CN105004776B - A kind of method and its application that network structure modified electrode is built based on DNA golden nanometer particles - Google Patents
A kind of method and its application that network structure modified electrode is built based on DNA golden nanometer particles Download PDFInfo
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Abstract
The invention discloses a kind of method and its application that network structure modified electrode is built based on DNA golden nanometer particles, golden nanometer particle (AuNPs) has been synthesized using reduction of sodium citrate method, utilize the complementary pairing effect of base between DNA sequence dna, construct the DNA gold nanos of a network structure, pass through dithiothreitol (DTT) connection electrode and network structure, double-stranded DNA is may be inserted into using methylene blue to be detected as a signaling molecule, is prepared DNA golden nanometer particles and is built network structure modified electrode.Compared with prior art, the detection for the electrode pair DNA methyltransferase that this present invention is provided has the characteristics of sensitivity is high, test limit is low, selectivity is good, stability is good.
Description
Technical field
The invention belongs to biosensor technology field, and in particular to build network structure based on DNA- golden nanometer particles and repair
Adorn the method and its application of electrode, it is possible to achieve to the Sensitive Detection of dnmt rna activity.
Background technology
In recent years, many research shows DNA methylation in terms of gene expression regulation, growth adjustment, genomic imprinting
Play a significant role, the generation with some tumours and hereditary disease is closely related.It is fast with embryology and oncology basic research
Speed development, DNA methylation is received more and more attention as one of important mechanisms of gene table science of heredity.
For the research of DNA methylation, there are many methods at present, can substantially be divided into two classes：One class is turned from DNA methyl
The angle of enzyme is moved, another kind of studied from the angle of DNA methylation level；The latter is divided into overall dna methylation level again
With 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, its with
The removing and reconstruction of trace, the inactivation of x chromosomes, germline differentiation etc. have substantial connection.And the generation of tumour may be with suppression
Oncogene promoter CpG island methylates, and then it is relevant to cause tumor suppressor gene to be closed.
Therefore, the research about DNA methylation turns into the study hotspot of current embryonic development and tumorigenicity, energy
It is enough to realize that to the Sensitive Detection of dnmt rna activity be the problem of solving first.
The content of the invention
In order to solve the above technical problems, building network structure modification based on DNA- golden nanometer particles the invention provides one kind
The method of electrode, golden nanometer particle (AuNPs) has been synthesized using reduction of sodium citrate method, using between DNA sequence dna base it is mutual
Recruit to effect, construct the DNA- gold nanos of a network structure, pass through dithiothreitol (DTT) connection electrode and network structure, profit
Double-stranded DNA is may be inserted into methylene blue to be detected as a signaling molecule, prepares DNA- golden nanometer particles structure netted
Structural modification electrode.
Present invention also offers a kind of application that network structure modified electrode is built based on DNA- golden nanometer particles, realize
Detection to the sensitivity, specificity, stability of DNA methyltransferase.
A kind of method that network structure modified electrode is built based on DNA- golden nanometer particles that the present invention is provided, including it is following
Step：
(1), 2.5OD DNAS1, DNA S2 sequences are dissolved in cushioning liquid respectively, DNA S1 cushioning liquid is obtained
With DNA S2 cushioning liquid；
(2), DNA S1 cushioning liquid and DNA S2 cushioning liquid are added separately in solution of gold nanoparticles, cultivated,
DNA S1- solution of gold nanoparticles and DNA S2- solution of gold nanoparticles are prepared into respectively；
(3) DNA S1- solution of gold nanoparticles and DNA S2- the solution of gold nanoparticles mixing for, obtaining step (2),
Hybridization, obtains the solution of DNA- golden nanometer particle network structures；
(4), the gold electrode after polishing is immersed in the Tris-HCl cushioning liquid containing dithiothreitol (DTT), taken out,
Cleaning, has been modified the gold electrode of dithiothreitol (DTT)；
(5) gold electrode for having modified dithiothreitol (DTT) that step (4) is obtained, is immersed into DNA- golden nanometer particle network structures
Solution, culture, obtain based on DNA- golden nanometer particles build network structure modified electrode.
