BIOSENSING USING NANOMATERIALS

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1 BIOSENSING USING NANOMATERIALS Edited by Arben Merkoçi

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3 BIOSENSING USING NANOMATERIALS

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5 BIOSENSING USING NANOMATERIALS Edited by Arben Merkoçi

6 Copyright # 2009 by John Wiley & Sons, Inc. All rights reserved. Published by John Wiley & Sons, Inc., Hoboken, New Jersey. Published simultaneously in Canada. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except as permitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, (978) , fax (978) , or on the web at Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, (201) , fax (201) , or online at Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives or written sales materials. The advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor author shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages. For general information on our other products and services or for technical support, please contact our Customer Care Department within the United States at (800) , outside the United States at (317) or fax (317) Wiley also publishes its books in a variety of electronic formats. Some content mat appears in print may not be available in electronic formats. For more information about Wiley products, visit our web site at www. wiley.com. Library of Congress Cataioging-in-Publication Data: Merkoçi, A. (Arben) Biosensing using nanomaterials / Arben Merkoçi. p. cm. - - (Wiley series in nanoscience and nanotechnology) Includes index. ISBN (cloth) 1. Biosensors. 2. Nanostructured materials. I. Title. R857.B54M dc Printed in the United States of America

7 CONTENTS CONTRIBUTORS SERIES PREFACE PREFACE xi xv xvii PART I CARBON NANOTUBES 1 1. Carbon Nanotube Based Sensors and Biosensors 3 Richard G. Compton, Gregory G. Wildgoose, and Elicia L. S. Wong 1.1. Introduction to the Structure of Carbon Nanotubes Electroanalysis Using CNT-Modified Electrodes Advantageous Application of CNTs in Sensors: ph Sensing Carbon Nanotube Based Biosensors Using CNTs in Biosensor Production for Medical Diagnostics and Environmental Applications 25 References Isotropic Display of Biomolecules on CNT-Arrayed Nanostructures 39 Mark R. Contarino, Gary Withey, and Irwin Chaiken 2.1. Introduction: CNT Arrays for Biosensing Functionalization of CNTs: Controlling Display Through Covalent Attachment Self-Assembling Interfaces: Anchor-Probe Approach Molecular Wiring of Redox Enzymes Multiplexing Biomolecules on Nanoscale CNT Arrays Conclusions 59 References Interaction of DNA with CNTs: Properties and Prospects for Electronic Sequencing 67 Sheng Meng and Efthimios Kaxiras 3.1. Introduction Structural Properties of Combined DNA CNT Systems Electronic Structure 79 v

8 vi CONTENTS 3.4. Optical Properties Biosensing and Sequencing of DNA Using CNTs Summary 92 References 93 PART II NANOPARTICLES Improved Electrochemistry of Biomolecules Using Nanomaterials 99 Jianxiu Wang, Andrew J. Wain, Xu Zhu, and Feimeng Zhou 4.1. Introduction CNT-Based Electrochemical Biosensors Nanoparticle-Based Electrochemical Biosensors Quantum Dot Based Electrochemical Biosensors Conclusions and Outlook 123 References The Metal Nanoparticle Plasmon Band as a Powerful Tool for Chemo- and Biosensing 137 Audrey Moores and Pascal Le Floch 5.1. Introduction The SPB: An Optical Property of Metal NPs Plasmon Band Variation Upon Aggregation of Nanoparticles Plasmon Band Variation on the Environment or Ligand Alteration Metal Nanoparticles as Labels Conclusions 169 References Gold Nanoparticles: A Versatile Label for Affinity Electrochemical Biosensors 177 Adriano Ambrosi, Alfredo de la Escosura-Muñiz, Maria Teresa Castañeda, and Arben Merkoçi 6.1. Introduction Synthesis of AuNPs Characterization of AuNPs AuNPs as Detecting Labels for Affinity Biosensors Conclusions 191 References Quantum Dots for the Development of Optical Biosensors Based on Fluorescence 199 W. Russ Algar and Ulrich J. Krull 7.1. Introduction 200

