Bioanalytical Chemistry

Susan R. Mikkelsen, PhD, is a Professor in the Department of Chemistry at the University of Waterloo, Ontario, Canada. She has presented at numerous conferences and is the author of over 35 peer-reviewed research articles and 115 presentations. She has organized and supervised a wide variety of bioanalytical research projects and has participated in local and international collaborations in this field. She is the co-author of the first edition of Bioanalytical Chemistry. Eduardo Cortón, PhD, is Head of the Bioan?lisis and Biosensors Laboratory in the Biochemistry Department at the University of Buenos Aires, Argentina. He is also an Adjunct Professor in the Department of Biological Chemistry at the University of Buenos Aires as well as an active Independent Researcher at the National Council of Scientific and Technical Research (CONICET). He has published over 35 peer-reviewed research articles and presented at 80 conferences. He is the co-author of the first edition of Bioanalytical Chemistry.

• Preface to Second Edition xixPreface to First Edition xxiAcknowledgments xxiii1. Quantitative Instrumental Measurements 11.1. Introduction 11.2. Optical Measurements 21.2.1. UV-Visible Absorbance 31.2.2. Turbidimetry (Light-Scattering) 51.2.3. Fluorescence 51.2.4. Chemiluminescence and Bioluminescence 71.3. Electrochemical Measurements 81.3.1. Potentiometry 101.3.2. Amperometry 101.3.3. Impedimetry 111.4. Radiochemical Measurements 121.4.1. Scintillation Counting 121.4.2. Geiger Counting 121.5. Surface Plasmon Resonance 131.6. Calorimetry 141.6.1. Differential Scanning Calorimetry (DSC) 151.6.2. Isothermal Titration Calorimetry (ITC) 161.7. Automation: Microplates, Multiwell Liquid Dispensers and Microplate Readers 161.8. Calibration of Instrumental Measurements 181.8.1. External Standards 181.8.2. Internal Standards 191.8.3. Standard Additions 201.9. Quantitative and Semi-Quantitative Measurements 211.9.1. Exact Concentration 211.9.2. Positive or Negative Result 21Suggested Reading 22Problems 222. Spectroscopic Methods for the Quantitation of Classes of Biomolecules 232.1. Introduction 232.2. Total Protein 242.2.1. Lowry Method 242.2.2. Smith (BCA) Method 242.2.3. Bradford Method 262.2.4. Ninhydrin-Based Assay 272.2.5. Other Protein Quantitation Methods 282.3. Total DNA 312.3.1. Diaminobenzoic Acid (DABA) Method 322.3.2. Diphenylamine (DPA) Method 322.3.3. Other Fluorimetric Methods 332.4. Total RNA 342.5. Total Carbohydrate 352.5.1. Ferricyanide Method 352.5.2. Phenol-Sulfuric Acid Method 362.5.3. 2-Aminothiophenol Method 362.5.4. Purpald Assay for Bacterial Polysaccharides 372.6. Free Fatty Acids 37References 38Problems 393. Enzymes 413.1. Introduction 413.2. Enzyme Nomenclature 423.3. Enzyme Commission Numbers 433.4. Enzymes in Bioanalytical Chemistry 453.5. Enzyme Kinetics 463.5.1. Simple One-Substrate Enzyme Kinetics 483.5.2. Experimental Determination of Michaelis-Menten Parameters 503.5.2.1. Eadie-Hofstee Method 503.5.2.2. Hanes Method 503.5.2.3. Lineweaver-Burk Method 513.5.2.4. Cornish-Bowden-Eisenthal Method 523.5.3. Comparison of Methods for the Determination of KM Values 523.5.4. One-Substrate, Two-Product Enzyme Kinetics 543.5.5. Two-Substrate Enzyme Kinetics 543.5.6. Examples of Enzyme-Catalyzed Reactions and their Treatment 563.5.7. Curve Fitting for Enzyme