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Single Biomolecule Detection and Analysis: Concepts, Applications, and Future Prospects

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This collection discusses various micro/nanodevice design and fabrication for single-biomolecules detection. It will be an ideal reference text for graduate students and professionals in diverse subject areas including materials science, biomedical engineering, chemical engineering, mechanical engineering, and nanoscience.

This book-

    • Discusses techniques of single-biomolecule detection, their advantages, limitations, and applications.

    • Covers comprehensively several electrochemical detection techniques.

    • Provides single-molecule separation, sensing, imaging, sequencing, and analysis in detail.

    • Examines different types of cantilever-based biomolecule sensing, and its limitations.

    Single Biomolecule Detection and Analysis covers single-biomolecule detection and characterization using micro/nanotechnologies and micro/nanofluidic devices, electrical and magnetic detection technologies, microscopy and spectroscopy techniques, single biomolecule optical, and nanopore devices. The text covers key important biosensors-based detection, stochastic optical reconstruction microscopy-based detection, electrochemical detection, metabolic engineering of animal cells, single-molecule intracellular delivery and tracking, terahertz spectroscopy-based detection, total internal reflection fluorescence (TIFR) detection, and Fluorescence Correlation Spectroscopy (FCS) detection. The text will be useful for graduate students and professionals in diverse subject areas including materials science, biomedical engineering, chemical engineering, mechanical engineering, and nanoscience. Discussing chemical process, physical process, separation, sensing, imaging, sequencing, and analysis of single-molecule detection, this text will be useful for graduate students and professionals in diverse subject areas including materials science, biomedical engineering, chemical engineering, mechanical engineering, and nanoscience. It covers microscopy and spectroscopy techniques for single-biomolecule detection, analysis, and their biomedical engineering applications.

    Author(s): Tuhin Subhra Santra, Fan-Gang Tseng
    Publisher: CRC Press
    Year: 2023

