Principles of Bacterial Detection:
Biosensors, Recognition Receptors
and Microsystems


Principles of Bacterial Detection:
Biosensors, Recognition
Receptors and Microsystems
Edited by

MOHAMMED ZOUROB
Biophage Pharma Inc.
Montreal, Canada

SOUNA ELWARY
Consultant to Biophage Pharma Inc.
Montreal, Canada

ANTHONY TURNER
Cranfield University
Bedfordshire, UK

13


Editors
Mohammed Zourob
Biophage Pharma Inc.
Montreal
Canada

Souna Elwary
Consultant to Biophage Pharma Inc.
Montreal
Canada


Anthony Turner
Cranfield University
Bedfordshire
UK


ISBN: 978-0-387-75112-2

e-ISBN: 978-0-387-75113-9

Library of Congress Control Number: 2007941938
© 2008 Springer Science+Business Media, LLC
All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the
publisher (Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief
excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and
retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter
developed is forbidden.
The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified
as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights.
Printed on acid-free paper
9 8 7 6 5 4 3 2 1
springer.com



Preface

Bacterial contamination of food and water resources, as well as the increasing incidence of
nosocomial infections, has us on our toes, looking for ways of recognizing these elements. In
addition, the recent and growing threats to personal and territorial securities make this task even
more urgent. Therefore, accurate assessment of the state of current technologies is a prerequisite
for undertaking any course of action towards future improvements. In particular, development
of new detection and identification technologies for the plethora of bacterial agents has become
increasingly important to scientists and to regulatory agencies. In recent years, there has been
much progress in the field of bacterial agents detection, resulting in the development of more
accurate, fast, analyte-specific, robust, and cost effective techniques by incorporating emerging
technologies from various disciplines.
Principles of Bacterial Detection: Biosensors, Recognition Receptors and Microsystems
presents a significant and up-to-date review of various integrated approaches for bacterial
detection by distinguished engineers and scientists. This work is a comprehensive approach to
bacterial detection, presenting a thorough knowledge of the subject and an effective integration
of disciplines in order to appropriately convey the state-of the-art fundamentals and applications
of the most innovative approaches.
The book consists of four parts. The first part (Chapters 1–4) is an introduction to
pathogenic bacteria and sampling techniques and provides an overview of the rapid microbiological methods. The second part (Chapters 5–20) describes the different transducers used for
bacterial detection. It covers the theory behind each technique and delivers a detailed stateof-the-art review for all the new technologies used. The third part (Chapters 21–29) covers
the different recognition receptors used in the latest methods for the detection of bacteria.
It describes in detail the use of immunoassays, nucleic acids, oligonucleotide microarrays,
carbohydrates, aptamers, protein microarrays, bacteriophage, phage display, and molecular
imprinted polymers as recognition elements. The fourth part (Chapters 30–36) covers the
different microsystems used for detection/identification and bacterial manipulations, mainly
bacteria lysis in microfluidics, PCR in microfluidics, dielectrophoresis, ultrasonic manipulation
techniques, and mass spectrometry.

We anticipate that the book will be helpful to academicians, practitioners, and
professionals working in various fields, including biomedical sciences, physical sciences,
microsystems engineering, nanotechnology, veterinary science and medicine, food QA, bioterrorism and security as well as allied health, healthcare and surveillance. Since the fundamentals
are also reviewed, we believe that the book will appeal to advanced undergraduate and graduate
students who study in areas related to bacterial detection.
We gratefully acknowledge all authors for their participation and contributions, which
made this book a reality. We give many thanks to Olivier Laczka and Joseph Piliero for the
book cover design.
Mohammed Zourob
Souna Elwary
Anthony Turner
June 2008
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Contents

Part I

Introduction

1. Introduction to Pathogenic Bacteria
Tracey Elizabeth Love and Barbara Jones
1. Pathogenic Microorganisms ...............................................................................................................
1.1. Toxins .........................................................................................................................................
1.2. Adherence ...................................................................................................................................
1.3. Invasion.......................................................................................................................................
1.4. Evasion of the Host Immune Response .....................................................................................
1.5. Iron Acquisition ..........................................................................................................................
1.6. Regulation of Virulence Factors ................................................................................................

2. Sources and Routes of Infection........................................................................................................
2.1. Natural Infection .........................................................................................................................
2.2. Food and Water ..........................................................................................................................
2.3. Hospital Acquired Infections......................................................................................................
2.4. Intentional Infection—Biological Warfare.................................................................................
3. Detection of Pathogenic Microorganisms..........................................................................................
4. Conclusions ........................................................................................................................................
References...........................................................................................................................................

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2. Sample Preparation: An Essential Prerequisite for High-Quality Bacteria
Detection
Jan W. Kretzer, Manfred Biebl and Stefan Miller
1. Introduction ........................................................................................................................................
2. The Sample.........................................................................................................................................

3. Sampling.............................................................................................................................................
3.1. Sample drawing ..........................................................................................................................
4. Microbiological Examination of Foods .............................................................................................
5. Microbiological Examination of Surfaces .........................................................................................
6. Microbiological Examination of Air..................................................................................................
7. Sample Handling ................................................................................................................................
8. Sample Preparation ............................................................................................................................
9. Sample Preparation for Detection of Intact Bacterial Cells..............................................................
10. Sample Preparation for Detection of Bacterial Nucleic Acids .........................................................
11. Conclusions and Future Perspectives ................................................................................................
References...........................................................................................................................................

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3. Detection of Bacterial Pathogens in Different Matrices: Current Practices
and Challenges
Ahmed E. Yousef
1. Introduction ........................................................................................................................................

2. Analytical Tools and Methods: A Historical Perspective .................................................................
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3. Defining the Terms ............................................................................................................................
4. Matrix Complexity and Pathogen Detection .....................................................................................
5. Techniques Currently Used in Pathogen Detection Methods ...........................................................
5.1. Culture Techniques .....................................................................................................................
5.2. Enzyme-Linked Immunoassay....................................................................................................
5.3. Polymerase Chain Reaction (PCR) ............................................................................................
6. Basics of Pathogen Detection ............................................................................................................
6.1. Sampling .....................................................................................................................................
6.1.1. Air Sampling.....................................................................................................................
6.1.2. Surfaces Sampling ............................................................................................................
6.1.3. Bulk Sampling ..................................................................................................................
6.2. Sample Preparation .....................................................................................................................
6.3. Pathogen Amplification ..............................................................................................................
6.4. Selection and Screening..............................................................................................................
6.5. Identification ...............................................................................................................................
6.5.1. Morphological Characteristics..........................................................................................
6.5.2. Biochemical and Physiological Traits..............................................................................
6.5.3. Serological Properties .......................................................................................................
6.5.4. Genetic Characteristics .....................................................................................................

6.6. Pathogenicity Testing..................................................................................................................
6.6.1. Koch’s Postulates..............................................................................................................
6.6.2. Mammalian Cell Culture (Tissue Culture).......................................................................
6.6.3. Virulence Genes and Gene Expression Products.............................................................
6.7. Testing for Specific Traits ..........................................................................................................
7. Challenges to Current Detection Methods.........................................................................................
7.1. Pathogen Quantification Problems .............................................................................................
7.2. Can a Small Bacterial Population be Detected Rapidly and Reliably?.....................................
7.3. Which Traits to Analyze, and How Many Tests are Needed for Identifying a Bacterial
Pathogen? ....................................................................................................................................
7.4. Real-Time Detection ...................................................................................................................
References...........................................................................................................................................

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4. Overview of Rapid Microbiological Methods
Jeanne Moldenhauer
1. Introduction ........................................................................................................................................
2. A History of Rapid Microbiological Methods: Industry Reluctance to Accept
These Methods ..................................................................................................................................
3. Types of Microbial Testing Performed .............................................................................................
4. Types of Rapid Microbiological Methods.........................................................................................
4.1. Growth-Based Technologies.......................................................................................................
4.2. Viability-Based Technologies.....................................................................................................
4.3. Cellular Component or Artifact-Based Technologies................................................................
4.4. Nucleic Acid-Based Technologies .............................................................................................
4.5. Automated Methods....................................................................................................................
4.6. Combination Methods.................................................................................................................
5. Overview of Rapid Technologies and How They Work ..................................................................
5.1. Adenosine Tri-Phosphate (ATP) Bioluminescence ...................................................................
5.2. Adenylate Kinase........................................................................................................................