Kinetic Data 573.6. Enzyme Activators 583.7. Enzyme Inhibitors 593.7.1. Competitive Inhibition 603.7.2. Noncompetitive Inhibition 603.7.3. Uncompetitive Inhibition 623.8. Enzyme Units and Concentrations 62Suggested Reading 64References 64Problems 644. Quantitation of Enzymes and Their Substrates 674.1. Introduction 674.2. Substrate Depletion or Product Accumulation 684.3. Direct and Coupled Measurements 694.4. Classification of Methods 714.5. Instrumental Methods 734.5.1. Optical Detection 734.5.1.1. Absorbance 734.5.1.2. Fluorescence 754.5.1.3. Luminescence 774.5.1.4. Nephelometry 794.5.2. Electrochemical Detection 794.5.2.1. Amperometry 794.5.2.2. Potentiometry 804.5.2.3. Conductimetry 804.5.3. Other Detection Methods 814.5.3.1. Radiochemical 814.5.3.2. Manometry 814.5.3.3. Calorimetry 824.6. High-Throughput Assays for Enzymes and Inhibitors 824.7. Assays for Enzymatic Reporter Gene Products 844.8. Practical Considerations for Enzymatic Assays 85Suggested Reading 86References 86Problems 875. Immobilized Enzymes 905.1. Introduction 905.2. Immobilization Methods 905.2.1. Nonpolymerizing Covalent Immobilization 915.2.1.1. Controlled-Pore Glass 925.2.1.2. Polysaccharides 935.2.1.3. Polyacrylamide 955.2.1.4. Acidic Supports 955.2.1.5. Anhydride Groups 965.2.1.6. Thiol Groups 975.2.2. Crosslinking with Bifunctional Reagents 975.2.3. Adsorption 985.2.4. Entrapment 995.2.5. Microencapsulation 1005.3. Properties of Immobilized Enzymes 1015.4. Immobilized Enzyme Reactors 1075.5. Theoretical Treatment of Packed-Bed Enzyme Reactors 109Suggested Reading 113References 113Problems 1146. Antibodies 1176.1. Introduction 1176.2. Structural and Functional Properties of Antibodies 1186.3. Polyclonal and Monoclonal Antibodies 1216.4. Antibody-Antigen Interactions 1226.5. Analytical Applications of Secondary Antibody-Antigen Interactions 1246.5.1. Agglutination Reactions 1246.5.2. Precipitation Reactions 126Suggested Reading 129References 129Problems 1297. Quantitative Immunoassays with Labels 1317.1. Introduction 1317.2. Labeling Reactions 1327.3. Heterogeneous Immunoassays 1347.3.1. Labeled-Antibody Methods 1367.3.2. Labeled-Ligand Assays 1367.3.3. Radioisotopes 1397.3.4. Fluorophores 1397.3.4.1. Indirect Fluorescence 1407.3.4.2. Competitive Fluorescence 1407.3.4.3. Sandwich Fluorescence 1407.3.4.4. Fluorescence Excitation Transfer 1407.3.4.5. Time-Resolved Fluorescence 1417.3.5. Quantum Dots 1427.3.6. Chemiluminescent Labels 1437.3.7. Enzyme Labels 1457.3.8. Lateral Flow Immunoassay 1487.4. Homogeneous Immunoassays 1497.4.1. Fluorescent Labels 1497.4.1.1. Enhancement Fluorescence 1497.4.1.2. Direct Quenching Fluorescence 1507.4.1.3. Indirect Quenching Fluorescence 1507.4.1.4. Fluorescence Polarization Immunoassay 1517.4.1.5. Fluorescence Excitation Transfer 1517.4.2. Enzyme Labels 1527.4.2.1. Enzyme-Multiplied Immunoassay Technique 1527.4.2.2. Substrate-Labelled Fluorescein Immunoassay 1537.4.2.3. Apoenzyme Reactivation Immunoassay 1537.4.2.4. Cloned Enzyme Donor Immunoassay 1547.4.2.5. Enzyme Inhibitory Homogeneous Immunoassay 1547.5. Evaluation of New Immunoassay Methods 155Suggested Reading 160References 160Problems 1618. Biosensors 1668.1. Introduction 1668.2. Biosensor Diversity and Classification 1698.3. Recognition Agents 1718.3.1. Natural Recognition Agents 1718.3.2. Artificial Recognition Agents 