Detection method of poly(adenosine diphosphate-ribose) polymerase

A polyadenosine diphosphate-ribose and detection method technology, which is 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 effect of simple and fast method

Inactive Publication Date: 2016-05-25
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
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  • Claims
  • Application Information

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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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  • Detection method of poly(adenosine diphosphate-ribose) polymerase
  • Detection method of poly(adenosine diphosphate-ribose) polymerase
  • Detection method of poly(adenosine diphosphate-ribose) polymerase

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Embodiment 1

[0023] The determination of embodiment 1.PARP standard solution electrochemical signal value-concentration standard curve figure

[0024] 100 μL of PARP standard solutions with different concentrations were incubated with the c-kit-1 modified gold electrode according to the above steps, catalyzed and the electrochemical signal was measured. Such as figure 2 The change relationship curve between the electrochemical signal value (ip) and the concentration of PARP is shown. In 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] The determination of embodiment 2.3-AB standard solution electrochemical signal value-concentration standard curve figure

[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, catalyze, and measure electrochemical signals with c-kit-1 modified gold electrodes according to the above steps. Such as image 3 The change relationship curve between the electrochemical signal value (ip) and the concentration of 3-AB, 3-AB is in the range of 1nM-50nM, there is a linear relationship between ip and concentration, and 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 (nM) of 3-AB.

[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 detection method of polyadenosine diphosphate-ribose polymerase (PARP), in particular to an electrochemical method for PARP detection, which belongs to the field of analytical chemistry. Background technique [0002] Poly ADP-ribosylation is an essential protein post-translational modification process catalyzed by the PARP family. In this process, PARP is activated by specific DNA, cleaves the substrate nicotinamide adenine dinucleotide (NAD), splits it into nicotinamide and adenosine diphosphate ribose (ADP ribose), and converts the latter Polymerized to the receptor protein, through several repeated reactions, a linear or branched ADP-ribose polymer (PAR) can be formed. 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 role in DNA repair, t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/26G01N27/48
CPCG01N27/26G01N27/48
Inventor 许媛媛孙杨杨卢辰赫
Owner NANJING AGRICULTURAL UNIVERSITY
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