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Method for analyzing quantum dot-enhanced high-sensitivity DNA adduct

A DNA adduct and analysis method technology, applied in the direction of analysis materials, measuring devices, instruments, etc., can solve the problems of large amount of DNA, low sensitivity, cumbersome operation, etc. tedious effect

Inactive Publication Date: 2010-05-12
RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Usually the DNA adducts formed in the human body are very low (per 10 8 ~10 9 Each normal base contains 1 adduct), therefore, the sensitivity of DNA adduct detection has always been a bottleneck restricting the development of related research
Existing DNA adduct detection methods have shortcomings such as low sensitivity, large amount of DNA, cumbersome operation, time-consuming and labor-consuming, and are rarely used in the detection of DNA adducts in environmental biological samples.

Method used

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  • Method for analyzing quantum dot-enhanced high-sensitivity DNA adduct
  • Method for analyzing quantum dot-enhanced high-sensitivity DNA adduct
  • Method for analyzing quantum dot-enhanced high-sensitivity DNA adduct

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Example 1. Detection and analysis of BPDE-DNA adduct standard substance by quantum dot-enhanced immunocapillary electrophoresis-laser-induced fluorescence analysis technology.

[0031] In this example, the BPDE-DNA adduct is the target of analysis. BPDE-DNA adduct is the final metabolite o-diol epoxybenzo(a)pyrene (BPDE) activated by the highly carcinogenic environmental pollutant benzo(a)pyrene (B(a)P) in vivo. formed by covalent binding of DNA. The primary antibody used is a mouse-derived monoclonal antibody against BPDE-dG adduct, which can specifically recognize and bind to BPDE-dG adduct in single-stranded DNA; the secondary antibody is labeled with quantum dots Qdot 625 of anti-mouse IgG.

[0032] The DNA sample to be tested was heated at 95°C for 10 minutes to denature the double-stranded DNA into single-stranded DNA, and placed on ice for 10 minutes to prevent the single-stranded DNA from renaturing. The quenched DNA sample was mixed with the primary antibody...

Embodiment 2

[0035] Example 2. Detection and analysis of BPDE-DNA adducts in genomic DNA of A549 cells after A549 cells were exposed to different concentrations of BPDE by quantum dot-enhanced immunocapillary electrophoresis-laser-induced fluorescence analysis.

[0036] A549 cells were exposed to different concentrations of BPDE for 2 hours, and their genomic DNA was extracted as a sample to be tested. 100 μg / mL of cellular genomic DNA was quenched and then denatured into single-stranded DNA, mixed with primary antibody and quantum dot-labeled secondary antibody, and subjected to immunocapillary electrophoresis-laser-induced fluorescence analysis according to the conditions described in Example 1. attached Figure 4 Capillary electrophoresis chromatogram of the detection of BPDE-DNA adducts in genomic DNA of A549 cells exposed to 0.1 nMBPDE, wherein peak 1 is the peak of BPDE-DNA adduct-quantum dot-labeled antibody complexes, and peak 2 is the excess free The peak of the antibody (includi...

Embodiment 3

[0037] Example 3. Detection and analysis of BPDE-DNA adducts in genomic DNA of A549 cells exposed to different concentrations of B(a)P by quantum dot-enhanced immunocapillary electrophoresis-laser-induced fluorescence analysis technology.

[0038] After exposure of different concentrations of B(a)P to A549 cells for 16 hours, the genomic DNA was extracted and used as a test sample. 100 μg / mL of cellular genomic DNA was quenched and then denatured into single-stranded DNA, mixed with primary antibody and quantum dot-labeled secondary antibody, and subjected to immunocapillary electrophoresis-laser-induced fluorescence analysis according to the conditions described in Example 1. attached Figure 5 Capillary electrophoresis-laser-induced fluorescence analysis spectrum for the detection of BPDE-DNA adducts in genomic DNA of A549 cells exposed to 1 nM B(a)P, in which peak 1 is the complex of BPDE-DNA adduct-quantum dot-labeled antibody Peak 2 is the peak of excess free antibody (i...

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Abstract

The invention relates to a method for analyzing quantum dot-enhanced high-sensitivity DNA adduct, comprising the following steps: using an antibody marked by a quantum dot as a fluorescence probe, combining with the immuno-capillary electrophoresis (CE)-laser induced fluorescence (LIF) detection technology and developing the method for analyzing high-sensitivity DNA adduct. The invention adopts the immuno-capillary electrophoresis (CE)-laser induced fluorescence (LIF) detection technology to separate and detect the compound formed by the antibody marked by the quantum dot and the DNA adduct to determine the content of the DNA adduct in the sample, and has the advantages of high detection sensitivity, little DNA usage amount, rapid analysis speed, strong specificity and the like. Therefore, the invention can be used for quantitative detection on trace DNA adduct generated by organism in an interlocking manner under the condition of environmental pollutant exposion, can be used for studying formation and restoration mechanism of the damaged DNA under the condition of lower-level exposion, and evaluates potential ecological risk for environmental carcinogens and heath hazard for human beings.

Description

technical field [0001] The invention relates to an antibody labeled with quantum dots as a fluorescent probe, and develops a high-sensitivity DNA adduct analysis method based on immunocapillary electrophoresis-laser-induced fluorescence detection technology. Background technique [0002] Quantum dots are semiconductor nanocrystals composed of II-VI or III-V elements (such as CdSe, CdTe, ZnSe, InP, InAs, etc.). Compared with traditional organic fluorescent dyes, quantum dots, as a new type of fluorescent probe, have many excellent optical properties: narrow and symmetrical emission wavelength, wide and continuous absorption wavelength, large molar absorption coefficient, high quantum yield, and fluorescence lifetime. It is durable and resistant to photobleaching, and has good biocompatibility. Therefore, quantum dots have shown extremely broad application prospects in biology, chemistry and other fields. [0003] DNA adducts refer to the covalent bonds formed by exogenous o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/533
Inventor 汪海林王智鑫吕美玲
Owner RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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