Specifically, step (1) is that the respectively 2.5OD of purchase DNAS1, DNA S2 sequences are dissolved in into Tris- respectively
It is 100 μM of DNA S2 cushioning liquid that DNA S1 cushioning liquid that concentration is 100 μM and concentration are respectively obtained in HCl cushioning liquid,
Saved backup at 4 DEG C；
Further, DNA S1 in step (1):SH-GTCTGATCCCTGTGTA, DNA S2:SH-
CAGACTAGGGACACCA；
Further, cushioning liquid Tris-HCl pH is 7.4 in step (1), and concentration is 0.1M.
Specifically, step (2) is：15 μ L, 100 μM of DNA S1 cushioning liquid and 15 μ L100 μM DNA S2 bufferings are molten
Liquid is added separately in 500 μ L solution of gold nanoparticles, culture, and DNA S1- solution of gold nanoparticles and DNA are prepared into respectively
S2- solution of gold nanoparticles；
Further, in step (2), the culture is specifically：10h is cultivated at 20~50 DEG C.
The preparation of golden nanometer particle in step (2)：Using reduction of sodium citrate method, first synthesize the AuNPs of size uniform,
This nano particle has good bio-compatibility, can combine a plurality of designed probe, therefore should by the nano particle of preparation
Detection for biology sensor has good amplification.
The method of the preparation of golden nanometer particle is specially in step (2)：The ultrapure of 50mL is added in 100mL round-bottomed flask
Water, adds 0.5mL, 1% aqueous solution of chloraurate is into round-bottomed flask so that the concentration of Chlorine in Solution auric acid drops to 0.01% (w/
V), by this solution in oil bath heated constant temperature in 92 ± 4 DEG C.Under being stirred vigorously of magneton, it is rapidly added a certain amount of prior
The citric acid three sodium solution (1%) prepared, continues to stir.Last solution becomes the orange-red solution of clarification, is just made
16nm or so gold nano solution.
Further, hybridization is specifically described in step (3)：Hybridize 2h at 37 DEG C.
Further, in step (3), DNA S1- solution of gold nanoparticles and DNA S2- solution of gold nanoparticles mixing
Volume ratio is 1：1.
Further, in step (4), in cushioning liquid of the gold electrode immersion containing dithiothreitol (DTT) after polishing
Take out, cleaned with Tris-HCl cushioning liquid after 12h；Concentration of the dithiothreitol (DTT) in Tris-HCl cushioning liquid is 2mM.
Further, in step (4), the gold electrode after the polishing refers to：Gold electrode is first successively with 0.3 He
0.5mm aluminium powder is processed by shot blasting, then is sequentially placed into volume ratio HNO3:H2O=1:In 1 solution, ethanol solution, ultra-pure water, enter
Row ultrasonic wave is cleaned, and the time of ultrasonic cleaning is respectively 3~5min.
Further, cultivated described in step (5), be specifically：10h is cultivated at 20~50 DEG C.
A kind of application that network structure modified electrode is built based on DNA- golden nanometer particles that the present invention is provided, is turned to DNA
The application of the detection of methylase.
It is a kind of based on DNA- golden nanometer particles build network structure modified electrode application process be：
A, modified electrode immersed into the Tris-HCl containing Dam MTase (transmethylase) and S- adenosylmethionines
37 DEG C of culture 0.5-3h, the electrode of the DNA- gold nano network structures methylated in cushioning liquid；
B, the electrode of the DNA- gold nano network structures methylated is immersed in the Tris-HCl cushioning liquid containing Mbo
In；
C, by step b processing after electrode be immersed in the Tris-HCl cushioning liquid containing methylenum careuleum, carry out differential arteries and veins
Rush voltammetry detection.
Further, in step a, the concentration that Tris-HCl cushioning liquid contains Dam Mtase is respectively 0,0.05,
The concentration of 0.75,2.5,5,10,20,30,40U/mL, S- adenosylmethionine is 32mM.
Further, step b is specially：The DNA- gold nano network structures methylated are immersed in containing 40U/mL's
In Mbo Tris-HCl cushioning liquid, 37 DEG C of immersion 2h.
Further, step c is specially：Electrode is immersed in the Tris-HCl cushioning liquid that 10mL contains 20 μM of methylenum careuleum
In, 15min is cultivated at 37 DEG C, differential pulse voltametry detection is carried out.
The pH of cushioning liquid used is 5-9 in this experiment, and optimal pH is 7.4.