    Language: English
    Pages: 354
    City: Boca Raton

    Cover
    Half Title
    Title Page
    Copyright Page
    Table of Contents
    Acknowledgements
    Preface
    Editors
    List of Contributors
    Chapter 1 Microfluidics-Based DNA Detection
    1.1 Introduction
    1.2 Design of Microfluidics Device
    1.3 Materials for Microfluidics
    1.4 DNA Structure
    1.5 DNA Extraction
    1.5.1 Cell Lysis
    1.5.2 DNA Purification
    1.5.2.1 Liquid-Phase Purification
    1.5.2.2 Solid-Phase Purification
    1.5.2.3 Validation of DNA Extract
    1.5.2.4 Summary
    1.6 Microfluidic DNA Detection Using DNA Separation
    1.7 Microfluidic DNA Detection Using Affinity Probes
    1.7.1 Synthesis of DNA Probes
    1.7.1.1 Phosphoramidite Method
    1.7.1.2 Column-Based Oligonucleotide Synthesis
    1.7.1.3 Microarray Based Oligonucleotide Synthesis
    1.7.2 Signal Generation
    1.7.2.1 Optical
    1.7.2.2 Electrochemistry
    1.7.2.3 Other Signaling Platforms
    1.7.3 Signal Amplification
    1.7.3.1 Enzyme-mediated Methods with Thermocycles
    1.7.3.2 Enzyme-Mediated Methods with Isothermal Process
    1.7.4 Loop-Mediated Isothermal Amplification
    1.7.4.1 Enzyme Free Methods
    1.8 Conclusions
    References
    Chapter 2 Single-Molecule Detection by Solid-State Nanopores
    2.1 Nanopore Technologies
    2.1.1 Introduction
    2.1.2 Types of Solid-State Nanopores
    2.1.3 Fabrication of Solid-State Nanopores
    2.1.4 Single-Molecule Detection by Solid-State Nanopores
    2.2 Fundamental Electrokinetic Transport Phenomena in Solid-State Nanopores
    2.2.1 Electrostatics
    2.2.2 Electric Double Layer
    2.2.3 Electroosmosis in a Cylindrical Pore and Pore Conductance
    2.2.4 Electrophoresis
    2.2.5 Steric Effects, Dielectric Saturation, and Viscoelectric Effects
    2.2.6 Computational Simulation
    2.3 Nanopore Electrokinetics in the Presence of a Concentration Gradient
    2.3.1 Salt Concentration Gradients in Nanopores
    2.3.2 Nonuniform Electroosmotic Flow (EOF)
    2.3.3 Transport-Induced-Charge Electroosmosis
    2.3.4 Diffusiophoresis and Electrodiffusiophoresis
    2.4 Conclusions and Future Outlook
    Acknowledgement
    References
    Chapter 3 Confocal Microscope-Based Detection
    3.1 Introduction
    3.2 Laser Scanning Confocal Microscope (LSCM)
    3.2.1 Fluorescent Laser Scanning Confocal Microscope
    3.2.1.1 Two-Photon and Multi-Photon FLSCM
    3.2.1.2 Fiber-Based and Miniaturized FLSCM
    3.2.1.3 Super-Resolution FLSCM
    3.2.2 Label-Free Laser Scanning Confocal Microscope (LLSCM)
    3.2.2.1 Laser Scanning Coherent Raman Scattering Microscope (LSCRSM)
    3.2.2.2 Second Harmonic Generation Microscope
    3.2.2.3 Summary and Prospect
    3.2.3 Laser Scanning Confocal Microscope for Single-Molecule Detection
    3.3 Fluorescence Resonance Energy Transfer Based on Confocal Microscope
    3.3.1 Principle of FRET
    3.3.2 Single Molecule Fluorescence Resonance Energy Transfer Measurement
    3.3.3 Alternating Laser Excitation smFRET
    3.3.4 High Order FRET
    3.3.5 Application of smFRET
    3.3.5.1 DNA (Holliday Junction, Flip-Flop Motion, Branch Migration)
    3.3.5.2 Protein (Protein Folding, Protein Motor (Movement of Myosin's Heads and Kinesin's Neck Linker))
    3.3.5.3 Enzymatic Reaction
    3.3.6 Advantages and Disadvantages
    3.4 Conclusions and Prospect
    Acknowledgment
    References
    Chapter 4 Flow Cytometry-Based Detection
    4.1 Introduction
    4.1.1 Flow Cytometry and Principles of "Single-Cell Analysis"
    4.1.2 Early History and Evolution of Cytometry
    4.1.3 High-Speed, Multiparameter Flow Cytometry
    4.1.4 Time-Resolved Cytometry
    4.1.5 Microfluidic Cytometry
    4.1.6 CyTOF
    4.2 Flow Cytometry and Single-Cell Analysis Technologies
    4.2.1 Optics
    4.2.2 Optical Measurements on Single Cells
    4.2.3 "Label-Free" Detection of Cells
    4.2.3.1 Conventional Light Scatter
    4.2.3.2 Multi-Angle Light Scatter
