阅读说明：本技术 基于高阶g4和乙酰基抗体检测乙酰转移酶活性的电化学传感器制备及应用 (Preparation and application of electrochemical sensor for detecting acetyltransferase activity based on high-order G4 and acetyl antibody ) 是由 胡宇芳 张青青 胡丹丹 詹甜玉 王邃 郭智勇 于 2019-10-14 设计创作，主要内容包括：本发明公开了一种基于高阶G4和乙酰基抗体检测乙酰转移酶活性的电化学传感器制备及应用,具体步骤如下：金电极预处理,底物肽溶液涂覆Au电极。将底物肽修饰电极置于HAT p300反应混合物中孵育,滴加DNA探针-乙酰化抗体混合液,加入TdT反应混合液使DNA延长生成随机排布的富G的DNA长链,利用钾离子使其形成N个随机G4结构,然后再加入大量DNAG链使其在镁离子的状态下叠加到随机G4结构上形成高阶G4。然后滴加hemin,使高阶G4具有辣根过氧化物酶活性,通过TMB+H<Sub>2</Sub>O<Sub>2</Sub>实现电化学信号输出,并用来检测乙酰转移酶活性及其抑制剂的筛选。优点是特异性好、灵敏度高、检测速度快、结果准确可靠、成本低。(The invention discloses preparation and application of an electrochemical sensor for detecting activity of acetyl transferase based on high-order G4 and acetyl antibody, which comprises the following specific steps: pretreating a gold electrode, and coating an Au electrode with a substrate peptide solution. Placing a substrate peptide modified electrode in an HAT p300 reaction mixture for incubation, dropwise adding a DNA probe-acetylated antibody mixed solution, adding a TdT reaction mixed solution to extend DNA to generate randomly arranged G-rich DNA long chains, forming N random G4 structures by using potassium ions, and then adding a large number of DNAG chains to be superposed on the random G4 structures in a magnesium ion state to form high-order G4. Then hemin was added dropwise to make higher order G4 having horseradish peroxidase activity, through TMB + H 2 O 2 Realizes electrochemical signal output and is used for detecting the activity of the acetyltransferase and screening inhibitors thereof. The method has the advantages of good specificity, high sensitivity, high detection speed, accurate and reliable result and low cost.)

1. Preparation and application of an electrochemical sensor for detecting acetyl transferase activity based on high-order G4 and acetyl antibody are characterized in that the mechanism is as follows: firstly, modifying sulfhydryl-containing substrate polypeptide to the surface of a gold electrode, transferring acetyl to a specific lysine residue of the substrate polypeptide by acetyl coenzyme A under the action of acetyltransferase after acetylation reaction to obtain acetylated polypeptide, solidifying an acetyl antibody-DNA probe compound to the surface of the electrode by utilizing the specific binding action of acetyl and acetyl antibodies, and then forming a G-rich DNA long chain by utilizing the TdT extension amplification action of the DNA probe, forming N G-quadruplexes randomly in the presence of potassium ions, introducing a DNA G chain containing a plurality of G bases, the DNAG chain can rapidly form G-quadruplex in the presence of magnesium ions and is orderly stacked on the TdT extended G-quadruplex, these G-quadruplex structures can form DNase with the catalytic performance of horseradish peroxidase-like enzyme by combining hemin. Hydrogen peroxide (H)₂O₂) Chemical reactions occur in the presence of 3, 3 ', 5, 5' -Tetramethylbenzidine (TMB), which produces a significant electrochemical response.

2. Preparation and application of an electrochemical sensor for detecting acetyl transferase activity based on high-order G4 and acetyl antibody comprise the following specific steps:

(1) pretreatment of a gold electrode: polishing gold electrode (Au) on chamois leather with aluminium oxide powder (0.05 μm) for 0.5-5 min, and ultrasonically cleaning the electrode with secondary distilled water in an ultrasonic cleaner for 0.5-5 min after polishing. Then, beads on the surface of the gold electrode are blown off by an ear washing ball, and the outside of the electrode is wiped by filter paper for later use.

