CN105004776A - Method for constructing modified electrode of net structure on basis of DNA-AuNPs and application of modified electrode of net structure - Google Patents
Method for constructing modified electrode of net structure on basis of DNA-AuNPs and application of modified electrode of net structure Download PDFInfo
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Abstract
The invention discloses a method for constructing a modified electrode of a net structure on the basis of DNA-AuNPs and application of the modified electrode of the net structure. According to the method, AuNPs are synthesized through a sodium citrate reduction method, a DNA-gold nanoparticle of the net structure is constructed through complementary pairing effects of basic groups between DNA sequences, the electrode is connected with the net structure through dithiothreitol, and methylene blue in which double-stranded DNA can be inserted is used as a point signal molecule for detection, so that the modified electrode of the net structure is prepared on the basis of the DNA-AuNPs. Compared with the prior art, the electrode provided by the invention has the characteristics of high sensitivity, low detection limit, good selectivity and good stability on the aspect of DNA transmethylase detection.
Description
Technical field
The invention belongs to biosensor technology field, be specifically related to the method and the application thereof that build reticulate texture modified electrode based on DNA-golden nanometer particle, the Sensitive Detection to dnmt rna activity can be realized.
Background technology
In recent years, many research shows that DNA methylation plays a significant role in gene expression regulation, Growth adjustment, genomic imprinting etc., closely related with the generation of some tumour and hereditary disease.Along with developing rapidly of embryology and oncology fundamental research, DNA methylation, as one of genetic important mechanisms of gene table, receives increasing concern.
For the research of DNA methylation, have a lot of method at present, roughly can be divided into two classes: a class is the angle from dnmt rna, another kind of is study from the angle of DNA methylation level; The latter is divided into again the detection of overall dna methylation level and 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 the removing of itself and trace and reconstruction, the chromosomal inactivation of x, germline are broken up etc. substantial connection.And the generation of tumour may methylate with tumor suppressor gene promoter CpG island, and then it is relevant to cause tumor suppressor gene to be closed.
Therefore, the methylated research of relative dna has become the study hotspot of current embryonic development and tumorigenicity, and can realize the Sensitive Detection of dnmt rna activity is the problem that first should solve.
Summary of the invention
For solving the problems of the technologies described above, the invention provides a kind of method building reticulate texture modified electrode based on DNA-golden nanometer particle, reduction of sodium citrate method is used to synthesize golden nanometer particle (AuNPs), utilize the complementary pairing effect of base between DNA sequence dna, construct a cancellated DNA-gold nano, by dithiothreitol (DTT) connecting electrode and reticulate texture, utilize methylene blue can insert double-stranded DNA to detect as a signaling molecule, prepare DNA-golden nanometer particle and build reticulate texture modified electrode.
Present invention also offers a kind of application building reticulate texture modified electrode based on DNA-golden nanometer particle, achieve the detection of the sensitivity to DNA methyltransferase, specificity, stability.
A kind of method building reticulate texture modified electrode based on DNA-golden nanometer particle provided by the invention, comprises the following steps:
(1), by DNAS1, DNA S2 sequence of 2.5OD be dissolved in respectively in buffer solution, obtain DNA S1 buffer solution and DNA S2 buffer solution;
(2), by DNA S1 buffer solution and DNA S2 buffer solution join respectively in solution of gold nanoparticles, cultivate, be prepared into DNA S1-solution of gold nanoparticles and DNA S2-solution of gold nanoparticles respectively;
(3) the DNA S1-solution of gold nanoparticles, by step (2) obtained and the mixing of DNA S2-solution of gold nanoparticles, hybridization, obtains the cancellated solution of DNA-golden nanometer particle;
(4), by the gold electrode after polishing immerse in the Tris-HCl buffer solution containing dithiothreitol (DTT), take out, cleaning, obtains the gold electrode having modified dithiothreitol (DTT);
(5) gold electrode having modified dithiothreitol (DTT), by step (4) obtained immerses the cancellated solution of DNA-golden nanometer particle, cultivates, and obtains building reticulate texture modified electrode based on DNA-golden nanometer particle.
