Bioanalytical Chemistry

Mikkelsen, S. — Cortón, E.

2ª Edición Abril 2016

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488 pags

789 gr

16 x 24 x 3 cm

ISBN 9781118302545

Editorial WILEY

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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

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