Contents

Cover

Title Page

Preface to Second Edition

Preface to First Edition

Acknowledgments

1: Quantitative Instrumental Measurements

1.1 Introduction
1.2 Optical Measurements
1.3 Electrochemical Measurements
1.4 Radiochemical Measurements
1.5 Surface Plasmon Resonance
1.6 Calorimetry
1.7 Automation: Microplates, Multiwell Liquid Dispensers and Microplate Readers
1.8 Calibration of Instrumental Measurements
1.9 Quantitative and Semi-Quantitative Measurements
Suggested Reading
Problems

2: Spectroscopic Methods for the Quantitation of Classes of Biomolecules

2.1 Introduction
2.2 Total Protein
2.3 Total DNA
2.4 Total RNA
2.5 Total Carbohydrate
2.6 Free Fatty Acids
References
Problems

3: Enzymes

3.1 Introduction
3.2 Enzyme Nomenclature
3.3 Enzyme Commission Numbers
3.4 Enzymes in Bioanalytical Chemistry
3.5 Enzyme Kinetics
3.6 Enzyme Activators
3.7 Enzyme Inhibitors
3.8 Enzyme Units and Concentrations
Suggested Reading
References
Problems

4: Quantitation of Enzymes and Their Substrates

4.1 Introduction
4.2 Substrate Depletion or Product Accumulation
4.3 Direct and Coupled Measurements
4.4 Classification of Methods
4.5 Instrumental Methods
4.6 High-Throughput Assays for Enzymes and Inhibitors
4.7. Assays for Enzymatic Reporter Gene Products27-30
4.8 Practical Considerations for Enzymatic Assays
Suggested Reading
References
Problems

5: Immobilized Enzymes

5.1 Introduction
5.2 Immobilization Methods
5.3 Properties of Immobilized Enzymes
5.4 Immobilized Enzyme Reactors
5.5 Theoretical Treatment of Packed-Bed Enzyme Reactors34-37
Suggested Reading
References
Problems

6: Antibodies

6.1 Introduction
6.2 Structural and Functional Properties of Antibodies
6.3 Polyclonal and Monoclonal Antibodies
6.4 Antibody-Antigen Interactions
6.5 Analytical Applications of Secondary Antibody-Antigen Interactions
Suggested Reading
References
Problems

7: Quantitative Immunoassays with Labels

7.1 Introduction
7.2 Labeling Reactions
7.3 Heterogeneous Immunoassays
7.4 Homogeneous Immunoassays
7.5 Evaluation of New Immunoassay Methods33,34
Suggested Reading
References
Problems

8: Biosensors

8.1 Introduction
8.2 Biosensor Diversity and Classification
8.3 Recognition Agents
8.4 Response of Enzyme-Based Biosensors5
8.5 Examples of Biosensor Configurations
8.6 Evaluation of Biosensor Perfomance
8.7 In VIVO Applications of Biosensors
Suggested Reading
References
Problems

9: Directed Evolution for the Design of Macromolecular Reagents

9.1 Introduction
9.2 Rational Design and Directed Evolution
9.3 Generation of Genetic Diversity
9.4 Linking Genotype and Phenotype
9.5 Identification and Selection of Successful Variants
9.6 Examples of Directed Evolution Experiments
Suggested Reading
References
Problems

10: Image-Based Bioanalysis

10.1 Introduction
10.2 Magnification and Resolution
10.3 Optical Microscopy
10.4 Electron Microscopy
10.5 Scanning Tunneling Microscopy
10.6 Atomic Force Microscopy (AFM)
10.7 Scanning Electrochemical Microscopy (SECM)
Suggested Reading
References
Problems

11: Principles of Electrophoresis

11.1 Introduction
11.2 Electrophoretic Support Media
11.3 Effect Of Experimental Conditions Onelectrophoretic Separations
11.4 Electric Field Strength Gradients
11.5 Pulsed Field Gel Electrophoresis (PFGE)
11.6 Detection of Proteins and Nucleic Acids After Electrophoretic Separation
Suggested Reading
References
Problems