5.3. Autofluorescence ........................................................................................................................
5.4. Biochemical Assays and Physiological Reactions.....................................................................
5.5. Biosensors and Immunosensors .................................................................................................
5.6. Carbon Dioxide Detection..........................................................................................................
5.7. Changes in Headspace Pressure .................................................................................................

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5.8. Colorimetric Detection of Carbon Dioxide Production.............................................................
5.9. Concentric Arcs of Photovoltaic Detectors with Laser Scanning.............................................

5.10. Direct Epifluorescent Filter Technique (DEFT)........................................................................
5.11. DNA Sequencing........................................................................................................................
5.12. Endospore Detection ..................................................................................................................
5.13. Enzyme Linked Immunosorbent Assay (ELISA)......................................................................
5.14. Flow Cytometry..........................................................................................................................
5.15. Fluorescent Probe Detection ......................................................................................................
5.16. Fatty Acid Profiles (Fatty Acid Methyl Esters, FAMEs)..........................................................
5.17. Fourier Transformed Infrared Spectroscopy (FTIR) .................................................................
5.18. Gram Stains (Rapid Method) .....................................................................................................
5.19. Impedance...................................................................................................................................
5.20. Immunological Methods.............................................................................................................
5.21. Lab-on-a-Chip (LOC), Arrays, Microarrays and Microchips ...................................................
5.22. Limulus Amebocyte Lysate (LAL) Endotoxin Testing.............................................................
5.23. Mass Spectrometry (Matrix-Assisted Laser Desorption-Time of Flight (MALTI-TOF))........
5.24. Microcalorimetry ........................................................................................................................
5.25. Micro-Electro-Mechanical Systems (MEMS) ...........................................................................
5.26. Nanotechnology ..........................................................................................................................
5.27. Near Infrared Spectroscopy (NIRS)...........................................................................................
5.28. Nucleic Acid Probes...................................................................................................................
5.29. Optical Particle Detection ..........................................................................................................
5.30. Polymerase Chain Reaction (PCR) ............................................................................................
5.31. Rep-PCR .....................................................................................................................................
5.32. Raman Spectroscopy ..................................................................................................................
5.33. Ribotyping/Molecular Typing ....................................................................................................
5.34. Solid Phase Laser Scanning Cytometry.....................................................................................
5.35. Southern Blotting/Restriction Fragment Length Polymorphism ...............................................
5.36. Spiral Plating ..............................................................................................................................
5.37. Turbidimetry ...............................................................................................................................
6. Potential Areas of Application of Rapid Microbiological Methods .................................................
7. Disclaimer...........................................................................................................................................

8. Conclusions ........................................................................................................................................
References...........................................................................................................................................

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Part II Biosensors
5. Surface Plasmon Resonance (SPR) Sensors for the Detection of Bacterial
Pathogens
Allen D. Taylor, Jon Ladd, Jiˇrí Homola and Shaoyi Jiang
1.
2.
3.
4.
5.
6.

Introduction ........................................................................................................................................
Fundamentals of Surface Plasmon Resonance Biosensing ...............................................................
SPR Sensor Instrumentation ..............................................................................................................
Surface Chemistries and Molecular Recognition Elements ..............................................................
Detection Formats ..............................................................................................................................
Quantification of Bacteria Cells ........................................................................................................
6.1. Challenges for the Detection of Whole Bacteria by SPR..........................................................
6.2. Effect of Bacteria Sample Treatment .........................................................................................
6.3. Examples of Bacteria Detection .................................................................................................

6.3.1. Escherichia coli.................................................................................................................
6.3.2. Salmonella spp. ................................................................................................................
6.3.3. Listeria monocytogenes.....................................................................................................

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6.3.4. Other Bacteria ..................................................................................................................
6.3.5. Detection of Multiple Bacteria ........................................................................................
7. Genetic Markers .................................................................................................................................
8. Antibody Biomarkers .........................................................................................................................
9. Conclusions and Future Perspectives ................................................................................................
References...........................................................................................................................................

6. Bacterial Detection

Biosensors

Using

Evanescent

Wave-Based

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Fluorescent

Kim E. Sapsford and Lisa C. Shriver-Lake
1. Introduction ........................................................................................................................................
2. Current State of Bacterial Fluorescent TIRF Biosensors..................................................................
2.1. Non-Planar Substrates.................................................................................................................
2.1.1. Fiber Optics ......................................................................................................................
2.1.2. Capillaries.........................................................................................................................
2.2. Planar Substrates .........................................................................................................................
2.2.1. NRL Array Biosensor ......................................................................................................
2.2.2. Other Optical Waveguides ...............................................................................................
2.2.3. TIRF-Microscopy .............................................................................................................
3. Future Aspects of Bacterial Fluorescent TIRF Biosensors ...............................................................
4. Conclusions ........................................................................................................................................
References...........................................................................................................................................


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7. Fiber Optic Biosensors for Bacterial Detection
Ryan B. Hayman
1. Fiber Optic Biosensors.......................................................................................................................
1.1. Whole-Cell Detection .................................................................................................................
1.1.1. Evanescent-Field Sensing ................................................................................................
1.1.2. Sandwich Immunoassays .................................................................................................
1.2. Bead-Based Arrays .....................................................................................................................
1.3. Nucleic Acid Sandwich Assays..................................................................................................
1.4. Nucleic Acid Direct Hybridization.............................................................................................
1.5. Extension Reactions....................................................................................................................
2. Conclusions and Future Perspectives ................................................................................................
References...........................................................................................................................................

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8. Integrated Deep-Probe Optical Waveguides for Label Free Bacterial
Detection
Mohammed Zourob, Nina Skivesen, Robert Horvath, Stephan Mohr, Martin B. McDonnell
and Nicholas J. Goddard
1. Introduction ........................................................................................................................................
1.1. Planar Optical Waveguides.........................................................................................................
1.2. Total Internal Reflection and Evanescent Waves ......................................................................
1.3. Waveguide Modes ......................................................................................................................
1.4. Frustrated Total Internal Reflection, Leaky Modes ...................................................................
1.5. Literature on Waveguides for Bacterial Detection ....................................................................
2. Deep-Probe Optical Waveguide Sensors with Tunable Evanescent Field .......................................
2.1. Waveguide Modes, Light Coupling and Sensing Depths of Evanescent Waves......................
2.1.1. Light Coupling Techniques..............................................................................................

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2.2. Waveguide Designs Based on Low-Index Substrates................................................................
2.2.1. Bacteria Detection Using Reverse Symmetry Waveguides ............................................
2.3. Waveguide Designs Based on Metal- and Dye-Clad Substrates—Leaky Modes.....................
2.3.1. Results ..............................................................................................................................
3. Integrated Deep-Probe Optical Waveguides Systems .......................................................................
3.1. Integration with Electric Field....................................................................................................
3.2. Integration with Ultrasound Standing Waves (USW)................................................................
4. Conclusions and Future Perspectives ................................................................................................
References...........................................................................................................................................

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9. Interferometric Biosensors
Daniel P. Campbell
1. Principles of Optical Interferometry ..................................................................................................

1.1. Optical Waveguides ....................................................................................................................
1.2. Planar Waveguide Operation ......................................................................................................
1.3. Types of Waveguides .................................................................................................................
2. Light Coupling Methods ....................................................................................................................
2.1. Interferometers ............................................................................................................................
2.2. Collinear or Single Channel Interferometers .............................................................................
2.3. Two-Channel Interferometers .....................................................................................................
3. Interferometric Array Sensors............................................................................................................
4. Surface Plasmon Interferometry ........................................................................................................
5. Other Interferometric Methods and Designs .....................................................................................
6. Surface Functionalization...................................................................................................................
7. Sample Collection Systems................................................................................................................
8. Interferometric Applications for Whole-Cell Detection....................................................................
9. Advantages and Limitations...............................................................................................................
10. Potential for Improving Current Performance...................................................................................
References...........................................................................................................................................

