BIOANALYTICAL CHEMISTRY -- CONTENTS -- Preface -- Acknowledgments -- 1. Spectroscopic Methods for Matrix Characterization -- 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 -- 2.5.2.2 Hanes Method -- 2.5.2.3 Lineweaver-Burk Method -- 2.5.2.4 Cornish-Bowden-Eisenthal Method -- 2.5.3 Comparison of Methods for the Determination of K(m) Values -- 2.5.4 One-Substrate, Two-Product Enzyme Kinetics -- 2.5.5 Two-Substrate Enzyme Kinetics -- 2.5.6 Examples of Enzyme-Catalyzed Reactions and Their Treatment -- 2.6 Enzyme Activators -- 2.7 Enzyme Inhibitors -- 2.7.1 Competitive Inhibition -- 2.7.2 Noncompetitive Inhibition -- 2.7.3 Uncompetitive Inhibition -- 2.8 Enzyme Units and Concentrations -- Suggested References -- References -- Problems -- 3. Quantitation of Enzymes and Their Substrates -- 3.1 Introduction -- 3.2 Substrate Depletion or Product Accumulation -- 3.3 Direct and Coupled Measurements -- 3.4 Classification of Methods -- 3.5 Instrumental Methods -- 3.5.1 Optical Detection -- 3.5.1.1 Absorbance -- 3.5.1.2 Fluorescence -- 3.5.1.3 Luminescence.

3.5.1.4 Nephelometry -- 3.5.2 Electrochemical Detection -- 3.5.2.1 Amperometry -- 3.5.2.2 Potentiometry -- 3.5.2.3 Conductimetry -- 3.5.3 Other Detection Methods -- 3.5.3.1 Radiochemical -- 3.5.3.2 Manometry -- 3.5.3.3 Calorimetry -- 3.6 Ultra-High-Throughput Assays (HTA) -- 3.7 Practical Considerations for Enzymatic Assays -- Suggested References -- References -- Problems -- 4. Immobilized Enzymes -- 4.1 Introduction -- 4.2 Immobilization Methods -- 4.2.1 Nonpolymerizing Covalent Immobilization -- 4.2.1.1 Controlled-Pore Glass -- 4.2.1.2 Polysaccharides -- 4.2.1.3 Polyacrylamide -- 4.2.1.4 Acidic Supports -- 4.2.1.5 Anhydride Groups -- 4.2.1.6 Thiol Groups -- 4.2.2 Cross-Linking with Bifunctional Reagents -- 4.2.3 Adsorption -- 4.2.4 Entrapment -- 4.2.5 Microencapsulation -- 4.3 Properties of Immobilized Enzymes -- 4.4 Immobilized Enzyme Reactors -- 4.5 Theoretical Treatment of Packed-Bed Enzyme Reactors -- Suggested References -- References -- Problems -- 5. Antibodies -- 5.1 Introduction -- 5.2 Structural and Functional Properties of Antibodies -- 5.3 Polyclonal and Monoclonal Antibodies -- 5.4 Antibody-Antigen Interactions -- 5.5 Analytical Applications of Secondary Antibody-Antigen Interactions -- 5.5.1 Agglutination Reactions -- 5.5.2 Precipitation Reactions -- Suggested References -- References -- Problems -- 6. Quantitative Immunoassays with Labels -- 6.1 Introduction -- 6.2 Labeling Reactions -- 6.3 Heterogeneous Immunoassays -- 6.3.1 Labeled-Antibody Methods -- 6.3.2 Labeled-Ligand Assays -- 6.3.3 Radioisotopes -- 6.3.4 Fluorophores -- 6.3.4.1 Indirect Fluorescence -- 6.3.4.2 Competitive Fluorescence -- 6.3.4.3 Sandwich Fluorescence -- 6.3.4.4 Fluorescence Excitation Transfer -- 6.3.4.5 Time-Resolved Fluorescence -- 6.3.5 Chemiluminescent Labels -- 6.3.6 Enzyme Labels -- 6.4 Homogeneous Immunoassays -- 6.4.1 Fluorescent Labels.

6.4.1.1 Enhancement Fluorescence -- 6.4.1.2 Direct Quenching Fluorescence -- 6.4.1.3 Indirect Quenching Fluorescence -- 6.4.1.4 Fluorescence Polarization Immunoassay -- 6.4.1.5 Fluorescence Excitation Transfer -- 6.4.2 Enzyme Labels -- 6.4.2.1 Enzyme-Multiplied Immunoassay Technique -- 6.4.2.2 Substrate-Labeled Fluorescein Immunoassay -- 6.4.2.3 Apoenzyme Reactivation Immunoassay (ARIS) -- 6.4.2.4 Cloned Enzyme Donor Immunoassay -- 6.4.2.5 Enzyme Inhibitory Homogeneous Immunoassay -- 6.5 Evaluation of New Immunoassay Methods -- Suggested References -- References -- Problems -- 7. Biosensors -- 7.1 Introduction -- 7.2 Response of Enzyme-Based Biosensors -- 7.3 Examples of Biosensor Configurations -- 7.3.1 Ferrocene-Mediated Amperometric Glucose Sensor -- 7.3.2 Potentiometric Biosensor for Phenyl Acetate -- 7.3.3 Potentiometric Immunosensor for Digoxin -- 7.3.4 Evanescent-Wave Fluorescence Biosensor for Bungarotoxin -- 7.3.5 Optical Biosensor for Glucose Based on Fluorescence Energy Transfer -- 7.3.6 Piezoelectric Sensor for Nucleic Acid Detection -- 7.3.7 Enzyme Thermistors -- 7.4 Evaluation of Biosensor Performance -- Suggested References -- References -- Problems -- 8. Directed Evolution for the Design of Macromolecular Bioassay Reagents -- 8.1 Introduction -- 8.2 Rational Design and Directed Evolution -- 8.3 Generation of Genetic Diversity -- 8.3.1 Polymerase Chain Reaction and Error-Prone PCR -- 8.3.2 DNA Shuffling -- 8.4 Linking Genotype and Phenotype -- 8.4.1 Cell Expression and Cell Surface Display (in vivo) -- 8.4.2 Phage Display (in vivo) -- 8.4.3 Ribosome Display (in vitro) -- 8.4.4 mRNA-Peptide Fusion (in vitro) -- 8.4.5 Microcompartmentalization (in vitro) -- 8.5 Identification and Selection of Successful Variants -- 8.5.1 Identification of Successful Variants Based on Binding Properties.