9 CONTENTS vii 7.2. Quantum Dots Basic Photophysics and Quantum Confinement Quantum Dot Surface Chemistry and Bioconjugation Bioanalytical Applications of Quantum Dots as Fluorescent Labels Fluorescence Resonance Energy Transfer and Quantum Dot Biosensing Summary 238 References Nanoparticle-Based Delivery and Biosensing Systems: An Example 247 Almudena Muñoz Javier, Pablo del Pino, Stefan Kudera, and Wolfgang J. Parak 8.1. Introduction Functional Colloidal Nanoparticles Polyelectrolyte Capsules as a Functional Carrier System Uptake of Capsules by Cells Delivery and Sensing with Polyelectrolyte Capsules Conclusions 270 References Luminescent Quantum Dot FRET-Based Probes in Cellular and Biological Assays 275 Lifang Shi, Nitsa Rosenzweig, and Zeev Rosenzweig 9.1. Introduction Luminescent Quantum Dots Fluorescence Resonance Energy Transfer Quantum Dot FRET-Based Protease Probes Summary and Conclusions 283 References Quantum Dot Polymer Bead Composites for Biological Sensing Applications 291 Jonathan M. Behrendt and Andrew J. Sutherland Introduction Quantum Dot Composite Construction Applications of QD Composites Future Directions 325 References Quantum Dot Applications in Biomolecule Assays 333 Ying Xu, Pingang He, and Yuzhi Fang Introduction to QDs and Their Applications Preparation of QDs for Conjugation with Biomolecules and Cells 337

10 viii CONTENTS Special Optoelectronic Properties in the Bioemployment of QDs Employment of QDs as Biosensing Indicators 344 References Nanoparticles and Inductively Coupled Plasma Mass Spectroscopy Based Biosensing 355 Arben Merkoçi, Roza Allabashi, and Alfredo de la Escosura-Muñiz ICP-MS and Application Possibilities Detection of Metal Ions Detection of Nanoparticles Analysis of Metal-Containing Biomolecules Bioanalysis Based on Labeling with Metal Nanoparticles Conclusions 372 References 373 PART III NANOSTRUCTURED SURFACES Integration Between Template-Based Nanostructured Surfaces and Biosensors 379 Walter Vastarella, Jan Maly, Mihaela Ilie, and Roberto Pilloton Introduction Nanosphere Lithography Nanoelectrodes Ensemble for Biosensing Devices Concluding Remarks 406 References Nanostructured Affinity Surfaces for MALDI-TOF-MS Based Protein Profiling and Biomarker Discovery 421 R. M. Vallant, M. Rainer, M. Najam-Ul-Haq, R. Bakry, C. Petter, N. Heigl, G. K. Bonn, and C. W. Huck Proteomics and Biomarkers MALDI in Theory and Practice Carbon Nanomaterials Near-Infrared Diffuse Reflection Spectroscopy of Carbon Nanomaterials 448 References 451 PART IV NANOPORES Biosensing with Nanopores 459 Ivan Vlassiouk and Sergei Smirnov Nanoporous Materials in Sensing 459

11 CONTENTS ix Nanochannel and Nanopore Fabrication Surface Modification Chemistry Nonelectrical Nanoporous Biosensors Electrical Nanoporous Biosensors Summary 486 References 486 INDEX 491

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13 CONTRIBUTORS W. Russ Algar, Department of Chemical and Physical Sciences, University of Toronto Mississauga, Mississauga, Ontario, Canada Roza Allabashi, University of Natural Resources and Applied Life Sciences, Vienna, Austria Adriano Ambrosi, Nanobioelectronics and Biosensors Group, Institut Catala de Nanotecnologia, Barcelona, Spain R. Bakry, Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University, Innsbruck, Austria Jonathan M. Behrendt, Department of Chemical Engineering and Applied Chemistry, Aston University, Birmingham, United Kingdom G. K. Bonn, Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University, Innsbruck, Austria Maria Teresa Castañeda, Nanobioelectronics and Biosensors Group, Institut Catala de Nanotecnologia, Barcelona, Spain, and Grup de Sensors i Biosensors, Departamento de Quımica, Universitat Autònoma de Barcelona, Bellaterra, Catalonia, Spain; on leave from Departamento de Ciencias Basicas, Universidad Autónoma Metropolitana-Azcapotzalco, Mexico D.F., Mexico Irwin Chaiken, Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania Richard G. Compton, Physical and Theoretical Chemistry Laboratory, Oxford University, Oxford, United Kingdom Mark R. Contarino, School of Biomedical Engineering, Science and Health Systems, Drexel University, and Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania Alfredo de la Escosura-Muñiz, Nanobioelectronics and Biosensors Group, Institut Catala de Nanotecnologia, Barcelona, Spain, and Instituto de Nanociencia de Aragón, Universidad de Zaragoza, Zaragoza, Spain Pablo del Pino, Fachbereich Physik, Philipps Universit at Marburg, Marburg, Germany Yuzhi Fang, Department of Chemistry, East China Normal University, Shanghai, China xi