Concrete, step (1) is that DNAS1, DNA S2 sequence being respectively 2.5OD bought to be dissolved in Tris-HCl buffer solution respectively to obtain concentration be respectively the DNAS1 buffer solution of 100 μMs and concentration be 100 μMs of DNA S2 buffer solution, saves backup at 4 DEG C;
Further, DNA S1:SH-GTCTGATCCCTGTGTA, DNA S2:SH-CAGACTAGGGACACCA in step (1);
Further, in step (1), the pH of buffer solution Tris-HCl is 7.4, and concentration is 0.1M.
Concrete, step (2) is: join in 500 μ L solution of gold nanoparticles respectively by 15 μ L, 100 μMs of DNA S1 buffer solution and 15 μ L100 μMs of DNA S2 buffer solution, cultivate, be prepared into DNA S1-solution of gold nanoparticles and DNA S2-solution of gold nanoparticles respectively;
Further, in step (2), described cultivation, specifically: at 20 ~ 50 DEG C, cultivate 10h.
The preparation of golden nanometer particle in step (2): utilize reduction of sodium citrate method, first synthesize the AuNPs of size uniform, this nano particle has good bio-compatibility, the probe that can design in conjunction with many, the detection therefore nano particle of preparation being applied to biology sensor has good amplification.
In step (2), the method for the preparation of golden nanometer particle is specially: the ultrapure water adding 50mL in the round-bottomed flask of 100mL, add 0.5mL, 1% aqueous solution of chloraurate is in round-bottomed flask, make the concentration of Chlorine in Solution auric acid drop to 0.01% (w/v), by this solution in oil bath heated constant temperature in 92 ± 4 DEG C.Under the vigorous stirring of magneton, add rapidly a certain amount of citric acid three sodium solution (1%) prepared in advance, continue to stir.Last solution becomes the orange-red solution of clarification, has just obtained the gold nano solution of about 16nm.
Further, hybridize specifically described in step (3): at 37 DEG C, hybridize 2h.
Further, in step (3), the volume ratio of DNA S1-solution of gold nanoparticles and the mixing of DNA S2-solution of gold nanoparticles is 1:1.
Further, in step (4), the gold electrode after polishing immerses in the buffer solution containing dithiothreitol (DTT) and takes out after 12h, cleans with Tris-HCl buffer solution; The concentration of dithiothreitol (DTT) in Tris-HCl buffer solution is 2mM.
Further, in step (4), the gold electrode after described polishing refers to: gold electrode first carries out polishing with the aluminium powder of 0.3 and 0.5mm successively, then puts into volume ratio HNO successively
3: H
2in O=1:1 solution, ethanolic solution, ultrapure water, carry out Ultrasonic Cleaning, the time of ultrasonic cleaning is respectively 3 ~ 5min.
Further, cultivate described in step (5), specifically: at 20 ~ 50 DEG C, cultivate 10h.
A kind of application building reticulate texture modified electrode based on DNA-golden nanometer particle provided by the invention, to the application of the detection of DNA methyltransferase.
A kind of application process based on DNA-golden nanometer particle structure reticulate texture modified electrode is:
A, immerse modified electrode containing Dam MTase (transmethylase) and S-adenosylmethionine Tris-HCl buffer solution in 37 DEG C of cultivation 0.5-3h, obtain the cancellated electrode of methylated DNA-gold nano;
B, the cancellated electrode of methylated DNA-gold nano is immersed in the Tris-HCl buffer solution containing Mbo;
C, be immersed in the electrode after step b process containing methylenum careuleum Tris-HCl buffer solution, carry out differential pulse voltametry detection.
Further, in step a, the concentration that Tris-HCl buffer solution contains Dam Mtase is respectively 0,0.05,0.75,2.5,5,10,20,30,40U/mL, and the concentration of S-adenosylmethionine is 32mM.
Further, step b is specially: be immersed in the Tris-HCl buffer solution of the Mbo containing 40U/mL by methylated DNA-gold nano reticulate texture, and 37 DEG C immerse 2h.
Further, step c is specially: electrode is immersed in 10mL and contains in the Tris-HCl buffer solution of 20 μMs of methylenum careuleum, cultivates 15min, carry out differential pulse voltametry detection at 37 DEG C.
In this experiment, the pH of buffer solution used is 5-9, and optimum pH is 7.4.
