CN113607794A - Graphene-modified electrochemical biosensor and preparation method and application thereof - Google Patents
Graphene-modified electrochemical biosensor and preparation method and application thereof Download PDFInfo
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
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- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry
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Abstract
The invention provides a graphene-modified electrochemical biosensor and a preparation method and application thereof, and belongs to the technical field of biosensors. The biosensor provided by the invention can modify the surface of the gold electrode by using only one DNA sequence in the whole process, and does not need to perform any additional modification on DNA. The sensor has stable system and simple operation, can conveniently and rapidly judge the methyltransferase in the solution to be detected in a qualitative and quantitative manner, and realizes that the detection limit of the methyltransferase can reach 0.7U/mL.
Description
Technical Field
The invention belongs to the technical field of biosensors, and particularly relates to a graphene-modified electrochemical biosensor and a preparation method and application thereof.
Background
Methylation transfers a methyl group from adenosylmethionine (SAM) in CpG dinucleotides to the C-5 position of cytosine. DNA methylation is a chemical modification process involving DNA methyltransferase (DNMT), which transfers a methyl group to a corresponding base by providing the methyl group through S-adenosylmethionine (SAM) under the catalysis of DNMT.
In mammals, DNA methylation occurs predominantly at the 5 'cytosine of CpG islands, resulting in a 5' methylcytosine. CpG islands are DNA regions rich in G, C bases and are located mainly in the promoter of a gene. Human CpG exists in 2 forms, one of which is dispersed in DNA and is mostly methylation-modified in normal tissues; the other is a CpG island with an excessively aggregated CpG structure, which is often present in the promoter region in an unmethylated state. In normal cells, CpG dinucleotides spanning the promoter region are largely unmethylated, whereas aberrant DNA methylation can lead to transcriptional silencing of gene expression.
DNA methylation plays a crucial role in various biological processes such as gene expression, gene imprinting, embryonic development, etc., and overexpression of methylated DNA is closely related to various cancers. Tumor type-specific hypermethylation in the promoter region of tumor suppressor genes has been observed and correlated with cancer subtypes. In mammals, DNA methylation occurs almost exclusively in CpG dinucleotides, and DNA methyltransferases catalyse the transfer of a methyl group from s-adenosylmethionine to cytosine. Therefore, it would be a prospective approach for those skilled in the art how to be able to determine the presence or absence of methyltransferases by reading the change in electrical signal with a sensor in a convenient and rapid manner.
Disclosure of Invention
The invention provides a graphene-modified electrochemical biosensor, and a preparation method and application thereof.
In order to achieve the above purpose, the invention provides a graphene-modified electrochemical biosensor and a preparation method and application thereof, comprising the following steps:
cleaning and pretreating the gold electrode;
fixing the nucleotide sequence on a gold electrode and using mercaptohexanol to seal the part of the nucleotide sequence which is not combined with the gold electrode to obtain the gold electrode with the nucleotide sequence fixed;
and carrying out methylation sensitive restriction endonuclease treatment on the obtained gold electrode with the fixed nucleotide sequence, extending by using terminal transferase, and immersing into a buffer solution containing graphene oxide for full action to obtain the graphene-modified electrochemical biosensor.
In the above step, the restriction enzyme is a sequence-specific dnase that prevents the expression of foreign DNA in bacterial cells by cleaving unprotected invasive DNA into small fragments. The above procedure used a methylation sensitive restriction endonuclease that specifically recognizes the 5'-CCGG-3' sequence. When methylated nucleotides are present in the recognition site, they are not recognized by methylation sensitive restriction endonucleases.
The terminal DNA transferase catalyzes the 3' -OH end of the probe DNA to realize the growth of the DNA and obtain a long single-stranded DNA as a growth product, and the catalysis does not need a template and has the characteristics of extremely simple preparation process, flexible design, multifunctional application and the like.
The graphene oxide is a two-dimensional carbon material with single atom thickness, and has the excellent characteristics of high heat conductivity coefficient, strong mechanical strength, good electron transport performance, large surface area and the like. The graphene oxide can adsorb single-stranded DNA, and compared with a single-stranded DNA modified gold electrode which does not adsorb graphene oxide, the single-stranded DNA modified electrode which adsorbs graphene oxide has obvious change of electrode impedance.
Preferably, the nucleotide sequence contains a CpG sequence 5'-CCGG-3', and the two ends of the nucleotide sequence are modified with sulfydryl.