14 xii CONTRIBUTORS Pingang He, Department of Chemistry, East China Normal University, Shanghai, China N. Heigl, Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University, Innsbruck, Austria C. W. Huck, Institute of Analytical Chemistry and Radiochemistry, Leopold- Franzens University, Innsbruck, Austria Mihaela Ilie, Department of Applied Electronics and Information Engineering, LAPI, Universitatea Politehnica Bucureşti, Bucharest, Romania Efthimios Kaxiras, Department of Physics and School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts Ulrich J. Krull, Department of Chemical and Physical Sciences, University of Toronto Mississauga, Mississauga, Ontario, Canada Stefan Kudera, Department of New Materials and Biosystems, Max Planck Institute for Metals Research, and Department of Biophysical Chemistry, University of Heidelberg, Stuttgart, Germany Pascal Le Floch, Heteroelements et Coordination, Ecole Polytechnique, Palaiseau, France Jan Maly, Department of Biology, University of J.E. Purkyne, Usti nad Labem, Czech Republic Sheng Meng, Department of Physics and School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts Arben Merkoçi, ICREA and Nanobioelectronics and Biosensors Group, Institut Catala de Nanotecnologia, Barcelona, Spain Audrey Moores, Department of Chemistry, McGill University, Montreal, Quebec, Canada Almudena Muñoz Javier, Fachbereich Physik, Philipps Universit at Marburg, Marburg, Germany M. Najam-Ul-Haq, Institute of Analytical Chemistry and Radiochemistry, Leopold- Franzens University, Innsbruck, Austria Wolfgang J. Parak, Fachbereich Physik, Philipps Universit at Marburg, Marburg, Germany C. Petter, Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University, Innsbruck, Austria Roberto Pilloton, ENEA C.R. Casaccia, Rome, Italy M. Rainer, Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University, Innsbruck, Austria

15 CONTRIBUTORS xiii Nitsa Rosenzweig, Department of Chemistry and the Advanced Materials Research Institute, University of New Orleans, New Orleans, Louisiana Zeev Rosenzweig, Department of Chemistry and the Advanced Materials Research Institute, University of New Orleans, New Orleans, Louisiana Lifang Shi, Department of Chemistry and the Advanced Materials Research Institute, University of New Orleans, New Orleans, Louisiana Sergei Smirnov, Department of Chemistry and Biochemistry, New Mexico State University, Las Cruces, New Mexico Andrew J. Sutherland, Department of Chemical Engineering and Applied Chemistry, Aston University, Birmingham, United Kingdom R. M. Vallant, Institute of Analytical Chemistry and Radiochemistry, Leopold- Franzens University, Innsbruck, Austria Walter Vastarella, ENEA C.R. Casaccia, Rome, Italy Ivan Vlassiouk, Department of Physics and Astronomy, University of California, Irvine, California Andrew J. Wain, Department of Chemistry and Biochemistry, California State University, Los Angeles, California Jianxiu Wang, School of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan, China Gregory G. Wildgoose, Physical and Theoretical Chemistry Laboratory, Oxford University, Oxford, United Kingdom Gary Withey, Engineering Division, Brown University, Providence, Rhode Island Elicia L. S. Wong, Physical and Theoretical Chemistry Laboratory, Oxford University, Oxford, United Kingdom Ying Xu, Department of Chemistry, East China Normal University, Shanghai, China Feimeng Zhou, Department of Chemistry and Biochemistry, California State University, Los Angeles, California Xu Zhu, School of Chemistry and Chemical Engineering, Central South University, Changsha, Hunan, China

