Bioanalytical chemistry

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.2.1 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 CONTENTS4.1 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 1026.3.1 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 1447.4 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 1819.5.2 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