CN107314981B - Method for analyzing and detecting PARP activity based on hemin-graphene composite material - Google Patents

Method for analyzing and detecting PARP activity based on hemin-graphene composite material Download PDF

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CN107314981B
CN107314981B CN201710638454.5A CN201710638454A CN107314981B CN 107314981 B CN107314981 B CN 107314981B CN 201710638454 A CN201710638454 A CN 201710638454A CN 107314981 B CN107314981 B CN 107314981B
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刘勇
徐晓林
卫伟
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Abstract

The invention discloses a method for analyzing and detecting PARP activity based on hemin-graphene composite material, which comprises the following steps: (1) selecting activated DNA (2) to synthesize H-GNs (hemin-graphene) composite material; (3) activating DNA, PARP, Nicotinamide Adenine Dinucleotide (NAD)+) Mixing reaction, PARP catalyzes and synthesizes PAR polymer (poly ADP-ribose) with a large amount of negative charge; (4) reacting the H-GNs with the PAR polymer product, adding a salt solution, and recording the agglomeration change of the H-GNs; (5) and detecting the product solution by using an ultraviolet-visible spectrometer. The invention utilizes the electrostatic interaction between H-GNs and a product PAR polymer to obtain the colorimetric reaction caused by the agglomeration change of the H-GNs in a salt solution, and can quantitatively detect the activity of PARP. The invention has the advantages of simplicity, convenience, rapidness, high sensitivity and no need of marking a DNA probe.

Description

Method for analyzing and detecting PARP activity based on hemin-graphene composite material
Technical Field
The invention belongs to a technology for quantitatively detecting the activity of PARP (poly (adenosine diphosphate) ribose polymerase), relates to the application of a graphene composite material in clinical detection, and particularly relates to the field of the application of the graphene composite material in quantitatively detecting biological enzymes.
Background
PARP, also known as poly ADP-ribose polymerase, is a group of cellular ribozymes that are present in most eukaryotes and catalyze poly ADP ribosylation, and the structure mainly includes three regions: DNA binding domain, self-modifying domain, catalytic domain. PARP is capable of binding various DNA structures and nucleosomes and has NAD+Depending on the catalytic activity, poly (ADP-ribose) polymers can be synthesized on the target protein. The formation of poly (ADP-ribose) polymers significantly alters the charge characteristics of receptor proteins, triggering DNA damage control and repair processes. PARP plays an important regulatory role in gene stabilization and development of tumors, inflammation and stress responses, metabolism and energy consumption, circadian rhythms, and the like. In addition, there is a difference in the expression of PARP in malignant tumor and normal tissue, and the difference may play an important role in the occurrence and development of diseases. In recent years, the role of PARP in DNA damage repair and maintenance of genomic stability has attracted attention. On the basis, the inhibitor is used for inhibiting PARP from participating in mediated tumor cell DNA damage repair, and is more current researchOne of the hot spots.
The conventional technology for detecting PARP activity mainly comprises an enzyme-linked immunosorbent assay, wherein an anti-PAR monoclonal antibody and an HRP-labeled goat anti-mouse lgG secondary antibody are used for establishing a colorimetric method or a chemiluminescence method for detecting PAR deposited on immune histone; or with radioactive NAD+The method of (3) to determine the enzymatic activity of PARP. However, such methods require labeling, have the disadvantages of high cost, large sample size, poor sensitivity, long detection time, and sometimes give false positive results. Currently, Hergenrother et al use chemoquantitation of NAD+The method indirectly measures the enzyme activity of the PARP-1, and can be used for high-throughput screening of small molecule inhibitors; and an ADP-ribose-pNP chromogenic substrate is synthesized, and a simple and sensitive colorimetric method is developed to evaluate the activity of PARP. However, this method requires a cumbersome synthesis. Thus, there are many limitations to this approach.
