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ggrk

至尊木虫 (职业作家)

理论与实践相结合

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专业: 生物、医学光子学

[交流] Bioanalytical chemistry-John Wiley & Sons 已有10人参与

Bioanalytical chemistry

CONTENTS
Preface xv
Acknowledgments xvii
1. Spectroscopic Methods for Matrix Characterization 1
1.1 Introduction 1
1.2 Total Protein 2
1.2.1 Lowry Method 3
1.2.2 Smith (BCA) Method 3
1.2.3 Bradford Method 4
1.2.4 Ninhydrin-Based Assay 5
1.2.5 Other Protein Quantitation Methods 5
1.3 Total DNA 7
1.3.1 Diaminobenzoic Acid Method 7
1.3.2 Diphenylamine Method 9
1.3.3 Other Fluorometric Methods 9
1.4 Total RNA 10
1.5 Total Carbohydrate 11
1.5.1 Ferricyanide Method 11
1.5.2 Phenol–Sulfuric Acid Method 11
1.5.3 2-Aminothiophenol Method 12
1.5.4 Purpald Assay for Bacterial Polysaccharides 12
1.6 Free Fatty Acids 13
References 14
Problems 15
2. Enzymes 16
2.1 Introduction 16
2.2 Enzyme Nomenclature 17
2.3 Enzyme Commission Numbers 18
2.4 Enzymes in Bioanalytical Chemistry 19
2.5 Enzyme Kinetics 21
2.5.1 Simple One-Substrate Enzyme Kinetics 23
2.5.2 Experimental Determination of Michaelis–Menten
Parameters 24
v
2.5.2.1 Eadie–Hofstee Method 25
2.5.2.2 Hanes Method 25
2.5.2.3 Lineweaver–Burk Method 26
2.5.2.4 Cornish–Bowden–Eisenthal Method 27
2.5.3 Comparison of Methods for the Determination
of Km Values 28
2.5.4 One-Substrate, Two-Product Enzyme Kinetics 29
2.5.5 Two-Substrate Enzyme Kinetics 29
2.5.6 Examples of Enzyme-Catalyzed Reactions and
Their Treatment 31
2.6 Enzyme Activators 32
2.7 Enzyme Inhibitors 33
2.7.1 Competitive Inhibition 34
2.7.2 Noncompetitive Inhibition 35
2.7.3 Uncompetitive Inhibition 35
2.8 Enzyme Units and Concentrations 36
Suggested References 38
References 38
Problems 38
3. Quantitation of Enzymes and Their Substrates 41
3.1 Introduction 41
3.2 Substrate Depletion or Product Accumulation 42
3.3 Direct and Coupled Measurements 43
3.4 Classification of Methods 45
3.5 Instrumental Methods 47
3.5.1 Optical Detection 47
3.5.1.1 Absorbance 47
3.5.1.2 Fluorescence 49
3.5.1.3 Luminescence 51
3.5.1.4 Nephelometry 53
3.5.2 Electrochemical Detection 53
3.5.2.1 Amperometry 53
3.5.2.2 Potentiometry 54
3.5.2.3 Conductimetry 54
3.5.3 Other Detection Methods 55
3.5.3.1 Radiochemical 55
3.5.3.2 Manometry 55
3.5.3.3 Calorimetry 56
3.6 Ultra-High-Throughput Assays (HTA) 56
3.7 Practical Considerations for Enzymatic Assays 57
Suggested References 57
vi CONTENTS
References 57
Problems 58
4. Immobilized Enzymes 61
4.1 Introduction 61
4.2 Immobilization Methods 61
4.2.1 Nonpolymerizing Covalent Immobilization 62
4.2.1.1 Controlled-Pore Glass 63
4.2.1.2 Polysaccharides 64
4.2.1.3 Polyacrylamide 65
4.2.1.4 Acidic Supports 66
4.2.1.5 Anhydride Groups 67
4.2.1.6 Thiol Groups 67
4.2.2 Cross-Linking with Bifunctional Reagents 68
4.2.3 Adsorption 69
4.2.4 Entrapment 69
4.2.5 Microencapsulation 70
4.3 Properties of Immobilized Enzymes 71
4.4 Immobilized Enzyme Reactors 76
4.5 Theoretical Treatment of Packed-Bed Enzyme Reactors 79
Suggested References 82
References 82
Problems 83
5. Antibodies 86
5.1 Introduction 86
5.2 Structural and Functional Properties of Antibodies 87
5.3 Polyclonal and Monoclonal Antibodies 90
5.4 Antibody–Antigen Interactions 91
5.5 Analytical Applications of Secondary Antibody–Antigen
Interactions 93
5.5.1 Agglutination Reactions 93
5.5.2 Precipitation Reactions 94
Suggested References 97
References 97
Problems 98
6. Quantitative Immunoassays with Labels 99
6.1 Introduction 99
6.2 Labeling Reactions 101
6.3 Heterogeneous Immunoassays 102
CONTENTS vii
6.3.1 Labeled-Antibody Methods 104
6.3.2 Labeled-Ligand Assays 104
6.3.3 Radioisotopes 106
6.3.4 Fluorophores 107
6.3.4.1 Indirect Fluorescence 108
6.3.4.2 Competitive Fluorescence 108
6.3.4.3 Sandwich Fluorescence 108
6.3.4.4 Fluorescence Excitation Transfer 108
6.3.4.5 Time-Resolved Fluorescence 109
6.3.5 Chemiluminescent Labels 110
6.3.6 Enzyme Labels 112
