Annual Review of Nano Research, Volume 3

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Volume 3

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ANNUAL REVIEW OF NANO RESEARCH Series Editors: Guozhong Cao (University of Washington, USA) C Jeffrey Brinker (University of New Mexico and Sandia National Laboratories, USA)

Vol. 1: ISBN-13 ISBN-10 ISBN-13 ISBN-10

978-981-270-564-8 981-270-564-3 978-981-270-600-3 (pbk) 981-270-600-3 (pbk)

Vol. 2: ISBN-13 978-981-279-022-4 ISBN-10 981-279-022-5 ISBN-13 978-981-279-023-1 (pbk) ISBN-10 981-279-023-3 (pbk) Vol. 3: ISBN-13 978-981-4280-51-8 ISBN-10 981-4280-51-8

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Volume 3

Editors

Guozhong Cao Qifeng Zhang University of Washington, USA

C. Jeffrey Brinker University of New Mexico and Sandia National Laboratories, USA

World Scientific NEW JERSEY

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TABLE OF CONTENTS

Preface Contributing Authors

xv xvii

Chapter 1. Nanoscale Biosensors and Biochips Wayne R. Leifert, Richard V. Glatz, Kelly Bailey, Tamara Cooper, Marta Bally, Brigitte Maria Stadler, Erik Reimhult and Joseph G. Shapter 1. General Introduction 2. Biological Detectors Used in Biosensing and Biochips 2.1. G-Protein Coupled Receptor Biosensors (GPCRs) 2.2. Pore-Forming Proteins 2.3. Cell- and Viral-Based Sensing 3. Lipid Supports for Biosensor and Biochip Fabrication 3.1. Why Functionalize Biosensors with Lipid Membranes? 3.2. Methods to Assemble Supported Lipid Membranes 3.3. Supported Lipid Membrane Platforms 3.4. Advanced Sensors Functionalized with Lipid Membranes 3.5. Future Perspectives 4. Nanopatterning for Biosensing and Biochip Fabrication 4.1. Parallel Nanopatterning Methods 4.2. Serial Nanopatterning Methods 5. Sensing Substrates: A Closer Look at Nanotubes 5.1. Carbon Nanotube Electrodes for Communicating with Redox Proteins 5.2. Aligned Carbon Nanotube Electrodes for Direct Electron Transfer to Enzymes 6. Reporter Technologies: Nano-Sized Labels for Biosensing Applications 6.1. Biosensors Utilizing Optical Reporting 6.2. Biosensors Utilising Electrochemical Reporting

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1 3 3 13 16 25 25 27 29 32 33 34 34 38 40 40 43 45 46 50

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7. Biosensing Applications 7.1. Medical 7.2. Food and Wine 7.3. Explosives and Biowarfare 7.4. Environmental 8. Conclusion References

53 53 55 56 57 59 60

Chapter 2. Surface Modifications and Applications of Magnetic and Selective Nonmagnetic Nanoparticles Rui Shen and Hong Yang

83

1. Introduction 2. General Approaches to Surface Modification of Nanostructures 2.1. Adsorption and Self-Assembly 2.2. Surface Modification Based on Organic Reactions 2.3. Surface Modification Based on Polymerization 2.4. Surface Modification with Inorganic Layers Based on Sol-Gel Approaches 2.5. Surface Modification with Multiple or Composite Layers 2.6. Experimental Designs 2.7. Surface Modification in the Synthesis of Hollow Spheres 3. Surface Modification of Magnetic Nanostructures 3.1. Oxides 3.2. Metals 3.3. Metal Alloys 4. Surface Modification in the Synthesis of Higher-Ordered and Complex Nanostructures 4.1. Hollow and Yolk-Shell Nanostructures 4.2. Anisotropic and Onion-Like Nanostructures 4.3. Other Higher Ordered Nanostructures 5. Applications of Surface-Modified Magnetic Nanoparticles 5.1. Surface Modifications in Nonbiological Applications 5.2. Surface Modifications in Biological Applications 6. Conclusion

83 86 87 90 92 94 99 100 102 103 104 108 111 114 115 120 122 127 127 129 137

Table of Contents

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Acknowledgments References

137 137

Chapter 3. Progress in Bionanocomposite Materials Eduardo Ruiz-Hitzky, Margarita Darder and Pilar Aranda

149

1. Introduction 2. Bionanocomposites for Bioplastics 3. Bionanocomposites for Biomedical Applications 4. Bionanocomposites for Sensor Devices and Other Applications 5. Concluding Remarks Acknowledgments References

149 152 162 171 180 181 181

Chapter 4. Mesoporous Silica Nanoparticles: Synthesis and Applications Juan L. Vivero-Escoto, Brian G. Trewyn and Victor S.-Y. Lin 1. Introduction 2. Synthesis of Mesoporous Silica Nanoparticles 2.1. Control of Morphology 2.2. Control of Surface Functionalization 3. Catalysis 3.1. Cooperative Catalysis (Acid/Base) 3.2. Gatekeeping Effect 3.3. Other Applications in Catalysis 4. Biotechnological and Biomedical Applications 4.1. Uptake and Intracellular Performance of MSNs 4.2. Controlled Delivery Systems 4.3. Biosensors 4.4. Multimodal Cell Imaging 5. Conclusions and Outlook Acknowledgments References

191

191 193 194 199 202 202 204 206 208 209 212 219 222 225 226 226

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Chapter 5. Nanostructured Mesoporous Materials as Drug Delivery Systems Isabel Izquierdo-Barba, Daniel Arcos and Maria Vallet-Regí

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1. Introduction 2. Cytotoxicity, Biocompatibility and Bioactivity of Silica Mesoporous Materials 3. Tailoring Mesoporous Drug Delivery Systems-Textural Properties Considerations 3.1. Pore Diameter 3.2. Surface Area 3.3. Pore Volume 3.4. Increasing the Surface Area - The Hybrid Route 4. Surface Functionalization of Mesoporous Drug Delivery Systems 5. Dosage in Mesoporous Materials 6. Mesoporous Materials for Intracellular Targeting 6.1. Cell Mechanism for Particles Internalization 6.2. Microstructural Considerations for SiO2 Nanoparticles Intracellular Targeting 7. Stimuli-Responsive Mesoporous Materials 7.1. Drug Release Mediated by Chemical Stimuli 7.2. Drug Release Mediated by Thermal Stimuli 7.3. Drug Release Mediated by Photo-Chemical Stimuli 7.4. Drug Release Mediated by Magnetic Stimuli 8. Conclusions and Outlook Acknowledgments References

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247 250 254 254

Chapter 6. Chemical Synthesis, Self-Assembly and Applications of Magnetic Nanoparticles Sheng Peng, Jaemin Kim and Shouheng Sun

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1. Introduction 1.1. General Background 1.2. Chemical Syntheses of Nanoparticles

275 275 277

236 239 239 244 244 245

256 259 260 263 263 264 267 269 269

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2. Ferrite Nanoparticles: MFe2O4 (M = Fe, Mn, Co) 2.1. Chemical Syntheses of Spherical Ferrite Nanoparticles 2.2. Shape-Controlled Synthesis and Self-Assembly 2.3. Surface Modification for Biological Applications 3. Metallic Iron, Cobalt and Iron-Cobalt Alloy Nanoparticles 3.1. Synthesis and Stabilization of Metallic Fe, Co, and FeCo Particles 3.2. Self-Assembly, Shape-Controlled Synthesis of Fe and Co 4. Tetragonal (L10-Phase) Hard Magnetic FePt Nanoparticles and Their Applications 4.1. General Chemical Syntheses of fcc-FePt Nanoparticles and the Phase Change via Thermal Treatment 4.2. Shape Controlled FePt Nanoparticles and Their Self-Assembly 4.3. Synthesis of Dispersible fct-FePt Nanoparticles 5. Rare-Earth Hard Magnets: Going Into Nanoscale 6. Summary and Outlook Acknowledgments References Chapter 7. Recent Development and Applications of Nanoimprint Technology Xing Cheng and L. Jay Guo 1. Introduction 2. Material Flow Behavior and the Associated Polymer Chain Alignment in NIL 2.1. Polymer Chain Alignment in Nanoimprinted Polymer Micro- and Nanostructures 2.2. Improving the Performance of Polymer Electronics by Nanoimprint-Induced Chain Orientation 3. Reversal Nanoimprint Lithography 3.1. Principles of Reversal Nanoimprint 3.2. Residual Layer Removal in Reversal Nanoimprint 3.3. Building 3D Polymer Nanostructures 3.4. Process Yield of Reversal Nanoimprint

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279 280 283 286 288 289 295 298 299 299 301 303 307 307 307

317 317 320 320 323 325 325 326 328 332

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4. Recent Applications of NIL 4.1. Organic Solar Cells with Imprinted Nanoscale Morphology 4.2. Nanoimprinting Nafion® Film for Micro Fuel Cell Applications 5. Roll-To-Roll Nanoimprint Lithography (R2RNIL) 6. Conclusion Acknowledgments References Chapter 8. Three-Dimensional Nanostructure Fabrication by Focused-Ion-Beam Chemical-Vapor-Deposition Shinji Matsui 1. Introduction 2. Three-Dimensional Nanostructure Fabrication 2.1. Fabrication Process 2.2. Three-Dimensional Pattern Generating System 3. Nanoeletromechanics 3.1. Young’s Modulus Measurement 3.2. Free-Space-Nanowiring 3.3. Nanoelectrostatic Actuator 4. Nanooptics: Brilliant Blue Observation from a Morpho-Butterfly-Scale Quasi-Structure 5. Nanobiology 5.1. Nanoinjector 5.2. Nanomanipulator 6. Summary References

335 335 339 341 346 348 348

351 351 352 353 356 359 359 364 371 373 376 376 379 382 382

Chapter 9. Dye-Sensitized Solar Cells Based on Nano-Structured Zinc Oxide Qifeng Zhang and Guozhong Cao

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1. Introduction 2. Nanostructures Offering Large Specific Surface Area

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2.1. ZnO Nanoparticulate Films 2.2. Nanoporous Structured ZnO Films 2.3. Other Nano-Structured ZnO Films 3. Nanostructures with Direct Pathway for Electron Transport 3.1. ZnO Nanowires 3.2. ZnO Nanotubes 3.3. ZnO Nanotips 3.4. ZnO Nanoflowers 3.5. Dendritic ZnO Nanowires 4. Core-shell Structures with ZnO Shell for Reduced Recombination Rate 4.1. Fabrication of Core-Shell Structures and Influence of Shell Thickness 4.2. The Role of ZnO Shell 5. Light Scattering Enhancement Effect 5.1. ZnO Aggregates 5.2. One-Dimensional ZnO Nanostructures for Light Scattering 6. Limitation on ZnO-Based DSSCs 6.1. Instability of ZnO in Acidic Dyes 6.2. Low Electron Injection Efficiency 6.3. New Types of Photosensitizers for ZnO 7. Conclusion and Outlook 7.1. Surface Modification of ZnO Aggregates - An Indirect Method for TiO2 Aggregates 7.2. Hydrothermal Growth of TiO2 Nanoparticle Aggregates 7.3. Emulsion-Assisted Synthesis of TiO2 Nanostructure Aggregation 7.4. Electrostatic Spray Deposition Fabrication of TiO2 Aggregates 7.5. Synthesis of Porous-Structured TiO2 Spheres Acknowledgments References

