CN105675687A - Method for preparing electrochemical biosensors and method for detecting activity of DNA (deoxyribonucleic acid) methyl transferase - Google Patents
Method for preparing electrochemical biosensors and method for detecting activity of DNA (deoxyribonucleic acid) methyl transferase Download PDFInfo
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- G—PHYSICS
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- 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
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- 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
Abstract
The invention discloses a method for preparing electrochemical biosensors and a method for detecting the activity of DNA (deoxyribonucleic acid) methyl transferase. Compared with the prior art, the methods have the advantages that methylated DNA is loaded on electrodes in a modification manner, so that coupled ferrocene and anti-methylation antibodies can be loaded on the electrodes in a modification manner, methylene blue signals and ferrocene signals can be converted, linear relations with the concentration of the DNA methyl transferase are constructed by the aid of proportions of the electric ferrocene signals to the electric methylene blue signals, and DNA methylation behavior can be detected; the method for detecting the activity of the DNA methyl transferase is high in sensitivity, low in detection limit and good in selectivity.
Description
Technical field
The invention belongs to biosensor technology field, be specifically related to the preparation method of a kind of electrochemica biological sensor and the method for detection dnmt rna activity, it is possible to achieve the Sensitive Detection to dnmt rna activity.
Background technology
Tumor epigenetics is an emerging subject, mainly includes several aspects such as DNA methylation, histone modification, chromatin modification, genomic imprinting and genetic regulation by non-coding RNAs. Wherein, DNA methylation is the focus studied with the relation of tumor. In recent years, many research shows that DNA methylation plays a significant role in gene expression regulation, Growth adjustment, genomic imprinting etc., and the generation with some tumor and heredopathia is closely related. Along with developing rapidly of fetology and oncology's basic research, DNA methylation receives more and more attention.
For the research of DNA methylation, having a lot of method at present, substantially can be divided into two classes: a class is the angle from dnmt rna, another kind of is that angle from DNA methylation level is studied; The latter is divided into again overall dna methylation level and the detection of special gene sequence DNA methylation level.
There are some researches show, the change of the DNA methylation pattern in embryo development procedure is ontogenetic key, and its removing with trace and reconstruction, the chromosomal inactivation of x, germline differentiation etc. have substantial connection. Through the research of nearly half a century, a kind of typical case that the change of DNA methylation level has been human cancer at present changes, and changes along with the Emergence and Development of malignant cell.
Therefore, the change of detection DNA methylation level is all most important to the diagnosis of tumor, generation, development fast and effectively. Detecting the method effort of DNA methylation at present, time-consuming, sensitivity is low and accurate not. Therefore, inquiring into the mechanism formed and change that methylates, set up accuracy good, highly sensitive, DNA methylation detection method simple to operate is significant.
Summary of the invention
For solving above-mentioned technical problem, the present invention provides the preparation method of a kind of electrochemica biological sensor and the method for detection dnmt rna activity, utilize the complementary pairing effect of base between DNA sequence, hair clip DNA1 after methylating forms double-strand with the hair clip DNA2 on electrode, the methylene blue molecule modified on hair clip DNA2 is away from electrode surface, the methylation sites on hair clip DNA1 is modified after adding ferrocene-anti-antibody that methylates, thus constructing the electrochemica biological sensor of a signal conversion, by the signal transition detection dnmt rna activity detected before and after methylating, achieve the susceptiveness to dnmt rna, specificity, the detection of stability.
The preparation method of a kind of electrochemica biological sensor provided by the invention, comprises the following steps:
(1) ferrocene-anti-antibody that methylates is prepared;
(2) hair clip DNA1 and hair clip DNA2 buffer solution are prepared;
(3) hair clip DNA1 is methylated;
(4) the hair clip DNA2 gold electrode modified is prepared;
(5) structure obtains electrochemica biological sensor.
Step (1) comprises the following steps: add in the PBS buffer solution containing dicarboxyl ferrocene by carbodiimide and N-hydroxy-succinamide, reactant at room temperature shakes 1～2 hour, the product obtained is dispersed in again in the PBS buffer solution containing anti-5-methylcytosine antibody, concussion reaction under room temperature, preserve overnight at 4 DEG C again, thus obtaining ferrocene-anti-antibody that methylates.
