Enzymes as Sensors

Chapter One: Recent Advances in Development of Genetically Encoded Fluorescent Sensors

• Abstract
• 1 Introduction
• 2 Fluorescent Proteins
• 3 Sensor Platforms
• 4 Types of Sensors
• 5 Conclusion

Chapter Two: Engineering Rugged Field Assays to Detect Hazardous Chemicals Using Spore-Based Bacterial Biosensors

• Abstract
• 1 Introduction
• 2 Whole Cell-Based Biosensors
• 3 Design, Creation, and Use of WCBs
• 4 The Development of Spore-Based Bacterial Biosensors
• 5 Challenges, Innovations, and Future Directions
• 6 Conclusions
• Acknowledgments

Chapter Three: Engineering BRET-Sensor Proteins

• Abstract
• 1 Introduction
• 2 LUCIDs and LUMABS: BRET-Sensors for Diagnostic Applications
• 3 BRET/FRET-Sensors for Intracellular Measurements
• 4 A Bright Future for BRET-Sensors
• Acknowledgments

Chapter Four: Enzymes as Sensors

• Abstract
• 1 Introduction
• 2 Optical Biosensors
• 3 Label-Free Detection and Fluorescence-Based Detection
• 4 Why Choose Biomolecules for Sensing?
• 5 Enzymes as MREs
• 6 Thermophilic Enzymes
• 7 Biological Engineering
• 8 Conclusions
• Acknowledgments

Chapter Five: Integrated Strategies to Gain a Systems-Level View of Dynamic Signaling Networks

• Abstract
• 1 Introduction
• 2 Strategies to Characterize Global Changes in the Phosphorylation Status of Cellular Proteins
• 3 Strategies to Track Kinase and Phosphatase Activity Profiles in Living Cells
• 4 Conclusions
• Acknowledgments

Chapter Six: Probing Cdc42 Polarization Dynamics in Budding Yeast Using a Biosensor

• Abstract
• 1 Introduction
• 2 Generation of a Biosensor of Active Cdc42
• 3 Detection and Quantification of Cdc42 Activation In Vivo
• 4 Application of the Cdc42 Biosensor
• 5 Concluding Remarks
• Acknowledgments

Chapter Seven: Novel Fluorescence-Based Biosensors Incorporating Unnatural Amino Acids

• Abstract
• 1 Introduction
• 2 Site-Specific Incorporation of Unnatural Amino Acids Into Proteins in Live Cells
• 3 An Unnatural Fluorescent Protein Biosensor for Hydrogen Peroxide
• 4 Concluding Remarks
• Acknowledgments

Chapter Eight: Folding- and Dynamics-Based Electrochemical DNA Sensors

• Abstract
• 1 Introduction
• 2 “Signal-Off” E-DNA Sensors
• 3 “Signal-On” E-DNA Sensors
• 4 Alternate Electrode Substrates
• 5 Alternate Sensor Fabrication Approaches
• 6 Conclusion and Outlook
• Acknowledgment

Chapter Nine: Construction of Protein-Based Biosensors Using Ligand-Directed Chemistry for Detecting Analyte Binding

• Abstract
• 1 Introduction
• 2 Expression of GABA_AR Consisting of α/β/γ Subunits in HEK293T Cells
• 3 Design and Synthesis of Labeling Reagents for GABA_AR
• 4 Chemical Labeling of GABA_AR Using LDAI Reagent
• 5 Construction of Fluorescent Biosensors Using Labeled GABA_ARs
• 6 Quantitative Analysis of Ligand Affinity Using BFQR-Based Biosensors
• 7 Summary and Future Directions
• Acknowledgments

Chapter Ten: Measuring and Imaging Metal Ions With Fluorescence-Based Biosensors: Speciation, Selectivity, Kinetics, and Other Issues

• Abstract
• 1 Introduction
• 2 Speciation of Metal Ions of Biological Interest
• 3 Metal Ion Buffers and Their Formulation
• 4 Selectivity of Metal Ion Binding to Indicators and Its Assessment
• 5 Advantages of Biomolecules for Metal Ion Sensing
• 6 Kinetics and Mechanism of Metal Ion Binding
• 7 Distribution of Metal Ions Throughout the Cell and Organism
• 8 Conclusion

Chapter Eleven: Bioapplications of Electrochemical Sensors and Biosensors

• Abstract
• 1 Introduction
• 2 Electrochemical Sensing in Biological Systems
• 3 Recent Advancements in Electrode Designs for Biological Monitoring
• 4 Conclusions and Future Trends
• Acknowledgments

Chapter Twelve: A Highly Sensitive Biosensor for ATP Using a Chimeric Firefly Luciferase

• Abstract
• 1 Introduction
• 2 ATP Detection Protocol
• 3 Conclusions
• Acknowledgments

Chapter Thirteen: Highly Modular Bioluminescent Sensors for Small Molecules and Proteins

• Abstract
• 1 Introduction
• 2 Sensing Small Molecules
• 3 Sensing Proteins
• 4 Toward Application in POC Settings
• 5 Conclusions

Chapter Fourteen: Sensitive Protein Detection and Quantification in Paper-Based Microfluidics for the Point of Care

• Abstract
• 1 Introduction
• 2 Paper-Based Devices and Sandwich Lateral Flow Assays
• 3 Assay Analysis Framework
• 4 Binding Kinetics in Porous Networks
• 5 Mechanisms for Protein Binding
• 6 Optical Methods in Paper
• 7 Sensitivity Enhancements (LOD)
• 8 Summary and Next Steps

Chapter Fifteen: Microneedle Enzyme Sensor Arrays for Continuous In Vivo Monitoring

• Abstract
• 1 Introduction
• 2 Approaches to the Fabrication of Microneedles for Sensing
• 3 Functionalization of Microneedles for Sensing
• 4 Methods
• Acknowledgments

Chapter Sixteen: Visualization of the Genomic Loci That Are Bound by Specific Multiprotein Complexes by Bimolecular Fluorescence Complementation Analysis on Drosophila Polytene Chromosomes

• Abstract
• 1 Introduction
• 2 Design of Fusion Proteins for BiFC Analysis
• 3 Construction and Testing of Plasmid Vectors for BiFC Analysis
• 4 Production of Drosophila Lines That Express BiFC Fusion Proteins
• 5 Comparison of the Levels of BiFC Fusion Protein Expression
• 6 Preparation of Drosophila Larvae for Salivary Gland Isolation
• 7 Preparation of Polytene Chromosome Spreads
• 8 Staining and Imaging of Polytene Chromosomes
• 9 Interpretation of BiFC Complex Binding on Polytene Chromosomes
• 10 Strategies to Limit the Artifacts Caused by the Differences Between Fusion Proteins and Endogenous Counterparts
• 11 Effects of BiFC Complex Formation on the Specificity of Chromatin Binding
• 12 Materials and Solutions