principles of bacterial detection: biosensors, recognition receptors and microsystems

Principles of Bacterial Detection:Biosensors, RecognitionReceptors and Microsystems

Edited by

MOHAMMED ZOUROBBiophage Pharma Inc.Montreal, Canada

SOUNA ELWARYConsultant to Biophage Pharma Inc.Montreal, Canada

ANTHONY TURNERCranfield UniversityBedfordshire, UK

123

EditorsMohammed Zourob Souna ElwaryBiophage Pharma Inc. Consultant to Biophage Pharma Inc.Montreal MontrealCanada [email protected] [email protected]

Anthony TurnerCranfield [email protected]

ISBN: 978-0-387-75112-2 e-ISBN: 978-0-387-75113-9

Library of Congress Control Number: 2007941938

2008 Springer Science+Business Media, LLCAll rights reserved. This work may not be translated or copied in whole or in part without the written permission of thepublisher (Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for briefexcerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage andretrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafterdeveloped is forbidden.The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identifiedas 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

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Preface

Bacterial contamination of food and water resources, as well as the increasing incidence ofnosocomial infections, has us on our toes, looking for ways of recognizing these elements. Inaddition, the recent and growing threats to personal and territorial securities make this task evenmore urgent. Therefore, accurate assessment of the state of current technologies is a prerequisitefor undertaking any course of action towards future improvements. In particular, developmentof new detection and identification technologies for the plethora of bacterial agents has becomeincreasingly important to scientists and to regulatory agencies. In recent years, there has beenmuch progress in the field of bacterial agents detection, resulting in the development of moreaccurate, fast, analyte-specific, robust, and cost effective techniques by incorporating emergingtechnologies from various disciplines.

Principles of Bacterial Detection: Biosensors, Recognition Receptors and Microsystemspresents a significant and up-to-date review of various integrated approaches for bacterialdetection by distinguished engineers and scientists. This work is a comprehensive approach tobacterial detection, presenting a thorough knowledge of the subject and an effective integrationof disciplines in order to appropriately convey the state-of the-art fundamentals and applicationsof the most innovative approaches.

The book consists of four parts. The first part (Chapters 14) is an introduction topathogenic bacteria and sampling techniques and provides an overview of the rapid microbio-logical methods. The second part (Chapters 520) describes the different transducers used forbacterial detection. It covers the theory behind each technique and delivers a detailed state-of-the-art review for all the new technologies used. The third part (Chapters 2129) coversthe 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 molecularimprinted polymers as recognition elements. The fourth part (Chapters 3036) covers thedifferent microsystems used for detection/identification and bacterial manipulations, mainlybacteria lysis in microfluidics, PCR in microfluidics, dielectrophoresis, ultrasonic manipulationtechniques, and mass spectrometry.

We anticipate that the book will be helpful to academicians, practitioners, andprofessionals working in various fields, including biomedical sciences, physical sciences,microsystems engineering, nanotechnology, veterinary science and medicine, food QA, bioter-rorism and security as well as allied health, healthcare and surveillance. Since the fundamentalsare also reviewed, we believe that the book will appeal to advanced undergraduate and graduatestudents who study in areas related to bacterial detection.

We gratefully acknowledge all authors for their participation and contributions, whichmade this book a reality. We give many thanks to Olivier Laczka and Joseph Piliero for thebook cover design.

Mohammed ZourobSouna Elwary

Anthony TurnerJune 2008

v

Contents

Part I Introduction

1. Introduction to Pathogenic Bacteria

Tracey Elizabeth Love and Barbara Jones

1. Pathogenic Microorganisms............................................................................................................... 31.1. Toxins ......................................................................................................................................... 41.2. Adherence ................................................................................................................................... 41.3. Invasion....................................................................................................................................... 71.4. Evasion of the Host Immune Response ..................................................................................... 71.5. Iron Acquisition .......................................................................................................................... 81.6. Regulation of Virulence Factors ................................................................................................ 8

2. Sources and Routes of Infection........................................................................................................ 92.1. Natural Infection ......................................................................................................................... 92.2. Food and Water .......................................................................................................................... 92.3. Hospital Acquired Infections...................................................................................................... 102.4. Intentional InfectionBiological Warfare................................................................................. 10

3. Detection of Pathogenic Microorganisms.......................................................................................... 114. Conclusions ........................................................................................................................................ 12

References........................................................................................................................................... 12

2. Sample Preparation: An Essential Prerequisite for High-Quality BacteriaDetection

Jan W. Kretzer, Manfred Biebl and Stefan Miller

1. Introduction ........................................................................................................................................ 152. The Sample......................................................................................................................................... 163. Sampling............................................................................................................................................. 17

3.1. Sample drawing .......................................................................................................................... 174. Microbiological Examination of Foods ............................................................................................. 175. Microbiological Examination of Surfaces ......................................................................................... 176. Microbiological Examination of Air.................................................................................................. 187. Sample Handling ................................................................................................................................ 208. Sample Preparation ............................................................................................................................ 219. Sample Preparation for Detection of Intact Bacterial Cells.............................................................. 2110. Sample Preparation for Detection of Bacterial Nucleic Acids ......................................................... 2311. Conclusions and Future Perspectives ................................................................................................ 27

References........................................................................................................................................... 28

3. Detection of Bacterial Pathogens in Different Matrices: Current Practicesand Challenges

Ahmed E. Yousef

1. Introduction ........................................................................................................................................ 312. Analytical Tools and Methods: A Historical Perspective ................................................................. 32

vii

viii Contents

3. Defining the Terms ............................................................................................................................ 324. Matrix Complexity and Pathogen Detection ..................................................................................... 325. Techniques Currently Used in Pathogen Detection Methods ........................................................... 33

5.1. Culture Techniques ..................................................................................................................... 335.2. Enzyme-Linked Immunoassay.................................................................................................... 355.3. Polymerase Chain Reaction (PCR) ............................................................................................ 36

6. Basics of Pathogen Detection ............................................................................................................ 366.1. Sampling ..................................................................................................................................... 37

