXS over other methods like crystallography is that no additional sample preparation is needed. The particle shape analysis is especially popular in biological SAXS, where the shapes of proteins and other natural colloidal polymers are determined. The intensity, position, and width of Bragg reflections from crystalline organic or inorganic components in biological materials contain useful information about local crystallographic and nanostructural features. Moreover, crystalline entities in biological tissues are seldom randomly oriented, nor are they perfectly single-crystalline. Using WAXS, the orientation and orientation distribution of crystalline or semicrystalline entities in tissues can be obtained and mapped as a function of position by employing quantitative texture analysis.

9.2.1

Example: X-ray characterization of zein–fatty acid films

Biodegradable plastics have been of high interest to researchers because of increasing concerns about pollution and the effect synthetic plastics have on the environment. There are several choices for the base of biopolymers, such as starch and proteins. Zein, the corn protein, is one such choice. It is alcohol-soluble and naturally occurs as a globular protein internally linked by disulfide bonds. Zein is capable of thin-film formation, and the zein film is tough, glossy, scuffproof and greaseproof.5 A plasticizer is added to increase film flexibility, since zein alone makes the film brittle.6 Three fatty acids, oleic acid, stearic acid and linoleic acid, have been used as plasticizers.7 The effects of different plasticizers on the molecular spacing were examined, and the effects of zein dissolving times, heating times and heating temperatures on denaturing and unfolding of the protein were investigated. Zein was dissolved in 75% aqueous ethanol for 40 minutes, 4 hours or 20 hours at a ratio of 20 g zein/100 ml ethanol with a stirring speed of 1200 rpm for mixing. Oleic acid at 10 or 18 g/20 g zein, stearic acid at 10 g/20 g zein and linoleic acid at 10 g/20 g zein were added as plasticizers. The solution was stirred at 60 or 80°C for 10 or 30 minutes with a mixing speed of 1200 rpm. It was then poured into ice water, where it precipitated into a soft solid. The soft solid was kneaded by hand for 30 minutes to form a dough/resin, from which 2D biaxially stretched films were drawn by hand. Stretched films were allowed to dry under ambient conditions at 23°C. The composition of plasticizer has been described as the amount of fatty acid compared to the amount of zein. Therefore, films labeled as having 50 and 90% fatty acid content actually contain 33 and 50% fatty acid by mass. To make things simple, the sample solutions were labeled by the plasticizer concentration, mixing time under stirring, heating temperature and heating time under

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Fig. 9.1 WAXS patterns of a biaxially stretched oleic acid zein film (50%, 40 min, 60°C, 10 min) with X-ray beam direction (a) normal and (b) parallel to the film surface.

stirring. For example, “oleic acid zein film (50%, 40 min, 60°C, 10 min)” means the sample solution from which the resin was made and stretched into film was prepared by dissolving 20 g zein and 10 g oleic acid in 100 ml 75% aqueous ethanol with a mixing speed of 1200 rpm and heating at 60°C for 10 minutes with a stirring speed of 1200 rpm. WAXS data were collected with a Bruker General Area Detector Diffraction System (GADDS, Bruker AXS, Inc., Madison, WI). The X-ray wavelength was Kα(1) = 1.54 056 Å. The system was equipped with a four-circle diffractometer and HiStar multiwire area detector. The distance between the sample and the detector was kept at 8.61 cm. The system was operated at a voltage of 40 kV and a current of 60 mA. SAXS data were also collected by the same setup, but with the HiStar area detector 103.70 cm away from the sample. The measured zein films were placed horizontally, and the normal direction to the film was the vertical direction. WAXS diffraction patterns from an oleic acid zein film sample (50%, 40 min, 60°C, 10 min) are depicted in Figs 9.1a and b with X-ray beam directions normal and parallel to the film surface, respectively. Two diffuse rings at approximately 4.6 and 9.9 Å are shown in the WAXS patterns. The 4.6 Å spacing is believed to represent the backbone spacing along the α-helix axis, and the 9.9 Å spacing is thought to represent the lateral α-helix spacing. No orientation was observed through the film thickness or parallel to the film surface. SAXS diffraction patterns from the same sample are depicted in Figs 9.2a and b, with X-ray beam directions normal and parallel to the film surface, respectively. The rings in Figs 9.2a and b indicate a periodicity of ∼130 Å (2θ = 0.68°) in the film. An intense vertical streak is observed in Fig. 9.2b. This streak was previously attributed to voids elongated parallel to the surface.6 No orientation is observed through the film thickness or parallel to the film surface. In Fig. 9.3, azimuthally integrated SAXS intensity plots of three 50% oleic acid film samples are shown to demonstrate the effect of increasing zein dissolving

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(a)

(b)

130 Å

130 Å Vertical streak

Fig. 9.2 SAXS patterns of biaxially stretched oleic acid zein film (50%, 40 min, 60°C, 10 min) with X-ray beam direction (a) normal and (b) parallel to the film surface. 600 a b c

500

c

Intensity

400 b a 300 200 100 0 0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

2θ Fig. 9.3 SAXS intensity plots of 50% oleic acid biaxially stretched films with different dissolving times: (a) (50%, 40 min, 60°C, 10 min); (b) (50%, 4 hr, 60°C, 10 min); (c) (50%, 20 hr, 60°C, 10 min).

time: (a) (50%, 40 min, 60°C, 10 min); (b) (50%, 4 hr, 60°C, 10 min); (c) (50%, 20 hr, 60°C, 10 min). As shown, there is no significant difference in molecular spacing between samples (a) and (b). Sample (c), which was dissolved for 20 hours, shifted to a larger molecular spacing. Upon comparing the results for increasing dissolving time of 50% oleic acid zein films, it can be concluded that increasing zein dissolving time in an ethanol solution causes a shift towards larger molecular spacing. Differences between sample intensities can be attributed to variations in sample thickness; thicker film samples will produce higher-intensity diffraction patterns up to an optimum thickness.

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Fig. 9.4 SAXS patterns of biaxially stretched 90% oleic acid film samples (90%, 40 min, 60°C, 10 min) with the X-ray beam parallel to the edge, (a) without and (b) with dipping in oleic acid.

As previously mentioned, an intense vertical streak is observed in Fig. 9.2b. Figs 9.4a and b depict biaxially stretched 90% oleic acid film samples (90%, 40 min, 60°C, 10 min) with the X-ray beam parallel to the edge, without and with dipping in oleic acid, respectively. Film-edge samples were dipped in oleic acid to determine whether this streak should be attributed to voids elongated parallel to the surface. As Fig. 9.4b shows, the vertical streak disappears upon dipping stacked film-edge samples in oleic acid. Thus it is suggested that the vertical streak should be attributed to voids between stacked layers in film-edge samples. When the film-edge samples were dipped in oleic acid, the fatty acid filled the air space between film layers, causing the vertical streak to disappear. Stearic acid was used as a plasticizer for zein films to determine the feasibility of using other long-chain fatty acids. Stearic acid contains no double bonds in its hydrocarbon chain. Stiffer and more brittle films were the result of using stearic acid as a plasticizer due to the lack of double bonds. WAXS diffraction patterns from a biaxially stretched stearic acid zein film sample (50%, 40 min, 80°C, 10 min) are depicted in Figs 9.5a and b with X-ray beam directions normal and parallel to the film surface, respectively. In Fig. 9.5a, the characteristic diffuse rings at approximately 4.6 and 9.9 Å are present. The additional sharp rings, at approximately 4.35, 4.15 and 3.70 Å, are due to stearic acid crystals. There is no observed orientation through the film thickness, as would be expected for a biaxially oriented film. In Fig. 9.5b, the arcs at 20.30, 13.60, 10.12, 8.19 and 5.78 Å are due to the layers of the stearic acid being oriented parallel to the film surface. The arcs at 4.35, 4.15 and 3.70 Å are intermolecular spacings. The arcs at 4.15 and 3.70 Å are split due to the tilt of the stearic acid molecules in the layers. The stearic acid crystal is in the B form, with the 4.15 and 3.70 Å split arcs being due to the 110 and 020 planes, respectively, while the 4.35 Å arc on the equator is 112.

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Fig. 9.5 WAXS patterns of biaxially stretched stearic acid zein film sample (50%, 40 min, 80°C, 10 min) with X-ray beam directions (a) normal and (b) parallel to the film surface.

Fig. 9.6 SAXS patterns of biaxially stretched stearic acid zein film sample (50%, 40 min, 80°C, 10 min) with X-ray beam directions (a) normal and (b) parallel to the film surface.

SAXS diffraction patterns from the same film sample are depicted in Figs 9.6a and b with X-ray beam directions normal and parallel to the film surface, respectively. Figure 9.6b shows a SAXS spacing, in the form of an arc, at 20.30 Å for the stearic acid film with the X-ray beam parallel to the film surface. Again, it is believed that this result demonstrates layers of stearic acid with orientation parallel to the surface of the film. Linoleic acid was also used to determine the feasibility of using other long-chain fatty acids as plasticizers for zein films. Linoleic acid has a melting point of −5°C and contains two double bonds in its hydrocarbon chain. A WAXS diffraction pattern from a biaxially stretched linoleic acid zein film sample (50%, 40 min, 60°C, 10 min) is depicted in Fig. 9.7, with the X-ray beam normal to the film surface. The characteristic diffuse rings at approximately 4.6 and 9.9 Å are

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Fig. 9.7 WAXS pattern of a biaxially stretched linoleic acid zein film sample (50%, 40 min, 60°C, 10 min) with X-ray beam normal to the film surface.

Fig. 9.8 SAXS patterns of a biaxially stretched linoleic acid zein film sample (50%, 40 min, 60°C, 10 min) with X-ray beam directions (a) normal and (b) parallel to the film surface.

present, and no orientation is apparent. SAXS diffraction patterns from the same film sample are depicted in Figs 9.8a and b with X-ray beam directions normal and parallel to the film surface, respectively. Both Figs 9.8a and b show that there is no SAXS spacing in the linoleic film sample.

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Fig. 9.9 WAXS patterns of biaxially stretched oleic acid zein film sample (50%, 40 min, 60°C, 10 min) measured at 9°C with X-ray beam directions (a) normal, (b) normal and angled and (c) parallel to the film surface.

9.2.2

Temperature-controlled WAXS

All of the samples that were examined using temperature-controlled WAXS had the detector set at 18.05 cm. The detector was moved further away from the temperaturecontrolled air stream to prevent the risk of damaging it. Because the detector was further away from the sample, the entire diffraction pattern could not be seen at one time. In order to view the 4.6 Å ring, the detector needed to be angled at 2θ = −15°. WAXS diffraction patterns from a biaxially stretched oleic acid zein film sample (50%, 40 min, 60°C, 10 min) measured at 9°C are depicted in Fig. 9.9a–c with X-ray beam directions normal, normal and angled, and parallel to the film surface, respectively. The 50% oleic acid film sample was held at 9°C, the minimum achievable, in order to try to crystallize the oleic acid in the film. The purpose of crystallizing the

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Fig. 9.10 WAXS patterns of biaxially stretched oleic acid zein film sample (90%, 40 min, 60°C, 10 min) measured at 9°C with X-ray beam directions (a) normal and angled, (b) parallel and (c) parallel and angled to the film surface.

oleic acid was to determine if it had become oriented similarly to the stearic acid crystals. The characteristic diffuse rings at approximately 4.6 and 9.9 Å are present, and no orientation is apparent normal or parallel to the surface of the film. WAXS diffraction patterns at 9°C from a biaxially stretched oleic acid zein film sample (90%, 40 min, 60°C, 10 min) are depicted in Fig. 9.10a–c with X-ray beam directions normal and angled, parallel, and parallel and angled to the film surface, respectively. The characteristic diffuse rings at approximately 4.6 and 9.9 Å are present. In Fig. 9.10a, two sharp rings at 4.97 and 4.45 Å are apparent in the diffuse 4.6 Å ring. This indicates that the oleic acid in the film has crystallized. In Fig. 9.10b, three arcs at 22.3, 14.02 and 8.42 Å surround the diffuse ring at 9.9 Å. These spacings correspond to 002, 003 and 005 of oleic acid. This means that oleic acid bilayers are oriented parallel to the surface of the film. In Fig. 9.10c, two arcs at 4.97 and 4.45 Å are again apparent in the diffuse 4.6 Å ring. These spacings are the same as the sharp rings seen when the X-ray beam was normal to the film surface. These arcs also

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Fig. 9.11 WAXS patterns of biaxially stretched stearic acid zein film sample (50%, 40 min, 60°C, 10 min) measured at 73°C with X-ray beam directions (a) normal, (b) normal and angled, (c) parallel and (d) parallel and angled to the film surface.

indicate that oleic acid molecules are oriented with their backbones normal to the film surface. WAXS diffraction patterns at 73°C from a biaxially stretched stearic acid zein film sample (50%, 40 min, 60°C, 10 min) are depicted in Fig. 9.11a–d with X-ray beam directions normal, normal and angled, parallel, and parallel and angled to the film surface, respectively. The purpose of melting the stearic acid was to confirm the lack of orientation of the zein and determine if the stearic acid remained oriented. The characteristic diffuse zein rings at approximately 4.6 and 9.9 Å are present. In Figs 9.11a and b, the additional rings at approximately 4.35, 4.15 and 3.70 Å are similar to those in the previous stearic acid film samples (Fig. 9.5) and are believed to be due to stearic acid crystals. There is no observed orientation of either the stearic acid or the zein with the beam through the film thickness. If the zein were oriented, an arc on

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the vertical axis of the 9.9 Å and another on the horizontal axis of the 4.6 Å ring would be present. Figs 9.11c and d show the same diffuse rings, but orientation is apparent parallel to the film-thickness direction. In Fig. 9.11c, the additional arcs at 20.28, 13.61, 10.75 and 8.17 Å correspond to layers of stearic acid oriented parallel to the film surface. In Fig. 9.11d, the arcs at 4.35, 4.15 and 3.70 Å, as at room temperature, are due to intermolecular spacings. The arcs at 4.15 and 3.70 Å are split due to the tilt of the stearic acid molecules in the layers. The stearic acid crystal is in the B form, with the 4.15 and 3.70 Å split arcs being the 110 and 020 indices, respectively, and the 4.35 Å arc on the equator being 112.

References 1. Novelline, R.A. (1997) Squire’s Fundamentals of Radiology, Harvard: Harvard University Press. 2. Whaites, E. and Cawson, R. (2002) Essentials of Dental Radiography and Radiology, 3 edn, Elsevier Health Sciences. 3. Bushburg, J., Seibert, A., Leidholdt, E. and Boone, J. (2002) The Essential Physics of Medical Imaging, Lippincott Williams & Wilkins. 4. Glatter, O. and Kratky, O. (1982) Small Angle X-ray Scattering, Academic Press. 5. Reiners, R.A., Wall, J.S. and Inglett, G.E. (1973) Corn proteins: potential for their industrial use. In: Pomeranz, Y. (ed.) Industrial Uses of Cereals, St Paul, MN: American Association of Cereal Chemistry, pp. 286–302. 6. Lai, H.M., Geil, P.H. and Padua, G.W. (1999) X-ray diffraction characterization of the structure of zein-oleic acid films. J Appl Polym Sci 71, 1267–1281. 7. Santosa, F.X.B. and Padua, G.W. (1999) Tensile properties and water absorption of zein sheets plasticized with oleic and linoleic acids. J Agric Food Chem 47, 2070–2074.

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10

Quartz crystal microbalance with dissipation

Boce Zhang and Qin Wang

Abstract: Quartz crystal microbalance with dissipation (QCM-D) is a novel technology for the analysis of surface phenomena, which provides a real-time and label-free method of studying macromolecule adsorption and/or interaction on various surfaces with high sensitivity (1 ng/cm2). In recent years, the development of QCM-D instruments and mathematical modeling techniques has enabled a dramatic boost in QCM-D’s novel applications in biomaterial research. In this chapter, we first explain the instrumentation and theory behind the QCM-D platform. Then some well-studied areas of application are introduced, including real-time monitoring adsorption and desorption kinetics; thickness, hydration and structural changes of attached biopolymers layers; mechanisms and kinetic studies of specific immunoassays; studies of the affinity of nanoscaled materials to biomolecules; and so on. Plenty of studies have also reported the design of a QCM-D sensor for immunosensing, with high specificity and sensitivity for food, agricultural and pharmaceutical applications. Keywords: quartz crystal microbalance with dissipation; QCM-D; biomolecule; interaction; adsorption kinetic; immunosensor

10.1

Background and principles

QCM-D stands for “quartz crystal microbalance with dissipation monitoring”, which is a novel technology for surface analysis. QCM-D provides a real-time and labelfree method for studying molecular adsorption and/or interaction on various surfaces. The traditional QCM-D technique enables a precise measurement of adsorbed mass with high sensitivity (0.1 μg/cm2) by capturing a tiny change in quartz crystal oscillation frequency, and is mostly carried out on a rigid surface in either air or vacuum. It has been used for over 50 years. A quartz crystal microbalance (QCM) relies on a piezoelectric quartz crystal, which oscillates under a voltage at a specific frequency. When molecules are adsorbed on a quartz crystal surface, the slight mass Nanotechnology Research Methods for Foods and Bioproducts, First Edition. Edited by Graciela W. Padua and Qin Wang. © 2012 John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc.

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increase causes a proportional increase in oscillation frequency through the Sauerbrey relationship: 1 Δm = −C Δf n

(10.1)

where Δm is mass change, Δf is oscillation frequency change, C is a constant that equals 17.7 ng/(s1cm2) for a 5 MHz quartz crystal and n represents overtone, which has to be an odd number (i.e. 1, 3, 5, 7, 11, 13). By estimating the adhering layer’s effective density (ρeff), it is also possible to calculate the layer’s effective thickness (deff): d eff =

Δm ρeff

(10.2)

However, because the Sauerbrey relationship can only apply to a rigid surface, traditional QCM is limited to measuring small and rigid molecules (e.g. metal oxide) only. Novel QCM-D technology measures both frequency and dissipation change of the quartz crystal. The dissipation parameter (D) is mathematically related to the structure and viscoelastic properties of the adsorbed layer. The sensitivity of QCM-D is 1 ng/ cm2, which is approximately 100 times higher than traditional QCM. By collecting dissipation, it is capable of characterizing a viscoelastic (soft) surface of a quartz crystal in a liquid environment, such as a protein adhering layer in aqueous solution. D is defined as: D=

E lost 2 πE stored

(10.3)

where Elost represents the energy lost in one oscillation circle and Estored is the total energy stored in the quartz crystal. Figure 10.1 illustrates a similar phenomenon to the Sauerbrey relationship, where the oscillating frequency decreases with adherence to either a rigid or a soft layer. As shown in the figure, the soft film usually has more interaction with a liquid environment, and hence generates more resistant force, which results in a faster

Crystal oscillation

Clean surface

Energy does not dissipate – ΔD = 0

Crystal oscillation

Rigid film

Energy dissipates slowly – ΔD > 0, small

Crystal oscillation

Soft film

Energy dissipates rapidly – ΔD > 0, large

Fig. 10.1 Energy dissipation changes when adhering to rigid or soft materials. To see a color version of this figure, see Plate 10.1.