Because methylenum careuleum molecule can be adsorbed onto in double-stranded DNA, contain in the Tris-HCl buffer solution of 20 μMs of methylenum careuleum so immerse 10mL at the modified electrode built, methylenum careuleum, as signaling molecule, detects by differential volt-ampere impulse method.The signal intensity of methylene blue is relevant to double-stranded DNA amount, also just relevant with the concentration of dnmt rna.Along with the increase of dnmt rna concentration, the peak intensity of differential volt-ampere impulse method and the signal intensity of methylene blue can increase thereupon, and therefore, this modified electrode quantitatively can detect variable concentrations dnmt rna.
The preparation method of this biology sensor, use golden nanometer particle synthesis simple, consume energy low, cost is low, good biocompatibility, by DNA, golden nanometer particle carries out coupling, and then utilize DNA base pair complementarity principle construction to become a DNA-golden nanometer particle reticulate texture, therefore insert the methylene blue signaling molecule more with electrochemical activity, thus electrochemical detection signal is amplified.The differential pulse voltametry strength signal utilizing methylene blue to produce, builds the linear relationship with DNA methyltransferase concentration, realizes the detection to DNA methylation behavior.Therefore to the detection of DNA methyltransferase have highly sensitive, detectability is low, selectivity good, the feature of good stability.
Accompanying drawing explanation
Fig. 1 be detect the modified electrode of dnmt rna activity based on DNA-golden nanometer particle reticulate texture amplification prepare schematic diagram;
Fig. 2 A is the scanning electron microscope phenogram of golden nanometer particle;
Fig. 2 B is the cancellated scanning electron microscope phenogram of DNA-golden nanometer particle;
Fig. 2 C is ultraviolet-visible absorption spectroscopy figure;
A is the ultraviolet-visible absorption spectroscopy figure of golden nanometer particle;
B is the cancellated ultraviolet-visible absorption spectroscopy figure of DNA-golden nanometer particle;
Fig. 3 A is 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 for using Mbo I process DNA-golden nanometer particle reticulate texture modified electrode;
D is for using Dam MTase (transmethylase) and Mbo I process DNA-golden nanometer particle reticulate texture modified electrode;
E is DNA-golden nanometer particle reticulate texture modified electrode;
Fig. 3 B is the impedance diagram of each step of modified electrode;
A is naked gold electrode;
B is for using Mbo I process DNA-golden nanometer particle reticulate texture modified electrode;
C is the gold electrode that dithiothreitol (DTT) is modified;
D is DNA-golden nanometer particle reticulate texture modified electrode;
E is for using Dam MTase process DNA-golden nanometer particle reticulate texture modified electrode;
F is for using Dam MTase and Mbo I process DNA-golden nanometer particle reticulate texture modified electrode;
Fig. 4 is the differential pulse voltammogram of each step of modified electrode;
A is naked gold electrode;
B is for using Mbo I process DNA-golden nanometer particle reticulate texture modified electrode;
C is DNA-golden nanometer particle reticulate texture modified electrode;
D is for using Dam MTase and Mbo I process DNA-golden nanometer particle reticulate texture modified electrode;
Fig. 5 A is the effect diagram of pH value to this experiment.
Fig. 5 B is the optimization figure of experimental temperature;
Fig. 5 C is the optimization figure of the time that methylates;
Fig. 6 A is based on the differential pulse voltammogram of the Electrochemical Modification electrode pair variable concentrations Dam MTase of DNA-golden nanometer particle reticulate texture amplification;
The Dam MTase of the Dam MTase of the Dam MTase of a to be the Dam MTase of 0.05U/mL, b be 0.75U/mL, c to be the Dam MTase of 2.5U/mL, d be 5U/mL, e to be the Dam MTase of 10U/mL, f be 20U/mL, g is the Dam MTase of 30U/mL,
Fig. 6 B passes the typical curve of modified electrode to variable concentrations Dam MTase catalysis for this reason.
