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Biosensing method for detecting activity of histone modification enzyme and screening inhibitor thereof based on non-modified antibody mediate nano-gold assembly

A biosensing and antibody modification technology, applied in the field of biosensing, can solve the problems of low sensitivity, poor reliability, and inability to meet accurate and rapid detection of detection technology, and achieve the effect of quick and easy operation and simple design

Inactive Publication Date: 2012-07-18
HUNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These detection techniques have low sensitivity, poor reliability, and require sophisticated instruments, which cannot meet the needs of accurate and rapid detection

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Detection of histone methyltransferases based on unlabeled antibody-mediated visual analysis of gold nanoparticle assembly

[0024] 1) Preparation of gold nanoparticles modified by histone methyltransferase substrate

[0025] Take a solution of 50 μM methyltransferase substrate peptide and control peptide, mix them at an optimal ratio (1:1) to form 500 μL, add this 500 μL solution to 500 μL of citric acid-protected gold nanoparticles solution, and store at room temperature Stirring was continued for 24 hours. Then the above solution was centrifuged at 12,000 rpm for 15 minutes to remove excess peptide, and the lower precipitate was redispersed in 500 μL with a concentration of 10 mM phosphate buffer (0.01M NaH 2 PO 4 , pH 7.2). This centrifugal washing process was repeated 3 times, and finally the lower sediment was dispersed in 500 μL with a concentration of 10 mM phosphate buffer (0.01M NaH 2 PO 4 , pH 7.2), and stored in a refrigerator at 4°C for late...

Embodiment 2

[0030] Example 2: Detection of histone acetyltransferase based on visual analysis of unlabeled antibody-mediated assembly of gold nanoparticles

[0031] 1) Preparation of gold nanoparticles modified by histone acetyltransferase substrate

[0032] The solutions of the acetyltransferase substrate peptide and the control peptide with a concentration of 50 μM were respectively taken and mixed at an optimal ratio (1:1) to form 500 μL. Add this 500 μL solution into 500 μL citric acid-protected gold nanoparticles solution, and stir at room temperature for 24 hours. Then the above reaction solution was centrifuged at 12,000 rpm for 15 minutes to remove excess peptide, and the lower precipitate was redispersed in 500 μL with a concentration of 10 mM phosphate buffer (0.01M NaH 2 PO 4 , pH 7.2). This centrifugal washing process was repeated 3 times, and finally the lower sediment was dispersed in 500 μL with a concentration of 10 mM phosphate buffer (0.01M NaH 2 PO 4 , pH 7.2), and...

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PUM

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Abstract

The invention discloses a colorimetric biosensing method for analyzing and detecting activity of histone modification enzyme and screening inhibitor thereof based on non-modified antibody mediate nano-gold. The method comprises the following steps of: designing substrate peptide of histone modification enzyme; determining the mixing self-assembly ratio of the substrate peptide of the histone modification enzyme to control peptide; and quantitatively detecting the activity of the histone modification enzyme and screening the corresponding inhibitor by colorimetry based on nano-gold assembly of non-labeled antibody mediate modified histone modification enzyme substrate peptide. The specificity interaction of the histone modification enzyme and the substrate peptide of the histone modification enzyme is detected, and the substrate peptide modified on the nano-gold whch is modified by the histone modification enzyme can be subjected to specific binding with the corresponding monoclonal antibodies to ensure that the nano-gold is assembled, the activity of the histone modification enzyme is detected, and the inhibitor of the histone modification enzyme is screened by ultraviolet quantitative analysis on the nano-gold which modifies the substrate peptide of the histone modification enzyme. The method is high in sensitivity and specificity, and is convenient and easy to operate and can be used for biological research, medical research, medicine screening and the like.

Description

technical field [0001] The invention belongs to a biosensing technology for detecting the activity of histone-modifying enzymes and screening their inhibitors, which specifically refers to the design of histone-modifying enzyme substrates, the mixing ratio of histone-modifying enzyme substrate peptides and control peptides, And a colorimetric quantitative detection method based on unmodified antibody-mediated self-assembly of gold nanoparticles. Background technique [0002] The detection and rapid screening of histone modifying enzymes and their inhibitors are of great significance to biological research, medical research, drug screening and other fields. At present, the commonly used detection technology for the activity of histone modifying enzymes is based on the detection of coenzyme by-products in enzyme-catalyzed reactions and the direct detection of modified products. The detection methods mainly include colorimetry, fluorescence resonance energy transfer, fluorescen...

Claims

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Application Information

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IPC IPC(8): G01N33/573G01N21/33
Inventor 甄珍蒋健晖楚霞唐丽娟俞汝勤
Owner HUNAN UNIV
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