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17 SERIES PREFACE Nanoscience and nanotechnology refer broadly to a field of applied science and technology whose unifying theme is the control of matter on the molecular level in scales smaller than 1 micrometer, normally 1 to 100 nanometers, and the fabrication of devices within that size range. Nanotechnology also refers to the manufacture of nanosized systems that perform specific electrical, mechanical, biological, chemical, or computing tasks. Nanotechnology is based on the fact that nanostructures, nanodevices, and nanosystems exhibit novel properties and functions as a result of their small size. It is a highly multidisciplinary field, drawing from such subjects as applied physics, materials science, colloidal science, device physics, supramolecular chemistry, and mechanical and electrical engineering. Given the inherent nanoscale functions of the biological components of living cells, it was inevitable that nanotechnology would also be related and applied to the life sciences through the application of nanoscaled tools to biological systems as well as the uses of biological systems as templates in the development of novel nanoscaled products. Much speculation exists related to what new science and technology may result from the synergy of the disciplines mentioned. Nanotechnology can be seen as an extension of existing sciences to the nanoscale level. Two primary approaches are used in nanotechnology. In the bottom-up approach, materials and devices are built from molecular components which assemble themselves chemically by principles of molecular composition. In the top-down approach, nanoobjects are constructed from larger entities without atomic-level control. The impetus for nanotechnology comes from a renewed interest in colloidal science, coupled with a new generation of analytical tools such as the atomic force microscope and the scanning tunneling miccroscope. Combined with refined processes such as electron-beam lithography and molecular-beam epitaxy, these instruments allow the deliberate manipulation of nanostructures and have led to the observation of novel phenomena. Examples of nanotechnology in modern use are the manufacture of polymers based on molecular structure and the design of computer chip layouts based on surface science. Despite the great promise of numerous nanotechnologies, such as quantum dots and nanotubes, real commercial applications have mainly used the advantages of colloidal nanoparticles in bulk form, such as in suntan lotion, cosmetics, protective coatings, and stain-resistant clothing. xv

18 xvi SERIES PREFACE Research in the field of nanoscience and technology is very active and is expected to remain so for the foreseeable future. The Wiley Series in Nanoscience and Nanotechnology will be focused on the following important topics: * Basic nanoscience * Nanotechnology tools * Nanomaterials * Nanobiosensors * Nanobiotechnology * Nanotechnology for environment. * Nanotechnology and energy * Nanotechnology and electronics/computers * Nanotechnology and ethical issues We welcome coverage of other topics in response to issues that arise in coming months. All topics will be edited by experts the various nanoscience and nanotechnology fields and will serve as a reference source for this new and exciting science and technology. Collaborations are welcome! Bellaterra, Catalonia, Spain July 2008 ARBEN MERKOÇI Series Editor

19 PREFACE The implementation of nanoscience and nanotechnology achievements in bioanalysis is the main objective of this book, whose aim is to explaining to readers several strategies related to the integration of nanomaterials with bioanalytical systems as one of the hottest topics of today s nanotechnology and nanoscience. Novel concepts are shown, together with practical aspects of nanoscale material s integration into biosensing systems. This integration is due to the capacity of nanomaterials to provide special optical or electrical properties as well as stability and to minimize surface fouling of the sensing systems where they are being integrated. Various nanomaterials, including carbon nanotubes, nanoparticles, nanomagnetic beads, and nanocomposites, are being used to develop highly sensitive and robust biosensors and biosensing systems. The materials mentioned are attractive probe candidates because of their (1) small size (1 to 100 nm) and correspondingly large surface-to-volume ratio; (2) chemically tailorable physical properties, which relate directly to size, composition, and shape; (3) unusual target binding properties; and (4) overall structural robustness. This is an interdisciplinary book dedicated to professionals who have an interest in the improving the current bioanalytical techniques and methodologies by implementing nanoscience and nanotechnology in general and nanomaterials in particular. The goal is to present the most recent scientific and technological advances as well as practical bioanalytical applications based on the use of nanomaterials. It will be an important reference source for a broad audience involved in the research, teaching, learning, and practice of nanomaterial integration into biosensing systems for clinical, environmental, and industrial applications. Bioanalysis in general and biosensor fields in particular are showing special interest in nanobiomaterials. Nanomaterials bring several advantages to bioanalysis. Their immobilization on sensing devices generates novel interfaces that enable sensitive optical or electrochemical detection of molecular and biomolecular analytes. Moreover, they are being used as effective labels to amplify the analysis and to design novel biomaterial architectures with predesigned and controlled functions with interest for several applications. Achieving higher sensitivities and better and more reliable analysis are one of the objectives of DNA probes and immunoanalysis. Carbon nanotubes (CNTs) represent one of the building blocks of nanotechnology. With one hundred times the tensile strength of steel, thermal conductivity better than that of all but the purest diamond, and electrical conductivity similar to that of copper but with the ability to carry much higher currents, CNTs seem to be a very interesting xvii

