Bioanalytical Chemistry

Description

A timely, accessible survey of the multidisciplinary field of bioanalytical chemistry

• Provides an all in one approach for both beginners and experts, from a broad range of backgrounds, covering introductions, theory, advanced concepts and diverse applications for each method
• Each chapter progresses from basic concepts to applications involving real samples
• Includes three new chapters on Biomimetic Materials, Lab-on-Chip, and Analytical Methods
• Contains end-of-chapter problems and an appendix with selected answers

Contents

Preface
Acknowledgments

1. Quantitative Instrumental Measurements

• 1.1 Introduction 1
• 1.2 Optical Measurements
• 1.2.1 UV-Visible Absorbance
• 1.2.2 Turbidimetry (Light-Scattering)
• 1.2.3 Fluorescence
• 1.2.4 Chemiluminescence and Bioluminescence
• 1.3 Electrochemical Measurements
• 1.3.1 Potentiometry
• 1.3.2 Amperometry
• 1.3.3 Impedimetry
• 1.4 Radiochemical Measurements
• 1.4.1 Scintillation Counting
• 1.4.2 Geiger Counting
• 1.5 Surface Plasmon Resonance
• 1.6 Calorimetry
• 1.6.1 Differential Scanning Calorimetry (DSC)
• 1.6.2 Isothermal Titration Calorimetry (ITC)
• 1.7 Automation: Microplates, Multiwell Liquid Dispensers and Microplate Readers
• 1.8 Calibration of Instrumental Signals
• 1.8.1 External Standards
• 1.8.2 Internal Standards
• 1.8.3 Standard Additions
• 1.9 Quantitative and Semi-Quantitative Measurements
• 1.9.1 Exact Concentration
• 1.9.2 Positive or Negative Result
• Suggested Reading
• Problems

2. Spectroscopic Methods for the Quantitation of Classes of Biomolecules

• 2.1 Introduction 29
• 2.2 Total Protein
• 2.2.1 Lowry Method
• 2.2.2 Smith (BCA) Method
• 2.2.3 Bradford Method
• 2.2.4 Ninhydrin Method
• 2.2.5 Other Protein Quantitation Methods
• 2.3 Total DNA
• 2.3.1 Diaminobenzoic Acid (DABA) Method
• 2.3.2 Diphenylamine (DPA) Method
• 2.3.3 Other Fluorometric Methods
• 2.4 Total RNA
• 2.5 Total Carbohydrate
• 2.5.1 Ferricyanide Method
• 2.5.2 Phenol-Sulfuric Acid Method
• 2.5.3 2-Aminothiophenol Method
• 2.5.4 Purpald Assay for Bacterial Polysaccharides
• 2.6 Free Fatty Acids
• References
• Problems

3. Enzymes

• 3.1 Introduction 52
• 3.2 Enzyme Nomenclature
• 3.3 Enzyme Commission Numbers
• 3.4 Enzymes in Bioanalytical Chemistry
• 3.5 Enzyme Kinetics
• 3.5.1 Simple One-Substrate Enzyme Kinetics
• 3.5.2 Experimental Determination of Michaelis-Menten Parameters
• 3.5.2.1 Eadie-Hofstee Method
• 3.5.2.2 Hanes Method
• 3.5.2.3 Lineweaver-Burk Method
• 3.5.2.4 Cornish-Bowden-Eisenthal Method
• 3.5.3 Comparison of Methods for the Determination of Km Values
• 3.5.4 One-Substrate, Two-Product Enzyme Kinetics
• 3.5.5 Two-Substrate Enzyme Kinetics
• 3.5.6 Examples of Enzyme-Catalyzed Reactions and their Treatment
• 3.5.7 Curve-Fitting for Enzyme Kinetic Data
• 3.6 Enzyme Activators
• 3.7 Enzyme Inhibitors
• 3.7.1 Competitive Inhibition
• 3.7.2 Noncompetitive Inhibition
• 3.7.3 Uncompetitive Inhibition
• 3.8 Enzyme Units and Concentrations
• Suggested Reading
• References
• Problems