In recent years, to overcome the disadvantages of PARP analysis methods, researchers have developed many simple and executable assays and applied them to the detection of PARP activity, such as colorimetric detection, fluorescence detection, electrochemical detection, etc. For example, professor neyoho, university of Hunan, establishes a fluorescence detection method for PARP activity by utilizing the regulation effect of PAR on fluorescence resonance energy transfer efficiency between fluorescent protein scGFP and cationic polymer CCP; by using hexaammine ruthenium as an indicator, a novel electrochemical detection method for PARP is established according to the amount of PAR electrostatic adsorption hexaammine ruthenium by professor of Changjingshan university with Shihui Daiz Dahliang; the Li Gen xi professor of Nanjing university utilizes PARP to cause the aggregation of gold glue modified with coenzyme NAD, and establishes a colorimetric detection method of PARP activity. Inspired by the method, a hemin-graphene composite material based colorimetric detection method for PARP activity is established. The method does not need to be marked, is easy to operate, and has the advantages of high stability, low detection line and wide detection range compared with the colorimetric method.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the problems in the prior art, a method for detecting PARP activity based on hemin-graphene composite material analysis is provided. Poly ADP nucleus synthesized by PARP catalysis in the inventionThe large amount of negative charges on the sugar chains changes the degree of dispersion of H-GNs at a certain salt concentration. In TMB and H2O2In the presence of the enzyme, PARP activity is determined by the change in the degree of color development obtained. It has the advantages of high sensitivity, good accuracy, no mark, etc.
The technical scheme is as follows: in order to solve the technical problem, the invention provides a method for analyzing and detecting PARP activity based on hemin-graphene composite material, which comprises the following steps:
(1) selecting activating DNA to activate PARP (poly adenosine diphosphate ribose polymerase);
(2) synthesizing H-GNs (hemin-graphene) composite material;
(3) mixing activated DNA, PARP, NAD + (nicotinamide adenine dinucleotide) for reaction to synthesize PAR (poly ADP-ribose) polymer with a large amount of negative charges;
(4) mixing the H-GNs composite material with the PAR polymer for reaction, adding a salt solution, and recording the agglomeration change of the H-GNs and the PAR polymer product;
(5) and (4) quantitatively detecting the product solution obtained in the step (4) by using an ultraviolet-visible spectrometer.
As an improved technical scheme of the invention, the following technical measures can be adopted: the sequence of the activating DNA in the step (1) is selected from Shanghai bioengineering Co., Ltd:
single-stranded DNA1: 5'-CCCGTGCGTGCGCGAGTGAGTTGGTGTGTGTGTGTGTGTGT-3'
Single-stranded DNA2: 5'-CAACTCACTCGCGCACGCACGGG-3'
The method for forming the activated double-stranded DNA comprises the following steps: after 3-10 minutes of water bath reaction at 95 ℃, the activated DNA single strand is cooled to room temperature to form hybridized double-stranded DNA, and the double-stranded DNA reacts with the PARP to activate the activity of the PARP.
As an improved technical scheme of the invention, the following technical measures can be adopted: the step (2) is specifically as follows: weighing 10mg of graphite oxide, dissolving the graphite oxide in 10-40mL of secondary water, wherein the concentration of the graphite oxide is 0.2-1mg/mL, performing ultrasonic dispersion for 2-4 h for mechanical stripping, centrifuging at the rotating speed of 2000-4000 rpm for 20-30 min, removing the graphite oxide which is not stripped, taking supernatant, dialyzing in a dialysis bag (MW:8000-12000) for one week, and removing impurity micromolecules to obtain uniformly dispersed graphene oxide; fully mixing the graphene oxide solution with 10-40mL of hemin dissolved in 0.1M NaOH solution in a flask, wherein the concentration of the hemin is 0.2-1 mg/mL; slowly adding 150-200 mu L of ammonia solution, finally adding 20-50 mu L of hydrazine hydrate, violently stirring the mixed solution for 30-60 min, and placing the flask in a water bath at 60 ℃ for reaction for 3-24 h to obtain a stably dispersed black solution; and centrifuging the black solution at 12000-13000 rpm for 30-60 min to obtain black precipitate, and washing with water for 3-5 times to obtain the H-GNs composite material which is easy to redisperse in water.
As an improved technical scheme of the invention, the following technical measures can be adopted: the step (3) is specifically as follows: configuring the PARP to different concentrations with a reaction buffer solution containing the activating DNA, the NAD+The reaction buffer solution is dripped with PARP with different concentrations and reacts for 1-2 hours at the temperature of 30-38 ℃.
As an improved technical scheme of the invention, the following technical measures can be adopted: the concentration of the activated DNA is 100-200 nM.
As an improved technical scheme of the invention, the following technical measures can be adopted: the NAD+The concentration is 100 to 500. mu.M.
