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
1.2.1. UV-Visible Absorbance 3
1.2.2. Turbidimetry (Light-Scattering) 5
1.2.3. Fluorescence 5
1.2.4. Chemiluminescence and Bioluminescence 7
1.3. Electrochemical Measurements 8
1.3.1. Potentiometry 10
1.3.2. Amperometry 10
1.3.3. Impedimetry 11
1.4. Radiochemical Measurements 12
1.4.1. Scintillation Counting 12
1.4.2. Geiger Counting 12
1.5. Surface Plasmon Resonance 13
1.6. Calorimetry 14
1.6.1. Differential Scanning Calorimetry (DSC) 15
1.6.2. Isothermal Titration Calorimetry (ITC) 16
1.7. Automation: Microplates, Multiwell Liquid Dispensers and Microplate Readers 16
1.8. Calibration of Instrumental Measurements 18
1.8.1. External Standards 18
1.8.2. Internal Standards 19
1.8.3. Standard Additions 20
1.9. Quantitative and Semi-Quantitative Measurements 21
1.9.1. Exact Concentration 21
1.9.2. Positive or Negative Result 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
2.2.1. Lowry Method 24
2.2.2. Smith (BCA) Method 24
2.2.3. Bradford Method 26
2.2.4. Ninhydrin-Based Assay 27
2.2.5. Other Protein Quantitation Methods 28
2.3. Total DNA 31
2.3.1. Diaminobenzoic Acid (DABA) Method 32
2.3.2. Diphenylamine (DPA) Method 32
2.3.3. Other Fluorimetric Methods 33
2.4. Total RNA 34
2.5. Total Carbohydrate 35
2.5.1. Ferricyanide Method 35
2.5.2. Phenol-Sulfuric Acid Method 36
2.5.3. 2-Aminothiophenol Method 36
2.5.4. Purpald Assay for Bacterial Polysaccharides 37
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
3.5.1. Simple One-Substrate Enzyme Kinetics 48
3.5.2. Experimental Determination of Michaelis-Menten Parameters 50
3.5.2.1. Eadie-Hofstee Method 50
3.5.2.2. Hanes Method 50
3.5.2.3. Lineweaver-Burk Method 51
3.5.2.4. Cornish-Bowden-Eisenthal Method 52
3.5.3. Comparison of Methods for the Determination of KM Values 52
3.5.4. One-Substrate, Two-Product Enzyme Kinetics 54
3.5.5. Two-Substrate Enzyme Kinetics 54
3.5.6. Examples of Enzyme-Catalyzed Reactions and their Treatment 56
3.5.7. Curve Fitting for Enzyme Kinetic Data 57
3.6. Enzyme Activators 58
3.7. Enzyme Inhibitors 59
3.7.1. Competitive Inhibition 60
3.7.2. Noncompetitive Inhibition 60
3.7.3. Uncompetitive Inhibition 62
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
4.5.1. Optical Detection 73
4.5.1.1. Absorbance 73
4.5.1.2. Fluorescence 75
4.5.1.3. Luminescence 77
4.5.1.4. Nephelometry 79
4.5.2. Electrochemical Detection 79
4.5.2.1. Amperometry 79
4.5.2.2. Potentiometry 80
4.5.2.3. Conductimetry 80
4.5.3. Other Detection Methods 81
4.5.3.1. Radiochemical 81
4.5.3.2. Manometry 81
4.5.3.3. Calorimetry 82
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
5.2.1. Nonpolymerizing Covalent Immobilization 91
5.2.1.1. Controlled-Pore Glass 92
5.2.1.2. Polysaccharides 93
5.2.1.3. Polyacrylamide 95
5.2.1.4. Acidic Supports 95
5.2.1.5. Anhydride Groups 96
5.2.1.6. Thiol Groups 97
5.2.2. Crosslinking with Bifunctional Reagents 97
5.2.3. Adsorption 98
5.2.4. Entrapment 99
5.2.5. Microencapsulation 100
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
6.5.1. Agglutination Reactions 124
6.5.2. Precipitation Reactions 126
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
7.3.1. Labeled-Antibody Methods 136
7.3.2. Labeled-Ligand Assays 136
7.3.3. Radioisotopes 139
7.3.4. Fluorophores 139
7.3.4.1. Indirect Fluorescence 140
7.3.4.2. Competitive Fluorescence 140
7.3.4.3. Sandwich Fluorescence 140
7.3.4.4. Fluorescence Excitation Transfer 140
7.3.4.5. Time-Resolved Fluorescence 141
7.3.5. Quantum Dots 142
7.3.6. Chemiluminescent Labels 143
7.3.7. Enzyme Labels 145
7.3.8. Lateral Flow Immunoassay 148
7.4. Homogeneous Immunoassays 149
7.4.1. Fluorescent Labels 149
7.4.1.1. Enhancement Fluorescence 149
7.4.1.2. Direct Quenching Fluorescence 150
7.4.1.3. Indirect Quenching Fluorescence 150
7.4.1.4. Fluorescence Polarization Immunoassay 151
7.4.1.5. Fluorescence Excitation Transfer 151
7.4.2. Enzyme Labels 152
