Real-Time Protein Monitoring and Manipulation Using Total Internal Reflection Fluorescence Microscopy
Date Issued
2004
Date
2004
Author(s)
Yen, Yi-Kuang
DOI
zh-TW
Abstract
The study of biomolecular recognition has been becoming crucial to provide insights into molecular genetics, design of biosensor devices, drug design, and development of targeted drug delivery systems. The in-depth understanding of biomolecular recogntion involves adsorption, interaction and desorption as well as associated manipulation between biomolecules. In this present study, we have successfully demonstrated a single biomolecular detection and real-time tracking of anti-IgG in a microchannel using total internal reflection flurorescence (TIRF) microscopy for illustration of protein adsorption and recognition.
TIRF microscopy is a well-suited technique for real-time imaging and monitoring of a single protein molecule in nano-layer fluidics due to its unique evanencent wave at the optically index-mismatch interaface that may excites fluorescences at the transparent near-wall region. Recent advances in charge coupled device (CCD) camera detection efficiency and speed have enabled the microscopy of temporal and spatial resolutions to be far-reaching 0.033 ms and 0.3 μm, respectively. A modified inverted TIRF microscopy was newly established, which allows a directed laser beam underneath through the inverted microscopy to be incident in a critical angle into the surface inside the microchannel. In this microchannel, the conductive ITO film was deposited to be electrically feedthrough for electrical manipulation. Based on the fluorescent beads analysis, which are 1.1 μm in size, the capability of TIRFM for single molecules monitoring and tracking, even the measurement in lateral size of molecules was demonstrated.
In this study, the TIRFM incorporates a MEMS-based electrical control biochip to monitor and track the motion of a single antigen molecule in which the real-time position and velocity of each frames were tracked and measured. The motion of an antigen molecule was founded to be dominated in a hydrodynamic boundary layer. A further comparison of experimental results with theoretics appears to be deviated unexpectedly, which provokes a further thought that a nano-layer fluidics exhibits a novel non-classical fluidic characteristics.
At last, the motion of fluorescence nanobeads manipulated by the application of DC voltage was real-time monitored. The velocities of beads were shown to be in a linear accordance with the applied voltages. As a result of the accordance, the manipulation of nonobeads with electrical control was demonstrated and verified.
Subjects
全反射螢光顯微鏡
漸逝波
微機電技術
MEMS
TIRFM
evanescent wave
Type
thesis
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