Preparation and application of histone acetyltransferase electrochemical sensor based on "Cu(II) to Cu(II)" mechanism

Abstract

The present invention is a preparation and application of a histone acetyltransferase electrochemical sensor based on a "Cu(II) to Cu(II)" mechanism. First, the sulfhydryl group in CoA and Cu(II) generate a CoA-Cu(II) complex in situ, which can be regarded as a nanochain complex with multiple adenines as side groups. The complex can be combined with GO through its adenine side group through a π-π conjugation effect, so as to be combined and fixed on the electrode surface, and the voltage signal of Cu(II) is collected by FSCV technology. Because the polymerization of CoA and Cu(II) can realize the electrochemical sensing analysis of CoA, and because CoA is the product of the HAT catalytic reaction, the electrochemical sensing analysis of HAT can be realized. By changing the concentration of CoA or HAT, a series of FSCV signal sizes corresponding to CoA and HAT at different concentrations can be obtained; when the inhibitor anacardic acid / C646 is added, the HAT activity is inhibited, CoA cannot be generated, and the complex cannot be formed, thereby realizing the screening of HAT inhibitors. Based on this, a fast, simple, reliable, stable and highly sensitive FSCV analysis method is constructed.

Description

Technical Field

[0001] The present invention relates to a special "Cu(II) to Cu(II)" mechanism, in particular to the preparation of a histone acetyltransferase electrochemical sensor based on a coenzyme A-copper ion complex and its application in analytical sensing of coenzyme A and histone acetyltransferase, belonging to the field of functional biomaterials and biosensor technology. Background Art

[0002] Histone acetylation is an important modification process in the gene transcription process of eukaryotic organisms. It is regulated by histone acetyltransferase (HAT). It mainly regulates gene transcription activation by changing the distribution of histone acetyl marks in local chromatin regions, thereby affecting the local chromatin relaxation state. HAT is a class of enzymes that can catalyze the acetylation of histones. Acetyl coenzyme A (Ac-CoA) as an acetyl donor transfers the acetyl group from Ac-CoA to a specific lysine residue of the substrate peptide, regulating chromosome structure and regulating gene expression. Once the dynamic balance between acetylation / deacetylation in cells is destroyed, it will lead to various diseases, such as Parkinson's disease, leukemia, and even cancer. Transcription coactivator p300 is one of the most common histone acetyltransferases. It regulates gene expression by acetylated chromatin and mediates many cellular physiological activities, such as DNA damage repair, gene transcription regulation, cell differentiation and apoptosis. Therefore, detecting the activity of HAT p300 is of great research significance for studying related biological processes and the development of disease occurrence.

[0003] At present, there are many methods to detect the activity of HAT p300, such as fluorescence, electrochemiluminescence, electrochemistry, etc. Among them, electrochemical methods have attracted widespread attention due to their unique advantages such as high sensitivity, low cost, simple equipment, convenient operation, and miniaturization. However, the stability and sensitivity of traditional electrochemical methods are limited. In response to these two problems, our research group combined electrochemical detection technology with information engineering technology, modified and designed a set of rapid scanning devices, among which the most classic fast scanning cyclic voltammetry (FSCV) is a variant of cyclic voltammetry (CV). It is an electrochemical method that uses high scanning speed to complete the detection of neurotransmitters and trace amounts in specific parts, which can effectively improve the detection sensitivity and increase the reliability of the analysis results. As a powerful electrochemical analysis technique, the most common method of FSCV is to combine a specially designed constant potential instrument with an electrode to scan the applied potential at a high scanning speed, resulting in sub-second time resolution and sub-micron spatial resolution. At present, the FSCV method is only used for the detection of small molecules (such as metal ions, dopamine, etc.), but there is a "blank" phenomenon in the detection of macromolecules, especially biological macromolecules. The most important reason is that biological macromolecules can bring a large background signal to the system and there is no way to achieve direct detection. Therefore, in the field of biochemistry, the widespread use of the FCSV method is still a major issue that needs to be solved urgently.