Embodiment
Embodiment 1
Build a method for reticulate texture modified electrode based on DNA-golden nanometer particle, comprise the following steps:
(1) DNA sequence dna (sulfhydrylation: S1, S2, DNA S1:SH-GTCTGATCCCTGTGTA, the DNA S2:SH-CAGACTAGGGACACCA that, will buy; ) be dissolved in respectively in 0.1M Tris-HCl (pH 7.4) buffer solution, and save backup at 4 DEG C; DNA S1:SH-GTCTGATCCCTGTGTA, DNA S2:SH-CAGACTAGGGACACCA;
(2), 15 μ L, 100 μMs of DNA S1 buffer solution and 15 μ L, 100 μMs of DNA S2 buffer solution are joined in 500 μ L solution of gold nanoparticles respectively, cultivate 10h at 37 DEG C, be prepared into DNA S1-solution of gold nanoparticles and DNA S2-solution of gold nanoparticles respectively;
(3) the 250 μ L DNA S1-solution of gold nanoparticles, by step (2) obtained and the mixing of 250 μ LDNA S2-solution of gold nanoparticles, carry out hybridization 2h at 37 DEG C, obtain the cancellated solution of DNA-golden nanometer particle;
(4), by gold electrode first carry out polishing with the aluminium powder of 0.3 and 0.5mm successively, then put into volume ratio HNO successively
3: H
2in O=1:1 solution, ethanolic solution, ultrapure water, carry out Ultrasonic Cleaning, the time of ultrasonic cleaning is respectively 3 ~ 5min; Gold electrode after polishing being immersed 500 μ L contains in the Tris-HCl buffer solution of dithiothreitol (DTT) after 12h, takes out, and with the cleaning of Tris-HCl buffer solution, obtains the gold electrode having modified dithiothreitol (DTT); The concentration of dithiothreitol (DTT) in Tris-HCl buffer solution is 2mM.
(5) gold electrode having modified dithiothreitol (DTT), by step (4) obtained immerses the cancellated solution of DNA-golden nanometer particle, at 37 DEG C, cultivate 10h, obtains building reticulate texture modified electrode based on DNA-golden nanometer particle.
Build an application for reticulate texture modified electrode based on DNA-golden nanometer particle, concrete grammar is:
A, immersed by modified electrode in 400 μ LTris-HCl buffer solution of the S-adenosylmethionine containing Dam and 32mM respectively, 37 DEG C immerse 2h, obtain methylated DNA-gold nano reticulate texture; Dam concentration is respectively 0,0.05,0.75,2.5,5,10,20,30,40U/mL.
Test and can carry out when the Dam containing 0.05 ~ 40U/mL, but increase slowly from signal during 40UmL, so select 40U/mLDam as optimum experimental condition.
B, be immersed in the Tris-HCl buffer solution of the Mbo containing 40U/mL by methylated DNA-gold nano reticulate texture, 37 DEG C immerse 2h;
C, electrode prepared by step b and be immersed in 10mL and contain in the Tris-HCl buffer solution of 20 μMs of methylenum careuleum, cultivate 15min at 37 DEG C, carry out differential pulse voltametry detection.
Fig. 6 A is the differential pulse voltammogram of the Electrochemical Modification electrode pair variable concentrations Dam MTase based on DNA-golden nanometer particle reticulate texture amplification; Fig. 6 B is the typical curve of modified electrode to variable concentrations Dam MTase catalysis.
Embodiment 2
Build a method for reticulate texture modified electrode based on DNA-golden nanometer particle, comprise the following steps:
(1) DNA sequence dna (sulfhydrylation: S1, S2, DNA S1:SH-GTCTGATCCCTGTGTA, the DNA S2:SH-CAGACTAGGGACACCA that, will buy; ) be dissolved in respectively in 0.1M Tris-HCl (pH 7.4) buffer solution, and save backup at 4 DEG C; DNA S1:SH-GTCTGATCCCTGTGTA, DNA S2:SH-CAGACTAGGGACACCA;
(2), 15 μ L, 100 μMs of DNA S1 buffer solution and 15 μ L, 100 μMs of DNA S2 buffer solution are joined in 500 μ L solution of gold nanoparticles respectively, cultivate 10h at 28 DEG C, be prepared into DNA S1-solution of gold nanoparticles and DNA S2-solution of gold nanoparticles respectively;
(3) the 250 μ L DNA S1-solution of gold nanoparticles, by step (2) obtained and the mixing of 250 μ L DNA S2-solution of gold nanoparticles, carry out hybridization 2h at 28 DEG C, obtain the cancellated solution of DNA-golden nanometer particle;
(4), by gold electrode first carry out polishing with the aluminium powder of 0.3 and 0.5mm successively, then put into volume ratio HNO3:H2O=1:1 solution, ethanolic solution, ultrapure water successively, carry out Ultrasonic Cleaning, the time of ultrasonic cleaning is respectively 3 ~ 5min; Gold electrode after polishing is immersed in the Tris-HCl buffer solution containing dithiothreitol (DTT) after 12h, taking-up, with the cleaning of Tris-HCl buffer solution, obtain the gold electrode having modified dithiothreitol (DTT);
(5) gold electrode having modified dithiothreitol (DTT), by step (4) obtained immerses the cancellated solution of DNA-golden nanometer particle, at 28 DEG C, cultivate 10h, obtains building reticulate texture modified electrode based on DNA-golden nanometer particle.