Further, step (1) is particularly as follows: add containing in the PBS buffer solution (pH=7.4) of the 10mL0.1M of 2mM dicarboxyl ferrocene by the carbodiimide of 0.03834g20mM and the N-hydroxy-succinamide of 0.03702g32mM, , at room temperature shake 1～2 hour, after the carboxyl of activation dicarboxyl ferrocene, the product obtained by 2mL is dispersed in the 4mLPBS buffer solution containing the 10 anti-5-methylcytosine antibody of μ L10mg/mL again, this solution is at room temperature shaken 2 hours, afterwards, preserve overnight at 4 DEG C again, thus obtaining ferrocene-anti-antibody that methylates.
Step (2) comprises the following steps: be dissolved in Tris-HCl buffer solution respectively by the hair clip DNA1 of respectively 2.5OD bought, hair clip DNA2 sequence, respectively obtain hair clip DNA1 buffer solution and hair clip DNA2 buffer solution, save backup at 4 DEG C;
Further, in step (2), hair clip DNA1 sequence is:
5 '-CCGGGTAGAGCTCCCTTCAATCCAACATGATACCCGG-3 '; Hair clip DNA2 sequence is: 5 '-SH-CCGGGTATCATGTTGGATTGAAGGGAGCTCTACCCGG-MB-3 '.
Further, step (2) is particularly as follows: be dissolved in the Tris-HCl buffer solution of 0.1MpH=7.4 respectively by the hair clip DNA1 of respectively 2.5OD bought, hair clip DNA2 sequence, respectively obtain hair clip DNA1 buffer solution that concentration is 100 μMs and concentration is 100 μMs of hair clip DNA2 buffer solution, save backup at 4 DEG C;
Step (3) is: joined by hair clip DNA1 containing S-adenosylmethionine and M.SssI transmethylase storing solution, reacts 2 hours at 37 DEG C, cleans, and hair clip DNA1 realizes methylating;
Further, step (3) is particularly as follows: join in 200 μ L buffer solution by the hair clip DNA1 buffer solution of 1 μ L100 μM, obtain 0.5 μM of hair clip DNA1 buffer solution, join containing 160 μMs of S-adenosylmethionine and 200 μ LM.SssI transmethylase storing solutions, react 2 hours under 37 DEG C of wet condition, afterwards, cleaning 3 times with the Tris HCl (pH=7.4) of 10mM, hair clip DNA1 realizes methylating.
Step (4) comprises the following steps: hair clip DNA2 buffer solution is added drop-wise to clean gold electrode surfaces, room temperature reaction under dark surrounds, after ultra-pure water cleans, again the gold electrode that hair clip DNA2 modifies is saved in the Tris-HCl buffer solution containing 6-mercaptoethanol, to close remaining hole; Obtain the hair clip DNA2 gold electrode modified.
Step (4) particularly as follows: add in the 100mM thio-alcohol reducing agent TCEP of 0.1 μ L in 100 μMs of hair clip DNA2 solution of 100 μ L, react 1 hour under room temperature, prevent DNA self from forming disulfide bond, then with PBS buffer solution, hair clip DNA2 derived above is diluted to 0.5 μM, the hair clip DNA2 of 5 μ L0.5 μM is added drop-wise to clean gold electrode surfaces, room temperature reaction 2 hours under dark surrounds, clean with ultra-pure water, again the gold electrode that hair clip DNA2 modifies is saved in the 10mMTris-HCl (pH=7.4) containing 1.0mM6-mercaptoethanol to close remaining hole.
Described polishing is particularly as follows: gold electrode is first processed by shot blasting with the aluminium powder of 0.3 and 0.5mm successively, then is sequentially placed into volume ratio HNO3:H2In O=1:1 solution, alcoholic solution, ultra-pure water, carry out ultrasonic waves for cleaning, the time of ultrasonic cleaning respectively 3～5min.
Step (5) is: be added drop-wise to by methylated hair clip DNA1 on the hair clip DNA2 gold electrode modified, the gold electrode modified is obtained after cultivating under room temperature, ferrocene step (1) obtained-anti-antibody labeling thing that methylates is added drop-wise to the gold electrode surfaces of modification, cultivate under room temperature, clean, obtain electrochemica biological sensor, it is achieved the conversion of methylene blue and ferrocene signal.