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10. Luminescence Techniques for the Detection of Bacterial Pathogens
Leigh Farris, Mussie Y. Habteselassie, Lynda Perry, S. Yanyun Chen, Ronald Turco,
Brad Reuhs and Bruce Applegate
1.
2.
3.
4.
5.
6.
7.

Beyond Robert Boyle’s Chicken.......................................................................................................
The Bacterial (lux) Luminescent System for Direct Pathogen Detection ........................................
The Firefly (luc) Luminescent System for Direct Pathogen Detection............................................
The Use of Alternative Luciferases in Pathogen Detection .............................................................
Luminescent-Based Immunoassays ...................................................................................................
Chemiluminescence Detection Methods ...........................................................................................
Conclusions and Future Perspectives ................................................................................................
References..........................................................................................................................................

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11. Porous and Planar Silicon Sensors
Charles R. Mace and Benjamin L. Miller
1. Introduction ........................................................................................................................................
1.1. Porous Silicon: A Three-Dimensional Matrix for Biosensing ..................................................
1.2. Effect of PSi Immobilization on Probe Viability: Experiments with GST ..............................
1.3. Toward Larger Targets: The First Macroporous Microcavity Structures.................................
1.4. Porous Silicon Bandgap Sensors in Novel Formats: “Smart Bandages” and “Smart Dust” ...
2. Arrayed Imaging Reflectometry—A Planar Silicon Biosensor........................................................
2.1. Theory.........................................................................................................................................

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2.1.1. Physical Rationale............................................................................................................
2.1.2. Substrate Design ..............................................................................................................
2.1.3. Mathematical Model ........................................................................................................

2.1.4. Monitoring the Null Reflectance Condition....................................................................
2.2. Applications of AIR Biosensing ................................................................................................
2.2.1. Limitations .......................................................................................................................
2.2.2. Probe Immobilization ......................................................................................................
2.2.3. Pathogen Detection ..........................................................................................................
3. Conclusions and Future Perspectives ................................................................................................
References..........................................................................................................................................

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12. Acoustic Wave (TSM) Biosensors: Weighing Bacteria
Eric Olsen, Arnold Vainrub and Vitaly Vodyanoy
1. Introduction ........................................................................................................................................
2. Historical Perspective, Theory and Background...............................................................................
2.1. Piezoelectricity and Acoustic Waves .........................................................................................
2.2. Acoustic Wave Devices .............................................................................................................
3. TSM Biosensors.................................................................................................................................
3.1. Detection of Microorganisms.....................................................................................................
3.2. Measurement in Liquid ..............................................................................................................
3.3. TSM Biosensor Characteristics ..................................................................................................
3.4. Commercial TSM Microbalances ..............................................................................................

3.5. Immobilization of Probes onto Sensor Surface .........................................................................
3.5.1. Physical Adsorption .........................................................................................................
3.5.2. Other Coupling Methods .................................................................................................
3.5.3. Combined Langmuir-Blodgett/Molecular Assembling Method .....................................
3.5.4. Solvent-Free Purified Monolayers...................................................................................
3.5.5. Immobilization of Monolayers of Phage Coat Proteins .................................................
3.5.6. Immobilization of Molecular Probes onto Porous Substrates ........................................
4. Problem of “Negative Mass”.............................................................................................................
5. Coupled Oscillators Model ................................................................................................................
6. Conclusions ........................................................................................................................................
References..........................................................................................................................................

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13. Amperometric Biosensors for Pathogenic Bacteria Detection
Ilaria Palchetti and Marco Mascini
1. Introduction ........................................................................................................................................
2. Amperometric Biosensors..................................................................................................................
2.1. Microbial Metabolism-Based Biosensors ..................................................................................
2.2. Immunosensors ...........................................................................................................................
2.3. DNA-Based Biosensors..............................................................................................................
3. Conclusion and Future Perspectives..................................................................................................
References..........................................................................................................................................

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14. Microbial Genetic Analysis Based on Field Effect Transistors
Yuji Miyahara, Toshiya Sakata and Akira Matsumoto
1. Introduction ........................................................................................................................................
2. Fundamental Principles of Field Effect Devices ..............................................................................
2.1. Metal-Insulator-Semiconductor (MIS) Capacitor ......................................................................
2.2. Principles of Biologically Coupled Field Effect Transistors for Genetic Analysis
(Genetic FETS) ...........................................................................................................................

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3. Fundamentals of Genetic Analysis....................................................................................................
3.1. DNA............................................................................................................................................
3.2. Genetic Analysis.........................................................................................................................
3.3. DNA Chip / DNA Microarray ...................................................................................................
4. Immobilization of DNA Molecules on the Surfaces of Solid Substrates ........................................
4.1. Silanization .................................................................................................................................
4.2. Thiol-Gold Bonding ...................................................................................................................
4.3. Avidin, Streptavidin and Biotin .................................................................................................
4.4. Others..........................................................................................................................................
5. Genetic Analysis Based on Field Effect Devices .............................................................................
5.1. Fundamental Characteristics of Genetic Field Effect Devices..................................................
5.1.1. Detection of DNA Molecular Recognition Events .........................................................
5.1.2. Immobilization Density of Oligonucleotide Probes........................................................
5.2. Single Nucleotide Polymorphisms (SNPs) Analysis .................................................................
5.2.1. Controlling Hybridization Temperature for SNPs Analysis...........................................
5.2.2. SNPs Analysis Based on Primer Extension ....................................................................
5.3. DNA Sequencing........................................................................................................................
6. Conclusions and Future Perspectives ................................................................................................
References..........................................................................................................................................

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15. Impedance-Based Biosensors for Pathogen Detection
Xavier Muñoz-Berbel, Neus Godino, Olivier Laczka, Eva Baldrich, Francesc Xavier Muñoz
and Fco. Javier Del Campo
1. Introduction ........................................................................................................................................
2. Fundamentals of Electrochemical Impedance Spectroscopy............................................................
2.1. Data Analysis: Plotting...............................................................................................................
2.2. Data Analysis: Interpretation .....................................................................................................
2.2.1. Non-Faradaic Parameters.................................................................................................
2.2.2. Faradaic Parameters .........................................................................................................
2.3. Measuring at Impedimetric Biosensors......................................................................................
2.3.1. Measurement Modes........................................................................................................

2.4. Bacterial Parasitizing Effect on Electrode Surface....................................................................
3. Development of an Immunosensor....................................................................................................
3.1. Biological Recognition Elements in Biosensors for Pathogen Detection.................................
3.1.1. Antibodies ........................................................................................................................
3.1.2. Nucleic Acids...................................................................................................................
3.1.3. Aptamers ..........................................................................................................................
3.1.4. Other Recognition Strategies...........................................................................................
3.2. Surface Modification Methods...................................................................................................
3.2.1. Adsorption........................................................................................................................
3.2.2. Self-assembled Monolayers .............................................................................................
3.2.3. Silanisation.......................................................................................................................
3.2.4. Protein A and Protein G ..................................................................................................
3.2.5. The Biotin-(Strept)Avidin System...................................................................................
3.2.6. Chemical Conjugation .....................................................................................................
3.2.7. Entrapment .......................................................................................................................
3.2.8. Microencapsulation ..........................................................................................................
3.3. Blocking......................................................................................................................................
3.4. Signal Amplification ..................................................................................................................
3.5. The Need for Negative Controls................................................................................................
3.6. Development of Novel Strategies: Assessing Performance Using ELISA and Microscopy ...
4. Current EIS Biosensors for Pathogen Detection...............................................................................
4.1. Biosensors Based on Interfacial Capacitance Changes .............................................................

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4.2. Biosensors Based on Charge-Transfer Resistance Changes......................................................

