阅读说明：本技术 一种检测组蛋白乙酰转移酶的传感器及制备方法 (Sensor for detecting histone acetyltransferase and preparation method thereof ) 是由 李金龙 程文婷 胡亮 易永祥 许传军 胡凯 张兆利 于 2019-11-18 设计创作，主要内容包括：组蛋白乙酰转移酶(HAT)在肿瘤发生发展过程中发挥了重要的作用,因此本发明构建了一种利用降低G-Quadruplex-Cu(II)金属酶活性的电化学方法来检测组蛋白乙酰转移酶。G-Quadruplex-Cu(II)有金属酶活性,能够产生很强的电化学信号,在有HAT存在的情况下,HAT能够催化底物肽乙酰化,产生大量的乙酰辅酶A,由于G-Quadruplex和乙酰辅酶A竞争性结合Cu(II),使电化学信号减弱,从而可以直接定量检测HAT,利用该法可以使HAT的检出限达到0.14nM。另外,本发明设计的检测方法因其不需要标记和使用抗体,仅仅使用价廉的DNA和Cu<Sup>2+</Sup>,从而大大降低了成本,更重要的是,电化学法操作简单,不需要复杂的修饰过程。(Histone Acetyltransferase (HAT) plays an important role in the process of tumorigenesis and development, so the invention constructs an electrochemical method for detecting histone acetyltransferase by reducing the activity of G-Quadruplex-Cu (II) metalloenzyme. G-Quadroplex-Cu (II) has metalloenzyme activity and can generate a strong electrochemical signal, HAT can catalyze acetylation of substrate peptide to generate a large amount of acetyl coenzyme A in the presence of HAT, and the electrochemical signal is weakened due to competitive combination of G-Quadroplex and acetyl coenzyme A with Cu (II), so that HAT can be directly and quantitatively detected, and the detection limit of HAT can reach 0.14nM by using the method. In addition, the detection method designed by the invention does not need to be marked and usedWith antibodies, only inexpensive DNA and Cu are used 2+ Thereby greatly reducing the cost, and more importantly, the electrochemical method is simple to operate and does not need a complex modification process.)

1. A sensor for detecting histone acetyltransferase, characterized in that the sensor self-assembles a signal probe (DNA S1) rich in G base sequence on an electrode through Au-S bond when CuCl is used₂When the immobilized type gold nanoparticle is dripped on the surface of an electrode, G-quadriplex x-Cu (II) with metalloenzyme activity is formed, a stronger electrochemical signal can be generated, acetylation of substrate peptide can be catalyzed when HAT exists, a large amount of CoA is generated, the G-quadriplex-Cu (II) modified electrode is immersed in a reaction buffer solution, and the peak value of a current signal is weakened due to the fact that G-quadriplex and CoA are competitively combined with Cu (II), and quantitative detection of HAT is realized.

2. A method for preparing the sensor according to claim 1, comprising the following steps:

(1) gold electrode treatment

1) Polishing the gold electrode by using alumina powder to obtain a polished electrode;

2) soaking the electrode polished in the step 1) in the goby solution [ V (H)₂SO₄)：(30％H₂O₂)＝3：1]Removing adsorbed organic matters within 2-20min, and thoroughly cleaning with deionized water;

3) soaking the electrode prepared in the step 2) in nitric acid for 10-30min, treating the electrode with ethanol and deionized water for 2-8min, drying the electrode with nitrogen, soaking the electrode in sulfuric acid, and scanning from 0 to 1.6V by Cyclic Voltammetry (CV) until a stable signal is obtained;

(2) preparation of DNA self-assembled electrode

1) Immersing the gold electrode successfully prepared in the step 1 into a DNA fixing buffer solution for washing the electrode three times, wherein the DNA fixing buffer solution comprises 1-3 mu M of DNA S1(SEQ. ID. NO1,5 ″ -one of TGGGTAGGGCGGGTTGGGTTTTTT-3'), heating at 95 ℃, and cooling to room temperature to prepare a soaked electrode;