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390 394 397 399 400 403 403 404 405 406 407 408 410 412 415 416 416 420 422 423 426 427 428 429 429 430 430

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Chapter 10. Nanocomposites as High Efficiency Thermoelectric Materials Suraj Joottu Thiagarajan, Wei Wang and Ronggui Yang 1. Introduction to Thermoelectricity 2. Nanocomposites as Highly Efficient Thermoelectric Materials 2.1. Modeling of Phonon Transport 2.2. Modeling of Electron Transport 3. Synthesis of Thermoelectric Nanocomposites 3.1. Preparation of Nanocomposites by Compaction Techniques 3.2. Synthesis of Nanocomposites by Phase Separation 4. Recent Achievements in Thermoelectric Nanocomposites 4.1. Bi2Te3-Based Nanocomposites for Low Temperature Applications 4.2. Medium Temperature Materials 4.3. High Temperature Materials 5. Summary Acknowledgments References

441

442 450 452 456 459 460 467 469 470 473 477 479 480 481

Chapter 11. Nanostructured Materials for Hydrogen Storage Saghar Sepehri and Guozhong Cao

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1. Introduction 2. Hydrogen Storage by Physisorption 2.1. Nanostructured Carbon 2.2. Zeolites 2.3. Metal – Organic Frameworks 2.4. Clathrates 2.5. Polymers with Intrinsic Microporosity 3. Hydrogen Storage by Chemisorption 3.1. Metal and Complex Hydrides 3.2. Chemical Hydrides 3.3. Nanocomposites

487 490 491 493 494 495 497 498 499 502 504

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4. Summary Acknowledgments References

511 511 512

Chapter 12. Recent Advances in the Characterization of Mesoporous Materials by Physical Adsorption Matthias Thommes

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1. Introduction 2. General Aspects of Surface and Pore Size Analysis by Physisorption 3. Pore Condensation and Hysteresis in Mesoporous Materials 3.1. Pore Condensation 3.2. Interpretation of Adsorption Hysteresis 4. Comments to Mesopore Size Analysis 4.1. Classical Methods 4.2. Pore Size Analysis by Non Local Density Functional Theory (NLDFT) 4.3. Hysteresis and Pore Size Analysis 5. Summary and Conclusion Acknowledgment References

516 521 524 524 526 542 542 543 546 548 550 550

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PREFACE

Annual Review of Nano Research publishes excellent review articles in selected topic areas authored by those who are authorities in their own subfields of nanotechnology with two vital aims: (1) to present a comprehensive and coherent distilling of the state-of-the-art experimental results and understanding of theories detailed from the otherwise segmented and scattered literature, and (2) to offer critical opinions regarding the challenges, promises, and possible future directions of nano research. The third volume of Annual Review of Nano Research includes 11 articles offering a concise review detailing recent advancements in a few selected subfields in nanotechnology. The first topic to be focused upon in this volume is the bio-applications of and bio-inspired nanostructured materials. The second featured subfield is the recent advancement in the synthesis and fabrication of nanomaterials or nanostructures. Energy related applications of nanostructures and nanomaterials are the third focal topic in this volume. We want to thank all the contributing authors for their time and efforts devoted to the excellent review articles published in this volume. Mr. Yeow-Hwa Quek from World Scientific Publishing was responsible for much of the coordination necessary to make the publication of this volume possible.

Guozhong Cao Seattle, WA Qifeng Zhang Seattle, WA C. Jeffrey Brinker Albuquerque, NM

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CONTRIBUTING AUTHORS

Aranda, Pilar * Instituto de Ciencia de Materiales de Madrid, Spain Arcos, Daniel * Universidad Complutense de Madrid, Madrid, Spain * Networking Research Center on Bioengineering, CIBER-BBN, Spain Bailey, Kelly * Commonwealth Scientific and Industrial Research Organization (CSIRO), Australia * The University of Adelaide, Australia Bally, Marta * Institute for Biomedical Engineering, Switzerland Cao, Guozhong * University of Washington, USA Cheng, Xing * Texas A&M University, USA Cooper, Tamara * Commonwealth Scientific and Industrial Research Organization (CSIRO), Australia * The University of Adelaide, Australia Darder, Margarita * Instituto de Ciencia de Materiales de Madrid, Spain * Instituto Madrileño de Estudios Avanzados en Materiales (IMDEAMateriales), Spain Glatz, Richard V. * South Australian Research and Development Institute (SARDI), Australia xvii

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Guo, L. Jay * The University of Michigan, USA Izquierdo-Barba, Isabel * Universidad Complutense de Madrid; Madrid, Spain * Networking Research Center on Bioengineering, CIBER-BBN, Spain Kim, Jaemin * Brown University, USA Leifert, Wayne R. * Commonwealth Scientific and Industrial Research Organization (CSIRO), Australia Lin, Victor S.-Y. * U.S. Department of Energy Ames Laboratory, USA * Iowa State University, USA Matsui, Shinji * University of Hyogo 3-1-2 Koto, Japan Peng, Sheng * Brown University, USA Reimhult, Erik * Laboratory for Surface Science and Technology, Switzerland Ruiz-Hitzky, Eduardo * Instituto de Ciencia de Materiales de Madrid, Spain Sepehri, Saghar * University of Washington, USA Shapter, Joseph G. * Flinders University, Australia Shen, Rui * University of Rochester, USA

Contributing Authors

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Stadler, Brigitte Maria * The University of Melbourne, Australia Sun, Shouheng * Brown University, USA Thiagarajan, Suraj Joottu * University of Colorado, USA Thommes, Matthias * Quantachrome Instruments, USA Trewyn, Brian G. * U.S. Department of Energy Ames Laboratory, USA * Iowa State University, USA Wang, Wei * University of Colorado, USA Yang, Hong * University of Rochester, USA Yang, Ronggui * University of Colorado, USA Vallet-Regí, Maria * Universidad Complutense de Madrid; Madrid, Spain * Networking Research Center on Bioengineering, CIBER-BBN, Spain Vivero-Escoto, Juan L. * U.S. Department of Energy Ames Laboratory, USA * Iowa State University, USA Zhang, Qifeng * University of Washington, USA

CHAPTER 1 NANOSCALE BIOSENSORS AND BIOCHIPS

Wayne R. Leifert1,*, Richard V. Glatz2, Kelly Bailey3,4, Tamara Cooper3,4, Marta Bally5, Brigitte Maria Stadler6, Erik Reimhult7 and Joseph G. Shapter8 1

Commonwealth Scientific and Industrial Research Organization (CSIRO), Division of Human Nutrition, Adelaide, Australia; 2South Australian Research and Development Institute (SARDI), Department of Entomology, Adelaide, Australia; 3Commonwealth Scientific and Industrial Research Organization (CSIRO), Division of Molecular and Health Technologies, Adelaide, Australia; 4 School of Molecular and Biomedical Science, The University of Adelaide, Adelaide, Australia; 5Laboratory of Biosensors and Bioelectronics, Institute for Biomedical Engineering, ETH Zurich, Zurich, Switzerland; 6Centre for Nanoscience and Nanotechnology, Department of Chemical and Biomolecular Engineering, The University of Melbourne, Melbourne, Australia; 7Laboratory for Surface Science and Technology, Department of Materials, ETH Zurich, Zurich, Switzerland; 8School of Chemistry, Physics and Earth Sciences, Flinders University, Adelaide, Australia *Corresponding author, Email: [email protected]

1. General Introduction Recent advances in molecular biology, surface chemistry, protein purification, signal transduction/amplification, lipid chemistry and nanofabrication technologies have converged in a relatively new field of science, that of molecular biosensing. The growing level of interest in biosensing research has seen the formation of a range of specific journals reporting on advances in the field. Biosensors and biochips have many potential (and some current) applications including provision of point-ofcare diagnostic tools, high-throughput drug discovery tools and in-field sensing tools for a variety of compounds including toxins and/or contaminants. Biosensors are generally accepted as being analytical

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devices based on a biologically active or biologically mimetic compound (a detector) coupled to a physical signal transduction mechanism (a transducer/reporter). The interaction of analyte with its biomolecular detector is thus exploited to produce an effect that can be measured through the transducer. Biochips generally refer to an array of individual biosensors, and can also be referred to as nucleotide or protein microarrays. This review explores examples of the various approaches to biosensor and biochip fabrication, including examination of components important to each system. We discuss advantages and disadvantages of biosensors that exploit whole cells as detectors and those which make use of one or several specific molecules, the most widely utilized being membrane-associated proteins, such as G-protein coupled receptors (GPCRs) and ion-channels, due to their diversity and current importance for sensing and screening technologies. There are two major challenges to establishing functionally active biological components within a sensor or chip design, these being the controlled capture and positioning of the biological detector onto a surface and maintenance of the detector’s functional/structural integrity. In the case of membrane proteins, it is vital that an appropriate hydrophobic environment is available to maintain protein structure and function. The use of lipid supports for this purpose, is being widely studied, and is discussed in this review. In combination with appropriate surface compositions, various techniques of nanopatterning technologies are important in the fabrication of biosensors in order to control the location, distribution, amount and orientation of the biomolecules on the surface. There are also a range of physical and biological substrates on which sensor and chip platforms are built and we pay particular attention to nanotubes as physical substrates due to their promising application in electrochemical biosensing. In order to detect changes occurring at the sensor or chip surface, a variety of transduction techniques exist and we discuss a range of nanosized reporter labels for their potential for biosensing applications. Finally, we touch on some of the applications and specific analytes commonly monitored using biosensing today.

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2. Biological Detectors Used in Biosensing and Biochips Since the first description of the biosensor concept by Clark Jr. and Lyons in 1962 involving glucose oxidase and the oxygen electrode [1], a variety of front-end biological detectors have been investigated for use in biosensing. These bio-recognition elements include enzymes, membrane proteins such as cell-receptors and ion-channels, antibodies, nucleic acids, virus particles and intact cells. We begin the discussion with an investigation of membrane proteins, which as diverse biomolecules crucial in cellular signaling and communication as well as regulation of transport into and out of the cell are of significant interest in the fabrication of biosensors and biochips. Because of their diversity, biological importance and level of characterization, the two key classes of membrane proteins utilized are the GPCR family and ion-channels. 2.1. G-Protein Coupled Receptor Biosensors (GPCRs) 2.1.1. Importance of GPCRs Many disease processes involve aberrant or altered GPCR signaling dynamics and GPCRs represent the most significant target class for medicinal pharmaceuticals (≈50% of marketed drugs, see Table 1) [2]. GPCRs are associated with almost every major therapeutic category or disease class, including pain, asthma, inflammation, obesity, cancer, as well as cardiovascular, metabolic, gastrointestinal and central nervous system diseases [3]. It is this vitally important function of these cellsurface receptors combined with the huge diversity of specific ligands they bind, which makes GPCRs so physiologically significant and attractive for biosensing applications. Therefore, there is a need to develop sophisticated and appropriate GPCR biosensors for the detection of a variety of ligands (Figure 1). This section focuses on some of the available cell-free GPCR assay nanotechnologies [4] and describes some of the more sophisticated functional GPCR biosensors. Cell-free biosensors have the potential advantage of being applicable to a range of assay environments in which cells may be damaged.