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energy dissipation than that of the rigid film. This process circle for obtaining ΔD is repeated 200 times in 1 second, which enables QCM-D’s high sensitivity and broad application. By collecting Δf and ΔD of multiple overtones, it can be fitted into a viscoelastic modeling system to generate information on changes in the mass and viscoelastic property of the interfacial surface. There are many advantages to measuring ΔD. (i) The Sauerbrey relationship shows the linear relationship between Δm and Δf for rigid molecules, but does not work for viscoelastic or softer adhering layers. Thus, by measuring ΔD, it is possible to verify the validity of the Sauerbery relationship. When ΔD is less than 10−6, it is typically considered as a rigid surface. (ii) If ΔD is larger than 10−6 – it is normally 3 × 10−6 to 1.1 × 10−5 for most biopolymers – both Δf and ΔD are required to fit in a Voigt model to obtain structural and mass change information. (iii) In terms of structural information, different viscoelastic molecules have different extensibilities toward the liquid environment. The better the interaction between adhering and liquid molecule, the larger the increase in ΔD. Thus, ΔD can be used to monitor swelling or hydration of the adhering molecules. (iv) Besides providing information on mass, the Voigt modeling process can also quantitatively analyze the thickness, shear elastic modulus and viscosity of the adsorbed film.

10.2

Instrumentation and data analysis

10.2.1

Sensors

The most important part of a QCM-D instrument is the piezoelectric quartz crystal or sensor. The supplier of the QCM-D instrument provides a wide range of crystal surfaces with diameters of 1.4 cm. A typical quartz crystal is shown in Fig. 10.2. The coating layer may include a variety of materials, from spin-coated polymers such as as polystyrene, polycarbonate (PC), poly(methyl methacrylate) (PMMA), polyethylene (PE) and polyvinyl chloride (PVC) to metal and metal oxides such as Au, SiO2, Al2O3, Ti, Ag, Cu, Cr and Si3N4. The thickness of the coating layer is usually 100–300 nm and can be reduced to several nanometers. This provides a pool of choices when studying the interaction or affinity between a sample and a certain surface. It should also be noted that the crystal surfaces are smoothly coated and after

Bottom view Top view Coating layer Quartz crystal

Side view Coating layer Gold Quartz Gold

diam. 14 mm

Contact electrodes Contact electrodes

5MHz Fig. 10.2

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QCM-D sensor overview. To see a color version of this figure, see Plate 10.2.

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PNA-DNA hybridization DNA-DNA hybridization DNA –6

0.4 × 10 ΔD

PNA

DNA

DNA

0

–5

–10

–15

–20

Δf (Hz) Fig. 10.3 Comparison of two antibodies on an antigen-covered sensor.1 To see a color version of this figure, see Plate 10.3. Reprinted from Specificity of DNA Hybridisation on a Functionalised Lipid Bilayer (Q-Sense application note), with permission from Q-sense. Available on www.q-sense.com.

usage the sensor is suitable for further microscopical study using scanning electron microscopy (SEM) or atomic force microscopy (AFM). Cleanness of the surfaces is also critical to QCM-D measurements. In most common cases, a contaminated surface (by protein, for example) alters the affinity of the sample molecules to gold or a coating layer, which can lead to a false estimation of the mass, thickness and viscoelastic properties of the adhering sample layer. To ensure the sensor surface, especially of a reused sensor, is clean, it is necessary to follow the cleaning and immobilization protocols supplied by the manufacturer. Different coating layers require different cleaning methods, but most adopt a combination of UV/ozone and pirahna solution treatment. To verify the cleanness of a sensor surface, f and D values in pure water are obtained before experiments begin. Sensors are considered acceptable when the D value is within 20% of the typical value of a new sensor.

10.2.2

Data analysis

Data analysis methods include both qualitative and quantitative analyses. Qualitative analysis provides a brief understanding of sample molecular behaviors by generating several raw data plots. Δf and ΔD raw data plots illustrate how f and D values change over time, which reflects the procedure of multistep treatment. ΔD versus Δf is a time-independent raw data plot that indicates a single reaction’s “fingerprint” in a multistep experiment1 (see Fig. 10.3). The slope of ΔD versus Δf is considered an indicator of affinity: the larger the slope, the lower the affinity. Quantitative analysis is usually carried out by fitting the raw data (Δf and ΔD) to different modeling systems so that quantitative mass changes, thickness changes and the visoelastic properties of adsorbed layers can be obtained from them. Two different models are applied in quantitative analysis: the Sauerbrey model and the Voigt model.

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The Sauerbrey relationship relies on three assumptions: (i) added mass is small compared to the mass of the crystal; (ii) mass is rigidly adsorbed, with no slip or deformation imposed by the oscillating surface; and (iii) added mass is evenly distributed. In order to comply with these assumptions, it is necessary to keep ΔD below the aforementioned cutoff value of 10−6. Otherwise, the Sauerbrey model underestimates Δm. The Voigt model is therefore more accurate in converting Δf to Δm for soft materials or polymers. The Voigt model requires at least two sets of Δf and ΔD input from different overtones, and will output the density (ρ), thickness (δ), viscosity (η or G″/ω) and elasticity (μ or G′) of the adhering layer.2

10.3

Applications

QCM-D has a broad range of applications in qualitatively and quantitatively studying interactions among proteins, lipids, polymers, cells/bacteria and other biomolecules. QCM-D is now routinely used to characterize the interaction between biopolymers and surfaces in many aspects of life sciences. First, QCM-D is capable of real-time monitoring of adsorption and desorption kinetics.3 Osaki et al.3 investigated the major driving forces for fibronectin protein adsorption on copolymer film and found they were hydrophobic attraction and electrostatic interaction. QCM-D has also been applied to real-time monitoring of the formation of polyelectrolyte multilayers in situ,4 as mentioned in Chapter 3 (see Fig. 10.4). The study successfully built up a multilayer on an SiO2 surface, which was generated by alternately introducing anionic hyaluronan (A) and cationic chitosan (B) on to the surface. The polymer solution was added in the order A + B + A + B + A, for 5 minutes each. At each step, the adhering layer was rinsed by flowing water so that a nonspecific bound (non-electrostatic attraction) polymer would be washed off the attached layer. From Fig. 10.4, the researchers found out that the dissipation values and the differences between three overtones were relatively small before rinsing with NaCl, which indicated that the adhering layer could be considered a rigid film and applied to the Sauerbrey model. As well as monitoring the multilayer formation, QCM-D also captured the swelling of the adhering film by rinsing with NaCl. After adding NaCl, the frequency of all overtones increased significantly (1st evidence). However, their increment is different among difference overtones, and thus the frequency signals were separated (2nd evidence). These two pieces of evidence led to the conclusion that there was a layer transformation from rigid to viscoelastic, and therefore Sauerbrey was no longer valid and would underestimate mass change (see Fig. 10.4b). Second, the structural changes, including thickness and hydration, of protein films can be detected by QCM-D in association with AFM.5 An advantage of using QCM-D is that the sample molecules can first absorb on an evenly coated quartz crystal surface, which can then be directly used in AFM. In an earlier study,6 QCM-D was proven to be capable of monitoring thickness and hydration changes in adhered mussel-adhesive protein (Mefp-1). In Fig. 10.5, the addition of NaIO4 triggered the release of coupled water molecules from immobilized protein layers. The increased f value after NaIO4 treatment indicated the mass decrease of the adhered protein layer through loss of the water molecules. The D value decreased to almost zero. The

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(a)

8,5 8 7,5 7 6,5 6 5,5 5 4,5 4 3,5 3 2,5 2 1,5 1 0,5 0

0

Frequency (Hz)

–5 –10

A B

B

–15

B

A

–20

A

–25

A B f-1 f-3 f-5 D-1 D-3 D-5

–30 –35 –40

A

Dissipation (1E-6)

186

0 10 20 30 40 50 60 70 80 90 100 110 120 Time (min)

(b) 8,0 7,0

Sauerbrey Model

Thickness [nm]

6,0 5,0 4,0 3,0 2,0 1,0 0,0

A1st layer

A 2nd layer

A 3rd layer

A 4th layer

A 5th layer

NaCl

Water

Fig. 10.4 (a) Frequency and dissipation responses of a real-time polyelectrolyte multilayer formation. (The large buffer step is generated by different solution properties when changing from water to NaCl and back.) (b) Thickness data after each of five polymer A adsorptions, NaCl and water. To see a color version of this figure, see Plate 10.4. Reprinted from Real Time Thickness Monitoring of Polyelectrolyte Multilayer Formation in Situ (Q-Sense application note), with permission from Q-sense. Available on www.q-sense.com.

captured f and D changes were then fitted into the Kevin–Voigt viscoelastic model to generate information about mass and thickness changes. Höök6 also summarized that QCM-D technology enables the real-time study of the kinetics of protein structural changes such as cross-linking and folding/unfolding. Similar studies focused on monitoring protein cross-linking at a solid–liquid interface.7 Poly-L-lysine (PLL) and immobilized histone (lysine-rich fraction) were applied in this research, and a cross-linking of histone was observed, which was accompanied by the viscoelastic property change of the adsorbed protein layer. Third, QCM-D has also been developed as a tool of verification of specific interaction. Limson et al.8 reported using QCM-D as a substitute for surface plasmon

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Exposure to NaIO4 After rinsing Collapsed sturcture Fig. 10.5 Monitoring of thickness and hydration changes in an adhered protein layer. To see a color version of this figure, see Plate 10.5. Reprinted from Structural change of Adsorbed Protein Layer (Q-Sense application note), with permission from Q-sense. Available on www.q-sense.com.

resonance (SPR) in studying protein–protein specific interactions such as chaperone– cochaperone interactions and receptor–ligand interactions. An organizing protein was selected as a cochaperone protein, which requires a chaperone protein (a heatshock protein in this case) to be biologically functional. The QCM-D technique was then successfully applied to both qualitative and quantitative analysis of this specific interaction.8 In other studies, either the receptor or the ligand was immobilized on the crystal in advance, and then the other part of the interaction pair was dissolved in running solution, so that the frequency and dissipation changes could be captured by QCM-D.9,10 A recent study investigated “DNA hybridization on a functionalized lipid bilayer”.1 The study used surface-immobilized synthetic peptide nucleic acid (PNA) as a probe for DNA sensitivity. Using QCM-D, the results showed that even a single mismatch between a 15-mer mixed-sequence biotin-PNA and DNA led to a clearly detectable lower and reversible adsorption, instead of the greater and irreversible binding seen in a fully complementary DNA strand (Fig. 10.6). The binding rate (slope) of the fully complementary DNA (DNAfc) was different to the mismatched DNA (DNAmm). With no influence from unspecific binding, DNAfc oligomers hybridized with the immobilized PNA at a higher sensitivity. Besides, in the case of 15-mer mismatched DNAmm, the binding was weaker and became reversible when rinsing solution was flowing over the sensor surface. Thus, it can be seen that QCM-D is highly sensitive in detecting a slightly interaction difference, which could even be generated by a 15-mer mismatch. Fourth, QCM-D can be used to study interactions between proteins and other materials, including metals, polymers and even nanoscaled materials. One study looked at the interaction of blood fibrinogen protein with noble metals, such as Ag, Au, Pd, Ti and anti-infection material: Bactiguard.11 This study provided a protocol

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DNAfc Fig. 10.6 Binding of single-strand biotin-PNA to mismatched DNA (DNAmm) and fully complementary DNA (DNAfc). To see a color version of this figure, see Plate 10.6. Reprinted from Specificity of DNA Hybridisation on a Functionalised Lipid Bilayer (Q-Sense application note), with permission from Q-sense. Available on www.q-sense.com.

for an in vitro method of investigating the activation of the immune complement in a mammalian blood system. QCM-D can also be an ideal biocompatibility evaluation method.12 A series of implant surfaces was compared for biocompatibility with human sera that contained the complement factor C3. After different implant

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Δmass (ng/cm2) 1400 IgG

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Fig. 10.7 Binding of anti-C3c to various substrate surfaces. To see a color version of this figure, see Plate 10.7. Reprinted from Protein Adsorption as Biocompatability Evaluation Method for Implant Surfaces (Q-Sense application note), with permission from Q-sense. Available on www.q-sense.com.

layers were prepared, all surfaces were incubated with the specific human sera. Heatinactivated sera were used as a negative control, where no specific sera should bind to the surface, and immunoglobulin G (IgG) was treated as a positive control, which was supposed to be the most complementary to the specific human sera. After incubation, the amount of bound antibody (anti-C3c) was evaluated using QCM-D (Fig. 10.7). Adersson et al. then concluded that the affinity or complement activation of the specific human sera to the different implant surfaces was polyurethane urea (PUUR) > polystyrene = self-assembled monolayer (SAM) > Ti. Another study13 also reported studying the interactions of synthetic polymers and nanomaterial-coated quartz crystals with protein. Because QCM-D can be used to monitor mass and thickness changes and to collect viscoelastic information of the adhered layer at solid–liquid interfaces, it has been developed as a piezoelectric immunosensor to detect various contaminants in food systems, such as veterinary drugs, pathogenic bacteria, toxins, pesticides and so on. The QCM-D immunosensor usually consists of a quartz crystal with an antigen or antibody immobilized on its surface. The sensor is developed depending on specific chemical/biological reactions or antibody–antigen interactions. The first immunosensor was designed by Shons et al.14 The surface was coated with nyebar C and bovine serum albumin (BSA) to detect BSA antibodies on the QCM platform without a dissipation function. O’Sullivan15 criticized two main drawbacks of this sensor: (i) the sensor lacked reproducibility in antibody immobilization on a crystal surface; and (ii) the viscous drag experience in the liquid phase generated a large experimental error. Today, both of these disadvantages have been overcome by commercialized QCM-D technology. As described in Section 10.2, QCM-D manufacturers routinely or specifically produce various types of crystal sensor. The dissipation function also utilized the liquid viscous drag phenomenon to provide viscoelastic information on the adhered layer. In an earlier review,15 immunosensors were grouped according to their applications. Nevertheless, adsorption of antibodies has been prevalently adopted in these studies, regardless of

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the nature of application.16–19 For instance, protein A, as an antibody, was most widely used for the detection of pesticides,20–23 bacteria,24–28 viruses29,30 and so on. Protein G, contrarily, was designed to immobilize cocaine antibodies.31 Moreover, various polymer and copolymer coatings were also developed as immunosensors, such as polyethyleneimine (PEI) activated with glutaraldehyde20,25,26,32,33 and poly(hydroxyethyl methacrylate–co-methyl methacrylate) (HEMA-MMA) specifically responsive to human immunoglobulin M (IgM) and α-fetoprotein.34 Recently, with the increasing attention on food safety, the interests of QCM-D immunosensor design have focused on pathogenic bacteria detection. For Escherichia coli, a flow-type immunosensor system was developed by Kim et al.35 The sensor was coated with a broad-spectrum anti-E. coli antibody. The sensor was then found to produce a linear sensor response when the microbial suspensions increased from 1.7 × 105 to 8.7 × 107 CFU/ml. The method was sensitive with a detection limit of 102 CFU/ml. However, this study did not overcome the problem of bacteria growth, which created a difficulty in real quantification of the analyte. Therefore, the system can only be used as a screening tool.36 In another study,37 QCM-D was designed to detect Staphylococcal enterotoxins, which are a major food-poisoning contaminant. The sensor was developed for label-free immunoassay, which has a linear range up to 60 g/ml of Staphylococcal enterotoxin B (SEB), and the detection limit was 2.5 g/ ml. From these values, it can be seen that the sensitivity is still not optimal. A QCM-D immunosensor was also targeted to Salmonella typhimurium. In the study,38 resonant frequency (F) and motional resistance (R) were calculated from f and D. F and R values were then found to be proportional to bacterial concentration in the range 105–108 CFU/ml. After sample pretreatment using anti-Salmonella magnetic beads as a collector, the detection limit could be lowered to 102 CFU/ml, making it a sensitive detection method.

10.4

Advantages

Although QCM-D has some limitations, such as harsh working environmental conditions and complicated data analysis, its merit lies primarily in its sensitivity. Compared with other commonly used optical surface study methods, such as SPR and ellipsometry (ELM), QCM-D is more informative when exploring mechanisms. QCM-D is also suitable for measuring solution viscosity with a relatively small quantity of sample. Research was conducted to compare QCM-D and SPR in the study of lipid bilayer formation on an SiO2 surface.39 This research demonstrated that QCM-D could provide more detailed quantitative information, which can be useful in the study of the build-up mechanism. From Fig. 10.8, SPR results showed a monotonic increase of lipids on the SiO2 surface, whereas QCM-D mass change indicated that the total adhering mass reached a peak and then decreased to the theoretical value. The energy dissipation results in Fig. 10.8 also showed a marked peak and then leveled off to almost zero. Keller et al. therefore concluded that “the vesicles initially adsorb intact and do not rupture and form a bilayer until a critical surface concentration is reached”,39 and the peak in QCM-D data was attributed to water trapped inside and between adsorbed vesicles. The result was proven later by AFM images.

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Fig. 10.8 QCM-D and SPR data during the formation of a lipid bilayer.39 To see a color version of this figure, see Plate 10.8. Reprinted from Lipid Bilayer Formation; A Comparison Between QCM-D, SPR and AFM (Q-Sense application note), with permission from Q-sense. Available on www.q-sense.com.