20 xviii PREFACE material. Since their discovery in 1991, CNTs have generated great interest for future applications based on their field emission and electronic transport properties, high mechanical strength, and chemical properties. The structural and electronic properties of CNTs provide them with distinct properties for facilitating direct electrochemistry of proteins and enzymes compared to other types of materials used so far. The bioelectrochemistry and optical properties, along with some other interesting features of CNTs coupled to several bioanalytical procedures, are also presented. Nanoparticles of a variety of shapes, sizes, and compositions are changing the bioanalytical measurement landscape continuously and so are also included. Nanoparticles can be used, for example, in quantification or codification purposes, due to their chemical behavior, which is similar to that of small molecules. They also provide novel platforms for improving the activity of DNA probes, antibodies, or enzymes. Nanoparticles may be expected to be superior in several ways to other materials commonly used in bioanalysis. They are more stable and cheaper, allow more flexibility, have faster binding kinetics (similar to those in a homogeneous solution), and have high sensitivity and high reaction rates for many types of multiplexed assays, ranging from immunoassays to DNA analysis. Improving the current bionalytical techniques and methodologies and finding novel concepts and applications in bioanalysis are two of the most important objectives of nanotechnology and nanoscience today. Advances in nanotechnology are affecting existing technologies and leading to the development of novel bioanalytical tools and techniques through improvements in precision and speed, lower sample requirements, and the ability to perform multiple detections in smaller devices. Novel biosensing systems that require less sample material are being developed so as to perform sophisticated tests at the point of care (e.g., blood analysis using a handheld device within a few minutes) and make possible the multiplex analysis (i.e., simultaneous and fast analysis of more than one variable). Analysis of the structure of living cells in real time and in the intact organism (in vivo) as opposed to using laboratory-prepared samples (in vitro), as well as molecular analysis (DNA, RNA, and protein analysis), including biosensors based on nanomaterials, represent challenges not only for basic research in nanotechnology but for the bioanalysis community, which is willing to see new input from this novel area of research that is developing so fast. In this context the book introduces novel concepts that are being achieved in the area of bioanalysis based on the fact that nanomaterials are opening up new opportunities not only for basic research but overall, are offering new tools for real bioanalytical applications. The focus is on the latest tendencies in the field of nanoparticles, carbon nanotubes, and nanohannels: integration into bionalytical systems, including sensors and biosensors. The area of biosensing using nanotechnology is providing the information necessary for real applications of nanomaterials and related nanotechnologies. The book should act as a very interesting interface of information between complementary fields, thus opening up new opportunities for researchers and others. It will also support doctoral students and those involved in learning and teaching nanobiotechnological applications in bioanalysis.

21 PREFACE xix How does the bionalytical community implement the spectacularly bright future offered us by nanoscience? What are the challenges facing bioanalysis in our nanoscale future? How do we move from an almost science fiction level toward real-world outcomes in nanotechnology? The bioanalytical community will be better able to respond to these questions in the future after reading this book, which not only compares nanomaterials to conventional materials but also gets inside the response mechanisms related to such improvements. The book s fifteen chapters deal with the most successful nanomaterials used so far in biosensing: carbon nanotubes, nanoparticles, and nanochannels, including detection strategies ranging from optical to electrochemical techniques. Each chapter provides a theoretical overview topic of interest as well as a discussion of the published data with a selected list of references for further details. The book provides a comprehensive forum that integrates interdisciplinary research and development of interest to scientists, engineers, researchers, manufacturers, teachers, and students in order to present the most recent advances in the integration of nanomaterials with bioanalysis as they relate to everyday life. The book promotes the use of novel nanomaterials in biosensors and biosensing systems through introduction of the highest-quality research in the field of nanomaterial-based bioassay. ARBEN MERKOÇI Bellaterra, Catalonia, Spain July 2008

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23 PART I CARBON NANOTUBES 1