Build an application for reticulate texture modified electrode based on DNA-golden nanometer particle, concrete grammar is:
A, immersed by modified electrode in 400 μ LTris-HCl buffer solution of the S-adenosylmethionine containing Dam and 32mM respectively, 37 DEG C immerse 2h, obtain methylated DNA-gold nano reticulate texture; Dam concentration is respectively 0,0.05,0.75,2.5,5,10,20,30,40U/mL.
B, be immersed in the Tris-HCl buffer solution of the Mbo containing 40U/mL by methylated DNA-gold nano reticulate texture, 37 DEG C immerse 2h;
C, electrode prepared by step b and be immersed in 10mL and contain in the Tris-HCl buffer solution of 20 μMs of methylenum careuleum, cultivate 15min at 37 DEG C, carry out differential pulse voltametry detection.
Fig. 6 A is the differential pulse voltammogram of the Electrochemical Modification electrode pair variable concentrations Dam MTase based on DNA-golden nanometer particle reticulate texture amplification; Fig. 6 B is the typical curve of modified electrode to variable concentrations Dam MTase catalysis.
All cultivation temperature in experiment can between 20 ~ 50 DEG C, but through the comparison of experimental data, as Fig. 5 B, when the cultivation temperature of experiment is 37 DEG C, the effect of experiment is best.
Compared with prior art, result is as follows for the testing result of the application:
Embodiment 3
Build a method for reticulate texture modified electrode based on DNA-golden nanometer particle, comprise the following steps:
(1) DNA sequence dna (sulfhydrylation: S1, S2, DNA S1:SH-GTCTGATCCCTGTGTA, the DNA S2:SH-CAGACTAGGGACACCA that, will buy; ) be dissolved in respectively in 0.1M Tris-HCl (pH 7.4) buffer solution, and save backup at 4 DEG C; DNA S1:SH-GTCTGATCCCTGTGTA, DNA S2:SH-CAGACTAGGGACACCA;
(2), 15 μ L, 100 μMs of DNA S1 buffer solution and 15 μ L, 100 μMs of DNA S2 buffer solution are joined in 500 μ L solution of gold nanoparticles respectively, cultivate 10h at 37 DEG C, be prepared into DNA S1-solution of gold nanoparticles and DNA S2-solution of gold nanoparticles respectively;
(3) the 250 μ L DNA S1-solution of gold nanoparticles, by step (2) obtained and the mixing of 250 μ L DNA S2-solution of gold nanoparticles, carry out hybridization 2h at 37 DEG C, obtain the cancellated solution of DNA-golden nanometer particle;
(4), by gold electrode first carry out polishing with the aluminium powder of 0.3 and 0.5mm successively, then put into volume ratio HNO successively
3: H
2in O=1:1 solution, ethanolic solution, ultrapure water, carry out Ultrasonic Cleaning, the time of ultrasonic cleaning is respectively 3 ~ 5min; Gold electrode after polishing being immersed 500 μ L contains in the Tris-HCl buffer solution of dithiothreitol (DTT) after 12h, takes out, and with the cleaning of Tris-HCl buffer solution, obtains the gold electrode having modified dithiothreitol (DTT); The concentration of dithiothreitol (DTT) in Tris-HCl buffer solution is 2mM.