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Table 1. Examples of some pharmaceuticals which target GPCRs for the indicated condition or disease state.

Brand Name (generic)

G-protein coupled receptor(s)

Disease/Indication

Zyprexa (Olanzapine)

Serotonin 5-HT2 and Dopamine

Schizophrenia, Antipsychotic

Risperdal (Risperidone)

Serotonin 5-HT2

Schizophrenia

Claritin (Loratidine)

Histamine H1

Rhinitis, Allergies

Imigran (Sumatriptan)

Serotonin 5-HT1B/1D

Migraine

Cardura (Doxazosin)

α-adrenoceptor

Prostate hypertrophy

Tenormin (Atenolol)

β1-adrenoceptor

Coronary heart disease

Serevent (Salmeterol)

β2-adrenoceptor

Asthma

Duragesic (Fentanyl)

Opioid

Pain

Imodium (Loperamide)

Opioid

Diarrhea

Cozaar (Losartan)

Angiotensin II

Hypertension

Zantac (Ranitidine)

Histamine H2

Peptic ulcer

Cytotec (Misoprostol)

Prostaglandin PGE1

Ulcer

Zoladex (Goserelin)

Gonadotrophin-releasing factor

Prostate cancer

Requip (Ropinirole)

Dopamine

Parkinson’s disease

Atrovent (Ipratropium)

Muscarinic

Chronic obstructive pulmonary disease (COPD)

GPCR activation can be initiated by a wide variety of stimuli such as light, odorants, neurotransmitters and hormones (Figure 1). In cells, the extracellular ligand is specifically and sensitively detected by a cell surface GPCR. Once binding/recognition takes place, the GPCR triggers the activation of a cellular heterotrimeric G-protein (guanine nuceleotidebinding protein) complex consisting of Gα, Gβ and Gγ subunits (Figure 1). Finally, the “signal transduction” cascade (in whole cells at

Nanoscale Biosensors and Biochips

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least) involves the activated G-proteins altering the activity of downstream “effector” protein(s) to yield a response that can be used to detect the binding event. This is the basis of many existing screening assays and some biosensor designs have exploited this approach, combined with different surface-attachment and transduction technologies. In cell-free GPCR assays discussed in this section, host cells which have been transfected with DNA encoding a particular GPCR of interest allow the cellular expression of the GPCR. Subsequently, the cells are treated in such a way as to allow a partial purification of the GPCRs (in their cell membranes) to obtain an ongoing supply of GPCRs. The purification process can result in small (nanometer scale) crude membrane fragments containing the GPCRs, which are suitable as detector molecules for biosensor applications [5]. 2.1.2. Surface Capture of GPCRs The arraying of membrane GPCRs has required appropriate surface chemistry for the immobilization of the lipid phase containing the GPCR of interest [6-8]. Surface modification with γ-aminopropylsilane (an amine presenting surface) provided the best combination of properties to allow surface capture of the GPCR-G-protein complex from crude membrane preparations, resulting in microspots of approximately 100 µm diameter. Atomic force microscopy (AFM) demonstrated that the height of the supported lipid bilayer was approximately 5 nm, corresponding to GPCRs confined in a single, supported lipid layer scaffold [9]. Using these chemically-derivatized surfaces, it was possible to demonstrate capture of fluorescently labeled β1, β2, and α2A subtypes of the adrenergic receptor, as well as neurotensin-1 receptors and D1dopamine receptors. Dose-response curves using the fluorescentlylabeled ligands gave IC50 values in the nM range suggesting that the GPCR-G-protein complex was largely preserved and biologically intact in the microspot. Furthermore, good long-term stability was achieved. Waller et al. [10] conjugated dextran beads with dihydroalprenolol, an antagonist of β 2 -adrenergic receptors (β-AR). This allowed the capture of solubilized β-AR to this immobilized surface ligand. The βAR was expressed as a fusion protein with green fluorescent protein

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Leifert et al. GPCR ligands Acetylcholine

Ghrelin

Opioids

Adenosine

Glucagon

Orexin

Adrenaline

Glutamate

Oxytocin

Adrenocorticotropic hormone

Gonadotropin-releasing hormone

Parathyroid hormone

Angiotensin II

Growth hormone-releasing factor

Photons (light)

Bradykinin

Growth-hormone secretagogue

Platelet activating factor

Calcitonin

Histamine

Prolactin releasing peptide

Chemokines

Luteinising hormone

Prostaglandins

Cholecystokinin

Lymphotactin

Secretin

Corticotropin releasing factor

Lysophospholipids

Serotonin

Dopamine

Melanocortin

Somatostatin

Endorphins

Melanocyte-stimulating hormone

Substances P, K

Endothelin

Melatonin

Thrombin

Enkephalins

Neuromedin-K

Thromboxanes

Fatty acids

Neuromedin-U

Thyrotropin

Follitropin

Neuropeptide-FF

Thyrotropin releasing hormone

GABA

Neuropeptide-Y

Tyramine

Galanin

Neurotensin

Urotensin

Gastric inhibitory peptide

Noradrenaline

Vasoactive intestinal peptide

Gastrin

Odorants

Vasopressin

ligand

GPCR

lipid bilayer membrane

γ

α β

γ

Ni2+ SUPPORT

MATRIX

Figure 1. A list of some of the known endogenous and exogenous GPCR ligands and a schematic depicting the transmembrane topology of a typical “serpentine” G-protein coupled receptor (GPCR) with its associated heterotrimeric G-protein complex. The membrane patch containing the GPCR with associated G-proteins is schematically shown attached to a theoretical solid support matrix. The receptor polypeptide chain traverses the plane of the membrane phospholipid bilayer seven times. The hydrophobic transmembrane segments of the GPCR are indicated by spirals. The ligand can bind to the receptor from the “extracellular” (outer) surface or depending on the receptor type, to a site deep within the receptor, surrounded by the transmembrane regions of the receptor protein. In this way, the receptor can act as a detector of its ligands. The G-proteins (Gα and Gβγ) are shown to interact with the “cytoplasmic” side of the receptor.

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(GFP), thus allowing fluorescent measurement of bound receptor possible. Thus, it was possible to screen for ligands of the β-AR through the reduction in fluorescence as receptor was removed from the beads due to competition of free (added) ligand. Another successful bead-based approach used paramagnetic beads to capture CCR5 receptors from a cell lysate held within a lipid bilayer [11]. More recently, site directed immobilization of membrane extracts containing either the M2muscarinic receptor or H1-histamine receptor, using complementary oligonucleotides has been investigated (unpublished data). Sequencedirected immobilization of oligo-tagged vesicles carrying GPCRs could potentially lead to the development of a self-sorting array platform for a large number of different receptor sub-types, through the inherent selectivity of complementary strands of oligonucleotides. 2.1.3. Ligand-Binding at GPCRs Ligand binding to a GPCR attached to a surface has been reported for the chemokine CCR5 receptor using surface plasmon resonance (SPR) [12]. For such GPCR surface display, purification of the GPCR has not always been necessary and crude membrane preparations have either been fused with an alkylthiol monolayer (approximately 3 nm thickness) formed on a gold-coated glass surface, or onto a carboxymethyl modified dextran sensor surface [13]. One problem of surface based assays is the difficulty in obtaining the correct orientation of the receptor once attached to the surface. This problem was overcome by using conformationally-dependent antibodies [14]. In this biosensor application, SPR has a distinct advantage as a screening tool since this technique can detect the cognate ligand without requiring fluorescent or radio-labeling. This allows SPR to be used in complex fluids of natural origin thus simplifying the development of assay technologies. Martinez et al. [15] used total internal reflection fluorescence (TIRF) to demonstrate ligand binding to the neurokinin-1 GPCR by surface immobilization of membrane fragments containing this receptor. The GPCR was expressed as a biotinylated protein using mammalian cells and could be selectively immobilized on a quartz sensor surface coated

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with streptavidin (streptavidin binds biotin with extremely high affinity). Using this approach, it was not necessary to detergent-solubilize and reconstitute the neurokinin-1 receptors, thus avoiding the deleterious effect(s) associated with such processes. The preparation of the biotinylated receptors allowed for a high-affinity interaction between biotin and streptavidin and thus a template-directed and uniform orientation of the neurokinin-1 receptor on the support matrix. TIRF measurements were made using a fluorescent-labeled agonist (i.e., the cognate agonist substance-P labeled with fluorescein). The highly sensitive TIRF fluorescence detection methodology was able to resolve the binding of fluorescently-tagged ligand (agonist) to as little as one attomol of receptor molecules [15]. This sensitivity far exceeds that of current physical approaches to biosensing (e.g. gas chromatography, mass spectrometry) and is a major reason why biosensing has become an important research area. 2.1.4. Detecting GPCR Conformational Changes The detection of intrinsic conformational changes in the GPCR following ligand (agonist) activation generally involves the use of fluorescence-based techniques and has been limited to date. One study demonstrated the immobilization of β2-adrenergic receptors onto glass and gold surfaces [16]. The receptors were site-specifically labeled with the fluorophore tetramethyl-rhodamine-maleimide at Cysteine 265 (Cys265) and the agonist-induced signal was large enough to detect using a simple intensified charge-coupled device (ICCD) camera image. Therefore, it was suggested that the technique may be useful for drug screening with GPCR arrays. In a recent study, ligand binding to the β2-adrenergic receptor has been demonstrated using plasmon waveguide resonance (PWR) [17]. Using this technique, changes in the refractive index upon ligand binding to surface-immobilized receptor results in a shift in the PWR spectra. Previously, PWR technology was used for detection of conformational changes in a proteolipid membrane containing the human δ-opioid receptor following binding of several types of ligands [18]. Although the

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ligands in that study [18] were of similar molecular weight, there were distinctly different refractive index changes induced by ligand binding and these were too large to be accounted for by differences in the mass alone. The inference from this finding was that a ligand-specific conformation change in the receptor protein may have been detected, a phenomenon that further increases the specificity of ligand-mediated PWR spectral alterations. 2.1.5. GTP Binding at G-Protein Subunits GPCR biosensors can also utilize the use of non-hydrolyzable GTP-analogs such as radiolabeled [35S]GTPγS or fluorescent-tagged Europium-GTP, which bind to the receptor-activated form of the Gα subunit targeting the site of guanine nucleotide exchange (GDP for GTP on the Gα subunit of the Gαβγ heterotrimer). Guanine nucleotide exchange is a very early, generic event in the signal transduction process of GPCR activation which can be measured without the need for intact cells and is, therefore, an attractive event to monitor. The radiolabeled [35S]GTPγS or fluorescent Europium-GTP binding assays measure the accumulative level of G-protein activation following agonist activation of a GPCR by determining the binding of these non-hydrolyzable analogs of GTP to the Gα subunit. Therefore, they are defined as “functional” assays of GPCR activation because GTP-binding indicates that a cellular response will occur, not just that a binding event was detected at the receptor (which may or may not lead to a cellular response). This is important for screening applications to detect novel compounds which activate or block GPCRs. Ligand regulation of the binding of [35S]GTPγS is one of the most widely used assay methods to measure receptor activation of heterotrimeric G-proteins, as discussed elsewhere in detail [19,20]. The move toward a fluorescent based Europium-GTP assay partly overcomes some of the limitations of radioactive-based assays and has already been successfully used with the following GPCRs, motilin, neurotensin, M1-muscarinic and α2Aadrenergic receptors [21,22].