Another study conducted a comparison between QCM-D and ELM.5 The structural change of Mefp-1 was used studied when the adhering protein layer was cross-linked by adding NaIO4. Mefp-1 is attractive for this study because of its open and flexible conformation, which can easily be changed. For this specific area of study, the ELM technique can only output mass and thickness data prior to and after cross-linking. Since QCM-D can detect more tiny mass and thickness changes, it can also model other mechanical properties, such as viscosity (μ′) and elasticity (η). μ′ and η can then be converted to storage modulus (G′) and loss modulus (G″) by the following equation: (10.4) G′ = μ′ G ′′ = 2 πfη where f is the resonant frequency.40 By collecting this type of information, QCM-D has been shown to be a suitable “viscometer” for viscosity analysis of solutions, especially for high-concentration protein solutions, for which a low sample volume and nondestructive analysis are preferable.41–43

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References 1. Höök, F., Ray, A., Krave, U., Norden, B. and Kasemo, B. (2001) Characterisation of PNA and DNA immobilisation and subsequent hybridisation with DNA using acoustic-shearwave attenuation measurements. Langmuir 17, 8305–8312. 2. Liu, S. and Kim, J. (2009) Application of Kelvin-Voigt model in quantifying whey protein adsorption on polyethersulfone using QCM-D. Journal of the Association of Laboratory Automation 14, 213–220. 3. Osaki, T., Renner, L., Herklotz, M. and Werner, C. (2006) Hydrophobic and electrostatic interactions in the adsorption of fibronectin at maleic acid copolymer films. J Phys Chem B 110, 12119–12124. 4. Richert, L., Lavalle, P., Payan, E., Shu, X., Prestwich, G., Stoltz, J., Schaaf, P., Voegel, J. and Picart, P. (2004) Layer by layer buildup of polysaccharide films: physical chemistry and cellular adhesion aspects. Langmuir 20, 448–458. 5. Lubarsky, G., Davison, M. and Brandley, R. (2007) Hydration-degydration of adsorbed protein films studied by AFM and QCM-D. Biosens Bioelectron 22, 1275–1281. 6. HÖÖk, F., Kasemo, B., Nylander, T., Fant, C., Sott, K. and Elwing, H. (2001) Variations in coupled water, viscoelastic properties and film thickness of a MEFP-1 protein film during adsorption and cross-linking: a quartz crystal microbalance with dissipation monitoring, ellipsometry, and surface plasmon resonance study. Anal Chem 73, 5796–5804. 7. Duttaa, A., Nayaka, A. and Belfort, G. (2008) Viscoelastic properties of adsorbed and cross-linked polypeptide and protein layer at a solid-liquid interface. Journal of Colloid and Interface Science 324, 55–60. 8. Limson, J., Odunuga, O., Green, H., HÖÖk, F. and Blatch, G. (2004) The use of a quartz crystal microbalance with dissipation for the measurement of protetin-protein interactions: a qualitative and quantitative analysis of the interactions between molecular chaperones. South African Journal of Science 100, 678–682. 9. Saitakis, M., Tsortos, A. and Gizeli, E. (2010) Probing the interaction of a membrance receptor with a surface-attached ligand using whole cells on acoustic biosensors. Biosensor and Bioelectronics 25, 1688–1693. 10. Bailey, K., Bally, M., Leifert, W., Voros, J. and McMurchie, T. (2009) G-protein coupled receptor array technologies: site directed immobilisation of liposomes containing the H1-histamine or M2-muscarinic. Proteomics 9, 2052–2063. 11. Hulander, M., Hong, J., Andersson, M., Gerven, F., Ohrlander, M., Tengvall, P. and Elwing, H. (2009) Blood interactions with noble metals: coagulation and immune complement activation. ACS Appl Mater Interfaces 1, 1053–1062. 12. Sellborn, A., Andersson, M., Fant, C., Gretzer, C. and Elwing, H. (2003) Methods for research on immune complement activation on modified sensor surfaces. Colloids and Surfaces B: Biointerfaces 27, 295–301. 13. Chen, R., Choi, H., Bangsaruntip, S., Yenilmez, E., Tang, X., Wang, Q., Chang, Y. and Dai, H. (2004) An investigation of the mechanisms of electronic sensing of protein adsorption on carbon nanotube devices. J Am Chem Soc 126, 1563–1568. 14. Shons, A., Dorman, F. and Najarian, J. (1972) The piezoelectric quartz immunosensor. J Biomed Mater Res 6, 565–570. 15. O’Sullivan, C. and Guilbault, G. (1999) Commercial quartz crystal microbalances – theory and applications. Biosensors & Bioelectronics 14, 663–670. 16. Sakai, G., Sakai, T., Uda, T., Miura, N. and Yamazoe, N. (1995) Evaluation of binding of HAS to monoclonal and polyclonal antibody by PZ immunosensing. Sensors Actuators B 42, 89–94.

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17. Yun, K., Kobatake, E., Haruyuma, T., Laukkaned, M., Keinanen, K. and Aizawa, M. (1998) Use of a quartz crystal microbalance to monitor immunoliposome-antigen interaction. Anal Chem 70(2), 260–264. 18. Carter, R.M., Jacobs, M.B., Lubrano, G.J. and Guilbault, G.G. (1995) Piezoelectric detection of ricin and affinity purified goat anti-ricin. Anal Lett 28, 1379–1386. 19. Harteveld, J.L.N., Nieuwenhuizen, M.S. and Wils, E.R.J. (1997) Detection of staphylococcal enterotoxin B employing a PZ immunosensor. Biosensors Bioelectron 12(7), 661–667. 20. Guilbault, G.G., Hock, B. and Schmid, R. (1992) PZ immunosensor for atrazine in drinking water. Biosensors Bioelectron 7, 411–419. 21. Minunni, M., Guilbault, G.G. and Hock, B. (1995) Quartz crystal microbalance as a biosensor. Anal Lett 28, 749–764. 22. Minunni, M., Skladal, P. and Mascini, M. (1994) A piezoelectric quartz crystal biosensor as a direct affinity sensor. Anal Lett 27, 1475– 1487. 23. Minunni, M., Mascini, M., Carter, R.M., Jacobs, M.B., Lubrano, G.J. and Guilbault, G.G. (1996) A quartz crystal microbalance displacement assay for listeria monocytogenes. Anal Chim Acta 335, 169–174. 24. Plomer, M., Guilbault, G.G. and Hock, B. (1992) Development of a PZ immunosensor for detection of enterobacteria. Enzyme Microbial Technol 14, 230–235. 25. König, B. and Grätzel, M. (1993) Detection of viruses and bacteria with pezoimmunosensors. Anal Lett 26, 1567–1575. 26. König, B. and Grätzel, M. (1993) Human granulocytes detected with a piezoimmunosensor. Anal Lett 26, 2313–2328. 27. Jacobs, M.B., Carter, R.M., Lubrano, G.J. and Guilbault, G.G. (1995) Piezoelectric biosensor for listeria monocytogenes. Am Lab 27, 11–26. 28. Boveniser, J.S., Jacobs, M.B., Guilbault, G.G. and O’Sullivan, C.K. (1998) Detection of pseudomonas aeroginosa using the quartz crystal microbalance. Anal Lett 31(8), 1287–1295. 29. König, B. and Grätzel, M. (1994) A novel immunosensor for herpes virus. Anal Chem 66, 341–348. 30. Attili, B.S. and Suleiman, A.A. (1995) Piezoelectric immunosensor for the detection of cortisol. Anal Lett 28, 2149–2159. 31. Attili, B.S. and Suleiman, A.A. (1996) A piezoelectric immunosensor for detection of cocaine. Microchem J 54(2), 174–179. 32. Prusak-Sochazewski, E., Luong, J. and Guilbault, G.G. (1990) Development of a piezoelectric immunosensor for detection of salmonella. Enzyme Microbial Technol 12, 173–175. 33. Prusak-Sochazewski, E. and Luong, J. (1990) A new approach to development of a reusable PZ biosensor. Anal Lett 23, 401–410. 34. Chu, X., Jiang, J.H., Shen, G.L. and Yu, R.Q. (1996) Simultaneous immunoassay array and robust method. Anal Chim Acta 336(1–3), 185–193. 35. Kim, N. and Park, I. (2003) Application of a flow-type antibody sensor to the detection of Escherichia coli in various foods. Biosens. Bioelectron 18, 1101–1107. 36. Ricci, F., Volpe, G., Micheli, L. and Palleschi, G. (2007) A review on novel developments and applications of immunosensors in food analysis. Anal Chim Acta 605, 111–129. 37. Lin, H. and Tsai, W. (2003) Piezoelectric crystal immunosensor for the detection of staphylococcal enterotoxin B. Biosens Bioelectron 18, 1479–1483. 38. Su, X. and Li, Y. A QCM immunosensor for Salmonella detection with simultaneous measurements of resonant frequency and motional resistance. Biosens Bioelectron 21, 840–848.

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39. Keller, C., Glasmastar, K., Zhdanov, V. and Kasemo, B. (2000) Formation of supported membanes from vesicles. Phys Rev Lett 84, 5443–5446. 40. Voinova, M., Rodahl, M., Jonsson, M. and Kasemo, B. (1999) Viscoelast acoustic response of layered polymer films at fluid-solid interfaces: continuum mechanics approach. Physica Scripta 59, 391–396. 41. Kurosawa, S., Tawara, E., Kamo, N. and Kobatake, Y. (1990) Oscillating frequency of piezoelectric quartz crystal in solutions. Anal Chim Acta 230, 41–49. 42. James, C., Mulcahy, D. and Steel, B. (1984) Viscometer calibration standards: viscosities of water between 0 and 60 degrees C and of selected aqueous sucrose solutions at 25 degrees C from measurements with a flared capillary viscometer. Journal of Physics D: Applied Physics 17, 225. 43. Saluja, A. and Kalonia, D. (2004) Measurement of fluid viscosity at microliter volumes using quartz impedance analysis. AAPS Pham Sci Tech 5: article 47.

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11

Focused ion beams

Yi Wang

Abstract: Focused ion beam (FIB) is a technique used for site-specific analysis and fabrication of materials. The ion beam is produced from a liquid ion metal (such as Ga) source. In this chapter, we introduce the set up and the principles of an FIB system. The four basic functions of FIB – imaging, milling, etching/deposition and implantation – are discussed. Generally, FIB and scanning electron microscopy (SEM) are combined into one system to achieve the precise machining abilities of FIB with the high-resolution and lessdestructive SEM imaging. 3D real-time imaging and 3D nanostructure fabrication can be accomplished using FIB systems. FIB has been utilized for imaging and fabrication on semiconductors, metals, ceramic, polymers and biological materials. Here, we present the use of FIB in investigating the formation of protein nanospheres by evaporation-induced self-assembly. Finally, we introduce two limitations of FIB: surface damage and implantation, which may impact the quality of imaging and fabrication. Keywords: focused ion beam; FIB; fabrication; 3D imaging; nanostructure; zein; nanosphere; surface damage; implantation

11.1 11.1.1

Background Introduction to the focused ion beam system

A focused ion beam (FIB) is a technique for site-specific imaging, milling, etching, deposition and implantation of materials. The FIB system projects a focused highenergy ion beam, called the primary beam, to interact with a sample surface via sputtering ions and electrons, which form a secondary beam. With a low primary beam current, the FIB may be used to image the sample surface by collecting secondary ions (SIs) and secondary electrons (SEs) via detectors, in a similar fashion to a scanning electron microscope (SEM). The SIs and SEs are collected separately by the highly positive or negative voltage at the particle detector grids. The detector Nanotechnology Research Methods for Foods and Bioproducts, First Edition. Edited by Graciela W. Padua and Qin Wang. © 2012 John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc.

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signal is then amplified and a 2D distribution of electrons or ions is displayed as a digital image. Generally, SE detection is the preferred method for both SEM and FIB imaging, because it has a lower noise level than the SI imaging. Modern FIB systems can achieve very high resolutions in imaging: 5 nm, for example. When operated with a high primary beam current, the ion beam carries enough momentum to cause mass transfer on the sample surface. A large amount of material is sputtered, including neutral atoms, as well as SIs and SEs. This effect allows precision milling of a specimen down to the submicrometer scale. An FIB can also perform gas-assisted etching and deposition operations, where gas reacts with the sample in the presence of the ion beam. In the etching process, the products of the gas–sample interactions are all volatile and are removed by the vacuum system, leaving the sample etched. In the deposition process, nonvolatile compounds are produced, which form a thin film on the sample surface. The FIB system has been widely used, primarily in the semiconductor industry and materials science fields. The first FIB system was built in 1961 with a plasma ion source, which had a low brightness and only produced a low beam current at submicrometer resolution.1 The FIB was further developed with the introduction of field-emission ion sources. The electron beam, by comparison, was a slow process and had difficulty penetrating hard materials, which were limitations to the lithographic techniques of semiconductor fabrication. An ion beam has a shorter wavelength, higher resolution and higher mass than the electron beam. FIB technology offers surface deposition or removal of materials at micro- and nanoscales, useful in the fabrication of devices and sample preparation for transmission electron microscopy (TEM).

11.1.2

Overview of the FIB

The FIB system consists of a vacuum chamber, ion source, ion column, sample stage, detectors, signal analysis and instrument control software (Fig. 11.1). A liquid-metal ion source (LMIS) sits on the top of the FIB column. Ions are produced at the source and shaped into a beam by an electric field. After passing through various lenses and apertures, the FIB is scanned on the sample surface. The FIB system produces a finely focused beam of ions for imaging at low beam currents and for site-specific milling at high beam currents. The precise control of the ion beam allows highly localized process regions without affecting the overall sample integrity. For example, an FIB can be used to make micro- or nanoscale marks on a sample surface or to precisely cut a 3D cross-section out of a bulk sample.

11.1.3

Ion beam production

Ion beams and electron beams are based on the same principle and serve many of the same purposes. Both beams consist of a stream of charged particles, and they employ similar methods for particle production and acceleration and use lenses and apertures for beam focusing. Both systems can be used to image a sample as well as to perform etching and deposition. Ions are much heavier than electrons. They can carry momentum thousands of times higher than can electrons with the same energy. The greater mass of ions allows them to easily blast atoms on surfaces out of their original

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Ion source Suppressor Extractor Spray aperture First lens Computer system Upper octopole for signal processing Variable aperture

Ion column

Blanking deflector

Vacuum chamber

Blanking aperture Deflection octopole Second lens Ion beam Ion detector Gas injection system

Secondary electron detector Sample stage

Fig. 11.1 Schematic diagram of an FIB system. To see a color version of this figure, see Plate 11.1.

positions. While an electron beam barely affects a surface, the heavy particles in an ion beam can penetrate deeper into material, kicking out atoms as they go. An FIB is capable of removing atoms from a surface in a very precise and controlled manner. It is capable of milling through a sample without changing its overall structure or chemical composition. Ion beams have other uses as well, including gas-assisted etching and deposition of materials on sample surfaces, and implantation of other metal ions. The ion source and the ion column, which contains lenses and apertures, are the main components in ion beam production. The most widely used ion source in FIB systems is the LMIS, which has the ability to provide a beam focus spot of ∼5 nm in diameter. The most commonly used metal in LMIS is gallium (Ga). Ga has some advantages over other choices of element: it has a low melting point (29.8°C) and low volatility at that melting point. Once heated, Ga can remain in the liquid phase and yields a long source life of about 400 mA-hours/mg. A relatively low operating temperature minimizes the interdiffusion with the tungsten needle substrate. The Ga ion has a heavy mass and is capable of milling through heavy materials without destroying the sample at once. Ga is also easily distinguishable from other elements during sample analysis. Ion sources based on gold and iridium are also available.

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In the LMIS, Ga is placed in contact with a tungsten needle, with a tip radius of 2–5 mm, and heated to near evaporation. Liquid Ga wets the tungsten needle and an electrical field causes ionization and field emission of Ga atoms. Ions are extracted by an extraction electrode and accelerated to 5–50 keV by a potential down the ion column. The ion column typically has two lenses: a condenser and an objective. The condenser lens is for beam forming, while the objective lens is for beam focusing. While SEM uses magnetic lenses to focus its beam of electrons, the FIB uses electrostatic lenses instead, because ions are much heavier and slower than electrons. A set of apertures is also found in the ion column, to help define the beam size and provide a range of ion current settings for different applications. The LMIS produces high-current-density ion beams with a very small energy spread. A modern FIB system can image a sample with a spot size on the order of a few nanometers. The production and acceleration system, as well as the sample chamber, should be in high vacuum so as to avoid loss or scattering of ions by collisions with gas molecules.

11.1.4

Ion–target interaction

The ability to mill, image and deposit material using an FIB instrument depends critically on the nature of the ion beam–solid interactions. Unlike electrons, the relatively large ions have a hard time penetrating the surface of a sample because it is much harder for them to pass through individual atoms. When an ion beam hits the sample surface, a fraction of the beam is backscattered from the surface layers, while the rest is slowed down and penetrates into the solid. As the ion penetrates the solid, it collides with atoms of the sample material. Collisions cause atoms and electrons to shift slightly from their equilibrium positions and to generate phonons, which can dissipate energy as they propagate. If the energy transferred from the penetrating ion is higher than the binding energy of the electrons to the nuclei, the electrons can leave their positions, producing emission of SIs and SEs. If the energy is higher than the binding energy of an atom to a molecule, the atom can leave its position, resulting in emission of neutral atoms. If recoiled atoms or electrons have enough kinetic energy, they can transfer their energy to other atoms and electrons upon collision. This produces a cascade of collisions. Interactions between the incident ion and the substrate occur at the expense of the initial kinetic energy of the ion. Consequently, if the ion is not backscattered out of the target surface, it will eventually come to rest, implanted within the target at some depth below the sample surface. Sputtering is one of the results of an ion beam hitting a target. Sputtering results from a series of elastic collisions, where momentum is transferred from the incident ions to the target atoms in a cascade-collision process. A surface atom may be ejected as a sputtered particle if it receives a component of kinetic energy that is sufficient to overcome the surface binding energy of the target material. A portion of the ejected atoms may be ionized and collected either to contribute to an image or to be massanalyzed. Inelastic interactions also occur as a result of ion bombardment. Inelastic scattering events can result in the production of phonons and plasma, and the emission of SEs. SE detection is the standard mode for FIB imaging; however, as previously mentioned, SIs can also be detected and used to form images.

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Fig. 11.2

11.1.5

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SIM image of zein spheres.