4.3. Biosensors Based on Conductivity Changes .............................................................................
4.4. Other Approaches .......................................................................................................................
5. Conclusions and Future Perspectives ................................................................................................
References..........................................................................................................................................

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16. Label-Free Microbial Biosensors Using Molecular Nanowire Transducers
Evangelyn Alocilja and Zarini Muhammad-Tahir
1. Introduction ........................................................................................................................................
1.1. Rationale for Rapid Tests...........................................................................................................
1.2. Target Microorganisms and Matrices ........................................................................................
1.2.1. Escherichia coli ...............................................................................................................
1.2.2. Salmonella........................................................................................................................
1.2.3. Bovine Viral Diarrhea Virus ...........................................................................................
1.3. Food Safety Applications ...........................................................................................................
2. Biosensor Formats .............................................................................................................................
2.1. Definition ....................................................................................................................................
2.2. Antibodies as Biological Sensing Element................................................................................
2.3. DNA as Biological Sensing Element.........................................................................................
2.4. DNA-Based Biosensors..............................................................................................................
2.5. Antibody-Based Biosensors .......................................................................................................
2.6. Biosensor Transducing Element: Conducting Polymer.............................................................
2.6.1. Polyaniline .......................................................................................................................
2.6.2. Self-doped Polyaniline.....................................................................................................
2.6.3. Carbon Nanotubes............................................................................................................

2.7. Conducting Polymer-Based Biosensor for Microbial/Viral Detection......................................
3. Illustration: Biosensor Using Self-doped and Non-self-doped Pani.................................................
3.1. Pani Preparation..........................................................................................................................
3.2. Pani Characterization..................................................................................................................
3.2.1. Conductivity Measurement..............................................................................................
3.2.2. Biosensor Fabrication ......................................................................................................
3.2.3. Indium Tin Oxide/Pani Biosensor...................................................................................
3.2.4. Lateral Flow Conductometric Biosensor.........................................................................
3.2.5. Signal Measurement ........................................................................................................
3.3. Properties of Pani .......................................................................................................................
3.4. Detection Concept of the Biosensor ..........................................................................................
3.5. Biosensor Properties ...................................................................................................................
3.5.1. ITO-Pani Biosensor .........................................................................................................
3.6. Lateral Flow Conductometric Biosensor ...................................................................................
3.7. Biosensor Performance...............................................................................................................
3.7.1. ITO/Pani Biosensor..........................................................................................................
3.8. Conductometric Biosensor .........................................................................................................
4. Conclusions and Future Perspectives ................................................................................................
References..........................................................................................................................................

377
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382

384
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388
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392
392
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393
393
393
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398
399
399
403
404
404
404
406
406

17. Magnetic Techniques for Rapid Detection of Pathogens
Yousef Haik, Reyad Sawafta, Irina Ciubotaru, Ahmad Qablan, Ee Lim Tan and
Keat Ghee Ong

1. Introduction ........................................................................................................................................ 415
2. Synthesis of Magnetic Particles ........................................................................................................ 417
2.1. Effect of Particle Size ................................................................................................................ 418


Contents

2.2. Synthesis Techniques .................................................................................................................
2.3. Encapsulation of Magnetic Particles..........................................................................................
2.3.1. Methods of Preparing Polymer/Protein Coatings ...........................................................
2.3.2. Examples of Polymer/Protein Encapsulated Particles ....................................................
3. Immobilization Strategies ..................................................................................................................
3.1. Modification of Particle Surface with a Ligand ........................................................................
4. Biological Targets..............................................................................................................................
5. Magnetic Immunoassays....................................................................................................................
5.1. Direct Immunoassay Detection Using Magnetic Beads ............................................................
5.1.1. Superconducting Quantum Interference Devices............................................................
5.1.2. ABICAP Column .............................................................................................................
5.2. Indirect Immunoassay Detection Using Magnetic Beads..........................................................
5.2.1. ELISA ..............................................................................................................................
6. Handling Techniques .........................................................................................................................
7. Magnetic Separation ..........................................................................................................................
7.1. Magnetic Force ...........................................................................................................................
7.2. High-Field Electromagnets.........................................................................................................
7.3. Permanent Magnets ....................................................................................................................
7.4. Numerical Analysis for Permanent Magnet Arrangements.......................................................
8. Giant Magnetoresistive (GMR) Devices for Bacterial Detection.....................................................
9. Bacteria Detection with Magnetic Relaxation Signal.......................................................................
10. Magnetoelastic Sensors for Bacterial Detection ...............................................................................
10.1. E. coli Detection.........................................................................................................................

11. Conclusions and Future Perspectives ................................................................................................
References..........................................................................................................................................

xv

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426
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441
442
446
448
449
450
453
454


18. Cantilever Sensors for Pathogen Detection
Raj Mutharasan
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.

Introduction ........................................................................................................................................
Millimeter-Sized Cantilever Sensors.................................................................................................
Reported Work on Detecting Cells Using Cantilever Sensors.........................................................
Physics of Cantilever Sensors ...........................................................................................................
Resonance Modes ..............................................................................................................................
Characterization of PEMC Sensors...................................................................................................
Mass Change Sensitivity ...................................................................................................................
Antibody Immobilization Methods ...................................................................................................
Detection in Batch and Stagnant Samples ........................................................................................
Detection in Flowing Samples ..........................................................................................................
Selectivity of Detection .....................................................................................................................
Conclusions ........................................................................................................................................
References..........................................................................................................................................


459
460
461
463
466
468
468
469
470
473
475
477
478

19. Detection and Viability Assessment of Endospore-Forming Pathogens
Adrian Ponce, Stephanie A. Connon and Pun To Yung
1. Introduction ........................................................................................................................................
1.1. Historical Perspective .................................................................................................................
1.2. Endospore Dormancy, Resistance and Longevity .....................................................................
1.3. Endospores as Biodosimeters for Evaluating Sterilization Regimes ........................................
1.4. Endospore-Forming Pathogens ..................................................................................................
1.5. Bioweapons, Bioinsecticides and Probiotics .............................................................................

481
481
482
484
485
487



xvi

Contents

2. Detection of Endospore-Forming Pathogens and their Endospores .................................................
2.1. Phenotypic Identification............................................................................................................
2.1.1. Phenotypic Identification of Bacillus anthracis..............................................................
2.1.2. Phenotypic Identification of Clostridium perfringens ....................................................
2.2. Parameters of a Sensor ...............................................................................................................
2.3. Rapid Immunoassays..................................................................................................................
2.3.1. Enzyme-Linked Immunosorbent Assays.........................................................................
2.3.2. Lateral-Flow Immunoassays............................................................................................
2.3.3. Immunomagnetic Electrochemiluminescence .................................................................
2.3.4. Flow Cytometry ...............................................................................................................
2.3.5. Vegetative Cells ...............................................................................................................
2.4. Rapid Nucleic Acid Assays........................................................................................................
2.4.1. PCR Sample Preparation and Endospore Lysis ..............................................................
2.4.2. The PCR Reaction ...........................................................................................................
2.4.3. Specificity of PCR Primers for Bacillus anthracis Detection ........................................
2.4.4. Rapid PCR Detection Methods High Throughput and real-time PCR ..........................
2.4.5. Field Implementation of Rapid PCR for Analysis of Environmental Samples .............
2.4.6. Monitoring the Air for Bacillus anthracis Endospores by PCR ....................................
2.5. Rapid Detection of Endospores via Dipicolinic Acid Biomarker.............................................
2.5.1. Terbium Dipicolinic Acid Luminescence Assay ............................................................
2.5.2. Anthrax Smoke Detector .................................................................................................
3. Validation of Sterilization by Rapid Endospore Viability Assessment ...........................................
3.1. Measuring Endospore Viability and Inactivation ......................................................................
3.2. Measuring Endospore Inactivation using Germinability Assays ..............................................
3.2.1. Rapid Germinability Assays ............................................................................................

