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Fluorescence correlation spectrum detection method

A spectral detection and fluorescence correlation technology, applied in the field of fluorescence correlation spectral detection, can solve the problem of not being able to detect the complete process of dynamic reaction, and achieve the effect of broadening the time scale

Active Publication Date: 2018-08-24
TSINGHUA UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Since the range of the time scale is too narrow to detect the complete process of the dynamic response, it is necessary to develop new technologies with a wider time scale detection range

Method used

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  • Fluorescence correlation spectrum detection method

Examples

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Effect test

Embodiment 1

[0049] Example 1 Using the fluorescence correlation spectroscopy detection method, the fluorescence luminescence blinking kinetics of ATTO655 fluorescent dye molecule 5 was studied.

[0050] The ATTO655 fluorescent dye molecule 5 will blink between fluorescent and non-fluorescent states, and the presence of reducing and oxidizing agents such as ascorbic acid (AA) and methyl viologen (MV) will accelerate this process. Taking the blinking kinetics of ATTO655 fluorescent dye molecule 5 under different concentrations of AA and MV as an example, please refer to Figure 9, the fluorescence spectrum detection method described in this embodiment can realize the detection of intramolecular dynamic processes with a time scale from 1.1 ms to 168 ms.

[0051] The specific experiment is as follows:

[0052] Slide 1, suitable for microscopy imaging, was treated overnight at room temperature with an aminosilane reagent (1ml aminopropyltriethoxysilane, 5ml acetic acid, and 94ml methanol), th...

Embodiment 2

[0055] Example 2 uses the fluorescence correlation spectroscopy detection method to study the kinetics of DNA double strand formation.

[0056] In this embodiment, the fluorescence correlation spectroscopy detection method and fluorescence resonance energy transfer are combined, and the target molecule 3 single-stranded DNA molecule labeled with the Alexa488 fluorescent dye molecule 5 is used as a fluorescent donor, and the complementary effect of the Cy5 fluorescent dye molecule 5 label Molecule 3'single-stranded DNA molecules are used as fluorescent receptors to study the dynamics of the target molecule 3 single-stranded DNA and its complementary interaction molecule 3' forming double-stranded DNA and the respiratory effect of the DNA end. see Figure 10 , the fluorescence spectrum detection method described in this embodiment can realize the kinetic detection of intermolecular interactions in a large time scale spanning three orders of magnitude from 0.45 ms to 3.78 s.

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Abstract

The invention discloses a fluorescence correlation spectrum detection method comprising the following steps: a probe molecule and a target molecule are provided, and the probe molecule is capable of emitting fluorescence by fluorescence correlation spectrum detection and has a binding domain capable of binding to the target molecule; a solution of the probe molecule is applied to the surface of aslide for microscopic imaging, and the probe molecule is fixed on the surface of the slide; a solution to be detected containing the target molecule is applied to the surface of the slide, the targetmolecule and the binding domain are mutually recognized and combined; and change in fluorescence luminescence of the target molecule is detected.

Description

technical field [0001] The invention relates to the field of biological macromolecular spectrum detection and single molecule detection, in particular to a fluorescence correlation spectrum detection method. Background technique [0002] Single-molecule fluorescence technology can observe the dynamic process of biomolecules in complex systems in real time, and reveal important information hidden in traditional ensemble average measurements, including the differences between biomolecules and the transient intermediate state during the reaction process. The above advantages of the single-molecule fluorescence method have made it widely concerned and applied in recent years. Its rapid development has enabled many traditional biological problems to be solved with new experimental techniques (W.E.Moerner, D.P.Fromm, Rev SciInstrum 2003, 74, 3597- 3619). [0003] Biological processes such as conformational change dynamics and molecular interactions span a wide distribution of tim...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/6486
Inventor 王文娟陈春来彭思佳
Owner TSINGHUA UNIV
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