6.1.1. Air Sampling..................................................................................................................... 376.1.2. Surfaces Sampling ............................................................................................................ 376.1.3. Bulk Sampling .................................................................................................................. 39

6.2. Sample Preparation ..................................................................................................................... 396.3. Pathogen Amplification .............................................................................................................. 396.4. Selection and Screening.............................................................................................................. 406.5. Identification ............................................................................................................................... 40

6.5.1. Morphological Characteristics.......................................................................................... 416.5.2. Biochemical and Physiological Traits.............................................................................. 416.5.3. Serological Properties ....................................................................................................... 426.5.4. Genetic Characteristics ..................................................................................................... 42

6.6. Pathogenicity Testing.................................................................................................................. 436.6.1. Kochs Postulates.............................................................................................................. 436.6.2. Mammalian Cell Culture (Tissue Culture)....................................................................... 436.6.3. Virulence Genes and Gene Expression Products............................................................. 44

6.7. Testing for Specific Traits .......................................................................................................... 447. Challenges to Current Detection Methods......................................................................................... 44

7.1. Pathogen Quantification Problems ............................................................................................. 447.2. Can a Small Bacterial Population be Detected Rapidly and Reliably?..................................... 447.3. Which Traits to Analyze, and How Many Tests are Needed for Identifying a Bacterial

Pathogen? .................................................................................................................................... 457.4. Real-Time Detection ................................................................................................................... 46References........................................................................................................................................... 46

4. Overview of Rapid Microbiological Methods

Jeanne Moldenhauer

1. Introduction ........................................................................................................................................ 492. A History of Rapid Microbiological Methods: Industry Reluctance to Accept

These Methods .................................................................................................................................. 503. Types of Microbial Testing Performed ............................................................................................. 504. Types of Rapid Microbiological Methods......................................................................................... 50

4.1. Growth-Based Technologies....................................................................................................... 504.2. Viability-Based Technologies..................................................................................................... 504.3. Cellular Component or Artifact-Based Technologies................................................................ 514.4. Nucleic Acid-Based Technologies ............................................................................................. 514.5. Automated Methods.................................................................................................................... 514.6. Combination Methods................................................................................................................. 51

5. Overview of Rapid Technologies and How They Work .................................................................. 515.1. Adenosine Tri-Phosphate (ATP) Bioluminescence ................................................................... 515.2. Adenylate Kinase........................................................................................................................ 525.3. Autofluorescence ........................................................................................................................ 525.4. Biochemical Assays and Physiological Reactions..................................................................... 525.5. Biosensors and Immunosensors ................................................................................................. 535.6. Carbon Dioxide Detection.......................................................................................................... 535.7. Changes in Headspace Pressure ................................................................................................. 53

Contents ix

5.8. Colorimetric Detection of Carbon Dioxide Production............................................................. 535.9. Concentric Arcs of Photovoltaic Detectors with Laser Scanning............................................. 545.10. Direct Epifluorescent Filter Technique (DEFT)........................................................................ 545.11. DNA Sequencing........................................................................................................................ 545.12. Endospore Detection .................................................................................................................. 555.13. Enzyme Linked Immunosorbent Assay (ELISA)...................................................................... 555.14. Flow Cytometry.......................................................................................................................... 555.15. Fluorescent Probe Detection ...................................................................................................... 555.16. Fatty Acid Profiles (Fatty Acid Methyl Esters, FAMEs).......................................................... 565.17. Fourier Transformed Infrared Spectroscopy (FTIR) ................................................................. 565.18. Gram Stains (Rapid Method) ..................................................................................................... 565.19. Impedance................................................................................................................................... 575.20. Immunological Methods............................................................................................................. 575.21. Lab-on-a-Chip (LOC), Arrays, Microarrays and Microchips ................................................... 575.22. Limulus Amebocyte Lysate (LAL) Endotoxin Testing............................................................. 585.23. Mass Spectrometry (Matrix-Assisted Laser Desorption-Time of Flight (MALTI-TOF))........ 585.24. Microcalorimetry ........................................................................................................................ 585.25. Micro-Electro-Mechanical Systems (MEMS) ........................................................................... 595.26. Nanotechnology .......................................................................................................................... 595.27. Near Infrared Spectroscopy (NIRS)........................................................................................... 595.28. Nucleic Acid Probes................................................................................................................... 595.29. Optical Particle Detection .......................................................................................................... 595.30. Polymerase Chain Reaction (PCR) ............................................................................................ 605.31. Rep-PCR ..................................................................................................................................... 605.32. Raman Spectroscopy .................................................................................................................. 615.33. Ribotyping/Molecular Typing .................................................................................................... 615.34. Solid Phase Laser Scanning Cytometry..................................................................................... 615.35. Southern Blotting/Restriction Fragment Length Polymorphism............................................... 625.36. Spiral Plating .............................................................................................................................. 625.37. Turbidimetry ............................................................................................................................... 62

6. Potential Areas of Application of Rapid Microbiological Methods ................................................. 627. Disclaimer........................................................................................................................................... 758. Conclusions ........................................................................................................................................ 75

References........................................................................................................................................... 75

Part II Biosensors

5. Surface Plasmon Resonance (SPR) Sensors for the Detection of BacterialPathogens

Allen D. Taylor, Jon Ladd, Jir Homola and Shaoyi Jiang

1. Introduction ........................................................................................................................................ 832. Fundamentals of Surface Plasmon Resonance Biosensing ............................................................... 833. SPR Sensor Instrumentation .............................................................................................................. 854. Surface Chemistries and Molecular Recognition Elements .............................................................. 885. Detection Formats .............................................................................................................................. 906. Quantification of Bacteria Cells ........................................................................................................ 91

6.1. Challenges for the Detection of Whole Bacteria by SPR.......................................................... 916.2. Effect of Bacteria Sample Treatment ......................................................................................... 926.3. Examples of Bacteria Detection ................................................................................................. 92