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2.1.6. G-Protein Dissociation GPCR biosensors can involve the detection of the final stage of activation of the G-protein heterotrimeric complex, that being the putative dissociation or rearrangement of the subunits following GPCRinduced G-protein activation [23,24]. This level of GPCR activation has currently not been investigated in great detail and may prove to be extremely valuable in future functional biosensor applications as Gprotein signalling always involves alterations to the heterotrimer structure. An advantage of cell-free GPCR assays involving G-proteins is that GDP can be used to “reset” all GPCRs to the inactive state, thereby allowing for greater resolution of receptor activation by effectively removing background signalling. Bieri et al. [25] used carbohydrate-specific biotinylation chemistry to achieve appropriate orientation and functional immobilization of the solubilized bovine rhodopsin receptor with high contrast micropatterns of the receptor being used to spatially separate protein regions. This reconstituted GPCR:G-protein system provided relatively stable results (over hours) with the added advantage of obtaining repeated activation/deactivation cycles of the GPCR:G-protein system, as occurs in vivo. Measurements were made using SPR detection of G-protein dissociation from the receptor surface following the positioning of the biotinylated form of the rhodopsin receptor onto a self-assembled monolayer (SAM) containing streptavidin. Although SPR is useful for the study of G-protein interactions, it may not be well suited to detect binding of small ligand molecules directly due to its reliance on changes in mass concentration. An advantage of repeated activation/deactivation cycles of GPCRs is that different compounds may be tested serially with the same receptor preparation, allowing for discernment of differential activation of the same receptors by different ligands. The above approach appears promising for future applications of chip-based technologies in the area of GPCR biosensor applications. The well known and highly utilized interaction between Ni2+ and histidine residues (most often used for purification oligohistidixnetagged proteins) may be a useful means of attachment for GPCRs and/or

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G-proteins. To this end, different surface chemistries are being investigated to optimize the affinity interaction [26,27]. Modifying the surface of epoxy-activated dextran beads by forming a Ni2+-NTA conjugate was shown to produce beads with a surface capable of binding hexahistidine (his)-tagged β1γ2 subunits [28]. Tethered β 1γ2 subunits were then used to capture Gαs subunits which in turn were capable of binding membrane preparations containing a β2-adrenergic receptor-GFP fusion protein. Alternatively, a fluorescent labeled ligand binding to the tethered β2-adrenergic receptor could be detected; the whole complex being measured using flow cytometry. Flow cytometry’s greatest advantage is its ability to be multiplexed, where different molecular assemblies can be made in one sample and then be discriminated by their unique spectral characteristics [10,28-30]. Indeed, particle-based nanotechnologies, e.g. quantum-dots [5,31,32], constitute another emerging enabling technology for GPCR biosensor applications. 2.1.7. GPCRs as Biological Detectors of Volatiles Many organisms, from nematodes to mammals, use GPCRs to sense volatile compounds and the neural signal transduction due to GPCRvolatile interactions is the basis of smell [33-36]. Vertebrates are known to utilize olfactory receptors (ORs) that are similar to other metabolic GPCRs [35]. Invertebrate ORs were discovered relatively recently and it appears that these ORs are atypical GPCRs in that they have reversed membrane topology and that they apparently each form dimers with the same highly conserved OR-like receptor which can function as an ion channel, independent of ligand-mediated activation of the OR with which it has dimerized [37-39]. Due to the inherent OR-volatile specificity and high affinity of the OR-volatile interaction, ORs are obvious candidates as biomolecules that could be adapted to detect specific volatiles. Biosensors of volatiles would have a multitude of potential applications, particularly for sensing hidden entities (e.g. explosive screening) and in the agrifood industry (e.g. quality control, fermentation monitoring). The current attempts at volatile biosensing are not only limited by generic issues such as instability of membrane proteins but also by the poor level

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of functional characterization of the different ORs [40]. Therefore, current attempts to produce a sensor for volatile compounds are limited to the most highly characterized receptors, these being human OR17-40 [41], I7 from rat [42], and Odr10 from the nematode Caenorhabditis elegans [43,44]. Hou et al. [45] reported an attempt to produce a biosensor that utilized the membrane fraction of recombinant yeast expressing rat I7 receptor, which is known to be activated by heptanal and octanal. This design used a gold electrode that was functionalized with biotinylated I7specific antibody, to which the membrane fractions were applied. Specific odorants were applied to the I7-presenting surface and interactions were monitored through variation in polarization resistance. Ligand binding was discernable although a specific response was difficult to resolve at ligand concentrations below 10-12 M, which is nevertheless more sensitive than current detection technologies. A common difficulty of presenting such ligands to the biosensor surface is the need for an organic solvent to carry the ligand. In the case discussed, dimethyl sulphoxide was used for this purpose and was found to only alter polarization resistance by 10%, even at the highest concentration tested (0.1 mM). Whole yeast cells have also been utilized to produce a I7-based biosensor [42]. Yeast cells were engineered to express I7 and a mammalian G-protein capable of linking ligand-mediated receptor stimulation to activate a MAP kinase pathway that induced synthesis of luciferase. Thus, in the presence of the luciferin substrate, ligand binding was detected as a dose-dependent fluorescent response. Importantly, sensitivity of the response was altered by the type of G-protein used to couple the OR to downstream elements. Another design utilized the human OR1740 protein expressed in yeast and receptor-containing nanosomes were produced by sonication of recombinant yeast membranes [46]. The nanosomes were then captured on a SAM functionalized with biotinyl groups that were used for attachment of neutravidin and then a biotinylated monoclonal antibody specific to the receptor. Interestingly, the myc-tagged receptors were functional when immobilized via a C-terminal tag but not when attached by the N-terminus.

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A crude Odr10-based olfactory biosensor was produced by expressing the OR in bacteria, obtaining a membrane fraction and coating a quartz crystal microbalance (QCM) chip with the membranes [47]. The authors of this study reported that ligand application could be detected as a change in QCM frequency. Another attempt utilized mammalian (HEK-293) cells to express Odr10, and SPR was then used to detect binding of the applied ligand (diacetyl) [48]. 2.1.8. The Future of GPCR Biosensors With growing interest and commercial investment in GPCRs in areas such as drug targets, orphan receptors, high throughput screening of drugs etc., greater attention will focus on biosensor development to allow for miniaturization, ultra-high throughput, and, eventually, microarray/biochip assay formats that will require nanotechnology-based approaches. The production of stable, robust, cell-free signaling assemblie’s comprising receptor and appropriate molecular switching components will form the basis of future GPCR/G-protein platforms which should be adaptable for laboratory- and field-based applications as microarrays and biosensors. 2.2. Pore-Forming Proteins Ion-channels are transmembrane proteins that regulate the transport of ions and/or small molecules across the lipid membrane. They can exist in the open or closed state, which can be regulated by a range of stimuli, including a change in membrane potential, mechanical stress or the binding of a ligand [49]. Some of the most commonly investigated poreforming peptides, are bacterial porins (e.g. OmpF) [50], α-hemolysin from the human pathogen Staphylococcus [51] and Gramicidin, a polypeptide antibiotic [52]. These have all been studied for their adaptability to a sensor platform [53-57]. Lipid supports, which are essential for the functional capture of the channels, are discussed in section 3.

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a) (i)

(ii)

b) (i)

(ii)

Figure 2. Pore-based Sensing. (a) Schematic of an ion-channel switch (sandwich) developed by Cornell et al. [59] (i) The lipid bilayer is composed of archaebacterial membrane spanning lipids (MSL) and half membrane spanning tethered lipids (DLP). MAAD are spacer molecules attached to the gold surface via a sulfur-gold bond. The mobile lipids (DPEPC/GDPE) and ion channels (Gα) attached to antibodies (Fab) via a streptavidin linker (SA), can move throughout the bilayer, unlike the immobilized ion channels (GT). (ii) Mobile channels (Gα) become cross-linked to tethered antibodies (Fab) on the membrane spanning lipids (MSLα) in the presence of analyte (A), preventing formation of complete channels and therefore decreasing measured current. Reproduced with permission [59]. (b) Braha et al. [57] demonstrated simultaneous stochastic sensing for zinc, cobalt and cadmium ions with an engineered protein pore. (i) Schematic representation of the pore with the single metal binding site (metals are represented by different sized, or filled, balls) in the lumen of the channel. Each time metal ions bind to the pore, the current is modulated, as illustrated in the trace which reflects the currents flowing through the pores that were recorded during the application of a +40 mV membrane potential. Arrows indicate the current through the fully opened pore (ii). Reproduced with permission [57].

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Native or engineered membrane-bound ion-channels are a promising class of receptors for biosensing applications as they allow the sensitive detection of analytes and produce an output (electrical current) which is inherently suitable for digitization [58]. Ion-channel switches, first reported by Cornell et al. [59] (Figure 2a) and single-channel stochastic sensors [58] (Figure 2b), both demonstrate mechanisms by which analyte detection and quantitation can be determined by measured changes in current due to ion-channel activity. An important thing to consider when utilizing ion-channels is that the current, produced by the movement of ions through a pore in a lipid membrane, is dependent on the accessibility of a clear passage through the pore and therefore, can fluctuate when ion-channels do not traverse the membrane or when the pores are partially or fully blocked. 2.2.1. Ion-Channel Switch The ion-channel switch described by Cornell et al. [59] comprises a gold electrode, to which a lipid membrane carrying gramicidin ionchannels bound to antibodies, are tethered. A current is produced (turned on) when ions flow through the channel (in the presence of an applied potential). It is subsequently switched off when mobile channels diffusing in the outer half of the membrane, become cross-linked to antibodies immobilized at the membrane surface [59] (Figure 2). The ion-channel switch has since demonstrated specific signals derived from interactions with a range of analytes including bacteria, DNA, proteins and drugs [60]. Recently, Oh et al. [61] reported the detection of influenza A virus in clinical samples using the ion-channel switch biosensor. 2.2.2. Stochastic Sensing Stochastic sensing involves monitoring current that flows through a single pore and the alterations of this flow in response to analyte binding events (Figure 2). Each time an analyte binds to a binding site within the pore, the current flow is altered and during the on/off equilibrium of the binding analyte, a characteristic flow pattern is produced and monitored.