12: Applications of Zone Electrophoresis

12.1 Introduction
12.2 Determination of Protein Net Charge and Molecular Weight Using Page1
12.3 Determination of Protein Subunit Composition and Subunit Molecular Weights2
12.4 Molecular Weight of DNA By Agarose Gel Electrophoresis5
12.5 Identification of Isoenzymes
12.6 Diagnosis of Genetic (Inherited) Disorders
12.7 DNA Fingerprinting and Restriction Fragment Length Polymorphism10
12.8 DNA Sequencing With the Maxam-Gilbert Method14
12.9 Immunoelectrophoresis16
Suggested Reading
References
Problems

13: Isoelectric Focusing and 2D Electrophoresis

13.1 Introduction
13.2 Carrier Ampholytes
13.3 Modern IEF With Carrier Ampholytes
13.4 Immobilized pH Gradients (IPGs)9
13.5 Two-Dimensional Electrophoresis12
13.6 Difference Gel Electrophoresis (DIGE)
Suggested Reading
References
Problems

14: Capillary Electrophoresis

14.1 Introduction
14.2 Electroosmosis
14.3 Elution of Sample Components
14.4 Sample Introduction2
14.5 Detectors for Capillary Electrophoresis
14.6 Capillary Polyacrylamide Gel Electrophoresis (C-PAGE)12
14.7 Capillary Isoelectric Focusing (CIEF)14
Suggested Reading
References
Problems

15: Centrifugation Methods

15.1 Introduction
15.2 Sedimentation and Relative Centrifugal g Force
15.3 Centrifugal Forces in Different Rotor Types
15.4 Clearing Factor (k)
15.5 Density Gradients
15.6 Types of Centrifugation Techniques
15.7 Harvesting Samples
15.8 Analytical Ultracentrifugation
15.9 Selected Examples
Suggested Reading
References
Problems

16: Chromatography of Biomolecules

16.1 Introduction
16.2 Units and Definitions
16.3 Plate Theory of Chromatography1
16.4 Rate Theory of Chromatography2
16.5 Size Exclusion (Gel Filtration) Chromatography
16.6 Stationary Phases for Size Exclusion Chromatography
16.7 Affinity Chromatography
16.8 Ion-Exchange Chromatography
Suggested Reading
References
Problems

17: Mass Spectrometry of Biomolecules

17.1 Introduction
17.2 Basic Description of the Instrumentation
17.3 Interpretation of Mass Spectra
17.4 Biomolecule Molecular Weight Determination
17.5 Protein Identification
17.6 Protein-Peptide Sequencing
17.7 Nucleic Acid Applications
17.8 Bacterial Mass Spectrometry
17.9 Mass Spectrometry Imaging
Suggested Reading
References
Problems

18: Micro-TAS, Lab-on-a-Chip, and Microarray Devices

18.1 Introduction
18.2 Device Fabrication Materials and Methods
18.3 Microfluidics
18.4 Detectors
18.5 Examples of Bioanalytical Devices
Suggested Reading
References
Problems

19: Validation of New Bioanalytical Methods

19.1 Introduction
19.2 Precision and Accuracy
19.3 Mean and Variance
19.4 Relative Standard Deviation and Other Precision Estimators
19.5 Estimation of Accuracy
19.6 Qualitative (Screening) Assays
19.7 Examples of Validation Procedures
Suggested Reading
References

