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 xix

Preface to First Edition xxi

Acknowledgments xxiii

1. Quantitative Instrumental Measurements 1

1.1. Introduction 1

1.2. Optical Measurements 2

1.2.1. UV-Visible Absorbance 3

1.2.2. Turbidimetry (Light-Scattering) 5

1.2.3. Fluorescence 5

1.2.4. Chemiluminescence and Bioluminescence 7

1.3. Electrochemical Measurements 8

1.3.1. Potentiometry 10

1.3.2. Amperometry 10

1.3.3. Impedimetry 11

1.4. Radiochemical Measurements 12

1.4.1. Scintillation Counting 12

1.4.2. Geiger Counting 12

1.5. Surface Plasmon Resonance 13

1.6. Calorimetry 14

1.6.1. Differential Scanning Calorimetry (DSC) 15

1.6.2. Isothermal Titration Calorimetry (ITC) 16

1.7. Automation: Microplates, Multiwell Liquid Dispensers and Microplate Readers 16

1.8. Calibration of Instrumental Measurements 18

1.8.1. External Standards 18

1.8.2. Internal Standards 19

1.8.3. Standard Additions 20

1.9. Quantitative and Semi-Quantitative Measurements 21

1.9.1. Exact Concentration 21

1.9.2. Positive or Negative Result 21

Suggested Reading 22

Problems 22

2. Spectroscopic Methods for the Quantitation of Classes of Biomolecules 23

2.1. Introduction 23

2.2. Total Protein 24

2.2.1. Lowry Method 24

2.2.2. Smith (BCA) Method 24

2.2.3. Bradford Method 26

2.2.4. Ninhydrin-Based Assay 27

2.2.5. Other Protein Quantitation Methods 28

2.3. Total DNA 31

2.3.1. Diaminobenzoic Acid (DABA) Method 32

2.3.2. Diphenylamine (DPA) Method 32

2.3.3. Other Fluorimetric Methods 33

2.4. Total RNA 34

2.5. Total Carbohydrate 35

2.5.1. Ferricyanide Method 35

2.5.2. Phenol-Sulfuric Acid Method 36

2.5.3. 2-Aminothiophenol Method 36

2.5.4. Purpald Assay for Bacterial Polysaccharides 37

2.6. Free Fatty Acids 37

References 38

Problems 39

3. Enzymes 41

3.1. Introduction 41

3.2. Enzyme Nomenclature 42

3.3. Enzyme Commission Numbers 43

3.4. Enzymes in Bioanalytical Chemistry 45

3.5. Enzyme Kinetics 46

3.5.1. Simple One-Substrate Enzyme Kinetics 48

3.5.2. Experimental Determination of Michaelis-Menten Parameters 50

3.5.2.1. Eadie-Hofstee Method 50

3.5.2.2. Hanes Method 50

3.5.2.3. Lineweaver-Burk Method 51

3.5.2.4. Cornish-Bowden-Eisenthal Method 52

3.5.3. Comparison of Methods for the Determination of KM Values 52

3.5.4. One-Substrate, Two-Product Enzyme Kinetics 54

3.5.5. Two-Substrate Enzyme Kinetics 54

3.5.6. Examples of Enzyme-Catalyzed Reactions and their Treatment 56

3.5.7. Curve Fitting for Enzyme Kinetic Data 57

3.6. Enzyme Activators 58

3.7. Enzyme Inhibitors 59

3.7.1. Competitive Inhibition 60

3.7.2. Noncompetitive Inhibition 60

3.7.3. Uncompetitive Inhibition 62

3.8. Enzyme Units and Concentrations 62

Suggested Reading 64

References 64

Problems 64

4. Quantitation of Enzymes and Their Substrates 67

4.1. Introduction 67

4.2. Substrate Depletion or Product Accumulation 68

4.3. Direct and Coupled Measurements 69

4.4. Classification of Methods 71

4.5. Instrumental Methods 73

4.5.1. Optical Detection 73

4.5.1.1. Absorbance 73

4.5.1.2. Fluorescence 75

4.5.1.3. Luminescence 77

4.5.1.4. Nephelometry 79

4.5.2. Electrochemical Detection 79

4.5.2.1. Amperometry 79

4.5.2.2. Potentiometry 80

4.5.2.3. Conductimetry 80

4.5.3. Other Detection Methods 81

4.5.3.1. Radiochemical 81

4.5.3.2. Manometry 81