1728.4. Response of Enzyme-Based Biosensors 1758.5. Examples of Biosensor Configurations 1788.5.1. Ferrocene-Mediated Amperometric Glucose Sensor 1788.5.2. Potentiometric Biosensor for Phenyl Acetate 1808.5.3. Evanescent-Wave Fluorescence Biosensor for Bungarotoxin 1818.5.4. Optical Biosensor for Glucose Based on Fluorescence Resonance Energy Transfer 1838.5.5. Piezoelectric Sensor for Nucleic Acid Detection 1848.5.6. Enzyme Thermistors 1868.5.7. Fluorescence Sensor for Nitroaromatic Explosives Based on a Molecularly Imprinted Polymer 1878.5.8. Immunosensor Microwell Arrays from Gold Compact Disks 1888.5.9. Nanoparticle-Enhanced Detection of Thrombin by SPR 1908.5.10. Environmental BOD and Toxicity Biosensors Based on Viable Cells 1928.5.11. Detection of Viruses using a Surface Acoustic Wave (SAW) Biosensor 1938.5.12. MEMS Microcantilever Biosensor for Virus Detection 1968.5.13. DNA Microarrays 1988.6. Evaluation of Biosensor Perfomance 2018.7. In Vivo Applications of Biosensors 2028.7.1. Biocompatible Materials 2038.7.2. Physiological Environment of the Human Body 2038.7.3. The Artificial Pancreas 2058.7.4. An Enzymatic Fuel Cell as a Component of an Implanted Biosensing System 2058.7.5. Other Examples of Implantable Biosensors 206Suggested Reading 207References 207Problems 2099. Directed Evolution for the Design of Macromolecular Reagents 2109.1. Introduction 2109.2. Rational Design and Directed Evolution 2119.3. Generation of Genetic Diversity 2149.3.1. Polymerase Chain Reaction and Error-Prone PCR 2159.3.2. DNA Shuffling 2179.4. Linking Genotype and Phenotype 2179.4.1. Cell Expression and Cell Surface Display (In vivo) 2189.4.2. Phage Display (In vivo) 2189.4.3. Ribosome Display (In vitro) 2199.4.4. mRNA-Peptide Fusion (In vitro) 2209.4.5. Microcompartmentalization (In vitro) 2209.5. Identification and Selection of Successful Variants 2219.5.1. Identification of Successful Variants Based on Binding Properties 2229.5.2. Identification of Successful Variants Based on Catalytic Activity 2229.6. Examples of Directed Evolution Experiments 2249.6.1. Directed Evolution of Galactose Oxidase 2249.6.2. α-Hemolysin Evolution 225Suggested Reading 226References 226Problems 22710. Image-Based Bioanalysis 22910.1. Introduction 22910.2. Magnification and Resolution 23010.3. Optical Microscopy 23110.3.1. The Compound Light Microscope 23110.3.2. The Confocal Microscope 23110.3.3. Sample Preparation 23210.3.4. General and Selective Stains 23310.3.5. Fluorescence In situ Hybridization 23410.3.6. Green Fluorescent Protein and its Analogues 23410.4. Electron Microscopy 23410.4.1. Principles and Instrumentation 23410.4.2. Sample Preparation 23510.4.3. Transmission Electron Microscopy (TEM) 23610.4.4. Scanning Electron Microscopy (SEM) 23610.5. Scanning Tunneling Microscopy 23710.5.1. Principles and Instrumentation 23710.5.2. Biological Applications 23710.6. Atomic Force Microscopy (AFM) 23710.6.1. Cantilevers and Operational Modes 23710.6.2. Samples and Substrates 23910.6.3. Biological Applications 23910.6.4. Four-Dimensional (4D) Scanning 24010.7. Scanning Electrochemical Microscopy (SECM) 24010.7.1. Principles and Instrumentation 24010.7.2. Samples and Substrates 24110.7.3. Biological Applications 241Suggested Reading 242References 242Problems 24311. Principles of Electrophoresis 24411.1. Introduction 