CN110082403B - Histone Acetyltransferase Chrono-Amperometric Sensor Based on Gold-Palladium Nanoflower/Graphene Composites and Its Applications - Google Patents
Histone Acetyltransferase Chrono-Amperometric Sensor Based on Gold-Palladium Nanoflower/Graphene Composites and Its Applications Download PDFInfo
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Abstract
本发明公开了基于金钯纳米花/石墨烯复合材料的组蛋白乙酰转移酶计时‑电流传感器及其应用，首先将底物多肽利用Au‑S作用固定于金电极表面，通过乙酰化反应将乙酰辅酶A上的乙酰基转移至底物多肽的特定赖氨酸残基上，利用生成的乙酰化多肽将具有较强催化能力的乙酰基抗体‑金钯纳米花/石墨烯复合材料特异性吸附，在含有双氧水的电解质溶液中产生明显的电化学信号。在乙酰化反应中，改变p300浓度及其小分子抑制剂浓度，通过乙酰基和乙酰基抗体的特异性结合作用，探究对所制备的一系列传感器电化学信号的影响。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 a time-current sensor of histone acetyltransferase based on gold-palladium nanoflower/graphene composite material and its application. First, a substrate polypeptide is fixed on the surface of a gold electrode by the action of Au-S, and the acetylated The acetyl group on the coenzyme A is transferred to the specific lysine residue of the substrate polypeptide, and the acetylated antibody-gold palladium nanoflower/graphene composite material with strong catalytic ability is specifically adsorbed by the generated acetylated polypeptide, A distinct electrochemical signal is produced in an electrolyte solution containing hydrogen peroxide. In the acetylation reaction, the concentration of p300 and its small molecule inhibitor were changed, and the effects on the electrochemical signals of a series of sensors were explored through the specific binding of acetyl and acetyl antibodies. The advantages are good specificity, high sensitivity, fast detection speed, accurate and reliable results, and low cost.
Description
Technical Field
The invention relates to a timing-current sensor and application thereof, in particular to a histone acetyltransferase electrochemical detection method based on a gold-palladium nanoflower/graphene composite material, and belongs to the technical field of functional biological materials and biosensing.
Background
Post-translational modifications of histones, such as acetylation, phosphorylation, methylation, ADP-ribosylation, ubiquitination, etc., play important roles in the physiological and pathological processes of eukaryotic organisms. Wherein, the acetylation of histone is mainly catalyzed by Histone Acetyltransferase (HAT), acetyl-CoA is used as a reaction substrate to carry out enzymatic reaction, acetyl on the acetyl-CoA is hydrolyzed and transferred to a specific lysine residue at the tail part of the histone, and the process is a reversible process. p300 is a typical histone acetyltransferase, has wide biological functions, participates in the regulation of cell cycle and gene transcription, and maintains the functions and stability of certain proteins. It also plays an important role in transcription and translation of viral oncoproteins, development of embryos, and the like. In recent years, many studies have shown that modification changes in histones have been reported in many different tumors. In addition to tumors, lysine acetylase dysfunction can cause many other diseases, such as inflammatory reactions, heart disease, diabetes, etc. Therefore, the realization of the ultra-sensitive detection of the histone acetyltransferase activity is of great significance.
The method for early detection of histone acetyltransferase mainly depends on an autoradiography technology or a radioisotope labeling technology, but the two methods need labeling radioactive elements, and have the defects of time and labor consumption, high cost, high environmental pollution and the like. In recent years, researchers have tried to find some methods for detecting histone acetyltransferase without labeling radioisotopes, but the research on histone acetyltransferase is only "Bingshan", and is still in the early stage of research, so that the development of a sensitive, accurate, rapid and simple method for detecting histone acetyltransferase is urgently needed. Electrochemical sensors have received much attention due to their high sensitivity, simple preparation and operation, and fast response time. In order to improve the sensitivity of electrochemical sensors, a number of different signal amplification strategies have been proposed. Among them, enzyme catalysis has received a great deal of attention due to the high specificity and effectiveness of enzymes. Typically, the enzyme is labeled on a biological probe or nanomaterial. However, the process of enzyme labeling is complex and time consuming. Furthermore, enzymes are easily inactivated under extreme acid or alkaline conditions, which limits their use. The nano material has good conductivity, excellent stability and high mechanical strength as a material with great development prospect in the 21 st century, and has considerable electrochemical catalytic capability. Therefore, the enzyme-free electrochemical sensor provides a new opportunity for sensitive detection of histone acetyltransferase.
