FRET (Fluorescence Resonance Energy Transfer) efficiency quantitative detecting method based on partial acceptor photo-bleaching and donor-acceptor alternate excitation

A quantitative detection method and alternate excitation technology, applied in material excitation analysis, fluorescence/phosphorescence, etc., can solve the problems of long measurement time, expensive instruments, high requirements for lasers and personnel skills, etc.

Inactive Publication Date: 2012-08-15
SOUTH CHINA NORMAL UNIVERSITY
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Problems solved by technology

Fluorescence lifetime measurement is a relatively accurate method at present. Other calculation methods of E generally use the fluorescence lifetime method as a comparison. However, the instruments used in the fluorescence lifetime measurement method are relatively expensive, require high skills for lasers and personnel, and the measurement time is relatively long. Easy photobleaching of samples

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  • FRET (Fluorescence Resonance Energy Transfer) efficiency quantitative detecting method based on partial acceptor photo-bleaching and donor-acceptor alternate excitation
  • FRET (Fluorescence Resonance Energy Transfer) efficiency quantitative detecting method based on partial acceptor photo-bleaching and donor-acceptor alternate excitation
  • FRET (Fluorescence Resonance Energy Transfer) efficiency quantitative detecting method based on partial acceptor photo-bleaching and donor-acceptor alternate excitation

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Embodiment Construction

[0041] refer to figure 1 , a method for quantitative detection of FRET efficiency based on partial photobleaching of the acceptor and alternate excitation of the donor and acceptor, including the processing steps of detecting the donor first and then the acceptor or the acceptor first and then the donor, wherein the first detection Donor retesting acceptor method processing steps include:

[0042] A: Use the donor excitation light to excite the donor-acceptor pair, and use the donor channel to collect the fluorescence intensity emitted by the donor, which is recorded as I DD ';

[0043] B: Select the receptor excitation light to excite only the receptor, and use the receptor channel to collect the fluorescence intensity emitted by the receptor, which is recorded as I AA ';

[0044] C: Using the receptor with the maximum intensity to excite photobleaching part of the receptor;

[0045] D: Select the donor-excited light to excite the bleached donor-acceptor pair, and use the...

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Abstract

The invention discloses an FRET (Fluorescence Resonance Energy Transfer) efficiency quantitative detecting method based on partial acceptor photo-bleaching and donor-acceptor alternate excitation. The invention relates to a quantitative detecting method, and particularly relates to the FRET efficiency quantitative detecting method based on the partial acceptor photo-bleaching and the donor-acceptor alternate excitation. According to the FRET efficiency quantitative detecting method, FRET efficiency of a donor-acceptor pair can be quantitatively obtained by the steps of partially photo-bleaching an acceptor, alternately exciting a sample by using donor exciting light and acceptor exciting light, and collecting fluorescence intensity of a donor channel and an acceptor channel before and after the acceptor is partially photo-bleached. The method directly detects the sample to be detected without any auxiliary sample or extra system correction and compensation. Not only can the FRET efficiency be quantitatively detected when the concentration ratio of the donor to the acceptor is 1 to 1, but also the FRET efficiency quantitative detection can be effectively realized when one donor and a plurality of acceptors exist. The FRET efficiency quantitative detecting method is simple in operation and causes small optical damage to the sample, can be used for repeatedly detecting the same sample, and can be applied to the rapid quantitative detection of the FRET efficiency of the donor-acceptor pair in the sample.

Description

technical field [0001] The invention relates to a quantitative detection method, in particular to a quantitative detection method of FRET efficiency based on partial photobleaching of acceptor and alternate excitation of donor and acceptor. Background technique [0002] Fluorescence resonance energy transfer (FRET) is a distance-dependent photophysical process in which a fluorophore in an excited state transfers energy to a neighboring acceptor molecule in a non-radiative manner through the interaction between dipoles. FRET efficiency (E) is the percentage of energy transferred from the donor to the acceptor due to the FRET effect. Because E is inversely proportional to the sixth power of the distance between the donor and the receptor, it is very sensitive to changes in distance. The measurement scale is in the nanometer range. FRET technology is widely used in the study of biological, chemical, and medical issues such as protein-protein interactions and conformational chan...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
Inventor 陈同生于怀娜
Owner SOUTH CHINA NORMAL UNIVERSITY
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