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Method for quantitative detection of activity of poly(ADP-ribose)polymerase-1 (PARP-1) based on electrochemical sensing electrode with deposited polyaniline

A polyadenosine diphosphate-ribose, quantitative detection technology, applied in the field of biosensing, can solve the problems of high cost, affecting the measurement results, high requirements for instruments, etc., and achieve the effect of low cost and improved sensitivity

Inactive Publication Date: 2018-07-03
HENAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Enzyme-linked immunosorbent assay has high sensitivity and good repeatability. However, there is cross-reaction in this method, which may cause the detection value to be higher than the true value. The quantitative method is time-consuming and relatively expensive.
Detecting PAR by detecting radioactive intensity requires relatively high equipment requirements and low safety for operators
Biotinylated NAD + Changes in kinetic properties due to changes in structure affect assay results

Method used

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  • Method for quantitative detection of activity of poly(ADP-ribose)polymerase-1 (PARP-1) based on electrochemical sensing electrode with deposited polyaniline
  • Method for quantitative detection of activity of poly(ADP-ribose)polymerase-1 (PARP-1) based on electrochemical sensing electrode with deposited polyaniline
  • Method for quantitative detection of activity of poly(ADP-ribose)polymerase-1 (PARP-1) based on electrochemical sensing electrode with deposited polyaniline

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] The electrochemical sensing electrode based on polyaniline deposition quantitatively detects PARP-1 activity, and the detection steps are:

[0016] 1) Soak the gold electrode in piranha washing solution (H 2 o 2 and concentrated H 2 SO 4 The volume ratio is 3:7), after soaking for 10 minutes and cleaning, polishing with 0.3 μm and 0.05 μm alumina powder, rinsed with ultrapure water, and dried under nitrogen atmosphere to obtain a cleaned electrode;

[0017] 2) Dilute the specific double-stranded DNA-1 with a sulfhydryl group modified at the 5' end in 40-50 μL of fixative solution and 0.5-2 μL of tris(2-carboxyethyl)phosphine (TCEP) to prepare a final concentration of 0.5-2 μM Then add the prepared sample dropwise to the cleaned electrode surface, cover the beaker, and assemble it overnight at room temperature, then incubate the electrode in 0.5-2mM mercaptohexanol solution for 0.5-1 hour, and rinse with PBS buffer solution Finally, add specific double-stranded DNA-2...

Embodiment 2

[0028]The difference from Example 1 is: different concentrations of PARP (U, 4nM): (a) 0 , (b) 0.05 , (c) 0.1 , (d) 0.3 , (e) 0.5 , (f) 0.75 , ( g) 1 , (h) 2 , (i) 3, the UV-visible light curve obtained, it can be seen that within the range of 0-3 mmol, PARP has a good linear relationship between 0.05 U and 1 U. It can be seen that the sensitivity is greatly improved by the method of the present invention. The invention does not need to prepare complex materials, and has the advantages of low cost, quickness and simplicity. Moreover, the method can be used in the detection of actual samples and has certain clinical significance.

Embodiment 3

[0030] Different from Example 1, a certain amount of benzamide inhibitor is added after step 4), and the electrochemical signal of benzamide inhibiting PARP-1 electrochemical sensor is measured, such as Figure 4 A shows the standard curve diagram of the relationship between electrochemical signal and benzamide concentration after treating 0.5 UPARP-1 with different concentrations of benzamide. IC drawn from the figure 50 The value is 11.51 μM, which is consistent with the previously reported literature results. Therefore, this method can be used to evaluate and screen PARP-1 inhibitors, which is of great significance for the discovery of new anticancer drugs.

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Abstract

The invention provides a method for quantitative detection of the activity of PARP-1 based on an electrochemical sensing electrode with deposited polyaniline. The method comprises the following steps:modifying a gold electrode by using a specific DNA double-helix structure with a mercapto group so as to obtain a specific unit exciting PARP-1; with a part of PARP-1 as a catalyst for nicotinamide adenine dinucleotide (NAD<+>), cleaving NAD<+> into two parts, i.e., poly(ADP-ribose) (PAR) and nicotinamide, wherein one of the two parts is used as a linking agent for linking with a PAR unit; subjecting PARP-1 to repeated catalysis so as to form a negatively-charged PAR unit; preparation of the electrochemical sensing electrode with deposited polyaniline; and detecting the generated current signals of polyaniline by using an electrochemical process so as to detect the activity of PARP-1. Unlike conventional methods for determining residual NAD<+> substrates by using enzyme-linked immunosorbent assay, the method provided by the invention can quantitatively measuring the activity of PARP-1 and is greatly improved in sensitivity. The method does not need preparation of complex materials andhas the advantages of low cost, rapidness and easiness.

Description

technical field [0001] The invention relates to a technique for quantitatively detecting polyadenosine diphosphate-ribose polymerase-1, which belongs to the technical field of biosensing. Background technique [0002] Polyadenosine diphosphate polymerase-1 (PARP-1) is a multifunctional protein post-translational modification enzyme present in most eukaryotic cells. PARP-1 is involved in DNA repair and transcriptional regulation, and plays an important role in repairing DNA damage and maintaining gene integrity. PARP-1 is stimulated by some DNA, where nicotinamide adenine dinucleotide (NAD + ) in the presence of a part of PARP as NAD + catalyst, the NAD + It is split into two parts: poly ADP-ribose (PAR) and nicotinamide, and one part is used as a linker to connect PAR units. The PAR units are linear or branched, and there are about 200 of them, and they are negatively charged. Studies have shown that poly-ADP-ribosylation is one of the important post-translational modifi...

Claims

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Application Information

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IPC IPC(8): G01N27/327G01N27/48
CPCG01N27/3272G01N27/3277G01N27/48
Inventor 刘勇范佳慧卫伟
Owner HENAN UNIVERSITY
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