As an improved technical scheme of the invention, the following technical measures can be adopted: the reaction buffer solution contains KCl and MgCl2、Zn(OAc)250mM Tris-HCl of pH 7.2-7.4, the initial concentration of KCl is 50mM, MgCl2Initial concentration 2mM, Zn (OAc)2The initial concentration was 50. mu.M.
As an improved technical scheme of the invention, the following technical measures can be adopted: the step (4) is specifically as follows: adding the H-GNs composite material into the PAR polymer mixed reaction solution synthesized by the PARP catalytic reaction, reacting for 20-40 min, then adding a NaCl solution, reacting for 30-50 min at 20-30 ℃, centrifuging, taking supernatant, adding into a phosphoric acid buffer solution, and adding into TMB and H2O2Recording and UV curing the solution in the presence of-visible spectrum detection.
As an improved technical scheme of the invention, the following technical measures can be adopted: the phosphoric acid buffer solution is 25-50 mM phosphoric acid buffer solution with the pH value of 5-6.
As an improved technical scheme of the invention, the following technical measures can be adopted: the concentration of NaCl is 0.01-1.0M, and TMB and H2O2The final concentrations of (A) are 0.1 to 1.0mM and 5 to 30mM, respectively.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention does not need to be marked, simplifies the detection method, and avoids the defects of high detection cost, complicated operation and poor reproducibility caused by marking the DNA probe.
(2) The hemin-graphene composite material has inherent catalase-like activity, has adjustable dispersibility in a salt solution, and can adsorb single-stranded DNA through pi-pi action, so that the hemin-graphene composite material is a detection material with great potential.
(3) The invention effectively utilizes the characteristics of the nano material, does not need to be detected by a precise instrument, simplifies the detection method, greatly reduces the virus detection cost, and has the advantages of low cost, rapidness, simplicity, convenience, sensitivity and good specificity.
(4) The method can successfully detect the activity of PARP added into serum, and has certain clinical significance.
Drawings
FIG. 1 is a flow chart for colorimetric detection of PARP activity based on H-GNs.
FIG. 2 shows a graph representing the UV absorption spectrum of H-GNs: a, hemin ultraviolet absorption curve, b, graphite oxide ultraviolet absorption curve, and c.H-GNs ultraviolet absorption curve.
FIG. 3 is a graph showing the relationship between the solubility of H-GNs and the concentration (mol) of NaCl solution.
FIG. 4 shows a verification chart of telomerase activity assay: a.H-GNs with PARP, NAD+The ultraviolet absorption intensity of the mixed solution; b.H ultraviolet absorption intensity of mixed solution of GNs, PARP and dsDNA; c.H-GNs with dsDNA, NAD+Ultraviolet absorption intensity of (4); d.PARP catalysisUV absorption intensity of the chemically synthesized product when mixed with H-GNs.
Figure 5 shows a graph of the change in uv absorbance for the detection of PARP activity: A. the uv-vis curves obtained at different PARP concentrations (PARP concentration (U,1U ═ 45ng), (a)0(b)0.05(c)0.1(d)0.3(e)0.5(f)0.75(g)1(h)2(i) 3; b. standard curves of uv absorption intensity versus PARP concentration and a linear relationship between uv absorption intensity and PARP concentration, from which it can be seen that PARP is well linear from 0.05U to 1U.
Detailed Description
The present invention will be further described with reference to the accompanying drawings.
Reagents and instruments used in this experiment:
PARP (poly (adenosine diphosphate) ribosyl polymerase), NAD+(nicotinamide adenine dinucleotide), hemin (hemin), TMB (3, 3', 5, 5' -tetramethylbenzidine), H2O2Hydrazine hydrate, ammonia water, NaCl, and an ultraviolet-visible spectrometer.