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25 CHAPTER 1 Carbon Nanotube Based Sensors and Biosensors RICHARD G. COMPTON, GREGORY G. WILDGOOSE, and ELICIA L. S. WONG Physical and Theoretical Chemistry Laboratory, Oxford University, Oxford, United Kingdom 1.1 Introduction to the structure of carbon nanotubes 1.2 Electroanalysis using CNT-modified electrodes Historical background Electron transfer at graphitic carbon electrodes: The role of edge-plane defects Exceptions to the rule that edge-plane defects are the important electroactive sites on CNTs 1.3 Advantageous application of CNTs in sensors: ph sensing 1.4 Carbon nanotube based biosensors Introduction Surface functionalization of carbon nanotubes and configuration of a CNT-based electrochemical biosensor 1.5 Using CNTs in biosensor production for medical diagnostics and environmental applications Medical diagnostics Environmental applications 1.1 INTRODUCTION TO THE STRUCTURE OF CARBON NANOTUBES In recent years carbon nanotubes (CNTs) have attracted, and continue to attract, considerable interest in a wide variety of scientific fields, not least that of the electroanalytical and bioelectroanalytical communities. However, before we summarize some of the main properties of CNTs that make them an interesting and often ideal material to use in electrochemical sensors and biosensors, let us begin by highlighting perhaps the most common misconception found in the literature concerning Biosensing Using Nanomaterials, Edited by Arben Merkoçi Copyright Ó 2009 John Wiley & Sons, Inc. 3

26 4 CARBON NANOTUBE BASED SENSORS AND BIOSENSORS CNTs: who should actually be credited with their discovery. Numerous examples can be found among the many thousands of carbon nanotube papers that begin with an erroneous introductory phrase along the lines of: Since the discovery of CNTs in 1991 by S. Iijima..., with subsequent sentences proceeding to list the much vaunted electrical and mechanical properties of CNTs. Certainly, Iijima s 1991 high-profile publication in Nature [1] brought the idea of nanometer-sized carbon fibers back to the attention of a wider scientific audience, particularly as, after the excitement created over the discovery of fullerenes, scientists were starting to think about materials on the truly nano scale. In fact, Iijima and Ichihashi should more correctly be credited with the first convincing discovery of single-walled carbon nanotubes (consisting of a single sheet of graphite rolled into a seamless tube) in their 1993 paper [2]. The case for who discovered multiwalled carbon nanotubes is more complex, with the earliest possible claim dating from as far back as 1952! However, work from 1976 by Oberlin and Endo [3] and in 1978 by Wiles and Abrahamson [4] (subsequently republished in 1999 [5]) present arguably the earliest and clearest characterization of what would later be recognized as multiwalled CNTs. For a more detailed discussion of who should be credited with the discovery of CNTs, the interested reader is directed to the comprehensive editorial by Monthioux and Kuznetsov [6]. Structurally, CNTs can be approximated as rolled-up sheets of graphite. CNTs are formed in two principal types: single-walled carbon nanotubes (SWCNTs), which consist of a single tube of graphite, as shown in Figure 1.1, and multiwalled carbon nanotubes (MWCNTs), which consist of several concentric tubes of graphite fitted one inside the other. The diameters of CNTs can range from just a few nanometers in the case of SWCNTs to several tens of nanometers for MWCNTs. The lengths of the tubes are usually in the micrometer range. Conceptually, the way in which the graphite sheet is rolled up to form each nanotube affects the electronic properties of that CNT. In general, any lattice point in the graphite sheet can be described as a vector position (n,m) relative to any given origin. The graphite sheet can then be rolled into a tube such that the lattice point chosen is coincident with the origin (Figure 1.1). The orientation of this roll-up vector relative to the graphite sheet determines whether the tube forms a chiral, armchair, or zigzag SWCNT, terms that describe the manner in which the fused rings of a graphite sheet are arranged at the termini of an idealized tube. Alternatively, SWCNTs are more precisely described in the literature by the roll-up vector coordinates as [n,m]-swcnts. It has been shown that when n m ¼ 3q, where q is an integer, the CNT is metallic or semimetallic and the remaining CNTs are semiconducting [7,8]. Therefore, statistically, one-third of SWCNTs are metallic depending on the method and conditions used during their production [7] and can possess high conductivity, greater than that of metallic copper, due to the ballistic (unscattered) nature of electron transport along a SWCNT [9]. If one considers the structure of a perfect crystal of graphite, two crystallographic faces can be identified, as shown in Figure 1.2. One crystal face consists of a plane containing all the carbon atoms of one graphite sheet, which we call the basal plane; the other crystal face is a plane perpendicular to the basal plane, which we call the edge plane. By analogy to the structure of graphite, two regions on a CNT can be