6.3.1. Escherichia coli................................................................................................................. 936.3.2. Salmonella spp. ................................................................................................................ 976.3.3. Listeria monocytogenes..................................................................................................... 98

x Contents

6.3.4. Other Bacteria .................................................................................................................. 986.3.5. Detection of Multiple Bacteria ........................................................................................ 99

7. Genetic Markers ................................................................................................................................. 1018. Antibody Biomarkers ......................................................................................................................... 1039. Conclusions and Future Perspectives ................................................................................................ 103

References........................................................................................................................................... 104

6. Bacterial Detection Using Evanescent Wave-Based FluorescentBiosensors

Kim E. Sapsford and Lisa C. Shriver-Lake

1. Introduction ........................................................................................................................................ 1092. Current State of Bacterial Fluorescent TIRF Biosensors.................................................................. 112

2.1. Non-Planar Substrates................................................................................................................. 1122.1.1. Fiber Optics ...................................................................................................................... 1122.1.2. Capillaries......................................................................................................................... 112

2.2. Planar Substrates ......................................................................................................................... 1122.2.1. NRL Array Biosensor ...................................................................................................... 1132.2.2. Other Optical Waveguides ............................................................................................... 1152.2.3. TIRF-Microscopy ............................................................................................................. 116

3. Future Aspects of Bacterial Fluorescent TIRF Biosensors ............................................................... 1174. Conclusions ........................................................................................................................................ 119

References........................................................................................................................................... 120

7. Fiber Optic Biosensors for Bacterial Detection

Ryan B. Hayman

1. Fiber Optic Biosensors....................................................................................................................... 1251.1. Whole-Cell Detection ................................................................................................................. 126

1.1.1. Evanescent-Field Sensing ................................................................................................ 1261.1.2. Sandwich Immunoassays ................................................................................................. 127

1.2. Bead-Based Arrays ..................................................................................................................... 1281.3. Nucleic Acid Sandwich Assays.................................................................................................. 1291.4. Nucleic Acid Direct Hybridization............................................................................................. 1311.5. Extension Reactions.................................................................................................................... 134

2. Conclusions and Future Perspectives ................................................................................................ 134References........................................................................................................................................... 135

8. Integrated Deep-Probe Optical Waveguides for Label Free BacterialDetection

Mohammed Zourob, Nina Skivesen, Robert Horvath, Stephan Mohr, Martin B. McDonnelland Nicholas J. Goddard

1. Introduction ........................................................................................................................................ 1391.1. Planar Optical Waveguides......................................................................................................... 1411.2. Total Internal Reflection and Evanescent Waves ...................................................................... 1411.3. Waveguide Modes ...................................................................................................................... 1431.4. Frustrated Total Internal Reflection, Leaky Modes ................................................................... 1441.5. Literature on Waveguides for Bacterial Detection .................................................................... 144

2. Deep-Probe Optical Waveguide Sensors with Tunable Evanescent Field ....................................... 1452.1. Waveguide Modes, Light Coupling and Sensing Depths of Evanescent Waves...................... 146

2.1.1. Light Coupling Techniques.............................................................................................. 148

Contents xi

2.2. Waveguide Designs Based on Low-Index Substrates................................................................ 1502.2.1. Bacteria Detection Using Reverse Symmetry Waveguides ............................................ 151

2.3. Waveguide Designs Based on Metal- and Dye-Clad SubstratesLeaky Modes..................... 1522.3.1. Results .............................................................................................................................. 156

3. Integrated Deep-Probe Optical Waveguides Systems ....................................................................... 1603.1. Integration with Electric Field.................................................................................................... 1613.2. Integration with Ultrasound Standing Waves (USW)................................................................ 163

4. Conclusions and Future Perspectives ................................................................................................ 166References........................................................................................................................................... 166

9. Interferometric Biosensors

Daniel P. Campbell

1. Principles of Optical Interferometry .................................................................................................. 1691.1. Optical Waveguides .................................................................................................................... 1711.2. Planar Waveguide Operation...................................................................................................... 1721.3. Types of Waveguides ................................................................................................................. 175

2. Light Coupling Methods .................................................................................................................... 1782.1. Interferometers ............................................................................................................................ 1802.2. Collinear or Single Channel Interferometers ............................................................................. 1832.3. Two-Channel Interferometers ..................................................................................................... 186

3. Interferometric Array Sensors............................................................................................................ 1924. Surface Plasmon Interferometry ........................................................................................................ 1955. Other Interferometric Methods and Designs ..................................................................................... 1966. Surface Functionalization................................................................................................................... 1977. Sample Collection Systems................................................................................................................ 1988. Interferometric Applications for Whole-Cell Detection.................................................................... 1999. Advantages and Limitations............................................................................................................... 20610. Potential for Improving Current Performance................................................................................... 206

References........................................................................................................................................... 208

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. Beyond Robert Boyles Chicken....................................................................................................... 2142. The Bacterial (lux) Luminescent System for Direct Pathogen Detection........................................ 2153. The Firefly (luc) Luminescent System for Direct Pathogen Detection............................................ 2194. The Use of Alternative Luciferases in Pathogen Detection ............................................................. 2225. Luminescent-Based Immunoassays ................................................................................................... 2226. Chemiluminescence Detection Methods ........................................................................................... 2227. Conclusions and Future Perspectives ................................................................................................ 225

References.......................................................................................................................................... 226

11. Porous and Planar Silicon Sensors

Charles R. Mace and Benjamin L. Miller

1. Introduction ........................................................................................................................................ 2311.1. Porous Silicon: A Three-Dimensional Matrix for Biosensing .................................................. 2321.2. Effect of PSi Immobilization on Probe Viability: Experiments with GST .............................. 2331.3. Toward Larger Targets: The First Macroporous Microcavity Structures................................. 2351.4. Porous Silicon Bandgap Sensors in Novel Formats: Smart Bandages and Smart Dust ... 235

2. Arrayed Imaging ReflectometryA Planar Silicon Biosensor........................................................ 2362.1. Theory......................................................................................................................................... 236