    4.2.3.3 Impedance Measurements of Electronic Cell Volume
    4.2.3.4 Fluorescence
    4.3 Fluorescent Probe Technologies
    4.3.1 Fluorophores
    4.3.1.1 Conventional Fluorophores
    4.3.1.2 New "Designer" Fluorophores
    4.3.1.3 Energy Transfer Probes
    4.3.1.4 SERS
    4.3.2 Fluorescent Probes
    4.3.2.1 DNA Dyes
    4.3.2.2 Other Fluorescent Stains
    4.3.2.3 Antibodies
    4.3.2.4 "Nanobodies"
    4.3.2.5 Peptides, Aptamers, etc.
    4.3.2.6 Nanoparticles
    4.4 Fluidics
    4.4.1 Conventional Hydrodynamic Focusing
    4.4.2 Orienting Fluid Flow
    4.4.3 Acoustic Focusing
    4.4.4 High-speed Flow Cytometry
    4.4.5 In-Vivo Flow Cytometry
    4.4.6 Microfluidics
    4.5 Light Excitation Sources
    4.5.1 Arc-Lamp Systems
    4.5.2 Lasers
    4.5.2.1 Water-Cooled Gas Systems
    4.5.2.2 Dye Lasers
    4.5.2.3 Semiconductor Lasers
    4.5.2.4 Polarized or Unpolarized?
    4.5.2.5 Constant Power Lasers or Pulsed Lasers?
    4.5.2.6 Beam Shaping
    4.5.3 LEDs
    4.5.3.1 Superluminescent LEDs
    4.5.3.2 Multispectral Superluminescent LEDs
    4.6 Detectors
    4.6.1 Photodiodes
    4.6.2 PMTs
    4.6.3 SiPMTs
    4.6.4 Multispectral Detectors
    4.6.5 "Imaging-In-Flow"
    4.7 Signal Processing
    4.7.1 Old Analog-to-Digital Systems
    4.7.2 Digital Signal Processing Chips
    4.8 Data Analysis
    4.8.1 Conventional Data Analysis
    4.8.2 Bioinformatic Analyses
    4.9 Cell Sorting
    4.9.1 Mechanical Cell Sorting
    4.9.2 Conventional "Ink-Jet" Droplet Sorting of CellSubsets
    4.9.3 Single-Cell Sorting (Stovel and Sweet, 1979) for Cell Separation of Complex Mixtures of Cells
    4.9.4 Fluidic Switching
    4.9.5 On-Chip Magnetic Sorting
    4.10 Biological and Clinical Applications
    4.10.1 DNA
    4.10.1.1 Cell Cycle Analysis
    4.10.1.2 Ploidy
    4.10.1.3 Chromosome Analyses
    4.10.2 Detection of Molecules on the Surface of Cells
    4.10.2.1 Antibody Detection of "Biomarkers"
    4.10.2.2 Use of Peptides and Aptamers
    4.10.2.3 Multicolor, High-Dimensionality Detection of Phenotypes
    4.10.2.4 Immunophenotyping of Cancers in the Clinic
    4.10.2.5 Minimal Residual Disease Monitoring
    4.10.3 Detection of Intracellular Molecules
    4.10.4 Detection of Active or Inactive Genes with Phospho-Specific Antibodies
    4.10.5 Detection of Drug Delivery
    4.10.5.1 Conventional Drugs
    4.10.5.2 Nanodrug Delivery Using Flow Cytometry
    4.11 Conclusions
    4.12 Future Scope
    4.12.1 Small, Portable Cytometers for Point-of-Care Medical Applications
    4.12.2 Small Portable Cytometers for Environmental Monitoring
    4.12.3 Cytometers Everywhere!
    References
    Chapter 5 Single-Molecule Separation
    5.1 Introduction
    5.2 Technology Involved in Single-Molecule Separation
    5.2.1 Aqueous Two-Phase Systems
    5.2.2 Capillary Electrophoresis
    5.2.3 Chromatography
    5.2.4 Protein Crystallization
    5.2.5 Hydrodynamics-Based Separation
    5.2.6 Electrohydrodynamics-Based Separation
    5.2.7 Microfluidics/Nanofluidics-Assisted Separation
    5.3 Remarks
    Acknowledgements
    References
    Chapter 6 Single-Molecule and Single-Particle Tracking and Analysis in a Living Cell
    6.1 Biophysical Insights Revealed by Single-Particle Tracking
    6.2 Overview of SMT/SPT Techniques
    6.3 3D Rotational Tracking
    6.4 Applications of SMT/SPT in Studying Cell Membrane Dynamics
    6.5 Trajectory Analysis of EGFR Dynamics on the Plasma Membrane
    6.6 Phase Transition of EGFR Trafficking
    6.7 Active Transport on Microtubule
    6.8 Applications of 3D Rotational Tracking
    6.9 Conclusion
    References
    Chapter 7 Determining the Location and Movement of Biomolecules and Biomolecular Complexes in Single Microbial Cells
    7.1 Introduction
    7.2 Computerized Fluorescence Microscopy
    7.2.1 Fluorescent Proteins (FPs)
    7.2.2 Super-Resolution Microscopy (Nanoscopy)
    7.2.2.1 Methods of Single Molecule Localization Microscopy