(2) Electrode 1: an Au electrode was coated with a substrate peptide (P) solution (20-2000. mu.M, 2-12. mu.L) and kept at 4 ℃ overnight, followed by washing with a phosphate buffer solution (PBS, pH 7.0, 100 mM). Then, the electrode was immersed in 0.05-2 mM 1-hexanethiol (MCH) solution for 20-70 min, and then the surface was washed with PBS buffer. HAT p300 reaction mixture (20-2000. mu.L) comprises p300 with different concentrations, 100-5000. mu.M Ac-CoA and PBS buffer. And placing the electrode modified by the substrate polypeptide in a HATp300 reaction mixture and incubating for 20-150 min at 25-42 ℃. After incubation, the electrodes were washed with PBS buffer to remove excess reagents and dried in a nitrogen stream as previously described.

(3) Electrode 2: in a PBS solution (100. mu.L) containing 10 to 80mM N-hydroxysuccinimide (NHS) and 50 to 100mM 11-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC. HCl), a DNA probe (0.05 to 1. mu.M, 2 to 12. mu.L) and an acetylated antibody (Ab) were added to the solution using a pipette_Ac)(1×10^-4～1×10^-2mg/mL, 2-12 muL) and incubating for 0.5-1.6 h at 25-42 ℃ to obtain the acetylated antibody-DNA probe compound. Dripping 2-12 mu L of the compound on the surface of the electrode 1, and incubating for 0.1-1 h.

(4) Electrode 3: the electrode 2 is placed in a TdT reaction mixture (0.5-3 mu L of redistilled water + dATP (1-30 mM, 0.1-6 mu L) + dGTP (1-30 mM, 0.1-6 mu L) + TdT (1-100U/mL, 0.1-5 mu L) + 0.5-5 mu L of 5 xTdT buffer), incubated for 0.4-1.7 h at 28-40 ℃, and slowly washed by PBS buffer. Then, a KCl solution (0.05-2 mM, 1-7 mu L) is dripped on the electrode, and incubation is carried out for 20-90 min at normal temperature. Then dropwise adding a G4 chain mixed solution (MgCl)₂(0.05-0.5M, 1-5 muL) + G4 (0.5-10 muM, 1-10 muL), standing at normal temperature for 0.6-1.2 h, then dropwise adding hemin (0.05-0.8 mM, 1-10 muL), and standing at normal temperature for 15-45 min to enable G4 and hemin to fully react to form a G4-hemin compound with horseradish peroxidase activity.

3. An electrochemical process according to claims 1-2, characterized in that: the acetyltransferase activity is detected by combining the high-order G4, acetyl antibody and TdT extension amplification for the first time, so that the acetyltransferase HAT p300 with low concentration can be detected, and the detection limit is 1 pM.

4. An electrochemical process according to claims 1 to 3, characterized in that: can screen the inhibitor of acetyltransferase, namely anacardic acid, C646 and the like, and has important significance for screening cancer-related medicaments.

Technical Field

The invention relates to a preparation and detection method of a histone acetyltransferase electrochemical sensor, in particular to preparation and application of an electrochemical sensor for detecting acetyltransferase activity based on high-order G4 and acetyl antibody, belonging to the technical field of functional biological materials and biosensing.

Background

Histone modifications are covalent post-translational modifications of histone proteins, which are critical in epigenetic regulation. Due to histone alterations, a variety of biological events can be controllably performed, such as transcriptional activation/inactivation, chromosomal packaging, and DNA damage/repair. In particular, the reversible acetylation of lysine residues is one of the major mechanisms of histone modification. The level of histone lysine acetylation is precisely mediated by the antagonistic catalytic activities of Histone Acetyltransferase (HAT) and Histone Deacetylase (HDAC). By acetylation, HAT reduces electrostatic interactions between histones and their encapsulating DNA, creating an open chromatin structure for regulating protein accessibility. In contrast, HDACs catalyze the removal of acetyl groups and enhance the attachment of histones and genes, resulting in closed chromosome configuration and transcriptional repression. The abnormal activity of HAT, interference in the induction of histone acetylation, has been shown to be associated with the etiology of many diseases, including cancer, neurological disorders, chronic inflammation, and aids infection. To date, HAT has become a new class of drug targets, and HAT-targeted inhibitors are also being extensively studied. In order to meet the requirement of HAT-related analysis and detection, a simple HAT activity detection method needs to be developed, which is of great significance for drug development and drug research in clinical diagnosis and is essential for better understanding of epigenetic related research.