Basic functions of the FIB system

The FIB system has four basic functions: imaging, milling, etching/deposition and implantation. The most common function is imaging. Like the SEs collected in an SEM, the SEs generated by ion beam sputtering can also be collected to form a scanning ion microscope (SIM) image. The ions generated by the ion source are accelerated by the ion column and hit the sample on the stage. The sputtered SEs are collected by the detectors. An image is formed based on the signal information given by the detectors. Figure 11.2 is an example of an SIM image. The sample in the image consists of zein spheres formed by evaporation-induced self-assembly. Since incident ions are abrasive compared to electrons, one can see several voids on the thin film of zein caused by ion beam radiation. The damage can be avoided by reducing the ion beam current. The second function of FIB systems is milling. Milling is a process that uses relatively heavy ions in the beam to bombard the sample surface. Heavy ions hit the surface of the sample and cause sputtering. FIB is a site-specific milling technique. With the help of the imaging function mentioned above, the FIB can produce very accurate and high-precision milling patterns with appropriate sample preparation. It can control the milling accurately with very little damage to the material outside of the milling pattern. At high currents, FIB can achieve high-resolution milling at relatively rapid speed with excellent reproducibility. Figure 11.3a is an SIM image taken after milling on the zein spheres in Fig. 11.2. The image was taken with the same imaging parameters as in Fig. 11.2. Figure 11.3b

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Fig. 11.3 (a) Top-view SIM image of zein spheres after milling. (b) 52° angle-view SEM image of the same spheres after milling.

is an angle-view SEM image of the milling result. Because the electron beam is installed at 52° from the ion beam in this particular system, it is capable of taking cross-section images after ion beam milling without stage movement. This feature affords accurate positioning in complicated fabrication procedures. Figure 11.3b’s cross-section view shows cavities in these two zein spheres, which would be invisible with traditional SEM imaging. The third function of FIB is etching/deposition. Gas-assisted FIB etching or deposition is a direct process where gas is supplied to a local area on which the controlled ion beam is focused. A cloud of a chemically reactive gas is supplied above the sample through a fine capillary tube, and molecules in the gas can be transported on to the sample by ion beam striking. If the products are all volatile, they will be removed by the system vacuum, leaving an etched surface. If there are nonvolatile compounds in the reaction products, those will be left as a thin film on the sample surface. The fourth function, ion implantation, is another method for surface modification. Ion implantation allows direct and controlled introduction of impurities into solids. A beam of dopant ions is aimed at a target material so that the ions are incident, with sufficient energy to become permanently embedded.

11.1.6

SEM and SIM

An SEM uses an electron beam as its primary beam. An SIM uses an ion beam. Both consist of a beam source, beam column, vacuum chamber, sample stage, detectors and computer system. They operate with the same principle: the beam scans the sample and the detectors collect SEs via their highly positive electrical potentials. On the detector, the SEs are converted to photons and photoelectrons for high amplification with low noise. The intensity distribution of the SEs can be viewed and displayed as an analogue image or further digitized. The electron beam and the ion

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beam have unique characteristics. Ions can be either positively or negatively charged, while electrons are always negative. Ions are much larger and more massive than electrons. The larger ions have a higher probability of interactions with atoms on the sample than electrons do, causing a rapid loss of energy. As a result, ion beams cause more localized atomic ionization of surface atoms and emissions of SIs and SEs. Higher ion beam current may remove atoms from beneath the sample surface, causing internal structural changes and even surface damage.

11.1.7

SEM and FIB combined system

The FIB system by itself has a wide range of functions and applications. However, FIB may cause undesired damage to samples. Combining the precise machining abilities of FIB with the higher-resolution and less destructive SEM imaging has offered an excellent solution. Both electron beams and ion beams are usually integrated into an SEM/FIB combined system. The electron beam is mainly for imaging, while the ion beam is mainly for milling, etching/deposition and implantation. This configuration enables low alternation of the sample during imaging as well as the benefit of surface modification by heavy ion beam. In a combined system, which is sometimes called a dual-beam system, the ion beam and the electron beam are placed at concentric positions, with an angle of 45–52° between the two for best performance. The two beams are focused on the so-called “coincidence point”, an optimized position for the majority of operations taking place within the system, with a typical working distance of several millimeters. The combination of SEM and FIB in a dual-beam system allows the electron and ion beams to work symbiotically to achieve tasks beyond the limitations of either system alone. The dual-beam system allows SEM imaging and FIB sample modification without sample-stage movement. In addition, the stage can be tilted, allowing changes in the sample–beam orientation. As a result, very creative ion beam milling and characterization can be achieved. In addition, electron beamassisted deposition of materials can be used to produce very low-energy deposition that will not affect the underlying surface of interest as dramatically as would ion beam-assisted deposition.

11.1.8 3D nanotomography with application of real-time imaging during FIB milling It is possible to integrate the electron and ion beam operation to provide 3D information by sputtering the sample in increments and obtaining near-real-time SEM images of the sample after each sputtering cycle. For example, following the preparation of any cross-section, the steps to obtain serial sections are relatively straightforward. After opening up the inside of the bulk substrate by milling a trench and imaging a polished sidewall of that trench, polish off a thin layer of material and open up a second surface for imaging, and so on. In this manner, one may analyze a selected volume of the sample instead of a single cross-sectional surface. This type of analysis is called 3D characterization.

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11.1.9

3D nanostructure fabrication by FIB

Comparing chemical vapor deposition (CVD) for 3D fabrication by laser, electron beam and FIB,2 the electron beam and FIB have advantages in spatial resolution and beam-scan control over laser. Because of the mass difference between electrons and ions, FIB CVD has a much higher deposition rate than electron beam CVD. Ions have a smaller penetration depth than electrons, which allows FIB CVD to produce complicated 3D nanostructures. Wargners et al.3 have demonstrated the fabrication of high-aspect-ratio pillars and walls with FIB CVD.

11.2

Applications

The FIB system has been utilized for imaging and fabrication in various fields. Target materials include semiconductors, metals, ceramics, polymers and biological materials. In the semiconductor industry, the FIB’s milling and lithography capabilities make it a useful tool for mask repairing, device modification, failure analysis and integrated circuit debugging. Indeed, FIB development has been driven by its applications in the semiconductor industry. It was first used as a direct device-fabrication instrument and photomask repair tool. Later, FIB was used in circuit microsurgery to perform rapid prototyping, circuit diagnostics and failure analysis. Gas chemistry was also added into the FIB chamber for selective etching and deposition. FIB is used to make lightemitting devices of porous Si for semiconductor materials.4 The small beam size and imaging capabilities of the FIB make it ideal for preparing site-specific SEM or TEM specimens of a wide range of materials, including polymers, steel, surface coatings, catalysts and semiconductors. The electrons transmitted through the sample can be used for image formation. TEM analysis requires the preparation of very thin samples. FIB milling can more accurately select the position of a cross-section specimen than can the conventional labor-intensive preparation.5 For milling, FIB should be operated with the proper beam size, shape, current and energy in order to remove a required amount material from a predefined location in a controllable manner.6 FIB has a very short wavelength and a very large energy density, which is important for direct fabrication of structures that have feature sizes at or below 1 mm.

11.2.1

Polymers

FIB has a variety of applications in polymer-related research. To fully understand material properties and establish structure–property relationships in polymer nanocomposites, it is helpful to visualize their 3D structures, the dispersion/distribution of nanofiller within the polymer matrix and the nature of the interfacial interaction between matrix and filler. FIB is capable of 3D analysis of nanolayer dispersion in the polymer matrix and may give information on material properties. FIB is a leadingedge tool in the field of nanotechnology, especially nanofabrication. Its capabilities are advantageous for stable control of subpicoampere currents for microprocessing, drift correction for beam irradiation position stabilization, 3D-CAD linkage,

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application of pattern-recognition technology and effective use of gas. Nanofabrication aims at building nanoscale structures, which in turn can be used as components, devices or systems. FIB is used in the fabrication of polymer-based optical fibers, light-emitting diodes, blend films, nanocontainers and optoelectronic devices.

11.2.2

Biological products

FIB has also been successfully applied to the analysis of the structure of biological specimens. Biological organisms are inherently complex. They are also in a continuous state of flux, rather than attaining equilibrium. FIB can examine biological samples quickly and efficiently without the artifacts resulting from other sectioning methods such as ultramicrotomy or freeze-fracturing. In situ imaging, provided by FIB through the collection of SEs in a manner similar to that of SEM, allows quasireal-time observation of the surface, thus improving sectioning precision. The ability to quickly mill cross-sections in various planes offers a powerful tool for understanding the relations between cell structure and morphology. Imaging and sectioning of biological specimens from a single yeast cell to small athropods have provided highresolution images of biological structures from the subcellular level to the microstructural level of tissues and organs. Preliminary results indicate that sample preparation is of primary concern during the imaging and manipulation of biological samples. In general, sample preparation involves chemical fixation, dehydration, embedment and staining or coating. An alternative to chemical fixation is the use of cryotechniques, in which structural preservation is accomplished through the freezing of cellular components.

11.2.3

Example: self-assembled protein structures

Zein is a major protein in corn. It is insoluble in water, but can be dispersed in ethanol solutions. It is an amphiphilic molecule because its amino acid sequence contains more than 50% hydrophobic residues. Zein’s structural model resembles a 3D prism 1.3 × 1.2 × 3 nm3. The lateral faces of the prism are hydrophobic, while the top and bottom surfaces are hydrophilic. Because of its amphiphilic character and regular geometric shape, zein is capable of self-assembly. Zein can form organized phases including spheres and bicontinous and lamellar structures from ethanol solutions.7 The structure of zein spheres was investigated by SEM and FIB. SEM images show that zein formed spheres ranging from several hundred nanometers to several microns. The interior structure of the spheres was investigated by FIB. The sitespecific precision of FIB allowed the selection of any sphere from the entire field for milling operations. FIB is easy to handle and efficient in the milling of zein spheres, since it has a range of beam currents to suit different sphere sizes. The operation of FIB milling on zein spheres is fairly straightforward. In this particular SEM/FIB combined system, the electron beam and ion beam are fixed at an angle of 52°, which makes the imaging of cross-sections efficient and accurate. At the beginning, the sample stage is set perpendicular to the electron beam. A real-time SEM image helps select the specific site for ion beam milling. When the target sphere is chosen, or at least when the small area of interest is in focus, the stage will rotate

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Fig. 11.4

SEM images of cross-sections of zein microspheres after FIB milling.

52° to become perpendicular to the ion beam. Then after being checked by both the angle-view real-time SEM image and the top-view real-time SIM image, a pattern with preset size, depth and shape is milled by the ion beam. The milling result can be recorded again by both SIM and SEM. To investigate the internal structures of zein spheres, FIB was used to mill half the sphere off. The ion beam milled a pattern, layer by layer, from top to bottom. Within a layer, the beam milled the material point by point, beginning from one corner and ending at the diagonal. After the top layer was milled out, the ion beam started from the corner of the subjacent layer. The milling time depends not only on the size of the pattern but also on the ion beam current. Beam current selection is available from 10 to 1000 pA, or even higher. It depends on sample area, milling depth and milling pattern. In an SEM/FIB combined system, the ion beam can run the milling process, while the electron beam can take SEM images of the cross-section without any position adjustment. Figure 11.4a shows an SEM image of a milling result. The size of the milling pattern was 2 × 1 × 1 mm3. There were originally two adjacent spheres; the left one has two cavities, while the smaller sphere to the right is solid. FIB milling

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Sequence of SEM images showing the FIB milling process on zein microspheres.

clearly shows a blade-like cut cross-section with no further structural damage. Figs 11.4b and c show additional examples of milling operations. Figure 11.5 highlights the steps of the FIB milling process on zein spheres. Figure 11.5a is an SIM image showing the sphere before milling. At this time, the sample stage is perpendicular to the ion beam, and the ion beam takes the image from the top. Figure 11.5b is an SIM image showing the same zein particle after FIB milling. The sample stage and the ion beam, as well as the electron beam, do not change their positions, which allows the ion beam to take the SIM image while keeping all the operation parameters the same as they were before milling. This feature makes FIB fast and easy to operate and makes precise targeting possible for much more complicated procedures, including deposition and implantation. Figure 11.5c is an SEM image showing the same zein particle after FIB milling. The SEM image clearly depicts the structure of the zein sphere, showing an internal cavity. The combined FIB/ SEM system with the ion beam and electron beam fixed at an angle has clear advantages in cross-sectional studies. The ion beam, from the top, can precisely mill the

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Fig. 11.6

SEM and SIM images taken before and after milling of zein microspheres by FIB.

target material in a customized pattern to show cross-sections. The electron beam, at 52° to the vertical, can image the cross-sections to show the internal structure. Figure 11.6 records the complete procedure of FIB milling on zein spheres. Figure 11.6a is an SEM image showing the spheres before milling from a side view. At that time, the positions of the ion beam and the electron beam are fixed, and there is no stage movement during the milling process. Figure 11.6b is an SIM image showing the spheres before milling from a top view. After taking Fig. 11.6a by SEM, the only operation is the switch from electron beam to ion beam to take Fig. 11.6b by SIM. In Fig. 11.6b, radiation damage can be seen on the ground of the sample because of the ion beam scanning. Figure 11.6c is an SIM image showing the particles after milling from a top view. The fixed ion beam, electron beam and sample stage make Figs 11.6b and c a perfect comparison, with no change or shift in the background. Figure 11.6d is an SEM image of the particles after milling. The angle view clearly shows the interior structure of the spheres. The ion beam has several pattern styles, such as rectangle, trapezoid and random quadrangle, from which to choose for sample milling. Figure 11.7 shows a sample

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Fig. 11.7

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SIM and SEM images of zein microspheres after FIB milling.

with quadrangle-pattern milling. Figure 11.7a is an SIM image taken by the ion beam from the top, while Fig. 11.7b is an SEM image taken by the electron beam from the side. The sample stage was not changed: the size differences between Figs 11.7a and b come from the different magnifications. The three milled spheres in the SIM and SEM images are around 1 mm in size. Figure 11.7a shows that the pattern selected for milling was a randomly drawn quadrangle, which was fitted to mill the three spheres together and to show their cross-sections in one single image for SEM. Another challenge is to take images of the interior structure of thin films. Figure 11.8a shows an SEM image of the sample surface taken from the side view before FIB milling. The side view by SEM has the advantage of showing the surface morphology of the sample. In Fig. 11.8a, the surface of the zein film is not flat and has lots of small concaves. Figure 11.8b is an SIM image of the sample surface after milling. The three rectangles were selected and milled one by one. The milling sites were chosen at the concaves, where the sample was suspected of having an interior structure under the surface. Figure 11.8c shows an SEM image of the cross-section of one of the milled concaves. A small cavity is shown.

11.3

Limitations

The drawbacks to FIB are the surface damage and implantation. Unlike with an electron beam, collisions resulting from the use of a Ga ion beam induce many secondary processes such as recoil and sputtering of constituent atoms, defect formation, electron excitation and emission, and photon emission. Thermal- and radiation-induced diffusion resulting from these collisions contribute to various phenomena of interdiffusion of constituent elements, phase transformation, amorphization, crystallization, track formation and permanent damage. Further, processes such as ion implantation and sputtering will change the surface morphology of the sample, possibly creating craters, facets, grooves, ridges, pyramids, blistering, exfoliation or a spongy surface. Damage can be minimized by FIB milling at lower voltage or by further milling with a low-voltage argon ion beam after completion of the FIB process.

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(a)

(b)

(c)

Fig. 11.8

SEM and SIM images taken before and after milling of zein microfilms by FIB.

Figure 11.9 shows an image with vertical strips. There are several cavities inside the sphere, as seen from the cross-section. The milling rate at the solid material is much lower than at the cavities. The large differences in milling rate cause the formation of the vertical strips.8 The section above the cavity shows no vertical strips, while the section below has them. Because the milling by ion beam operates from top to bottom and layer by layer, there is no milling-rate difference for the parts at a higher layer than the cavities and the vertical strips only appear at layers below the cavities. Figure 11.10 shows SEM images of two spheres after FIB milling. The spheres had a hardness/softness distribution. The core area was hard while the outer area was soft, possibly because zein spheres grow by adsorption of molecules in solution. If the selected beam current is not high enough, the milling will not run through the hard area inside the particle. As seen in Fig. 11.10a, the core of the sphere is hard and the ion beam current is not high enough to sputter and mill the center part. The residual material is in a fan shape because the bottom part of the sphere is also hard due to the deformation when the sphere settles down at the end of the sphere

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SEM image of zein microspheres, showing vertical strips after FIB milling.

Fig. 11.10 SEM images showing the texture and hardness differences of the interior structures of zein microspheres after FIB milling.

formation process. The outer area is soft and is milled off as expected. Figure 11.10b shows another sphere in the same sample. There is a thin cylinder left in the center of the sphere, sitting on another thick and short cylinder. This indicates that the hard areas are the core and the bottom of the sphere. The ion beam is milling from the top of the sample, so if the beam current is not strong enough and is blocked by the hard material in the sample, the material below it will also be left.

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Fig. 11.11 SEM image showing the texture and hardness differences of the interior structures of zein microspheres after FIB milling.

It is difficult to identify whether the material is hard or soft from the above SEM and SIM images. However, in the SEM image shown in Fig. 11.11, the dark and bright contrasts characterize the material as either hard or soft. There is residual material left in all three spheres after milling, and all residues start from the core position down to the bottom. Also, the film under the spheres has the sections under the cores visible, while the material under the shells was milled out. Thus, the cores of the spheres were harder than the shells and the cores covered the materials below them from milling, both the shell and the bottom film.9 Figure 11.12 shows an SEM image of the sample after FIB milling. There were two particles milled. The one in the front has its cross-section milled, showing four cavities irregularly distributed within the particle. The one in the back shows a crosssection with two parts. The top shows a smooth face and solid spheres without cavities. The bottom shows multiple zein columns. It is believed that the bottom part was formed by differential milling, which means the milling rates were different at different milling positions. During FIB milling, the cavities had a much higher milling rate than the solid, which caused differential milling, shown here as zein columns. Figure 11.13a shows an SEM image of the sample surface after FIB milling. Around the milling rectangle, there is a square with a dark color inside it and a bright color outside it. The color change is caused by the ion beam scanning after the switch from the electron beam and before the ion beam milling started. The dark square area corresponds to the penetration of the ions into the films while the ion beam was scanning. The boundary of the square, which is also the boundary of the dark and bright

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Fig. 11.12

SEM image showing differential milling on zein microspheres by FIB.

Fig. 11.13

SEM images showing radiation damage on zein materials after FIB milling.