Preferably, the nucleotide sequence is immobilized on a gold electrode and the part of the nucleotide sequence not bound to the gold electrode is blocked with mercaptohexanol by:
uniformly mixing the nucleotide sequence solution with tris (2-carboxyethyl) phosphine, adding a dried gold electrode, soaking at room temperature for 10-12 hours, taking out, and drying to obtain the gold electrode with the fixed nucleotide sequence;
the gold electrode fixed with the nucleotide sequence is immersed in a mercaptohexanol solution for 1-2 hours at room temperature to block the part of the nucleotide sequence which is not combined with the gold electrode.
Preferably, the nucleotide sequence solution is added at a concentration of 1. mu.M and tris (2-carboxyethyl) phosphine is added at a concentration of 10 mM.
Preferably, the steps of subjecting the obtained nucleotide sequence-immobilized gold electrode to methylation-sensitive restriction endonuclease treatment, extending by using terminal transferase, and immersing in a graphene oxide-containing buffer solution for full action are specifically as follows:
immersing the obtained gold electrode with the fixed nucleotide sequence into a 10mM first Tris-HCl buffer solution containing methylation sensitive restriction endonuclease, and treating at the temperature of 36-38 ℃ for 60-70 minutes to obtain a first intermediate electrode;
washing the intermediate electrode with PBS buffer solution, soaking in 20mM second Tris-HCl buffer solution containing 40U/mL TdTase and 10 μ M dNTP or dATP, and treating at 36-38 deg.C for 60-70min to obtain second intermediate electrode;
and (3) washing the obtained second intermediate electrode, immersing the second intermediate electrode into 20mM second Tris-HCl buffer solution containing 100ppm of graphene oxide, and treating the second intermediate electrode at the temperature of 36-38 ℃ for 60-70 minutes to obtain the graphene modified electrochemical biosensor.
Preferably, the first Tris-HCl buffer solution has a pH of 7.4 and is prepared from 50mM NaCl, 10mM MgCl21mM DTT, and a second Tris-HCl buffer solution having a pH of 7.4 and consisting of 50mM NaCl and 10mM MgCl2And (4) forming.
The invention also provides the graphene-modified electrochemical biosensor prepared by the preparation method according to any one of the technical schemes.
The invention also provides application of the graphene-modified electrochemical biosensor in the detection of the methyltransferase, which can detect the methyltransferase within the range of 0.7-12U/mL.
Preferably, when the methylation transferase exists in a detected buffer system, the methylation transferase can catalyze the methylation of a CpG sequence 5'-CCGG-3' in a nucleotide sequence fixed on a gold electrode, so that the methylation sensitive restriction endonuclease cannot perform catalysis and subsequent reactions, and the detected electrode impedance modified by the methylation transferase is also obviously greater than that of the graphene-modified electrochemical biosensor, so that the detection aim is fulfilled.
Preferably, the obtained gold electrode with the fixed nucleotide sequence is immersed into a third 10mM Tris-HCl buffer solution containing the methyltransferase for methylation at the temperature of 36-38 ℃ for 75-85 minutes during detection;
wherein the third Tris-HCl buffer has a pH of 7.4 and is prepared from 50mM NaCl and 10mM MgCl21mM DTT and 160. mu.M SAM, and the concentration of the methyltransferase added was 40U/mL.
The invention also provides application of the graphene modified electrochemical biosensor in gene expression/gene imprinting/embryonic development/tumor detection according to the technical scheme.
The invention also provides an electric signal reading method which can be combined with the electrochemical functional material to cause the electrochemical impedance of the electrode to change.
Preferably, the electrochemically functional material comprises a graphene material.
Compared with the prior art, the invention has the advantages and positive effects that:
the invention provides a graphene-modified electrochemical DNA biosensor and a preparation method and application thereof. The sensor has the principle that the methylation transferase can catalyze the methylation of cytosine deoxyribonucleotide in a DNA CpG sequence, so that the methylation sensitive restriction endonuclease cannot complete the catalysis and further cannot perform subsequent reaction, and the finally modified electrode impedance is increased, so that the aim of detection is fulfilled.
The sensor provided by the invention can only use a section of DNA sequence to modify the surface of the gold electrode in the whole process, and the rest reactions are carried out on the surface of the gold electrode without any additional modification of DNA. The sensor system is stable, the operation is simple, whether the methyltransferase exists can be conveniently and rapidly judged, the identification of the methyltransferase can be realized in a qualitative and quantitative mode, and the detection limit of the methyltransferase can reach 0.7U/mL.