Further, step (5) particularly as follows: be added drop-wise on the gold electrode of hair clip DNA2 modification prepared by step (4) by methylated hair clip DNA1 prepared by 5 μ L step (3), hair clip DNA1 and hair clip DNA2 realizes base pair complementarity, after cultivating 2 hours under room temperature, obtain the gold electrode modified, the ferrocene obtained by 5 μ L step (1)-anti-antibody labeling thing that methylates is added drop-wise under the gold electrode surfaces room temperature of modification and cultivates 1h, label may identify which and is connected on methylation sites, again with 0.1MPBS buffer solution cleaning electrode, obtain electrochemica biological sensor, realize methylene blue and the conversion of ferrocene signal.
A kind of method detecting dnmt rna activity, comprises the following steps:
A, prepare ferrocene-anti-antibody that methylates;
B, prepare hair clip DNA1 and hair clip DNA2 buffer solution;
C, hair clip DNA1 is methylated;
D, prepare hair clip DNA2 modify gold electrode;
E, structure electrochemica biological sensor, build the linear relationship with DNA methyltransferase concentration by the signal of telecommunication ratio of ferrocene with methylene blue, it is achieved the detection detection dnmt rna of DNA methylation behavior is active.
Step a is particularly as follows: add containing in the PBS buffer solution (pH=7.4) of the 10mL0.1M of 2mM dicarboxyl ferrocene by the carbodiimide of 0.03834g20mM and the N-hydroxy-succinamide of 0.03702g32mM, at room temperature shake 1～2 hour, after the carboxyl of activation dicarboxyl ferrocene, the product obtained by 2mL is dispersed in the 4mLPBS buffer solution containing the 10 anti-5-methylcytosine antibody of μ L10mg/mL again, this solution is at room temperature shaken 2 hours, afterwards, preserve overnight at 4 DEG C again, thus obtaining ferrocene-anti-antibody that methylates.
Further, step b particularly as follows:
The hair clip DNA1 of respectively 2.5OD bought, hair clip DNA2 sequence are dissolved in the Tris-HCl buffer solution of 0.1MpH=7.4 respectively, respectively obtain hair clip DNA1 buffer solution that concentration is 100 μMs and concentration is 100 μMs of hair clip DNA2 buffer solution, save backup at 4 DEG C;
In step b, hair clip DNA1 sequence is:
5 '-CCGGGTAGAGCTCCCTTCAATCCAACATGATACCCGG-3 '; Hair clip DNA2 sequence is: 5 '-SH-CCGGGTATCATGTTGGATTGAAGGGAGCTCTACCCGG-MB-3 '.
Step c, particularly as follows: hair clip DNA1 joins the M.SssI transmethylase storing solution containing S-adenosylmethionine and variable concentrations, reacts 2 hours at 37 DEG C, cleans, and hair clip DNA1 realizes methylating.
M.SssI transmethylase storing solution concentration is: 0U/mL, 0.25U/mL, 0.375U/mL, 0.5U/mL, 1U/mL, 10U/mL, 20U/mL, 40U/mL.
Step d particularly as follows: add in the 100mM thio-alcohol reducing agent TCEP of 0.1 μ L in 100 μMs of hair clip DNA2 solution of 100 μ L, react 1 hour under room temperature, prevent DNA self from forming disulfide bond, then with PBS buffer solution, hair clip DNA2 derived above is diluted to 0.5 μM, the hair clip DNA2 of 5 μ L0.5 μM is added drop-wise to clean gold electrode surfaces, room temperature reaction 2 hours under dark surrounds, clean with ultra-pure water, again the gold electrode that hair clip DNA2 modifies is saved in the 10mMTris-HCl (pH=7.4) containing 1.0mM6-mercaptoethanol to close remaining hole.