The invention constructs a novel enzyme-free electrochemical sensor based on a gold-palladium nanoflower/graphene composite material by using a timing-current method, and the novel enzyme-free electrochemical sensor is used for detecting the activity of histone acetyltransferase (taking HATp300 as an example). The gold-palladium nanoflower/graphene composite material has two functions: the immobilization amount of the acetyl antibody is increased through the large specific surface area; the signal amplification is realized by constructing a mimic enzyme by utilizing the catalytic property of the nano material. The magnitude of the electrochemical output signal can be adjusted by controlling the amount of HATp300, and thus, detection of the activity of HATp300 and its inhibitors can be achieved. At present, no report related to preparation and application of a histone acetyltransferase timing-current sensor based on a gold-palladium nanoflower/graphene composite material is found at home and abroad.
Disclosure of Invention
The invention aims to provide a histone acetyltransferase timing-current sensor based on a gold-palladium nanoflower/graphene composite material, which has the advantages of good specificity, high sensitivity, high detection speed, accurate and reliable result and low cost, and an application thereof.
The technical scheme adopted by the invention for solving the technical problems is as follows: the histone acetyltransferase timing-current sensor based on the gold-palladium nanoflower/graphene composite material and the application thereof comprise the following specific steps:
(1) acetyl antibody-gold palladium nanoflower/graphiteAlkene composite material (Ab)Ac-AuPd @ GO) preparation
Dispersing 0.1-1 g of Graphene (GO) in 10-30 mL of deionized water for 0.1-1 h by ultrasonic treatment, and then adding 1-5 mL of 0.1-3 g/L chloroauric acid (HAuCl)4) The solution and 1-5 mL of 0.1-3 g/L chloropalladite (H)2PdCl4) The solution is uniformly stirred for 0.1-1 h by magnetic force, placed under a xenon arc lamp for irradiation for 0.1-1 h, and then transferred into a drying oven for reaction for 10-36 h at 30-100 ℃. And finally, placing the sample in a hydrogen tube furnace to react for 0.5-5 h at the temperature of 100-500 ℃. And dispersing the prepared AuPd @ GO in deionized water to obtain 0.1-5 mg/mL AuPd @ GO dispersion liquid for later use.
Collecting 1-300 μ L of 0.1-5 g/L acetyl antibody (Ab)Ac) And (3) irradiating the mixture in a helium neon laser therapeutic instrument for 5-60 s, adding the mixture into 0.1-3 mL of the AuPd @ GO dispersion liquid, and reacting for 1-8 h at 30-42 ℃.
(2) Preparation of histone acetyltransferase chrono-amperometric sensors
a.Au
Before using the gold electrode (Au), Al with the diameter of 0.1-0.5 μm and 0.01-0.1 μm are respectively used2O3Polishing the powder, ultrasonically cleaning the powder for 1-5 times by using ultrapure water, then drying the powder in nitrogen flow, and soaking the powder in 0.01-0.5M H2SO4In the solution, cyclic voltammetry scanning is carried out for 5-30 min within the range of (-0.1-0.5) V to (+ 0.5-1.5) V, and finally, the solution is cleaned by ultrapure water and dried by nitrogen for standby.
b.peptide/Au
Dripping 5-20 mu L of 0.01-1 mM substrate polypeptide (peptide) on the surface of treated Au for reaction for 1-8 h, self-assembling a monomolecular layer of the substrate polypeptide fixed by Au-S bonds, then slowly washing an electrode with distilled water, dripping 5-20 mu L of histone acetyltransferase reaction solution [ HATp300 (200-1000 nM, 1-5 mu L) and acetyl coenzyme A (1-5 mM, 1-5 mu L) to be fully mixed in Phosphate Buffer Solution (PBS), and putting the electrode in a thermostat at 25-37 ℃ for incubation for 30-120 min.
c.AbAc-AuPd@GO/peptide/Au
Taking Ab prepared in (1)AcDripping 2-12 mu L of-AuPd @ GO solution on a peptide/Au electrode, and standing at 30-42 DEG CAnd (3) placing for 10-60 min, then slowly flushing the electrode with distilled water, and then detecting the time-current.
In the acetylation reaction in the electrode preparation process, the concentration of p300 is changed, and the influence of the concentration on electrochemical signals is researched. The electrochemical parameter conditions are as follows: chrono-current method, voltage: -0.3V; time: for 100 s. The Phosphate Buffer Solution (PBS) (O.1M, pH7.0) used in the experiment was prepared from 0.1M sodium dihydrogenphosphate and 0.1M disodium hydrogenphosphate, and the pH was controlled by adjusting the volume ratio, and when used as an electrolyte solution, it contained 5mM hydrogen peroxide solution.