Because methylene cyan molecule can be adsorbed onto in double-stranded DNA, so containing in the modified electrode immersion 10mL built
In the Tris-HCl cushioning liquid of 20 μM of methylenum careuleum, methylenum careuleum is detected as signaling molecule with differential volt-ampere impulse method.It is sub-
The signal intensity of methyl blue is related to double-stranded DNA amount, also just relevant with the concentration of dnmt rna.As DNA methyl is shifted
The increase of enzyme concentration, the peak intensity of differential volt-ampere impulse method is that the signal intensity of methylene blue can increase therewith, therefore, this modification
Electrode can quantitatively be detected to various concentrations dnmt rna.
The preparation method of this biology sensor, synthesizes simple using golden nanometer particle, consumes energy low, cost is low, bio-compatible
Property it is good, by DNA, golden nanometer particle is coupled, and then using DNA base complementary pairing principle construction into a DNA- gold nano
Particle network structure, therefore the more methylene blue signaling molecules with electro-chemical activity of insertion, so that electrochemistry is examined
Signal is surveyed to be amplified.The differential pulse voltametry strength signal produced using methylene blue, is built and DNA methyltransferase concentration
Linear relationship, realize detection to DNA methylation behavior.Therefore the detection to DNA methyltransferase has sensitivity height, detection
The characteristics of limit is low, the good, stability of selectivity is good.
Brief description of the drawings
Fig. 1 is the modification electricity that dnmt rna activity is detected based on DNA- golden nanometer particle network structures amplification
Pole prepares schematic diagram；
Fig. 2A is the SEM phenogram of golden nanometer particle；
Fig. 2 B are the SEM phenogram of DNA- golden nanometer particle network structures；
Fig. 2 C are ultraviolet-visible absorption spectroscopy figure；
A is the ultraviolet-visible absorption spectroscopy figure of golden nanometer particle；
B is the ultraviolet-visible absorption spectroscopy figure of DNA- golden nanometer particle network structures；
Fig. 3 A are the cyclic voltammogram of each step of modified electrode；
A is naked gold electrode；
B is the gold electrode that dithiothreitol (DTT) is modified；
C is with Mbo I processing DNA- golden nanometer particle network structure modified electrodes；
D is with Dam MTase (transmethylase) and Mbo I processing DNA- golden nanometer particle network structure modified electrodes；
E is DNA- golden nanometer particle network structure modified electrodes；
Fig. 3 B are the impedance diagram of each step of modified electrode；
A is naked gold electrode；
B is with Mbo I processing DNA- golden nanometer particle network structure modified electrodes；
C is the gold electrode that dithiothreitol (DTT) is modified；
D is DNA- golden nanometer particle network structure modified electrodes；
E is with Dam MTase processing DNA- golden nanometer particle network structure modified electrodes；
F is with Dam MTase and Mbo I processing DNA- golden nanometer particle network structure modified electrodes；
Fig. 4 is the differential pulse voltammogram of each step of modified electrode；
A is naked gold electrode；
B is with Mbo I processing DNA- golden nanometer particle network structure modified electrodes；
C is DNA- golden nanometer particle network structure modified electrodes；
D is with Dam MTase and Mbo I processing DNA- golden nanometer particle network structure modified electrodes；
Fig. 5 A are influence figure of the pH value to this experiment.
Fig. 5 B are the optimization figure of experimental temperature；
Fig. 5 C are the optimization figure for the time of methylating；
Electrochemical Modification electrode pair various concentrations Dams of Fig. 6 A based on DNA- golden nanometer particle network structure amplifications
MTase differential pulse voltammogram；
A is 2.5U/mL Dam MTase, d for 0.05U/mL Dam MTase, b the Dam MTase, c for being 0.75U/mL
It is 30U/mL Dam for 5U/mL Dam MTase, e the Dam MTase, f for being 10U/mL the Dam MTase, g for being 20U/mL
MTase,
Fig. 6 B pass the standard curve that modified electrode is catalyzed to various concentrations Dam MTase for this.