Drawings
FIG. 1 is a representation of experimental feasibility provided by an embodiment of the present invention (a) EIS; (b) CV, Cv;
FIG. 2 illustrates the effect of a methyltransferase enzyme on modifying the impedance of an electrode;
FIG. 3 provides the time optimization of methyltransferase action;
FIG. 4 shows the optimization of the time for treating the electrode with HpaII restriction endonuclease provided by the embodiment of the present invention;
FIG. 5 is a graph of electrode impedance as a function of methyltransferase concentration as provided by an embodiment of the invention;
FIG. 6 is a graph showing the change in electrode impedance with the concentration of methyltransferase.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Reagent and apparatus
Nucleic acid sequence:
the HPLC-purified nucleic acid sequence used in this experiment, Shanghai Bioengineering Co., Ltd., base sequence 5 '-3' is:
TTATCCAGAGTGCCGGACGCAGCAAAGCGTCCGGCACTCTGGATTT, both ends of which are modified with sulfhydryl.
Experimental reagent:
dithiothreitol (DTT), Sigma-Aldrich; dNTP, Shanghai Biotechnology engineering Co., Ltd; dATP, Shanghai Biotech Co., Ltd; disodium hydrogen phosphate (Na)2HPO4) Chemical agents of the national drug group, ltd; sodium chloride (NaCl), national drug group chemical agents ltd; CPG methyltransferase (M.SssI Mta)se), NEB (beijing) ltd; terminal deoxynucleotide transfer, (TdTase) Thermo fisher, Inc. (USA); absolute ethanol, pure chromatography, chemical reagents of national drug group limited; acetone, chromato-pure, chemical reagents of national drug group, ltd; 6-mercaptohex-1-ol (MCH), Sigma-Aldrich (USA); tris (2-carboxyethyl) phosphine (TCEP), bailingwei technologies ltd, beijing; S-adenosyl-L-methionine, Sigma-Aldrich (USA); the water used in the experiment meets the first grade water standard of laboratory water index (GB-6682-92). The liquid reagents used in the experiments were filtered using a 0.22 μm syringe filter before use.
An experimental instrument:
electrochemical workstation, model CHI660E electrochemical workstation, shanghai chenhua instrument factory; vortex Mixer, Mixer4K speed-adjustable mini vortex Mixer, shanghai bio-engineering ltd; a thermostat, DNP-9052BS-III electric heating thermostat; shanghai New Miao medical device manufacturing, Inc.; an ultrasonic cleaner, a KQ-100B ultrasonic cleaner, a working frequency of 40KHz, an ultrasonic electric power of 100w, Kunshan City ultrasonic instruments Limited; a constant temperature water bath, a Tianjin Tester electric heating constant temperature water bath DK-98-II constant temperature groove; centrifuge, TGL-16B desk centrifuge, maximum rotation 16000rpm, power 145W, Shanghai' an pavilion scientific instruments and plants.
Example 1 graphene-modified electrochemical biosensor
1.1 pretreatment of gold electrodes
Polishing the gold electrode with 0.05 mu m alumina powder, cleaning with deionized water after polishing until no white powder is observed visually, then placing the polished gold electrode in an ultrasonic cleaning machine, cleaning with analytically pure absolute ethyl alcohol for 3min, then replacing the ethyl alcohol, and continuing cleaning, and thus, performing the operation for three times. And then rinsed with deionized water. Then blowing the mixture by using nitrogen. And carrying out electrochemical analysis on the polished and cleaned gold electrode. The gold electrode was placed in 0.5M H2SO4The treatment is carried out in the solution, the working parameter of an electrochemical workstation is-0.2V to +1.5V, and the scanning speed is 100mV-1. The process simultaneously scans the electrodes until a stable cyclic voltammogram is obtained at the electrochemical workstation. After thatThe electrodes were rinsed with ultrapure water and dried with nitrogen, awaiting experimental use.
1.2 immobilization sequence on electrodes
Adding the mixed solution of the nucleotide sequence solution (diluted by sterilized/non-sterilized deionized water) and the tris (2-carboxyethyl) phosphine into a 2ml centrifuge tube, putting a blow-dried electrode into the centrifuge tube, and soaking for 12 hours at normal temperature, wherein the sequence concentration is 1 mu M, and the concentration of the tris (2-carboxyethyl) phosphine is 10 mM. The gold electrode with the fixed DNA strand is taken out, washed with buffer solution gently, wiped to dry the periphery of the electrode, and cut to make sure that the surface of the gold electrode is not touched.