The invention principle is as follows: the histone acetyltransferase timing-current sensor based on the gold-palladium nanoflower/graphene composite material and the application thereof are utilized, and a novel enzyme-free electrochemical sensor is constructed by a timing-current method based on an acetyl antibody-gold-palladium nanoflower/graphene composite material and used for detecting the activity of histone acetyltransferase. Firstly, substrate polypeptide is fixed on the surface of a gold electrode by utilizing Au-S action, then acetylation reaction is carried out by dripping the acetylation reaction on the surface of the electrode, acetyl on acetyl coenzyme A is transferred to specific lysine residue of the substrate polypeptide to generate acetylated polypeptide, then an acetyl antibody-gold palladium nanoflower/graphene composite material with stronger catalytic capability is dripped on the surface of the electrode, and the prepared electrode is placed in electrolyte solution containing hydrogen peroxide to generate an obvious electrochemical catalytic signal. In the acetylation reaction process, as the concentration of p300 is increased, the amount of acetylated substrate polypeptide is increased, and the load capacity of the acetyl antibody-gold palladium nanoflower/graphene composite material can be increased through the specific binding effect of acetyl and acetyl antibodies. 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. The linear correlation equation of the electrochemical response of the sensor to the p300 concentration logarithm value is obtained through experiments, and the equation is that y is 10.084lgCp300+40.988，R20.9889 linear range of 0.001-1000 nM and detection limit of 0.37pM, thus demonstratingThe sensor can realize high-sensitivity detection on the p 300.
(2) High specificity. Other common related enzymes do not interfere with the detection system. The reason is that: according to the method, acetyl on acetyl coenzyme A is transferred to lysine residues of substrate polypeptide based on p300 catalytic acetylation reaction, the generation of acetyl affects the load of an acetyl antibody-gold palladium nanoflower/graphene composite material, so that the generation of an electrochemical signal is affected, 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 timing-current sensor to hydrogen peroxide is utilized to realize the detection of the HATp300 inhibitor (such as C646), and the correlation between the electrochemical response of the sensor and the HATp300 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 histone acetyltransferase timing-current sensor based on the gold-palladium nanoflower/graphene composite material and the application thereof have the advantages of high sensitivity, good selectivity, simplicity in operation, quickness in analysis, easiness in operation and the like, can realize detection of low-concentration HATp300 and screening of small-molecule inhibitors thereof, and have good application prospects.
Drawings
FIG. 1 is a diagram of a feasibility experiment of the sensor of the present invention;
FIG. 2 is an electrochemical response of a sensor of the present invention to the presence or absence of p 300;
FIG. 3 is a graph of the logarithmic calibration of the electrochemical response of the sensor of the present invention to different concentrations of p300 versus p300 concentration;
FIG. 4 is a graph of selectivity and interference performance experiments for a sensor of the present invention;
FIG. 5 is a graph of the inhibition of p300 activity by various concentrations of C646.
Detailed Description
The invention is described in further detail below with reference to the accompanying examples.
EXAMPLE 1 preparation of the sensor
(1) Acetyl antibody-gold palladium nanoflower/graphene composite material (Ab)Ac-AuPd @ GO) preparation
Dispersing 0.5g of Graphene (GO) in 20mL of deionized water, performing ultrasonic treatment for 0.5h, and adding 2.5mL of 1g/L chloroauric acid (HAuCl)4) Solution and 2.5mL of 1g/L chloropalladite (H)2PdCl4) The solution is evenly stirred for 0.5h by magnetic force, is placed under a xenon arc lamp for irradiation for 0.5h, and is transferred into a drying oven for reaction for 24h at the temperature of 60 ℃. Finally, the sample was placed in a hydrogen tube furnace for 2h at 300 ℃. And dispersing the prepared AuPd @ GO in deionized water to obtain 1mg/mL AuPd @ GO dispersion for later use.
100 μ L of 1g/L acetyl antibody (Ab)Ac) Irradiating the mixture in a helium neon laser therapeutic instrument for 30s, adding the mixture into 1mL of the AuPd @ GO dispersion liquid, and reacting for 4h at 37 ℃.