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2.1.1. Physical Rationale............................................................................................................ 2362.1.2. Substrate Design .............................................................................................................. 2372.1.3. Mathematical Model ........................................................................................................ 2382.1.4. Monitoring the Null Reflectance Condition.................................................................... 240

2.2. Applications of AIR Biosensing ................................................................................................ 2422.2.1. Limitations ....................................................................................................................... 2422.2.2. Probe Immobilization ...................................................................................................... 2442.2.3. Pathogen Detection .......................................................................................................... 246

3. Conclusions and Future Perspectives ................................................................................................ 250References.......................................................................................................................................... 251

12. Acoustic Wave (TSM) Biosensors: Weighing Bacteria

Eric Olsen, Arnold Vainrub and Vitaly Vodyanoy

1. Introduction ........................................................................................................................................ 2552. Historical Perspective, Theory and Background............................................................................... 256

2.1. Piezoelectricity and Acoustic Waves......................................................................................... 2562.2. Acoustic Wave Devices ............................................................................................................. 256

3. TSM Biosensors................................................................................................................................. 2593.1. Detection of Microorganisms..................................................................................................... 2613.2. Measurement in Liquid .............................................................................................................. 2633.3. TSM Biosensor Characteristics .................................................................................................. 2643.4. Commercial TSM Microbalances .............................................................................................. 2673.5. Immobilization of Probes onto Sensor Surface ......................................................................... 269

3.5.1. Physical Adsorption ......................................................................................................... 2713.5.2. Other Coupling Methods ................................................................................................. 2723.5.3. Combined Langmuir-Blodgett/Molecular Assembling Method ..................................... 2723.5.4. Solvent-Free Purified Monolayers................................................................................... 2753.5.5. Immobilization of Monolayers of Phage Coat Proteins ................................................. 2763.5.6. Immobilization of Molecular Probes onto Porous Substrates ........................................ 281

4. Problem of Negative Mass............................................................................................................. 2825. Coupled Oscillators Model ................................................................................................................ 2866. Conclusions ........................................................................................................................................ 290

References.......................................................................................................................................... 291

13. Amperometric Biosensors for Pathogenic Bacteria Detection

Ilaria Palchetti and Marco Mascini

1. Introduction ........................................................................................................................................ 2992. Amperometric Biosensors.................................................................................................................. 300

2.1. Microbial Metabolism-Based Biosensors .................................................................................. 3022.2. Immunosensors ........................................................................................................................... 3032.3. DNA-Based Biosensors.............................................................................................................. 306

3. Conclusion and Future Perspectives.................................................................................................. 310References.......................................................................................................................................... 310

14. Microbial Genetic Analysis Based on Field Effect Transistors

Yuji Miyahara, Toshiya Sakata and Akira Matsumoto

1. Introduction ........................................................................................................................................ 3132. Fundamental Principles of Field Effect Devices .............................................................................. 314

2.1. Metal-Insulator-Semiconductor (MIS) Capacitor ...................................................................... 3142.2. Principles of Biologically Coupled Field Effect Transistors for Genetic Analysis

(Genetic FETS) ........................................................................................................................... 315

Contents xiii

3. Fundamentals of Genetic Analysis.................................................................................................... 3173.1. DNA............................................................................................................................................ 3173.2. Genetic Analysis......................................................................................................................... 3173.3. DNA Chip / DNA Microarray ................................................................................................... 318

4. Immobilization of DNA Molecules on the Surfaces of Solid Substrates ........................................ 3184.1. Silanization ................................................................................................................................. 3184.2. Thiol-Gold Bonding ................................................................................................................... 3204.3. Avidin, Streptavidin and Biotin ................................................................................................. 3204.4. Others.......................................................................................................................................... 321

5. Genetic Analysis Based on Field Effect Devices ............................................................................. 3225.1. Fundamental Characteristics of Genetic Field Effect Devices.................................................. 322

5.1.1. Detection of DNA Molecular Recognition Events ......................................................... 3225.1.2. Immobilization Density of Oligonucleotide Probes........................................................ 326

5.2. Single Nucleotide Polymorphisms (SNPs) Analysis ................................................................. 3275.2.1. Controlling Hybridization Temperature for SNPs Analysis........................................... 3285.2.2. SNPs Analysis Based on Primer Extension .................................................................... 329

5.3. DNA Sequencing........................................................................................................................ 3316. Conclusions and Future Perspectives ................................................................................................ 335

References.......................................................................................................................................... 336

15. Impedance-Based Biosensors for Pathogen Detection

Xavier Muoz-Berbel, Neus Godino, Olivier Laczka, Eva Baldrich, Francesc Xavier Muozand Fco. Javier Del Campo

1. Introduction ........................................................................................................................................ 3412. Fundamentals of Electrochemical Impedance Spectroscopy............................................................ 342

2.1. Data Analysis: Plotting............................................................................................................... 3442.2. Data Analysis: Interpretation ..................................................................................................... 344

2.2.1. Non-Faradaic Parameters................................................................................................. 3452.2.2. Faradaic Parameters ......................................................................................................... 347

2.3. Measuring at Impedimetric Biosensors...................................................................................... 3502.3.1. Measurement Modes........................................................................................................ 350

2.4. Bacterial Parasitizing Effect on Electrode Surface.................................................................... 3533. Development of an Immunosensor.................................................................................................... 354

3.1. Biological Recognition Elements in Biosensors for Pathogen Detection................................. 3543.1.1. Antibodies ........................................................................................................................ 3553.1.2. Nucleic Acids................................................................................................................... 3553.1.3. Aptamers .......................................................................................................................... 3563.1.4. Other Recognition Strategies........................................................................................... 356

3.2. Surface Modification Methods................................................................................................... 3573.2.1. Adsorption........................................................................................................................ 3573.2.2. Self-assembled Monolayers ............................................................................................. 3583.2.3. Silanisation....................................................................................................................... 3593.2.4. Protein A and Protein G .................................................................................................. 3603.2.5. The Biotin-(Strept)Avidin System................................................................................... 3603.2.6. Chemical Conjugation ..................................................................................................... 3613.2.7. Entrapment ....................................................................................................................... 3623.2.8. Microencapsulation .......................................................................................................... 362

