Preface to Second Edition xix

Preface to First Edition xxi

Acknowledgments xxiii

1. Quantitative Instrumental Measurements 1

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

2.1. Introduction 23

2.2. Total Protein 24

2.3. Total DNA 31

2.4. Total RNA 34

2.5. Total Carbohydrate 35

2.6. Free Fatty Acids 37

References 38

Problems 39

3. Enzymes 41

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 67

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 90

5.1. Introduction 90

5.2. Immobilization Methods 90

5.3. Properties of Immobilized Enzymes 101

5.4. Immobilized Enzyme Reactors 107

5.5. Theoretical Treatment of Packed-Bed Enzyme Reactors 109

Suggested Reading 113

References 113

Problems 114

6. Antibodies 117

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 131

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 166

8.1. Introduction 166

8.2. Biosensor Diversity and Classification 169

8.3. Recognition Agents 171

8.4. Response of Enzyme-Based Biosensors 175

8.5. Examples of Biosensor Configurations 178

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 229

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 244

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 270

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

13.1. Introduction 290

13.2. Carrier Ampholytes 291

13.3. Modern IEF with Carrier Ampholytes 293

13.4. Immobilized pH Gradients (IPGs) 296

13.5. Two-Dimensional Electrophoresis 299

13.6. Difference Gel Electrophoresis (DIGE) 301

Suggested Reading 303

References 303

Problems 304

14. Capillary Electrophoresis 306

14.1. Introduction 306

14.2. Electroosmosis 307

14.3. Elution of Sample Components 308

14.4. Sample Introduction 309

14.5. Detectors for Capillary Electrophoresis 310

14.6. Capillary Polyacrylamide Gel Electrophoresis (C-PAGE) 319

14.7. Capillary Isoelectric Focusing (CIEF) 321

Suggested Reading 322

References 323

Problems 323

15. Centrifugation Methods 325

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 349

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 377

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 393

17.7. Nucleic Acid Applications 397

17.8. Bacterial Mass Spectrometry 398

17.9. Mass Spectrometry Imaging 399

Suggested Reading 401

References 401

Problems 402

18. Micro-TAS, Lab-on-a-Chip, and Microarray Devices 404

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 414

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