Bioanalytical chemistry / Susan R. Mikkelsen, Eduardo Corton. -- 2nd ed. -- Hoboken, New Jersey : John Wiley & Sons, Inc., c2016. – (58.1734/M636/2nd ed.)

Contents

Preface to Second Edition

Preface to First Edition

Acknowledgments

1. Quantitative Instrumental Measurements

1.1. Introduction    1

1.2. Optical Measurements    2

1.3. Electrochemical Measurements    8

1.4. Radiochemical Measurements    12

1.5. Surface Plasmon Resonance    13

1.6. Calorimetry    14

1.7. Automation: Microplates, Multiwell Liquid Dispensers and Microplate Readers    16

1.8. Calibration of Instrumental Measurements    18

1.9. Quantitative and Semi-Quantitative Measurements    21

Suggested Reading    22

Problems    22

2. Spectroscopic Methods for the Quantitation of Classes of Biomolecules  23

3.1. Introduction    41

3.2. Enzyme Nomenclature    42

3.3. Enzyme Commission Numbers    43

3.4. Enzymes in Bioanalytical Chemistry    45

3.5. Enzyme Kinetics    46

3.6. Enzyme Activators    58

3.7. Enzyme Inhibitors    59

3.8. Enzyme Units and Concentrations    62

Suggested Reading    64

References    64

Problems    64

4. Quantitation of Enzymes and Their Substrates

4.1. Introduction    67

4.2. Substrate Depletion or Product Accumulation    68

4.3. Direct and Coupled Measurements    69

4.4. Classification of Methods    71

4.5. Instrumental Methods    73

4.6. High-Throughput Assays for Enzymes and Inhibitors    82

4.7. Assays for Enzymatic Reporter Gene Products    84

4.8. Practical Considerations for Enzymatic Assays    85

Suggested Reading    86

References    86

Problems    87

5. Immobilized Enzymes

6. Antibodies

6.1. Introduction    117

6.2. Structural and Functional Properties of Antibodies    118

6.3. Polyclonal and Monoclonal Antibodies    121

6.4. Antibody-Antigen Interactions    122

6.5. Analytical Applications of Secondary Antibody-Antigen Interactions    124

Suggested Reading    129

References    129

Problems    129

7. Quantitative Immunoassays with Labels

7.1. Introduction    131

7.2. Labeling Reactions    132

7.3. Heterogeneous Immunoassays    134

7.4. Homogeneous Immunoassays    149

7.5. Evaluation of New Immunoassay Methods    155

Suggested Reading    160

References    160

Problems    161

8. Biosensors

8.1. Introduction    166

8.2. Biosensor Diversity and Classification    169

8.3. Recognition Agents    171

8.4. Response of Enzyme-Based Biosensors

8.5. Examples of Biosensor Configurations

8.6. Evaluation of Biosensor Perfomance    201

8.7. In Vivo Applications of Biosensors    202

Suggested Reading    207

References    207

Problems    209

9. Directed Evolution for the Design of Macromolecular Reagents        210

9.1. Introduction    210

9.2. Rational Design and Directed Evolution    211

9.3. Generation of Genetic Diversity    214

9.4. Linking Genotype and Phenotype    217

9.5. Identification and Selection of Successful Variants    221

9.6. Examples of Directed Evolution Experiments    224

Suggested Reading    226

References    226

Problems    227

10. Image-Based Bioanalysis

10.1. Introduction    229

10.2. Magnification and Resolution    230

10.3. Optical Microscopy    231

10.4. Electron Microscopy    234

10.5. Scanning Tunneling Microscopy    237

10.6. Atomic Force Microscopy (AFM)    237

10.7. Scanning Electrochemical Microscopy (SECM)    240

Suggested Reading    242

References    242

Problems    243

11. Principles of Electrophoresis

11.1. Introduction    244

11.2. Electrophoretic Support Media    248

11.3. Effect of Experimental Conditions Onelectrophoretic Separations    254

11.4. Electric Field Strength Gradients    255

11.5. Pulsed Field Gel Electrophoresis (PFGE)    256

11.6. Detection of Proteins and Nucleic Acids After Electrophoretic Separation 258

Suggested Reading    265

References    266

Problems    266

12. Applications of Zone Electrophoresis                               268

12.1. Introduction    268

12.2. Determination of Protein Net Charge and Molecular Weight Using PAGE   268

12.3. Determination of Protein Subunit Composition and Subunit Molecular Weights

12.4. Molecular Weight of DNA by Agarose Gel Electrophoresis    272

12.5. Identification of Isoenzymes    273

12.6. Diagnosis of Genetic (Inherited) Disorders    274

12.7. DNA Fingerprinting and Restriction Fragment Length Polymorphism    275

12.8. DNA Sequencing with the Maxam-Gilbert Method    279

12.9. Immunoelectrophoresis    282

Suggested Reading    287

References    287

Problems    288

13. Isoelectric Focusing and 2D Electrophoresis                         290

14. Capillary Electrophoresis

15.1. Introduction    325

15.2. Sedimentation and Relative Centrifugal g Force    325

15.3. Centrifugal Forces in Different Rotor Types    327

15.4. Clearing Factor (K)    329

15.5. Density Gradients    330

15.6. Types of Centrifugation Techniques    333

15.7. Harvesting Samples    336

15.8. Analytical Ultracentrifugation    336

15.9. Selected Examples    342

Suggested Reading    346

References    346

Problems    347

16. Chromatography of Biomolecules

16.1. Introduction    349

16.2. Units and Definitions    350

16.3. Plate Theory of Chromatography    350

16.4. Rate Theory of Chromatography    351

16.5. Size Exclusion (Gel Filtration) Chromatography    353

16.6. Stationary Phases For Size Exclusion Chromatography    358

16.7. Affinity Chromatography    360

16.8. Ion-exchange Chromatography    368

Suggested Reading    374

References    374

Problems    375

17. Mass Spectrometry of Biomolecules

17.1. Introduction    377

17.2. Basic Description of the Instrumentation    379

17.3. Interpretation of Mass Spectra    386

17.4. Biomolecule Molecular Weight Determination    388

17.5. Protein Identification    392

17.6. Protein-Peptide Sequencing

17.7. Nucleic Acid Applications

17.8. Bacterial Mass Spectrometry

17.9. Mass Spectrometry Imaging

Suggested Reading    401

References    401

Problems    402

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

18.1. Introduction    404

18.2. Device Fabrication Materials and Methods    405

18.3. Microfluidics    405

18.4. Detectors    407

18.5. Examples of Bioanalytical Devices    407

Suggested Reading    412

References    412

Problems    413

19. Validation of New Bioanalytical Methods

19.1. Introduction    414

19.2. Precision and Accuracy    415

19.3. Mean and Variance    416

19.4. Relative Standard Deviation and Other Precision Estimators    417

19.5. Estimation of Accuracy    424

19.6. Qualitative (Screening) Assays    427

19.7. Examples of Validation Procedures    428

Suggested Reading    435

References    436

Answers to Selected Problems    437

Index    449