Step e particularly as follows: be added drop-wise on the gold electrode of hair clip DNA2 modification prepared by step (4) by methylated hair clip DNA1 prepared by 5 μ L step (3), hair clip DNA1 and hair clip DNA2 realizes base pair complementarity, after cultivating 2 hours under room temperature, obtain the gold electrode modified, the ferrocene obtained by 5 μ L step (1)-anti-antibody labeling thing that methylates is added drop-wise under the gold electrode surfaces room temperature of modification and cultivates 1h, label may identify which and is connected on methylation sites, again with 0.1MPBS buffer solution cleaning electrode, obtain electrochemica biological sensor, realize methylene blue and the conversion of ferrocene signal, the signal of telecommunication ratio of ferrocene with methylene blue is built the linear relationship with DNA methyltransferase concentration, realize the detection to DNA methylation behavior.
Compared with prior art, the present invention passes through methylated DNA modification to electrode, so that having coupled ferrocene-anti-to methylate antibody modification to electrode, realize methylene blue and the conversion of ferrocene signal, the signal of telecommunication ratio of ferrocene with methylene blue is built the linear relationship with DNA methyltransferase concentration, it is achieved the detection to DNA methylation behavior. The detection of DNA methyltransferase is had feature highly sensitive, that detection limit is low, selectivity is good by detection method provided by the invention.
Accompanying drawing explanation
Fig. 1 be based on signal transition detection dnmt rna activity electrochemica biological sensor prepare schematic diagram;
Fig. 2 is the square wave voltammetry figure under the electrochemica biological sensor different experimental conditions of preparation; Wherein a is hair clip DNA1/ hair clip DNA2/ gold electrode; B is methylated hair clip DNA1/ hair clip DNA2/ gold electrode; C is ferrocene-anti-antibody/hair clip DNA1/ hair clip DNA2/ gold electrode that methylates;
Fig. 3 A is the electrochemical impedance figure of different modifying electrode;
A is bare electrode; B is hair clip DNA2/ gold electrode; C is hair clip DNA1/ hair clip DNA2/ gold electrode; D is ferrocene-anti-antibody/hair clip DNA1/ hair clip DNA2/ gold electrode that methylates;
Fig. 3 B is the cyclic voltammogram of different modifying electrode;
A is bare electrode; B is hair clip DNA2/ gold electrode; C is hair clip DNA1/ hair clip DNA2/ gold electrode; D is ferrocene-anti-antibody/hair clip DNA1/ hair clip DNA2/ gold electrode that methylates;
Fig. 4 A is optimum experimental information drawing, temperature;
Fig. 4 B is optimum experimental information drawing, incubation time;
Fig. 4 C is optimum experimental information drawing, pH;
Fig. 5 A is the square wave voltammetry curve chart of the dnmt rna M.SssI of detection variable concentrations; Concentration is followed successively by 0U/mL, 0.25U/mL, 0.375U/mL, 0.5U/mL, 1U/mL, 10U/mL, 20U/mL, 40U/mL from a to h;
Fig. 5 B is based on to methylene blue IMBThe figure that makees of electrochemical signals and dnmt rna M.SssI concentration;
Fig. 5 C is based on to ferrocene IFcThe figure that makees of electrochemical signals and dnmt rna M.SssI concentration;
Fig. 5 D is based on IFc/|IMB| the figure that makees of electrochemical signals and dnmt rna M.SssI concentration
Fig. 6 is the system selectivity analysis to dnmt rna M.SssI.
Detailed description of the invention
Embodiment 1
A kind of method building electrochemica biological sensor detection dnmt rna activity based on signal conversion, comprises the following steps:
(1) N-hydroxy-succinamide of the carbodiimide of 0.03834g20mM and 0.03702g32mM is added containing in the PBS buffer solution (pH=7.4) of the 10mL0.1M of 2mM dicarboxyl ferrocene, at room temperature shake 1～2 hour, after the carboxyl of activation dicarboxyl ferrocene, the product obtained by 2mL is dispersed in the 4mLPBS buffer solution containing the 10 anti-5-methylcytosine antibody of μ L10mg/mL again, this solution is at room temperature shaken 2 hours, afterwards, preserve overnight at 4 DEG C again, thus obtaining ferrocene-anti-antibody that methylates.