3.2.2. Nucleic Acid-Based Amplification Methods for Detecting Germinable, Viable
Bacillus anthracis Spores..............................................................................................
3.2.3. Germination Observed via Loss of Phase Brightness.....................................................
3.2.4. Germination Observed via DPA release .........................................................................
3.3. Measuring Endospore Inactivation Using Metabolic Activity Assays .....................................
4. Conclusions and Future Perspectives ................................................................................................
References..........................................................................................................................................

489
489
490
490
491
492
492
493
493
494
494
495
495
496
496
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498
500
501
501
503
505

505
508
508
508
509
510
512
513
514

20. Label-Free Fingerprinting of Pathogens by Raman Spectroscopy
Techniques
Ann E. Grow
1.
2.
3.
4.

Introduction ........................................................................................................................................
Raman Microscopy for Whole-Organism Fingerprinting.................................................................
Surface-Enhanced Raman Scattering (SERS) for Whole-Organism Fingerprinting........................
MicroSERS for the Detection and Identification of Pathogens and Toxins ....................................
4.1. MicroSERS Detection of Bacteria .............................................................................................
4.1.1. SERS Fingerprinting of Bacteria.....................................................................................
4.1.2. Impact of Growth Conditions on Bacterial Fingerprints ................................................
4.1.3. Viable vs. Nonviable Bacteria.........................................................................................
4.1.4. Integrated MicroSERS Detection and Identification of Bacteria ...................................
4.1.5. Impact of Growth Conditions on Biomolecule Capture .................................................
4.1.6. Analysis of Bacteria in Complex Samples......................................................................
4.2. MicroSERS Detection of Spores................................................................................................

4.2.1. SERS Fingerprinting of Spores .......................................................................................
4.2.2. Impact of Growth Conditions on Spore Fingerprints .....................................................
4.2.3. Viable vs. Nonviable Spores ...........................................................................................
4.2.4. Integrated MicroSERS Detection and Identification of Spores......................................

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535
535
536
539
542
544
544
545
545
547
550
551


Contents

xvii

4.3. MicroSERS Detection of Bacterial Toxins................................................................................
4.3.1. SERS Fingerprinting of Toxins.......................................................................................
4.3.2. Analysis of Toxins in Complex Samples........................................................................

5. Conclusion and Future Perspectives..................................................................................................
References..........................................................................................................................................

Part III

554
555
558
559
560

Recognition Receptors

21. Antibodies and Immunoassays for Detection of Bacterial Pathogens
Padmapriya P. Banada and Arun. K. Bhunia
1. Introduction ........................................................................................................................................
2. Antibodies ..........................................................................................................................................
2.1. Polyclonal Antibody ...................................................................................................................
2.2. Monoclonal Antibody.................................................................................................................
2.3. Use of Synthetic Peptides for Antibody Production .................................................................
2.4. Recombinant DNA Technology.................................................................................................
2.4.1. Phage Display ..................................................................................................................
3. Capture and Concentration of Cells by Immunomagnetic Separation .............................................
3.1. Automated IMS Systems............................................................................................................
4. Immunoassays for Pathogen Detection .............................................................................................
4.1. Radioimmunoassay.....................................................................................................................
4.2. Enzyme Immunoassays ..............................................................................................................
4.2.1. Escherichia coli ................................................................................................................
4.2.2. Listeria monocytogenes ....................................................................................................
4.2.3. Salmonella.........................................................................................................................

4.2.4. Staphylococcal Enterotoxins............................................................................................
4.2.5. Clostridium botulinum Toxins.........................................................................................
4.3. Lateral Flow Immunoassay ........................................................................................................
4.4. Other Immunoassays ..................................................................................................................
4.4.1. Latex Agglutination (LA) and Reverse Passive Latex Agglutination (RPLA) Tests ....
4.4.2. Enzyme-Linked Fluorescent Assay .................................................................................
4.4.3. Time-Resolved Fluorescence Immunoassay ...................................................................
4.4.4. Chemiluminescent Immunoassay ....................................................................................
4.4.5. Capillary Microbead (Spheres) Immunoassay ................................................................
4.4.6. Electrochemical-Immunoassay ........................................................................................
4.5. Optical Biosensors......................................................................................................................
4.5.1. Surface Plasmon Resonance ............................................................................................
4.5.2. Fiber-Optic Biosensors ....................................................................................................
4.5.3. Antibody-Based Microfluidic Sensors ............................................................................
4.5.4. Serodiagnosis ...................................................................................................................
5. Recent Developments in Immunoassays...........................................................................................
5.1. Protein/Antibody Microarrays....................................................................................................
5.2. Mass Spectrometric Immunodetection.......................................................................................
5.3. SERS Biochip Technology ......................................................................................................
6. Limitations and Challenges ...............................................................................................................
6.1. Specificity and Sensitivity..........................................................................................................
6.2. Effect of Physical and Chemical Stresses on the Expression Profile of Antigens in Bacteria
6.2.1. Effect of Media Composition on the Expression of Proteins in Bacteria......................
6.2.2. Effect of Stress on the Expression of Proteins in Bacteria ............................................
7. Conclusions and Future Perspectives ................................................................................................
References..........................................................................................................................................

567
568
570

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573
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577
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580
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587
587
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588
589
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591

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xviii

Contents

22. Rapid Nucleic Acid-Based Diagnostics Methods for the Detection
of Bacterial Pathogens
Barry Glynn
1. Introduction ........................................................................................................................................
1.1. Detection of Pathogenic Bacteria from Clinical Samples .........................................................
1.2. NAD Assays for the Detection of Respiratory Infection, Sepsis and Sexually
Transmitted Infection ..................................................................................................................
1.3. Profiling of Multi-drug Resistance ............................................................................................
1.4. Bioterrorism ................................................................................................................................
2. Detection of Bacterial Food-Borne Pathogens..................................................................................
2.1. Recent Outbreaks........................................................................................................................
2.2. Benefits and Limitations of Conventional Methods..................................................................
2.3. Development of Rapid Diagnostics Methods ............................................................................
3. Rapid Nucleic Acid Diagnostics for Bacterial Food-Borne Pathogens............................................
3.1. In Vitro Nucleic Acid Amplification-Based Detection of Food-Borne Pathogens ..................
3.2. Requirements for a NAD-Based Food Assay............................................................................

3.3. Polymerase Chain Reaction (PCR) ............................................................................................
3.4. Application of PCR-Based Tests to Pathogen Detection in Food Samples .............................
3.5. Use of RNA as an Alternative Nucleic Acid Diagnostic Target ..............................................
3.6. Sample Preparation for NAD from Clinical Sample Types......................................................
3.7. Limitations of NAD in Clinical Settings ...................................................................................
4. Formats of NAD Assays for Food Pathogen Detection ...................................................................
4.1. Nucleic Acid-Based Diagnostics Based on In Vitro Amplification Technologies...................
4.2. PCR-ELISA and PCR-DNA Probe Membrane Based Assays
for Campylobacter and Salmonella ............................................................................................
4.3. Specific Examples of Nucleic Acid Diagnostics Assays for the Detection of Bacterial
Food-Borne Pathogens ................................................................................................................
4.3.1. Commercially Available Conventional NAD Assays
for Food-Borne Bacterial Pathogens.............................................................................
4.3.2. Alternative In Vitro Amplification Technologies ...........................................................
4.4. Standardisation of In Vitro Amplification-Based NAD Assays
and Inter-Laboratory Validation Studies ....................................................................................
4.5. Real-Time In Vitro Amplification-Based Nucleic Acid Diagnostics .......................................
4.5.1. Specific Examples of Real-Time PCR Assays for the Detection of Bacterial
Food-Borne Pathogens ..................................................................................................
4.5.2. Alternative Real-Time In Vitro Amplification-Based Diagnostics Technologies .........
4.6. Limitations and Other Considerations for In Vitro Amplification NAD Tests ........................
4.7. Non-Amplified Direct DNA Probe-Based Nucleic Acid Diagnostics ......................................
4.8. DNA-Probe Based Detection Methods ......................................................................................
5. Conclusions and Future Perspectives ................................................................................................
5.1. Emerging Nucleic Acid Diagnostic Technologies for Food-Borne Pathogen Detection .........
5.1.1. Biosensors ........................................................................................................................
5.1.2. Microarrays ......................................................................................................................
References..........................................................................................................................................