4.5.3.3. Calorimetry 82

4.6. High-Throughput Assays for Enzymes and Inhibitors 82

4.7. Assays for Enzymatic Reporter Gene Products 84

4.8. Practical Considerations for Enzymatic Assays 85

Suggested Reading 86

References 86

Problems 87

5. Immobilized Enzymes 90

5.1. Introduction 90

5.2. Immobilization Methods 90

5.2.1. Nonpolymerizing Covalent Immobilization 91

5.2.1.1. Controlled-Pore Glass 92

5.2.1.2. Polysaccharides 93

5.2.1.3. Polyacrylamide 95

5.2.1.4. Acidic Supports 95

5.2.1.5. Anhydride Groups 96

5.2.1.6. Thiol Groups 97

5.2.2. Crosslinking with Bifunctional Reagents 97

5.2.3. Adsorption 98

5.2.4. Entrapment 99

5.2.5. Microencapsulation 100

5.3. Properties of Immobilized Enzymes 101

5.4. Immobilized Enzyme Reactors 107

5.5. Theoretical Treatment of Packed-Bed Enzyme Reactors 109

Suggested Reading 113

References 113

Problems 114

6. Antibodies 117

6.1. Introduction 117

6.2. Structural and Functional Properties of Antibodies 118

6.3. Polyclonal and Monoclonal Antibodies 121

6.4. Antibody-Antigen Interactions 122

6.5. Analytical Applications of Secondary Antibody-Antigen Interactions 124

6.5.1. Agglutination Reactions 124

6.5.2. Precipitation Reactions 126

Suggested Reading 129

References 129

Problems 129

7. Quantitative Immunoassays with Labels 131

7.1. Introduction 131

7.2. Labeling Reactions 132

7.3. Heterogeneous Immunoassays 134

7.3.1. Labeled-Antibody Methods 136

7.3.2. Labeled-Ligand Assays 136

7.3.3. Radioisotopes 139

7.3.4. Fluorophores 139

7.3.4.1. Indirect Fluorescence 140

7.3.4.2. Competitive Fluorescence 140

7.3.4.3. Sandwich Fluorescence 140

7.3.4.4. Fluorescence Excitation Transfer 140

7.3.4.5. Time-Resolved Fluorescence 141

7.3.5. Quantum Dots 142

7.3.6. Chemiluminescent Labels 143

7.3.7. Enzyme Labels 145

7.3.8. Lateral Flow Immunoassay 148

7.4. Homogeneous Immunoassays 149

7.4.1. Fluorescent Labels 149

7.4.1.1. Enhancement Fluorescence 149

7.4.1.2. Direct Quenching Fluorescence 150

7.4.1.3. Indirect Quenching Fluorescence 150

7.4.1.4. Fluorescence Polarization Immunoassay 151

7.4.1.5. Fluorescence Excitation Transfer 151

7.4.2. Enzyme Labels 152

7.4.2.1. Enzyme-Multiplied Immunoassay Technique 152

7.4.2.2. Substrate-Labelled Fluorescein Immunoassay 153

7.4.2.3. Apoenzyme Reactivation Immunoassay 153

7.4.2.4. Cloned Enzyme Donor Immunoassay 154

7.4.2.5. Enzyme Inhibitory Homogeneous Immunoassay 154

7.5. Evaluation of New Immunoassay Methods 155

Suggested Reading 160

References 160

Problems 161

8. Biosensors 166

8.1. Introduction 166

8.2. Biosensor Diversity and Classification 169

8.3. Recognition Agents 171

8.3.1. Natural Recognition Agents 171

8.3.2. Artificial Recognition Agents 172

8.4. Response of Enzyme-Based Biosensors 175

8.5. Examples of Biosensor Configurations 178

8.5.1. Ferrocene-Mediated Amperometric Glucose Sensor 178

8.5.2. Potentiometric Biosensor for Phenyl Acetate 180

8.5.3. Evanescent-Wave Fluorescence Biosensor for Bungarotoxin 181

8.5.4. Optical Biosensor for Glucose Based on Fluorescence Resonance Energy Transfer 183

8.5.5. Piezoelectric Sensor for Nucleic Acid Detection 184

8.5.6. Enzyme Thermistors 186

8.5.7. Fluorescence Sensor for Nitroaromatic Explosives Based on a Molecularly Imprinted Polymer 187

8.5.8. Immunosensor Microwell Arrays from Gold Compact Disks 188