(5) gold electrode having modified dithiothreitol (DTT), by step (4) obtained immerses the cancellated solution of DNA-golden nanometer particle, at 37 DEG C, cultivate 10h, obtains building reticulate texture modified electrode based on DNA-golden nanometer particle.
A kind of application building reticulate texture modified electrode based on DNA-golden nanometer particle:
A, immersed by modified electrode in the 400 μ LTris-HCl buffer solution of S-adenosylmethionine of Dam and 32mM containing 40U/mL respectively, 37 DEG C immerse cultivations 0.5,1,2,3h, obtain methylated DNA-gold nano reticulate texture;
B, be immersed in the Tris-HCl buffer solution of the Mbo containing 40U/mL by methylated DNA-gold nano reticulate texture, 37 DEG C immerse 2h;
C, electrode are immersed in 10mL and contain in the Tris-HCl buffer solution of 20 μMs of methylenum careuleum, cultivate 15min, carry out differential pulse voltametry detection at 37 DEG C.
As Fig. 5 C, showing that experiment incubation time should be 2h is Best Times
Embodiment 4
Build a method for reticulate texture modified electrode based on DNA-golden nanometer particle, comprise the following steps:
(1) DNA sequence dna (sulfhydrylation: S1, S2, DNA S1:SH-GTCTGATCCCTGTGTA, the DNA S2:SH-CAGACTAGGGACACCA that, will buy; ) be dissolved in respectively in 0.1M Tris-HCl (pH 9.0) buffer solution, and save backup at 4 DEG C; DNA S1:SH-GTCTGATCCCTGTGTA, DNA S2:SH-CAGACTAGGGACACCA;
(2), 15 μ L, 100 μMs of DNA S1 buffer solution and 15 μ L, 100 μMs of DNA S2 buffer solution are joined in 500 μ L solution of gold nanoparticles respectively, cultivate 10h at 37 DEG C, be prepared into DNA S1-solution of gold nanoparticles and DNA S2-solution of gold nanoparticles respectively;
(3) the 250 μ L DNA S1-solution of gold nanoparticles, by step (2) obtained and the mixing of 250 μ L DNA S2-solution of gold nanoparticles, carry out hybridization 2h at 37 DEG C, obtain the cancellated solution of DNA-golden nanometer particle;
(4), by gold electrode first carry out polishing with the aluminium powder of 0.3 and 0.5mm successively, then put into volume ratio HNO3:H2O=1:1 solution, ethanolic solution, ultrapure water successively, carry out Ultrasonic Cleaning, the time of ultrasonic cleaning is respectively 3 ~ 5min; Gold electrode after polishing being immersed 500 μ L contains in the Tris-HCl buffer solution of dithiothreitol (DTT) after 12h, takes out, and with the cleaning of Tris-HCl buffer solution, obtains the gold electrode having modified dithiothreitol (DTT); The concentration of dithiothreitol (DTT) in Tris-HCl buffer solution is 2mM.
(5) gold electrode having modified dithiothreitol (DTT), by step (4) obtained immerses the cancellated solution of DNA-golden nanometer particle, at 37 DEG C, cultivate 10h, obtains building reticulate texture modified electrode based on DNA-golden nanometer particle.
A kind of application building reticulate texture modified electrode based on DNA-golden nanometer particle:
A, immersed by modified electrode in the 400 μ LTris-HCl buffer solution of S-adenosylmethionine of Dam and 32mM containing 40U/mL respectively, 37 DEG C immerse cultivations 0.5,1,2,3h, obtain methylated DNA-gold nano reticulate texture;
B, be immersed in the Tris-HCl buffer solution of the Mbo containing 40U/mL by methylated DNA-gold nano reticulate texture, 37 DEG C immerse 2h;
C, electrode are immersed in 10mL and contain in the Tris-HCl buffer solution of 20 μMs of methylenum careuleum, cultivate 15min, carry out differential pulse voltametry detection at 37 DEG C.
The pH walking buffer solution used as each in Fig. 5 experiment can regulate within the scope of 5-9, but our experiments show that experiment effect is best when pH is 7.4.