Embodiment
Embodiment 1
A kind of method that network structure modified electrode is built based on DNA- golden nanometer particles, is comprised the following steps：
(1), by the DNA sequence dna (sulfhydrylation of purchase：S1, S2, DNA S1:SH-GTCTGATCCCTGTGTA, DNA S2:
SH-CAGACTAGGGACACCA；) be dissolved in respectively in 0.1M Tris-HCl (pH 7.4) cushioning liquid, and preserved at 4 DEG C
It is standby；DNA S1:SH-GTCTGATCCCTGTGTA, DNA S2:SH-CAGACTAGGGACACCA；
(2), 100 μM of DNA S2 cushioning liquid of 15 μ L, 100 μM of DNA S1 cushioning liquid and 15 μ L are added separately to
In 500 μ L solution of gold nanoparticles, 10h is cultivated at 37 DEG C, DNA S1- solution of gold nanoparticles and DNA S2- are prepared into respectively
Solution of gold nanoparticles；
(3) the 250 μ L DNA S1- solution of gold nanoparticles for, obtaining step (2) and 250 μ L DNA S2- Jenner's grain of rices
Sub- solution mixing, carries out hybridization 2h at 37 DEG C, obtains the solution of DNA- golden nanometer particle network structures；
(4), gold electrode is first processed by shot blasting with 0.3 and 0.5mm aluminium powder successively, then is sequentially placed into volume ratio
HNO3:H2O=1:In 1 solution, ethanol solution, ultra-pure water, ultrasonic wave cleaning is carried out, time of ultrasonic cleaning is respectively 3~
5min；Gold electrode after polishing is immersed into 500 μ L to contain in the Tris-HCl cushioning liquid of dithiothreitol (DTT) after 12h, taken
Go out, cleaned with Tris-HCl cushioning liquid, modified the gold electrode of dithiothreitol (DTT)；Dithiothreitol (DTT) is slow in Tris-HCl
The concentration rushed in solution is 2mM.
(5) gold electrode for having modified dithiothreitol (DTT) that step (4) is obtained, is immersed into DNA- golden nanometer particle network structures
Solution, cultivate 10h at 37 DEG C, obtain based on DNA- golden nanometer particles build network structure modified electrode.
A kind of application that network structure modified electrode is built based on DNA- golden nanometer particles, specific method is：
A, the 400 μ LTris-HCl that modified electrode immerses to the S- adenosylmethionines containing Dam and 32mM respectively delay
Rush in solution, 37 DEG C of immersion 2h, the DNA- gold nano network structures methylated；Dam concentration is respectively 0,0.05,0.75,
2.5,5,10,20,30,40U/mL。
Experiment can be carried out in the Dam containing 0.05~40U/mL, but from 40UmL when signal increase it is slow, institute
To select 40U/mLDam as optimum experimental condition.
B, the Tris-HCl cushioning liquid that the DNA- gold nano network structures methylated are immersed in the Mbo containing 40U/mL
In, 37 DEG C of immersion 2h；
C, step b is prepared into electrode be immersed in the Tris-HCl cushioning liquid that 10mL contains 20 μM of methylenum careuleum, at 37 DEG C
15min is cultivated, differential pulse voltametry detection is carried out.
Fig. 6 A are the Electrochemical Modification electrode pair various concentrations Dam based on DNA- golden nanometer particle network structure amplifications
MTase differential pulse voltammogram；Fig. 6 B are the standard curve that modified electrode is catalyzed to various concentrations Dam MTase.