4. Quantitation of Enzymes and their Substrates

• 4.1 Introduction 87
• 4.2 Substrate Depletion or Product Accumulation
• 4.3 Direct and Coupled Measurements
• 4.4 Classification of Methods
• 4.5 Instrumental Methods
• 4.5.1 Optical Detection
• 4.5.1.1 Absorbance
• 4.5.1.2 Fluorescence
• 4.5.1.3 Luminescence
• 4.5.1.4 Nephelometry
• 4.5.2 Electrochemical Detection
• 4.5.2.1 Amperometry
• 4.5.2.2 Potentiometry
• 4.5.2.3 Conductimetry
• 4.5.3 Other Detection Methods
• 4.5.3.1 Radiochemical
• 4.5.3.2 Manometry
• 4.5.3.3 Calorimetry
• 4.6 High-Throughput Assays for Enzymes and Inhibitors
• 4.7 Assays for Enzymatic Reporter Gene Products
• 4.8 Practical Considerations for Enzymatic Assays
• Suggested Reading
• References
• Problems

5. Immobilized Enzymes

• 5.1 Introduction 117
• 5.2 Immobilization Methods
• 5.2.1 Nonpolymerizing Covalent Immobilization
• 5.2.1.1 Controlled-Pore Glass
• 5.2.1.2 Polysaccharides
• 5.2.1.3 Polyacrylamide
• 5.2.1.4 Acidic Supports
• 5.2.1.5 Anhydride Groups
• 5.2.1.6 Thiol Groups
• 5.2.2 Crosslinking with Bifunctional Reagents
• 5.2.3 Adsorption
• 5.2.4 Entrapment
• 5.2.5 Microencapsulation
• 5.3 Properties of Immobilized Enzymes
• 5.4 Immobilized Enzyme Reactors
• 5.5 Theoretical Treatment of Packed-Bed Enzyme Reactors
• 5.6 Suggested Reading
• 5.7 References
• 5.8 Problems

6. Antibodies

• 6.1 Introduction 152
• 6.2 Structural and Functional Properties of Antibodies
• 6.3 Polyclonal and Monoclonal Antibodies
• 6.4 Antibody-Antigen Interactions
• 6.5 Analytical Applications of Secondary Antibody-Antigen Interactions
• 6.5.1 Agglutination Reactions
• 6.5.2 Precipitation Reactions
• Suggested Reading
• References
• Problems

7. Quantitative Immunoassays with Labels

• 7.1 Introduction 171
• 7.2 Labelling Reactions
• 7.3 Heterogeneous Immunoassays
• 7.3.1 Labelled-Antibody Methods
• 7.3.2 Labelled-Ligand Assays
• 7.3.3 Radioisotopes
• 7.3.4 Fluorophores
• 7.3.5 Chemiluminescent Labels
• 7.3.6 Enzyme Labels
• 7.4 Homogeneous Immunoassays
• 7.4.1 Fluorescent Labels
• 7.4.2 Enzyme Labels
• 7.5 Evaluation of New Immunoassay Methods
• Suggested Reading
• References
• Problems