3.3. Blocking...................................................................................................................................... 3623.4. Signal Amplification .................................................................................................................. 3633.5. The Need for Negative Controls ................................................................................................ 3643.6. Development of Novel Strategies: Assessing Performance Using ELISA and Microscopy ... 365

4. Current EIS Biosensors for Pathogen Detection............................................................................... 3654.1. Biosensors Based on Interfacial Capacitance Changes ............................................................. 366

xiv Contents

4.2. Biosensors Based on Charge-Transfer Resistance Changes...................................................... 3674.3. Biosensors Based on Conductivity Changes ............................................................................. 3694.4. Other Approaches ....................................................................................................................... 370


16. Label-Free Microbial Biosensors Using Molecular Nanowire Transducers

Evangelyn Alocilja and Zarini Muhammad-Tahir

1. Introduction ........................................................................................................................................ 3771.1. Rationale for Rapid Tests........................................................................................................... 3771.2. Target Microorganisms and Matrices ........................................................................................ 378

1.2.1. Escherichia coli ............................................................................................................... 3781.2.2. Salmonella........................................................................................................................ 3791.2.3. Bovine Viral Diarrhea Virus ........................................................................................... 380

1.3. Food Safety Applications ........................................................................................................... 3812. Biosensor Formats ............................................................................................................................. 382

2.1. Definition .................................................................................................................................... 3822.2. Antibodies as Biological Sensing Element................................................................................ 3822.3. DNA as Biological Sensing Element......................................................................................... 3842.4. DNA-Based Biosensors.............................................................................................................. 3852.5. Antibody-Based Biosensors ....................................................................................................... 3872.6. Biosensor Transducing Element: Conducting Polymer............................................................. 388

2.6.1. Polyaniline ....................................................................................................................... 3902.6.2. Self-doped Polyaniline..................................................................................................... 3912.6.3. Carbon Nanotubes............................................................................................................ 391

2.7. Conducting Polymer-Based Biosensor for Microbial/Viral Detection...................................... 3923. Illustration: Biosensor Using Self-doped and Non-self-doped Pani................................................. 392

3.1. Pani Preparation.......................................................................................................................... 3923.2. Pani Characterization.................................................................................................................. 392

3.2.1. Conductivity Measurement.............................................................................................. 3923.2.2. Biosensor Fabrication ...................................................................................................... 3933.2.3. Indium Tin Oxide/Pani Biosensor ................................................................................... 3933.2.4. Lateral Flow Conductometric Biosensor......................................................................... 3933.2.5. Signal Measurement ........................................................................................................ 393

3.3. Properties of Pani ....................................................................................................................... 3943.4. Detection Concept of the Biosensor .......................................................................................... 3983.5. Biosensor Properties ................................................................................................................... 399

3.5.1. ITO-Pani Biosensor ......................................................................................................... 3993.6. Lateral Flow Conductometric Biosensor ................................................................................... 4033.7. Biosensor Performance............................................................................................................... 404

3.7.1. ITO/Pani Biosensor.......................................................................................................... 4043.8. Conductometric Biosensor ......................................................................................................... 404


17. Magnetic Techniques for Rapid Detection of Pathogens

Yousef Haik, Reyad Sawafta, Irina Ciubotaru, Ahmad Qablan, Ee Lim Tan andKeat Ghee Ong

1. Introduction ........................................................................................................................................ 4152. Synthesis of Magnetic Particles ........................................................................................................ 417

2.1. Effect of Particle Size ................................................................................................................ 418

Contents xv

2.2. Synthesis Techniques ................................................................................................................. 4232.3. Encapsulation of Magnetic Particles.......................................................................................... 423

2.3.1. Methods of Preparing Polymer/Protein Coatings ........................................................... 4242.3.2. Examples of Polymer/Protein Encapsulated Particles .................................................... 426

3. Immobilization Strategies .................................................................................................................. 4263.1. Modification of Particle Surface with a Ligand ........................................................................ 430

4. Biological Targets.............................................................................................................................. 4305. Magnetic Immunoassays.................................................................................................................... 430

5.1. Direct Immunoassay Detection Using Magnetic Beads ............................................................ 4305.1.1. Superconducting Quantum Interference Devices............................................................ 4315.1.2. ABICAP Column............................................................................................................. 432

5.2. Indirect Immunoassay Detection Using Magnetic Beads.......................................................... 4335.2.1. ELISA .............................................................................................................................. 433

6. Handling Techniques ......................................................................................................................... 4387. Magnetic Separation .......................................................................................................................... 439

7.1. Magnetic Force ........................................................................................................................... 4397.2. High-Field Electromagnets......................................................................................................... 4407.3. Permanent Magnets .................................................................................................................... 4417.4. Numerical Analysis for Permanent Magnet Arrangements....................................................... 442

8. Giant Magnetoresistive (GMR) Devices for Bacterial Detection..................................................... 4469. Bacteria Detection with Magnetic Relaxation Signal....................................................................... 44810. Magnetoelastic Sensors for Bacterial Detection ............................................................................... 449

10.1. E. coli Detection......................................................................................................................... 45011. Conclusions and Future Perspectives ................................................................................................ 453

References.......................................................................................................................................... 454

18. Cantilever Sensors for Pathogen Detection

Raj Mutharasan

1. Introduction ........................................................................................................................................ 4592. Millimeter-Sized Cantilever Sensors................................................................................................. 4603. Reported Work on Detecting Cells Using Cantilever Sensors......................................................... 4614. Physics of Cantilever Sensors ........................................................................................................... 4635. Resonance Modes .............................................................................................................................. 4666. Characterization of PEMC Sensors ................................................................................................... 4687. Mass Change Sensitivity ................................................................................................................... 4688. Antibody Immobilization Methods ................................................................................................... 4699. Detection in Batch and Stagnant Samples ........................................................................................ 47010. Detection in Flowing Samples .......................................................................................................... 47311. Selectivity of Detection ..................................................................................................................... 47512. Conclusions ........................................................................................................................................ 477