Embodiment 2
A kind of method that network structure modified electrode is built based on DNA- golden nanometer particles, is comprised the following steps：
(1), by the DNA sequence dna (sulfhydrylation of purchase：S1, S2, DNA S1:SH-GTCTGATCCCTGTGTA, DNA S2:
SH-CAGACTAGGGACACCA；) be dissolved in respectively in 0.1M Tris-HCl (pH 7.4) cushioning liquid, and preserved at 4 DEG C
It is standby；DNA S1:SH-GTCTGATCCCTGTGTA, DNA S2:SH-CAGACTAGGGACACCA；
(2), 100 μM of DNA S2 cushioning liquid of 15 μ L, 100 μM of DNA S1 cushioning liquid and 15 μ L are added separately to
In 500 μ L solution of gold nanoparticles, 10h is cultivated at 28 DEG C, DNA S1- solution of gold nanoparticles and DNA S2- are prepared into respectively
Solution of gold nanoparticles；
(3) the 250 μ L DNA S1- solution of gold nanoparticles for, obtaining step (2) and 250 μ L DNA S2- Jenner's grain of rices
Sub- solution mixing, carries out hybridization 2h at 28 DEG C, obtains the solution of DNA- golden nanometer particle network structures；
(4), gold electrode is first processed by shot blasting with 0.3 and 0.5mm aluminium powder successively, then is sequentially placed into volume ratio
HNO3:H2O=1:In 1 solution, ethanol solution, ultra-pure water, ultrasonic wave cleaning is carried out, time of ultrasonic cleaning is respectively 3~
5min；After 12h in Tris-HCl cushioning liquid of the gold electrode immersion containing dithiothreitol (DTT) after polishing, take out, use
Tris-HCl cushioning liquid is cleaned, and has been modified the gold electrode of dithiothreitol (DTT)；
(5) gold electrode for having modified dithiothreitol (DTT) that step (4) is obtained, is immersed into DNA- golden nanometer particle network structures
Solution, cultivate 10h at 28 DEG C, obtain based on DNA- golden nanometer particles build network structure modified electrode.
A kind of application that network structure modified electrode is built based on DNA- golden nanometer particles, specific method is：
A, the 400 μ LTris-HCl that modified electrode immerses to the S- adenosylmethionines containing Dam and 32mM respectively delay
Rush in solution, 37 DEG C of immersion 2h, the DNA- gold nano network structures methylated；Dam concentration is respectively 0,0.05,0.75,
2.5,5,10,20,30,40U/mL。
B, the Tris-HCl cushioning liquid that the DNA- gold nano network structures methylated are immersed in the Mbo containing 40U/mL
In, 37 DEG C of immersion 2h；
C, step b is prepared into electrode be immersed in the Tris-HCl cushioning liquid that 10mL contains 20 μM of methylenum careuleum, at 37 DEG C
15min is cultivated, differential pulse voltametry detection is carried out.
Fig. 6 A are the Electrochemical Modification electrode pair various concentrations Dam based on DNA- golden nanometer particle network structure amplifications
MTase differential pulse voltammogram；Fig. 6 B are the standard curve that modified electrode is catalyzed to various concentrations Dam MTase.
All cultivation temperatures in experiment can be between 20~50 DEG C, but the comparison Jing Guo experimental data, such as Fig. 5 B, experiment
Cultivation temperature be 37 DEG C when, the effect of experiment is best.
The testing result of the application is compared with prior art, as a result as follows：
Embodiment 3
A kind of method that network structure modified electrode is built based on DNA- golden nanometer particles, is comprised the following steps：
(1), by the DNA sequence dna (sulfhydrylation of purchase：S1, S2, DNA S1:SH-GTCTGATCCCTGTGTA, DNA S2:
SH-CAGACTAGGGACACCA；) be dissolved in respectively in 0.1M Tris-HCl (pH 7.4) cushioning liquid, and preserved at 4 DEG C
It is standby；DNA S1:SH-GTCTGATCCCTGTGTA, DNA S2:SH-CAGACTAGGGACACCA；
(2), 100 μM of DNA S2 cushioning liquid of 15 μ L, 100 μM of DNA S1 cushioning liquid and 15 μ L are added separately to
In 500 μ L solution of gold nanoparticles, 10h is cultivated at 37 DEG C, DNA S1- solution of gold nanoparticles and DNA S2- are prepared into respectively
Solution of gold nanoparticles；
(3) the 250 μ L DNA S1- solution of gold nanoparticles for, obtaining step (2) and 250 μ L DNA S2- Jenner's grain of rices
Sub- solution mixing, carries out hybridization 2h at 37 DEG C, obtains the solution of DNA- golden nanometer particle network structures；
(4), gold electrode is first processed by shot blasting with 0.3 and 0.5mm aluminium powder successively, then is sequentially placed into volume ratio
HNO3:H2O=1:In 1 solution, ethanol solution, ultra-pure water, ultrasonic wave cleaning is carried out, time of ultrasonic cleaning is respectively 3~
5min；Gold electrode after polishing is immersed into 500 μ L to contain in the Tris-HCl cushioning liquid of dithiothreitol (DTT) after 12h, taken
Go out, cleaned with Tris-HCl cushioning liquid, modified the gold electrode of dithiothreitol (DTT)；Dithiothreitol (DTT) is slow in Tris-HCl
The concentration rushed in solution is 2mM.
