Photoelectrochemical biosensor for detecting histone acetyltransferase activity and preparation method thereof

A technology of acetyltransferase and biosensor, applied in the direction of material electrochemical variables, scientific instruments, instruments, etc., can solve the problems of health hazards for operators, expensive antibody reagents, cumbersome sample processing, etc., to improve detection sensitivity and realize small instruments The effect of simple chemicalization and detection method

Inactive Publication Date: 2019-08-23
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Radioactive technology has the hazard of radioactive contamination, which is also harmful to the health of the operator
ELISA is limited by the effectiveness of antibodies and false positives, and antibody reagents are relatively expensive
Chromatography and mass spectrometry require expensive large-scale instruments, cumbersome sample pretreatment, and professional operators

Method used

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  • Photoelectrochemical biosensor for detecting histone acetyltransferase activity and preparation method thereof
  • Photoelectrochemical biosensor for detecting histone acetyltransferase activity and preparation method thereof
  • Photoelectrochemical biosensor for detecting histone acetyltransferase activity and preparation method thereof

Examples

Experimental program
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Effect test

preparation example Construction

[0052] (1) Preparation of catalytic reaction solution: mix 20-50 μL of different concentrations of histone acetyltransferase, 20-50 μL of substrate peptide (concentration of 10-40 μM), 10-40 μL of acetyl-CoA (concentration of 100-300 μM) well mixed. The mixture is then incubated at 20-50°C for 1-5 hours. The resulting solution was labeled as catalytic reaction solution.

[0053] (2) Preparation of exfoliated tungsten sulfide: weigh 50-300mg WS 2 And 50-300mg of polyacrylic acid dissolved in 20-60mL of water, ultrasonic 2-6h. Centrifuge the green dispersion at 2000-10000rpm for 5-30min, collect the supernatant and centrifuge at 5000-10000rpm for 5-30min, wash the obtained precipitate with deionized water several times to remove the residue, and finally dry it under vacuum.

[0054] (3) Preparation of polydopamine: Dissolve 100-200mg of dopamine hydrochloride in 50-100mL of deionized water, under vigorous stirring, take 500-1000μL of 1-3mol / L NaOH solution and add it to the a...

Embodiment 1

[0072] Example 1: Histone acetyltransferases catalyze acetylation of substrate peptides

[0073] Mix 20 μL of 50 nM histone acetyltransferase, 20 μL of substrate peptide (concentration of 20 μM), and 20 μL of acetyl-CoA (concentration of 100 μM). The mixture was then incubated at 37°C for 1 hour. The resulting solution was labeled as catalytic reaction solution.

Embodiment 2

[0074] Embodiment 2: Preparation of exfoliated tungsten sulfide

[0075] Weigh 50mg WS 2 And 300mg of polyacrylic acid dissolved in 60mL of water, ultrasonic 6h. The green dispersion was centrifuged at 3000rpm for 10min, the supernatant was collected and centrifuged at 5000rpm for 15min, the obtained precipitate was washed several times with deionized water to remove the residue, and finally dried under vacuum.

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Abstract

The invention discloses a photoelectrochemical biosensor for detecting histone acetyltransferase activity and a preparation method thereof. The photoelectrochemical biosensor comprises an ITO electrode, stripped WS2, polydopamine, SMCC, coenzyme A, phos-tag-biotin, and streptavidin, wherein the stripped WS2, polydopamine, SMCC, coenzyme A, phos-tag-biotin, and streptavidin are sequentially modified on the surface of the electrode. According to the photoelectrochemical biosensor and the method in the invention, by utilizing good biocompatibility and conductivity of WS2 and specifically recognized avidin and biotin, quenching of a photoelectric signal is realized, and the detection sensitivity of the histone acetyltransferase is improved. The specificity of activity detection of the histoneacetyltransferase is improved by utilizing the specific binding and identification effects of maleimide in the SMCC to sulfydryl in the CoA. The detection method disclosed by the invention is simple,miniaturization of the instrument is realized, the method is easy to operate, and the detection of the activity of the histone acetyltransferase can be realized only by simply processing the surface of the ITO electrode.

Description

technical field [0001] The invention relates to the technical field of photoelectrochemical analysis, in particular to a photoelectrochemical biosensor for detecting histone acetyltransferase and a preparation method thereof. Background technique [0002] In eukaryotic chromatin, histones and DNA can form its basic building blocks. Histones can be acetylated in the presence of histone acetyltransferases (HATs), which are not only an essential regulatory mechanism in epigenetic systems, but also control the transcriptional and structural state of chromatin. Acetylation reactions play crucial roles in many fundamental biological processes such as genome stability, histone deposition, DNA replication repair, and nutrient metabolism. Lysine residues on histones are important acetylation sites, and HATs can acetylate them. This phenomenon affects chromatin structure and gene expression. [0003] Studies have shown that the pathogenesis of many clinical diseases, such as cancer...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/26G01N27/30G01N27/327
CPCG01N27/26G01N27/30G01N27/3271
Inventor 殷焕顺陈燕李菲周云雷艾仕云
Owner SHANDONG AGRICULTURAL UNIVERSITY
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