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The frequency of the current fluctuations is related to concentration of the analyte, whereas the current signature (revealed by fluctuation duration and amplitude) is related to the analyte’s identity (reviewed by Bayley and Cremer [58] as well as Schmidt [62]). Some reported desirable attributes of stochastic sensing include fast detection, as well as the sensitivity and reversibility of sensor elements [57]. Current fluctuations monitored through a single pore, have demonstrated the capability for a variety of analytes, including metals [57], organic molecules [63] and oligonucleotides [64]. Shim and Gu [65] and Kang et al. [66] recently demonstrated increased stability of a single pore chip by encapsulation of the associated lipid bilayer within an agarose gel. This resulted in a more robust chip that can be stored for longer periods than bilayers alone. Pore-based transduction systems such as these have great potential in the field of biosensing. 2.3. Cell- and Viral-Based Sensing The biological component of a biosensor is currently most often an enzyme, antibody or other sub cellular component (such as the receptors and ion-channels mentioned in previous sections). The purification of these proteins can be labor intensive, expensive and the resulting product incompletely purified or unstable. Whole-cell sensors preserve the localization and temporal control of protein function and can utilize reporting processes that may involve multiple enzymes and signaling cascades. These types of reporting system are advantageous when biological/metabolic relevance of an analyte is important rather than simply its detection. Thus, unlike purified enzymes and antibodies, cell biosensors can report on bioavailability, metabolic regulation, toxicity, genotoxicity (DNA damage) etc. The key challenge for cell-based biosensors is the maintenance of cell viability under assay conditions. This requirement may lessen the utility of cell-based systems in field applications or environments that are deleterious to cell viability. Cellular responses can be specific to a substance (e.g. GPCR-ligand interactions) or a general response to adverse environmental conditions (e.g. regulated apoptosis) and each of these could be monitored depending on the specific biosensor design. Often cells are genetically

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modified with a reporter protein such as luciferase, GFP or βgalactosidase. The genes for these reporters are placed under the control of a promoter that responds to the analyte of interest resulting in the expression of the reporter protein, which can be detected. This approach has been exploited in identifying many environmental pollutants that induce particular promoters and for measuring stress responses. With advances in nanotechnology, cell-based nanobiosensors are now emerging with increasing sophistication, sensitivity and detection methods that are available, although most are still in the proof-of-concept stage. Different cell types can be more applicable to certain applications and/or measurements. In the following sections we discuss nanobiosensors utilizing bacterial, yeast, fungal, algal and mammalian cells. Several examples of cellular GPCR-based (olfactory) biosensor designs are discussed earlier (see 2.1.7) 2.3.1. Bacterial Biosensors Bacteria are particularly exploited in biosensors since they contain many defence mechanisms against analytes of interest such as environmental pollutants, including mercury and arsenic [67]. Bacteria can be easily produced at low cost and genetically manipulated and as mentioned previously. Reporter genes are often fused to DNA elements that respond to the presence of these analytes to produce a signal [67]. For example, bacteria can be used to detect genotoxic agents since increased DNA damage (due to the presence of a genotoxin) results in degradation of the endogenous repressor of SOS genes and subsequent expression of genes associated with DNA repair [68]. Other stress responses such as oxidation, nutrient starvation, membrane damage, heat shock and apoptotic responses can also be measured [67,68]. In a “lab-on-a-chip” format, E. coli were genetically engineered such that the activation of the fabA, dnaK or grpE promoters produced the enzyme β-galactosidase [69]. These bacteria were applied to electrochemical cells (100 nL capacities) on a silicon chip as a broth or immobilized within agar. The cells contained embedded electrodes for electrochemical measurements. Upon exposure to the representative toxicant phenol, and in the presence of the substrate

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p-aminophenyl-β-D-galactopyranoside, β-galactosidase was expressed because of activation of the relevant promoters. β-galactosidase cleaved p-aminophenyl-β-D-galactopyranoside into p-aminophenol and β-Dgalactopyranoside. p-aminophenol is electrochemically active and the application of a 220 mV potential produces oxidation of p-aminophenol molecules, which is converted to a current that can be monitored to detect phenol concentrations as low as 1.6 ppm. The fabA promoter gave the largest response to phenol exposure which corresponded to the increased sensitivity of this promoter towards membrane damage inflicted by phenol. It is hoped that advantages such as the small sampling requirement, high signal to noise ratio, potential for highthroughput and high degree of robustness will combine to make this platform suitable for field applications. Carbon nanotubes are also being exploited in bacterial nanobiosensors. Pseudomonas putida have been coated onto an osmium redox polymer on a carbon nanotube-modified electrode and covered by a dialysis membrane to form an amperometric biosensor with increased electron transfer efficiency [70]. The respiratory activity of the cells was correlated with the oxidation of glucose, measured via the osmium redox polymer that acted as an electron acceptor. The system was then modified to measure phenol, which could be detected in an artificial wastewater sample, using phenol adapted P. putida. 2.3.2. Fungal and Algae Cell Biosensors The use of fungal cells (such as yeasts) in biosensors can provide the advantages of using bacteria but being eukaryotic cells they may provide information that is more relevant to higher eukaryotic organisms. This is a particularly important attribute for toxicity and drug screening. Fungal cells remain relatively easy to culture and genetically manipulate, and can be more robust with regard to pH, ionic strength and temperature than mammalian cells [71] due to their resistant cell walls. Wild-type cells can be used as biological oxygen demand sensors or to detect catabolic substrates. Oxygen consumption can be correlated to many physiologically relevant processes such as cell viability, protein synthesis and mitochondria function. Saccharomyces cerevisiae cells have been

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immobilized onto amine functionalized polystyrene nanobeads that were loaded with the oxygen-sensitive fluorescent ruthenium (II) [72]. This allowed optical detection of oxygen consumption since in the presence of molecular oxygen, the fluorescence of ruthenium (II) was quenched. In the presence of high concentrations of glucose, there was increased fluorescence indicating that cellular respiration and oxygen consumption was increased compared to when glucose was absent. Genetically modified cells can report on gene regulation in response to environmental factors or can be engineered to express and monitor the activation of other receptors such as those involved with olfaction or disease processes. An olfactory receptor (a GPCR) that responds to 2,4dinitrotoluene (DNT), a mimic for the explosive trinitrotoluene (TNT), has been identified and yeast were engineered to express the receptor and its associated signaling components including the G-proteins, adenylyl cyclase and a cAMP responsive DNA element that promoted the expression of GFP upon stimulation of the receptor [73] (for more discussion of the application of explosives detection see section 7.3). S. cerevisiae has also been engineered to express the human olfactory receptor, OR17-40. These cells could be immobilized on interdigitated gold microelectrodes coated with poly-L-lysine and the conductance on the surface of the electrode was shown to be modified by receptor-ligand interactions [74]. Algae could potentially be exploited for environmental biosensing since the inhibition of algal photosynthesis can be correlated to toxic effects of pollutants such as herbicides. (for further discussion on environmental monitoring with biosensors, see section 7.4). Inhibition of photosynthesis by photosystem II (PSII)-inhibiting herbicides (e.g. atrazine) can be measured as a change in chlorophyll fluorescence, caused by these compounds blocking the PSII quinone-binding site thereby inhibiting photosynthetic electron flow. This approach was demonstrated by using Chlorell vulgaris entrapped on quartz microfiber filters, and using a fibre-optic bundle to monitor chlorophyll fluorescence, which increased in the presence of atrazine [75]. Changes in chlorophyll fluorescence in response to exposure to formaldehyde and methanol vapor have also been monitored in a biochip platform that

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could be used to simultaneously test many different algal strains with different sensitivities to toxicants [76]. 2.3.3. Mammalian Cell Biosensors Mammalian cell biosensors are finding particular usefulness within the pharmaceutical industry since using whole cells can maximize the information content of the assay and allow examination of a compound’s action on cells close to the intended target, within the context of all cellular machinery. This is particularly useful in the pharmaceutical industry that requires technologies that can provide reliable predictive information on lead compounds early in their development to reduce unwarranted development costs [77]. Impedance-based technologies (reviewed by McGuiness in 2007 [78]) can be used to detect cell death or proliferation, as well as smaller changes caused by receptor signaling and resulting in cytoskeletal rearrangements or changes in cell-cell interactions and adherence. Sensor chip-based impedance spectroscopy has been applied to measure the activation of GPCRs binding with neuropeptide Y (these receptors are implicated in human breast carcinoma [79]). Adenylyl cyclase activity in MCF-7 mamma carcinoma cells adhered to a microelectrode array, was stimulated with forskolin resulting in reduced impedance [79]. This effect could be blocked by pre-treatment with neuropeptide Y, which is known to inhibit adenylyl cyclase activity. Ligand-binding to various GPCRs in adherent cells has also been characterized using mass redistribution cell assay technologies (MRCAT) and resonant waveguide grating (RWG) [80]. Another example of a mammalian cell biosensor used malignant cells taken from a specific patient and exposed to chemotherapeutics to predict if that patient’s response will be favorable. Live metastatic human mammary cancer cells have been adhered to the gold surface of a QCM to sense for disruption of microtubules within the tumor cells in response to anti-tumor agents taxol and nocodazole [81]. To detect the excretion of interleukin-2 from mouse T-cells, silica nanoparticles were used to form a nanoparticle layer between two layers of gold, and antibodies specific to interleukin-2 were immobilized onto the sensor

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surface. Concanavalin-A was used to trigger secretion of interleukin-2 from the cells which was detected by the antibodies with a limit of 10 pg/ml using localized SPR [82]. The authors suggest that this technique has potential to be applied to high-throughput cell analysis systems for reporting on various cell activities and functions. These examples do not require cell engineering and are label-free, making them less invasive and therefore possibly more physiologically relevant. Other label-free technologies for whole cells have recently been more thoroughly reviewed [77]. However, most mammalian cell biosensors do rely on engineering cells to produce signals such as fluorescence, or over expression of a target that can produce an observable change in cellular physiology. These genetically encodable fluorescent biosensors have recently been reviewed [83] and will not be covered in detail here. Nanotechnologies are also being developed to sense changes within whole cells. Plasmonic biosensors or gold nanoparticles (20 nm) have been functionalized with an anti-actin antibody and a TAT-HA2 peptide, which mediated the endocytotic uptake of the nanoparticles into the cell, and their subsequent release from endosomes into the cytoplasm [84]. Binding of the nanosensors to actin could be measured since this brought the probes into such close proximity that the plasmon resonance became red-shifted, which could be detected by darkfield reflectance imaging or confocal microscopy, with detection limited to between 623-643 nm. It is hoped that further advances will enable monitoring of cytoskeletal rearrangements made as a biological response. Other optical sensing components can be entrapped within inert nanosized polymer particles termed PEBBLEs (probes encapsulated by biologically localized embedding) [85]. Possible probes include calcium sensitive dyes, pH sensitive dyes or enzymes such as horseradish peroxidase which can be used to detect reactive oxygen species [86] leading to applications such as analysis of effects of drugs, toxins or environment, on cell physiology. The inert polymer is permeable allowing the encapsulated probe to interact with analytes, and protects both the dye from interference by biological conditions, and the cell from any dye-associated toxicity. PEBBLEs are introduced into cells through surface modification with peptides that mediate cellular uptake, transfection using lipid reagents, picoinjection or gene gun

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bombardment. PEBBLEs containing the BME-44 ionophore, ETH 5350 chromophore, and ionic exchanger KTFPB, have been used to measure increases in potassium in rat C6 Glioma cells treated with kainic acid to open potassium channels [87]. Calcium-sensing PEBBLEs have also been use in SY5Y neuroblastoma cells to report on calcium released from mitochondria in response to exposure to the neurotoxin m-dinitrobenzene [88]. Currently, cell-based approaches usually utilize a large population of cells within which different responses are occurring, and the responses are averaged. However, the response of individual cells can be different in an environment that is free of influences from neighboring cells. Optical nanosensors are being developed to allow intracellular measurement of biological processes within single live cells. Tapered optical fibres with nanosized tips (30-50 nm) have been applied to a wide range of applications (reviewed in Leung et al. [89]) and can be used to probe conditions inside a cell. The tip of the nanofibre is approximately 10-fold smaller than the wavelength of excitation light transmitted along the fibre. Photons travel as far along the fibre as possible but cannot escape from the tip although an evanescent field continues to travel a short distance through the remainder of tip providing excitation light for molecules that are in close proximity ( Eb

(4)

where d is the size of nanoparticles, Eb is the energy barrier height. Figure 8 shows the temperature dependent electrical conductivity, Seebeck coefficient, and power factor for compacted nanoparticle composites made of Si0.8Ge0.2 alloy as a function of energy barrier height. The nanoparticle diameter is assumed to be 20 nm and the doping concentration is assumed to be 1.0x1020 cm-3. As shown in Fig. 8(a), the electrical conductivity decreases with energy barrier height and the Seebeck coefficient increases with energy barrier height due to the low

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energy carrier filtering. Figure 8(b) shows that there exists an optimum barrier height for the power factor enhancement. Overall the enhancement is effective at low temperature and becomes less effective at high temperature, which is very similar to experiment observations [52, 53]. This simplified model could be a good tool to guide the material synthesis since it predicts the dependence of thermoelectric transport properties on carrier concentration, temperature, grain (nanoparticle) size, and energy barrier height after the input parameters are optimized with experimental data.