(2) Preparation of histone acetyltransferase chrono-amperometric sensors
a.Au
Gold electrode (Au) was coated with 0.3 μm and 0.05 μm diameter Al before use2O3Grinding the powder, ultrasonically cleaning with ultrapure water for 3 times, drying in nitrogen flow, and soaking in 0.1M H2SO4In the solution, cyclic voltammetry scanning is carried out for 5min within the range of-0.3V- +1.2V, and finally, the solution is cleaned by ultrapure water and then is dried by nitrogen for standby.
b.peptide/Au
mu.L of 0.2mM substrate polypeptide (peptide) was dropped on the treated Au surface for reaction for 4h, the substrate polypeptide self-assembled monolayer immobilized by Au-S bond was then slowly washed with distilled water, 10. mu.L of histone acetyltransferase reaction solution [ HATp300(500nM, 2. mu.L) and acetyl coenzyme A (2.5mM, 2. mu.L) were thoroughly mixed in Phosphate Buffered Saline (PBS) ] and the electrode was incubated in 30 ℃ incubator for 80 min.
c.AbAc-AuPd@GO/peptide/Au
Taking Ab prepared in (1)Ac6 μ L of the-AuPd @ GO solution was applied drop-wise to a peptide/Au electrode, allowed to stand at 37 ℃ for 30min, then the electrode was rinsed slowly with distilled water and then used for chrono-amperometric detection.
The electrochemical response of the prepared three electrodes in a PBS (0.1M, pH7.0) electrolyte solution was examined, see FIG. 1. Ab produced can be seenAcThe electrochemical response of AuPd @ GO/peptide/Au is very clear compared with the other two electrodes, demonstrating the successful assembly of the desired electrode, also indicating AbAc-electrocatalytic capacity of AuPd @ GO.
Example 2 electrochemical response with and without p300
Histone acetyltransferase timing-current sensors based on gold palladium nanoflower/graphene composite and their applications, the detection of histone acetyltransferase was investigated using our biosensor prepared in example 1. Referring to fig. 2, in the absence of p300, the sensor had essentially no electrochemical response in PBS (0.1M, pH7.0), while in the presence of p300, there was a significant electrochemical response, demonstrating that the sensor could be used for p300 activity detection.
Example 3 p300 Activity assay
The histone acetyltransferase timing-current sensor based on the gold-palladium nanoflower/graphene composite material and the application thereof have the same preparation steps as the specific example 1, and the concentration of p300 is changed in turn during the acetylation reaction process, wherein the concentration of p300 is as follows: 0, 0.001, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 150, 200, 300, 500, 700, 1000nM, and then used to prepare a sensor. Recording the electrochemical response of the sensor in PBS (0.1M, pH7.0), obtaining a series of electrochemical response curves corresponding to p300 with different concentrations according to the experimental result, establishing a quantitative relation between the magnitude of the electrochemical response current and the concentration of p300, and determining the concentration of p300 in the sample to be detected according to the quantitative relation between the magnitude of the electrochemical response current and the concentration of p 300. The experimental results are shown in fig. 3, which illustrates that the electrochemical response of the sensor is more obvious as the concentration of p300 is increased, and the linear correlation equation is that y is 10.084lgCp300+40.988，R2The linear range is 0.001-1000 nM and the detection limit is 0.37pM, which shows that the sensor can realize high-sensitivity detection on the activity of p 300.
Example 4 specific and interference-resistant assays
The concentrations of p300 and other enzymes in the selectivity experiments were all 100nM, and the abbreviations for the other enzymes used are as follows: protein Kinase (PKA), acetylcholinesterase (AChE), terminal transferase (TdT), alkaline phosphatase (ALP). A sensor was prepared according to the sensor preparation procedure of example 1 above, substituting p300 for the same concentration of enzyme in the acetylation reaction. The results are shown in fig. 4, and compared with p300, the electrochemical response of the sensor to other enzymes is very small, and is basically close to a blank signal, which indicates that the sensor has good selectivity for detecting p 300. Meanwhile, the four enzymes are respectively added into a p300 reaction system (coexisting with p 300), and the influence on electrochemical signals is negligible, so that the strong anti-interference capability of the sensor is illustrated.
Example 5 detection of the p300 inhibitor C646
The histone acetyltransferase timing-current sensor based on the gold-palladium nanoflower/graphene composite material and the application thereof have the same preparation steps as the specific example 1, the concentration of p300 is 100nM, inhibitor C646 with different concentrations is sequentially added, and the concentration of C646 is 0, 0.1, 0.5, 1, 5, 10, 20, 30, 50, 70, 100, 150, 200, 300, 400, 500, 600, 700, 800 and 1000 MuM, and then the sensor is prepared. The electrochemical response of the sensor in PBS (0.1M, pH7.0) was recorded. From the experimental results (see fig. 5), the higher the concentration of the inhibitor C646, the weaker the corresponding current response, indicating that C646 inhibits p300 activity more strongly, and the half inhibitory concentration is 29.5 μ M.