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areas, is not sharp, because the selected ion beam current is not high enough to damage the surface. In Fig. 11.13b the dark square is again caused by ion beam scanning. The penetration of ions not only causes the dark color but damages the morphology of the sample surface. Originally, there were pores on the surface of the sample. The penetration and radiation caused by the ions made the pores larger, which can be seen by comparing the pores inside the dark square with the pore outside. Similar phenomena are observed in Fig. 11.14, showing SEM images of the sample surface after ion beam scanning. In Fig. 11.14a, a dark square and radiated pores

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can be seen, similar to those in Fig. 11.13. Fig. 11.14b shows a magnified part of the dark square area in Fig. 11.14a. Several rounded particles on the upper image are caused by radiation damage. Under ion beam scanning, the normal zein spheres melt and shrink due to sputtering. Meanwhile, the sphere boundaries are rounded off and become sharp and bright. Figure 11.15 shows two SEM images of zein spheres before and after FIB milling. The FIB makes the scanned area appear darker due to ion penetration. It also causes radiation damage to the zein spheres and surrounding material. In Fig. 11.15a, the spheres are round and bright, with a smooth surface, and the film under the spheres is smooth and clear as well. However, in Fig. 11.15b the spheres are dark and appear damaged at the surface. Also, the film under the spheres is dark and shows small pores produced by the ion beam scanning.

Fig. 11.14 SEM images showing the radiation damage on zein materials after FIB milling. (b) shows a magnified section of (a).

Fig. 11.15 SEM images showing the morphology differences in the sample surface before and after FIB milling on a zein microsphere.

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Materials absorbing radiation may undergo structural damage. The energy deposition of the energetic ions on the sample can result in radiation damage. In Fig. 11.16, a zein film was examined by the FIB system for an SEM/FIB investigation. After targeting using the electron beam, the system was switched to the ion beam for milling. However, due to radiation damage, the electron beam was switched back on

Fig. 11.16 milling.

SEM image showing the radiation damage on zein materials caused by FIB

Fig. 11.17 SEM image showing the contamination caused by the ion beam on zein microspheres during FIB milling.

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after seconds of ion beam scanning on the sample. Figure 11.16 shows an SEM image of the sample surface after radiation damage. The ion beam current was 10 pA, which is low and safe enough for zein spheres. However, the structure of the film sustained radiation damage, producing a film of high porosity. The ion beam seemed to enlarge the pores originally present at the film surface. Because of the low beam current selected, the radiation effect only damaged one thin layer on the surface of the sample. The square on the surface in Fig. 11.16 shows the scanned area. The structure below the surface layer is shown inside the square, while outside the square is the surface structure. The FIB milling can cause contamination of the samples. Figure 11.17 shows a small bright particle like a drop on the face of the cross-section. The small drop does not belong to the original sample but was made by the ion beam during milling. The small drop is Ga, which was implanted by the primary ion beam. In the process of FIB milling, the Ga ion from the original ion beam recoiled one or more atoms in the sample, which resulted in the recoiling of constituent atoms, leading to the creation of atomic defects along the path of the ion beam. The Ga has been collected into droplets by diffusion. The diffusion rate was sufficiently large to allow the Ga to “pool” into droplets.

References 1. Orloff, J. (1993) High-resolution focused ion-beams. Rev Sci Instrum 64, 1105–1130. 2. Matsui, S., Kaito, T., Fujita, J., Komuro, M., Kanda, K. and Haruyama, Y. (2000) Threedimensional nanostructure fabrication by focused-ion-beam chemical vapor deposition. Journal of Vacuum Science & Technology B 18, 3181–3184. 3. Wagner, A., Levin, J.P., Mauer, J.L., Blauner, P.G., Kirch, S.J. and Longo, P. (1990) X-ray mask repair with focused ion-beams. Journal of Vacuum Science & Technology B 8, 1557–1564. 4. Schmuki, P., Erickson, L.E. and Lockwood, D.J. (1998) Light emitting micropatterns of porous Si created at surface defects. Phys Rev Lett 80, 4060–4063. 5. Overwijk, M.H.F., Vandenheuvel, F.C. and Bullelieuwma, C.W.T. (1993) Novel scheme for the preparation of transmission electron-microscopy specimens with a focused ionbeam. Journal of Vacuum Science & Technology B 11, 2021–2024. 6. Tseng, A.A. (2004) Recent developments in micromilling using focused ion beam technology. Journal of Micromechanics and Microengineering 14, R15–R34. 7. Wang, Y. and Padua, G.W. (2010) Formation of zein microphases in ethanol-water. Langmuir 26, 12897–12901. 8. Orloff, J., Swanson, L.W. and Utlaut, M. (1996) Fundamental limits to imaging resolution for focused ion beams. Journal of Vacuum Science & Technology B 14, 3759–3763. 9. Wong, K.C., Haslauer, C.M., Anantharamaiah, N., Pourdeyhimi, B., Batchelor, A.D. and Griffis, D.P. (2010) Focused ion beam characterization of bicomponent polymer fibers. Microsc Microanal 16, 282–290.

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12 X-ray computerized microtomography Leilei Yin

Abstract: X-rays have been used widely to study internal structures in various applications. Combined with computed tomography (CT), X-ray CT is able to acquire 3D volumetric images in a totally nondestructive way. The mechanisms of X-ray absorption contrast are briefly introduced in this chapter, as are the critical components in an X-ray CT system. Common artifacts observed in X-ray CT practice are also discussed. Keywords: X-ray; computed tomography; 3D; nondestructive; contrast; artifacts

12.1

Introduction

X-ray computerized microtomography (CT) is a combination of imaging and computing methods used to acquire 3D images of a sample with internal structures. An X-ray microscope takes multiple projection images at different viewing angles, then a computer reconstructs these 2D projection images into 3D volumetric data. Because X-rays can penetrate a large depth into most materials, X-ray micro-CT has the advantage of being noninvasive and nondestructive in revealing very detailed internal structures of a sample without physically openening it up. An X-ray micro-CT is in principle similar to a medical CAT scan machine, but with specially developed optics. The micro-CT can achieve much higher resolution (submicron versus ∼1 mm) and can adapt to a wide range of sample materials other than bones and body tissues.

12.2

X-ray generation

X-rays can be generated by strong acceleration of electrons or transition from a high energy level to a lower energy level in atoms. The most popular X-ray source is a tube system, shown in Fig. 12.1, in which a high voltage (up to several hundred thousand Nanotechnology Research Methods for Foods and Bioproducts, First Edition. Edited by Graciela W. Padua and Qin Wang. © 2012 John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc.

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Electron beam Tube housing Filament (cathode) Fig. 12.1

Simplified structure of an X-ray tube.

X-ray emission

X-ray energy Transition line Fig. 12.2 Bremsstrahlung spectra of X-ray tube emission. The electron beam energy increases from dashed line to dot–dash line to solid line.

volts) accelerates electrons emitted from a filament (cathode) to a very high energy in order to bombard a heavy metal target (anode). Upon collision with the anode, the high-energy electrons are rapidly slowed down or deflected. During the process, most of the electrons’ energy is lost to heat, and the rest turns into X-ray radiation, called Bremsstrahlung radiation.1 Because the deceleration process for each electron is different, the Bremsstrahlung spectrum is a continuous wide band of radiation, as demonstrated in Fig. 12.2. Some of the incident electrons may collide with low-energy-level electrons in the anode and excite them into a higher energy level. Those excited electrons then fall back to a lower energy level and release the difference in energy into X-rays. Because the energy levels in a

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specific metal are fixed, X-rays generated by these transitions have distinctive energy characters. Commonly used anode metals are copper (Cu), tungsten (W) and molybdenum (Mo). If the accelerating potential is high enough to exceed some of the transition energy of the anode material, both the Bremsstrahlung and the transition line will contribute to the generated X-rays. The typical output power of a tube system ranges from a few watts to tens of watts, while the generated X-ray energy level runs from a few keV (103 eV) to a couple of hundred keV. The X-ray radiation from a tube system has some characteristics that are worth noting for users of the micro-CT instrument. First, the radiation has a wide-band spectrum, which means the X-ray contains all different energy levels. A low-energy X-ray is often called a “soft” X-ray, while a highenergy X-ray is called a “hard” X-ray. Second, the radiation expands to higher energy with an increase of accelerating potential. Third, when the accelerating potential exceeds a certain transition energy of the anode material, a transition line shows up in the spectrum. Even the intensity of the transition line is very high compared to the continuous Bremsstrahlung; the total power of a transition line remains low, however, because the width of the line is still very narrow. In most cases, the high-intensity transition line does not make any significant difference to the micro-CT images. Higher-power X-rays can be generated by a synchrotron radiation source.2,3 A synchrotron source is a rather large facility instead of a lab instrument, where very highenergy electrons fly at near light speed inside a ring of vacuum tube called a storage ring. The energy of electrons inside the storage ring is as high as several GeV (109 eV). The high-energy electrons are guided through a vertical magnetic field between a pair or many pairs of magnets. The magnetic field forces the electrons to change direction in the plane of the storage ring due to the Lorentz force between moving electrons and the magnetic field. During changes in flying direction, the electrons release a small portion of their kinetic energy into high-power X-ray radiation. Because the speed of the high-energy electrons is very close to the speed of light, the X-ray radiation is confined to a very small solid angle due to specific relativity. The result is an extremely high-power (up to several kilowatts), highly collimated and near-chromatic X-ray beam. A comparison of an X-ray tube and a synchrotron source would be similar to a light bulb and a high-power laser. The laser emits light of pure color, highly directional and with high intensity, while the light bulb gives out mild, unidirectional white light. There are several advantages to using a synchrotron X-ray source for micro-CT imaging, but such a facility is not easily accessible to most researchers for daily use. The X-ray tube remains the popular source for microCT systems.

12.3 X-ray images Understanding how X-rays interact with materials is critical to interpreting X-ray images. The attenuation of X-rays by a uniform material is described by the wellknown Lambert–Beer law:4 I = I o exp( −mx )

(12.1)

where I is the transmitted intensity, I0 is the incident intensity, x is the thickness of the material and m is the linear attenuation coefficient. The linear attenuation coefficient

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is determined mostly by two factors related to the sample material: the elemental composition and the density. If we apply a little mathematic transformation to the equation to separate the two factors, we get: ⎛ m ⎞ I = I o exp ⎜ − rx ⎟ ⎝ r ⎠

(12.2)

m the mass attenuation coefficient, is determined by the material’s eler mental composition and the wavelength of the X-ray used. Over a wide range of wavelengths of X-rays, except at the energy of the electron transitions, the mass attenuation coefficient is proportional to: The term, −

m ∝ Z mln r

(12.3)

where Z is the atomic number of the interacting element and l is the wavelength of the X-ray. m is in the range of 3–4, and n is near 3.5 The mass attenuation coefficients of carbon, silicon and copper are shown in Fig. 12.3.

105 C Si Cu

4

Mass attenuation coefficient (cm^2/g)

10

1000

100

10

1

0.1 1000

10000 X-ray energy (eV)

100000

Fig. 12.3 Mass attenuation coefficients of carbon, silicon and copper at X-ray energies from 1 to 100 keV. In most ranges of the plot, X-ray absorption follows an exponential relation to the energy. Copper has a transition line near 8.979 keV and silicon has a transition line near 1.839 keV. Plot based on data from Grodzins.6

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From (12.3), we can summarize that the attenuation of an X-ray depends on the atomic number of the elements inside the sample, the wavelength of the X-ray and the density of the sample. Heavy elements, high-density samples and low-energy X-rays can all contribute to a high absorption of X-rays. If absorption images are used in the micro-CT scan, the above three factors will determine the brightness and contrast. Figure 12.4 shows a single section from a micro-CT scan of a sugar/salt mixture. These two granules have a similar size and color, and are difficult to identify by eye. X-ray micro-CT images are able to clearly distinguish salt from sugar. Sodium and chlorine in salt have higher atomic numbers than carbon and oxygen in sugar, which gives salt a higher absorption contrast under X-ray. The choice of the X-ray energy used for transmission imaging has a great effect on the contrast and signal-to-noise (S/N) level. The fluctuation in the number of X-ray photons that reach the detector is an important source of noise. If the X-ray photons transmitted through the sample follow a random process, termed the Poisson process, the statistical S/N level is: S /N = n

n

= n

(12.4)

Fig. 12.4 A single section of CT data of a mixture of granular sugar and table salt filled inside a plastic straw. Grayscale indicates the X-ray absorption contrast. The bright granules are salt crystals, gray ones are sugar crystals. The data are reconstructed from 931 1024 × 1024-pixel projection images, isotropic voxel size 4 μm. Scanned with Xradia MXCT-400, 40 keV, 200 μA, 5-second exposure time.

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Fig. 12.5 Transmission images of PDMS sheets with an internal cavity and channel structure, 1024 × 1024 pixels, 5 μm pixel size. X-ray settings are (left) 20 keV, 200 μA, 2-second exposure time; (right) 80 keV, 50 μA, 2-second exposure time.

where n is the number of X-ray photons reaching the detector. As one can easily see, increasing the transmission (larger n) will improve the S/N level. Using a higher X-ray energy will effectively increase the transmission because the mass attenuation coefficient decreases for higher X-ray energies. However, higher transmission through the sample also makes it difficult to differentiate the absorptions of features inside it. Figure 12.5 shows X-ray transmission images of a thin slab of polydimethylsiloxane (PDMS) with internal structures. The image, captured with a 20 keV X-ray, has adequate contrast to reveal internal channels (horizontal and vertical lines). In the 80 keV image, the contrast becomes so low that the channels are barely visible. One must find an appropriate energy level to balance between satisfying contrast and a high S/N level in an image. Grodzins found that through the longest path through the sample, at least 13∼14% of transmission was required to optimize contrast and noise in an absorption image.6 In the periodic table of elements, metals occupy most high-atomic-number positions, and usually have high densities as well. Metals are thus very visible in X-ray absorption images. In materials such as fat, proteins, carbohydrates and polymers, the major elements are hydrogen, oxygen, carbon and nitrogen. Those are lowatomic-number elements, and their compounds usually have a low density, so soft X-rays are often used to achieve better contrasts. But some heavy elements are frequently present in food-related products or samples, such as sulfur, chlorine, sodium and even iodide. These elements can increase the contrast in X-ray images even at relatively low concentrations.

12.4

X-ray micro-CT systems

A complete X-ray micro-CT system includes two major subsystems: hardware (X-ray micro-CT scanner) and software (reconstruction software). In this section, the hardware will be given a brief introduction. An X-ray micro-CT scanner includes

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Projection image Object Line detector

Rotation Translation X-ray tube Projection image

Object

Area detector Rotation X-ray tube Fig. 12.6

(a) Fan-beam-configuration CT scanner. (b) Cone-beam-configuration CT scanner.

these major components: X-ray tube (nonsynchrotron system), sample rotating stage, imaging detector and computer for control and image acquisition. Two configurations of X-ray beam profile are commonly used in micro-CT systems (see Fig. 12.6). The first is a fan beam, which the X-ray beam diverges in one dimension and confines in the perpendicular dimension. The projection of such a beam is a thin line. Accordingly, a line detector is positioned in the beam plane to acquire projection images. During imaging, one thin section across the rotational axis of the sample is scanned at all viewing angles, then the sample translates along the rotational axis for the next section. There are several advantages to this configuration: (i) it uses a relatively low-cost line detector for imaging; (ii) it enables the parallel process of the reconstruction of one slice and data acquisition from the next slice; and (iii) the length of the sample to be scanned is very flexible. It can be a single slice, a few slices or very long, provided the computer is capable of processing and storing data. The imaging time is nearly proportional to the number of sections scanned. The second configuration is a cone-shaped beam, in which the beam expands in both directions. The projection is thus a round pattern. An area detector is used to capture the full projection image of the X-ray beam. With this configuration, the sample only rotates without any translation in the axial direction. However, the reconstruction of volumetric data has to start after all projection images are acquired.

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Nanotechnology Research Methods for Foods and Bioproducts Projection image

(a)

Object

L

l X-ray microfocus tube

Projection image

(b)

Object

X-ray microfocus tube Fig. 12.7 Variable magnification in an X-ray CT imaging setup. (a) The object is placed close to the X-ray tube and far from the imaging detector for high magnification. (b) Magnification is reduced as the object is placed closer to the imaging detector and farther from the X-ray source.

An X-ray photon’s wavelength can be calculated from Einstein’s photoelectric equation:7 E = hv =

hc hc ⇒l= l E

(12.5)

where c is the speed of light and h is Planck’s constant. h = 6.626 × 10−34J sec or h = 4.136 × 10−15 eV sec. A 10 keV X-ray photon’s wavelength is as short as 1.24 Å. Most X-ray tubes’ emissions range from around ten to a few hundred keV. With the extremely short wavelength and the polychromatic, incoherent nature of the emission from a tube source, scattering or diffraction of X-rays is usually not a concern for resolutions in X-ray microscopy. The path of an X-ray from a source through an object to the imaging plane can be considered a straight line. The diverging character of the X-ray beam in either a fan-beam or a cone-beam configuration adds magnification to the X-ray projection image of the object. The magnification of the projection image is a simple geometrical relation. As shown in Fig. 12.7, the projection magnification MP is: MP =

l+L L = 1+ l l

(12.6)

where l is the source–object distance and L is the object–imaging plane distance. The projection magnification helps reveal tiny features in an object on the projection L images. And MP can easily be increased by increasing the ratio of . However, one l

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(a)

Projection image

X-ray tube with an infinitesimal beam spot

Object

(b)

Projection image Object X-ray tube with a real beam spot

(c) Projection image Object X-ray tube with a real beam spot Fig. 12.8 The effect of X-ray beam spot size on the imaging resolution. (a) An ideal coneshaped X-ray source with infinitesimal spot size. The projection is a perfectly magnified image of the object. (b) With a real-beam spot size, a high imaging magnification suffers from a large degree of blurring (gray area), which degrades the resolution in the projection image. (c) A low-magnification image suffers less from blurring due to the beam spot size.