The treated electrode was immersed in a 2mL disposable centrifuge tube containing 1mM mercaptohexanol solution in a volume appropriate to immerse the electrode surface, 50. mu.L for this experiment. The electrode was immersed at room temperature for 1 hour. Used for blocking the part of the DNA chain which is not combined with the gold electrode.
1.3 restriction endonuclease treatment
The electrode obtained under 1.2 was immersed in a 10mM Tris-HCl buffer (pH7.4, 50mM NaCl, 10mM MgCl) containing restriction endonuclease (HpaII)21mM DTT) at a temperature of 36-38 ℃ for 60-70 minutes.
1.4 terminal transferase extension
After washing the electrode with 10mM PBS buffer (pH7.4, 0.2M NaCl), the electrode was immersed in 20mM Tris-HCl buffer (pH7.4, 50mM NaCl, 10mM MgCl) containing 40U/mL TdTase and 10. mu.M dNTP or dATP2) Treating at 36-38 deg.C for 60-70 min. The electrodes were then washed with 10mM PBS buffer (pH7.4, 0.2M NaCl).
1.5 graphene oxide treatment
The electrode was immersed in 20mM Tris-HCl buffer (pH7.4, 50mM NaCl, 10mM MgCl2) containing 100ppm of graphene oxide and treated at 36-38 ℃ for 60-70 minutes. The electrode was then washed with 10mM PBS buffer (pH7.4, 0.2M NaCl) to give a graphene-modified electrochemical biosensor.
Example 2 electrochemical biosensor modified with methyltransferase
In this example, the gold electrode pretreatment and immobilization sequence steps on the electrode are the same as 1.1 and 1.2 under example 1 above.
1.3 Methyltransferase treatment
The electrode, immobilized with the sequence and blocked with mercaptohexanol, was immersed in 10mM Tris-HCl buffer (pH7.4, 50mM NaCl, 10mM MgCl) containing methyltransferase (M.SssIMtase) at a concentration of 40U/mL21mM DTT, 160. mu.M SAM) at 37 ℃ for 75-85 minutes, preferably 80 minutes, to finally obtain the methyltransferase-modified electrochemical biosensor.
Example 3 electrochemical detection
The sensors obtained in examples 1 and 2 were subjected to cyclic voltammetry in 10mM Tris-HCl (pH7.4, 1mM EDTA, 0.1M NaCl, 1mM MgCl) respectively2) The electrolyte solution was thoroughly deoxygenated with high purity nitrogen for 20 minutes prior to each measurement, measured in buffer. The alternating current impedance (EIS) is in a range of 5mM [ Fe (CN)6]3-Is measured. The alternating voltage amplitude and the voltage frequency for EIS measurement are respectively 5mV and 0.1 Hz-10 KHz.
Example 4 Experimental results and discussion
4.1 principle of the experiment
The single-stranded DNA base is exposed and can form hydrogen bonds with graphene, so that the graphene is adsorbed, and the charge transfer resistance (Rct) of the electrode is greatly reduced under the condition that the graphene exists. And fixing the immobilization Chain (CP) for marking sulfydryl on the surface of the gold electrode through a gold-sulfur bond. The sequence contains a 5'-CCGG-3' sequence, a neck ring which is not treated by a methyltransferase and is fixed on an electrode can be cut by a methylation sensitive restriction endonuclease to form a cohesive end, and then the cohesive end is extended by a terminal deoxynucleotidyl transferase to form a single-stranded DNA sequence, and graphene can be combined with the single-stranded DNA sequence. If the methyltransferase is present in the buffer system, the subsequent reaction will not continue because the methylated CpG sequence 5'-CCGG-3' is not cleaved by the HpaII methylation-sensitive restriction endonuclease. The electrode impedance is large compared to that of the electrode which has not been modified by the methyltransferase.
4.2 feasibility characterization
Feasibility verification of the above sensor was performed using Electrochemical Impedance (EIS) (FIG. 1-a) and Cyclic Voltammetry (CV) (FIG. 1-b), and both sets of characterizations showed that the impedance of the electrode treated with methyltransferase was greater than the impedance of the electrode not treated with methyltransferase in the presence of methyltransferase M.SssIMtase. The preliminary test conforms to the experimental prediction result, and the next test is carried out.