Claims (10)
1. build a method for reticulate texture modified electrode based on DNA-golden nanometer particle, it is characterized in that, said method comprising the steps of:
(1), by DNAS1, DNA S2 sequence of 2.5OD be dissolved in respectively in buffer solution, obtain DNA S1 buffer solution and DNA S2 buffer solution;
(2), by DNA S1 buffer solution and DNA S2 buffer solution join respectively in solution of gold nanoparticles, cultivate, be prepared into DNA S1-solution of gold nanoparticles and DNA S2-solution of gold nanoparticles respectively;
(3) the DNA S1-solution of gold nanoparticles, by step (2) obtained and the mixing of DNA S2-solution of gold nanoparticles, hybridization, obtains the cancellated solution of DNA-golden nanometer particle;
(4), by the gold electrode after polishing immerse in the Tris-HCl buffer solution containing dithiothreitol (DTT), take out, cleaning, obtains the gold electrode having modified dithiothreitol (DTT);
(5) gold electrode having modified dithiothreitol (DTT), by step (4) obtained immerses the cancellated solution of DNA-golden nanometer particle, cultivates, and obtains building reticulate texture modified electrode based on DNA-golden nanometer particle.
2. the method building reticulate texture modified electrode based on DNA-golden nanometer particle according to claim 1, it is characterized in that, step (1) is: DNAS1, DNA S2 sequence being respectively 2.5OD bought being dissolved in respectively in Tris-HCl buffer solution and obtaining concentration be respectively the DNA S1 buffer solution of 100 μMs and concentration is 100 μMs of DNA S2 buffer solution, saves backup at 4 DEG C.
3. the method building reticulate texture modified electrode based on DNA-golden nanometer particle according to claim 1 and 2, is characterized in that, DNA S1:SH-GTCTGATCCCTGTGTA, DNA S2:SH-CAGACTAGGGACACCA in step (1).
4. the method building reticulate texture modified electrode based on DNA-golden nanometer particle according to claim 1 and 2, is characterized in that, in step (2), and described cultivation, specifically: at 20 ~ 50 DEG C, cultivate 10h.
5. the method building reticulate texture modified electrode based on DNA-golden nanometer particle according to claim 1, it is characterized in that, step is hybridized specifically described in (3): at 37 DEG C of hybridization 2h; The volume ratio of DNA S1-solution of gold nanoparticles and the mixing of DNA S2-solution of gold nanoparticles is 1:1.
6. the method building reticulate texture modified electrode based on DNA-golden nanometer particle according to claim 1, it is characterized in that, in step (4), the gold electrode after polishing immerses in the buffer solution containing dithiothreitol (DTT) and takes out after 12h, cleans with Tris-HCl buffer solution; The concentration of dithiothreitol (DTT) in Tris-HCl buffer solution is 2mM.
7. the method building reticulate texture modified electrode based on DNA-golden nanometer particle according to claim 1, cultivates described in step (5), specifically: at 37 DEG C, cultivate 10h.
8. any one of claim 1-7 builds the application of reticulate texture modified electrode based on DNA-golden nanometer particle, it is characterized in that, to the application of the detection of DNA methyltransferase.
9. the application building reticulate texture modified electrode based on DNA-golden nanometer particle according to claim 8, it is characterized in that, embody rule method is:
A, immerse modified electrode containing Dam MTase and S-adenosylmethionine Tris-HCl buffer solution in 37 DEG C cultivate 0.5-3h, obtain the cancellated electrode of methylated DNA-gold nano;
B, the cancellated electrode of methylated DNA-gold nano is immersed in the Tris-HCl buffer solution containing Mbo;
C, by step (7) process after electrode be immersed in the Tris-HCl buffer solution containing methylenum careuleum, carry out differential pulse voltametry detection.
10. the application building reticulate texture modified electrode based on DNA-golden nanometer particle according to claim 9, it is characterized in that, step c is specially: electrode is immersed in 10mL and contains in the Tris-HCl buffer solution of 20 μMs of methylenum careuleum, cultivate 15min at 37 DEG C, carry out differential pulse voltametry detection.