L in the hope of revealing smaller and smaller features l inside the object. Every X-ray tube has a real beam spot size which degrades spatial resolution at high projection magnifications. An ideal X-ray tube with an infinitesimal beam spot size is a perfect point source; therefore, the projection image is just as sharp as the features inside the object, as shown in Fig. 12.8. But with a real beam spot size Ds, simple geometry shows the projection of every point in the object L spreads into a round blurring of Db = Ds. The blurring in the projection image, l which degrades the spatial resolution, rises nearly proportionally with the projection magnification MP. Microfocus X-ray tubes are widely used in lab systems in both fan-beam and conebeam configurations. In a microfocus tube, the X-ray is generated from a small spot on the anode, on which the beam of electrons is focused. A spot size of 5∼10 microns cannot keep increasing

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is achievable with optimized design and a balance between beam quality, tube lifetime and performance stability. By varying the accelerating voltage and current of the electron beam, the energy level and output power of a microfocus tube can be conveniently adjusted. However, most of the electron beam’s energy turns into heat that is dissipated on the anode. The heat load on such a tiny focus spot practically limits the power that can be sustainably applied on the anode, and therefore the output of X-rays. A higher-power microfocus usually has a larger beam spot size, which works against the spatial resolution of the micro-CT system. Detectors in a micro-CT are very important components. Most imaging detectors in micro-CT systems consist of scintillator and optical detectors like charge-coupled device (CCD) cameras or photodiode arrays. A scintillator is a type of material that generates visible light when illuminated by high-energy photons such as X-rays or gamma rays. Silicon-based semiconductors are not effective at directly detecting high-energy photons. Semiconductor materials generate electron–hole pairs when photons are captured in the P–N junction region, which then become detectable electrical signals. But pure or lightly-doped Si has a fairly low atomic weight and density, which means it has a low mass attenuation coefficient. Most X-ray photons will penetrate the thin P–N junction without producing electron–hole pairs. Scintillator materials have higher atomic weights and densities, and can be made with larger thickness to maximize the probability of capturing X-ray photons.8 The integration of scintillator and semiconductor photodetectors combines the advantages of both components for high-efficiency detection of X-rays. Line or area optical detectors are well developed and readily available with a wide selection of pixel numbers, pixel sizes and spectral sensitivities. CCD cameras, for example, are available in 1 megapixel (1024 × 1024) or 4 megapixel (2048 × 2048), with a pixel size up to tens of microns. The spectral response covers the full range of visible light. A larger pixel size enables more electric charge to be stored in each pixel for a higher dynamic range, which is very favorable in acquiring precise grayscale projection images for the accurate reconstruction of 3D volumetric data. CCD chips can be cooled to low temperatures to reduce thermal noise. If a photodetector with a large pixel size is used to directly capture an image, the smallest feature that can be identified from the image is no smaller than the size of the pixel. To acquire both high-spatial-resolution and high-dynamic-range images, some coupling device is necessary to bridge the high-resolution projection images on the scintillator plate and the large pixels on the high-performance CCD detector. Since X-ray projection images are transformed into visible light patterns on the scintillator, traditional optical components can be directly used to change the magnification and focus. Imaging-transfer optical fiber bundles and optical lenses are the most popular coupling devices between the scintillator plate and the photodetector (see Fig. 12.9). Thousands of optical fibers are fused together in a highly ordered way to make a precise point-to-point transfer of light from one end to the other. This special bundle is not only able to transfer light with low loss but can also transfer images. By making the bundle into a tapered shape, it can change the size of the images being transferred as well. Only light within the numerical aperture can be effectively coupled to the fiber, which reduces crosslink between neighbor pixels. However, the optical fiber bundle is not very useful in high-resolution image coupling. If the projection images on the scintillator plate have features below a couple of microns, a highfidelity transfer would require the diameter of each individual fiber in the bundle to

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(a)

225

Visible light Scintillator plate Area photodetector

X-ray

Tapered fiber bundle

(b)

Scintillator plate

Visible light

X-ray

Optical lens

Area photodetector

Fig. 12.9 (a) Tapered optical fiber bundle coupling images from the scintillator plate to the photodetector. (b) Optical lens used as a coupling device. Both coupling devices provide magnification to the optical images, not the X-ray.

be equal to or less than the feature size. When the optical fiber’s core size (core size is a fraction of the overall diameter) is reduced below a couple of microns, the transfer loss rises rapidly. If the core size is further reduced to near or below the wavelength of light, light is practically prohibited from propagating in the thin fiber. An optical lens is another choice for the coupling device between the scintillator plate and the photodetector. The selection of lenses, with different imaging sizes and resolutions, is almost unlimited. It is very practical to have an array of lenses in a single micro-CT system for variable fields of view. The resolution of the optical lens conversion is diffraction-limited by the emission wavelength of the scintillator. A high-magnification lens also has a short focus depth. It is favorable for high-resolutionimage coupling to use a thin scintillator plate and a high-magnification lens. Since the front focus plane of the lens is on the scintillator plate instead of on the object, high-resolution can be achieved on a thick object. Anyone with experience of optical microscopy knows that a higher-magnification objective has a shorter working distance, as well as a narrower focus depth. To achieve ∼1 micron resolution, a typical 40× objective will have less than 1 mm working distance and the focal depth will only be a couple of microns. This means that if a specimen is too thick, the area well below the surface won’t be able to be imaged with high resolution. On the other hand, a high-resolution micro-CT system with a scintillator and high-magnification lens does not have such a restriction. This is because the lens and the photodetector are not directly imaging the object, but rather “staring at” the projected images on the scintillator, as demonstrated in Fig. 12.10. The wavelength of the X-ray is extremely short compared to the features to be imaged, so the diffraction limit of the X-ray can be practically neglected. Even with a large-sized sample and a long distance between

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(a)

Optical image

X-ray projection image Optical lens Object

X-ray microfocus tube Scintillator plate

(b)

Photodetector Optical image

Optical lens Object

X-ray microfocus tube Scintillator plate

Photodetector

Fig. 12.10 Optical lens coupling in X-ray imaging of variable magnification. The scintillator is kept at the lens’s front focus plane regardless of the object’s position and projection magnification.

the sample and the scintillator plate, the projection image on the scintillator still has a very high resolution. As a matter of fact, the resolution of most micro-CT systems is limited by the resolution of the optical components, not the X-ray components. The size of each pixel on the detector combined with the magnification of the lens adds another factor that affects the spatial resolution of a micro-CT system. One should consider both the physical resolution of the lens and the equivalent pixel size of the digital image; whichever is larger will determine the spatial resolution of the micro-CT data. For example, a 20× lens with a 10 micron pixel-sized CCD camera gives an equivalent 0.5 micron pixel size on its images; but the physical resolution of the 20× lens is, say, 2 microns. So 2 microns will be the size of the smallest feature that can be clearly identified. Any feature smaller than 2 microns will appear blurred on the CCD camera due to the diffraction limit of the optical lens. Even if each pixel on the CCD is equal to 0.5 μm on the scintillator, the lens simply is not capable of converting that high spatial resolution on to the camera. In practice, one should keep the equivalent pixel size less than the physical resolution of the lens if the imaging resolution is to be maximized.

12.5

Data reconstructions

The goal of data reconstruction of micro-CT scans is to convert all the information from hundreds or even thousands of X-ray transmission images into a 3D matrix – which contains the whole viewing volume of the micro-CT scan – of the attenuation coefficient.

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Fig. 12.11

227

An X-ray transmits through all voxels in its path.

Let’s take a moment and apply the Lambert–Beer law: I = I o exp( −mx )

(12.7)

to a real sample which consists of non-uniform material. We have “digitized” the sample into many little cubes. Each cube, called a voxel, represents the smallest volumetric unit in the digital dataset. In an X-ray transmission image, each pixel represents the transmission of an X-ray through a row of voxels along its path, as shown in Fig. 12.11. The Lambert–Beer law turns into an accumulation form: ⎛ ⎞ I = I o exp ⎜ ∑ − m (i ) ⋅ Δx ⎟ ⎝ i ⎠

(12.8)

Therefore: 1

⎛ I⎞ ⎟ 0⎠

∑ − m (i ) = Δx log ⎜⎝ I i

(12.9)

where Δx is the size of the voxel. Data reconstruction aims to find the correct value of m(i) for each and every voxel. Of course, it is impossible to find all values of m(i) from a single transmission image. There is no way to know which voxel along the path contributes to how much absorption. But through many transmission images from different viewing angles, computers and software are able to spatially correlate the attenuation coefficients of all voxels. A simplified case is shown in Fig. 12.12. One sphere feature with a high absorption coefficient (represented in a darker color) and another diamond-shaped feature with a lower absorption coefficient are to be scanned. Three projection measurements are shown in the figure, at different viewing angles. The sphere projects the same shadows on all measurements. The diamond-shaped feature leaves triangles, flat tops with different widths and heights. The reconstruction software spatially correlates the object’s shape and the absorption coefficients. Of course, real samples are much more complex than this extremely simplified case. The software and computer need to reconstruct 3D datasets containing up to

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Fig. 12.12

Basic principle of 3D reconstruction from projection images at multiple angles.

billions of voxels from hundreds or thousands of multimegapixel projection images. The efficiency of the algorithm and the computing capacity of the hardware are critical to system performance. The details of the reconstruction algorithm are beyond the scope of this chapter. Interested readers may find Lewitt and Bates,9,10 Kak and Slaney11 and Natterer12 very educational. Even by the standards of the fast-developing computer industry, one trend is providing stunning computing capability to the reconstruction task. In recent years the shifting of reconstruction calculations from the central processing unit (CPU) to the graphics processing unit (GPU) on the graphics card has boosted efficiency dramatically. A reconstruction that used to take hours on a cluster of CPUs can be finished on a single professional-grade graphics card in less than 1 hour. With the parallel processing on multiple graphics accelerators that can be fit into one computer, such capability can be multiplied at a reasonable cost. It is now possible to reconstruct 3D data from several thousand projections of 16 megapixel (4096 × 4096) images in a couple of hours on a single workstation. Researchers should not miss any opportunity to utilize this new technology for micro-CT data reconstruction.

12.6

Artifacts in micro-CT images

Even with an adequate number of high-quality transmission images and an efficient reconstruction algorithm, error-free 3D volumetric data should not be taken for granted. There are still several artifact-causing issues associated with the X-ray source, the mechanical parts that manipulate the sample, imaging parts including the photodetector and scintillator, and the versatile materials that may be present in samples.

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Fig. 12.13 Single section of CT scan data on a jalapeño pepper stem. (a) Ring artifacts are easily visible even after bright-field correction is applied. (b) With dynamic ring reduction, there is almost no visible ring artifact. Scanned with Xradia MicroXCT-400, 30 keV, 200 μA, 1-second exposure time. 3D data are reconstructed from 809 projections with a pixel size of 19 μm.

12.6.1

Ring artifacts

Ring artifacts originate from a non-uniform response on imaging components, the photodetector and the scintillator plate. Defects on the scintillator or dead pixels on the CCD sensor will result in an abnormal signal on particular pixels in all transmission images. When the micro-CT system takes hundreds of images at all viewing angles, the abnormal pixels form a series of concentric rings. Without specific knowledge or calibration, the reconstruction software is unable to distinguish the fault in the hardware from real features inside the sample. The result in a 3D dataset is a series rings with mass attenuation coefficients higher or lower than the accurate values, as shown in Fig. 12.13. Several methods can reduce ring artifacts from aspects of hardware, software and operating procedures. Bright-field correction can effectively reduce ring artifacts. A blank image without the presence of a sample indicates defects or faulty pixels. This image is then used to calibrate all transmission images of a sample. However, a nonlinear response of a photodetector still leaves weak ring artifacts after bright-field correction. Reconstruction software specially designed to recognize the circular character of ring artifacts can be very effective in reducing this as well. Recently, dynamic reduction of ring artifacts in hardware design has shown excellent results. During a micro-CT scan, in addition to simply rotating the sample, the sample stage also randomly translates the sample laterally by a small registered distance. Later, the reconstruction software shifts all images back according to the registered distance. This is equivalent to averaging any abnormal pixel with surrounding pixels during scanning. All abnormal pixels are no longer fixed to particular positions in all images. In this way, the origin of ring artifacts is effectively weakened. Bright-field correction is usually used in combination with dynamic ring reduction to achieve the best results.

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Fig. 12.14 Reconstructed section images of the pepper stem shown in Fig. 12.13 with and without correction of center errors. The central image has undergone center error correction. A point-shaped feature is indicated by an arrow. Center errors equivalent to five pixel lengths to the left or right turn the point feature into the “(” and “)” shapes visible in the left and right images.

12.6.2

Center errors

Reconstruction software assumes the sample’s rotational axis in the center of all transmission images. This assumption carries some errors in almost all micro-CT systems. If the construction software assumes a wrong rotating axis, the spatial correlation for all features results in the same error as the wrongfully assumed axis. The center error thus carries a distinctive character and is fairly easy to identify. With a significant center error, all features in the reconstructed 3D data present a full circle (from a 360º scan) or a semicircle (from a 180º scan) with the same size, as demonstrated in Fig. 12.14. All semicircles have their opening points in the same direction. Fortunately, the origin of a center error is very simple and consistent in all transmission images. The correction for a center error is to find the fixed shift and apply it on all images before reconstruction. The simplest process is usually an iteration of tests judged by the human eye. The reconstruction software processes the data with a range of shifts and workers find the one with the least center error.

12.6.3

Beam-hardening artifacts

Beam-hardening artifacts are mostly contributed by the polychromatic character of the Bremsstrahlung spectrum and by higher attenuation to the low-energy part of the X-ray emission. When an X-ray transmits through a sample, the front portion of the sample absorbs a higher percentage of the low-energy X-ray than of the high-energy part. This is because the attenuation coefficient is higher for lower-energy X-rays, as shown in (12.3). Only relatively high-energy parts of the polychromatic X-ray, the so-called “hard X-ray”, can reach deep inside the sample. The high-pass filtering effect effectively “hardens” the X-ray that penetrates the sample. With transmission images from all viewing angles, the reconstruction software will find the inner part of the sample with lower mass attenuation coefficient, not because it is true, but because the outer part of the sample has taken away more of the low-energy X-ray before it can reach the inner part of the sample. Beam-hardening artifacts are rare in X-ray micro-CT data acquired at synchrotron X-ray facilities because synchrotron

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Fig. 12.15 A single section image of a glass rod. A line measurement across the center is shown as an insert. The high peaks on both edges are caused by the phase-contrast artifacts. The low reading of the absorption coefficient on the center region is due to beam-hardening artifacts. Scanned with Xradia MicroXCT-200, 100 keV, 100 μA, 5-second exposure time. 3D data reconstructed from 951 projections of 1024 × 1024 image. Isotropic voxel size is 12 μm.

X-rays have a very narrow spectrum, and thus all emissions have almost the same energy. In a tube-based micro-CT system, beam-hardening artifacts can be reduced by filtering the X-ray with a pre-hardening filter. The filter is usually a thin slab of uniform material with its atomic number close to the average atomic number of the sample material. The filter is placed between the X-ray tube and the sample. It removes most of the low-energy part of the X-ray and only leaves the high-energy X-ray for micro-CT scanning. The trade-off of using a pre-hardening filter is that less X-ray will be utilized for imaging, so a longer imaging time may be necessary to maintain the S/N level in all transmission images.

12.6.4

Phase-contrast artifacts

X-ray micro-CT data of samples with high contrast features often contain artifacts like “hot corners” or “hot edges”. These kinds of artifact present phase contrast instead of absorption contrast. But even with an absorption-contrast configuration, there are phase contrast artifacts. This is unintentional. When an X-ray transmits through two materials with a high difference in attenuation coefficients, not only are their transmitted intensities very different, but so are their phases. The result is a slightly bent X-ray beam near the boundary of the sudden change of materials. The deflected X-ray interferes with the straight beam to create a halo ring around the high-contrast features in transmitted images. Reconstructed data therefore contain faulty high attenuation coefficients around high-contrast features; see the demonstration in Fig. 12.15. Since the deflection of X-rays varies with energy, the

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phase-contrast artifacts in data from a tube micro-CT system are less intense than are those from a synchrotron system. This is a rare case in which a tube system is superior to a synchrotron system.

12.7

A couple of issues in X-ray micro-CT practice

Besides the operation of any particular X-ray micro-CT system, a couple of issues should be considered before every experiment in order to achieve the best result at a reasonable cost:1 the spatial resolution, and associated issues of contrast and field of view;2 and localized imaging and sample-size reduction.

12.7.1

The spatial resolution, and associated issues of contrast and field of view

Most researchers will no doubt prefer the highest spatial resolution for their data whenever possible. And many commercial or custom-built micro-CT systems do allow adjustable resolutions. However, in practice, operating a micro-CT instrument at the highest-resolution configuration does not always reveal fine features in the sample. There are associated issues with high-spatial-resolution settings. Using a high spatial resolution (small voxel size) reduces the field of view. The digital volumetric data of the micro-CT scan are a cylinder space with its diameter, measured in pixel numbers, determined by the horizontal pixel number of the imaging detector. A small pixel size, which is necessary for high spatial resolution, proportionally means a small field of view. For example, an area detector with 1024 × 1024 pixels can cover a ∼1 × 1 cm viewing area when set up at a 10 micron pixel size on the sample. Reducing the pixel size to 1 micron leaves only a 1 × 1 mm area to be imaged in a single projection. The volume of the final 3D data is proportional to the 3rd order of the voxel size. Simply pursuing high spatial resolution may severely reduce the imaging volume. On the other hand, to cover a large-sized sample in a single scan, one has to compromise on the voxel size, as well as the spatial resolution. The finest feature that can be distinguished in the final 3D data is not only determined by the micro-CT system’s resolution setting, but also, more often, by the sample material’s absorption contrast. As the size of a feature reduces, the contrast on the projection image also suffers because of the reduced absorption along the path of an X-ray according to the Lambert–Beer law. A small feature inside a sample may become too “transparent” to be seen before it is too “small” to be seen. Without enough contrast in projection images, unlikely small features will be identified in 3D reconstructed data. The highest resolution is only achievable on high-contrast features. The choice of imaging resolution should be made with knowledge of the sample material.

12.7.2

Localized imaging and sample-size reduction

Sample-size reduction here means physically trimming the sample size. In most cases, the X-ray micro-CT is a noninvasive imaging technique, able to reveal features buried inside an object. Some micro-CT systems offer the capability of a localized

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X-ray computerized microtomography

233

Detector

Whole object

A

B

Imaged volume

X-ray beam Fig. 12.16

Localized scan on a small region inside a large object.

scan, which scans specified small regions inside a relatively large object. This capability is very helpful for delicate samples in which any alternation is strictly off limits, as well as being very convenient for researchers. However, what is convenient is not always good. Figure 12.16 demonstrates localized micro-CT scanning. The field of view is smaller than the dimension of the whole object, so only a portion is included in the final 3D volumetric data. Only the gray area in the figure is imaged in all projection images and reconstructed into the 3D data. However, areas that fall into the X-ray beam, marked A and B, will absorb the X-ray as well. These volumes reduce the transmitted signal and add unintended background to the data. The reconstructed data quality degrades with the presence of extra surrounding material. Although localized scanning may tolerate an oversized sample, it is good practice to trim the sample down as long as it can maintain its structural integrity and include all the desired volume.

