Bioanalytical Chemistry

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