8.5.9. Nanoparticle-Enhanced Detection of Thrombin by SPR 190

8.5.10. Environmental BOD and Toxicity Biosensors Based on Viable Cells 192

8.5.11. Detection of Viruses using a Surface Acoustic Wave (SAW) Biosensor 193

8.5.12. MEMS Microcantilever Biosensor for Virus Detection 196

8.5.13. DNA Microarrays 198

8.6. Evaluation of Biosensor Perfomance 201

8.7. In Vivo Applications of Biosensors 202

8.7.1. Biocompatible Materials 203

8.7.2. Physiological Environment of the Human Body 203

8.7.3. The Artificial Pancreas 205

8.7.4. An Enzymatic Fuel Cell as a Component of an Implanted Biosensing System 205

8.7.5. Other Examples of Implantable Biosensors 206

Suggested Reading 207

References 207

Problems 209

9. Directed Evolution for the Design of Macromolecular Reagents 210

9.1. Introduction 210

9.2. Rational Design and Directed Evolution 211

9.3. Generation of Genetic Diversity 214

9.3.1. Polymerase Chain Reaction and Error-Prone PCR 215

9.3.2. DNA Shuffling 217

9.4. Linking Genotype and Phenotype 217

9.4.1. Cell Expression and Cell Surface Display (In vivo) 218

9.4.2. Phage Display (In vivo) 218

9.4.3. Ribosome Display (In vitro) 219

9.4.4. mRNA-Peptide Fusion (In vitro) 220

9.4.5. Microcompartmentalization (In vitro) 220

9.5. Identification and Selection of Successful Variants 221

9.5.1. Identification of Successful Variants Based on Binding Properties 222

9.5.2. Identification of Successful Variants Based on Catalytic Activity 222

9.6. Examples of Directed Evolution Experiments 224

9.6.1. Directed Evolution of Galactose Oxidase 224

9.6.2. α-Hemolysin Evolution 225

Suggested Reading 226

References 226

Problems 227

10. Image-Based Bioanalysis 229

10.1. Introduction 229

10.2. Magnification and Resolution 230

10.3. Optical Microscopy 231

10.3.1. The Compound Light Microscope 231

10.3.2. The Confocal Microscope 231

10.3.3. Sample Preparation 232

10.3.4. General and Selective Stains 233

10.3.5. Fluorescence In situ Hybridization 234

10.3.6. Green Fluorescent Protein and its Analogues 234

10.4. Electron Microscopy 234

10.4.1. Principles and Instrumentation 234

10.4.2. Sample Preparation 235

10.4.3. Transmission Electron Microscopy (TEM) 236

10.4.4. Scanning Electron Microscopy (SEM) 236

10.5. Scanning Tunneling Microscopy 237

10.5.1. Principles and Instrumentation 237

10.5.2. Biological Applications 237

10.6. Atomic Force Microscopy (AFM) 237

10.6.1. Cantilevers and Operational Modes 237

10.6.2. Samples and Substrates 239

10.6.3. Biological Applications 239

10.6.4. Four-Dimensional (4D) Scanning 240

10.7. Scanning Electrochemical Microscopy (SECM) 240

10.7.1. Principles and Instrumentation 240

10.7.2. Samples and Substrates 241

10.7.3. Biological Applications 241

Suggested Reading 242

References 242

Problems 243

11. Principles of Electrophoresis 244

11.1. Introduction 244

11.2. Electrophoretic Support Media 248

11.2.1. Paper 248

11.2.2. Starch Gels 249

11.2.3. Polyacrylamide Gels 250

11.2.4. Agarose Gels 254

11.2.5. Polyacrylamide-Agarose Gels 254

11.3. Effect of Experimental Conditions Onelectrophoretic Separations 254

11.4. Electric Field Strength Gradients 255

11.5. Pulsed Field Gel Electrophoresis (PFGE) 256

11.6. Detection of Proteins and Nucleic Acids After Electrophoretic Separation 258