8. Biosensors

• 8.1 Introduction 217
• 8.2 Biosensor Diversity and Classification
• 8.3 Recognition Agents
• 8.3.1 Natural Recognition Agents
• 8.3.2 Artificial Recognition Agents
• 8.4 Response of Enzyme-Based Biosensors
• 8.5 Examples of Biosensor Configurations
• 8.5.1 Ferrocene-Mediated Amperometric Glucose Sensor
• 8.5.2 Potentiometric Biosensor for Phenyl Acetate
• 8.5.3 Evanescent-Wave Fluorescence Biosensor for Bungarotoxin
• 8.5.4 Optical Biosensor for Glucose Based on Fluorescence Energy Transfer
• 8.5.5 Piezoelectric Sensor for Nucleic Acid Detection
• 8.5.6 Enzyme Thermistors
• 8.5.7 Fluorescence Sensor for Nitroaromatic Explosives Based on a Molecularly Imprinted Polymer
• 8.5.8 Immunosensor Microwell Arrays from Gold Compact Disks
• 8.5.9 Nanoparticle-Enhanced Detection of Thrombin by SPR
• 8.5.10 Environmental BOD and Toxicity Biosensors Based on Viable Cells
• 8.5.11 Detection of Viruses using a Surface Acoustic Wave (SAW) Biosensor
• 8.5.13 DNA Microarrays
• 8.5.12 MEMS Microcantilever Biosensor for Virus Detection
• 8.6 Evaluation of Biosensor Perfomance
• 8.7 In Vivo Applications of Biosensors
• 8.7.1 Biocompatible Materials
• 8.7.2 Physiological Environment of the Human Body
• 8.7.3 The Artificial Pancreas
• 8.7.4 An Enzymatic Fuel Cell as a Component of an Implanted Biosensing System
• 8.7.5 Other Examples of Implantable Biosensors
• Suggested Reading
• References
• Problems

9. Directed Evolution for the Design of Macromolecular Reagents

• 9.1 Introduction 273
• 9.2 Rational Design and Directed Evolution
• 9.3 Generation of Genetic Diversity
• 9.3.1 Polymerase Chain Reaction and Error-Prone PCR
• 9.3.2 DNA Shuffling
• 9.4 Linking Genotype and Phenotype
• 9.4.1 Cell Expression and Cell Surface Display (in vivo)
• 9.4.2 Phage Display (in vivo)
• 9.4.3 Ribosome Display (in vitro)
• 9.4.4 mRNA-Peptide Fusion (in vitro)
• 9.4.5 Microcompartmentalization (in vitro)
• 9.5 Identification and Selection of Successful Variants
• 9.5.1 Identification of Successful Variants Based on Binding Properties
• 9.5.2 Identification of Successful Variants Based on Catalytic Activity
• 9.6 Examples of Directed Evolution Experiments
• 9.6.1 Directed Evolution of Galactose Oxidase
• 9.6.2 a-Hemolysin Evolution
• Suggested Reading
• References
• Problems

10. Image-Based Bioanalysis

• 10.1 Introduction 298
• 10.2 Magnification and Resolution
• 10.3 Optical Microscopy
• 10.3.1 The Compound Light Microscope
• 10.3.2 The Confocal Microscope
• 10.3.3 Sample Preparation
• 10.3.4 General and Selective Stains
• 10.3.5 Fluorescence In situ Hybridization
• 10.3.6 Green Fluorescent Protein and its Analogues
• 10.4 Electron Microscopy
• 10.4.1 Principles and Instrumentation
• 10.4.2 Sample Preparation
• 10.4.3 Transmission Electron Microscopy (TEM)
• 10.4.4 Scanning Electron Microscopy (SEM)
• 10.5 Scanning Tunneling Microscopy
• 10.5.1 Principles and Instrumentation
• 10.5.2 Biological Applications
• 10.6 Atomic Force Microscopy (AFM)
• 10.6.1 Cantilevers and Operational Modes
• 10.6.2 Samples and Substrates
• 10.6.3 Biological Applications
• 10.6.4 Four-Dimensional (4D) Scanning
• 10.7 Scanning Electrochemical Microscopy (SECM)
• 10.7.1 Principles and Instrumentation
• 10.7.2 Samples and Substrates
• 10.7.3 Biological Applications
• Suggested Reading
• References
• Problems