References 1. Cowley, J.M. (1995) Diffraction Physics, 3 edn, Amsterdam: North-Holland Personal Library, Elsevier Science. 2. Winick, H. (1994) Synchrotron Radiation Source: A Primer, Singapore: World Scientific. 3. Hofmann, A. (2004) The Physics of Synchrotron Tadiation, Cambridge: Cambridge University Press. 4. Ingle, J.D. and Crouch, S.R. (1988) Spectrochemical Analysis, Englewood Cliffs, NJ: Prentice Hall. 5. Stock, S.R. (2009) MicroComputed Tomography Methodology and Applications, Boca Raton, FL: CRC Press. 6. Grodzins, L. (1983) Optimum energies for X-ray transmission tomography of small samples: applications of synchrotron radiation to computerized-tomography. Nuclear Instruments & Methods in Physics Research 206, 541–545. 7. Jackson, J.D. (1999) Classical Electrodynamics, 3 edn, New York: John Wiley & Sons. 8. Martin, T. and Koch, A. (2006) Recent developments in X-ray imaging with micrometer spatial resolution. Journal of Synchrotron Radiation 13, 180–194.

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9. Lewitt, R.M. and Bates, R.H.T. (1978) Image-reconstruction from projections. 3. Projection completion methods (theory). Optik 50, 189–204. 10. Lewitt, R.M. and Bates, R.H.T. (1978) Image-reconstruction from projections. 4. Projection completion methods (computational examples). Optik 50, 269–278. 11. Kak, A.C. and Slaney, M. (2001) Principle of Computerized Tomography Imaging, Philadelphia, PA: Society for Industrial and Applied Mathematics. 12. Natterer, F. (2001) The Mathematics of Computerized Tomography, Philadelphia, PA: Society for Industrial and Applied Mathematics.

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Index

Page numbers in italics denote figures, those in bold denote tables. actin microtubules 6, 24 adsorption/desorption kinetics, QCM-D monitoring 185–6, 186 aggregation 6–7, 74, 82 alcalase 24 alginate 19, 25 alumina 67 amphiphiles 7 liquid crystals 14 amyloidogenic proteins 8–9 nanotubes 24 analytical electron microscopy 128, 139–42, 139, 141 annular dark-field images 137 high-angle 137–8, 138 anthocyanins 61 antibiotics 64 antioxidants 58 aquasomes 84 arachaesomes 73 aragonite 45–6 argon lasers 153 artifacts SEM charging 124–6, 124–6 chemical 124–6, 124–6 X-ray micro-CT 228–32 beam-hardening artifacts 230–1 center errors 230, 230 phase-contrast artifacts 231–2, 231 ring artifacts 229, 229 artificial organ implants 22 ascorbic acid 56 astigmatism in SEM lenses 110, 110 atomic force microscopy (AFM) 26, 45, 183 applications 185–6, 187

Auger electrons 104 autocorrelation 150–1, 150 photon correlation spectroscopy 147, 151–2, 151 avalanche photodiode 155–6, 156 Bacillus licheniformis 24 backscattered electrons 104, 105, 106 bacteria pathogenic, detection of 190 probiotic 58 Bactiguard 187 beam-hardening artifacts 230–1 bilayers 6, 14, 15, 24–5, 26, 72 bioadhesion 67, 71 bioavailability 60–1 biobased nanocomposites 45–50 biodegradable plastics 170 biological barriers 64–5 biological samples, FIB analysis 203 biopharmaceutics classification system 60 biopolymers 2, 19, 20, 22, 23, 33 see also individual materials biosensors 75 antigen-detecting 2 block copolymers 7, 76 blood-brain barrier 66 Boltzmann constant 149 bone 46 bovine serum albumin 11, 67 electrospinning 23 Bragg equation 131 Bragg reflections 170 Bremsstrahlung radiation 216 beam-hardening artifacts 230–1 bright-field correction 229

Nanotechnology Research Methods for Foods and Bioproducts, First Edition. Edited by Graciela W. Padua and Qin Wang. © 2012 John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc.

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236

Index

bright-field images 133, 136–7 Brillouin doublet 146–7, 147 Brillouin, Leon 146 bromelain 74 Brownian motion 146 frequency 146–7, 147 phase 148–9, 148, 149 CAC see critical aggregate concentration calcium 56, 59, 74 calcium phosphate nanoparticles 71 camptosor 67 cancer therapy 79 targeted drug delivery 71–2 canola protein–k-carrageenan 14 capsid proteins 24 Carbitol 32 carbohydrate ceramic nanoparticles 84 carbohydrates 11–13 cellulose whiskers 12–13 cyclodextrins 11–12 carbon mass attenuation coefficient 218 nanoparticles 67 nanotubes 23, 26, 42 carbopol 33 carboxymethylcellulose 25 nanocomposites 46 L-carnitine 59 beta-carotene 58, 59 carotenoids 58, 59 bioavailability 61 oxidization of 61 carrageenan 13, 19, 25, 26 casein 9, 10, 19, 48 casein micelles 5, 6 self-assembly 7 catalase 11 catechins 59 bioavailability 61 cathode-ray tube 128 cellulose nanocomposites 47 nanowhiskers 47 whiskers 12–13 center errors 230, 230 central processing unit 228 ceramic nanoparticles 67, 84 channel width 152 chaperone-cochaperone interactions 187 charge-coupled devices 128, 224

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charging artifacts in SEM 124–6, 124–6 chemical vapor deposition 202 chi-squared test 157 chitosan 19, 26, 47, 73, 78 nanospheres 33 self-assembly 21 cholesterol 74 choline 59 chromium 59 chymotrypsin 11 clays kaolin 42, 49 montmorillonite 42, 49 nanoclays 42 silicate nanocomposites 44 coacervates 67 nanoparticles 82–3 coalescence 80 coatings conductive 111 nanocoatings 27–8, 27 spin coating 27 sputter-coating 126 see also layer-by-layer assembly cochleates 67, 74–5 coenzyme Q10 64 collagen 6, 9, 48 colloidal particles 65 colloidosomes 83 colloids 6 Compton scattering 104, 164 condensation 32 conductive coatings 111 contamination in SEM 122–4, 123 controlled release drug delivery 69–70, 70 conventional TEM 127–8, 130–6, 131, 132, 134, 135 copolymers 20 copper, mass attenuation coefficient 218 core(polyester)-shell(polysaccharides) 79 critical aggregate concentration (CAC) 21 critical packing parameter 14 cryo-TEM 143 cubosomes 84 cumulant expansion DLS 159 curcumin 31, 58, 60 cyclodextrins 11–12 uses of 12 daidzein 59 dark-field images 133, 137

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Index

237

annular see annular dark-field images delivery systems 64–72 biological barriers 64–5 layer-by-layer thin films 25 modes of action 68, 69–72 controlled release 69–70, 70 targeting delivery 70–2 nanoemulsions 32 nanofibers 22 nanoscale see nanoscale delivery systems nanospheres 33 types and design principles 67–9, 68, 69 see also individual vehicles dendrimers 1, 67, 77–8 dendrosomes 77 dextran 11, 66, 73 dextran sulfate 25 diffusion coefficient 149 discriminator 152 DLS see dynamic light scattering Doppler effect 146 double emulsions 81 doxorubicin 67 drug delivery systems controlled release 69–70, 70 nanofibers 22 targeted 71–2 see also delivery systems dynamic light scattering 2, 145–61 apparatus 152–6, 154–6 cumulant method 159 data analysis 156–8 maximum-entropy method 159 multiple-decay methods 158 nonspherical particles 157–8 photon correlation spectroscopy 147, 151–2, 151 principle 145–51 regularization methods 158–9 dynodes 154

secondary 104, 105–6, 106, 155, 195 electron beams 196–7 electron energy-loss spectrum 140, 141 electron microscopy analytical 128, 139–42, 139, 141 high-resolution 133 scanning see scanning electron microscopy transmission see transmission electron microscopy electron sources 108, 108 electron-target interaction SEM 104–5, 105, 106 TEM 129–30, 129 electrospinning 9, 10, 21–2, 22 electrostatic interactions 7, 20 ellipsometry 190 emulsification high-energy 30–1 low-energy 31–3 ultrasonic 30–1 emulsifiers 30 emulsions double 81 foods 30 lycopene 56 microemulsions 6 nanoemulsions 6, 29–30, 32, 80–1 oil-in-water 80 water-in-oil 80 endothelium 71 energy-dispersive spectrometers 140 enthalpy 7 entropy 7 environmental SEMs 111 enzymes 11 epigallocatechin gallate 31, 60 Escherichia coli, detection of 190 exfoliated structures 43–4, 44 extracellular matrix 7–8

egg white protein 48 eggshell membrane protein 9, 10–11 Einstein’s photoelectric equation 222 elastic scattering 104, 164 see also X-ray diffraction electromagnetic lenses 109 electrons 104 Auger 104 backscattered 104, 105, 106 extranuclear 164

Fabry–Perot interferometer 147 fibrous assemblies 7 field-emission electron guns SEM 108, 108 TEM 128 fish myofibrillar protein 48 flavonoids 59, 64 bioavailability 61 flavor delivery 2 flocculation 80

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238

Index

5-fluorouracil 67, 77 focused ion beam (FIB) technique 142, 195–214 3D nanostructure fabrication 202 applications 202–7 biological samples 203 polymers 202–3 self-assembled protein structures 203–7, 204–8 etching/deposition 200 imaging 199, 199 ion beam production 196–8 ion implantation 200 ion-target interaction 198 limitations 207–14, 209–13 milling 199–200, 200 3D nanotomography with real-time imaging 201 overview 196, 197 principles 195–6 SEM dual-beam system 201 folate receptors, drug targeting 71–2 food matrix 56–8, 57 nutraceutical interaction with 61–4, 62, 63 food microstructure 56–8, 57 food packaging 2 nanocomposites 41–54 food quality 2 food safety 2 formaldehyde 28 Fourier transform 134, 164 fullerene 23 functional foods 55–6 delivery systems 64–72 biological barriers 64–5 modes of action 68, 69–72 nano-scale 65–7 nanoscale 72–85 types and design principles 67–9, 68, 69 food matrix 56–8, 57 nanoscale delivery systems 72–85 dendrimers 77–8 hydrogels 75 lipid nanoparticles 81–3 liposomes 1, 2, 6, 72–4 nano-cochleates 88–9 nanocrystalline particles 83–5 nanoemulsions 80–1 polymeric micelles 75–6 polymeric nanoparticles 78–80

Padua_bindex.indd 238

nutraceuticals 2, 25, 55–101, 59 delivery systems 64–72 health benefits and dose levels 59 interaction with food matrix 61–4, 62, 63 solubility and bioavailability 60–1 gallium ion sources 197–8 Gantrez 33 gate period 152 Gaussian distribution 158 gelatin 9, 19, 78 gene therapy 77 genistein 59 Gibbs free energy 32 gliadin 10 glutenin 10 glycolipids 14 gold microparticles colloidal 65 electron diffraction 132 graphene nanosheets 42–3 graphics processing unit 228 gravitational separation 80 guest molecules 78 hard X-rays 217, 230 helium-neon lasers 153 hemoglobin 6 high-angle annular dark field images 137–8, 138 high-resolution electron microscopy 133 hollow capsules 84 homogenization 5 high-pressure 30, 31 Huyghens wavelets 134 hyaluronan 26 hydrocolloids 13 hydrogels 1, 23, 67 nanoparticles 75 smart/intelligent 75 hydrogen bonds 7, 20 hydrophilic films 26 hydrophilicity 26 hydrophilic–lipophilic balance (HLB) 30 hydrophobic compounds 26 delivery systems 66 hydrophobic films 26 hydrophobic interactions 7, 20 hydroxyapatite 46 hydroxyapatite/chitosan nanocapsules 34 hydroxypropyl methylcellulose (HPMC) 47

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Index

image capture 224–6, 225 image resolution pixels 224, 225–6, 227 voxels 227, 227 imaging annular dark-field images 137–8, 138 bright-field images 133, 136–7 dark-field images 133, 137 FIB 199, 199 real-time 201 X-ray detection 168–9, 217–19, 218, 219 inclusion complexes 12 inelastic scattering 104, 164 infinite periodic minimal surfaces (IPMS) 14 infrared spectroscopy 45 inorganic materials 14–15 inositol 59 intercalated structures 43–4, 44 intestinal absorption 60 inverse Laplace transformation 157, 159 ill condition 157 iodine 59 ion beams 196–8 iron 59 bioavailability 61 iron oxide crystals 65 isoelectric point 22 isoflavones 59 kaemferol 59 kaolin clays 42, 49 kaolinite nanoplates 43 α-lactalbumin nanotubes 8–9 lactic acid 47 β-lactoglobulin–high-methoxyl pectin 14 Lambert-Beer law 217–18, 227, 232 Langmuir-Blodgett technique 27 Laplace transformation 156 inverse 157, 159 laser light sources 152–3 TEM00 mode 153 lauric acid 33 layer-by-layer assembly 15, 19, 24–9 bilayers 24–5 hollow nanocapsules 28–9 nanocoatings 27–8, 27 nanofilms on planar surfaces 25–7, 26 see also self-assembly lectins 67, 71, 79

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239

binding to carbohydrates 73 length-scale 5–17 lenses electromagnetic 109 SEM 109 aberrations 110, 110 TEM 130–1, 131 X-ray micro-CT 225–6, 226 light scattering dynamic see dynamic light scattering frequency 146–7, 147 phase 148–9, 148, 149 static 146 lignans 59 linoleic acid zein films 174–5, 175 lipase 11 lipid bilayers 190, 191 lipid drug conjugates 82 lipid nanoparticles 81–3 coacervates 82–3 lipid drug conjugates 82 nanostructure lipid carriers 82 solid 81–2 lipopolyplexes 74 liposome-in-micro spheres 73 liposomes 1, 2, 6, 72–4 arachaeosomes 73 classification 72 magnetoliposomes 73 niosomes 73 PEGylated 74 photosensitizer-based 74 polyplexes/lipopolyplexes 74 proteosomes 73 self-assembly 7 stealth 72 targeted drug delivery 71–2 transferosomes 73 virosomes 73 liquid crystals 14 liquid-metal ion source (LMIS) 196, 197–8 localized imaging 232–3, 233 lutein 59 lycopene 59 bioavailability 61 emulsion 56 macrophages 66 magnesium 56, 59 magnesium aluminum silicate 42, 43 magnetoliposomes 73

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240

Index

manganese 59 mass attenuation coefficient 218, 218 maximum-entropy DLS 159 melamine 28 membrane channels 6 metal oxides 26 micelles 1, 2, 5–17 casein 5, 6, 7 polymeric 67, 75–6 self-assembly 20–1 uses of 6 micro-CT see X-ray micro-CT microemulsions 6 microfluidization 31 microfocus X-ray tubes 223–4 microtubules, actin 6, 24 Mie scattering 146 milk nanostructures in 5 proteins see casein; whey protein milk fat globule membrane 74 milling 199–200, 200 3D nanotomography with real-time imaging 201 minerals 59 see also individual minerals molecular necklaces 12 monoclonal antibodies 72, 79 monoglycerides 14 mononuclear phagocytic system (MPS) 72 montmorillonite 15, 26 clays 42, 49 multiple-decay DLS 158 myristic acid 33 nano-cochleates 88–9 nano-scale delivery systems 65–7 nanocapsules 28–9, 78 nanocellulose 12–13 nanoclays see nanocomposites nanocoatings 27–8, 27 nanocomposites 1, 41–54 biobased 45–50 carboxymethylcellulose 46 cellulose 47 formation of 43–4, 44 pectin 46–7 polylactic acid 47–8 polymers 42–3 protein 48–9, 50 starch 46

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structure 44–5 zinc oxide 46 nanocrystalline particles 83–5 aquasomes 84 ceramic nanoparticles 84 colloidosomes 83 cubosomes 84 hollow capsules 84 nanotubes and nanowires 85 polyelectrolytes 85 nanoemulsions 6, 29–30, 80–1 double emulsions 81 high-energy emulsification 30–1 low-energy emulsification 31–3 in parenteral nutrition 6 phase-inversion 30, 32–3 nanofibers collagen 9 drug delivery systems 22 peptide 7 self-assembly 21–3 electrospinning 21–3, 22 hydrogels 1, 23 nanofibrils 8–9 nanofilms 25–7, 26 growth mode 27 on planar surfaces 25–7, 26 nanoparticles 33–4 calcium phosphate 71 carbohydrate ceramic 84 carbon 67 ceramic 67, 84 classification 65 delivery systems see delivery systems hydrogels 75 lipid 81–3 coacervates 82–3 lipid drug conjugates 82 solid 81–2 polymeric 78–80 SEM 117–19, 118, 119 nanoscale delivery systems 72–85 dendrimers 77–8 hydrogels 75 lipid nanoparticles 81–3 liposomes 1, 2, 6, 72–4 nano-cochleates 88–9 nanocrystalline particles 83–5 nanoemulsions 80–1 polymeric micelles 75–6 polymeric nanoparticles 78–80

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Index

nanospheres 33 nanostructures 5–17 3D fabrication 202 lipid carriers 82 self-assembly 19–40 nanotomography, 3D 201 nanotubes 1, 85 amyloidogenic protein 24 carbon 23, 26, 42 alpha-lactalbumin 8–9 self-assembly 23–4 whey protein 5 nanowhiskers 27 cellulose 47 nanowires 85 niosomes 73 nisin 74 nutraceuticals 2, 25, 55–101, 59 delivery systems 64–72 biological barriers 64–5 modes of action 67–72, 68 nano-scale 65–7 types and design principles 67–9, 68, 69 health benefits and dose levels 59 interaction with food matrix 61–4, 62, 63 solubility and bioavailability 60–1 see also functional foods nutrition 1–2 oil-in-water emulsions 80 oleic acid zein films SAXS 171–2, 172, 173 WAXS 171, 171 temperature-controlled 176–7, 176, 177 oligonucleotide 78 omega-3 fatty acids 58, 59 opsonization 66 osteopontin 10 Ostwald ripening 32, 80 oxygen transmission rate 14 paclitaxel 67 palmitic acid 33 parenteral nutrition 6 particle sizing 145–61 pectin 13, 25 nanocomposites 46–7 PEGylated liposomes 74 PEGylation 66, 72, 78