24411.2. Electrophoretic Support Media 24811.2.1. Paper 24811.2.2. Starch Gels 24911.2.3. Polyacrylamide Gels 25011.2.4. Agarose Gels 25411.2.5. Polyacrylamide-Agarose Gels 25411.3. Effect of Experimental Conditions Onelectrophoretic Separations 25411.4. Electric Field Strength Gradients 25511.5. Pulsed Field Gel Electrophoresis (PFGE) 25611.6. Detection of Proteins and Nucleic Acids After Electrophoretic Separation 25811.6.1. Stains and Dyes 25811.6.2. Detection of Enzymes by Substrate Staining 26011.6.3. The Southern Blot 26011.6.4. The Northern Blot 26211.6.5. The Western Blot 26211.6.6. Detection of DNA Fragments on Membranes with DNA Probes 263Suggested Reading 265References 266Problems 26612. Applications of Zone Electrophoresis 26812.1. Introduction 26812.2. Determination of Protein Net Charge and Molecular Weight Using PAGE 26812.3. Determination of Protein Subunit Composition and Subunit Molecular Weights 27012.4. Molecular Weight of DNA by Agarose Gel Electrophoresis 27212.5. Identification of Isoenzymes 27312.6. Diagnosis of Genetic (Inherited) Disorders 27412.7. DNA Fingerprinting and Restriction Fragment Length Polymorphism 27512.8. DNA Sequencing with the Maxam-Gilbert Method 27912.9. Immunoelectrophoresis 282Suggested Reading 287References 287Problems 28813. Isoelectric Focusing and 2D Electrophoresis 29013.1. Introduction 29013.2. Carrier Ampholytes 29113.3. Modern IEF with Carrier Ampholytes 29313.4. Immobilized pH Gradients (IPGs) 29613.5. Two-Dimensional Electrophoresis 29913.6. Difference Gel Electrophoresis (DIGE) 301Suggested Reading 303References 303Problems 30414. Capillary Electrophoresis 30614.1. Introduction 30614.2. Electroosmosis 30714.3. Elution of Sample Components 30814.4. Sample Introduction 30914.5. Detectors for Capillary Electrophoresis 31014.5.1. Laser-Induced Fluorescence Detection 31114.5.2. Mass Spectrometric Detection 31314.5.3. Amperometric Detection 31514.5.4. Radiochemical Detection 31814.6. Capillary Polyacrylamide Gel Electrophoresis (C-PAGE) 31914.7. Capillary Isoelectric Focusing (CIEF) 321Suggested Reading 322References 323Problems 32315. Centrifugation Methods 32515.1. Introduction 32515.2. Sedimentation and Relative Centrifugal g Force 32515.3. Centrifugal Forces in Different Rotor Types 32715.3.1. Swinging-Bucket Rotors 32715.3.2. Fixed-Angle Rotors 32815.3.3. Vertical Rotors 32815.4. Clearing Factor (K) 32915.5. Density Gradients 33015.5.1. Materials Used to Generate a Gradient 33115.5.2. Constructing Pre-Formed and Self-Generated Gradients 33115.5.3. Redistribution of the Gradient in Fixed-Angle and Vertical Rotors 33315.6. Types of Centrifugation Techniques 33315.6.1. Differential Centrifugation 33415.6.2. Rate-Zonal Centrifugation 33415.6.3. Isopycnic Centrifugation 33615.7. Harvesting Samples 33615.8. Analytical Ultracentrifugation 33615.8.1. Instrumentation 33715.8.2. Sedimentation Velocity Analysis 33815.8.3. Sedimentation Equilibrium Analysis 34115.9. Selected Examples 34215.9.1. Analytical Ultracentrifugation for Quaternary Structure Elucidation 34215.9.2. Isolation of Retroviruses by Self-Generated Gradients 34315.9.3. Isolation of Lipoproteins from Human Plasma 34415.9.4. Centrifugal Microfluidic Analysis 344Suggested Reading 346References 346Problems 34716.Chromatography of Biomolecules 34916.1. Introduction 34916.2. Units and Definitions 