(2) the hair clip DNA1 of respectively 2.5OD bought, hair clip DNA2 sequence are dissolved in the Tris-HCl buffer solution of 0.1MpH=7.4 respectively, respectively obtain hair clip DNA1 buffer solution that concentration is 100 μMs and concentration is 100 μMs of hair clip DNA2 buffer solution, save backup at 4 DEG C; Hair clip DNA1 sequence is:
5 '-CCGGGTAGAGCTCCCTTCAATCCAACATGATACCCGG-3 '; Hair clip DNA2 sequence is: 5 '-SH-CCGGGTATCATGTTGGATTGAAGGGAGCTCTACCCGG-MB-3 ';
(3) the hair clip DNA1 buffer solution of 1 μ L100 μM is joined in 200 μ L buffer solution, obtain 0.5 μM of hair clip DNA1 buffer solution, join containing 160 μMs of S-adenosylmethionine and 0U/mL, 0.25U/mL, 0.375U/mL, 0.5U/mL, the 200 μ LM.SssI transmethylase storing solutions of 1U/mL, 10U/mL, 20U/mL, 40U/mL, react 2 hours under 37 DEG C of wet condition, afterwards, cleaning 3 times with the Tris HCl (pH=7.4) of 10mM, hair clip DNA1 realizes methylating.
(4) add in the 100mM thio-alcohol reducing agent TCEP of 0.1 μ L in 100 μMs of hair clip DNA2 solution of 100 μ L, react 1 hour under room temperature, prevent DNA self from forming disulfide bond, then with PBS buffer solution, hair clip DNA2 derived above is diluted to 0.5 μM, the hair clip DNA2 of 5 μ L0.5 μM is added drop-wise to clean gold electrode surfaces, room temperature reaction 2 hours under dark surrounds, clean with ultra-pure water, then the gold electrode that hair clip DNA2 modifies is saved in the 10mMTris-HCl (pH=7.4) containing 1.0mM6-mercaptoethanol to close remaining hole.
(5) methylated hair clip DNA1 prepared by 5 μ L step (3) is added drop-wise on the gold electrode of hair clip DNA2 modification prepared by step (4), hair clip DNA1 and hair clip DNA2 realizes base pair complementarity, after cultivating 2 hours under room temperature, obtain the gold electrode modified, the ferrocene obtained by 5 μ L step (1)-anti-antibody labeling thing that methylates is added drop-wise under the gold electrode surfaces room temperature of modification and cultivates 1h, label may identify which and is connected on methylation sites, again with 0.1MPBS buffer solution cleaning electrode, obtain electrochemica biological sensor, realize methylene blue and the conversion of ferrocene signal, the signal of telecommunication ratio of ferrocene with methylene blue is built the linear relationship with DNA methyltransferase concentration, realize the detection to DNA methylation behavior.
Embodiment 2
A kind of method building electrochemica biological sensor detection dnmt rna activity based on signal conversion, comprises the following steps:
(1) N-hydroxy-succinamide of the carbodiimide of 0.03834g20mM and 0.03702g32mM is added containing in the PBS buffer solution (pH=7.4) of the 10mL0.1M of 2mM dicarboxyl ferrocene, at room temperature shake 1～2 hour, after the carboxyl of activation dicarboxyl ferrocene, the product obtained by 2mL is dispersed in the 4mLPBS buffer solution containing the 10 anti-5-methylcytosine antibody of μ L10mg/mL again, this solution is shaken 2 hours at 20 DEG C, afterwards, preserve overnight at 4 DEG C again, thus obtaining ferrocene-anti-antibody that methylates.
(2) the hair clip DNA1 of respectively 2.5OD bought, hair clip DNA2 sequence are dissolved in the Tris-HCl buffer solution of 0.1MpH=7.4 respectively, respectively obtain hair clip DNA1 buffer solution that concentration is 100 μMs and concentration is 100 μMs of hair clip DNA2 buffer solution, save backup at 4 DEG C; Hair clip DNA1 sequence is:
5 '-CCGGGTAGAGCTCCCTTCAATCCAACATGATACCCGG-3 ';Hair clip DNA2 sequence is: 5 '-SH-CCGGGTATCATGTTGGATTGAAGGGAGCTCTACCCGG-MB-3 ';
(3) the hair clip DNA1 buffer solution of 1 μ L100 μM is joined in 200 μ L buffer solution, obtain 0.5 μM of hair clip DNA1 buffer solution, join the 200 μ LM.SssI transmethylase storing solutions containing 160 μMs of S-adenosylmethionine and 40U/mL, react 2 hours under room temperature wet condition, afterwards, cleaning 3 times with the Tris HCl (pH=7.4) of 10mM, hair clip DNA1 realizes methylating.