    7.2.2.2 Methods Based on Special Illumination of Samples
    7.2.3 Examples of Studies with CFM
    7.2.3.1 Yeast
    7.2.3.2 Bacteria
    7.3 Atomic Force Microscopy
    7.3.1 AFM Imaging
    7.3.2 Single-Molecule Force Spectroscopy (SMFS)
    7.3.3 AFM with Functionalized Tip
    7.3.4 AFM of Isolated Microbial Supramolecular Complexes
    7.3.5 Combined Use of AFM
    7.4 Limitations and Perspectives
    7.5 Conclusions
    Funding
    References
    Chapter 8 Pull Down Assay-Based Protein Analysis
    8.1 Enzyme Linked Immuno-Sorbent Immunoassay
    8.1.1 Direct ELISA
    8.1.2 Indirect ELISA
    8.1.3 Sandwich ELISA
    8.1.4 Competitive ELISA
    8.1.5 Commonly Used Substrate and Enzymatic Markers
    8.1.6 Checkerboard ELISA
    8.1.6.1 Data Analysis
    8.1.6.2 Troubleshooting
    8.1.7 Applications of ELISA
    8.1.7.1 Food Industry
    8.1.7.2 Immunology
    8.1.7.3 Diagnosis
    8.1.7.4 Cell Cytotoxicity
    8.1.7.5 Pharmaceutical Industry
    8.2 Digital Elisa
    8.3 Pull-Down Assay
    8.3.1 Basic Components Needed for Pull Down Assay
    8.3.1.1 Binding Ligands and Fusion Tags
    8.3.1.2 Binding Parameter
    8.3.1.3 Elution of Protein Complexes
    8.3.1.4 Detection of Bait-Prey Complex
    8.3.2 Advantages
    8.4 Significance of Biomolecule Interaction
    8.4.1 Single Molecule Pull Down Assay (SiMPull Assay)
    8.4.2 Concepts Behind SiMpull
    8.4.3 Steps Involved During Preparation
    8.4.3.1 Preparation of Flow Chamber
    8.4.3.2 Sample Preparation
    8.4.3.3 Antibody Immobilization
    8.4.3.4 Single Molecule Pull Down Assay
    8.4.3.5 Fluorophore Labeling
    8.4.4 Advantages of SiMPull Assay
    8.4.5 Limitations
    8.4.6 Applications of SiMPull Assay
    8.4.6.1 Receptor Pull Down
    8.4.6.2 Mitochondrial Protein Pull Down
    8.4.6.3 Pull Down of Endogenous Complex in Native Tissues
    8.4.6.4 Single Molecule Localization and Tracking
    8.5 SiMPull as Biosensor
    8.5.1 SiMPull in Contrast to ELISA
    8.5.2 SiMpull in Contrast to Surface Plasmon Resonance (SPR)
    8.6 Alpha-Synuclein (a-SYN) Protein and Oligomer Detection
    8.7 Conclusion and Future Prospect
    References
    Chapter 9 Atomic Force Microscopy for Single Molecule Detection and Analysis
    9.1 Introduction
    9.2 AFM Working Principle and Imaging Modes
    9.2.1 Contact and Tapping Mode AFM
    9.2.2 Non-contact Mode AFM
    9.2.3 AFM Force Spectroscopy
    9.2.4 Multiparametric AFM
    9.2.5 Multifrequency AFM
    9.2.6 High-Speed AFM
    9.2.7 TREC Mode AFM
    9.3 Requirements for Single-Molecule AFM
    9.3.1 Frequency Modulation AFM at a Constant Height
    9.3.2 Ultrahigh Vacuum and Ultralow Temperature
    9.3.3 Tip Functionalization
    9.3.4 qPlus Sensor
    9.3.5 Vertical and Lateral Resolution
    9.4 AFM Assisted Single-Molecule Imaging
    9.4.1 Structural Determination of DNA
    9.4.2 Dynamics of Protein-Nucleic Acids Complexes
    9.4.3 Nanoscale Visualization of Protein-Based Interactions
    9.5 Single-Molecule Adhesion Force Kinetics
    9.6 Summary and Perspective
    9.7 Conclusion
    Acknowledgment
    References
    Chapter 10 SERS Analysis for Single-Molecule Detection of Disease Biomarkers
    10.1 Introduction to Single-Molecule Surface-Enhanced Raman Spectroscopy (SM-SERS)
    10.2 Fabrication of SM-SERS Substrates
    10.3 SM SERS for Sensing Disease Biomarkers
    10.3.1 Cancer Diagnosis
    10.3.2 Cardiovascular Disease
    10.3.3 Infectious Disease
    10.4 Summary and Outlook
    References
    Chapter 11 Single Molecule Imaging Using State-of-the-Art Microscopy Techniques
    11.1 Introduction
    11.2 Techniques that Enable Single-Molecule Fluorescence-Based Detection
    11.2.1 Confocal Microscopy
    11.2.2 Total Internal Reflection Fluorescence Microscopy (TIRFM)
    11.2.3 Fluorescence Resonance Energy Transfer (FRET)
    11.2.4 Fluorescence Recovery After Photobleaching (FRAP)
    11.2.5 Super-Resolution Microscopy
    11.3 Conclusion
    References
    Index