Electrochemical techniques are receiving attention because of their advantages such as excellent sensitivity and ease of operation. Today, nucleic acid nanostructures such as DNA tetramer structures, nucleic acid-like structures, show significant applications in the field of post-translational modification of electrochemical sensing. Recently, functional nucleic acids of the G-quadruplex (G4) have attracted considerable interest as a universal molecular kit. In general, G4 is a unique four-stranded nucleic acid structure formed by stacking Hoogsteen base-paired G-quadruplexes, and this phenomenon is prevalent in G-rich sequences. The electrochemical technology has the advantages of simple operation, quick response, low background, high sensitivity and low instrument requirement, and is a promising technology for biosensing at present.

The invention provides a preparation method of an electrochemical sensor for detecting acetyltransferase activity based on high-order G4 and acetyl antibody, which comprises the steps of firstly modifying a substrate polypeptide containing sulfydryl to the surface of a gold electrode, transferring acetyl to a specific lysine residue of the substrate polypeptide by acetyl coenzyme A under the action of acetyltransferase after acetylation reaction to obtain acetylated polypeptide, solidifying an acetyl antibody-DNA probe compound to the surface of the electrode by utilizing the specific binding action of acetyl and acetyl antibody, forming G-rich DNA long chains by using TdT extension amplification action to form N G-tetrads at random in the presence of potassium ions, introducing a DNA G chain containing a plurality of G bases, rapidly forming the G-tetrads by using the DNA G chain in the presence of magnesium ions, and orderly stacking the G-tetrads extended by using the TdT extension, these G-quadruplex structures can form DNase with the catalytic performance of horseradish peroxidase-like enzyme by combining hemin. Hydrogen peroxide (H)₂O₂) Chemical reactions occur in the presence of 3, 3 ', 5, 5' -Tetramethylbenzidine (TMB), which produces a significant electrochemical response. At present, a preparation method and application of an electrochemical sensor for detecting the activity of acetyltransferase based on high-order G4 and acetyl antibody are not found at home and abroad.

Disclosure of Invention

The invention aims to solve the technical problem of providing the preparation and application of the electrochemical sensor for detecting the activity of the acetyl transferase based on the high-order G4 and the acetyl antibody, which has the advantages of good specificity, high sensitivity, high detection speed, accurate and reliable result and low cost.

The technical scheme adopted by the invention for solving the technical problems is as follows: preparation and application of an electrochemical sensor for detecting acetyl transferase activity based on high-order G4 and acetyl antibody comprise the following specific steps:

(1) pretreatment of a gold electrode: polishing gold electrode (Au) on chamois leather with aluminium oxide powder (0.05 μm) for 0.5-5 min, and ultrasonically cleaning the electrode with secondary distilled water in an ultrasonic cleaner for 0.5-5 min after polishing. Then, beads on the surface of the gold electrode are blown off by an ear washing ball, and the outside of the electrode is wiped by filter paper for later use.

(2) Electrode 1: an Au electrode was coated with a substrate peptide (P) solution (20-2000. mu.M, 2-12. mu.L) and kept at 4 ℃ overnight, followed by washing with a phosphate buffer solution (PBS, pH 7.0, 100 mM). Then, the electrode was immersed in 0.05-2 mM 1-hexanethiol (MCH) solution for 20-70 min, and then the surface was washed with PBS buffer. HAT p300 reaction mixture (20-2000. mu.L) comprises p300 with different concentrations, 100-5000. mu.M Ac-CoA and PBS buffer. And placing the electrode modified by the substrate polypeptide in a HATp300 reaction mixture and incubating for 20-150 min at 25-42 ℃. After incubation, the electrodes were washed with PBS buffer to remove excess reagents and dried in a nitrogen stream as previously described.

(3) Electrode 2: in a PBS solution (100. mu.L) containing 10 to 80mM N-hydroxysuccinimide (NHS) and 50 to 100mM 11-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC. HCl), a DNA probe (0.05 to 1. mu.M, 2 to 12. mu.L) and an acetylated antibody (Ab) were added to the solution using a pipette_Ac)(1×10^-4～1×10^-2mg/mL, 2-12 muL) and incubating for 0.5-1.6 h at 25-42 ℃ to obtain the acetylated antibody-DNA probe compound. Dripping 2-12 mu L of the compound on the surface of the electrode 1, and incubating for 0.1-1 h.