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| CN105606671A (en) * | 2016-01-18 | 2016-05-25 | 南京农业大学 | Detection method of poly(adenosine diphosphate-ribose) polymerase |
| CN105675687A (en) * | 2016-03-23 | 2016-06-15 | 安徽师范大学 | Method for preparing electrochemical biosensors and method for detecting activity of DNA (deoxyribonucleic acid) methyl transferase |
| CN105973963A (en) * | 2016-04-26 | 2016-09-28 | 中南大学 | Construction method for hairpin DNA supported dual-signal molecular sensing interface and application of sensing interface |
| CN106442670A (en) * | 2016-09-12 | 2017-02-22 | 安徽师范大学 | Homogeneous-phase electrochemical sensor, preparation method and purpose thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030027195A1 (en) * | 2001-08-03 | 2003-02-06 | Ford William E. | Metallisation of nucleic acids via metal nanoparticles produced ex-situ |
| US20050064446A1 (en) * | 2001-11-01 | 2005-03-24 | Minerva Biotechnologies Corporation | Detection of binding species with colloidal and non-colloidal structures |
| CN102183503A (en) * | 2011-01-24 | 2011-09-14 | 中国科学院合肥物质科学研究院 | Light irradiation preparation method and use of ultra-sensitive surface enhanced Raman scattering active base |
| CN102435662A (en) * | 2011-09-16 | 2012-05-02 | 南京工业大学 | Method for detecting target mercury ions in water body |
| CN103305881A (en) * | 2013-03-05 | 2013-09-18 | 浙江师范大学 | Porous reticular gold nano-sheet array and preparation method thereof |
-
2015
- 2015-08-06 CN CN201510491487.2A patent/CN105004776B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030027195A1 (en) * | 2001-08-03 | 2003-02-06 | Ford William E. | Metallisation of nucleic acids via metal nanoparticles produced ex-situ |
| US20050064446A1 (en) * | 2001-11-01 | 2005-03-24 | Minerva Biotechnologies Corporation | Detection of binding species with colloidal and non-colloidal structures |
| CN102183503A (en) * | 2011-01-24 | 2011-09-14 | 中国科学院合肥物质科学研究院 | Light irradiation preparation method and use of ultra-sensitive surface enhanced Raman scattering active base |
| CN102435662A (en) * | 2011-09-16 | 2012-05-02 | 南京工业大学 | Method for detecting target mercury ions in water body |
| CN103305881A (en) * | 2013-03-05 | 2013-09-18 | 浙江师范大学 | Porous reticular gold nano-sheet array and preparation method thereof |
Non-Patent Citations (5)
| Title |
|---|
| JUEWEN LIU & YI LU: "Preparation of aptamer-linked gold nanoparticle purple aggregates for colorimetric sensing of analytes", 《NATURE PROTOCOLS》 * |
| XIAODONG CAO 等: "Gold nanoparticle-based signal amplification for biosensing", 《ANALYTICAL BIOCHEMISTRY》 * |
| XIAOXIAO HE 等: "A sensitive signal-on electrochemical assay for MTase activity using AuNPs amplification", 《BIOSENSORS AND BIOELECTRONICS》 * |
| XIAOYING JING 等: "DNA-AuNPs based signal amplification for highly sensitive detection of DNA methylation,methyltransferase activity and inhibitor screening", 《BIOSENSORS AND BIOELECTRONICS》 * |
| 赵凯 等: "DNA-金纳米粒子网状结构的合成及在癌胚抗原检测中的初步应用", 《生物学杂志》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105606671A (en) * | 2016-01-18 | 2016-05-25 | 南京农业大学 | Detection method of poly(adenosine diphosphate-ribose) polymerase |
| CN105606671B (en) * | 2016-01-18 | 2018-06-26 | 南京农业大学 | A kind of detection method of polyadenosine diphosphate ribose polymerase |
| CN105675687A (en) * | 2016-03-23 | 2016-06-15 | 安徽师范大学 | Method for preparing electrochemical biosensors and method for detecting activity of DNA (deoxyribonucleic acid) methyl transferase |
| CN105675687B (en) * | 2016-03-23 | 2018-04-13 | 安徽师范大学 | A kind of preparation method of electrochemica biological sensor and the method for detecting dnmt rna activity |
| CN105973963A (en) * | 2016-04-26 | 2016-09-28 | 中南大学 | Construction method for hairpin DNA supported dual-signal molecular sensing interface and application of sensing interface |
| CN106442670A (en) * | 2016-09-12 | 2017-02-22 | 安徽师范大学 | Homogeneous-phase electrochemical sensor, preparation method and purpose thereof |
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