7.4.2.1. Enzyme-Multiplied Immunoassay Technique 152
7.4.2.2. Substrate-Labelled Fluorescein Immunoassay 153
7.4.2.3. Apoenzyme Reactivation Immunoassay 153
7.4.2.4. Cloned Enzyme Donor Immunoassay 154
7.4.2.5. Enzyme Inhibitory Homogeneous Immunoassay 154
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
8.3.1. Natural Recognition Agents 171
8.3.2. Artificial Recognition Agents 172
8.4. Response of Enzyme-Based Biosensors 175
8.5. Examples of Biosensor Configurations 178
8.5.1. Ferrocene-Mediated Amperometric Glucose Sensor 178
8.5.2. Potentiometric Biosensor for Phenyl Acetate 180
8.5.3. Evanescent-Wave Fluorescence Biosensor for Bungarotoxin 181
8.5.4. Optical Biosensor for Glucose Based on Fluorescence Resonance Energy Transfer 183
8.5.5. Piezoelectric Sensor for Nucleic Acid Detection 184
8.5.6. Enzyme Thermistors 186
8.5.7. Fluorescence Sensor for Nitroaromatic Explosives Based on a Molecularly Imprinted Polymer 187
8.5.8. Immunosensor Microwell Arrays from Gold Compact Disks 188
8.5.9. Nanoparticle-Enhanced Detection of Thrombin by SPR 190
8.5.10. Environmental BOD and Toxicity Biosensors Based on Viable Cells 192
8.5.11. Detection of Viruses using a Surface Acoustic Wave (SAW) Biosensor 193
8.5.12. MEMS Microcantilever Biosensor for Virus Detection 196
8.5.13. DNA Microarrays 198
8.6. Evaluation of Biosensor Perfomance 201
8.7. In Vivo Applications of Biosensors 202
8.7.1. Biocompatible Materials 203
8.7.2. Physiological Environment of the Human Body 203
8.7.3. The Artificial Pancreas 205
8.7.4. An Enzymatic Fuel Cell as a Component of an Implanted Biosensing System 205
8.7.5. Other Examples of Implantable Biosensors 206
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
9.3.1. Polymerase Chain Reaction and Error-Prone PCR 215
9.3.2. DNA Shuffling 217
9.4. Linking Genotype and Phenotype 217
9.4.1. Cell Expression and Cell Surface Display (In vivo) 218
9.4.2. Phage Display (In vivo) 218
9.4.3. Ribosome Display (In vitro) 219
9.4.4. mRNA-Peptide Fusion (In vitro) 220
9.4.5. Microcompartmentalization (In vitro) 220
9.5. Identification and Selection of Successful Variants 221
9.5.1. Identification of Successful Variants Based on Binding Properties 222
9.5.2. Identification of Successful Variants Based on Catalytic Activity 222
9.6. Examples of Directed Evolution Experiments 224
9.6.1. Directed Evolution of Galactose Oxidase 224
9.6.2. -Hemolysin Evolution 225
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
10.3.1. The Compound Light Microscope 231
10.3.2. The Confocal Microscope 231
10.3.3. Sample Preparation 232
10.3.4. General and Selective Stains 233
10.3.5. Fluorescence In situ Hybridization 234
10.3.6. Green Fluorescent Protein and its Analogues 234
10.4. Electron Microscopy 234
10.4.1. Principles and Instrumentation 234
10.4.2. Sample Preparation 235
10.4.3. Transmission Electron Microscopy (TEM) 236
10.4.4. Scanning Electron Microscopy (SEM) 236
10.5. Scanning Tunneling Microscopy 237
10.5.1. Principles and Instrumentation 237
10.5.2. Biological Applications 237
10.6. Atomic Force Microscopy (AFM) 237
10.6.1. Cantilevers and Operational Modes 237
10.6.2. Samples and Substrates 239
10.6.3. Biological Applications 239
10.6.4. Four-Dimensional (4D) Scanning 240
10.7. Scanning Electrochemical Microscopy (SECM) 240
10.7.1. Principles and Instrumentation 240
10.7.2. Samples and Substrates 241
10.7.3. Biological Applications 241
Suggested Reading 242
References 242
Problems 243
11. Principles of Electrophoresis 244
11.1. Introduction 244
11.2. Electrophoretic Support Media 248
11.2.1. Paper 248
11.2.2. Starch Gels 249
11.2.3. Polyacrylamide Gels 250
11.2.4. Agarose Gels 254
11.2.5. Polyacrylamide-Agarose Gels 254
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
11.6.1. Stains and Dyes 258
11.6.2. Detection of Enzymes by Substrate Staining 260
11.6.3. The Southern Blot 260
11.6.4. The Northern Blot 262