11. Principles of Electrophoresis

• 11.1 Introduction 319
• 11.2 Electrophoretic Support Media
• 11.2.1 Paper
• 11.2.2 Starch Gels
• 11.2.3 Polyacrylamide Gels
• 11.2.4 Agarose Gels
• 11.2.5 Polyacrylamide-Agarose Gels
• 11.3 Effect of Experimental Conditions on Electrophoretic Separations
• 11.4 Electric Field Strength Gradients
• 11.5 Detection of Proteins and Nucleic Acids after Electrophoretic Separation
• 11.5.1 Stains and Dyes
• 11.5.2 Detection of Enzymes by Substrate Staining
• 11.5.3 The Southern Blot
• 11.5.4 The Northern Blot
• 11.5.5 The Western Blot
• 11.5.6 Detection of DNA Fragments on Membranes with DNA Probes
• Suggested Reading
• References
• Problems

12. Applications of Zone Electrophoresis

• 12.1 Introduction 350
• 12.2 Determination of Protein Net Charge and Molecular Weight using PAGE
• 12.3 Determination of Protein Subunit Composition and Subunit Molecular Weights
• 12.4 Molecular Weight of DNA by Agarose Gel Electrophoresis
• 12.5 Identification of Isoenzymes
• 12.6 Diagnosis of Genetic (Inherited) Disease
• 12.7 DNA Fingerprinting and Restriction Fragment Length Polymorphism
• 12.8 DNA Sequencing with the Maxam-Gilbert Method
• 12.9 Immunoelectrophoresis
• Suggested Reading
• References
• Problems

13. Isoelectric Focussing and 2D Electrophoresis

• 13.1 Introduction 378
• 13.2 Carrier Ampholytes
• 13.3 Modern IEF with Carrier Ampholytes
• 13.4 Immobilized pH Gradients (IPGs)
• 13.5 Two-Dimensional Electrophoresis
• Suggested Reading
• References
• Problems

14. Capillary Electrophoresis

• 14.1 Introduction 399
• 14.2 Electroosmosis
• 14.3 Elution of Sample Components
• 14.4 Sample Introduction
• 14.5 Detectors for Capillary Electrophoresis
• 14.5.1 Laser-Induced Fluorescence Detection
• 14.5.2 Mass Spectrometric Detection
• 14.5.3 Amperometric Detection
• 14.5.4 Radiochemical Detection
• 14.6 Capillary Polyacrylamide Gel Electrophoresis (C-PAGE)
• 14.7 Capillary Isoelectric Focussing (CIEF)
• Suggested Reading
• References
• Problems

15. Centrifugation Methods

• 15.1 Introduction 425
• 15.2 Sedimentation and Relative Centrifugal g Force
• 15.2.1 Biological Particles
• 15.3 Centrifugal Forces in Different Rotor Types
• 15.3.1 Swinging-Bucket Rotors
• 15.3.2 Fixed-Angle Rotors
• 15.3.3 Vertical Rotors
• 15.4 Clearing Factor (k)
• 15.5 Density Gradients
• 15.5.1 Materials Used to Generate a Gradient
• 15.5.2 Constructing Pre-Formed and Self-Generated Gradients
• 15.5.3 Redistribution of the Gradient in Fixed-Angle and Vertical Rotors
• 15.6 Types of Centrifugation Techniques
• 15.6.1 Differential Centrifugation
• 15.6.2 Rate-Zonal Centrifugation
• 15.6.3 Isopycnic Centrifugation
• 15.7 Harvesting Samples
• 15.8 Analytical Ultracentrifugation
• 15.8.1 Instrumentation
• 15.8.2 Sedimentation Velocity Analysis
• 15.8.3 Sedimentation Equilibrium Analysis
• 15.9 Selected Examples
• 15.9.1 Analytical Ultracentrifugation for Quaternary Structure Elucidation
• 15.9.2 Isolation of Retroviruses by Self-Generated Gradients
• 15.9.3 Isolation of Lipoproteins from Human Plasma
• Suggested Reading
• References
• Problems