603

604
604
606
606
606
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607
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608
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609
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611
611
612
612
612
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616
617
617
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619
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620
621

621
621
622
623

23. Oligonucleotide and DNA Microarrays: Versatile Tools for Rapid Bacterial
Diagnostics
Tanja Kostic, Patrice Francois, Levente Bodrossy and Jacques Schrenzel
1. Introduction ........................................................................................................................................
2. Microarray Technology .....................................................................................................................
3. Technical Aspects of Microarray Technology..................................................................................
3.1. Probes..........................................................................................................................................
3.1.1. Genome Fragments ..........................................................................................................

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632
632
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Contents

3.1.2. PCR Products ...................................................................................................................
3.1.3. Oligonucleotide Probes....................................................................................................
3.2. Substrates for Printing ................................................................................................................
3.2.1. Slides with Poly-L-Lysine Coating .................................................................................
3.2.2. Slides with Amino Silane Coating ..................................................................................
3.2.3. Slides with Aldehyde Coating.........................................................................................
3.2.4. Slides with Epoxy Coating ..............................................................................................

3.2.5. Proprietary Surface Chemistries ......................................................................................
3.2.6. Probe Spacers...................................................................................................................
3.3. Targets for Microarray Analysis ................................................................................................
3.3.1. Target Amplifications and Sensitivity Issues..................................................................
3.3.2. Labeling of the Targets....................................................................................................
3.3.3. Hybridization and Wash Conditions ...............................................................................
3.4. Classical Commercially-Available Microarray Formats ...........................................................
3.4.1. Spotting Approaches........................................................................................................
3.4.2. In Situ Synthesis ..............................................................................................................
3.5. Alternative Methods for Improving Microarray-Based Detection Sensitivity..........................
3.5.1. Resonance-Light Scattering (RLS)..................................................................................
3.5.2. Planar-Waveguide Technology (PWT) ...........................................................................
3.5.3. Liquid Arrays ...................................................................................................................
3.5.4. Three-Dimensional Microarray Formats .........................................................................
3.6. Marker Genes Used on Microbial Diagnostic Microarrays (MDMs).......................................
4. Analysis and QC Aspects ..................................................................................................................
5. Applications of Microarray Technology in Microbial Diagnostics..................................................
5.1. Gene Expression Studies ............................................................................................................
5.2. Comparative Genome Hybridizations (CGH)............................................................................
5.3. Generic or Universal Microarrays..............................................................................................
5.4. Microarrays for Sequence Analysis ...........................................................................................
5.5. Microbial Diagnostic Microarrays .............................................................................................
6. Conclusions ........................................................................................................................................
References..........................................................................................................................................

xix

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639
639
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641
641
641
642
643
643
644
644
645
646
647
648
649
649

24. Pathogenic Bacterial Sensors Based on Carbohydrates as Sensing

Elements
Haiying Liu
1.
2.
3.
4.
5.
6.
7.
8.
9.

Introduction ........................................................................................................................................
Bacterial Surface Lectins...................................................................................................................
Surface Carbohydrate Structures of Pathogenic Bacteria.................................................................
Carbohydrate Microarrays for Detection of Bacteria .......................................................................
Lectin Microarrays for Detection of Bacteria...................................................................................
Conjugated Fluorescent Glycopolymers for Detection of Bacteria..................................................
Glyconanoparticles for Detection of Bacteria...................................................................................
Carbohydrate-Functionalized Carbon Nanotubes for Detection of Bacteria....................................
Conclusions and Future Perspectives ................................................................................................
References..........................................................................................................................................

660
661
664
668
670
672
676

678
680
681

25. Aptamers and Their Potential as Recognition Elements for the Detection
of Bacteria
Casey C. Fowler, Naveen K. Navani, Eric D. Brown and Yingfu Li
1. Functional Nucleic Acids .................................................................................................................. 689
1.1. Properties of Nucleic Acids ....................................................................................................... 690
1.2. Synthesizing, Sequencing and Modifying Nucleic Acids ......................................................... 692


xx

2.

3.

4.

5.

6.

Contents

1.2.1. DNA Polymerase and Polymerase Chain Reaction ........................................................
1.2.2. RNA Polymerase and In Vitro Transcription .................................................................
1.2.3. Reverse Transcription ......................................................................................................
1.2.4. Other Modifications .........................................................................................................

Isolation of Functional Nucleic Acids...............................................................................................
2.1. Introduction to SELEX...............................................................................................................
2.2. Selection Methods ......................................................................................................................
2.2.1. Bead and Column Based Selections................................................................................
2.2.2. Polyacrylamide Gel Electrophoresis (PAGE) Based Selections ....................................
2.2.3. Capillary Electrophoresis (CE) Based Selections ...........................................................
2.3. Optimizing Functional Nucleic Acids........................................................................................
Aptamers: Properties and Targets .....................................................................................................
3.1. The Growing Aptamer Catalogue ..............................................................................................
3.2. Aptamer Specificity....................................................................................................................
3.3. Aptamer–Ligand Interactions .....................................................................................................
3.4. Aptamers vs. Other Recognition Elements................................................................................
Applications of Aptamers ..................................................................................................................
4.1. Aptamers for Purification...........................................................................................................
4.2. Aptamers with Therapeutic Potential.........................................................................................
4.3. Aptamers as Sensing Elements ..................................................................................................
4.3.1. Conformation-Dependent Fluorescent Sensors ...............................................................
4.3.2. Quantum Dot Sensors ......................................................................................................
4.3.3. Target Detection by Fluorescence Anisotropy................................................................
4.3.4. Enzyme Linked Aptamer Assays ....................................................................................
4.3.5. Acoustic Sensors..............................................................................................................
4.3.6. Electrochemical Sensors ..................................................................................................
Aptamers for Detection of Pathogenic Bacteria ...............................................................................
5.1. Categories of Microbial Agents to be Detected ........................................................................
5.1.1. Gram-Positive Bacteria....................................................................................................
5.1.2. Gram-Negative Bacteria ..................................................................................................
5.2. Traditional Pathogen Detection Methods ..................................................................................
5.3. Aptamers in Pathogen Detection................................................................................................
Conclusions ........................................................................................................................................
References..........................................................................................................................................


692
692
693
693
694
694
694
696
696
697
697
697
698
698
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700
701
701
702
702
703
703
704
705
705
706
706
707
707

708
708
709
710
710

26. Protein Microarray Technologies for Detection and Identification
of Bacterial and Protein Analytes
Christer Wingren and Carl AK Borrebaeck
1. Introduction ........................................................................................................................................
1.1. Definition and Classification of Protein Microarrays................................................................
1.2. Functional Protein Microarrays..................................................................................................
1.3. Affinity Protein Microarrays ......................................................................................................
1.4. Alternative Microarray Setups ...................................................................................................
2. Detection of Bacteria and Bacterial Protein Analytes ......................................................................
2.1. Serotyping of Bacteria................................................................................................................
2.2. Detection of Pathogenic Organisms...........................................................................................
2.3. Detection of Multiple Toxins.....................................................................................................
2.4. Simultaneous Detection and Identification of Bacterial Proteins and Bacteria........................
3. Detection of Diagnostic Markers, Toxin Regulators and Associated Protein Expression Profiles.
3.1. Identification of Potential Diagnostic Markers and/or Vaccine Candidates.............................
3.2. Disease State Differentiation and Identification of Diagnostic Markers ..................................
3.3. Identification of Potential Toxin Modulators/Regulators..........................................................
3.4. Screening of Protein Expression Signatures Associated with Bacterial Infection ...................