References.......................................................................................................................................... 478

19. Detection and Viability Assessment of Endospore-Forming Pathogens

Adrian Ponce, Stephanie A. Connon and Pun To Yung

1. Introduction ........................................................................................................................................ 4811.1. Historical Perspective ................................................................................................................. 4811.2. Endospore Dormancy, Resistance and Longevity ..................................................................... 4821.3. Endospores as Biodosimeters for Evaluating Sterilization Regimes ........................................ 4841.4. Endospore-Forming Pathogens .................................................................................................. 4851.5. Bioweapons, Bioinsecticides and Probiotics ............................................................................. 487

xvi Contents

2. Detection of Endospore-Forming Pathogens and their Endospores ................................................. 4892.1. Phenotypic Identification............................................................................................................ 489

2.1.1. Phenotypic Identification of Bacillus anthracis.............................................................. 4902.1.2. Phenotypic Identification of Clostridium perfringens .................................................... 490

2.2. Parameters of a Sensor ............................................................................................................... 4912.3. Rapid Immunoassays.................................................................................................................. 492

2.3.1. Enzyme-Linked Immunosorbent Assays......................................................................... 4922.3.2. Lateral-Flow Immunoassays............................................................................................ 4932.3.3. Immunomagnetic Electrochemiluminescence ................................................................. 4932.3.4. Flow Cytometry ............................................................................................................... 4942.3.5. Vegetative Cells ............................................................................................................... 494

2.4. Rapid Nucleic Acid Assays........................................................................................................ 4952.4.1. PCR Sample Preparation and Endospore Lysis .............................................................. 4952.4.2. The PCR Reaction ........................................................................................................... 4962.4.3. Specificity of PCR Primers for Bacillus anthracis Detection........................................ 4962.4.4. Rapid PCR Detection Methods High Throughput and real-time PCR .......................... 4972.4.5. Field Implementation of Rapid PCR for Analysis of Environmental Samples ............. 4982.4.6. Monitoring the Air for Bacillus anthracis Endospores by PCR .................................... 500

2.5. Rapid Detection of Endospores via Dipicolinic Acid Biomarker............................................. 5012.5.1. Terbium Dipicolinic Acid Luminescence Assay ............................................................ 5012.5.2. Anthrax Smoke Detector ................................................................................................. 503

3. Validation of Sterilization by Rapid Endospore Viability Assessment ........................................... 5053.1. Measuring Endospore Viability and Inactivation ...................................................................... 5053.2. Measuring Endospore Inactivation using Germinability Assays .............................................. 508

3.2.1. Rapid Germinability Assays ............................................................................................ 5083.2.2. Nucleic Acid-Based Amplification Methods for Detecting Germinable, Viable

Bacillus anthracis Spores.............................................................................................. 5083.2.3. Germination Observed via Loss of Phase Brightness..................................................... 5093.2.4. Germination Observed via DPA release ......................................................................... 510

3.3. Measuring Endospore Inactivation Using Metabolic Activity Assays ..................................... 5124. Conclusions and Future Perspectives ................................................................................................ 513

References.......................................................................................................................................... 514

20. Label-Free Fingerprinting of Pathogens by Raman SpectroscopyTechniques

Ann E. Grow

1. Introduction ........................................................................................................................................ 5252. Raman Microscopy for Whole-Organism Fingerprinting................................................................. 5273. Surface-Enhanced Raman Scattering (SERS) for Whole-Organism Fingerprinting........................ 5314. MicroSERS for the Detection and Identification of Pathogens and Toxins .................................... 534

4.1. MicroSERS Detection of Bacteria ............................................................................................. 5354.1.1. SERS Fingerprinting of Bacteria..................................................................................... 5354.1.2. Impact of Growth Conditions on Bacterial Fingerprints ................................................ 5364.1.3. Viable vs. Nonviable Bacteria......................................................................................... 5394.1.4. Integrated MicroSERS Detection and Identification of Bacteria ................................... 5424.1.5. Impact of Growth Conditions on Biomolecule Capture ................................................. 5444.1.6. Analysis of Bacteria in Complex Samples...................................................................... 544

4.2. MicroSERS Detection of Spores................................................................................................ 5454.2.1. SERS Fingerprinting of Spores ....................................................................................... 5454.2.2. Impact of Growth Conditions on Spore Fingerprints ..................................................... 5474.2.3. Viable vs. Nonviable Spores ........................................................................................... 5504.2.4. Integrated MicroSERS Detection and Identification of Spores...................................... 551

Contents xvii

4.3. MicroSERS Detection of Bacterial Toxins................................................................................ 5544.3.1. SERS Fingerprinting of Toxins....................................................................................... 5554.3.2. Analysis of Toxins in Complex Samples........................................................................ 558

5. Conclusion and Future Perspectives.................................................................................................. 559References.......................................................................................................................................... 560

Part III Recognition Receptors

21. Antibodies and Immunoassays for Detection of Bacterial Pathogens

Padmapriya P. Banada and Arun. K. Bhunia

1. Introduction ........................................................................................................................................ 5672. Antibodies .......................................................................................................................................... 568

2.1. Polyclonal Antibody................................................................................................................... 5702.2. Monoclonal Antibody................................................................................................................. 5702.3. Use of Synthetic Peptides for Antibody Production ................................................................. 5712.4. Recombinant DNA Technology................................................................................................. 573

2.4.1. Phage Display .................................................................................................................. 5733. Capture and Concentration of Cells by Immunomagnetic Separation ............................................. 575

3.1. Automated IMS Systems............................................................................................................ 5774. Immunoassays for Pathogen Detection ............................................................................................. 577

4.1. Radioimmunoassay..................................................................................................................... 5774.2. Enzyme Immunoassays .............................................................................................................. 577