(4) add in the 100mM thio-alcohol reducing agent TCEP of 0.1 μ L in 100 μMs of hair clip DNA2 solution of 100 μ L, react 1 hour at 20～44 DEG C, prevent DNA self from forming disulfide bond, then with PBS buffer solution, hair clip DNA2 derived above is diluted to 0.5 μM, the hair clip DNA2 of 5 μ L0.5 μM is added drop-wise to clean gold electrode surfaces, room temperature reaction 2 hours under dark surrounds, clean with ultra-pure water, then the gold electrode that hair clip DNA2 modifies is saved in the 10mMTris-HCl (pH=7.4) containing 1.0mM6-mercaptoethanol to close remaining hole.
(5) methylated hair clip DNA1 prepared by 5 μ L step (3) is added drop-wise on the gold electrode of hair clip DNA2 modification prepared by step (4), hair clip DNA1 and hair clip DNA2 realizes base pair complementarity, after cultivating 2 hours at 20 DEG C, obtain the gold electrode modified, the ferrocene obtained by 5 μ L step (1)-anti-antibody labeling thing that methylates is added drop-wise under the gold electrode surfaces room temperature of modification and cultivates 1h, label may identify which and is connected on methylation sites, again with 0.1MPBS buffer solution cleaning electrode, obtain electrochemica biological sensor, realize methylene blue and the conversion of ferrocene signal, the signal of telecommunication ratio of ferrocene with methylene blue is built the linear relationship with DNA methyltransferase concentration, realize the detection to DNA methylation behavior.
Embodiment 3
Only changing cultivation temperature from 20 DEG C-44 DEG C, test respectively successively, other reaction conditions, with embodiment 2, obtain temperature optimization figure as shown in Figure 4 A;
Embodiment 4
Only change the response time in step (3), tested respectively successively from 0.5～3 hour, other reaction conditions, with embodiment 2, obtain response time optimization figure as shown in Figure 4 B;
Embodiment 5
Only changing the pH of buffer solution, test respectively successively from 6-11, other reaction conditions, with embodiment 2, obtain pH optimization figure as shown in Figure 4 C;
Embodiment 6
Only change the detection object in step (3), respectively contain the 200 μ LM.SssI transmethylase storing solutions of 160 μMs of S-adenosylmethionine and 40U/mL, the 200 μ LDam transmethylase storing solutions containing 160 μMs of S-adenosylmethionine and 40U/mL, the 200 μ LHae III transmethylase storing solutions containing 160 μMs of S-adenosylmethionine and 40U/mL, test respectively successively, other reaction conditions, with embodiment 2, obtain selectivity analysis chart as shown in Figure 6.
SEQUENCELISTING
<110>Anhui Normal University
<120>a kind of electrochemica biological sensor preparation method and detection dnmt rna activity
<130>1
<160>2
<170>PatentInversion3.3
<210>1
<211>37
<212>DNA
<213>hair clip DNA1 sequence
<400>1
5'-ccgggtagagctcccttcaatccaacatgatacccgg-3'37
<210>2
<211>37
<212>DNA
<213>hair clip DNA2 sequence
<400>2
5'-SH-ccgggtatcatgttggattgaagggagctctacccgg-MB-3'37
Claims (9)
1. the preparation method of an electrochemica biological sensor, it is characterised in that described preparation method comprises the following steps:
(1) ferrocene-anti-antibody that methylates is prepared;
(2) hair clip DNA1 and hair clip DNA2 buffer solution are prepared;
(3) hair clip DNA1 is methylated;
(4) the hair clip DNA2 gold electrode modified is prepared;
(5) structure obtains electrochemica biological sensor.
2. preparation method according to claim 1, it is characterized in that, step (1) comprises the following steps: add in the PBS buffer solution containing dicarboxyl ferrocene by carbodiimide and N-hydroxy-succinamide, reactant at room temperature shakes 1～2 hour, the product obtained is dispersed in again in the PBS buffer solution containing anti-5-methylcytosine antibody, concussion reaction under room temperature, then preserve overnight at 4 DEG C, thus obtaining ferrocene-anti-antibody that methylates.