16. Chromatography of Biological Macromolecules

• 16.1 Introduction 457
• 16.2 Units and Definitions
• 16.3 Plate Theory of Chromatography
• 16.4 Rate Theory of Chromatography
• 16.5 Size Exclusion (Gel Filtration) Chromatography
• 16.6 Gel Matrices for Size Exclusion Chromatography
• 16.7 Affinity Chromatography
• 16.7.1 Immobilization of Affinity Ligands
• 16.7.2 Elution Methods
• 16.7.3 Determination of Association Constants by High Performance Affinity Chromatography
• 16.8 Ion-Exchange Chromatography
• 16.8.1 Retention Model for Ion-Exchange Chromatography of Polyelectrolytes
• Suggested Reading
• References
• Problems

17. Mass Spectrometry of Biomolecules

• 17.1 Introduction 492
• 17.2 Basic Description of the Intrumentation
• 17.2.1 Soft Ionization Sources
• 17.2.1.1 Fast Atom/Ion Bombardment (FAB)
• 17.2.1.2 Electrospray Ionization (ESI)
• 17.2.1.3 Matrix-Assisted Laser Desorption/Ionization (MALDI)
• 17.2.2 Mass Analyzers
• 17.2.3 Detectors
• 17.3 Interpretation of Mass Spectra
• 17.4 Biomolecule Molecular Weight Determination
• 17.5 Protein Identification
• 17.6 Protein-Peptide Sequencing
• 17.7 Nucleic Acid Applications
• 17.8 Bacterial Mass Spectrometry
• Suggested Reading
• References
• Problems

18. Micro-TAS, Lab-on-a-Chip and Microarray Devices

• 18.1 Introduction 528
• 18.2 Device Fabrication Materials and Methods
• 18.3 Microfluidics
• 18.3.1 Fluid Transport
• 18.3.2 Valves and Reservoirs
• 18.3.3 Mixing and Sample Separation
• 18.4 Detectors
• 18.5 Examples of Bioanalytical Devices
• 18.5.1 DNA Separation Using a Nanofence Array Microfluidic Device
• 18.5.2 Two Dimensional Electrophoresis on a Microfluidic Chip
• 18.5.3 Microfluidic Antibody Capture for Single-Cell Proteomics
• 18.5.4 Multiplexed PCR Amplification and DNA Detection on a Microfluidic Chip
• 18.5.5 Silicone Protein Separation Chip Based on a Grafted Ion-Exchange Polymer
• 18.5.6 Circular, Biofunctionalized PEG Microchannels for Cell Adhesion Studies
• Suggested Reading
• References
• Problems

19. Validation of New Bioanalytical Methods

• 19.1 Introduction 541
• 19.2 Precision and Accuracy
• 19.3 Mean and Variance
• 19.4 RSD and Other Precision Estimators
• 19.4.1 Distribution of Errors and Confidence Limits
• 19.4.2 Linear Regression and Calibration
• 19.4.3 Precision Profiles
• 19.4.4 Limit of Quantitiation and Detection
• 19.4.5 Linearizing Sigmoidal Curves (Four-Parameter Log-Logit Model)
• 19.4.6 Effective Dose Method
• 19.5 Estimation of Accuracy
• 19.5.1 Standardization
• 19.5.2 Matrix Effects
• 19.5.2.1 Recovery
• 19.5.2.2 Parallelism
• 19.5.3 Interferences
• 19.6 Qualitative (Screening) Assays
• 19.6.1 Figures of Merit for Qualitative (Screening) Assays
• 19.7 Examples of Validation Procedures
• 19.7.1 Validation of a Qualitative Antibiotic Susceptibility Test
• 19.7.2 Measurement of Plasma Homocysteine byFluorescence Polarization Immunoassay (FPIA) Methodology
• 19.7.3 Determination of Enzymatic Activity of ß-Galactosidase
• 19.7.4 Establishment of a Cutoff Value for

Semi-Quantitative Assays for Cannabinoids
Suggested Reading
References
Answers to Selected Problems 571
Index