Activating DNA sequence (selected from shanghai bioengineering limited):
single-stranded DNA1: 5'-CCCGTGCGTGCGCGAGTGAGTTGGTGTGTGTGTGTGTGTGT-3'
Single-stranded DNA2: 5'-CAACTCACTCGCGCACGCACGGG-3'
Example 1:
an analysis method for detecting PARP activity based on H-GNs composite material analysis comprises the following detection steps:
the synthesis steps of the H-GNs composite material are as follows: weighing 10mg of graphite oxide, dissolving the graphite oxide in 20mL of secondary water (0.5mg/mL), ultrasonically dispersing for 2h, mechanically stripping, centrifuging at 3000rpm for 30min, removing the graphite oxide which is not stripped, taking supernatant, dialyzing in a dialysis bag (MW:8000-12000) for one week, and removing impurity micromolecules to obtain the uniformly dispersed graphene oxide. The resulting graphene oxide solution was mixed well with 20mL of hemin (0.5mg/mL) dissolved in 0.1M NaOH solution in a flask. After completion, 200. mu.L ammonia solution was added slowly and finally 30. mu.L hydrazine hydrate was added. The mixed solution was stirred vigorously for 60 minutes, and the flask was placed in a water bath (60 ℃ C.) to react for 20 hours to give a stably dispersed black solution. The resulting black solution was centrifuged at 13000rpm for 30 minutes to give a black precipitate, which was washed three times with water to give H-GNs. The resulting H-GNs can be easily redispersed in water.
DNA hybridization step: two activated DNA single strands were added to a DNA hybridization buffer (10mM Tris-HCl, pH 7.4,0.1M NaCl) and slowly cooled to room temperature in a water bath at 95 ℃ for 5 minutes to form hybridized activated DNA.
PARP catalyzes the synthesis of PAR: the PARP is prepared in different concentrations by reaction buffer solution containing activated DNA, NAD+Reaction buffer (50mM Tris-HCl, pH 7.4,50mM KCl,2mM MgCl)2,50μM Zn(OAc)2) 0.1U of PARP was added dropwise thereto, and the reaction was carried out at 37 ℃ for 1 hour.
PARP Activity detection step: 150 μ L of 50 μ g/mL H-GNs composite material was added to the PAR-produced solution, and 0.4M NaCl solution was added to react at 28 ℃ for 30 min. After completion of the reaction, the reaction mixture was centrifuged, and the supernatant was added to a phosphate buffer solution (25mM pH 5.0) at 0.8mM TMB and 25mM H2O2In the presence of the solvent, the solution is recorded and detected by ultraviolet-visible spectrum. The experimental result is shown in the curve d in fig. 4, the PARP is in a good linear relationship between 0.05 and 1U, and the detection limit is 0.034U.
Reference example
The graphite oxide ultraviolet absorption curve, the hemin ultraviolet absorption curve and the H-GNs ultraviolet absorption curve are respectively shown as curves a-c in FIG. 2.
Comparative example
To demonstrate the necessity of activating DNA for PARP activity testing, a blank experiment was performed, unlike example 1, in which the UV absorption of PARP was performed in the absence of activating DNA (i.e., H-GNs were performed in combination with PARP, NAD) under otherwise identical conditions+Uv absorption of the mixed solution), the test results are shown in curve a of fig. 4.
To demonstrate NAD+The necessity of testing for PARP Activity, a blank test was performed, unlike example 1, in the absence of NAD under otherwise unchanged conditions+The UV absorption of PARP (i.e. the UV absorption of the mixed solution of H-GNs and PARP, activated DNA) was performed, and the results are shown in the curve b of FIG. 4。
The results of the ultraviolet absorption test in the absence of the reactive detector PARP blank test are shown in figure 4, curve c.
Example 2
The difference from example 1 is: different concentrations of PARP were used (U,1U ═ 45 ng): (a)0(b)0.05(c)0.1(d)0.3(e)0.5(f)0.75(g)1(h)2(i)3, and the ultraviolet-visible light curve obtained is shown in FIG. 5, it can be seen that PARP shows a good linear relationship between 0.05U and 1U in the range of 0-3U.
In conclusion, the H-GNs composite material can quantitatively detect PARP, and has the advantages that the H-GNs composite material has inherent catalase-like activity and adjustable dispersibility in a salt solution, the characteristics of the nano material are effectively utilized, a precise instrument is not needed for detection, the detection method is simplified, the virus detection cost is greatly reduced, the activity of the PARP added into serum can be successfully detected, and the clinical significance is good.
Although the present invention has been described with reference to the above embodiments, the present invention is not limited to the above embodiments, which are only illustrative and not restrictive, and those skilled in the art can make various modifications without departing from the spirit and scope of the present invention as claimed in the claims.