(5) gold electrode for having modified dithiothreitol (DTT) that step (4) is obtained, is immersed into DNA- golden nanometer particle network structures
Solution, cultivate 10h at 37 DEG C, obtain based on DNA- golden nanometer particles build network structure modified electrode.
A kind of application that network structure modified electrode is built based on DNA- golden nanometer particles：
A, modified electrode immersed respectively the Dam containing 40U/mL and 32mM S- adenosylmethionines 400 μ
In LTris-HCl cushioning liquid, 37 DEG C of immersion cultures 0.5,1,2,3h, the DNA- gold nano network structures methylated；
B, the Tris-HCl cushioning liquid that the DNA- gold nano network structures methylated are immersed in the Mbo containing 40U/mL
In, 37 DEG C of immersion 2h；
C, electrode are immersed in the Tris-HCl cushioning liquid that 10mL contains 20 μM of methylenum careuleum, are cultivated 15min at 37 DEG C, are entered
Row differential pulse voltametry is detected.
Such as Fig. 5 C, show that experiment incubation time should be 2h for Best Times
Embodiment 4
A kind of method that network structure modified electrode is built based on DNA- golden nanometer particles, is comprised the following steps：
(1), by the DNA sequence dna (sulfhydrylation of purchase：S1, S2, DNA S1:SH-GTCTGATCCCTGTGTA, DNA S2:
SH-CAGACTAGGGACACCA；) be dissolved in respectively in 0.1M Tris-HCl (pH 9.0) cushioning liquid, and preserved at 4 DEG C
It is standby；DNA S1:SH-GTCTGATCCCTGTGTA, DNA S2:SH-CAGACTAGGGACACCA；
(2), 100 μM of DNA S2 cushioning liquid of 15 μ L, 100 μM of DNA S1 cushioning liquid and 15 μ L are added separately to
In 500 μ L solution of gold nanoparticles, 10h is cultivated at 37 DEG C, DNA S1- solution of gold nanoparticles and DNA S2- are prepared into respectively
Solution of gold nanoparticles；
(3) the 250 μ L DNA S1- solution of gold nanoparticles for, obtaining step (2) and 250 μ L DNA S2- Jenner's grain of rices
Sub- solution mixing, carries out hybridization 2h at 37 DEG C, obtains the solution of DNA- golden nanometer particle network structures；
(4), gold electrode is first processed by shot blasting with 0.3 and 0.5mm aluminium powder successively, then is sequentially placed into volume ratio
HNO3:H2O=1:In 1 solution, ethanol solution, ultra-pure water, ultrasonic wave cleaning is carried out, time of ultrasonic cleaning is respectively 3~
5min；Gold electrode after polishing is immersed into 500 μ L to contain in the Tris-HCl cushioning liquid of dithiothreitol (DTT) after 12h, taken
Go out, cleaned with Tris-HCl cushioning liquid, modified the gold electrode of dithiothreitol (DTT)；Dithiothreitol (DTT) is slow in Tris-HCl
The concentration rushed in solution is 2mM.
(5) gold electrode for having modified dithiothreitol (DTT) that step (4) is obtained, is immersed into DNA- golden nanometer particle network structures
Solution, cultivate 10h at 37 DEG C, obtain based on DNA- golden nanometer particles build network structure modified electrode.
A kind of application that network structure modified electrode is built based on DNA- golden nanometer particles：
A, modified electrode immersed respectively the Dam containing 40U/mL and 32mM S- adenosylmethionines 400 μ
In LTris-HCl cushioning liquid, 37 DEG C of immersion cultures 0.5,1,2,3h, the DNA- gold nano network structures methylated；
B, the Tris-HCl cushioning liquid that the DNA- gold nano network structures methylated are immersed in the Mbo containing 40U/mL
In, 37 DEG C of immersion 2h；
C, electrode are immersed in the Tris-HCl cushioning liquid that 10mL contains 20 μM of methylenum careuleum, are cultivated 15min at 37 DEG C, are entered
Row differential pulse voltametry is detected.
The pH of cushioning liquid used in each step in being tested such as Fig. 5 can be adjusted in the range of 5-9, but be our experiments show that and worked as pH
For 7.4 when, experiment effect is best.
