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Manufactured histone acetyltransferase electrochemiluminescence biosensor based on DNA nano-prism and application of histone acetyltransferase electrochemiluminescence biosensor

An acetyltransferase and luminescent biology technology, which is applied in the fields of functional biological materials and biosensing, and achieves the effects of good application prospects, easy operation and simple operation.

Active Publication Date: 2019-04-12
山东博科诊断科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no related reports on the construction of HAT p300 electrochemiluminescence sensor by combining prism, HCR amplification technology and CoA aptamer at home and abroad

Method used

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  • Manufactured histone acetyltransferase electrochemiluminescence biosensor based on DNA nano-prism and application of histone acetyltransferase electrochemiluminescence biosensor
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  • Manufactured histone acetyltransferase electrochemiluminescence biosensor based on DNA nano-prism and application of histone acetyltransferase electrochemiluminescence biosensor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1 Preparation of sensor

[0039] (1) HAT-aptamer reaction

[0040] ①Acetylation reaction: Take HAT p300 (1000nM, 0.2μL) and peptide (1mM, 0.4μL), acetyl-CoA (1mM, 1μL) in phosphate buffer solution (PBS, 10mM, pH 7.0) and mix thoroughly, the total volume 2 μL. Place the reaction solution in a constant temperature water bath at 30°C and incubate for 30 min; ②Add CoA aptamer (2 μL, 1.25 μM) to the solution in ①, shake and mix evenly, and react in a water bath at 30°C for 2 h; ③Add cDNA (2 μL, 1.25 μM) to ② μM) were shaken and mixed evenly, and the total volume was 6 μL, and reacted in a water bath at 30° C. for 1 hour, and set aside.

[0041] (2) Preparation of electrochemiluminescence sensor

[0042] a.Au electrode

[0043] Gold electrodes (Au) were used with Al with diameters of 0.3 μm and 0.05 μm, respectively. 2 o 3 The powder was ground, ultrasonically cleaned three times with ultrapure water, dried in a nitrogen stream, and soaked in 0.1M H 2 SO 4 ...

Embodiment 2

[0054] Example 2 Electrochemiluminescence response with or without p300

[0055] Construction of histone acetyltransferase electrochemiluminescent biosensor based on DNA nano-prism and its application, our biosensor was prepared based on Example 1. See figure 2 , In the absence of p300, the sensor has basically no ECL response in PBS (0.1M, pH 7.0), while in the presence of p300, there is an obvious ECL response, which proves that the sensor can be used for p300 activity detection.

Embodiment 3

[0056] Example 3 Detection of p300 activity

[0057] Construction of histone acetyltransferase electrochemiluminescence biosensor and its application based on DNA nano triangular prisms. The preparation steps of the sensor are the same as in Example 1. During the acetylation reaction, the concentration of p300 is changed sequentially. The concentration of p300 is: 0, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 500 nM were subsequently used to prepare sensors. Record the electrochemiluminescence response of the sensor in PBS (0.1M, pH 7.0). According to the experimental results, a series of electrochemical response curves corresponding to different concentrations of p300 are obtained, and the quantitative relationship between the electrochemical response current and the concentration of p300 is established. According to the quantitative relationship between the two, determine the concentration of p300 in the sample to be tested. Experimental results such as image 3 As shown, it...

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Abstract

The invention discloses a manufactured histone acetyltransferase electrochemiluminescence biosensor based on a DNA nano-prism and application of the histone acetyltransferase electrochemiluminescencebiosensor. The histone acetyltransferase electrochemiluminescence biosensor based on the DNA nano-prism is manufactured through the specific steps that (1) HAT-aptamer reaction is carried out, specifically, firstly, acetylation reaction is carried out, HATp300 and polypeptide are separately selected, and acetylcoenzyme A is fully mixed in a phosphate buffer solution (PBS, 10mM, pH7.0); secondly, CoA aptamer is added to the reaction solution in the first step; and thirdly, cDNA is added to the solution in the second step; and (2) the electrochemiluminescence sensor is manufacture through a. Auelectrode, b. prism / Au electrode, c. cDNA / prism / Au electrode, d. H1-H2 / cDNA / prism / Au electrode and e. Ru / H1-H2 / cDNA / prism / Au electrode. In the acetylation reaction, the concentration of p300 and the small molecule inhibitor concentration thereof are changed, and the effect of a series of sensors on an electrochemiluminescence signal is investigated. The manufactured histone acetyltransferase electrochemiluminescence biosensor based on the DNA nano-prism and the application of the histone acetyltransferase electrochemiluminescence biosensor have the advantages of good specificity, high sensitivity, fast detection speed, accurate and reliable results and low cost.

Description

technical field [0001] The invention relates to an electrochemiluminescence sensor and a detection method thereof, in particular to the construction of a histone acetyltransferase electrochemiluminescence biosensor based on DNA nano triangular prisms and its application, and belongs to the technical field of functional biomaterials and biosensing. Background technique [0002] Histone acetyltransferase (HAT) is a typical biological enzyme, which transfers the acetyl group on acetyl-CoA to specific lysine residues of substrate histone or non-histone substrate polypeptide, To achieve the purpose of regulating the structure of chromosomes and then regulating the expression of genes. Dysfunction of histone acetylation, or abnormal action of acetyltransferases, is associated with a range of diseases, such as cancer, metabolic syndrome, and neurological disorders. Measuring the activity of HATs and the potency of their inhibitors will greatly facilitate the biochemical study of g...

Claims

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Application Information

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IPC IPC(8): G01N27/327G01N27/48
CPCG01N27/3277G01N27/3278G01N27/48
Inventor 胡宇芳张青青胡丹丹马少华郭智勇王邃
Owner 山东博科诊断科技有限公司
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