4.2.1. Escherichia coli ................................................................................................................ 5804.2.2. Listeria monocytogenes .................................................................................................... 5834.2.3. Salmonella......................................................................................................................... 5834.2.4. Staphylococcal Enterotoxins............................................................................................ 5834.2.5. Clostridium botulinum Toxins......................................................................................... 584

4.3. Lateral Flow Immunoassay ........................................................................................................ 5844.4. Other Immunoassays .................................................................................................................. 585

4.4.1. Latex Agglutination (LA) and Reverse Passive Latex Agglutination (RPLA) Tests .... 5854.4.2. Enzyme-Linked Fluorescent Assay ................................................................................. 5854.4.3. Time-Resolved Fluorescence Immunoassay ................................................................... 5854.4.4. Chemiluminescent Immunoassay .................................................................................... 5864.4.5. Capillary Microbead (Spheres) Immunoassay ................................................................ 5864.4.6. Electrochemical-Immunoassay ........................................................................................ 586

4.5. Optical Biosensors...................................................................................................................... 5874.5.1. Surface Plasmon Resonance............................................................................................ 5874.5.2. Fiber-Optic Biosensors .................................................................................................... 5874.5.3. Antibody-Based Microfluidic Sensors ............................................................................ 5884.5.4. Serodiagnosis ................................................................................................................... 589

5. Recent Developments in Immunoassays........................................................................................... 5905.1. Protein/Antibody Microarrays.................................................................................................... 5905.2. Mass Spectrometric Immunodetection....................................................................................... 5915.3. SERS Biochip Technology ...................................................................................................... 591

6. Limitations and Challenges ............................................................................................................... 5916.1. Specificity and Sensitivity.......................................................................................................... 5916.2. Effect of Physical and Chemical Stresses on the Expression Profile of Antigens in Bacteria 592

6.2.1. Effect of Media Composition on the Expression of Proteins in Bacteria...................... 5926.2.2. Effect of Stress on the Expression of Proteins in Bacteria ............................................ 593


xviii Contents

22. Rapid Nucleic Acid-Based Diagnostics Methods for the Detectionof Bacterial Pathogens

Barry Glynn

1. Introduction ........................................................................................................................................ 6031.1. Detection of Pathogenic Bacteria from Clinical Samples ......................................................... 6041.2. NAD Assays for the Detection of Respiratory Infection, Sepsis and Sexually

Transmitted Infection .................................................................................................................. 6041.3. Profiling of Multi-drug Resistance ............................................................................................ 6061.4. Bioterrorism................................................................................................................................ 606

2. Detection of Bacterial Food-Borne Pathogens.................................................................................. 6062.1. Recent Outbreaks........................................................................................................................ 6062.2. Benefits and Limitations of Conventional Methods.................................................................. 6072.3. Development of Rapid Diagnostics Methods ............................................................................ 607

3. Rapid Nucleic Acid Diagnostics for Bacterial Food-Borne Pathogens............................................ 6073.1. In Vitro Nucleic Acid Amplification-Based Detection of Food-Borne Pathogens .................. 6073.2. Requirements for a NAD-Based Food Assay............................................................................ 6083.3. Polymerase Chain Reaction (PCR) ............................................................................................ 6083.4. Application of PCR-Based Tests to Pathogen Detection in Food Samples ............................. 6093.5. Use of RNA as an Alternative Nucleic Acid Diagnostic Target .............................................. 6103.6. Sample Preparation for NAD from Clinical Sample Types...................................................... 6113.7. Limitations of NAD in Clinical Settings ................................................................................... 611

4. Formats of NAD Assays for Food Pathogen Detection ................................................................... 6124.1. Nucleic Acid-Based Diagnostics Based on In Vitro Amplification Technologies................... 6124.2. PCR-ELISA and PCR-DNA Probe Membrane Based Assays

for Campylobacter and Salmonella ............................................................................................ 6124.3. Specific Examples of Nucleic Acid Diagnostics Assays for the Detection of Bacterial

Food-Borne Pathogens ................................................................................................................ 6134.3.1. Commercially Available Conventional NAD Assays

for Food-Borne Bacterial Pathogens............................................................................. 6144.3.2. Alternative In Vitro Amplification Technologies ........................................................... 615

4.4. Standardisation of In Vitro Amplification-Based NAD Assaysand Inter-Laboratory Validation Studies .................................................................................... 616

4.5. Real-Time In Vitro Amplification-Based Nucleic Acid Diagnostics ....................................... 6174.5.1. Specific Examples of Real-Time PCR Assays for the Detection of Bacterial

Food-Borne Pathogens .................................................................................................. 6174.5.2. Alternative Real-Time In Vitro Amplification-Based Diagnostics Technologies ......... 618

4.6. Limitations and Other Considerations for In Vitro Amplification NAD Tests ........................ 6194.7. Non-Amplified Direct DNA Probe-Based Nucleic Acid Diagnostics ...................................... 6204.8. DNA-Probe Based Detection Methods ...................................................................................... 620

5. Conclusions and Future Perspectives ................................................................................................ 6215.1. Emerging Nucleic Acid Diagnostic Technologies for Food-Borne Pathogen Detection ......... 621

5.1.1. Biosensors ........................................................................................................................ 6215.1.2. Microarrays ...................................................................................................................... 622

References.......................................................................................................................................... 623

23. Oligonucleotide and DNA Microarrays: Versatile Tools for Rapid BacterialDiagnostics

Tanja Kostic, Patrice Francois, Levente Bodrossy and Jacques Schrenzel

1. Introduction ........................................................................................................................................ 6292. Microarray Technology ..................................................................................................................... 6303. Technical Aspects of Microarray Technology.................................................................................. 632

3.1. Probes.......................................................................................................................................... 6323.1.1. Genome Fragments .......................................................................................................... 632

Contents xix

3.1.2. PCR Products ................................................................................................................... 6323.1.3. Oligonucleotide Probes.................................................................................................... 632