2) treating the prepared electrode in step (2) -1) with an aqueous solution containing 0.5-2mM MCH for 10-30 minutes to avoid non-specific adsorption, and then adding 10. mu.L of 50. mu.M CuCl₂The solution is dripped on the surface of the electrode to be incubated for 90 minutes, and an incubation electrode is prepared;

3) washing the incubation electrode prepared in step (2) -2) with Tris-HCl (10mM) and deionized water to remove excess CuCl₂Blowing the membrane by nitrogen to prepare the DNA self-assembly electrode;

(3) HAT Activity assay

1) 1 × reaction buffer solution (pH value 7.4) containing p300,500 μ MAc-CoA and 200 μ M substrate peptide with different concentrations is placed at 20-35 ℃ for incubation for 50-100 minutes to obtain reaction buffer solution;

2) and (3) immersing the DNA self-assembly electrode prepared in the step (2) into the reaction buffer solution prepared in the step (3) -2) for 10-40min, and finally washing the electrode with 5-20mM Tris-HCl to prepare the sensor for detecting the histone acetyltransferase, which can be used for electrochemical detection of the histone acetyltransferase.

3. The sensor of claim 2, wherein in step (1) the gold electrode process:

the diameter of the gold electrode is 3.0 mm;

the nitric acid is specifically 50% nitric acid;

the concentration of the sulfuric acid is 0.5M.

4. The sensor of claim 2, wherein step (2) prepares the DNA self-assembled electrode in which:

the concentration of the DNA S1 is 2 mu M;

the concentration of the MCH is 1 mM;

the electrode was treated with an aqueous MCH solution for 15 minutes as described.

5. The sensor of claim 2, wherein in step (3) the HAT activity assay:

the reaction buffer solution is 50-200 mu L;

100 mu L of the reaction buffer solution;

the pH of the reaction buffer solution is 7.4;

the incubation temperature is 30 ℃;

the incubation time was 80 minutes;

the prepared DNA self-assembly electrode is immersed in the reaction buffer solution in the step (3) -2) for 30 min;

the Tris-HCl concentration of the electrode washed by the Tris-HCl is 10 mM.

6. A method for detecting protein acetyltransferase using the sensor of claim 1, wherein electrochemical detection is carried out using a conventional three-electrode system using a CHI660D potentiostat.

7. The sensor of claim 6,

specifically, Differential Pulse Voltammetry (DPV) was performed in 0.1 mPBS;

specifically, the PBS solution specifically contained 1.0mM hydrogen peroxide and 0.2mM hydroquinone;

specifically, DPV: pulse period, 0.2 s; amplitude, 0.05; the scanning range is-0.4-0.1V;

specifically, Electrochemical Impedance Spectroscopy (EIS) experiments: bias potential, 0.232V;

specifically, the frequency range: 0.1 Hz-10 kHz; amplitude: 5 mV.

8. Use of the sensor according to claim 1 and the method for detecting a protein acetyltransferase according to claim 6 for detecting a histone acetyltransferase.

Technical Field

The invention relates to the field of histone acetyltransferase detection, in particular to a sensor for detecting histone acetyltransferase and a preparation method thereof.

Background

Histone Acetyltransferases (HATs) acetylate histone lysines, a typical "histone code" in epigenetic marker systems, which cause transcriptional activation, DNA replication, histone deposition and DNA repair. Abnormal gene silencing by HAT activation is closely related to the pathogenesis of many diseases such as neurological diseases, cancer, metabolic syndrome, and the like, and therefore, determination of the activity of HAT and the efficacy of their inhibitors is of great significance for the biochemical mechanism study of gene transcription and the development of anti-cancer drugs.

Conventional methods for detecting HAT activity rely primarily on autoradiography and radioisotopes, both of which are compromised by radioactive materials. Several nonradioactive methods for detecting HAT have been developed, such as fluorescence, colorimetric and electrochemical methods, which mostly rely on antibody recognition at the acetylation site, and thus have some inherent disadvantages, such as high cost of labeled antibody and complicated preparation of probe by modification of nanomaterial.