(a)

(b)

Figure 8. The temperature dependent electrical conductivity and Seebeck coefficient (a) and power factor (b) for Si0.8Ge0.2 alloy compacted nanoparticle composites as a function of energy barrier height Eb. Reprinted with permission from Ref. [50]: R. Yang and G. Chen, in SAE World Congress (Society of Automotive Engineers, 2006), Article # 2006-01-0289. Copyright @ Society of Automotive Engineers

Faleev and Leonard [54] developed a model for predicting the Seebeck coefficient, electrical conductivity and ZT of materials with nanoscale metallic inclusions, using the idea of the band bending at the metal-semiconductor interface acting as energy filters. They found that the Seebeck coefficient of the nanocomposite material is always enhanced compared to the inclusion-free system, and that the smaller the

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nanoinclusion, the greater the enhancement. Similar to Yang and Chen [50], they also found that the power factor is optimized for certain values of the boundary potential. The enhancement of the ZT is dominated by the reduction in the lattice thermal conductivity for low carrier density, while at high carrier density the electronic contribution becomes important. In summary, electrical and energy transport in nanostructures differs significantly from macrostructures because of classical and quantum size effects on energy carriers. Both thermal conductivity reduction and the possibility to maintain—and even enhance—the electronic power factor in nanocomposites render cost-effective random nanocomposites as a promising alternative to expensive superlattices for high ZT material development. The key to thermal conductivity reduction is to have high interface density where nanocomposites can have much higher interface density than simple 1D stacks such as superlattices, thus nanocomposites benefits ZT enhancement in terms of thermal conductivity reduction. In the meantime, the interfaces can be viewed as energy filters for electrons which allow only electrons having higher energy to pass through the barrier, and thus enhance the Seebeck coefficient. Overall there exists an optimum barrier height and nanoparticle (grain) size for the electronic power factor enhancement due to the electrical conductivity reduction at the same time.

3. Synthesis of Thermoelectric Nanocomposites A thermoelectric nanocomposite is a composite constructed by incorporating thermoelectric nanostructures in a matrix of a bulk thermoelectric material or compacting various thermoelectric nanostructures into bulk form. Several methods for the preparation of thermoelectric nanocomposites have been exercised. These methods to obtaining bulk samples with nanoscale features can be broadly classified into two categories: (i) compaction of nanoscale constituents (nanoparticles, nanowires, etc.) into bulk samples, (ii) in situ precipitation of nanoscale constituents by means of phase separation. In the following, the two routes are briefly described.

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3.1. Preparation of Nanocomposites by Compaction Techniques Several different compaction techniques have been utilized in the recent past to obtain bulk thermoelectric samples from nanoscale constituents that are synthesized by an array of physical and chemical methods. The essence of all compaction techniques is to apply high pressure for densification, and often a rather high temperature to soften the material so that plastic deformation allows better filling and material flow by diffusion to remove the remaining porosity. The challenge is in achieving high density (and low porosity) without losing the nanoscale microstructure and keeping the material chemically pure. 3.1.1. Compaction Methods Cold compaction is a process in which powder materials are compressed in a temperature range where high temperature deformation mechanics like dislocation or diffusional creep can be neglected. Cold compressing is the most important compaction method in powder metallurgy. Thus, cold sintering offers the potential for retaining the metastable nanoscale constituents. Despite this, the nanopowders may not bond very well, leading to lower carrier mobility and therefore low ZT. A more common way of consolidation of nanopowders is hot pressing, where, in addition to the high pressure, moderate to high temperature is applied to the sample simultaneously. This results in a better particle-particle bonding, and higher carrier mobility in the final sample. However, it is a challenge to retain the nanometer-sized crystal grains in the final sample because the grains can grow significantly. Figure 9 shows the TEM images of nanocomposites of BixSb2-xTe3 prepared by ball milling and hot pressing [55]. Evidently, under the right conditions hot pressing can preserve the nanostructure and lead to enhanced thermoelectric performance.

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Figure 9. TEM images showing the microstructure of a hot pressed nanocomposite bulk sample of Bi-Sb-Te. (A) Low magnification image showing the nanograins. (B) High magnification image showing the nanosize, high crystallinity, random orientation and clean grain boundaries. The nanostructure is seen to be preserved even after hot pressing. Reprinted with permission from Ref. [55] B. Poudel, Q. Hao, Y. Ma, Y. Lan, A. Minnich, B. Yu, X. Yan, D. Wang, A. Muto, D. Vashaee, X. Chen, J. Liu, M. S. Dresselhaus, G. Chen, and Z. Ren, Science 320, 634 (2008) Copyright @ American Association for the Advancement of Science.

Spark Plasma Sintering (SPS), also known as Field Assisted Sintering Technique or Pulsed Electric Current Sintering, is a novel sintering technique which is gaining increasing popularity for making thermoelectric nanocomposites [56]. In the SPS technique, the sample is heated by pulsed electric current which flows through the punch-die-sample-assembly under a low voltage. It is expected that due to the high current, at the comparatively small gaps between the powder particles, electrical discharges will occur. These discharges result in microscopic electric arcs, leading to high temperatures and pressures locally, forming a good contact between the particles. And additional advantage is that, gases and moisture that have been adsorbed on surfaces of the nanoparticles are eliminated, and oxide layers can be broken due to the arcs. Subsequently, Joule heating occurs in the compact due to the current flow, especially at spots of high electrical resistance. This temporarily overheats the sample while the overall sintering temperature is relatively low. As the heat is generated internally in the SPS, in contrast to the conventional hot pressing where the heat is provided by external heating elements, very high heating rates

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(>300°C/min) and short sintering times in the range of a few minutes can be achieved resulting in a fast throughput. Also, the high speed of the process ensures it has the potential of densifying powders with nanosize or nanostructure while avoiding coarsening which accompanies standard densification routes. Densities very close to theoretical densities and excellent thermoelectric performances have been achieved in samples treated by SPS process. In view of these advantages over the other compaction methods, it is being increasingly utilized for making thermoelectric nanocomposites [57-59]. 3.1.2. Synthesis of Thermoelectric Nanostructures There are numerous techniques available to synthesize the nanoscale constituents, such as nanoparticles, nanoplates, nanowires, nanobelts and nanotubes etc. Some of the popular techniques for synthesizing thermoelectric nanostructures are described below. Mechanical Attrition Mechanical attrition is one of the most popular methods for synthesis of nanostructures from bulk raw materials, due not only to the convenience and minimal requirement for complex equipment, but also, to the versatility in terms of the number of different systems of materials that can be prepared this way. Mechanical attrition produces its nanostructures by the structural decomposition of coarse grains into finer structures as a result of plastic deformation and can be carried out at room temperature. The process can be performed on high energy mills, centrifugal type mill and vibratory type mill, and low energy tumbling mill. Nanoparticles, of sizes ranging from 200 nm to as low as 5-10 nm, can be prepared by the use of attritors, vibratory mills and horizontal ball mills [60]. As the process is sensitive to contamination from the milling environment, tight atmospheric control is essential to maintain the purity of the material, in particular to avoid oxidation. Consequently, an argon or nitrogen gas atmosphere is used for preparation of thermoelectric materials. Contamination from wear debris of the milling media is also a problem with mechanical attrition that may negatively impact the quality

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of the alloy, requiring a judicious selection of processing time and milling speed [2]. Mechanical attrition has been used for the preparation of nanopowders of Fe-Si alloys [61], Si-Ge alloys [52, 62], PbTe [63, 64] and PbSbTe alloys[65], BiSbTe alloys [55, 66, 67], MgSiSn[68], CoSb3 [69], and materials such as La3-xTe4 [70] that are challenging to synthesize using melt synthesis and other traditional methods. Wet Chemistry Synthesis Wet chemistry method is the powerful tool to generate various nanostructures in different shapes. For example, solvothermal (including hydrothermal) method synthesizes the nanostructures by using the solubility in water (or a suitable solvent) of inorganic precursors at elevated temperatures (above the critical point of the solvent) and selfformed pressures in an autoclave, and the subsequent crystallization of the dissolved material from the fluid. Compared with other synthesis routes performed at atmospheric pressure, the increased reaction temperature in the solvothermal technique may lead to an accelerated crystal growth accompanied by a narrow particle size distribution and better crystallinity. Another advantage of this method is that nanostructures of different morphologies such as nanopowders, nanorods, polygonal nanosheets, polyhedral nanoparticles and sheet-rods can be synthesized. Also, as most materials can be dissolved in the solvent by heating and pressurizing close to the critical point, this approach is suitable for synthesizing nanostructures of a wide variety of solid materials. Hydrothermal synthesis has been used to obtain nanostructures of Bi2Te3 [71-73], Sb2Te3 [74], PbX (X=S, Se, Te) [75], CoSb3 [76, 77], etc. Figure 10 shows TEM images of Bi2Te3 nanotubes synthesized by hydrothermal processs. On the other hand, ambient solution phase method can be operated in mild conditions to fabricate different kinds of nanostructures through an anisotropic growth process by adding different surfactants or tuning reaction conditions, such as temperature, pH value etc. Bi2Te3 nanoplates and nanorods have been successfully fabricated using this method [78, 79], and using a two step process, Te/Bi2Te3 coreshell nanowires can be obtained [80]. Furthermore, rough silicon

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nanowires can be synthesized through a wet etching process and were recently reported with enhanced thermoelectric performance [81].

Figure 10. TEM photos of hydrothermally synthesized Bi2Te3 nanotubes. Reprinted with permission from Ref. [71] X. B. Zhao, X. H. Ji, Y. H. Zhang, T. J. Zhu, J. P. Tu, and X. B. Zhang, Applied Physics Letters 86, 062111 (2005), Copyright @ American Physical Society.