[0004] For classical electrochemistry, the metal ion divalent copper (Cu(II)) undergoes a "Cu(II) to Cu(0)" transition under low scan rate conditions due to its own redox activity, resulting in extremely unstable electrochemical signals and affected sensitivity. In view of this, the present invention proposes a "Cu(II) to Cu(II)" mechanism based on rapid scanning cyclic voltammetry, based on the binding effect of CoA (containing 1 thiol group) and Cu(II), and utilizes the conjugation effect of the adenine structure on CoA and graphene two-dimensional material (GO) to make an electrochemical substrate, and realizes the detection of CoA bio-small molecules through the continuous voltage signal output of Cu(II); since CoA is the product of the HAT p300 catalytic reaction, the sensing method can be used to indirectly detect the activity of HAT p300 and the screening of its small molecule inhibitors, thereby avoiding the influence of bio-macromolecules on the background signal. At present, there is no electrochemical sensor that combines electrochemical rapid scanning technology, "Cu(II) to Cu(II)" mechanism, and CoA-Cu(II) complex to detect CoA and HAT p300. This method has lower sensitivity, better signal stability, and better novelty. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a preparation and application of a histone acetyltransferase electrochemical sensor based on the "Cu(II) to Cu(II)" mechanism which is simple to operate, has high sensitivity, good specificity, fast detection speed and low cost.

[0006] The technical solution adopted by the present invention to solve the above technical problems is: preparation and application of histone acetyltransferase electrochemical sensor based on the "Cu(II) to Cu(II)" mechanism, and the specific steps are as follows:

[0007] (1) Preparation of CoA-Cu(II) complex

[0008] Take 1-10 μL of Cu(NO3)2 solution with a final concentration of 50-500 μM and 1-20 μL of CoA solution with a final concentration of 0.01-150 μM in sequence, and mix them with 100 mM pH 7.0 PBS buffer solution to make a 0.1-1 mL solution. Vigorously shake the solution for 5-15 minutes to obtain a CoA-Cu(II) complex.

[0009] (2) Preparation of electrochemical biosensors

[0010] a. GCE: A glassy carbon electrode (GCE, 3 mm in diameter) was polished on suede with 1, 0.3 and 0.05 μm aluminum oxide powder for 1 to 5 min, then ultrasonically washed with water and ethanol for 1 to 5 min, and then dried with nitrogen for later use. The electrode was marked as GCE.

[0011] b. GO / GCE: 4.0-8.0 mg of graphene was dispersed in 2.0-10.0 mL of 0.1-0.3 M acetic acid buffer solution with a pH of 5.0-6.0, and ultrasonically dispersed in an ultrasonic cleaner for 2-5 h to obtain a graphene dispersion. Using cyclic voltammetry, the potential range was -2-2 V, the scan rate was 10 mV / s, and the scan was repeated 10-20 times, and the graphene was electrodeposited onto GCE to obtain a graphene-modified glassy carbon electrode, which was labeled as GO / GCE.

[0012] c. CoA-Cu(II) / GO / GCE: 5.0-10 μL of the CoA-Cu(II) complex synthesized in step (1) was added dropwise onto the surface of GO / GCE and allowed to stand at room temperature for 0.1-1.0 h. The electrode was slowly rinsed with PBS buffer solution (10 mM, pH 7.0) to remove excess complex. The electrode was labeled as CoA-Cu(II) / GO / GCE.

[0013] Subsequently, electrochemical fast scan cyclic voltammetry (FSCV) was used for scanning, with the initial potential set at -1.5 to -0.3 V, the termination potential set at -0.2 to 0.4 V, the scanning speed set at 10 to 400 V / s, and PBS buffer solution (100 mM, pH 7.0) as the electrolyte solution to achieve the detection of CoA and HAT activity and the screening of their inhibitors.

[0014] CoA analysis test:

[0015] In step (1), the CoA concentration is changed (final concentration range: 0-150 μM), and coordination polymerization is carried out with Cu(NO3)2 solution in different proportions to form a number of CoA-Cu(II) complexes, while keeping step (2) unchanged. Based on this, a series of CoA electrochemical sensors can be obtained and applied to CoA electrochemical sensing analysis.

[0016] HAT analysis test:

[0017] Step (1) is updated as follows: sequentially add 1 to 5 μL of HAT solution with a final concentration of 0 to 500 ng / mL, 1 to 5 μL of acetyl coenzyme A (Ac-CoA) with a final concentration of 50 to 250 μM, 1 to 5 μL of substrate polypeptide solution with a final concentration of 20 to 100 μM, add 75 to 96 μL of 100 mM pH 7.0 PBS buffer solution, stir vigorously for 2 to 5 min in a stirrer, then react gently at 25 to 35°C for 0.5 to 2 h, add 1 to 10 μL of Cu(NO3)2 solution with a final concentration of 50 to 500 μM, mix well, incubate at 25 to 50°C for 5 to 15 min, and store in a refrigerator at 4°C. Step (2) remains unchanged, and a series of HAT electrochemical sensors are obtained based on this, and applied to HAT electrochemical sensing analysis.