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Contents

xxi

4. Conclusions and Future Perspectives ................................................................................................ 726
References.......................................................................................................................................... 726

27. Bacteriophage: Powerful Tools for the Detection of Bacterial Pathogens
Mathias Schmelcher and Martin J. Loessner
1. Introduction ........................................................................................................................................
2. Detection by Phage Amplification ....................................................................................................
3. Detection Through Phage-Mediated Cell Lysis................................................................................
3.1. Measurement of ATP Release....................................................................................................
3.2. Detection of Other Cytoplasmic Markers..................................................................................
3.3. Measurement of Impedance .......................................................................................................
4. Detection Through Cell Wall Recognition, Phage Adsorption and DNA Injection........................
4.1. Immobilized Phage .....................................................................................................................
4.2. Detection Through Phage-Encoded Affinity Molecules ...........................................................

4.3. Fluorescently Labeled Phage .....................................................................................................
5. Detection by Reporter Phage.............................................................................................................
5.1. Luciferase Reporter Phage (LRP) ..............................................................................................
5.2. Fluorescent Protein Reporter Phage...........................................................................................
5.3. Other Reporter Phages ...............................................................................................................
6. Other Detection Methods Using Phage.............................................................................................
6.1. Phage Display for Production of Highly Specific Binding Molecules .....................................
6.2. Dual Phage Technology .............................................................................................................
7. Conclusions and Future Perspectives ................................................................................................
References..........................................................................................................................................

731
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734
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746
747
747
749
750
750


28. Phage Display Methods for Detection of Bacterial Pathogens
Paul A. Gulig, Julio L. Martin, Harald G. Messer, Beverly L. Deffense and
Crystal J. Harpley
1. Introduction ........................................................................................................................................
1.1. Why Detect Bacteria and What Tools Are Available? .............................................................
1.2. Immunological Tools..................................................................................................................
1.3. Nucleic Acid-Based Tools .........................................................................................................
2. What Types of Antigen Detection Methods Are Being Developed? ...............................................
3. Phage Display ....................................................................................................................................
3.1. Phage M13..................................................................................................................................
3.2. Principles of Phage Display .......................................................................................................
3.3. Phages Versus Phagemids ..........................................................................................................
3.4. Phage Display Formats...............................................................................................................
3.4.1. Random Peptides .............................................................................................................
3.4.2. Antibody Fragments ........................................................................................................
3.5. The Phages Themselves Are Not the Ultimate Tool.................................................................
3.6. Using Phage Display ..................................................................................................................
4. Review of Literature on Phage Display Against Bacterial Pathogens .............................................
4.1. Random Peptide Phage Display.................................................................................................
4.2. scFv Libraries .............................................................................................................................
4.3. Single Domain Antibodies (sdAbs) and Domain Antibodies (dAbs) .......................................
5. Summary of Our Results Using and Developing Phage Display scFv and Peptides ......................
5.1. Panning Methods ........................................................................................................................
5.2. Screening Methods .....................................................................................................................
5.3. Genetic Modification of Phagemid Clones................................................................................
5.4. Random Peptide Phage Libraries...............................................................................................

756
756

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762
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xxii

Contents

6. New Directions ..................................................................................................................................
6.1. Proteins Based on Phage Display ..............................................................................................
6.1.1. Affibodies.........................................................................................................................

6.1.2. Anticalins .........................................................................................................................
6.1.3. Ankyrins...........................................................................................................................
6.1.4. Trinectins..........................................................................................................................
6.2. Alternatives to Phage Display....................................................................................................
6.2.1. Aptamers ..........................................................................................................................
6.2.2. Ribosome Display............................................................................................................
6.2.3. mRNA Display ................................................................................................................
7. Conclusions ........................................................................................................................................
References..........................................................................................................................................

778
778
778
778
778
779
779
779
779
780
780
780

29. Molecular Imprinted Polymers for Biorecognition of Bioagents
Keith Warriner, Edward P.C. Lai, Azadeh Namvar, Daniel M. Hawkins
and Subrayal M. Reddy
1. Introduction ........................................................................................................................................
2. Principles of Molecular Imprinting ...................................................................................................
2.1. Imprinting Considerations ..........................................................................................................
2.1.1. Versatility.........................................................................................................................

2.1.2. Template Molecule ..........................................................................................................
2.1.3. Functional Monomer........................................................................................................
2.1.4. Cross-Linking...................................................................................................................
2.1.5. Polymerization .................................................................................................................
2.1.6. Solvent .............................................................................................................................
2.2. Aqueous Phase MIP ...................................................................................................................
2.2.1. Hydrogels .........................................................................................................................
2.2.2. MIP Within Hydrogels ....................................................................................................
2.2.3. Polyacrylamide Gels—HydroMIPs .................................................................................
3. Solid Phase Extraction Based on MIPs for Concentrating Bioagents..............................................
3.1. Antibiotics...................................................................................................................................
3.2. Mycotoxins .................................................................................................................................
3.3. Nano-Sized Structures ................................................................................................................
3.4. Peptides and Proteins .................................................................................................................
3.5. Viruses ........................................................................................................................................
3.6. Bacterial Cells and Endospores..................................................................................................
4. Biosensors Based on MIPs ................................................................................................................
4.1. MIP-based Sensors for Detection of Amino Acids ...................................................................
4.2. Molecular-Imprinted Films for Toxins ......................................................................................
4.3. Microbial Imprinted Polymers ...................................................................................................
5. Conclusions and Future Perspectives ................................................................................................
References..........................................................................................................................................

Part IV

785
786
787
787
788

788
789
790
790
791
792
793
793
795
795
798
799
800
801
802
803
804
805
806
808
809

Microsystems

30. Microfluidics-Based Lysis of Bacteria and Spores for Detection
and Analysis
Ning Bao and Chang Lu
1. Introduction ........................................................................................................................................ 817
2. Bench Scale Methods for Bacteria/Spore Lysis................................................................................ 818
3. Bacteria/Spore Lysis Based on Microfluidic Systems...................................................................... 820



Contents

3.1. Mechanical Lysis........................................................................................................................
3.2. Chemical Lysis ...........................................................................................................................
3.3. Thermal Lysis .............................................................................................................................
3.4. Laser-Based Lysis ......................................................................................................................
3.5. Electrical Lysis ...........................................................................................................................
4. Conclusions and Future Perspectives ................................................................................................
References..........................................................................................................................................

xxiii

820
821
823
826
827
829
829

31. Detection of Pathogens by On-Chip PCR
Pierre-Alain Auroux
1. Introduction ........................................................................................................................................
2. Microfluidics ......................................................................................................................................
2.1. History of Miniaturized Total Analysis System ( TAS) ..........................................................
2.2. Advantages of Miniaturized Analysis Systems .........................................................................
3. DNA Amplification ...........................................................................................................................
3.1. A Brief History of DNA ............................................................................................................

3.2. PCR Characteristics and Applications .......................................................................................
3.3. Components to Perform PCR.....................................................................................................
3.4. PCR Process ...............................................................................................................................
3.5. Conventional PCR ......................................................................................................................
3.6. Real-Time PCR: Apparatus and Detection Techniques ............................................................
3.7. On-Chip PCR..............................................................................................................................
3.7.1. Capillary-Based Thermocyclers ......................................................................................
3.7.2. Microdevice-Based Thermocyclers .................................................................................
3.7.3. Static-Sample Systems.....................................................................................................
3.7.4. Dynamic-Sample Systems ...............................................................................................
4. Minireview .........................................................................................................................................
5. Conclusions ........................................................................................................................................
References..........................................................................................................................................