(4) Electrode 3: the electrode 2 is placed in a TdT reaction mixture (0.5-3 mu L of redistilled water + dATP (1-30 mM, 0.1-6 mu L) + dGTP (1-30 mM, 0.1-6 mu L) + TdT (1-100U/mL, 0.1-5 mu L) + 0.5-5 mu L5 xTdTbuffer), incubated for 0.4-1.7 h at 28-440 ℃, and slowly washed by PBS buffer. Then, a KCl solution (0.05-2 mM, 1-7 mu L) is dripped on the electrode, and incubation is carried out for 20-90 min at normal temperature. Then dropwise adding a G4 chain mixed solution (MgCl)₂(0.05-0.5M, 1-5 muL) + G4 (0.5-10 muM, 1-10 muL), standing at normal temperature for 0.6-1.2 h, then dropwise adding hemin (0.05-0.8 mM, 1-10 muL), and standing at normal temperature for 15-45 min to enable G4 and hemin to fully react to form a G4-hemin compound with horseradish peroxidase activity.

As not suggested in the experimental procedure, these modified electrodes were each rinsed slowly with PBS buffer.

Substrate polypeptide sequence: CRGKGGKGLKGGAKA are provided.

DNA Probe: 5' -NH₂-ACGGTT-3’。

DNA G chain: 5'-GGGTGGGTGGGTGGGT-3' are provided.

The invention principle is as follows: the electrochemical sensor for detecting the activity of the acetyl transferase based on the high-order G4 and the acetyl antibody is prepared and applied by adopting a gold electrode Au, fixing a substrate peptide through an Au-S bond, transferring acetyl from Ac-CoA to a lysine residue of the substrate polypeptide through acetylation reaction, preparing the sensor through antigen-antibody interaction, using TdT extension and amplification for multiple purposes, using G4 stacking effect, and using the sensor to perform TMB + H₂O₂The electrochemical response of (a) achieves an electrochemical output. Clearly, the greater the concentration of the target, the more pronounced the current response, within a certain range of concentrations. The experimental result shows that the current magnitude and the concentration of the target object are in a linear relationship in a certain range, and the detection of the target object is realized. The advantages are that:

(1) high sensitivity. Experiments show that the linear correlation equation of the electrochemiluminescence response of the sensor to the HAT p300 concentration logarithm value is that y is-11.37 lgC_p300-24.58，R²0.9947, the linear range is 0.01-100 nM, and the detection limit is 1pM, thus the sensor can realize high-sensitivity detection on HAT p 300.

(2) High specificity. Other common related enzymes do not interfere with the detection system. The reason is that: the invention is based on that HAT p300 catalyzes acetylation reaction to generate, the amount of generated acetyl affects the combination with a signal unit, and other enzymes cannot catalyze acetylation reaction, so that the detection system is not interfered.

(3) The result is accurate. The recovery rate is between 90% and 110%.

(4) And (3) an inhibitor. The electrochemical response of the electrochemical luminescence biosensor to the inserted luminophor is utilized to realize the detection of HAT p300 inhibitor (such as Anacardic Acid (Anacardic Acid) and C646), and the correlation between the electrochemical luminescence response of the sensor and the HAT p300 inhibitor can be obtained.

(5) The preparation and detection method has the advantages of less reagent dosage, high detection speed and low cost.

In conclusion, the electrochemical sensor for detecting the activity of the acetyltransferase based on the high-order G4 and the acetyl antibody is prepared and applied, has the advantages of high sensitivity, good selectivity, simplicity in operation, rapidness in analysis, easiness in operation and the like, can realize the detection of low-concentration HAT p300 and the screening of small-molecule inhibitors thereof, and has good application prospects.

Drawings

FIG. 1 is a graph showing comparative electrochemical responses of different modified electrodes of a sensor of the present invention;

FIG. 2 is a diagram of a feasibility experiment of the sensor of the present invention;

FIG. 3 is a graph of the electrochemical response of a sensor of the present invention to different concentrations of HAT p300 versus the log calibration of the concentration of HAT p 300;

FIG. 4 is a graph of a selectivity experiment of a sensor of the present invention;

FIG. 5 is an anti-interference experimental graph of the sensor of the present invention;

FIG. 6 shows the inhibition of HAT p300 activity by anacardic acid at various concentrations;

FIG. 7 is a graph of the inhibition of HAT p300 activity by various concentrations of C646.

Detailed Description

The invention is described in further detail below with reference to the accompanying examples.