Fluorescence correlation spectroscopy detection method

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

Active Publication Date: 2020-04-10
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 spectroscopy detection method
  • Fluorescence correlation spectroscopy detection method
  • Fluorescence correlation spectroscopy detection method

Examples

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Effect test

Embodiment 1

[0049] Example 1 The fluorescence luminescence scintillation kinetics of ATTO655 fluorescent dye molecule 5 was studied by using the fluorescence correlation spectroscopy detection method.

[0050] The ATTO655 fluorescent dye molecule 5 flashes between a fluorescent and non-fluorescent state, and the presence of reducing agents and oxidizing agents such as ascorbic acid (AA) and methyl viologen (MV) accelerates this process. Take the scintillation kinetics of ATTO655 fluorescent dye molecule 5 under different concentrations of AA and MV as an example, see Figure 9, the fluorescence spectrum detection method described in this embodiment can realize the detection of the intramolecular dynamics process on the time scale from 1.1ms to 168ms.

[0051] The specific experiments are as follows:

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

Embodiment 2

[0055] Example 2 The kinetics of DNA double-strand formation was studied by using the fluorescence correlation spectroscopy detection method.

[0056] In this example, the fluorescence correlation spectroscopy detection method is combined with fluorescence resonance energy transfer, the target molecule 3 single-stranded DNA molecule labeled with Alexa488 fluorescent dye molecule 5 is used as a fluorescent donor, and the complementary effect of Cy5 fluorescent dye molecule 5 labeled Molecular 3' single-stranded DNA molecule is used as a fluorescent receptor to study the kinetics of target molecule 3 single-stranded DNA and its complementary action molecule 3' to form double-stranded DNA and DNA end respiration. see Figure 10 , the fluorescence spectroscopy detection method described in this embodiment can realize the kinetic detection of the intermolecular interaction in a large time scale range spanning three orders of magnitude from 0.45ms to 3.78s.

[0057] The specific ex...

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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 spectral detection and single-molecule detection, in particular to a fluorescence correlation spectral 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 averaging measurements, including differences between biomolecules and transient intermediate states in the reaction process. The above advantages of single-molecule fluorescence methods have made it widely concerned and applied in recent years, and its rapid development has enabled many traditional biological problems to be solved by 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 have a wide distribution of time scales, th...

Claims

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Application Information

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Patent Type & Authority Patents(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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