Electrochemical Deposition Electrochemical deposition provides a facile and effective route to fabricate various nanostructured metal alloys for thermoelectric applications [82, 83]. Stacy’s group made a breakthrough by fabricating high quality Bi2Te3 nanowire arrays for the first time using the porous anodic alumina (PAA) template assisted electrodeposition process. This technique has been quickly developed as a popular method to obtain various thermoelectric nanowire arrays, such as: Bi2Te3, Sb2Te3, Bi-SbTe, Bi-Te-Se, CoSb3, PbTe etc. [84-89]. A high degree of control in the diameter and length of the nanowires can be exercised in this method. The diameter of as-obtained nanowires ranges from 20 to 300 nm and is related to the template pore size, while the length depends on the electrodeposition time. Moreover, the alloy composition can be adjusted by changing the content of electrolyte solution [90], and the orientation of the nanowire arrays can be changed

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by tuning the deposition potential or using pulsed electrodeposition process [91]. Uniformity of growth can also be achieved by electroplating in low temperature [92]. Furthermore, in some special electrodeposition conditions, novel hollow thermoelectric nanostructures can be obtained. Li et al. reported the successful fabrication of Bi nanotube arrays [93], and Zhu’s group was able to synthesize Bi2Te3 and relative compounds nanotube arrays [94]. On the other hand, even without the assistance of templates, one-dimensional chinelike Bi-Sb nanostructure was fabricated through a template-free electrodeposition process by Zhou et al. [95], and PbTe cubes can be directly deposited on the polycrystalline gold substrate by Xiao et al. [96]. Using the cyclic electrodeposition/stripping method, significant amounts of long polycrystalline Bi-Te nanowires were obtained on highly oriented pyrolytic graphite (HOPG) surface [97].

Figure 11. (a) and (b) show the TEM images of multilayered Bi2Te3/Sb nanowires deposited in different conditions labeled in the bottom of each corresponding figure. (c) and (d) are the corresponding high magnification TEM images. Reprinted with persmission from Ref. [100]: W. Wang, G. Q. Zhang, and X. G. Li, Journal of Physical Chemistry C 112, 15190 (2008) Copyright @ American Chemical Society.

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Electrodeposition can also be used to synthesize various heterostructured nanomaterials. Using template assisted pulsed electrodeposition, different kinds of thermoelectric materials can be deposited alternately and periodically, by applying different deposition potentials [98, 99]. Wang et al. reported a detailed study of Bi-Sb-Te system and successfully manipulated the growth of Bi2Te3/Sb heterostructure nanowires with desired period, and the minimum period of as-synthesized Bi2Te3/Sb heterostructure nanowires to as low as 10 nm (Fig. 11) [100]. They also fabricated Bi2Te3/Te heterostructured nanowire arrays through a nanoconfined precipitation process [101]. Inert Gas Condensation Inert gas condensation is a versatile process in use today for synthesizing experimental quantities of nanostructured metallic and intermetallic powders. A feature of the process is its ability to generate non-agglomerated nanopowders, which can be sintered at relatively low temperatures. An evaporative source is used to generate the powder particles, which are convectively transported to and collected on a cold substrate. The nanoparticles develop in a thermalizing zone just above the evaporative source, due to interactions between the hot vapor species and the much colder inert gas atoms (typically 1-20 mbar pressure) in the chamber. Recently, this method has been utilized for making Si-Ge nanocomposites [102]. Sonochemical Synthesis The underlying mechanism of sonochemistry arises from the acoustic cavitation phenomenon, that is the formation, growth and implosive collapse of bubbles in a liquid medium due to irradiation with ultrasonic waves. Extremely high temperatures (>5000 K), pressures (>20 MPa), and very high cooling rates (>107 K/s) can be attained locally during acoustic cavitation that lead to many unique properties in the irradiated solution [103]. The remarkable advantages of this method include a rapid reaction rate, the controllable reaction condition and the ability to form nanoparticles with uniform shapes, narrow size distributions and high

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purities. Sonochemical synthesis method has been used to obtain nanocrystals of Bi2Se3 [104], Bi2Te3 and intermediate compounds [105], and other metal tellurides and selenides [106]. Figure 12 shows a TEM micrograph of Bi2Se3 nanocrystals made by sonochemical synthesis.

Figure 12. TEM image and a plot of the size distribution of nanocrystals of Bi2Se3 prepared by sonochemical synthesis. The scale of the TEM image is 100 nm. Reprinted with permission from Ref. [104] X. F. Qiu, J. J. Zhu, L. Pu, Y. Shi, Y. D. Zheng, and H. Y. Chen, Inorganic Chemistry Communications 7, 319 (2004) Copyright @ Elsevier.

In addition to the above methods, chemical vapor deposition [107], and sol–gel process [108] have also been explored to synthesize thermoelectric nanostructures.

3.2. Synthesis of Nanocomposites by Phase Separation The phase separation method of synthesizing nanostructures in situ in a bulk sample is inspired by precipitation hardening of aluminum. Basically, in this process, different kinds of metals will be heated up to the liquid phase, and then quenched to obtain a homogenous solid solution. According to the miscibility gap in the phase diagram, the asobtained metastable solid solution will decompose into different phases A and B (or phases rich in A and B during the spinodal decomposition) after a nucleation and growth process by annealing at certain duration, and thus forms the embedded precipitates in bulk matrix. The size of the precipitates increases as the duration and the temperature of the annealing process increase [109-114]. For example, according to the pseudo-binary PbTe-Sb2Te3 phase diagram (Fig. 13), Ikeda et al. were able to produce the self-assembled lamellae PbTe and Sb2Te3 with

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epitaxy-like interfaces by annealing the metastable Pb2Sb6Te11 alloy. Such spontaneous formation of nanoscale features is desirable because it minimizes the possibility of oxidation and the introduction of other forms of impurities, which would lead to degradation of electrical performance.

Figure 13. Pseudo-binary phase diagram of PbTe-Sb2Te3, with the Pb2Sb6Te11 phases shown as a metastable phase. The region near the eutectic composition is enlarged in (b). Reprinted with permission from Ref. [114] : T. Ikeda, L. A. Collins, V. A. Ravi, F. S. Gascoin, S. M. Haile, and G. J. Snyder, Chemistry of Materials 19, 763 (2007) Copyright @ American Chemical Society.

The phase separation method has also been used to obtain PbTe nanocomposites with Ag, Pb, and Sb nanoprecipitates [110, 115], AgSbTe2 in PbTe [111] and in PbSnTe [112], and PbS in PbTe [113]. Figure 14(a) shows a TEM micrograph of LAST-18 sample (AgPb18SbTe20) obtained by phase separation process. The sample shows nano-sized region of the crystal structure that is Ag-Sb–rich in composition. The surrounding structure is epitaxially related to this feature, but is Ag-Sb–poor in composition, and closer to that of PbTe. Figure 14(b) shows the TEM image of the lamellar nanostructure

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formed spontaneously by the separation of PbSbTe into Sb2Te3-rich and PbTe-rich phases.

(a)

(b)

Figure 14. (a) TEM image of a AgPb18SbTe20 sample showing a nano-sized region (a “nanodot” shown in the enclosed area) of the crystal structure that is Ag-Sb–rich in composition. The surrounding structure, which is epitaxially related to this feature, is Ag-Sb–poor in composition, and closer to that of PbTe. Reprinted with permission from Ref. [111] : K. F. Hsu, et al. Science 303, 818 (2004) Copyright @ American Association for the Advancement of Science. (b) Microstructure of metastable phase Pb2Sb6Te11 transformed into self-assembled lamellae of Sb2Te3 and PbTe regions by annealing. The lighter regions are PbTe, and the darker regions are Sb2Te. Reprinted with permission from Ref. [114] : T. Ikeda, L. A. Collins, V. A. Ravi, F. S. Gascoin, S. M. Haile, and G. J. Snyder, Chemistry of Materials 19, 763 (2007) Copyright @ American Chemical Society.

4. Recent Achievements in Thermoelectric Nanocomposites As the Seebeck coefficient, the electrical conductivity and thermal conductivity are strongly temperature dependent, any thermoelectric material is suitable for operation over a limited temperature range. Corresponding to the conventional bulk thermoelectric materials as shown in Fig. 2, nanostructured bulk thermoelectric materials with enhanced ZT have been developed over the past 5-10 years. Figure 15 summarizes some nanostructured bulk thermoelectric materials appropriated for different temperature ranges.

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Figure 15. Plots showing the temperature dependence of the dimensionless figure of merit ZT of several nanocomposite materials. In comparison with Fig. 2, it may be noted that that many of the nanocomposites show enhanced ZTs significantly higher than 1.

4.1. Bi2Te3-Based Nanocomposites for Low Temperature Applications Thermoelectric materials that operate in the range 200 K to 400 K are considered as low temperature materials. The primary application of these materials is refrigeration and temperature control of laboratory instruments. Another application of materials that operate at this temperature range lies in the recovery of low-quality waste heat from automobile radiators (~400 K) or even from electronic chips. Currently, most of the thermoelectric devices commercially available and commonly used for applications around room temperature are based on Bi2Te3-Sb2Te3 alloys, due to the fact they have the highest ZT (~1) among any bulk materials around room temperature. However, the temperature range over which these devices can efficiently operate is rather small (-20°C to 100°C) due to the fast deterioration of thermoelectric properties with variation of temperature. Recently, Poudel

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et al. [55] made nanostructured samples of BixSb2-xTe3 by hot pressing nanopowders obtained by ball milling of crystalline ingots of Bi2Te3 and Sb2Te3 under inert conditions. Figure 9 shows the micrograph of a sample obtained using TEM. Nanoscale crystalline features, randomly oriented with each other can be clearly seen. As shown in Fig. 15, ZT of about 1.2 has been reached at room temperature, and as high as 0.8 at 250°C. Comparing this data with the ZT of bulk BixSb2-xTe3 materials shown in Fig. 2, it can be seen that the nanocomposite has extended the operational range of the material to a considerable extent, making it useful for both cooling and power generation applications. The high ZT is the result of low lattice thermal conductivity, due to the increased phonon scattering with the interfaces of nanostructures and dislocations. The nanocomposites also show a comparable or higher power factor throughout the temperature range than the bulk ingots. These samples do not suffer from cleavage problem that is common in ingots prepared by traditional zone-melting, which leads to easier device fabrication and integration. Alternative to starting from the alloyed crystalline ingots of BixSb2-xTe3 as in Poudel’s work, nanocomposites could also be made by starting from elemental chunks of Bi, Sb and Te, which are ball milled to get nanopowders [116]. These nanopowders are then hot pressed to obtain samples that show similarly high ZT. The direct route from elements to nanostructured alloy-compounds is more cost-effective and environmentally friendly. The ZT obtained by this method are only about 10% lower than those obtained by using the compounds of Bi2Te3 and Sb2Te3 as the starting materials, apparently due to some microstructural differences and absence of minority elements like Zn, Cd. Hydrothermal method has also been used to synthesize Bi2Te3 nanocomposites [73, 117-119]. Ni et al. synthesized nanopowders of Bi2Te3 using this method, which were then hot pressed with zone-melted alloy in a 10:90 ratio [73]. It was found that the nanosized powders reduce the thermal conductivity much stronger than the electrical conductivity, which results in an enhanced thermoelectric figure of merit of a nanocomposite. ZT value of up to 0.83 has been obtained. Further improvement on the figure of merit of the nanocomposites should be

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possible by appropriate doping of the nanopowders and optimization the composition of the base alloys. Cao et al. [117] synthesized nanosized binary Bi2Te3 and Sb2Te3 powders by hydrothermal route, which were then hot pressed in 1:1, 1:3 and 1:7 ratios. TEM images show that the composites have a laminated structure composed of Bi2Te3 and Sb2Te3 nanolayers with the thickness varying alternately between 5 and 50 nm. The nanoscale laminated structure improves the thermoelectric performance in comparison with bulk samples of similar compositions, reaching a high ZT of 1.47 at 450 K for the nanocomposite of 1:1 composition. Tang et al. prepared bulk p-type Bi2Te3 materials with layered nanostructures combining melt spinning with spark plasma sintering [120]. The lattice thermal conductivity measured was up to 60% lower than zone melt ingot, and ZT is enhanced up to 70%. Figure 16 shows the lattice thermal conductivity and electrical conductivity of these samples. While the lattice thermal conductivity of all the SPS samples was lower than that of the ingot, one of the samples showed a higher electrical conductivity than the ingot, resulting in the highest ZT of about 1.35 at 300 K.