8.5.2 Identification of Successful Variants Based on Catalytic Activity -- 8.6 Directed Evolution of Galactose Oxidase -- Suggested References -- References -- Problems -- 9. Principles of Electrophoresis -- 9.1 Introduction -- 9.2 Electrophoretic Support Media -- 9.2.1 Paper -- 9.2.2 Starch Gels -- 9.2.3 Polyacrylamide Gels -- 9.2.4 Agarose Gels -- 9.2.5 Polyacrylamide-Agarose Gels -- 9.3 Effect of Experimental Conditions on Electrophoretic Separations -- 9.4 Electric Field Strength Gradients -- 9.5 Detection of Proteins and Nucleic Acids After Electrophoretic Separation -- 9.5.1 Stains and Dyes -- 9.5.2 Detection of Enzymes by Substrate Staining -- 9.5.3 The Southern Blot -- 9.5.4 The Northern Blot -- 9.5.5 The Western Blot -- 9.5.6 Detection of DNA Fragments on Membranes with DNA Probes -- Suggested References -- References -- Problems -- 10. Applications of Zone Electrophoresis -- 10.1 Introduction -- 10.2 Determination of Protein Net Charge and Molecular Weight Using PAGE -- 10.3 Determination of Protein Subunit Composition and Subunit Molecular Weights -- 10.4 Molecular Weight of DNA by Agarose Gel Electrophoresis -- 10.5 Identification of Isoenzymes -- 10.6 Diagnosis of Genetic (Inherited) Disease -- 10.7 DNA Fingerprinting and Restriction Fragment Length Polymorphism -- 10.8 DNA Sequencing with the Maxam-Gilbert Method -- 10.9 Immunoelectrophoresis -- Suggested References -- References -- Problems -- 11. Isoelectric Focusing -- 11.1 Introduction -- 11.2 Carrier Ampholytes -- 11.3 Modern IEF with Carrier Ampholytes -- 11.4 Immobilized pH Gradients (IPGs) -- 11.5 Two-Dimensional Electrophoresis -- Suggested References -- References -- Problems -- 12. Capillary Electrophoresis -- 12.1 Introduction -- 12.2 Electroosmosis -- 12.3 Elution of Sample Components -- 12.4 Sample Introduction -- 12.5 Detectors for Capillary Electrophoresis.

12.5.1 Laser-Induced Fluorescence Detection -- 12.5.2 Mass Spectrometric Detection -- 12.5.3 Amperometric Detection -- 12.5.4 Radiochemical Detection -- 12.6 Capillary Polyacrylamide Gel Electrophoresis (C-PAGE) -- 12.7 Capillary Isoelectric Focusing (CIEF) -- Suggested References -- References -- Problems -- 13. Centrifugation Methods -- 13.1 Introduction -- 13.2 Sedimentation and Relative Centrifugal g Force -- 13.3 Centrifugal Forces in Different Rotor Types -- 13.3.1 Swinging-Bucket Rotors -- 13.3.2 Fixed-Angle Rotors -- 13.3.3 Vertical Rotors -- 13.4 Clearing Factor (k) -- 13.5 Density Gradients -- 13.5.1 Materials Used to Generate a Gradient -- 13.5.2 Constructing Pre-Formed and Self-Generated Gradients -- 13.5.3 Redistribution of the Gradient in Fixed-Angle and Vertical Rotors -- 13.6 Types of Centrifugation Techniques -- 13.6.1 Differential Centrifugation -- 13.6.2 Rate-Zonal Centrifugation -- 13.6.3 Isopycnic Centrifugation -- 13.7 Harvesting Samples -- 13.8 Analytical Ultracentrifugation -- 13.8.1 Instrumentation -- 13.8.2 Sedimentation Velocity Analysis -- 13.8.3 Sedimentation Equilibrium Analysis -- 13.9 Selected Examples -- 13.9.1 Analytical Ultracentrifugation for Quaternary Structure Elucidation -- 13.9.2 Isolation of Retroviruses by Self-Generated Gradients -- 13.9.3 Isolation of Lipoproteins from Human Plasma -- Suggested References -- References -- Problems -- 14. Chromatography of Biomolecules -- 14.1 Introduction -- 14.2 Units and Definitions -- 14.3 Plate Theory of Chromatography -- 14.4 Rate Theory of Chromatography -- 14.5 Size Exclusion (Gel Filtration) Chromatography -- 14.6 Gel Matrices for Size Exclusion Chromatography -- 14.7 Affinity Chromatography -- 14.7.1 Immobilization of Affinity Ligands -- 14.7.2 Elution Methods -- 14.7.3 Determination of Association Constants by High-Performance Affinity Chromatography.

14.8 Ion-Exchange Chromatography.