[0018] Utilizing the preparation and application of the histone acetyltransferase electrochemical sensor based on the "Cu(II) to Cu(II)" mechanism, fast scanning cyclic voltammetry was used, with the initial potential set at -1.5 to -0.3 V, the termination potential at -0.2 to 0.4 V, and the scanning speed at 10 to 400 V / s. The various modified electrodes prepared above were used as working electrodes, platinum electrodes as auxiliary electrodes, Ag / AgCl as reference electrodes, and PBS buffer solution (100 mM, pH 7.0) as electrolyte solutions to determine the FSCV electrochemical signals corresponding to different concentrations of CoA and HAT p300 activity, and to establish a quantitative relationship between concentration and FSCV response.

[0019] Principle of the invention: The present invention is a preparation and application of a histone acetyltransferase electrochemical sensor based on the "Cu(II) to Cu(II)" mechanism. First, the thiol group in CoA and Cu(II) generate a CoA-Cu(II) complex in situ, which can be regarded as a nanochain complex with multiple adenines as side groups. The complex can be combined with GO through its adenine side groups through the π-π conjugation effect, thereby being fixed on the electrode surface. The voltage signal of Cu(II) is collected using the FSCV technology. Because the polymerization of CoA and Cu(II) can realize the electrochemical sensing analysis of CoA, and because CoA is the product of the HAT catalytic reaction, the electrochemical sensing analysis of HAT can be realized. By changing the concentration of CoA or HAT, a series of FSCV signal sizes corresponding to CoA and HAT at different concentrations can be obtained. When the inhibitor anacardic acid / C646 is added, the HAT activity is inhibited, CoA cannot be generated, and the complex cannot be formed, thereby realizing the screening of HAT inhibitors. Through the relationship between the concentration of CoA, HAT, anacardic acid, C646 and the voltage signal output of FSCV technology, the quantitative analysis of CoA, HAT, anacardic acid, and C646 in the sample to be tested can be achieved. Based on this, a fast, simple, reliable, stable, and highly sensitive FSCV analysis method was constructed.

[0020] Compared with traditional electrochemical technology, the advantages of the present invention are: the present invention constructs a preparation and application of a histone acetyltransferase electrochemical sensor based on the "Cu(II) toCu(II)" mechanism. First, graphene is uniformly modified on the GCE surface by cyclic voltammetry, and then the synthesized CoA-Cu(II) complex is assembled on GO / GCE at room temperature, and a simple FSCV sensor is prepared by the "Cu(II) to Cu(II)" mechanism. Within a certain range, the greater the CoA concentration, the more Cu(II) is bound, that is, the more CoA-Cu(II) complexes are, and the more obvious the FSCV response is; the greater the HAT concentration, the more CoA is generated, the more CoA-Cu(II) complexes are generated, and the more obvious the FSCV response is. The experimental results show that the FSCV signal response size is linearly related to the logarithm of the CoA and HAT concentrations within a certain range, which better realizes the analysis and detection of CoA and HAT. Its advantages are:

[0021] (1) The results are highly reliable. The FSCV technology at high scan rates maintains the "Cu(II) to Cu(II)" operating mechanism. The electrode surface is approximately considered to be in a Cu(0)-free state. FSCV uses voltage as the ordinate and time as the abscissa. After several output cycles, it still has good signal stability, and the results are accurate and reliable.

[0022] (2) High sensitivity. The FSCV technology used in the present invention has no signal attenuation during the implementation process, so the sensitivity is higher than that of classical electrochemistry. Finally, we obtained a detection limit of 6.3 nM for CoA and 6.8 pg / mL for HAT.

[0023] (3) High specificity. Other common enzymes such as horseradish peroxidase (HRP), chymotrypsin (CHY), uracil DNA-glycosylase (UDG), acetylcholinesterase (AChE), protein kinase (PKA) and cholesterol oxidase (ChOx) have no interference with this detection system.

[0024] (4) Excellent stability. The relative standard deviation (RSD) is less than 5%.

[0025] (5) The actual amount of preparation and detection method is small. The detection speed is fast and the cost is low. The present invention uses a small amount of reagents in the preparation and detection process of the sensor, and the operation is convenient. Only a small amount of reagent materials is needed to achieve high-sensitivity detection of CoA and HAT.