4.3 Effect of methyltransferase on modified electrode impedance
FIG. 2 shows the impedance spectrum of the DNA sequence modified electrode at each stage of the treatment of immersing the DNA sequence modified electrode in a solution containing methyltransferase, and the impedance spectrum of the DNA sequence modified electrode at each stage of immersing the DNA sequence modified electrode in a solution not containing methyltransferase. Wherein, a and d are bare gold electrodes without modification, c and f curves are impedance curves of MCH sealed electrodes, and b and e are impedance curve solutions after subsequent treatment by methyltransferase. As can be seen from the graph, the resistance of the electrode treated with methyltransferase is significantly greater than that of the unmodified electrode. The next test can be performed.
4.4 optimization of the Experimental conditions
In order to achieve the best sensing performance and make the experimental result more scientific, the experiment also carries out corresponding optimization on the reaction conditions, and simultaneously can shorten the reaction time as much as possible and improve the efficiency, wherein the optimization comprises the optimization of the action time of the methyltransferase and the optimization of the processing time of the HpaII methylation sensitive restriction endonuclease.
As can be seen from FIG. 3, when the action time of the methyltransferase and the modified electrode is changed, the charge transfer impedance of the electrode gradually increases with the time, and after the reaction lasts for 75-85min, the telephone impedance tends to be balanced, which indicates that the reaction reaches the limit, and the DNA modified on the surface of the electrode basically reacts completely. The inability of the methylation-sensitive restriction endonuclease HpaII to cleave the DNA strand due to methylation of the DNA sequence does not result in the inability of the 3' TdTase to catalyze DNA strand extension, and thus the graphene cannot bind to the electrode to reduce impedance. Therefore, the reaction time of the methyltransferase is determined to be 75-85min, preferably 80 min.
As can be seen from FIG. 4, the impedance of the electrode decreased with increasing treatment time of the restriction endonuclease, and the electrochemical impedance value did not decrease any more and reached equilibrium by reaction for 60-70 min. It is shown that the specific sequence on the DNA modified electrode has been completely catalyzed by HpaII to form a state of exposing the 3' -OH end, and the reaction reaches the limit at this time, and does not increase with the duration of the reaction time, and the analysis reason may be due to the steric hindrance. Therefore, the treatment time of the HpaII enzyme is selected to be 60 to 70min, preferably 60 min.
Comparing fig. 3 and 4, it can be seen that when the DNA sequence ends are extended using dNTP and dATP, the impedance changes are not much from the impedance point of view when dNTP and dATP are used, but the analysis shows that the error of the electrode impedance value of the chain extension material using dATP is generally smaller than that of dNTP, and the analysis may be caused by that dNTP generates an indeterminate base complementary sequence during the chain growth process, and a spatial structure is more easily formed, which affects the binding of the generated DNA chain to graphene. Therefore, dATP is likely to produce less of such a condition than dNTPs, so in subsequent experiments dATP was used for strand growth.
4.5 sensitivity characterization
The sensitivity of the sensor obtained in example 1 was investigated by adjusting the methyltransferase concentration in the range of 0-40u/ml under the optimized experimental conditions described above, and the sensor was able to perform qualitative detection when the methyltransferase concentration is higher than 1u/ml, as shown in FIG. 5. The concentration is 0.7-12u/ml, and the linear relation is better. When the concentration is more than 15u/ml, the electrochemical impedance does not change greatly, but the quantitative analysis can still be carried out. At concentrations less than 0.5u/ml, the impedance of the modified electrode is not substantially changed, so that methyltransferases at concentrations less than 0.5u/ml cannot be detected.
Therefore, when analyzing the data with the concentration of methyltransferase set in the range of 0.7-12u/ml, as can be seen from FIG. 6, the impedance and the methyltransferase in the concentration interval are basically in a linear relationship, the linear equation is y 740+165.7x, the linear correlation coefficient is 0.9855, the detection limit is 0.7u/ml, x represents the unit of enzyme concentration u/ml, and y represents the unit of the value of the charge transfer impedance Ω.
Therefore, the application develops a biosensor which can qualitatively detect the methyltransferase and can quantitatively detect the methyltransferase in the concentration range of 0.7-12 u/ml. The biosensor utilizes the methylation transferase to catalyze the methylation of cytosine deoxyribonucleotide in a DNACG sequence, so that the methylation sensitivity of restriction endonuclease cannot be catalyzed, further subsequent reaction cannot be carried out, and finally the impedance of the modified electrode is increased, thereby achieving the purpose of detection. In the whole process of the sensor, only one section of DNA sequence is used for modifying the surface of the gold electrode, and the rest reactions are carried out on the surface of the gold electrode without any additional modification of DNA.