Answers to Selected Problems

Index

End User License Agreement

List of Tables

Table 2.1

Table 3.1

Table 3.2

Table 3.3

Table 3.4

Table 4.1

Table 5.1

Table 5.2

Table 6.1

Table 6.2

Table 7.1

Table 7.2

Table 8.1

Table 8.2

Table 9.1

Table 9.2

Table 9.3

Table 11.1

Table 11.2

Table 12.1

Table 12.2

Table 12.3

Table 12.4

Table 13.1

Table 13.2

Table 14.1

Table 14.2

Table 14.3

Table 15.1

Table 15.2

Table 16.1

Table 16.2

Table 16.3

Table 16.4

Table 16.5

Table 16.6

Table 16.7

Table 16.8

Table 17.1

Table 17.2

Table 17.3

Table 17.4

Table 17.5

Table 17.6

Table 17.7

Table 17.8

Table 17.9

Table 17.10

Table 19.1

Table 19.2

Table 19.3

Table 19.4

Table 19.5

Table 19.6

List of Illustrations

Figure 1.1

Figure 1.2

Figure 1.3

Figure 2.1

Figure 2.2

Figure 2.3

Figure 2.4

Figure 2.5

Figure 2.6

Figure 2.7

Figure 2.8

Figure 2.9

Figure 3.1

Figure 3.2

Figure 3.3

Figure 3.4

Figure 3.5

Figure 3.6

Figure 3.7

Figure 3.8

Figure 3.9

Figure 3.10

Figure 3.11

Figure 3.12

Figure 3.13

Figure 3.14

Figure 3.15

Figure 4.1

Figure 4.2

Figure 4.3

Figure 4.4

Figure 4.5

Figure 4.6

Figure 5.1

Figure 5.2

Figure 5.3

Figure 5.4

Figure 5.5

Figure 5.6

Figure 5.7

Figure 5.8

Figure 5.9

Figure 5.10

Figure 5.11

Figure 5.12

Figure 5.13

Figure 5.14

Figure 6.1

Figure 6.2

Figure 6.3

Figure 6.4

Figure 6.5

Figure 6.6

Figure 6.7

Figure 6.8

Figure 6.9

Figure 7.1

Figure 7.2

Figure 7.3

Figure 7.4

Figure 7.5

Figure 7.6

Figure 7.7

Figure 7.8

Figure 7.9

Figure 7.10

Figure 7.11

Figure 7.12

Figure 7.13

Figure 7.14

Figure 7.15

Figure 8.1

Figure 8.2

Figure 8.3

Figure 8.4

Figure 8.5

Figure 8.6

Figure 8.7

Figure 8.8

Figure 8.9

Figure 8.10

Figure 8.11

Figure 8.12

Figure 8.13

Figure 8.14

Figure 8.15

Figure 8.16

Figure 8.17

Figure 8.18

Figure 8.19

Figure 8.20

Figure 8.21

Figure 8.22

Figure 8.23

Figure 8.24

Figure 8.25

Figure 8.26

Figure 9.1

Figure 9.2

Figure 9.3

Figure 9.4

Figure 9.5

Figure 9.6

Figure 9.7

Figure 9.8

Figure 9.9

Figure 10.1

Figure 10.2

Figure 10.3

Figure 10.4

Figure 11.1

Figure 11.2

Figure 11.3

Figure 11.4

Figure 11.5

Figure 11.6

Figure 11.7

Figure 11.8

Figure 11.9

Figure 11.10

Figure 11.11

Figure 11.12

Figure 11.13

Figure 11.14

Figure 11.15

Figure 11.16

Figure 11.17

Figure 11.18

Figure 11.19

Figure 12.1

Figure 12.2

Figure 12.3

Figure 12.4

Figure 12.5

Figure 12.6

Figure 12.7

Figure 12.8

Figure 12.9

Figure 12.10

Figure 12.11

Figure 12.12

Figure 12.13

Figure 12.14

Figure 12.15

Figure 13.1

Figure 13.2

Figure 13.3

Figure 13.4

Figure 13.5

Figure 13.6

Figure 13.7

Figure 13.8

Figure 13.9

Figure 13.10

Figure 13.11

Figure 13.12

Figure 13.13

Figure 14.1

Figure 14.2

Figure 14.3

Figure 14.4

Figure 14.5

Figure 14.6

Figure 14.7

Figure 14.8

Figure 14.9

Figure 14.10

Figure 14.11

Figure 14.12

Figure 14.13

Figure 14.14

Figure 15.1

Figure 15.2

Figure 15.3

Figure 15.4

Figure 15.5

Figure 15.6

Figure 15.7

Figure 15.8

Figure 15.9

Figure 15.10

Figure 15.11

Figure 15.12

Figure 15.13

Figure 16.1

Figure 16.2

Figure 16.3

Figure 16.4

Figure 16.5

Figure 16.6

Figure 16.7

Figure 16.8

Figure 16.9

Figure 16.10

Figure 16.11

Figure 16.12

Figure 17.1

Figure 17.2

Figure 17.3

Figure 17.4

Figure 17.5

Figure 17.6

Figure 17.7

Figure 17.8

Figure 17.9

Figure 17.10

Figure 17.11

Figure 17.12

Figure 17.13

Figure 17.14

Figure 17.15

Figure 17.16

Figure 17.17

Figure 17.18

Figure 18.1

Figure 18.2

Figure 18.3

Figure 18.4

Figure 18.5

Figure 19.1

Figure 19.2

Figure 19.3

Figure 19.4

Figure 19.5

Figure 19.6

Figure 19.7

Figure 19.8

Figure 19.9

Published by John Wiley & Sons, Inc., Hoboken, New Jersey.