11.6.1. Stains and Dyes 258

11.6.2. Detection of Enzymes by Substrate Staining 260

11.6.3. The Southern Blot 260

11.6.4. The Northern Blot 262

11.6.5. The Western Blot 262

11.6.6. Detection of DNA Fragments on Membranes with DNA Probes 263

Suggested Reading 265

References 266

Problems 266

12. Applications of Zone Electrophoresis 268

12.1. Introduction 268

12.2. Determination of Protein Net Charge and Molecular Weight Using PAGE 268

12.3. Determination of Protein Subunit Composition and Subunit Molecular Weights 270

12.4. Molecular Weight of DNA by Agarose Gel Electrophoresis 272

12.5. Identification of Isoenzymes 273

12.6. Diagnosis of Genetic (Inherited) Disorders 274

12.7. DNA Fingerprinting and Restriction Fragment Length Polymorphism 275

12.8. DNA Sequencing with the Maxam-Gilbert Method 279

12.9. Immunoelectrophoresis 282

Suggested Reading 287

References 287

Problems 288

13. Isoelectric Focusing and 2D Electrophoresis 290

13.1. Introduction 290

13.2. Carrier Ampholytes 291

13.3. Modern IEF with Carrier Ampholytes 293

13.4. Immobilized pH Gradients (IPGs) 296

13.5. Two-Dimensional Electrophoresis 299

13.6. Difference Gel Electrophoresis (DIGE) 301

Suggested Reading 303

References 303

Problems 304

14. Capillary Electrophoresis 306

14.1. Introduction 306

14.2. Electroosmosis 307

14.3. Elution of Sample Components 308

14.4. Sample Introduction 309

14.5. Detectors for Capillary Electrophoresis 310

14.5.1. Laser-Induced Fluorescence Detection 311

14.5.2. Mass Spectrometric Detection 313

14.5.3. Amperometric Detection 315

14.5.4. Radiochemical Detection 318

14.6. Capillary Polyacrylamide Gel Electrophoresis (C-PAGE) 319

14.7. Capillary Isoelectric Focusing (CIEF) 321

Suggested Reading 322

References 323

Problems 323

15. Centrifugation Methods 325

15.1. Introduction 325

15.2. Sedimentation and Relative Centrifugal g Force 325

15.3. Centrifugal Forces in Different Rotor Types 327

15.3.1. Swinging-Bucket Rotors 327

15.3.2. Fixed-Angle Rotors 328

15.3.3. Vertical Rotors 328

15.4. Clearing Factor (K) 329

15.5. Density Gradients 330

15.5.1. Materials Used to Generate a Gradient 331

15.5.2. Constructing Pre-Formed and Self-Generated Gradients 331

15.5.3. Redistribution of the Gradient in Fixed-Angle and Vertical Rotors 333

15.6. Types of Centrifugation Techniques 333

15.6.1. Differential Centrifugation 334

15.6.2. Rate-Zonal Centrifugation 334

15.6.3. Isopycnic Centrifugation 336

15.7. Harvesting Samples 336

15.8. Analytical Ultracentrifugation 336

15.8.1. Instrumentation 337

15.8.2. Sedimentation Velocity Analysis 338

15.8.3. Sedimentation Equilibrium Analysis 341

15.9. Selected Examples 342

15.9.1. Analytical Ultracentrifugation for Quaternary Structure Elucidation 342

15.9.2. Isolation of Retroviruses by Self-Generated Gradients 343

15.9.3. Isolation of Lipoproteins from Human Plasma 344

15.9.4. Centrifugal Microfluidic Analysis 344

Suggested Reading 346

References 346

Problems 347

16.Chromatography of Biomolecules 349

16.1. Introduction 349

16.2. Units and Definitions 350

16.3. Plate Theory of Chromatography 350

16.4. Rate Theory of Chromatography 351

16.5. Size Exclusion (Gel Filtration) Chromatography 353

16.6. Stationary Phases For Size Exclusion Chromatography 358