11.6.5. The Western Blot 262
11.6.6. Detection of DNA Fragments on Membranes with DNA Probes 263
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
14.5.1. Laser-Induced Fluorescence Detection 311
14.5.2. Mass Spectrometric Detection 313
14.5.3. Amperometric Detection 315
14.5.4. Radiochemical Detection 318
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
15.3.1. Swinging-Bucket Rotors 327
15.3.2. Fixed-Angle Rotors 328
15.3.3. Vertical Rotors 328
15.4. Clearing Factor (K) 329
15.5. Density Gradients 330
15.5.1. Materials Used to Generate a Gradient 331
15.5.2. Constructing Pre-Formed and Self-Generated Gradients 331
15.5.3. Redistribution of the Gradient in Fixed-Angle and Vertical Rotors 333
15.6. Types of Centrifugation Techniques 333
15.6.1. Differential Centrifugation 334
15.6.2. Rate-Zonal Centrifugation 334
15.6.3. Isopycnic Centrifugation 336
15.7. Harvesting Samples 336
15.8. Analytical Ultracentrifugation 336
15.8.1. Instrumentation 337
15.8.2. Sedimentation Velocity Analysis 338
15.8.3. Sedimentation Equilibrium Analysis 341
15.9. Selected Examples 342
15.9.1. Analytical Ultracentrifugation for Quaternary Structure Elucidation 342
15.9.2. Isolation of Retroviruses by Self-Generated Gradients 343
15.9.3. Isolation of Lipoproteins from Human Plasma 344
15.9.4. Centrifugal Microfluidic Analysis 344
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
16.6.1. Particulate Gels 358
16.6.2. Monolithic Stationary Phases 360
16.7. Affinity Chromatography 360
16.7.1. Immobilization of Affinity Ligands 362
16.7.2. Elution Methods 364
16.7.3. Determination of Association Constants by High Performance Affinity Chromatography 364
16.8. Ion-exchange Chromatography 368
16.8.1. Retention Model for Ion-Exchange Chromatography of Polyelectrolytes 369
16.8.2. Further Advances in Ion-Exchange Chromatography 374
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
17.2.1. Soft Ionization Sources 379
17.2.1.1. Fast Atom/Ion Bombardment (FAB) 380
17.2.1.2. Electrospray Ionization (ESI) 380
17.2.1.3. Matrix-Assisted Laser Desorption/Ionization (MALDI) 381
17.2.2. Mass Analyzers 382
17.2.3. Detectors 385
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
18.3.1. Fluid Transport 405
18.3.2. Valves and Reservoirs 406
18.3.3. Mixing and Sample Separation 406
18.4. Detectors 407
18.5. Examples of Bioanalytical Devices 407
18.5.1. DNA Separation Using a Nanofence Array Microfluidic Device 408
18.5.2. Two Dimensional Electrophoresis on a Microfluidic Chip 409
18.5.3. Microfluidic Antibody Capture for Single-Cell Proteomics 410
18.5.4. Multiplexed PCR Amplification and DNA Detection on a Microfluidic Chip 410
18.5.5. Silicone Protein Separation Chip Based on a Grafted Ion-Exchange Polymer 411
18.5.6. Circular, Biofunctionalized PEG Microchannels for Cell Adhesion Studies 411
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
19.4.1. Distribution of Errors and Confidence Limits 418
19.4.2. Linear Regression and Calibration 419
19.4.3. Precision Profiles 420
19.4.4. Limit of Quantitiation and Detection 421
19.4.5. Linearizing Sigmoidal Curves (Four-Parameter Log-Logit Model) 422
19.4.6. Effective Dose Method 423
19.5. Estimation of Accuracy 424
19.5.1. Standardization 424
19.5.2. Matrix Effects 425
19.5.2.1. Recovery 425
19.5.2.2. Parallelism 426
19.5.3. Interferences 426
19.6. Qualitative (Screening) Assays 427
19.6.1. Figures of Merit for Qualitative (Screening) Assays 427
19.7. Examples of Validation Procedures 428
19.7.1. Validation of a Qualitative Antibiotic Susceptibility Test 428
19.7.2. Measurement of Plasma Homocysteine by Fluorescence Polarization Immunoassay (FPIA) Methodology 429
19.7.3. Determination of Enzymatic Activity of -Galactosidase 433
19.7.4. Establishment of a Cutoff Value for Semi-Quantitative Assays for Cannabinoids 434
Suggested Reading 435
References 436
Answers to Selected Problems 437
Index 449