3.2. Substrates for Printing................................................................................................................ 6343.2.1. Slides with Poly-L-Lysine Coating ................................................................................. 6343.2.2. Slides with Amino Silane Coating .................................................................................. 6353.2.3. Slides with Aldehyde Coating......................................................................................... 6353.2.4. Slides with Epoxy Coating .............................................................................................. 6353.2.5. Proprietary Surface Chemistries ...................................................................................... 6363.2.6. Probe Spacers................................................................................................................... 636

3.3. Targets for Microarray Analysis ................................................................................................ 6373.3.1. Target Amplifications and Sensitivity Issues.................................................................. 6373.3.2. Labeling of the Targets.................................................................................................... 6383.3.3. Hybridization and Wash Conditions ............................................................................... 638

3.4. Classical Commercially-Available Microarray Formats ........................................................... 6393.4.1. Spotting Approaches........................................................................................................ 6393.4.2. In Situ Synthesis .............................................................................................................. 639

3.5. Alternative Methods for Improving Microarray-Based Detection Sensitivity.......................... 6413.5.1. Resonance-Light Scattering (RLS).................................................................................. 6413.5.2. Planar-Waveguide Technology (PWT) ........................................................................... 6413.5.3. Liquid Arrays ................................................................................................................... 6413.5.4. Three-Dimensional Microarray Formats ......................................................................... 642

3.6. Marker Genes Used on Microbial Diagnostic Microarrays (MDMs)....................................... 6434. Analysis and QC Aspects .................................................................................................................. 6435. Applications of Microarray Technology in Microbial Diagnostics.................................................. 644

5.1. Gene Expression Studies ............................................................................................................ 6445.2. Comparative Genome Hybridizations (CGH)............................................................................ 6455.3. Generic or Universal Microarrays.............................................................................................. 6465.4. Microarrays for Sequence Analysis ........................................................................................... 6475.5. Microbial Diagnostic Microarrays ............................................................................................. 648

6. Conclusions ........................................................................................................................................ 649References.......................................................................................................................................... 649

24. Pathogenic Bacterial Sensors Based on Carbohydrates as SensingElements

Haiying Liu

1. Introduction ........................................................................................................................................ 6602. Bacterial Surface Lectins................................................................................................................... 6613. Surface Carbohydrate Structures of Pathogenic Bacteria................................................................. 6644. Carbohydrate Microarrays for Detection of Bacteria ....................................................................... 6685. Lectin Microarrays for Detection of Bacteria................................................................................... 6706. Conjugated Fluorescent Glycopolymers for Detection of Bacteria.................................................. 6727. Glyconanoparticles for Detection of Bacteria................................................................................... 6768. Carbohydrate-Functionalized Carbon Nanotubes for Detection of Bacteria.................................... 6789. Conclusions and Future Perspectives ................................................................................................ 680

References.......................................................................................................................................... 681

25. Aptamers and Their Potential as Recognition Elements for the Detectionof Bacteria

Casey C. Fowler, Naveen K. Navani, Eric D. Brown and Yingfu Li

1. Functional Nucleic Acids .................................................................................................................. 6891.1. Properties of Nucleic Acids ....................................................................................................... 6901.2. Synthesizing, Sequencing and Modifying Nucleic Acids ......................................................... 692

xx Contents

1.2.1. DNA Polymerase and Polymerase Chain Reaction ........................................................ 6921.2.2. RNA Polymerase and In Vitro Transcription ................................................................. 6921.2.3. Reverse Transcription ...................................................................................................... 6931.2.4. Other Modifications ......................................................................................................... 693

2. Isolation of Functional Nucleic Acids............................................................................................... 6942.1. Introduction to SELEX............................................................................................................... 6942.2. Selection Methods ...................................................................................................................... 694

2.2.1. Bead and Column Based Selections................................................................................ 6962.2.2. Polyacrylamide Gel Electrophoresis (PAGE) Based Selections .................................... 6962.2.3. Capillary Electrophoresis (CE) Based Selections ........................................................... 697

2.3. Optimizing Functional Nucleic Acids........................................................................................ 6973. Aptamers: Properties and Targets ..................................................................................................... 697

3.1. The Growing Aptamer Catalogue .............................................................................................. 6983.2. Aptamer Specificity.................................................................................................................... 6983.3. AptamerLigand Interactions ..................................................................................................... 7003.4. Aptamers vs. Other Recognition Elements................................................................................ 700

4. Applications of Aptamers .................................................................................................................. 7014.1. Aptamers for Purification........................................................................................................... 7014.2. Aptamers with Therapeutic Potential......................................................................................... 7024.3. Aptamers as Sensing Elements .................................................................................................. 702

4.3.1. Conformation-Dependent Fluorescent Sensors ............................................................... 7034.3.2. Quantum Dot Sensors ...................................................................................................... 7034.3.3. Target Detection by Fluorescence Anisotropy................................................................ 7044.3.4. Enzyme Linked Aptamer Assays .................................................................................... 7054.3.5. Acoustic Sensors.............................................................................................................. 7054.3.6. Electrochemical Sensors .................................................................................................. 706

5. Aptamers for Detection of Pathogenic Bacteria ............................................................................... 7065.1. Categories of Microbial Agents to be Detected ........................................................................ 707

5.1.1. Gram-Positive Bacteria.................................................................................................... 7075.1.2. Gram-Negative Bacteria .................................................................................................. 708

5.2. Traditional Pathogen Detection Methods .................................................................................. 7085.3. Aptamers in Pathogen Detection................................................................................................ 709

6. Conclusions ........................................................................................................................................ 710References.......................................................................................................................................... 710

26. Protein Microarray Technologies for Detection and Identificationof Bacterial and Protein Analytes

Christer Wingren and Carl AK Borrebaeck

1. Introduction ........................................................................................................................................ 7151.1. Definition and Classification of Protein Microarrays................................................................ 7161.2. Functional Protein Microarrays........................

principles of bacterial detection: biosensors, recognition receptors and microsystems

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