6.4 Homogeneous Immunoassays 116
6.4.1 Fluorescent Labels 116
6.4.1.1 Enhancement Fluorescence 116
6.4.1.2 Direct Quenching Fluorescence 116
6.4.1.3 Indirect Quenching Fluorescence 117
6.4.1.4 Fluorescence Polarization Immunoassay 117
6.4.1.5 Fluorescence Excitation Transfer 118
6.4.2 Enzyme Labels 118
6.4.2.1 Enzyme-Multiplied Immunoassay Technique 118
6.4.2.2 Substrate-Labeled Fluorescein Immunoassay 119
6.4.2.3 Apoenzyme Reactivation Immunoassay (ARIS) 119
6.4.2.4 Cloned Enzyme Donor Immunoassay 120
6.4.2.5 Enzyme Inhibitory Homogeneous Immunoassay 120
6.5 Evaluation of New Immunoassay Methods 121
Suggested References 126
References 126
Problems 127
7. Biosensors 131
7.1 Introduction 131
7.2 Response of Enzyme-Based Biosensors 132
7.3 Examples of Biosensor Configurations 135
7.3.1 Ferrocene-Mediated Amperometric Glucose Sensor 135
7.3.2 Potentiometric Biosensor for Phenyl Acetate 137
7.3.3 Potentiometric Immunosensor for Digoxin 138
7.3.4 Evanescent-Wave Fluorescence Biosensor
for Bungarotoxin 139
7.3.5 Optical Biosensor for Glucose Based on
Fluorescence Energy Transfer 141
7.3.6 Piezoelectric Sensor for Nucleic Acid Detection 142
7.3.7 Enzyme Thermistors 144
viii CONTENTS
7.4 Evaluation of Biosensor Performance 145
Suggested References 147
References 147
Problems 148
8. Directed Evolution for the Design of Macromolecular
Bioassay Reagents 150
8.1 Introduction 150
8.2 Rational Design and Directed Evolution 152
8.3 Generation of Genetic Diversity 154
8.3.1 Polymerase Chain Reaction and Error-Prone PCR 155
8.3.2 DNA Shuffling 157
8.4 Linking Genotype and Phenotype 158
8.4.1 Cell Expression and Cell Surface Display (in vivo) 158
8.4.2 Phage Display (in vivo) 159
8.4.3 Ribosome Display (in vitro) 160
8.4.4 mRNA-Peptide Fusion (in vitro) 160
8.4.5 Microcompartmentalization (in vitro) 160
8.5 Identification and Selection of Successful Variants 161
8.5.1 Identification of Successful Variants Based on
Binding Properties 162
8.5.2 Identification of Successful Variants Based on
Catalytic Activity 163
8.6 Directed Evolution of Galactose Oxidase 164
Suggested References 165
References 165
Problems 166
9. Principles of Electrophoresis 167
9.1 Introduction 167
9.2 Electrophoretic Support Media 171
9.2.1 Paper 171
9.2.2 Starch Gels 172
9.2.3 Polyacrylamide Gels 173
9.2.4 Agarose Gels 177
9.2.5 Polyacrylamide–Agarose Gels 177
9.3 Effect of Experimental Conditions on
Electrophoretic Separations 177
9.4 Electric Field Strength Gradients 178
9.5 Detection of Proteins and Nucleic Acids After
Electrophoretic Separation 180
9.5.1 Stains and Dyes 181
CONTENTS ix
9.5.2 Detection of Enzymes by Substrate Staining 183
9.5.3 The Southern Blot 184
9.5.4 The Northern Blot 184
9.5.5 The Western Blot 185
9.5.6 Detection of DNA Fragments on Membranes
with DNA Probes 185
Suggested References 188
References 188
Problems 189
10. Applications of Zone Electrophoresis 191
10.1 Introduction 191
10.2 Determination of Protein Net Charge and Molecular Weight
Using PAGE 191
10.3 Determination of Protein Subunit Composition and Subunit
Molecular Weights 193
10.4 Molecular Weight of DNA by Agarose Gel Electrophoresis 195
10.5 Identification of Isoenzymes 196
10.6 Diagnosis of Genetic (Inherited) Disease 197
10.7 DNA Fingerprinting and Restriction Fragment
Length Polymorphism 199
10.8 DNA Sequencing with the Maxam–Gilbert Method 202
10.9 Immunoelectrophoresis 206
Suggested References 210
References 211
Problems 211
11. Isoelectric Focusing 213
11.1 Introduction 213
11.2 Carrier Ampholytes 214
11.3 Modern IEF with Carrier Ampholytes 216
11.4 Immobilized pH Gradients (IPGs) 219
11.5 Two-Dimensional Electrophoresis 222
Suggested References 224
References 225
Problems 225
12. Capillary Electrophoresis 227
12.1 Introduction 227
12.2 Electroosmosis 229
12.3 Elution of Sample Components 229
12.4 Sample Introduction 230
x CONTENTS
12.5 Detectors for Capillary Electrophoresis 231
12.5.1 Laser-Induced Fluorescence Detection 232