Of course, the above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Variations, modifications, additions and substitutions which may occur to those skilled in the art and which fall within the spirit and scope of the invention are also considered to be within the scope of the invention.
Claims (2)
1. Histone acetyltransferase timing-current sensor Ab based on gold-palladium nanoflower/graphene composite materialAc-AuPd @ GO/peptide/Au application, characterized in that the application method is as follows:
(1) acetyl antibody-gold palladium nanoflower/graphene composite AbAcPreparation of (E) -AuPd @ GO
Dispersing 0.1-1 g of graphene GO in 10-30 mL of deionized water for 0.1-1 h by ultrasonic treatment, and then adding 1-5 mL of HAuCl chloroauric acid of 0.1-3 g/L4The solution and 1-5 mL of 0.1-3 g/L chloropalladate H2PdCl4Uniformly stirring the solution by magnetic force for 0.1-1 h, placing the solution under a xenon arc lamp for irradiating for 0.1-1 h, and transferring the solution into a drying oven to react for 10-36 h at the temperature of 30-100 ℃; finally, placing the sample in a hydrogen tube furnace to react for 0.5-5 h at 100-500 ℃, and dispersing the prepared AuPd @ GO in deionized water to obtain 0.1-5 mg/mL AuPd @ GO dispersion liquid for later use;
collecting 1-300 μ L of 0.1-5 g/L acetyl antibody AbAcIrradiating the mixture in a helium neon laser therapeutic apparatus for 5-60 s, adding the mixture into 0.1-3 mL of the AuPd @ GO dispersion liquid, and reacting for 1-8 h at 30-42 ℃;
(2) preparation of histone acetyltransferase chrono-amperometric sensors
a. Gold electrode Au
Before the Au electrode is used, Al with the diameter of 0.1-0.5 mu m and 0.01-0.1 mu m are respectively used2O3Polishing the powder, ultrasonically cleaning the powder for 1-5 times by using ultrapure water, then drying the powder in nitrogen flow, and soaking the powder in 0.01-0.5M H2SO4In the solution, cyclic voltammetry scanning is carried out for 5-30 min within the range of-0.3V- +1.2V, and finally, the solution is cleaned by ultrapure water and then dried by nitrogen for standby;
b.peptide/Au
dripping 5-20 mu L of 0.01-1 mM substrate polypeptide peptide on the surface of treated Au for reaction for 1-8 h, slowly flushing an electrode with distilled water after the substrate polypeptide self-assembled monolayer is fixed through an Au-S bond, dripping 5-20 mu L of histone acetyltransferase reaction solution, and incubating the electrode in a thermostat at 25-37 ℃ for 30-120 min;
wherein the composition of the reaction solution of histone acetyltransferase is that HAT p300 and acetyl coenzyme A are fully mixed in phosphate buffer solution PBS; wherein the concentration of HAT p300 is 200-1000 nM, and the dosage is 1-5 muL; the concentration of acetyl coenzyme A is 1-5 mM, and the dosage is 1-5 mu L;
c.AbAc-AuPd@GO/peptide/Au
taking Ab prepared in (1)Ac-AuPd @ GO solutionDripping 2-12 mu L of the solution on a peptide/Au electrode, standing for 10-60 min at 30-42 ℃, then slowly flushing the electrode with distilled water, and then detecting the timing-current to obtain the histone acetyltransferase timing-current sensor based on the gold-palladium nanoflower/graphene composite material; placing the prepared electrode in an electrolyte solution containing hydrogen peroxide to generate an obvious electrochemical catalytic signal; in the acetylation reaction process in the electrode preparation process, as the concentration of the HATp300 is increased, the acetylated substrate polypeptide is increased, and the load capacity of the acetyl antibody-gold palladium nanoflower/graphene composite material is increased through the specific binding action of acetyl and acetyl antibodies; the current magnitude and the concentration of the target HATp300 are in a linear relation in a certain range, and detection of HAT p300 with different concentrations is realized.
2. Use according to claim 1, characterized in that the detection limit of HAT p300 is as low as 0.37 pM.
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Application publication date: 20190802 Assignee: Ningbo Science and Technology Innovation Association Assignor: Ningbo University Contract record no.: X2023980033633 Denomination of invention: Histone acetyltransferase chronoamperometric sensor based on gold palladium nanoflower/graphene composite and its application Granted publication date: 20211102 License type: Common License Record date: 20230317 |