[0026] In summary, the present invention constructs a preparation and application of a histone acetyltransferase electrochemical sensor based on the "Cu(II) to Cu(II)" mechanism, which has the advantages of simple operation, high specificity, high sensitivity, low cost, etc., can realize the detection of lower concentrations of CoA and HAT, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a feasibility analysis diagram of the sensor of the present invention;

[0028] Figure 2 It is a linear relationship diagram of the potential response of the sensor of the present invention to CoA and the logarithm of the concentration;

[0029] Figure 3 It is a linear relationship diagram of the HAT potential response of the sensor of the present invention to the logarithm of the concentration;

[0030] Figure 4 It is a linear relationship diagram of the potential response of the sensor of the present invention to anacardic acid / C646 and the logarithm of the concentration;

[0031] Figure 5 This is a graph showing the selectivity of the sensor of the present invention to CoA;

[0032] Figure 6 This is an experimental diagram of the selectivity of the sensor of the present invention to HAT. DETAILED DESCRIPTION

[0033] The present invention is further described in detail below with reference to the accompanying drawings.

[0034] Example 1 Preparation of CoA-Cu(II) complex

[0035] Cu(NO3)2 solution (10 μL, 500 μM), CoA solution (10 μL, 1 mM) and 80 μL of PBS buffer solution (100 mM, pH 7.0) were mixed to prepare a coordination polymer, which was vigorously shaken at room temperature for 15 min to obtain a CoA-Cu(II) complex.

[0036] Example 2 Preparation of electrochemical biosensor

[0037] a. GCE: A glassy carbon electrode (GCE, 3 mm in diameter) was polished on suede with 1, 0.3 and 0.05 μm aluminum oxide powder for 2 min, then ultrasonically washed with water and ethanol for 5 min, and then dried with nitrogen for later use. The electrode was marked as GCE.

[0038] b. GO / GCE: 5.0 mg of graphene was dispersed in 5.0 mL of acetic acid-sodium acetate buffer solution (0.1 M, pH 5.0), and dispersed in an ultrasonic cleaner for 2 hours to obtain a graphene oxide dispersion. Then, cyclic voltammetry was used with the potential range set to -2.0 to 2.0 V, the scanning speed set to 10 mV / s, and the scanning was repeated for 10 to 20 circles to electrodeposit the above-mentioned graphene oxide onto GCE to obtain a graphene-modified glassy carbon electrode, which was labeled as GO / GCE.

[0039] c. CoA-Cu(II) / GO / GCE: Take 10 μL of the CoA-Cu(II) complex solution synthesized in step (1) and drop it on the GO / GCE surface. Incubate at room temperature for 0.5 h. Rinse the electrode slowly with PBS buffer solution (10 mM, pH 7.0) to remove unbound complexes. Mark the electrode as CoA-Cu(II) / GO / GCE.

[0040] d. CoA-Cu(II) / GCE: Take 10 μL of the CoA-Cu(II) complex solution synthesized in step (1) and add it dropwise on the surface of the GCE electrode. Incubate at room temperature for 0.5 h. Rinse the electrode slowly with PBS buffer solution (10 mM, pH 7.0) to remove the unbound CoA-Cu(II) complex. Mark the electrode as CoA-Cu(II) / GCE.

[0041] e.Cu(II) / GO / GCE: Take Cu(NO3)2 solution (10μL, 500μM) and drop it on the surface of GO / GCE electrode, incubate it at room temperature for 0.5h, and mark the electrode as Cu(II) / GO / GCE.

[0042] To prove that the sensor of the present invention can detect CoA, a biosensor was prepared based on Example 1 and Example 2. The electrochemical responses of various sensors were compared. Figure 1 The complex-modified CoA-Cu(II) / GO / GCE has a good signal output under FSCV, while the FSCV responses of the other four electrodes are very small, indicating that the complex formed by the thiol group contained in CoA and Cu(II) is effectively adsorbed on the surface of GO / GCE, but cannot be adsorbed on the surface of the GCE electrode without GO modification; the presence of GO can accelerate the electron transfer rate on the electrode surface; the adenine structure on the side chain of the CoA-Cu(II) complex can react with GO and thus be adsorbed on its surface, while the copper ion itself cannot react with graphene oxide to produce a strong signal, which proves the strong coordination ability of CoA and Cu(II), thereby proving that the experiment is feasible in theory and technology.