833
834
834
834
835
835
836
837
838
839
840
841
842
843
843
844

846
848
849

32. Micro- and Nanopatterning for Bacteria- and Virus-Based
Biosensing Applications
David Morrison, Kahp Y. Suh and Ali Khademhosseini
1. Introduction ........................................................................................................................................
2. Fundamentals of Bacterial and Viral Surface Interactions ...............................................................
3. Technologies for Patterning...............................................................................................................
3.1. Overview.....................................................................................................................................
3.2. Photolithography.........................................................................................................................
3.3. Micromolding (Soft Lithography)..............................................................................................
3.3.1. Replica Molding ..............................................................................................................
3.3.2. Microcontact Printing ......................................................................................................
3.3.3. Microtransfer Molding.....................................................................................................
3.3.4. Capillary Force Lithography ...........................................................................................
3.4. Scanning Probe Lithography ......................................................................................................
4. Biosensing Applications and Examples ............................................................................................
4.1. Overview.....................................................................................................................................
4.2. Healthcare Applications .............................................................................................................
4.3. Detection of Toxins in the Environment ...................................................................................
4.4. Real Devices and Challenges .....................................................................................................
5. Conclusions and Future Perspectives ................................................................................................
References..........................................................................................................................................

855
857
858
858

858
859
859
859
860
860
861
862
862
864
865
866
866
867


xxiv

Contents

33. Microfabricated Flow Cytometers for Bacterial Detection
Sung-Yi Yang and Gwo-Bin Lee
1. Introduction ........................................................................................................................................
1.1. Bio-MEMS .................................................................................................................................
1.2. Review of Microfabrication Techniques....................................................................................
1.2.1. Bulk Micromachining Technique ....................................................................................
1.2.2. Surface Micromachining Technique ...............................................................................
1.2.3. LIGA ................................................................................................................................
1.2.4. Polymer-Based Micromachining Techniques for Microfluidic Devices ........................
2. Operation Principles ..........................................................................................................................

2.1. Cell Transportation and Focusing ..............................................................................................
2.1.1. Hydrodynamic Approach.................................................................................................
2.1.2. Pneumatic Approach........................................................................................................
2.1.3. Electrokinetic Approach ..................................................................................................
2.2. Cell Detection .............................................................................................................................
2.2.1. Optical Waveguide Approach .........................................................................................
2.2.2. Buried Optical Fiber Approach .......................................................................................
2.2.3. Large-Scale Optical System Approach ...........................................................................
2.3. Cell Sorting.................................................................................................................................
2.3.1. Hydrodynamic Sorting.....................................................................................................
2.3.2. Pneumatic Sorting............................................................................................................
2.3.3. Electrokinetic Sorting ......................................................................................................
2.3.4. Magnetic Sorting..............................................................................................................
3. Applications .......................................................................................................................................
3.1. Environmental Monitoring .........................................................................................................
3.2. Rapid Assessment of Bacterial Viability ...................................................................................
3.3. Rapid Analysis of Bacteria Levels in Food...............................................................................
3.4. Antibiotic Susceptibility Testing................................................................................................
3.5. Bacterial Detection in Blood and Urine ....................................................................................
4. Conclusions and Future Perspectives ................................................................................................
References..........................................................................................................................................

869
871
872
872
872
872
873
874

875
875
878
879
880
881
882
882
883
883
884
885
885
885
886
888
888
889
889
889
890

34. Bacterial Concentration, Separation and Analysis by Dielectrophoresis
Michael Pycraft Hughes and Kai Friedrich Hoettges
1.
2.
3.
4.
5.


Introduction ........................................................................................................................................
Theory ................................................................................................................................................
Applications of Electrokinetics to Bacteria.......................................................................................
Toward an Integrated Detection System ...........................................................................................
Conclusions and Future Perspectives ................................................................................................
References..........................................................................................................................................

895
897
901
904
905
906

35. Ultrasonic Microsystems for Bacterial Cell Manipulation
Martyn Hill and Nicholas R. Harris
1. Introduction ........................................................................................................................................
1.1. Ultrasound and Bacterial Cells ..................................................................................................
1.1.1. Cell Viability....................................................................................................................
1.2. Ultrasound and Microfluidics.....................................................................................................
2. Relevant Ultrasonic Phenomena........................................................................................................
2.1. Axial Radiation Forces...............................................................................................................
2.2. Lateral and Secondary Radiation Forces ...................................................................................

909
910
910
910
910
910

912


Contents

2.3. Acoustic Streaming ....................................................................................................................
2.4. Cavitation....................................................................................................................................
3. Applications of Ultrasonic Particle Manipulation.............................................................................
3.1. Practical Considerations .............................................................................................................
3.1.1. Transduction.....................................................................................................................
3.1.2. Mechanical Effects ..........................................................................................................
3.1.3. Construction .....................................................................................................................
3.2. Filtration and Fractionation of Cells..........................................................................................
3.2.1. Filtration and Concentration ............................................................................................
3.2.2. Fractionation of Cells ......................................................................................................
3.2.3. Trapping of Cells .............................................................................................................
3.3. Biosensor Enhancement by Forcing Cells to a Surface ............................................................
4. Conclusions and Future Perspectives ................................................................................................
References..........................................................................................................................................

xxv

913
914
914
914
914
915
916
917

917
920
921
922
924
924

36. Recent Advances in Real-Time Mass Spectrometry
Detection of Bacteria
Arjan L. van Wuijckhuijse and Ben L.M. van Baar
1. Introduction ........................................................................................................................................
1.1. General........................................................................................................................................
1.2. Scope...........................................................................................................................................
1.3. MS in the Whole Cell Analysis of Bacteria ..............................................................................
1.3.1. The Definition of ‘Identity’ of Bacteria..........................................................................
1.3.2. Mass Spectrometry of Bacteria .......................................................................................
1.4. Aerosol MS.................................................................................................................................
1.4.1. MS of Deposited Aerosols ..............................................................................................
1.4.2. Direct MS of aerosols......................................................................................................
2. Current State of the Technology .......................................................................................................
2.1. Considerations on Aerosol MS of Bacteria ...............................................................................
2.2. Deposition and PyMS Based Technology .................................................................................
2.3. Deposition and MALDI MS Based Technology .......................................................................
2.4. Single Particle LDI MS Technology .........................................................................................
2.5. Single Particle MALDI MS Technology ...................................................................................
3. Conclusions and Future Perspectives ................................................................................................
References..........................................................................................................................................

929
929

930
930
930
931
936
936
938
939
939
940
941
941
943
946
947

Index ....................................................................................................................................................... 955


Contributors

Evangelyn Alocilja
Biosystems and Agricultural Engineering
Michigan State University
East Lansing, Michigan
USA

Manfred Biebl
Profos AG
Regensburg

Germany

Bruce Applegate
Department of Food Science
Purdue University
West Lafayette, Indiana
USA

Levente Bodrossy
Department of Bioresources
Austrian Research Centres
Seibersdorf, Austria

Pierre-Alain Auroux
National Institute for Standards and Technology
EEEL, Semiconductor Electronics Division
Gaithersburg, Maryland
USA

Carl AK Borrebaeck
Department of Immunotechnology
and
CREATE Health
Lund University
Lund, Sweden

Ben L. M. van Baar
TNO Defence
Security and Safety
Rijswijk, The Netherlands

Eva Baldrich
Centro Nacional de Microelectronica
IMB-CNM-CSIC
Esfera UAB
Campus Universidad Autónoma de Barcelona
Barcelona, Spain
Padmapriya P. Banada
Molecular Food Microbiology Laboratory
Department of Food Science
Purdue University
West Lafayette, Indiana
USA
Ning Bao
Department of Agricultural and Biological Engineering
School of Chemical Engineering
Birck Nanotechnology Center
Bindley Bioscience Center
Purdue University
West Lafayette, Indiana
USA
Arun K. Bhunia
Molecular Food Microbiology Laboratory
Department of Food Science
Purdue University
West Lafayette, Indiana
USA

Eric D. Brown
Department of Biochemistry and Biomedical
Sciences and

Department of Chemistry
McMaster University
Hamilton, Canada

Daniel P. Campbell
Georgia Tech Research Institute
Atlanta, Georgia
USA

Fco. Javier Del Campo
Instituto de Biotecnología y Biomedicina
Departamento de Microbiología y Genética
Universidad Autónoma de Barcelona
Barcelona, Spain

S. Yanyun Chen
Department of Food Service
Purdue University
West Lafayette, Indiana
USA

Irina Ciubotaru
QuarTek Corporation
Greensboro, North Carolina
USA
xxvii