27 INTRODUCTION TO THE STRUCTURE OF CARBON NANOTUBES 5 FIGURE 1.1 (a) How the lattice vectors a 1 and a 2 of a graphene sheet can be used to describe a roll-up vector to form a single-walled carbon nanotube; (b) space-filled model of a zigzag (n,0)-swcnt. identified (and are labeled in Figure 1.1) as (1) basal-plane-like regions comprising smooth, continuous tube walls and (2) edge-plane-like regions where the rolled-up graphite sheets terminate, typically located at the tube ends and around holes and defect sites along tube walls. As we discuss in Section 1.2, it is these edge-plane-like defects that are crucial to an understanding of some of the surface chemistry and the electrochemical behavior of CNT (MWCNT, in particular) based analytical and bioanalytical systems. FIGURE 1.2 Crystal faces of a highly ordered crystal of graphite and the formation of an edge-plane step defect.

28 6 CARBON NANOTUBE BASED SENSORS AND BIOSENSORS In the case of MWCNTs, a number of morphological variations are also possible, depending on the conditions and chosen method of CNT formation [e.g., chemical vapor deposition (CVD) or the high-voltage arc discharge method (ARC)] [10]. They can be formed as hollow-tube (h-mwcnts), herringbone (hb-mwcnts) [10], or bamboo-like (b-mwcnts) [11]. The different internal structures of h-mwcnts and b-mwcnts are clearly visible in the high-resolution transmission electron microscopic images shown in Figure 1.3. In h-mwcnts the graphitic tubes are oriented parallel to the tube axis and the central cavity remains open along the entire length of the h-mwcnt. The hb-mwcnts and b-mwcnts differ in that the graphitic tubes are oriented at an angle to the axis of the nanotube in both cases, but while the central cavity remains open in the hb-mwcnts, in the b-mwcnts the central cavity is periodically closed off into compartments similar to the structure of bamboo, from which the b-mwcnt name is derived. The interested reader is directed to a fascinating insight into the mechanism of b-mwcnt formation from metal nanoparticle catalyst seeds using the CVD method in real time by Lin et al. [12] using transmission electron microscopy. From Figure 1.3 it is also apparent that, due to the nonaxial arrangement of graphitic tubes in the hb-mwcnts and b-mwcnts, a greater number of edge-plane-like defect sites are formed along the length of the nanotubes compared to the h-mwcnts. In the case of MWCNTs it has recently been proposed that only the outer two or three graphitic tubes carry any significant current along the tube length except at the FIGURE 1.3 Three morphological variations of MWCNTs, and high-resolution transmission electron micrograph images of (a) bamboo-like and (b) hollow-tube MWCNTs.