[0043] CoA analysis test:

[0044] According to the above Example 1, the reaction system was controlled to be 100 μL, and 10 μL of CoA solutions of different concentrations (final concentrations: 0, 0.02, 0.05, 0.08, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 80, 100, 120, 150 μM) were added in sequence, 10 μL of Cu (II) solution with a final concentration of 50 μM was added, 80 μL of PBS buffer solution (100 mM, pH 7.0) was added, and the mixture was vigorously shaken at room temperature for 15 min, and then 10 μL of the above reaction solution was added dropwise to the surface of the GO / GCE electrode, and after incubation at 30°C for 0.5 h, the electrode surface was slowly rinsed with PBS buffer solution (10 mM, pH 7.0) to remove unbound molecules, and a series of CoA electrochemical sensors were obtained for CoA electrochemical sensing analysis. The results are shown in FIG. Figure 2 As shown, as the CoA concentration increases, the potential response continues to increase, and there is a good linear relationship between the logarithmic value of the CoA concentration in the range of 0.01 to 100 μM and the potential response, with a detection limit of 6.3 nM, indicating that the sensor achieves highly sensitive detection of CoA.

[0045] HAT analysis test:

[0046] 5 μL of HAT solution of different concentrations (final concentration: 0, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 150, 200, 300, 500 ng / mL), 1 μL of acetyl-CoA with a final concentration of 50 μM, 4 μL of substrate peptide with a final concentration of 20 μM and 80 μL of PBS buffer solution (100 mM, pH 7.0) were added in sequence. After mild reaction at 30°C for 80 min, 1 μL of 5 mM Cu(NO3)2 solution (final concentration: 50 μM) was added to the above solution to make the total volume of the system 100 μL. The solution was incubated at 30°C for 15 min. Subsequently, 10 μL of the above reaction solution was dripped on the surface of the GO / GCE electrode. After incubation at 30°C for 0.5 h, the electrode was heated with PBS buffer solution (10 mM, pH 7.0). 7.0) Slowly rinse the electrode surface to remove unbound molecules. Based on this, a series of HAT electrochemical sensors can be obtained for HAT electrochemical sensing analysis. Figure 3 As shown, within a certain range, the greater the HAT concentration, the stronger the potential response, and the potential response of the sensor to HAT shows a good linear relationship with the logarithm of the concentration. The linear range is 0.01-200 ng / mL, and the detection limit is 6.8 pg / mL, indicating that the sensor can achieve high-sensitivity detection of HAT.

[0047] For the inhibitor anacardic acid / C646:

[0048] According to the preparation steps of the HAT assay described above, 2 μL of HAT solution with a final concentration of 200 ng / mL, 1 μL of acetyl-CoA with a final concentration of 50 μM, 4 μL of substrate peptide with a final concentration of 20 μM, 2 μL of anacardic acid solution of different concentrations (final concentrations: 0, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000 μM) / C646 (final concentrations: 0, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 150, 200 μM) and 80 μL of PBS buffer solution (100 mM, pH 5.0) were added in sequence. 7.0), and after gentle stirring at 30°C for 80 minutes, 1 μL of 5 mM Cu(NO3)2 solution (final concentration: 50 μM) was added to the above solution, and the total volume of the system was maintained at 100 μL. Incubate at 30°C for 15 minutes. Take 10 μL of the above reaction solution on the GO / GCE electrode and incubate at room temperature for 15 minutes. Use PBS buffer solution (10 mM, pH 7.0) to slowly rinse the electrode surface to remove unbound complexes. The results are as follows Figure 4As shown in Figure 2, as the concentration of anacardic acid / C646 increases, the potential response decreases, indicating that anacardic acid / C646 has a good inhibitory effect on activity, IC 50 The concentrations of the two molecules were 1.77, 1.80, 2.29, 2.31 / 1.04, 1.05, 1.54, and 1.55 μM, realizing the screening of HAT inhibitors.

[0049] Example 3 Selective Detection:

[0050] In order to verify the selectivity of the sensor for CoA, according to the sensor preparation steps of Examples 1 and 2 above, ATP (adenosine triphosphate), ADP (adenosine diphosphate), ascorbic acid (AA), uric acid (UA), acetaminophen (AP), glutathione (GSH), cysteine ​​(Cys) and glucose (Glu) with the same concentration as CoA were added to replace CoA to participate in the reaction. The results are as follows Figure 5 As shown, it can be seen that compared with CoA, the potential responses of other small molecules are weak, indicating that the sensor has good selectivity for CoA.