At present, chinese patent CN201910501021.4 discloses a photoelectric chemical biosensor for detecting histone acetyltransferase activity and a preparation method thereof, comprising: the ITO electrode is sequentially modified with stripped WS2, polydopamine, SMCC, coenzyme A, phos-tag-biotin and streptavidin on the surface of the electrode, the invention utilizes good biocompatibility and conductivity of WS2 and specifically identified avidin and biotin to realize quenching of photoelectric signals and improve the detection sensitivity of histone acetyltransferase, and utilizes the specific binding and identification function of maleimide in SMCC on sulfhydryl in CoA to improve the specificity of histone acetyltransferase activity detection.

At present, there is a need to develop a simple method for detecting HAT activity without the use of antibodies and tedious modifications.

The G-quatruplex-Cu (II) metalloenzyme has good peroxidase performance and can catalyze H₂O₂And TMB, generating stronger electrochemical signals, and based on the result, the invention develops an electrochemical method, HAT activity is measured by reducing the activity of G-Quadruplex-Cu (II) metalloenzyme, HAT can catalyze protein acetylation to generate a large amount of CoA, and the CoA contains thiol groups and can be competitively combined with Cu (II) to reduce the activity of G-Quadruplex-Cu (II) metalloenzyme, thereby realizing the detection of HAT. Therefore, the method of the biosensor has high sensitivity for detecting HAT activity and does not need complicated modification.

Disclosure of Invention

In view of the above-described prior art, an object of the present invention is to provide a sensor for detecting histone acetyltransferase and a method for preparing the same.

First, the present invention provides a sensor for detecting histone acetyltransferase, which has the following principle: as shown in FIG. 1, a signal probe (DNAS1) rich in G base sequence was self-assembled on the electrode mainly through Au-S bond, and CuCl was added₂When the solution is dripped on the surface of an electrode, G-quadriplex x-Cu (II) is formed, the G-quadriplex x-Cu (II) has metalloenzyme activity and generates stronger electrochemical signals, when HAT exists, the acetylation of substrate peptide can be catalyzed, a large amount of CoA is generated, the G-quadriplex-Cu (II) modified electrode is immersed in reaction buffer solution, and the peak value of a current signal is weakened due to the fact that the G-quadriplex and the CoA are combined competitively with the Cu (II)And realizing the quantitative detection of HAT.

Then, the invention also provides a preparation method of the sensor, which comprises the following specific steps:

(1) gold electrode treatment

1) Polishing a gold electrode (phi ═ 3mm) with alumina powder to obtain a polished electrode;

2) soaking the electrode polished in the step 1) in the goby solution [ V (H)₂SO₄)：(30％H₂O₂)＝3：1]Removing adsorbed organic matters within 2-20min, and thoroughly cleaning with deionized water;

3) soaking the electrode prepared in the step 2) in nitric acid for 10-30min, treating the electrode with ethanol and deionized water for 2-8min, drying the electrode with nitrogen, soaking the electrode in sulfuric acid, and scanning from 0 to 1.6V by Cyclic Voltammetry (CV) until a stable signal is obtained;

preferably, the gold electrode has a diameter of 3.0 mm;

preferably, the nitric acid is specifically 50% nitric acid;

preferably, the concentration of sulfuric acid is 0.5M;

(2) preparation of DNA self-assembled electrode

1) Immersing the gold electrode successfully prepared in the step 1 into a DNA fixing buffer solution to wash the electrode for three times,

wherein the DNA fixing buffer solution comprises 1-3 mu M DNA S1(SEQ. ID. NO1,5 'and TGGGTAGGGCGGGTTGGGTTTTTT-3'), and the soaked electrode is prepared by heating at 95 ℃ for 5 minutes and then cooling to room temperature;

preferably, the concentration of DNA S1 is 2. mu.M;

2) treating the prepared electrode in step (2) -1) with an aqueous solution containing 0.5-2mM MCH for 10-30 minutes to avoid non-specific adsorption, and then adding 10. mu.L of 50. mu.M CuCl₂The solution is dripped on the surface of the electrode to be incubated for 90 minutes, and an incubation electrode is prepared;

preferably, the concentration of MCH is 1 mM;

preferably, the electrode is treated with an aqueous solution of MCH for 15 minutes;

