Biomedical Engineering Reference
In-Depth Information
2
Optical Methods of Single Molecule Detection and
Applications in Biosensors
Xiaomeng Wang, Melissa Massey, Paul A.E. Piunno, and Ulrich J. Krull
CONTENTS
2.1
Introduction ..........................................................................................................................84
2.2
The Principle of Single Molecule Detection Using Optical Methods ..........................85
2.2.1
Origins of Fluorescence ........................................................................................85
2.2.2
Measurement of Fluorescence ..............................................................................86
2.2.3
Parameters Affecting Fluorescence ....................................................................86
2.2.3.1
Molecular Structure: (Example—Degree of Conjugation) ................86
2.2.3.2
Substituent Effects: (Example—Electron-Donating and
Electron-Withdrawing Groups) ............................................................87
2.2.3.3
Heterocyclic Compounds ......................................................................87
2.2.3.4
Temperature ............................................................................................87
2.2.3.5
Charge Transfer and Internal Rotation ................................................87
2.2.3.6
Solvent Viscosity/Twist Angle: (Example—the Cyanine Dyes) ......88
2.2.3.7
Quenching ................................................................................................88
2.2.3.8
Dynamic Quenching: (Example—Stern-Volmer Relation) ..............88
2.2.3.9
Static Quenching ....................................................................................89
2.2.3.10
Perrin/Sphere of Effective Quenching ..............................................89
2.2.3.11
Formation of a Ground-State Nonfluorescent Complex
(Preassociation) ......................................................................................90
2.2.3.12
Fluorescence Resonance Energy Transfer ........................................91
2.2.4
Signal-to-Noise and the Practical Issues of Single Molecule Detection ........93
2.3
Methods of Detection ........................................................................................................94
2.3.1
Fluorescence Correlation Spectroscopy ..............................................................94
2.3.2
Near-Field Scanning Optical Microscopy ..........................................................96
2.3.3
Far-Field Confocal Microscopy ............................................................................97
2.3.4
Wide-Field Epi-Illumination Microscopy ..........................................................98
2.3.5
Total internal Reflection Microscopy ..................................................................99
2.4
Applications of Single Molecule Detection ....................................................................99
2.4.1
DNA Sequencing and DNA Fragment Sizing ..................................................100
2.4.2
Single-Pair Fluorescence Resonance Energy Transfer ....................................100
2.4.3
Single-Molecule Electrophoresis ........................................................................102
2.4.4
Single Molecule Detection in the Study of Dynamics ....................................102
2.4.5
Single Molecule Detection in Biomolecular Dynamics ..................................103
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