Figure 16. Electrical conductivity and thermal conductivity of layered nanostructure of Bi2Te3 using melt spinning combined with spark plasma sintering, in comparison with the zone melt ingot. The numbers in the sample name indicate the speed of the roller during the melt spin process in m/s. Reprinted with permission from Ref. [120] X. Tang, W. Xie, H. Li, W. Zhao, Q. Zhang and M. Niino, Appl. Phys. Lett. 90, 012102, (2007) Copyright @ American Physical Society.

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Theoretical studies briefly reviewed in Sec. 2 show that nanostructuring of a bulk material can lead to up to an order of magnitude decrease in the lattice thermal conductivity, as seen in Si-Ge material system for instance. However, in Bi2Te3 thin films, the lattice thermal conductivity could only be reduced by a factor of 2 with respect to bulk at room temperature. The reason is likely due to the presence of structural modulations (natural nanostructures) and dislocations in bulk Bi2Te3 which already reduce the lattice thermal conductivity [121], and make the effect of further scattering of phonons less pronounced.

4.2. Medium Temperature Materials Several nanocomposites materials with high ZT in the medium temperature range (400 K to 800 K) have been discovered. These materials could have significant impacts in waste heat recovery for both transportation sectors and industrial exhaust heat. The most prominent ones are based on alloys of PbTe, Mg2Si, skutterudites, etc. 4.2.1. Pbte-Based Nanocomposites Heremans et al. [122] prepared PbTe nanopowders by ball-milling, and then sintered the powders into bulk samples. The nanocomposites showed a slight increase in the Seebeck coefficient over bulk PbTe of the same carrier concentration. It was also found that the scattering parameter showed a slight increase as well, implying the possibility that the nanostructure was responsible for electron energy filtering. In a separate study [110], bulk PbTe samples were prepared in which nanoparticles of excess Pb or Ag metal were precipitated within the PbTe matrix by a tempering anneal process. These samples showed a remarkable enhancement (by up to 100%) in the Seebeck coefficient, and a simultaneous increase in the scattering parameter (which went from < 1 for bulk to about 3-4 in the nanoprecipitates samples). Though the origin of this increase in the scattering parameter is not clear, the effect probably is energy filtering of the electrons, resulting in the high Seebeck coefficient. On the other hand, because the mobility of the

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electrons was too low, no increase in the power factor and ZT was obtained. More recently, Sootsman et al. [115] prepared PbTe with nanoprecipitates of both Pb and Sb simultaneously. This resulted in large enhancement in the power factor over that of bulk PbTe. Remarkably, and rather inexplicably, when the concentration of Sb was 3% and Pb was 2% in the nanocomposite, the electron mobility actually increased with temperature (between 300 K and 450 K). Thought ionic impurity scattering can result in a rising mobility, it is not expected to be dominant at these temperatures. Moreover, simple nanostructuring with either Pb or Sb did not result in the enhancement. Thus, co-nanostructuring seems to result in a novel effect that could probably be extended to other material systems. Some of the highest values of the figure of merit in the medium temperature range have been obtained in the AgSbTe2-(PbTe)m (LAST-m) family of thermoelectric materials [111]. These materials have NaCl structure, with the tellurium occupying the Cl positions, and silver, lead and antimony occupying the Na positions. Thus the anions carry a net charge of -2, while each of the cations carries a net charge of +2. (One pair of Ag+ and Sb3+ may be considered to iso-electronically substitute for two Pb2+ ions in the lattice). Originally, the LAST compounds were considered to be solid solutions of AgSbTe2 and PbTe. Although, according to X-ray diffraction data such as shown in Fig. 17, bulk Ag1-xPbmSbTem+2 specimens with m from 6 to 18 are single-phase, the results of electron diffraction and HRTEM suggest that different microscopic phases co-exist in these specimens [123], which differ in composition. It is always found that a minority phase rich in Ag and Sb is endotaxially embedded in the majority phase poor in Ag and Sb (and rich in Pb). Thus, contrary to the previous understanding [124], AgSbTe2-PbTe do not form solid solutions but exhibit extensive nanostructures caused by compositions fluctuations. High ZT in the order of 2 or more has been demonstrated in the LAST materials at high temperatures. This enhancement of the figure of merit is the result of a very low lattice thermal conductivity, without much loss in the Seebeck coefficient and electrical conductivity. The spontaneously developed nanoscale inhomogeneities act as embedded nanoparticles that scatter phonons, thus reducing the lattice thermal

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conductivity. The low lattice thermal conductivity is caused by the increased phonon scattering due to the distribution of three types of atoms with different masses over the lattice positions of one kind. For different m, the compounds AgPbmSbTem+2 demonstrated close values of thermal conductivity, namely, below 0.5 W/mK at 700 K and 1.3 W/mK at room temperature. All compounds of the LAST family exhibit semiconductor properties with a narrow band gap of ~0.25 eV. The electrical conductivity of compounds increases with an increase in m (i.e. the PbTe content), and reaches a maximum at m=18. The LAST materials demonstrated are n-type. Electrons are the predominant charge carriers; hence, the Seebeck coefficient is negative. However p-type materials can be obtained by use for Na in place of Ag [125], or by using Sn in addition to the Ag, Pb, Sb and Te [112, 126].

Figure 17. Typical powder X-ray diffraction pattern obtained for LAST-m samples showing a single phase rock salt-like lattice structure. However, according to electron diffraction and HRTEM, depending upon m and the processing conditions, both nanophase separation and long-range atomic ordering within the nanophases exist. From Ref. [123]: E. Quarez, K. F. Hsu, R. Pcionek, N. Frangis, E. K. Polychroniadis, and M. G. Kanatzidis, Journal of the American Chemical Society 127, 9177 (2005).

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Ab initio electronic structure calculations [127] show that the high power factor of the LAST compounds can be explained by the appearance of resonance states in the lower part of the conduction band and in the upper part of the valence band. Ag atoms introduce new electronic states near the top of the valence band of PbTe; isolated Sb atoms introduce resonant electronic states near the bottom of the PbTe conduction band. The Ag–Sb pairs result in an increase in the density of states right around the band gap, compared to that of pure PbTe. As a result, the Seebeck coefficient and the power factor are increased. However, it is also found that the increase in the power factor is small when compared with the values typical of pure lead telluride; hence, the good thermoelectric ZT values for the LAST compounds were largely due to the nanostructure-induced thermal conductivity reduction [128]. The LAST materials were originally synthesized by mixing the constituent elements, melting them and then cooling slowly to room temperature, leading to the formation of the nanoscale features by phase separation. Later works have shown that it is possible to obtain similar crystallographic structure and thermoelectric performances by using a mechanical alloying and annealing [58, 129] or preparing nanoparticles by hydrothermal synthesis and then compacting via pressure-less sintering, hot pressing and spark plasma sintering [130]. As PbS is immiscible in PbTe, it is possible to use a similar method as used for the preparation of the LAST compounds to obtain phase separated PbTe-PbS alloys. Androulakis et al. [113] prepared (PbTe)1-x(PbS)x and (Pb0.95Sn0.05Te)1-x(PbS)x. These materails were found to contain nanoscale features rich in PbS, resulting in lattice thermal conductivity as low as ~0.4 W/mK at room temperature. As the mobility of the carriers stayed reasonably high (of the order of 100 cm2/Vs), the ZT reached 1.5 at 642 K for the sample with x=0.08. Ikeda et al. performed extensive microstructural studies in the immiscible PbTe-Sb2Te3 system. It was found that rapid solidification of off- and near-eutectic compositions yield a variety of microstructures, from dendritic to lamellar [131]. Starting with the metastable composition Pb2Sb6Te11 close to the eutectic, they were able to obtain nanometer lamellar structures that resemble thin film superlattices [114, 132]. Figures 13 and 14 (b) show the pseudo-binary phase diagram of the

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PbTe-Sb2Te3 system, and the naturally formed nanoscale multilayers, respectively. It was also shown that by adjusting the temperature and rate of the transformation process, it is possible to control the lamellar spacing. 4.2.2. Mg2Si-Based Nanocomposites An ideal thermoelectric material should not only have a high ZT, but should also be composed of elements that are abundant, non-toxic and light. That is why Mg2(Si,Sn) based materials have attracted much attention lately [133]. In fact, a reasonably high ZT value of ~1.1 was obtained at 800 K [134] in MgSi0.4Sn0.6 solid solutions, which is comparable to that of PbTe and filled skutterudites. Zhang et al. [135] undertook a microstructure study of high ZT Mg2Si0.4-xSn0.6Sbx alloys. The lattice thermal conductivity of these samples are about 1.5-2.1 W/mK at 300 K, as compared to 7.9 W/mK of Mg2Si and 5.9 W/mK of Mg2Sn. Interestingly, the samples showed in situ formed nanodots by phase separation, similar to that observed in the LAST materials. These naturally formed nanoscale compositional/structural modulations are believed to be responsible for the low value of thermal conductivity in these samples.

4.3. High Temperature Materials Thermoelectric devices that operate in the temperature range of above 800 K are primarily of interest to power generation modules in probes for deep space exploration. Silicon-germanium alloys have been used for making the space-exploration generators. 4.3.1. Si-Ge Nanocomposites Alloys of Si and Ge, which represent a solid solution SixGe1-x are among the very few thermoelectric materials that operate at temperatures of above 1000 K. Elemental silicon and germanium are crystallized in the diamond-like structure. As a result of the rigid and symmetric crystal structure, they exhibit thermal conductivity too high to become good

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thermoelectric materials (150 and 63 W/mK, respectively, at room temperature). However, their thermal conductivity can be reduced to approximately 5-10 W/mK by the formation of a solid solution alloying [136]. The chemical stability of SixGe1-x solid solutions at high temperatures, particularly, against oxidation, and the high figure of merit (