Bioanalytical chemistry - Mikkelsen & Corton

CONTENTS

1. Spectroscopic Methods for Matrix Characterization 1

1.1 Introduction 

1.2 Total Protein 

1.2.1 Lowry Method 

1.2.2 Smith (BCA) Method 

1.2.3 Bradford Method

1.2.4 Ninhydrin-Based Assay 

1.2.5 Other Protein Quantitation Methods 

1.3 Total DNA 

1.3.1 Diaminobenzoic Acid Method 

1.3.2 Diphenylamine Method 

1.3.3 Other Fluorometric Methods 

1.4 Total RNA 

1.5 Total Carbohydrate 

1.5.1 Ferricyanide Method 

1.5.2 Phenol–Sulfuric Acid Method 

1.5.3 2-Aminothiophenol Method 

1.5.4 Purpald Assay for Bacterial Polysaccharides 

1.6 Free Fatty Acids 

References 

Problems 

2. Enzymes 

2.1 Introduction 

2.2 Enzyme Nomenclature 

2.3 Enzyme Commission Numbers 

2.4 Enzymes in Bioanalytical Chemistry 

2.5 Enzyme Kinetics 

2.5.1 Simple One-Substrate Enzyme Kinetics 

2.5.2 Experimental Determination of Michaelis–Menten Parameters 

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

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

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

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

6. Quantitative Immunoassays with Labels 99

6.1 Introduction 99

6.2 Labeling Reactions 101

6.3 Heterogeneous Immunoassays 102

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

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

7.4 Evaluation of Biosensor Performance 145

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

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

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