3) washing the incubation electrode prepared in step (2) -2) with Tris-HCl (10mM) and deionized water to removeExcess CuCl₂Blowing the membrane by nitrogen to prepare the DNA self-assembly electrode;

preferably, the Tris-HCl concentration is 10 mM;

(3) HAT Activity assay

1) 1 × reaction buffer solution (pH value 7.4) containing p300,500 μ MAc-CoA and 200 μ M substrate peptide with different concentrations is placed at 20-35 ℃ for incubation for 50-100 minutes to obtain reaction buffer solution;

preferably, the reaction buffer is 50-200. mu.L;

more preferably, 100. mu.L of reaction buffer;

preferably, the reaction buffer pH is 7.4;

preferably, the incubation temperature is 30 ℃;

preferably, the incubation time is 80 minutes;

2) immersing the DNA self-assembly electrode prepared in the step (2) into the reaction buffer solution prepared in the step (3) -2) for 10-40min, and finally washing the electrode with 5-20mM Tris-HCl to prepare the sensor for detecting the histone acetyltransferase, which can be used for electrochemical detection of the histone acetyltransferase;

preferably, the prepared DNA self-assembly electrode is immersed in the reaction buffer solution in the step (3) -2) for 30 min;

preferably, the Tris-HCl concentration of the electrode is 10mM by washing with Tris-HCl;

the invention also provides a detection method for detecting protein acetyltransferase by using the sensor for detecting histone acetyltransferase, which comprises the following steps:

performing electrochemical detection on the CHI660D potentiostat by using a traditional three-electrode system;

preferably, Differential Pulse Voltammetry (DPV) is performed in 0.1 mPBS;

more preferably, the PBS solution contains in particular 1.0mM hydrogen peroxide and 0.2mM hydroquinone;

specifically, DPV: pulse period, 0.2 s; amplitude, 0.05; the scanning range is-0.4-0.1V;

specifically, Electrochemical Impedance Spectroscopy (EIS) experiments: bias potential, 0.232V;

specifically, the frequency range: 0.1 Hz-10 kHz; amplitude: 5 mV.

Finally, the present invention also provides the use of the above-mentioned sensor for detecting histone acetyltransferase and the detection method for detecting protein acetyltransferase by using the detector for detecting histone acetyltransferase in the detection of histone acetyltransferase.

The invention has the beneficial effects

(1) The invention can directly detect HAT quantitatively, and the detection limit of HAT can reach 0.14nM by using the method.

(2) The detection method designed by the invention only uses cheap DNA and Cu because the detection method does not need to label and use antibodies²⁺Thereby greatly reducing the cost.

(3) The electrochemical method is simple to operate and does not need a complex modification process.

Drawings

Fig. 1 is a schematic diagram of the operation of a sensing system.

FIG. 2 is an AC impedance spectrum in test example 1.

FIG. 3 is a graph showing the results of the test for optimizing the test conditions in test example 2.

FIG. 4 is a graph showing the optimum test results in test example 3.

FIG. 5 is a graph showing the results of the selectivity test in test example 4.

FIG. 6 is a graph showing the optimum test results in test example 5.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

The chemical reagents used in the invention are specifically:

the DNA oligonucleotide (RGKGGKGLGKGGAKA) used in the experiment is synthesized by Shanghai Biotechnology Limited company, and is in powder form, and the purity is more than 95%;

the oligonucleotide sequence purified by the high performance liquid chromatography is shown in SEQ ID No. 1: DNA S1, 5'-TGGGTAGGGCGGGTTGGGTTTTTT-3', synthesized by Shanghai Biotechnology Ltd;

CuCl₂human p300(HAT), MCH, ethylenediaminetetraacetic acid (EDTA), acetyl-CoA (Ac-CoA) and Lacquertree acid were purchased from Sigma, Shanghai, China;

the invention dissolves peptide powder in 10mM Tris-HCl buffer solution (PH7.4) for dilution; the oligonucleotides were diluted with 10mM Tris-HCl buffer (pH7.4) to make 100. mu.M stock solutions;

the ultrapure water used for diluting all solutions is produced from a pure water system (Milli-Q), and the purity after purification reaches 18M omega cm of gold standard resistance.