29 ELECTROANALYSIS USING CNT-MODIFIED ELECTRODES 7 ends of the tubes [13]. This is due to the mismatch and poor overlap of p-orbitals between different tubes within the multiwalled structure, where adjacent tubes may show different metallic or semiconducting properties. However, at the ends of the tubes, because of the higher energy density at these reactive edge-plane-like sites, intershell electron hopping is possible and the current is discharged by the entire ensemble of tube ends [13]. These inherent properties of CNTs, together with their high aspect ratio and the ability to incorporate them easily and rapidly onto or into an electrode substrate (as either random networks or as highly ordered and well-defined nanoscale structures such as vertical arrays, which are discussed in later sections), have resulted in CNTs being used in numerous electroanalytical and bioelectroanalytical applications and sensors. Furthermore, as the CNTs often possess chemical reactivity and surface functional groups similar to those of graphite, their properties can be tailored in an advantageous manner by chemically modifying the surface of the CNTs using an extraordinary variety of synthetic strategies, some of which are reviewed in later sections of this chapter. 1.2 ELECTROANALYSIS USING CNT-MODIFIED ELECTRODES Historical Background The first reported use of CNTs in electroanalysis was the pioneering work of Britto and co-workers in 1996 [14]. By incorporating CNTs into a paste electrode using bromoform as the binder, they were able to explore the electrochemical oxidation of dopamine. However, the number of papers using CNTs in electroanalysis exploded at around the turn of the twenty-first century, due in large part to the pioneering work of Joseph Wang, then at the University of New Mexico, whose article had, as of August 2007, received almost 260 citations [15]! By modifying a glassy carbon (GC) electrode with a sprinkling of CNTs, Wang et al. reported that the biologically important molecule nicotinamide adenine dinucleotide (NADH) could be detected at a much lower potential than at a bare GC electrode in the absence of nanotubes [16]. This work inspired a new trend within the electroanalytical community, and the race was on to modify electrode substrates, usually but not always a GC electrode, with CNTs which were then found to allow the electrocatalytic detection of literally hundreds of inorganic and biological analytes, such as insulin [17], uric acid [18], catechol [19], morphine [20], brucine [21], cytochrome c [22], galactose [23], glucose [24], nitric oxide [25], and horseradish peroxidase [26], as well as NADH [16] and hydrogen peroxide [27], the latter two of which comprise important substrates, cofactors, and products for more than 800 enzymes. The list of analytes that could be detected electrocatalytically at these CNT-modified electrodes was (and perhaps still is) increasing at a rate of more than a dozen or more new analytes each week. The apparent electrocatalytic benefit of using a CNT-modified electrode was typically manifested by a lower detection limit, increased sensitivity, and in particular,

30 8 CARBON NANOTUBE BASED SENSORS AND BIOSENSORS a lower overpotential at which the analyte was detected compared to the unmodified electrode substrate [28]. However, at the time, very few researchers were asking what the underlying physical cause of this apparent electrocatalytic behavior could be attributed to it was as if this effect was considered to be due to some intrinsic, almost magical property of the CNTs themselves! This question prompted further investigation of the electrochemical behavior of CNTs [29 31]. In order to understand the electrocatalytic behavior observed at CNT-modified electrodes, it is first necessary to understand the electrochemical behavior of an analogous electrode substrate: graphite, in particular where on a graphite electrode the electroactive sites for electron transfer are located Electron Transfer at Graphitic Carbon Electrodes: The Role of Edge-Plane Defects Over the past three decades there has been a strong interest in understanding the fundamentals of electron transfer at graphite electrodes. To do this, it is necessary to fabricate an electrode that has a well-defined structure, and as such, most research is usually carried out on highly ordered pyrolytic graphite (HOPG) electrodes. The basal-plane crystal face of a HOPG consists principally of atomically flat terraces of graphite. However, the main difficulty faced when trying to understand electron transfer at graphite electrode surfaces is that these surfaces are inherently spatially heterogeneous. Even the most carefully prepared surface of a HOPG possesses step defects (on a well-prepared surface these defects can comprise as little as 0.2% of the total surface coverage [32]) which reveal thin bands of the edge-plane face of the HOPG crystal, the height of which varies as multiples of nm. These nanobands of edge-plane regions break up the large, flat, basal-plane terraces into islands up to 1 to 10 mm in size, depending on the quality of the HOPG crystal and the surface preparation [33 35]. The rates of electron transfer at these two crystal faces are very different. Therefore, any attempt to simulate the electrochemical and coupled mass transport processes at such spatially heterogeneous electrode surfaces normally requires that we solve prohibitively computationally demanding three-dimensional problems. In Recent work by Davies et al. within the Compton group, a method of modeling such spatially heterogeneous electrode surfaces using a diffusion domain approximation was developed [29,36,37]. The mathematical and computational details of this model are left to the interested reader, but the resulting benefit of this powerful approach is that the physical model that one is attempting to simulate is reduced from an almost intractable three-dimensional problem to a much more manageable two-dimensional problem [29,36,37]. By simulating experimental data obtained at a HOPG electrode using standard redox probes such as ferrocyanide/ferricyanide and hexamineruthenium(iii)/(ii), Davies et al. suggested that while the electron transfer rate at the edge-plane step defects on the HOPG surface, kedge 0, was relatively fast (typically on the order of 10 2 cm=s), the electron transfer on the basal-plane regions of the HOPG surface was occurring so slowly ðkbasal 0 < 10 9 cm=sþ as to be almost zero. In other words, the entire faradaic electron transfer process occurs solely,