Construction method and application of electrochemical Faraday cage immunosensor for detecting activity of histone acetyltransferase

An immunosensor and acetyltransferase technology, applied in the field of functional materials and biosensing, can solve the problems of long response time, expensive instruments, false positives, etc., and achieve the effects of less reagent consumption, fast detection speed, and accurate results

Active Publication Date: 2018-09-28
NINGBO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods have certain sensitivity and accuracy, but they also have certain shortcomings: some instruments are expensive, complicated to operate, and require high technology; some have many steps and are prone to false positives and false negatives; Some reagents have low specificity and sensitivity, and some have long response time and cannot be measured repeatedly
There is no report on an electrochemical Faraday cage immunosensor based on nano-gold / graphene / acetyl antibody composites for detecting histone acetyltransferase activity

Method used

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  • Construction method and application of electrochemical Faraday cage immunosensor for detecting activity of histone acetyltransferase
  • Construction method and application of electrochemical Faraday cage immunosensor for detecting activity of histone acetyltransferase
  • Construction method and application of electrochemical Faraday cage immunosensor for detecting activity of histone acetyltransferase

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specific Embodiment 1

[0032] A construction method and application of an electrochemical Faraday cage immunosensor for detecting histone acetyltransferase activity, the specific steps are as follows:

[0033] (1) Preparation of peptide / gold electrode (peptide / Au)

[0034] Take acetyltransferase p300 (100nM, 2μL) and substrate polypeptide (1mM, 0.4μL), acetyl-CoA (1mM, 1μL) in phosphate buffered solution (PBS) (0.1M, pH7.0) and mix thoroughly, the total Volume 10 μL. The reaction solution was incubated in a constant temperature water bath at 30°C for 30 min. Take 5 μL of the p300 catalytic reaction solution, drop-coat it on the surface of the gold electrode, and incubate in a 4°C refrigerator for 4h.

[0035] (2) Preparation of methylene blue-cured gold nanoparticles / graphene / acetyl antibody composites (MB&AuNPs@GO-Ab)

[0036] a. Synthesis of gold nanoparticles (AuNPs)

[0037] Chlorauric acid (HAuCl 4 ) (0.01M, 0.25mL), cetyltrimethylammonium bromide (CTAB) (0.1M, 1mL) was mixed with water (8...

specific Embodiment 2

[0044] A construction method and application of an electrochemical Faraday cage immunosensor for detecting histone acetyltransferase activity, the specific steps are as follows:

[0045] (1) Preparation of peptide / gold electrode (peptide / Au)

[0046] Take acetyltransferase p300 (120nM, 1.7μL) and substrate polypeptide (1.2mM, 0.3μL), acetyl-CoA (1.2mM, 0.8μL) in phosphate buffer solution (PBS) (0.1M, pH7.0) Mix well to a total volume of 10 μL. The reaction solution was incubated in a constant temperature water bath at 32°C for 25 min. Take 4 μL of the p300 catalytic reaction solution, drop-coat it on the surface of the gold electrode, and incubate in a 4°C refrigerator for 5h.

[0047] (2) Preparation of methylene blue-cured gold nanoparticles / graphene / acetyl antibody composites (MB&AuNPs@GO-Ab)

[0048] a. Synthesis of gold nanoparticles (AuNPs)

[0049] Chlorauric acid (HAuCl 4 ) (0.1M, 0.025mL), cetyltrimethylammonium bromide (CTAB) (0.5M, 0.2mL) was mixed with water (...

specific Embodiment 3

[0056] A construction method and application of an electrochemical Faraday cage immunosensor for detecting histone acetyltransferase activity, the specific steps are as follows:

[0057] (1) Preparation of peptide / gold electrode (peptide / Au)

[0058] Take acetyltransferase p300 (80nM, 2.5μL) and substrate polypeptide (0.8mM, 0.5μL), acetyl-CoA (0.8mM, 1.25μL) in phosphate buffer solution (PBS) (0.1M, pH7.0) Mix well to a total volume of 10 μL. The reaction solution was incubated in a constant temperature water bath at 33 °C for 34 min. Take 4.8 μL of the p300 catalytic reaction solution, drop-coat it on the surface of the gold electrode, and incubate in a refrigerator at 4°C for 7 hours.

[0059] (2) Preparation of methylene blue-cured gold nanoparticles / graphene / acetyl antibody composites (MB&AuNPs@GO-Ab)

[0060] a. Synthesis of gold nanoparticles (AuNPs)

[0061] Chlorauric acid (HAuCl 4 ) (0.095M, 0.03mL), cetyltrimethylammonium bromide (CTAB) (0.4M, 0.25mL) was mixed...

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Abstract

The invention discloses a construction method and an application of an electrochemical Faraday cage immunosensor for detecting the activity of histone acetyltransferase. The construction method comprises the following steps: (1) preparing peptide/Au: respectively taking acetyltransferase p300, polypeptide and acetyl-CoA, fully mixing the taken substances in PBS (0.1 M, pH 7.0), incubating the obtained solution in a constant-temperature water bath, taking and dropwise applying a catalytic reaction to the surface of a gold electrode, and incubating the gold electrode in a 4 DEG C refrigerator; (2) preparing MB&AuNPs@GO-Ab: mixing HAuCl4 and CTAB with water, adding ascorbic acid to the obtained reaction mixture, adding NaOH to obtain CTAB covered AuNPs, centrifuging and purifying the CTAB covered AuNPs, dispersing the purified CTAB covered AuNPs in an equal amount of water, adding GO to the obtained solution, performing ultrasonic dispersion, standing the dispersed solution for later use,adding an acetyl antibody, performing incubation, adding MB, and performing vibration and uniform mixing; and (3) producing the electrochemical Faraday cage immunosensor: taking and dropwise applyingthe MB&AuNPs@GO-Ab to the surface of the peptide/Au, performing incubation at room temperature, and placing the produced sensor in the PBS (0.1 M, pH 7.0) to carry out electrochemical SWV test.

Description

technical field [0001] The invention relates to an electrochemical immunosensor, in particular to a construction method of an electrochemical Faraday cage immunosensor for detecting histone acetyltransferase activity and its detection application in a biological environment, belonging to the technical field of functional materials and biosensing. Background technique [0002] Cancer, also known as malignant tumor, is one of the three major diseases in today's human society, and its impact on human health is becoming more and more significant. Although encouraging progress has been made in the study of malignant tumors, the prevention and treatment of malignant tumors are not perfect, and multidisciplinary development and in-depth research are still needed. Therefore, the development of new analytical sensing methods and platforms, rapid, high-sensitivity, multi-channel dynamic monitoring of cancer-related markers and their occurrence and development process, provide directio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/48G01N33/535
CPCG01N27/48G01N33/535
Inventor 胡宇芳张青青徐利华王娇饶家佳郭智勇王邃
Owner NINGBO UNIV
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