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241

Peltier heat pump 154 peptides 8–11, 20 nanofibers 7 permeability nanocapsules 28–9 water vapor 46, 48 pH 28, 56 phagocytes 66 phase-contrast artifacts 231–2, 231 phase-inversion temperature (PIT) 30, 32–3 phonons 198 phosphatidylserine 74 phospholipid membranes 6 phospholipids 14 phosphorus 59 photodetectors 154–5, 155 photodiode arrays 224 photomultiplier tubes 154–5, 155 photon correlation spectroscopy 147, 151–2, 151 see also dynamic light scattering photosensitizer-based liposomes 74 N-phthaloyl-carboxymethylchitosan 21 phytochemicals 55, 58 phytoestrogens 59 pi-pi interactions 20 piezoelectric immunosensors 189 pathogenic bacteria detection 190 pixels 224, 225–6, 227 size and spatial resolution 232 plasma 198 plasticizers 49 Poisson process 219 poloxamers 79 poloxamines 79 poly(alkylcyanoacrylate) (PACA) 78 poly(allylamine hydrochloride) (PAH) 25 poly(caprolactone) (PCL) 78, 79 polycarbophil 33 poly(cyanoacrylate) 78, 79 polycyclic aromatic hydrocarbons 63 poly(dimethyldiallylammonium chloride) 26 polydimethylsiloxane 220, 220 polyelectrolyte multilayers (PEMs) 24 nanocapsules 28–9 polyelectrolytes 85 polyethylene 41 polyethylene glycol (PEG) 8, 11, 66, 71, 72, 78, 79 polyethylene oxide (PEO) 66

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242

Index

polyethylene terephthalate (PET) 15, 41 polyethyloxazoline 72 polyglutamic acid 25 polyisohexyl cyanoacrylate (PIHCA) 78–9 polylactic acid (PLA) 19, 28, 45, 78, 79 nanocomposites 47–8 polylactic-co-glycolic acid 28 poly(DL-lactic-co-glycolic acid) 34 poly(D,L, lactide)-b-polyethylene glycol (PLA-PEG) 77 polymeric micelles 67, 75–6 polymeric nanoparticles 78–80 polymers FIB analysis 202–3 multilayer technology 15 nanocomposites 42–3 see also individual materials poly(methyl methacrylate) (PMMA) 42–3, 47 poly(methyl vinyl ether-co-maleic anhydride) (PVM/MA) 71, 79 polymethyloxazoline 72 polyoxamines 66 polypeptides 21 polyplexes 74 polypropylene 41 polypropylene–clay nanocomposites 45 polysaccharides 20, 21, 79 polysorbate 80 79 polystyrene 28, 41 poly(styrene sulfonate) (PSS) 25 polyvinyl alcohol 11 polyvinyl chloride (PVC) 41 poly(vinyl pyrrolidone) 11 potassium 59 probiotic bacteria 58 projection magnification 222–3, 223 proloxamers 66 protein–polysaccharides 13–14 coacervates 7 proteins 8–11, 20 amyloidogenic 8–9, 24 capsid 24 casein 9, 10, 19, 48 egg white 48 eggshell membrane 9, 10–11 fish myofibrillar 48 folding 7 interactions, QCM-D monitoring 187, 189 nanocomposites 48–9, 50

Padua_bindex.indd 242

sialoprotein 10 soy 19 structural changes, QCM-D monitoring 185–6, 187 whey 5, 19, 48–9 proteosomes 73 QCM-D see quartz crystal microbalance with dissipation quantum dot semiconductor nanocrystals 65 quartz crystal microbalance with dissipation (QCM-D) 26, 181–94 advantages 190–1, 191 applications 185–90, 186–9 adsorption/desorption kinetics 185, 186 monitoring of specific interactions 186–7, 188 as piezoelectric immunosensor 189 protein interactions 187–9, 189 protein structural changes 185–6, 187 data analysis 184–5, 186 Sauerbrey model 182, 182, 185 sensors 183–4, 183 Voight model 183, 185 quercetin 58, 59 quinoline 64 radiation damage in SEM 120–2, 120–2 real-time imaging 201 receptor-ligand interactions 187 refraction index 145 regularization method DLS 158–9 reticulo-endothelial system (RES) 72 reversed bicontinuous cubic phases 14 ring artifacts in X-ray micro-CT 229, 229 rotating-anode X-ray tubes 167 Rutherford scattering 137 salicylic acid 33 Salmonella typhimurium, detection of 190 sample containers for DLS 153–4, 154 sample-size reduction 232–3 saponins 59 Sauerbrey relationship 182, 182 scanning electron microscopy (SEM) 2, 13, 45, 103–26, 183, 195, 200–1 applications 111–19 controlled magnifications 115–17, 116, 117 nanoparticles 117–19, 118, 119

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Index

zein microstructures 112–15, 112–15, 204–8 backscattered electrons 104, 105, 106 conductive coatings 111 electron beam scanning 109 electron sources 108, 108 electron-target interaction 104–5, 105, 106 environmental SEMs 111 FIB dual-beam system 201 lens aberrations 110, 110 lenses and apertures 109 limitations 119–26 charging 124–6, 124–6 contamination 122–4, 123 radiation damage 120–2, 120–2 principles 103–4 secondary electrons 104, 105–6, 106 system 107, 107 vacuum 111 X-rays 107 see also scanning ion microscopy scanning ion microscopy (SIM) 199–200, 199, 200–1, 200 see also scanning electron microscopy scanning TEM (STEM) 128, 136–9, 136, 138 scintillators 224 seashells 45–6 secondary electrons 104, 105–6, 106, 155, 195 emission coefficient 105–6 secondary ions 195 Sefsol 218 32 selective area diffraction (SAD) 131 aperture 133 selenium 58, 59 self-assembly 6–8, 19–40 CAC 21 factors influencing 7 hierarchical 8 layer-by-layer 15, 19, 24–9 bilayers 24–5 hollow nanocapsules 28–9 nanocoatings 27–8, 27 nanofilms on planar surfaces 25–7, 26 micelles 20–1 nanofibers 21–3, 22 electrospinning 21–3, 22 hydrogels 1, 23 nanotubes 23–4

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243

zein microstructures 203–7, 204–8 SEM see scanning electron microscopy semiconductor diode lasers 153 sialoprotein 10 signal-to-noise ratio 219–20 silica 6, 67 silica barrier films 14–15 silicate clay nanocomposites 44 silicon, mass attenuation coefficient 218 silicon dioxide 6 silicon-based semiconductors 224 SIM see scanning ion microscopy small-angle X-ray scattering (SAXS) 169 applications 170 linoleic acid zein films 175, 175 oleic acid zein films 171–2, 172, 173 stearic acid zein films 174, 174 sodium 59 sodium alginate 78 sodium caseinate–gum arabic 13 sodium caseinate–gellan gum 14 sodium caseinate–high-methoxyl pectin 14 sodium caseinate–low-methoxyl pectin 13–14 soft X-rays 217 solid lipid nanoparticles 81–2 solubility 60–1 solution casting 27 soy proteins 13, 19 bionanocomposite films 49 spatial resolution 232 spin coating 27 sputter-coating 126 sputtering 198 staphylococcal enterotoxins, detection of 190 starch nanocomposites 46 static light scattering 146 stealth liposomes 72 stealth particles 79 stearic acid 33, 173 stearic acid zein films SAXS 174, 174 WAXS 173, 174 temperature-controlled 178–9, 178 Stokes–Einstein equation 149 submicron emulsions see nanoemulsions sulfur 59 supramolecular complexes 12 surface plasmon resonance (SPR) 26, 186–7 lipid bilayer formation 190, 191

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244

Index

surfactants 30 synchrotron radiation sources 167, 217 tactoid structures 43–4, 44 tangeretin 59 targeted release drug delivery 70–2 taurine 59 tea catechins 58 tea polyphenols 60 TEM see transmission electron microscopy temperature-controlled WAXS 176–9, 176–8 oleic acid zein films 176–7, 176, 177 stearic acid zein films 178–9, 178 tetracycline 64 tetrahydrofuran 28 thermal deposition 27 thermionic electron gun 108, 108 Thomson scattering 164 tissue engineering 22 titania 48, 67 topotecan 67 transferosomes 73 transmission electron microscopy (TEM) 2, 45, 104, 127–44 analytical 128, 139–42, 139, 141 apertures 132–3 conventional 127–8, 130–6, 131, 132, 134, 135 electron-target interaction 129–30, 129 instrumentation 128–9 limitations 143 sample preparation 142–3, 196 scanning 128, 136–9, 136, 138 tubulin 24 tunicates 13 Tween 80 32 ultramicrotomy 142 ultrasonic emulsification 30–1 urease 11 vaccines, edible 58 vacuum for SEM 111 van der Waals forces 7, 20 vasoactive intestinal peptide receptors (VIP-R) 73 virosomes 73 viscosity 149–50 vitamin A 59 vitamin B1 59

Padua_bindex.indd 244

vitamin B2 59 vitamin B3 59 vitamin B5 59 vitamin B6 59 vitamin B7 59 vitamin B9 59 vitamin B12 59 absorption 71 vitamin C 58, 59 vitamin D 59 vitamin E 58, 59, 63 vitamin K 59 Voight relationship 183, 185 voxels 227, 227 size and spatial resolution 232 water vapor permeability 46, 48 water-in-oil emulsions 80 wheat gluten 10 whey protein 19 nanocomposites 48–9 nanotubes 5 whey protein–gum arabic 13 whey protein–carboxy methylcellulose 13 whey protein–xanthan gum 14 wide-angle X-ray scattering (WAXS) 169 applications 170 linoleic acid zein films 174–5, 175 oleic acid zein films 171 stearic acid zein films 173, 174 temperature-controlled 176–9, 176–8 oleic acid zein films 176–7, 176, 177 stearic acid zein films 178–9, 178 wound dressings 22 X-rays 107, 163–4 elastic scattering 104, 164 hard 217, 230 incident 164 inelastic scattering 104, 164 photon wavelength 222 soft 217 X-ray computerized microtomography see X-ray micro-CT X-ray detectors 168–9 image plates 168–9 photo count 168 small-angle X-ray scattering (SAXS) 169, 170 wide-angle X-ray scattering (WAXS) 169, 170

1/20/2012 12:02:18 PM

Index

X-ray diffraction (XRD) 2, 22, 45, 163–79 applications 169–79 temperature-controlled WAXS 176–9, 176–8 zein-fatty acid films 170–5, 171–5 set-up 165 small angle 45 wide angle 45 X-ray detectors 168–9 X-ray sources 165–7 X-ray images 217–19, 218, 219 X-ray micro-CT 215–34 artifacts 228–32 beam-hardening artifacts 230–1 center errors 230, 230 phase-contrast artifacts 231–2, 231 ring artifacts 229, 229 data reconstruction 226–8, 227, 228 localized imaging 232–3, 233 sample-size reduction 232–3 set-up 220–6, 221–3, 225, 226 beam profile 221, 221 detectors 224 image capture 224–6, 225 projection magnification 222–3, 223 scanner 220 spatial resolution 232

Padua_bindex.indd 245

245

X-ray generation 215–17, 216 X-ray images 217–20, 218–20 X-ray scattering 164–5 X-ray sources 165–7, 215–17, 216 synchrotrons 167, 217 X-ray tubes 166–7 X-ray tubes 166–7 microfocus 223–4 rotating-anode 167 sealed 166–7 yogurt 56 Young’s modulus 26, 49 zeaxanthin 59 zein microstructures 9, 10, 19, 49 FIB-SEM 204–8 limitations 209–13 self-assembly 203–7, 204–8 SEM 112–15, 112–15 charging artifacts 124–6 contamination 123 radiation damage 120–2 SIM 199–200, 199, 200 zein-fatty acid films, X-ray characterization 170–5, 171–5 zero loss peak 140 zinc oxide nanocomposites 46

1/20/2012 12:02:18 PM

Food Science and Technology G E N E R A L F O O D S C I E N C E & T E C H N O LO G Y, E N G I N E E R I N G A N D P R O C E S S I N G Organic Production and Food Quality: A Down to Earth Analysis Handbook of Vegetables and Vegetable Processing Nonthermal Processing Technologies for Food Thermal Procesing of Foods: Control and Automation Innovative Food Processing Technologies Handbook of Lean Manufacturing in the Food Industry Intelligent Agrifood Networks and Chains Practical Food Rheology Food Flavour Technology, 2nd edition Food Mixing: Principles and Applications Confectionery and Chocolate Engineering Industrial Chocolate Manufacture and Use, 4th edition Chocolate Science and Technology Essentials of Thermal Processing Calorimetry in Food Processing: Analysis and Design of Food Systems Fruit and Vegetable Phytochemicals Water Properties in Food, Health, Pharma and Biological Systems Food Science and Technology (textbook) IFIS Dictionary of Food Science and Technology, 2nd edition Drying Technologies in Food Processing Biotechnology in Flavor Production Frozen Food Science and Technology Sustainability in the Food Industry Kosher Food Production, 2nd edition

Blair Sinha Zhang Sandeep Knoerzer Dudbridge Bourlakis Norton Taylor Cullen Mohos Beckett Afoakwa Tucker Kaletunç de la Rosa Reid Campbell-Platt IFIS Chen Havkin-Frenkel Evans Baldwin Blech

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Paliyath Paeschke Mine Kneifel Smith Pasupuleti Mine Jardine Onwulata Cho

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F U N C T I O N A L F O O D S , N U T R AC E U T I C A L S & H E A LT H Functional Foods, Nutraceuticals and Degenerative Disease Prevention Nondigestible Carbohydrates and Digestive Health Bioactive Proteins and Peptides as Functional Foods and Nutraceuticals Probiotics and Health Claims Functional Food Product Development Nutraceuticals, Glycemic Health and Type 2 Diabetes Nutrigenomics and Proteomics in Health and Disease Prebiotics and Probiotics Handbook, 2nd edition Whey Processing, Functionality and Health Benefits Weight Control and Slimming Ingredients in Food Technology

INGREDIENTS Hydrocolloids in Food Processing Natural Food Flavors and Colorants Handbook of Vanilla Science and Technology Enzymes in Food Technology, 2nd edition Food Stabilisers, Thickeners and Gelling Agents Glucose Syrups – Technology and Applications Dictionary of Flavors, 2nd edition Vegetable Oils in Food Technology, 2nd edition Oils and Fats in the Food Industry Fish Oils Food Colours Handbook Sweeteners Handbook Sweeteners and Sugar Alternatives in Food Technology

F O O D S A F E T Y, Q UA L I T Y A N D M I C R O B I O LO G Y Food Safety for the 21st Century The Microbiology of Safe Food, 2nd edition Analysis of Endocrine Disrupting Compounds in Food Microbial Safety of Fresh Produce Biotechnology of Lactic Acid Bacteria: Novel Applications HACCP and ISO 22000 – Application to Foods of Animal Origin Food Microbiology: An Introduction, 2nd edition Management of Food Allergens Campylobacter Bioactive Compounds in Foods Color Atlas of Postharvest Quality of Fruits and Vegetables Microbiological Safety of Food in Health Care Settings Food Biodeterioration and Preservation Phycotoxins Advances in Food Diagnostics Advances in Thermal and Non-Thermal Food Preservation

For further details and ordering information, please visit www.wiley.com/go/food Padua_both.indd 246

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Food Science and Technology from Wiley-Blackwell S E N S O RY S C I E N C E , CO N S U M E R R E S E A R C H & N E W P R O D U C T D E V E LO P M E N T Sensory Evaluation: A Practical Handbook Statistical Methods for Food Science Concept Research in Food Product Design and Development Sensory and Consumer Research in Food Product Design and Development Sensory Discrimination Tests and Measurements Accelerating New Food Product Design and Development Handbook of Organic and Fair Trade Food Marketing Multivariate and Probabilistic Analyses of Sensory Science Problems

Kemp Bower Moskowitz Moskowitz Bi Beckley Wright Meullenet

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F O O D L AW S & R E G U L AT I O N S The BRC Global Standard for Food Safety: A Guide to a Successful Audit Food Labeling Compliance Review, 4th edition Guide to Food Laws and Regulations Regulation of Functional Foods and Nutraceuticals

D A I RY F O O D S Dairy Ingredients for Food Processing Processed Cheeses and Analogues Technology of Cheesemaking, 2nd edition Dairy Fats and Related Products Bioactive Components in Milk and Dairy Products Milk Processing and Quality Management Dairy Powders and Concentrated Products Cleaning-in-Place: Dairy, Food and Beverage Operations Advanced Dairy Science and Technology Dairy Processing and Quality Assurance Structure of Dairy Products Brined Cheeses Fermented Milks Manufacturing Yogurt and Fermented Milks Handbook of Milk of Non-Bovine Mammals Probiotic Dairy Products

S E A F O O D, M E AT A N D P O U LT RY Handbook of Seafood Quality, Safety and Health Applications Fish Canning Handbook Fish Processing – Sustainability and New Opportunities Fishery Products: Quality, safety and authenticity Thermal Processing for Ready-to-Eat Meat Products Handbook of Meat Processing Handbook of Meat, Poultry and Seafood Quality

B A K E RY & C E R E A L S Whole Grains and Health Gluten-Free Food Science and Technology Baked Products – Science, Technology and Practice Bakery Products: Science and Technology Bakery Food Manufacture and Quality, 2nd edition

B E V E R AG E S & F E R M E N T E D F O O D S / B E V E R AG E S Technology of Bottled Water, 3rd edition Wine Flavour Chemistry, 2nd edition Wine Quality: Tasting and Selection Beverage Industry Microfiltration Handbook of Fermented Meat and Poultry Microbiology and Technology of Fermented Foods Carbonated Soft Drinks Brewing Yeast and Fermentation Food, Fermentation and Micro-organisms Wine Production Chemistry and Technology of Soft Drinks and Fruit Juices, 2nd edition

PAC K AG I N G Food and Beverage Packaging Technology, 2nd edition Food Packaging Engineering Modified Atmosphere Packaging for Fresh-Cut Fruits and Vegetables Packaging Research in Food Product Design and Development Packaging for Nonthermal Processing of Food Packaging Closures and Sealing Systems Modified Atmospheric Processing and Packaging of Fish Paper and Paperboard Packaging Technology

For further details and ordering information, please visit www.wiley.com/go/food

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