12.5.2 Mass Spectrometric Detection 235
12.5.3 Amperometric Detection 236
12.5.4 Radiochemical Detection 239
12.6 Capillary Polyacrylamide Gel Electrophoresis (C-PAGE) 240
12.7 Capillary Isoelectric Focusing (CIEF) 242
Suggested References 244
References 244
Problems 244
13. Centrifugation Methods 247
13.1 Introduction 247
13.2 Sedimentation and Relative Centrifugal g Force 247
13.3 Centrifugal Forces in Different Rotor Types 249
13.3.1 Swinging-Bucket Rotors 249
13.3.2 Fixed-Angle Rotors 250
13.3.3 Vertical Rotors 250
13.4 Clearing Factor (k) 251
13.5 Density Gradients 252
13.5.1 Materials Used to Generate a Gradient 252
13.5.2 Constructing Pre-Formed and Self-Generated
Gradients 253
13.5.3 Redistribution of the Gradient in Fixed-Angle
and Vertical Rotors 254
13.6 Types of Centrifugation Techniques 255
13.6.1 Differential Centrifugation 255
13.6.2 Rate-Zonal Centrifugation 256
13.6.3 Isopycnic Centrifugation 257
13.7 Harvesting Samples 257
13.8 Analytical Ultracentrifugation 257
13.8.1 Instrumentation 258
13.8.2 Sedimentation Velocity Analysis 259
13.8.3 Sedimentation Equilibrium Analysis 262
13.9 Selected Examples 263
13.9.1 Analytical Ultracentrifugation for Quaternary
Structure Elucidation 263
13.9.2 Isolation of Retroviruses by Self-Generated Gradients 264
13.9.3 Isolation of Lipoproteins from Human Plasma 264
Suggested References 265
References 265
Problems 266
CONTENTS xi
14. Chromatography of Biomolecules 268
14.1 Introduction 268
14.2 Units and Definitions 268
14.3 Plate Theory of Chromatography 269
14.4 Rate Theory of Chromatography 270
14.5 Size Exclusion (Gel Filtration) Chromatography 272
14.6 Gel Matrices for Size Exclusion Chromatography 277
14.7 Affinity Chromatography 278
14.7.1 Immobilization of Affinity Ligands 280
14.7.2 Elution Methods 281
14.7.3 Determination of Association Constants by
High-Performance Affinity Chromatography 283
14.8 Ion-Exchange Chromatography 286
14.8.1 Retention Model for Ion-Exchange Chromatography
of Polyelectrolytes 288
Suggested References 292
References 293
Problems 293
15. Mass Spectrometry of Biomolecules 295
15.1 Introduction 295
15.2 Basic Description of the Instrumentation 297
15.2.1 Soft Ionization Sources 297
15.2.1.1 Fast Atom–Ion Bombardment 297
15.2.1.2 Electrospray Ionization 299
15.2.1.3 Matrix-Assisted Laser
Desorption/Ionization 299
15.2.2 Mass Analyzers 300
15.2.3 Detectors 303
15.3 Interpretation of Mass Spectra 304
15.4 Biomolecule Molecular Weight Determination 308
15.5 Protein Identification 310
15.6 Protein–Peptide Sequencing 312
15.7 Nucleic Acid Applications 315
15.8 Bacterial Mass Spectrometry 318
Suggested References 318
References 319
Problems 320
16. Validation of New Bioanalytical Methods 322
16.1 Introduction 322
16.2 Precision and Accuracy 323
xii CONTENTS
16.3 Mean and Variance 324
16.4 Relative Standard Deviation and Other Precision Estimators 325
16.4.1 Distribution of Errors and Confidence Limits 325
16.4.2 Linear Regression and Calibration 326
16.4.3 Precision Profiles 328
16.4.4 Limit of Quantitiation and Detection 329
16.4.5 Linearizing Sigmoidal Curves (Four-Parameter
Log-Logit Model) 329
16.4.6 Effective Dose Method 331
16.5 Estimation of Accuracy 332
16.5.1 Standardization 333
16.5.2 Matrix Effects 333
16.5.2.1 Recovery 333
16.5.2.2 Parallelism 333
16.5.3 Interferences 334
16.6 Qualitative (Screening) Assays 335
16.6.1 Figures of Merit for Qualitative (Screening) Assays 335
16.7 Examples of Validation Procedures 336
16.7.1 Validation of a Qualitative Antibiotic
Susceptibility Test 336
16.7.2 Measurement of Plasma Homocysteine by
Fluorescence Polarization Immunoassay
(FPIA) Methodology 337
16.7.3 Determination of Enzymatic Activity
of b-Galactosidase 340
16.7.4 Establishment of a Cutoff Value for Semi-Quantitative
Assays for Cannabinoids 342
Suggested References 344
References 345
Answers to Problems 346
Index 357

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