Claims (10)
1. a kind of method that network structure modified electrode is built based on DNA- golden nanometer particles, it is characterised in that methods described bag
Include following steps：
（1）, 2.5OD DNA S1, DNA S2 sequences be dissolved in cushioning liquid respectively, obtain DNA S1 cushioning liquid and
DNA S2 cushioning liquid；
（2）, DNA S1 cushioning liquid and DNA S2 cushioning liquid is added separately in solution of gold nanoparticles, cultivate, respectively
It is prepared into DNA S1- solution of gold nanoparticles and DNA S2- solution of gold nanoparticles；
（3）, by step（2）Obtained DNA S1- solution of gold nanoparticles and DNA S2- solution of gold nanoparticles mixing, hybridization,
Obtain the solution of DNA- golden nanometer particle network structures；
（4）, by after polishing gold electrode immerse the Tris-HCl cushioning liquid containing dithiothreitol (DTT) in, take out, cleaning,
The gold electrode of dithiothreitol (DTT) is modified；
（5）, by step（4）The obtained gold electrode for having modified dithiothreitol (DTT) immerses the molten of DNA- golden nanometer particle network structures
Liquid, culture obtains building network structure modified electrode based on DNA- golden nanometer particles.
2. the method according to claim 1 that network structure modified electrode is built based on DNA- golden nanometer particles, its feature
It is, step（1）For：The respectively 2.5OD of purchase DNAS1, DNA S2 sequences are dissolved in Tris-HCl bufferings respectively molten
It is 100 μM of DNA S2 cushioning liquid that DNA S1 cushioning liquid that concentration is 100 μM and concentration are respectively obtained in liquid, is protected at 4 DEG C
Deposit standby.
3. the method according to claim 1 or 2 that network structure modified electrode is built based on DNA- golden nanometer particles, it is special
Levy and be, step（1）Middle DNA S1:SH-GTCTGATCCCTGTGTA, DNA S2: SH-CAGACTAGGGACACCA.
4. the method according to claim 1 or 2 that network structure modified electrode is built based on DNA- golden nanometer particles, it is special
Levy and be, step（2）In, the culture is specifically：10h is cultivated at 20 ~ 50 DEG C.
5. the method according to claim 1 that network structure modified electrode is built based on DNA- golden nanometer particles, its feature
It is, step（3）Described in hybridization be specifically：37oC hybridizes 2h；DNA S1- solution of gold nanoparticles and DNA S2- Jenners
The volume ratio of rice corpuscles solution mixing is 1：1.
6. the method according to claim 1 that network structure modified electrode is built based on DNA- golden nanometer particles, its feature
It is, step（4）In, take out, use after 12h in cushioning liquid of the gold electrode immersion containing dithiothreitol (DTT) after polishing
Tris-HCl cushioning liquid is cleaned；Concentration of the dithiothreitol (DTT) in Tris-HCl cushioning liquid is 2mM.
7. the method according to claim 1 that network structure modified electrode is built based on DNA- golden nanometer particles, step（5）
Described in cultivate, be specifically：37o10h is cultivated under C.
8. any one of claim 1-7 builds the application of network structure modified electrode based on DNA- golden nanometer particles, its feature exists
In the application to the detection of DNA methyltransferase.
9. the application according to claim 8 that network structure modified electrode is built based on DNA- golden nanometer particles, its feature
It is, concrete application method is：
A, by modified electrode immerse the Tris-HCl cushioning liquid containing Dam MTase and S- adenosylmethionines in 37 DEG C training
Support 0.5-3h, the electrode of the DNA- gold nano network structures methylated；
B, the electrode of the DNA- gold nano network structures methylated is immersed in the Tris-HCl cushioning liquid containing Mbo；
C, by step b processing after electrode be immersed in the Tris-HCl cushioning liquid containing methylenum careuleum, carry out differential pulse volt
Peace method is detected.
10. the application according to claim 9 that network structure modified electrode is built based on DNA- golden nanometer particles, its feature
It is, step c is specially：Electrode is immersed in the Tris-HCl cushioning liquid that 10mL contains 20 μM of methylenum careuleum, in 37 DEG C of cultures
15min, carries out differential pulse voltametry detection.
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