Biosensing method for DNA (Deoxyribose Nucleic Acid) demethylase based on nanoparticle aggregation

A demethylase and nanoparticle technology, applied in the field of biosensing, can solve the problems of low sensitivity, complicated operation, long time consumption, etc., and achieve the effects of high SERS detection, enhanced Raman signal, and simple operation steps.

Active Publication Date: 2012-07-18
HUNAN UNIV
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AI Technical Summary

Problems solved by technology

At present, the detection techniques for DNA demethylases include sandwich immunoassay and phosphorus 32 radioisotope labeling method. There are many elution steps, complicated operation, long time-consuming, low sensitivity, poor stability of reagents, and isotope decay will affect the results. and radioactive contamination

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1: content analysis of DNA demethylase reference product

[0027] (1) Preparation of gold nanoparticle probes modified with Raman dyes and methylated DNA

[0028] Weigh 1.58mg 5,5'-dithiobis(2-nitrobenzoic acid) and dissolve in 4mL 50mM Na 2 Dilute the HPO4 solution with sterilized water to 500 μM (protect from light, spare), take 1 mL of nano-gold solution and centrifuge at 10,000 r / min for 10 min, dilute to 300 μL with sterilized water, add 500 μM 5,5′-disulfide while stirring 150 μL of bis(2-nitrobenzoic acid), after 30 minutes, add 2.8 nM DNA with terminal modified sulfhydryl groups, and place in a refrigerator at 4°C; after 24 hours, slowly add 57 μL of PB (100 mM, pH 7.4) while stirring, 10 Minutes later, add 30 μL of PBS (10 mM, containing 2M NaCl) to make the final concentration of NaCl in the solution reach 0.1 M, and place it in a refrigerator at 4 ° C; after 48 hours, slowly add 70 μL of PBS while stirring to make the concentration of NaCl in the ...

Embodiment 2

[0036]Example 2: Detection of the content of DNA demethylase extracted from lung cancer cells

[0037] (1) Extraction and purification of DNA demethylase from A549 (ATCC) lung cancer cells

[0038] According to the instructions of the nuclear protein extraction kit, the nuclear protein extract was obtained from A549 cells, and the nuclear protein was purified by affinity chromatography, and Tris-HCl buffer solution (10mM Tris, 10mM MgCl 2 , pH 7.5) for elution to obtain a higher purity DNA demethylase.

[0039] (2) Preparation of gold nanoparticle probes modified with methylated DNA

[0040] Weigh 1.58mg 5,5'-dithiobis(2-nitrobenzoic acid) and dissolve in 4mL 50mM Na 2 HPO4 solution, diluted with sterilized water to 500μM (protect from light, for use), take 1mL nano-gold solution and centrifuge at 10000r / min for 10min, dilute to 450μL with sterilized water, add 2.8nM end-modified DNA with sulfhydryl groups, place in 4 After 24 hours, slowly add 57 μL of PB (100 mM, pH 7.4) ...

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Abstract

The invention discloses a biosensing method for DNA (Deoxyribose Nucleic Acid) demethylase based on nanoparticle aggregation. According to the biosensing method, a nucleic acid functionalized nanoparticle probe is prepared; a specific bucleic sequence contains a cleavage site of restriction enzyme; the cleavage site is subjected to methylation treatment; the methylated DNA is enabled to generate a methylated action by the activated DNA demethylase, thereby simultaneously digesting and degrading the DNA for the methylation-sensitive restriction enzyme and exonuclease, causing the nanoparticle to aggregate, enhancing the coupling and realizing high-sensitivity SERS (Surface Enhanced Raman Spectroscopy) detection. Meanwhile, according to the method, the light absorption property is changed and the detection can be carried out by a colorimetric method. The method has the advantages of simple operation steps, quickness, high sensitivity and strong specificity and can become one of new methods for researching the DNA demethylation within the whole-genome range and apparent genetic rearrangement process and provide a technical means for function research, activity analysis and inhibitor investigation of large biological molecules.

Description

technical field [0001] The invention belongs to a biosensing technology for quantitatively analyzing DNA demethylases, specifically referring to DNA demethylation, enzymatic degradation and functionalized nanoparticle probes, surface plasmon resonance phenomena of nanoparticles, and nanoparticle Coupling enhancement and changes in light absorption properties resulting from particle agglomeration. Background technique [0002] DNA demethylases are of great significance for the study of genome-wide DNA demethylation and epigenetic reprogramming. At present, the detection techniques for DNA demethylases include sandwich immunoassay and phosphorus 32 radioisotope labeling method. There are many elution steps, complicated operation, long time-consuming, low sensitivity, poor stability of reagents, and isotope decay will affect the results. and radioactive contamination. Contents of the invention [0003] Aiming at the deficiencies in the prior art, the present invention propo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/25G01N21/33
Inventor 王玉蒋健晖楚霞唐丽娟俞汝勤谭蔚泓
Owner HUNAN UNIV
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