Published simultaneously in Canada.

No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except as permitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, (978) 750-8400, fax (978) 750-4470, or on the web at www.copyright.com. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, (201) 748-6011, fax (201) 748-6008, or online at http://www.wiley.com/go/permission.

Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. No warranty may be created or extended by sales representatives or written sales materials. The advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor author shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages.

For general information on our other products and services or for technical support, please contact our Customer Care Department within the United States at (800) 762-2974, outside the United States at (317) 572-3993 or fax (317) 572-4002.

Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic formats. For more information about Wiley products, visit our web site at www.wiley.com.

Library of Congress Cataloging-in-Publication Data:

Names: Mikkelsen, Susan R., 1960- | Cortón, Eduardo, 1962-Title: Bioanalytical chemistry / Susan R. Mikkelsen, Eduardo Cortón.

Description: Second edition. | Hoboken, New Jersey : John Wiley & Sons, Inc., [2016] | Includes index.

Identifiers: LCCN 2015041460 (print) | LCCN 2015043820 (ebook) | ISBN 9781118302545 (cloth) | ISBN 9781119057703 (pdf) | ISBN 9781119057635 (epub)

Subjects: LCSH: Analytical biochemistry. | Chemistry, Analytic.

Classification: LCC QP519.7 .M54 2016 (print) | LCC QP519.7 (ebook) | DDC 612/.01585–dc23

LC record available at http://lccn.loc.gov/2015041460

Preface to Second Edition

The success of our first edition, with reader feedback, has encouraged us to produce an expanded and updated second edition of Bioanalytical Chemistry. Over the past decade, we have both read, in the primary literature, about enormous advances in certain areas, including imaging and microfluidic devices. It had also been suggested that an introductory chapter involving instrumental measurement principles and methods would be helpful to readers. New chapters were warranted, and we have included these in our second edition.

Traditional areas of bioanalytical chemistry, such as electrophoresis, chromatography and immunoassay, have also seen surprising and significant developments. An enormous development in 2D electrophoresis, involving the simultaneous separation of two or more cell lysates on a single gel, has provided a quantum leap for proteomic studies. Nanoparticles, quantum dots and new polymers have seen many applications to biomolecule separation and quantitation. We have attempted to provide an introduction, as well as leading references, to these areas in our second edition.

The Argentine-Canadian collaboration of the authors began in the mid-1990s as a result of mutual interests in bioanalytical chemistry research and teaching. Over the years, despite the advantages of modern telecommunications technology, we have found that travel is both necessary and beneficial, especially when the final stages of manuscript preparation are underway. With the north-south travel, we have found that the climate is always pleasant somewhere.

Susan R. Mikkelsen
Eduardo Cortón
Buenos Aires, Argentina
August 2015

Preface to First Edition

The expanding role of bioanalytical chemistry in academic and industrial environments has made it important for students in chemistry and biochemistry to be introduced to this field during their undergraduate training. Upon introducing a bioanalytical chemistry course in 1990, I found that there was no suitable textbook that incorporated the diverse methods and applications in the depth appropriate to an advanced undergraduate course. Many specialized books and monographs exists that cover one or two topics in detail, and some of these are suggested at the end of each chapter as sources of further information.

This book is intended for use as a textbook by advanced undergraduate chemistry and biochemistry students, as well as bioanalytical chemistry graduate students. These students will have completed standard introductory analytical chemistry and biochemistry courses, as well as instrumental analysis. We have assumed familiarity with basic spectroscopic, electrochemical and chromatographic methods, as they apply to chemical analysis.

The subject material in each chapter has generally been organized as a progression from basic concepts to applications involving real samples. Mathematical descriptions and derivations have been limited to those that are believed essential for an understanding of each method, and are not intended to be comprehensive reviews. Problems given at the end of each chapter are included to allow students to assess their understanding of each topic; most of these problems have been used as examination questions by the authors.

As research in industrial, government and academic laboratories moves toward increasingly interdisciplinary programs, the authors hope that this book will be used to facilitate, and to prepare students for, collaborative scientific work.

Susan R. Mikkelsen
Eduardo Cortón
Waterloo, Canada
July 2003