16.6.1. Particulate Gels 358

16.6.2. Monolithic Stationary Phases 360

16.7. Affinity Chromatography 360

16.7.1. Immobilization of Affinity Ligands 362

16.7.2. Elution Methods 364

16.7.3. Determination of Association Constants by High Performance Affinity Chromatography 364

16.8. Ion-exchange Chromatography 368

16.8.1. Retention Model for Ion-Exchange Chromatography of Polyelectrolytes 369

16.8.2. Further Advances in Ion-Exchange Chromatography 374

Suggested Reading 374

References 374

Problems 375

17. Mass Spectrometry of Biomolecules 377

17.1. Introduction 377

17.2. Basic Description of the Instrumentation 379

17.2.1. Soft Ionization Sources 379

17.2.1.1. Fast Atom/Ion Bombardment (FAB) 380

17.2.1.2. Electrospray Ionization (ESI) 380

17.2.1.3. Matrix-Assisted Laser Desorption/Ionization (MALDI) 381

17.2.2. Mass Analyzers 382

17.2.3. Detectors 385

17.3. Interpretation of Mass Spectra 386

17.4. Biomolecule Molecular Weight Determination 388

17.5. Protein Identification 392

17.6. Protein-Peptide Sequencing 393

17.7. Nucleic Acid Applications 397

17.8. Bacterial Mass Spectrometry 398

17.9. Mass Spectrometry Imaging 399

Suggested Reading 401

References 401

Problems 402

18. Micro-TAS, Lab-on-a-Chip, and Microarray Devices 404

18.1. Introduction 404

18.2. Device Fabrication Materials and Methods 405

18.3. Microfluidics 405

18.3.1. Fluid Transport 405

18.3.2. Valves and Reservoirs 406

18.3.3. Mixing and Sample Separation 406

18.4. Detectors 407

18.5. Examples of Bioanalytical Devices 407

18.5.1. DNA Separation Using a Nanofence Array Microfluidic Device 408

18.5.2. Two Dimensional Electrophoresis on a Microfluidic Chip 409

18.5.3. Microfluidic Antibody Capture for Single-Cell Proteomics 410

18.5.4. Multiplexed PCR Amplification and DNA Detection on a Microfluidic Chip 410

18.5.5. Silicone Protein Separation Chip Based on a Grafted Ion-Exchange Polymer 411

18.5.6. Circular, Biofunctionalized PEG Microchannels for Cell Adhesion Studies 411

Suggested Reading 412

References 412

Problems 413

19. Validation of New Bioanalytical Methods 414

19.1. Introduction 414

19.2. Precision and Accuracy 415

19.3. Mean and Variance 416

19.4. Relative Standard Deviation and Other Precision Estimators 417

19.4.1. Distribution of Errors and Confidence Limits 418

19.4.2. Linear Regression and Calibration 419

19.4.3. Precision Profiles 420

19.4.4. Limit of Quantitiation and Detection 421

19.4.5. Linearizing Sigmoidal Curves (Four-Parameter Log-Logit Model) 422

19.4.6. Effective Dose Method 423

19.5. Estimation of Accuracy 424

19.5.1. Standardization 424

19.5.2. Matrix Effects 425

19.5.2.1. Recovery 425

19.5.2.2. Parallelism 426

19.5.3. Interferences 426

19.6. Qualitative (Screening) Assays 427

19.6.1. Figures of Merit for Qualitative (Screening) Assays 427

19.7. Examples of Validation Procedures 428

19.7.1. Validation of a Qualitative Antibiotic Susceptibility Test 428

19.7.2. Measurement of Plasma Homocysteine by Fluorescence Polarization Immunoassay (FPIA) Methodology 429

19.7.3. Determination of Enzymatic Activity of β-Galactosidase 433

19.7.4. Establishment of a Cutoff Value for Semi-Quantitative Assays for Cannabinoids 434

Suggested Reading 435

References 436

Answers to Selected Problems 437

Index 449