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Construction of electrochemical luminescence sensor for detecting acetyltransferase activity

An acetyltransferase and activity detection technology, applied in the field of electrochemiluminescent biosensors, can solve the problems of no sensors yet, and achieve the effects of high detection sensitivity, enhanced signal strength, and easy manipulation

Active Publication Date: 2017-08-29
青岛卓越海特信息技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, no patents and articles have been found on the application of such sensors in HAT activity

Method used

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  • Construction of electrochemical luminescence sensor for detecting acetyltransferase activity
  • Construction of electrochemical luminescence sensor for detecting acetyltransferase activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] (1) Gold electrode surface pretreatment and activation

[0059] The gold electrode was made of 0.3 μm Al 2 o 3 The powder was ground and polished on the suede, then ultrasonically cleaned with ethanol and water for 3 minutes, and the surface of the electrode was blown dry with pure nitrogen.

[0060] Cleaned and dried gold electrode was used as working electrode, Ag / AgCl was used as reference electrode, and platinum wire was used as counter electrode. 2 SO 4 In solution, -0.20~1.65V, 100mV / s, scan CV until stable.

[0061] Repeat this until the gold electrode reaches the activation standard, wash the gold electrode with water, and dry it with nitrogen.

[0062] (2) Gold electrode surface modification

[0063] Dip the activated gold electrode into 1 μM cDNA (5'-CTA AGT AAC TCT GCA CTC TTA TAT ATCATA GAA TTG GTA GAT-(CH 2 )6-SH-3') in PBS solution (50mM, pH 7.4), incubated at 35°C for 1h to form a cDNA-modified Au electrode (cDNA-Au).

[0064] After washing with wa...

Embodiment 2

[0070] (1) Gold electrode surface pretreatment and activation

[0071] The gold electrode was made of 0.3 μm Al 2 o 3 The powder was ground and polished on the suede, then ultrasonically cleaned with ethanol and water for 2 minutes, and the surface of the electrode was blown dry with pure nitrogen.

[0072] Cleaned and dried gold electrode was used as working electrode, Ag / AgCl was used as reference electrode, and platinum wire was used as counter electrode. 2 SO 4In solution, -0.20~1.65V, 100mV / s, scan CV until stable.

[0073] Repeat this until the gold electrode reaches the activation standard, wash the gold electrode with water, and dry it with nitrogen.

[0074] (2) Gold electrode surface modification

[0075] Dip the activated gold electrode into 5 μM cDNA (5'-CTA AGT AAC TCT GCA CTC TTA TAT ATCATA GAA TTG GTA GAT-(CH 2 )6-SH-3') in PBS solution (50mM, pH 7.4), incubated at 37°C for 1h to form a cDNA-modified Au electrode (cDNA-Au).

[0076] After washing with wat...

Embodiment 3

[0082] (1) Gold electrode surface pretreatment and activation

[0083] The gold electrode was made of 0.3 μm Al 2 o 3 The powder was ground and polished on the suede, then ultrasonically cleaned with ethanol and water for 2 minutes, and the surface of the electrode was blown dry with pure nitrogen.

[0084] Cleaned and dried gold electrode was used as working electrode, Ag / AgCl was used as reference electrode, and platinum wire was used as counter electrode. 2 SO 4 In solution, -0.20~1.65V, 100mV / s, scan CV until stable.

[0085] Repeat this until the gold electrode reaches the activation standard, wash the gold electrode with water, and dry it with nitrogen.

[0086] (2) Gold electrode surface modification

[0087] Dip the activated gold electrode into 10μM cDNA (5'-CTA AGT AAC TCT GCA CTC TTA TAT ATCATA GAA TTG GTA GAT-(CH 2 )6-SH-3') in PBS solution (50mM, pH 7.4), incubated at 36°C for 3h to form a cDNA-modified Au electrode (cDNA-Au).

[0088] After washing with wa...

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Abstract

The invention relates to a construction of an electrochemical luminescence sensor for detecting acetyltransferase activity. The construction comprises the following steps: firstly, polishing, cleaning and activating a gold electrode, assembling captured DNA on the surface of the electrode through action of gold-sulfur bond, and enclosing non-specific binding sites on the surface of the electrode with MCH; then adsorbing a polypeptide chains through electrostatic action, performing acetylation treatment on the polypeptide chains under the action of HATp300, so as to separate from the surface of the electrode; secondly, performing hybrid chain reaction on the electrode in a hybrid chain solution containing two hairpin DNA, and reducing a silver cluster; and finally, detecting electrochemical luminescence signal of the assembled electrochemical luminescence sensor in the solution. The ECL biosensor has remarkable advantages on the quantitative analysis of HAT p300 and the activity analysis on HAT p300 in complicated cell lysis buffering solution.

Description

technical field [0001] The invention belongs to the field of electrochemiluminescence biosensors, and in particular relates to the construction of an electrochemiluminescence sensor used for detecting acetyltransferase activity. Background technique [0002] The change of chromatin structure caused by histone modification plays an important role in the regulation of gene expression in eukaryotes. Histone modifications can include methylation, phosphorylation, ubiquitination, and acetylation, among others. Among them, acetylation is the most important modification method, and histone acetyltransferase (HAT) is a key enzyme that regulates the level of acetylation modification of histones and transcription factors, which makes the chromatin structure loose and activates transcription. Histone acetylation catalyzed by HAT plays an important role in biochemistry and pharmacology, because it plays an important role in epigenetic gene regulation, so the activity of HAT in the cont...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/76G01N27/327
CPCG01N21/76G01N27/3277
Inventor 张菲菲王宗花邹妍管嵩
Owner 青岛卓越海特信息技术有限公司
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