Claims (10)
1. The preparation method of the electrochemical biosensor modified by graphene is characterized by comprising the following steps:
cleaning and pretreating the gold electrode;
fixing the nucleotide sequence on a gold electrode and using mercaptohexanol to seal the part of the nucleotide sequence which is not combined with the gold electrode to obtain the gold electrode with the nucleotide sequence fixed;
and carrying out methylation sensitive restriction endonuclease treatment on the obtained gold electrode with the fixed nucleotide sequence, extending by using terminal transferase, and immersing into a buffer solution containing graphene oxide for full action to obtain the graphene-modified electrochemical biosensor.
2. The preparation method according to claim 1, wherein the nucleotide sequence comprises a CpG sequence 5'-CCGG-3', and thiol groups are modified at both ends of the nucleotide sequence.
3. The method according to claim 1, wherein the step of immobilizing the nucleotide sequence on the gold electrode and the step of blocking the portion of the nucleotide sequence not bound to the gold electrode with mercaptohexanol comprises:
uniformly mixing the nucleotide sequence solution with tris (2-carboxyethyl) phosphine, adding a dried gold electrode, soaking at room temperature for 10-12 hours, taking out, and drying to obtain the gold electrode with the fixed nucleotide sequence;
the gold electrode fixed with the nucleotide sequence is immersed in a mercaptohexanol solution for 1-2 hours at room temperature to block the part of the nucleotide sequence which is not combined with the gold electrode.
4. The method according to claim 3, wherein the nucleotide sequence solution is added at a concentration of 1. mu.M and the concentration of tris (2-carboxyethyl) phosphine is 10 mM.
5. The preparation method according to claim 1, wherein the steps of subjecting the obtained nucleotide sequence-immobilized gold electrode to methylation-sensitive restriction endonuclease treatment, extending by using terminal transferase, and immersing in a buffer solution containing graphene oxide for full action are specifically as follows:
immersing the obtained gold electrode with the fixed nucleotide sequence into a 10mM first Tris-HCl buffer solution containing methylation sensitive restriction endonuclease, and treating at the temperature of 36-38 ℃ for 60-70 minutes to obtain a first intermediate electrode;
washing the intermediate electrode with PBS buffer solution, soaking in 20mM second Tris-HCl buffer solution containing 40U/mL TdTase and 10 μ M dNTP or dATP, and treating at 36-38 deg.C for 60-70min to obtain second intermediate electrode;
and (3) washing the obtained second intermediate electrode, immersing the second intermediate electrode into 20mM second Tris-HCl buffer solution containing 100ppm of graphene oxide, and treating the second intermediate electrode at the temperature of 36-38 ℃ for 60-70 minutes to obtain the graphene modified electrochemical biosensor.
6. The method of claim 5, wherein the first Tris-HCl buffer solution has a pH of 7.4 and is prepared from 50mM NaCl, 10mM MgCl21mM DTT, and a second Tris-HCl buffer solution having a pH of 7.4 and consisting of 50mM NaCl and 10mM MgCl2And (4) forming.
7. The graphene-modified electrochemical biosensor prepared according to the preparation method of any one of claims 1 to 6.
8. The graphene-modified electrochemical biosensor according to claim 7, wherein the graphene-modified electrochemical biosensor is capable of detecting methyltransferases in the range of 0.7-12U/mL.
9. The application of claim 8, wherein in the application, when the methyltransferase exists in the detected buffer system, the methyltransferase can catalyze the methylation of the CpG sequence 5'-CCGG-3' in the nucleotide sequence immobilized on the gold electrode, so that the methylation-sensitive restriction endonuclease cannot perform catalysis and subsequent reactions, and the detected electrode impedance modified by the methyltransferase is significantly greater than that of the graphene-modified electrochemical biosensor, so as to achieve the detection purpose.
10. The use according to claim 9, wherein, in the detection, the obtained gold electrode having the nucleotide sequence immobilized thereon is immersed in 10mM third Tris-HCl buffer solution containing methyltransferase and methylated at a temperature of 36-38 ℃ for 75-85 minutes;
wherein the third Tris-HCl buffer has a pH of 7.4 and is prepared from 50mM NaCl and 10mM MgCl21mM DTT and 160. mu.M SAM, and the concentration of the methyltransferase added was 40U/mL.
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