[0051] In order to verify the selectivity of the sensor for HAT, the sensor preparation steps of Examples 1 and 2 were followed, and horseradish peroxidase (HRP), chymotrypsin (CHY), uracil DNA-glycosylase (UDG), acetylcholinesterase (AChE), protein kinase (PKA) and cholesterol oxidase (ChOx) at the same concentration as HAT were added to replace HAT to participate in the reaction. The results are shown in Figure 1. Figure 6 As shown, it can be seen that compared with HAT, the electrochemical signals of other enzymes are weak, indicating that the sensor has good selectivity for HAT.

[0052] It should also be noted that the above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Relevant technicians in the technical field can make changes, modifications, additions or substitutions within the essential scope of the present invention, which should also fall within the protection scope of the claims of the present invention.

Claims

1. A use of a histone acetyltransferase electrochemical sensor based on the "Cu(II) to Cu(II)" mechanism, characterized in that: Applied to the detection of CoA and HAT activity and the screening of their inhibitors. The specific steps are as follows: (1) Preparation of CoA-Cu(II) complex Take 1-10 μL of a Cu(NO3)2 solution with a final concentration of 50-500 μM and 1-20 μL of a CoA solution with a final concentration of 0.01-150 μM in sequence, and mix them with a 100 mM pH 7.0 PBS buffer solution to make a 0.1-1 mL solution, and shake the solution vigorously for 5-15 minutes to obtain a CoA-Cu(II) complex; (2) Preparation of electrochemical biosensors a. GCE: The glassy carbon electrode was polished on suede with 1, 0.3 and 0.05 μm aluminum oxide powder for 1 to 5 min, then ultrasonically washed with water and ethanol for 1 to 5 min, and then dried with nitrogen for later use. This electrode is marked as GCE. b. GO / GCE: 4.0-8.0 mg of graphene was dispersed in 2.0-10.0 mL of 0.1-0.3 M acetic acid buffer solution with a pH of 5.0-6.0, and ultrasonically dispersed in an ultrasonic cleaner for 2-5 h to obtain a graphene dispersion; using cyclic voltammetry, the potential range was -2-2 V, the scan rate was 10 mV / s, and the scan was repeated for 10-20 cycles, and the above graphene was electrodeposited onto GCE to obtain a graphene-modified glassy carbon electrode, which was labeled as GO / GCE; c. CoA-Cu(II) / GO / GCE: 5.0-10 μL of the CoA-Cu(II) complex synthesized in step (1) was added dropwise to the surface of GO / GCE, and allowed to stand at room temperature for 0.1-1.0 h. The electrode was slowly rinsed with PBS buffer solution to remove excess complex. The electrode was labeled as CoA-Cu(II) / GO / GCE. Electrochemical fast scan cyclic voltammetry (FSCV) was used for scanning, with the initial potential set at -1.5 to -0.3 V, the termination potential set at -0.2 to 0.4 V, the scanning speed set at 10 to 400 V / s, and PBS buffer solution used as the electrolyte solution to achieve the detection of CoA and HAT activity and the screening of their inhibitors.

2. The use according to claim 1, characterized in that The specific steps for applying to CoA activity detection are: by changing the CoA concentration in the preparation method step (1) in claim 1, and coordinating and polymerizing with the Cu(NO3)2 solution in different proportions to form a plurality of CoA-Cu(II) complexes, while keeping step (2) unchanged, a series of CoA electrochemical sensors can be obtained based on this, and applied to CoA electrochemical sensing analysis; wherein, the CoA concentration is changed so that the final CoA concentration ranges from 0 to 150 μM.

3. The use according to claim 1, characterized in that The specific steps for HAT activity detection are as follows: the preparation method step (1) in claim 1 is updated to sequentially add 1 to 5 μL of a HAT solution with a final concentration of 0 to 500 ng / mL, 1 to 5 μL of acetyl coenzyme A with a final concentration of 50 to 250 μM, 1 to 5 μL of a substrate polypeptide solution with a final concentration of 20 to 100 μM, and 75 to 96 μL of a 100 mM pH 7.0 PBS buffer solution, vigorously stir for 2 to 5 min in a stirrer, and then react gently at 25 to 35° C. for 0.5 to 2 h, then add 1 to 10 μL of a Cu(NO3)2 solution with a final concentration of 50 to 500 μM, mix well, incubate at 25 to 50° C. for 5 to 15 min, and store in a refrigerator at 4° C.; step (2) remains unchanged, and a series of HAT electrochemical sensors are obtained based on this, and applied to HAT electrochemical sensing analysis.

4. The use according to claim 2, characterized in that: The detection limit of CoA was 6.3 nM.

5. The use according to claim 3, characterized in that: The detection limit of HAT was 6.8 pg / mL.

Images