Claims (8)

1. A method for detecting PARP activity based on hemin-graphene composite analysis, which is characterized by comprising the following steps:
(1) selecting activated DNA to activate poly (A-bis (phosphoribosyl) phosphate polymerase (PARP);
(2) synthesizing hemin-graphene (H-GNs) composite materials;
(3) will activate DNA, PARP, Nicotinamide Adenine Dinucleotide (NAD)+) Mixing reaction to synthesize poly ADP-ribose (PAR) polymer with a large amount of negative charges;
(4) mixing and reacting the H-GNs composite material with the PAR polymer, adding salt solution, and recording the products of H-GNs and PAR polymerThe agglomeration change of the polymer is as follows: adding the H-GNs composite material into the PAR polymer mixed reaction solution synthesized by the PARP catalytic reaction, reacting for 20-40 min, then adding a NaCl solution, reacting for 30-50 min at 20-30 ℃, centrifuging, taking supernatant, adding into a phosphoric acid buffer solution, and adding into TMB and H2O2In the presence of the solvent, recording and detecting the solution by ultraviolet-visible spectrum;
(5) carrying out quantitative detection on the product solution obtained in the step (4) by using an ultraviolet-visible spectrometer;
the sequence of the activating DNA in the step (1) is as follows:
single-stranded DNA1: 5'-CCCGTGCGTGCGCGAGTGAGTTGGTGTGTGTGTGTGTGTGT-3'
Single-stranded DNA2: 5'-CAACTCACTCGCGCACGCACGGG-3'
The method for forming the activated double-stranded DNA comprises the following steps: after 3-10 minutes of water bath reaction at 95 ℃, the activated DNA single strand is cooled to room temperature to form hybridized double-stranded DNA, and the double-stranded DNA reacts with the PARP to activate the activity of the PARP.
2. The method for detecting PARP activity based on hemin-graphene composite assay as claimed in claim 1, wherein the step (2) is specifically as follows: weighing 10mg of graphite oxide, dissolving the graphite oxide in 10-40mL of secondary water, carrying out ultrasonic dispersion for 2-4 h for mechanical stripping, centrifuging at the rotating speed of 2000-4000 rpm for 20-30 min, removing the graphite oxide which is not stripped, taking supernatant, dialyzing for one week in a dialysis bag to remove impurity micromolecules to obtain uniformly dispersed graphene oxide, wherein the molecular weight range of the dialysis bag is 8000-12000; fully mixing the graphene oxide solution with 10-40mL of hemin dissolved in 0.1M NaOH solution in a flask, wherein the concentration of the hemin is 0.2-1 mg/mL; slowly adding 150-200 mu L of ammonia solution, finally adding 20-50 mu L of hydrazine hydrate, violently stirring the mixed solution for 30-60 min, and placing the flask in a water bath at 60 ℃ for reaction for 3-24 h to obtain a stably dispersed black solution; and centrifuging the black solution at 12000-13000 rpm for 30-60 min to obtain black precipitate, and washing with water for 3-5 times to obtain the H-GNs composite material which is easy to redisperse in water.
3. The method for detecting PARP activity based on hemin-graphene composite assay as claimed in claim 1, wherein the step (3) is specifically as follows: configuring the PARP to different concentrations with a reaction buffer solution containing the activating DNA, the NAD+The reaction buffer solution is dripped with PARP with different concentrations and reacts for 1-2 hours at the temperature of 30-38 ℃.
4. The method for detecting PARP activity based on hemin-graphene composite assay of claim 3, wherein the activating DNA concentration is 100-200 nM.
5. The method for detecting PARP activity based on hemin-graphene composite assay of claim 3, wherein said NAD+The concentration is 100 to 500. mu.M.
6. The method for detecting PARP activity based on hemin-graphene composite analysis of claim 3, wherein said reaction buffer solution is KCl-containing MgCl2、Zn(OAc)250mM Tris-HCl of pH 7.2-7.4, the initial concentration of KCl is 50mM, MgCl2Initial concentration 2mM, Zn (OAc)2The initial concentration was 50. mu.M.
7. The method for detecting PARP activity based on hemin-graphene composite assay of claim 1, wherein said phosphate buffer solution is 25-50 mM phosphate buffer solution with pH 5-6.
8. The method for detecting PARP activity based on hemin-graphene composite assay of claim 1, wherein the concentration of NaCl is 0.01-1.0M, and the TMB and H are2O2The final concentrations of (A) are 0.1 to 1.0mM and 5 to 30mM, respectively.
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