35016.3. Plate Theory of Chromatography 35016.4. Rate Theory of Chromatography 35116.5. Size Exclusion (Gel Filtration) Chromatography 35316.6. Stationary Phases For Size Exclusion Chromatography 35816.6.1. Particulate Gels 35816.6.2. Monolithic Stationary Phases 36016.7. Affinity Chromatography 36016.7.1. Immobilization of Affinity Ligands 36216.7.2. Elution Methods 36416.7.3. Determination of Association Constants by High Performance Affinity Chromatography 36416.8. Ion-exchange Chromatography 36816.8.1. Retention Model for Ion-Exchange Chromatography of Polyelectrolytes 36916.8.2. Further Advances in Ion-Exchange Chromatography 374Suggested Reading 374References 374Problems 37517. Mass Spectrometry of Biomolecules 37717.1. Introduction 37717.2. Basic Description of the Instrumentation 37917.2.1. Soft Ionization Sources 37917.2.1.1. Fast Atom/Ion Bombardment (FAB) 38017.2.1.2. Electrospray Ionization (ESI) 38017.2.1.3. Matrix-Assisted Laser Desorption/Ionization (MALDI) 38117.2.2. Mass Analyzers 38217.2.3. Detectors 38517.3. Interpretation of Mass Spectra 38617.4. Biomolecule Molecular Weight Determination 38817.5. Protein Identification 39217.6. Protein-Peptide Sequencing 39317.7. Nucleic Acid Applications 39717.8. Bacterial Mass Spectrometry 39817.9. Mass Spectrometry Imaging 399Suggested Reading 401References 401Problems 40218. Micro-TAS, Lab-on-a-Chip, and Microarray Devices 40418.1. Introduction 40418.2. Device Fabrication Materials and Methods 40518.3. Microfluidics 40518.3.1. Fluid Transport 40518.3.2. Valves and Reservoirs 40618.3.3. Mixing and Sample Separation 40618.4. Detectors 40718.5. Examples of Bioanalytical Devices 40718.5.1. DNA Separation Using a Nanofence Array Microfluidic Device 40818.5.2. Two Dimensional Electrophoresis on a Microfluidic Chip 40918.5.3. Microfluidic Antibody Capture for Single-Cell Proteomics 41018.5.4. Multiplexed PCR Amplification and DNA Detection on a Microfluidic Chip 41018.5.5. Silicone Protein Separation Chip Based on a Grafted Ion-Exchange Polymer 41118.5.6. Circular, Biofunctionalized PEG Microchannels for Cell Adhesion Studies 411Suggested Reading 412References 412Problems 41319. Validation of New Bioanalytical Methods 41419.1. Introduction 41419.2. Precision and Accuracy 41519.3. Mean and Variance 41619.4. Relative Standard Deviation and Other Precision Estimators 41719.4.1. Distribution of Errors and Confidence Limits 41819.4.2. Linear Regression and Calibration 41919.4.3. Precision Profiles 42019.4.4. Limit of Quantitiation and Detection 42119.4.5. Linearizing Sigmoidal Curves (Four-Parameter Log-Logit Model) 42219.4.6. Effective Dose Method 42319.5. Estimation of Accuracy 42419.5.1. Standardization 42419.5.2. Matrix Effects 42519.5.2.1. Recovery 42519.5.2.2. Parallelism 42619.5.3. Interferences 42619.6. Qualitative (Screening) Assays 42719.6.1. Figures of Merit for Qualitative (Screening) Assays 42719.7. Examples of Validation Procedures 42819.7.1. Validation of a Qualitative Antibiotic Susceptibility Test 42819.7.2. Measurement of Plasma Homocysteine by Fluorescence Polarization Immunoassay (FPIA) Methodology 42919.7.3. Determination of Enzymatic Activity of β-Galactosidase 43319.7.4. Establishment of a Cutoff Value for Semi-Quantitative Assays for Cannabinoids 434Suggested Reading 435References 436Answers to Selected Problems 437Index 449