3. preparation method according to claim 1 and 2, it is characterised in that in step (2), hair clip DNA1 sequence is: 5 '-CCGGGTAGAGCTCCCTTCAATCCAACATGATACCCGG-3 '; Hair clip DNA2 sequence is:
5’-SH-CCGGGTATCATGTTGGATTGAAGGGAGCTCTACCCGG-MB-3’。
4. preparation method according to claim 1 and 2, it is characterized in that, hair clip DNA1 is joined by step (3) containing S-adenosylmethionine and M.SssI transmethylase storing solution, react 2 hours at 37 DEG C, cleaning, hair clip DNA1 realizes methylating.
5. preparation method according to claim 1 and 2, it is characterized in that, step (4) comprises the following steps: hair clip DNA2 buffer solution is added drop-wise to clean gold electrode surfaces, room temperature reaction under dark surrounds, after ultra-pure water cleans, again the gold electrode that hair clip DNA2 modifies is saved in the Tris-HCl buffer solution containing 6-mercaptoethanol, to close remaining hole; Obtain the hair clip DNA2 gold electrode modified.
6. preparation method according to claim 1 and 2, it is characterized in that, step (5) is: be added drop-wise to by methylated hair clip DNA1 on the hair clip DNA2 gold electrode modified, the gold electrode modified is obtained after cultivating under room temperature, ferrocene step (1) obtained-anti-antibody labeling thing that methylates is added drop-wise to the gold electrode surfaces of modification, cultivates under room temperature, cleans, obtain electrochemica biological sensor, it is achieved the conversion of methylene blue and ferrocene signal.
7. the method detecting dnmt rna activity, it is characterised in that described method comprises the following steps:
A, prepare ferrocene-anti-antibody that methylates;
B, prepare hair clip DNA1 and hair clip DNA2 buffer solution;
C, hair clip DNA1 is methylated;
D, prepare hair clip DNA2 modify gold electrode;
E, structure electrochemica biological sensor, build the linear relationship with DNA methyltransferase concentration by the signal of telecommunication ratio of ferrocene with methylene blue, it is achieved the detection detection dnmt rna of DNA methylation behavior is active.
8. method according to claim 7, it is characterized in that, step c, particularly as follows: hair clip DNA1 joins the M.SssI transmethylase storing solution containing S-adenosylmethionine and variable concentrations, reacts 2 hours at 37 DEG C, cleaning, hair clip DNA1 realizes methylating.
9. the method according to claim 7 or 8, it is characterized in that, step e particularly as follows: be added drop-wise on the gold electrode of hair clip DNA2 modification prepared by step (4) by methylated hair clip DNA1 prepared by 5 μ L step (3), hair clip DNA1 and hair clip DNA2 realizes base pair complementarity, after cultivating 2 hours under room temperature, obtain the gold electrode modified, the ferrocene obtained by 5 μ L step (1)-anti-antibody labeling thing that methylates is added drop-wise under the gold electrode surfaces room temperature of modification and cultivates 1h, label may identify which and is connected on methylation sites, again with 0.1MPBS buffer solution cleaning electrode, obtain electrochemica biological sensor, realize methylene blue and the conversion of ferrocene signal, the signal of telecommunication ratio of ferrocene with methylene blue is built the linear relationship with DNA methyltransferase concentration, realize the detection to DNA methylation behavior.
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| CN107144603A (en) * | 2017-05-16 | 2017-09-08 | 安徽师范大学 | A kind of impedance type electrochemica biological sensor based on electrostatic interaction, preparation method and applications |
| CN113533477A (en) * | 2021-08-13 | 2021-10-22 | 中国人民解放军陆军军医大学 | Method for determining activity of DNA methyltransferase and application thereof |
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| CN107144603B (en) * | 2017-05-16 | 2019-10-01 | 安徽师范大学 | A kind of impedance type electrochemica biological sensor based on electrostatic interaction, preparation method and applications |
| CN113533477A (en) * | 2021-08-13 | 2021-10-22 | 中国人民解放军陆军军医大学 | Method for determining activity of DNA methyltransferase and application thereof |
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