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A detection method for polyadenosine diphosphate-ribose polymerase

A polyadenosine diphosphate-ribose and detection method technology, applied in the field of analytical chemistry, can solve the problems of biomolecular denaturation, expensive instruments, expensive and other problems, achieve high sensitivity, avoid the labeling process, and the method is simple and fast

Inactive Publication Date: 2018-06-26
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These assays often require expensive instrumentation and require radioactive or enzymatic labeling of the catalytic substrate
It is well known that the labeling process is often very time-consuming and expensive, and may even result in the denaturation of biomolecules

Method used

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  • A detection method for polyadenosine diphosphate-ribose polymerase
  • A detection method for polyadenosine diphosphate-ribose polymerase
  • A detection method for polyadenosine diphosphate-ribose polymerase

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Example 1. Determination of electrochemical signal value-concentration standard curve of PARP standard solution

[0024] 100 μL of PARP standard solutions of different concentrations were respectively incubated with c-kit-1 modified gold electrode according to the above steps, catalyzed and measured electrochemical signals. Such as figure 2 The curve showing the relationship between the electrochemical signal value (ip) and the concentration of PARP, within the range of PARP0.01U-1U, there is a linear relationship between ip and concentration, and the linear regression equation is y=-0.8912-2.17559x, R 2 =0.998, where y is the peak current ip (μA) of SWV, and x is the concentration of PARP (U).

Embodiment 23-A

[0025] Example 2.3 Determination of electrochemical signal value-concentration standard curve of AB standard solution

[0026] Different concentrations of 3-AB were incubated with 100 μL of the reaction solution containing 1 U of PARP at 4°C for 12 hours. Incubate with c-kit-1 modified gold electrode, catalyze and measure the electrochemical signal according to the above steps. Such as image 3 The curve showing the relationship between the electrochemical signal value (ip) and the concentration of 3-AB, 3-AB is in the range of 1nM-50nM, and there is a linear relationship between ip and the concentration. The linear regression equation is y=-3.32915+0.03898x, R 2 =0.997, where y is the peak current ip (μA) of SWV, and x is the concentration of 3-AB (nM).

[0027]

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Abstract

The invention belongs to the technical field of analytical chemistry and relates to a detection method and application of PARP [poly(adenosine diphosphate-ribose) polymerase]. The detection method mainly includes: modifying a single-stranded DNA (c-kit-1) capable of specifically binding with the PARP on the surface of a gold electrode in a classical mercapto self-assembly mode and enabling c-kit-1 to form a tetramer configuration through treatment; after the tetramer configuration is incubated with the PARP, adding a specific catalytic substrate, namely NAD (nicotinamide ademinedinucleotide) of the PARP, to enable the PARP to self-catalyze to produce PAR with high negative charges; using the negative charges of the PAR for adsorbing electrical signal molecules RuHex with positive charges, quantifying the RuHex adsorbed on the surface of the electrode through an electroanalysis method, and drawing a standard curve according to a relation between electrochemical signals and PARP concentration so as to achieve sensitive PARP detection by measuring the electrochemical signals of to-be-detected samples and calculating. The detection method is high in repeatability and sensitivity and can be applied to detection of the PARP and an inhibitor 3-AB (3-aminobenzamide) thereof.

Description

Technical field [0001] The invention relates to a method for detecting polyadenosine diphosphate ribose polymerase (PARP), in particular to an electrochemical method for detecting PARP, belonging to the field of analytical chemistry. Background technique [0002] Polyadenosine diphosphate ribosylation is an essential protein post-translational modification process catalyzed by the PARP family. In this process, PARP is activated by a specific DNA, cleaves the substrate nicotinamide adenine dinucleotide (NAD), cleaves it into nicotinamide and adenosine diphosphate ribose (ADP ribose), and combines the latter After polymerization to the receptor protein, a linear or branched ADP-ribose polymer (PAR) can be formed through several repeated reactions. The polymer generally contains about 200 ADP ribose units and has high electronegativity. About 90% of the polyadenosine diphosphate ribosylation modification reactions in the human body are completed by PARP, which plays an important r...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N27/26G01N27/48
CPCG01N27/26G01N27/48
Inventor 许媛媛苗晋锋张源淑邵营格孙杨杨卢辰赫
Owner NANJING AGRICULTURAL UNIVERSITY
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