CN104020198A - Method for detecting DNA by electrochemical transducer with signal amplification technology - Google Patents
Method for detecting DNA by electrochemical transducer with signal amplification technology Download PDFInfo
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- CN104020198A CN104020198A CN201410272431.3A CN201410272431A CN104020198A CN 104020198 A CN104020198 A CN 104020198A CN 201410272431 A CN201410272431 A CN 201410272431A CN 104020198 A CN104020198 A CN 104020198A
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- 230000003321 amplification Effects 0.000 title claims abstract description 11
- 238000003199 nucleic acid amplification method Methods 0.000 title claims abstract description 11
- 238000000034 method Methods 0.000 title claims description 31
- 238000005516 engineering process Methods 0.000 title abstract description 4
- 239000000523 sample Substances 0.000 claims abstract description 29
- 239000010931 gold Substances 0.000 claims abstract description 25
- 238000002360 preparation method Methods 0.000 claims abstract description 22
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 17
- 229910052737 gold Inorganic materials 0.000 claims abstract description 17
- 238000004064 recycling Methods 0.000 claims abstract description 4
- 108020004414 DNA Proteins 0.000 claims description 51
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 claims description 38
- 239000002245 particle Substances 0.000 claims description 21
- 229910052697 platinum Inorganic materials 0.000 claims description 19
- 239000008055 phosphate buffer solution Substances 0.000 claims description 15
- 239000000243 solution Substances 0.000 claims description 14
- 238000006243 chemical reaction Methods 0.000 claims description 13
- KUUVQVSHGLHAKZ-UHFFFAOYSA-N thionine Chemical compound C=1C=CC=CSC=CC=1 KUUVQVSHGLHAKZ-UHFFFAOYSA-N 0.000 claims description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- 102100033072 DNA replication ATP-dependent helicase DNA2 Human genes 0.000 claims description 7
- 101000927313 Homo sapiens DNA replication ATP-dependent helicase DNA2 Proteins 0.000 claims description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- 150000002343 gold Chemical class 0.000 claims description 6
- 238000012417 linear regression Methods 0.000 claims description 5
- PZBFGYYEXUXCOF-UHFFFAOYSA-N TCEP Chemical compound OC(=O)CCP(CCC(O)=O)CCC(O)=O PZBFGYYEXUXCOF-UHFFFAOYSA-N 0.000 claims description 4
- 238000002484 cyclic voltammetry Methods 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 3
- 239000007853 buffer solution Substances 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 claims description 3
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- 238000005498 polishing Methods 0.000 claims description 3
- 239000002244 precipitate Substances 0.000 claims description 3
- 238000001338 self-assembly Methods 0.000 claims description 3
- 238000002604 ultrasonography Methods 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims description 3
- 238000004364 calculation method Methods 0.000 claims description 2
- 238000013507 mapping Methods 0.000 claims description 2
- YNDXUCZADRHECN-JNQJZLCISA-N triamcinolone acetonide Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@H]3OC(C)(C)O[C@@]3(C(=O)CO)[C@@]1(C)C[C@@H]2O YNDXUCZADRHECN-JNQJZLCISA-N 0.000 claims description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 claims 1
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 6
- 238000001514 detection method Methods 0.000 abstract description 6
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 125000004122 cyclic group Chemical group 0.000 abstract description 3
- JUWSSMXCCAMYGX-UHFFFAOYSA-N gold platinum Chemical compound [Pt].[Au] JUWSSMXCCAMYGX-UHFFFAOYSA-N 0.000 abstract 4
- 239000002105 nanoparticle Substances 0.000 abstract 4
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 abstract 2
- 230000002860 competitive effect Effects 0.000 abstract 1
- 238000001179 sorption measurement Methods 0.000 description 5
- 238000009835 boiling Methods 0.000 description 4
- 230000035484 reaction time Effects 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 238000011161 development Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000001509 sodium citrate Substances 0.000 description 3
- 108020003215 DNA Probes Proteins 0.000 description 2
- 239000003298 DNA probe Substances 0.000 description 2
- 241000238370 Sepia Species 0.000 description 2
- 229910021607 Silver chloride Inorganic materials 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- NICDRCVJGXLKSF-UHFFFAOYSA-N nitric acid;trihydrochloride Chemical compound Cl.Cl.Cl.O[N+]([O-])=O NICDRCVJGXLKSF-UHFFFAOYSA-N 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- HKZLPVFGJNLROG-UHFFFAOYSA-M silver monochloride Chemical compound [Cl-].[Ag+] HKZLPVFGJNLROG-UHFFFAOYSA-M 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 2
- PBVAJRFEEOIAGW-UHFFFAOYSA-N 3-[bis(2-carboxyethyl)phosphanyl]propanoic acid;hydrochloride Chemical compound Cl.OC(=O)CCP(CCC(O)=O)CCC(O)=O PBVAJRFEEOIAGW-UHFFFAOYSA-N 0.000 description 1
- QEDXSHCYPROEOK-UHFFFAOYSA-N 3-phosphanylpropanoic acid Chemical class OC(=O)CCP QEDXSHCYPROEOK-UHFFFAOYSA-N 0.000 description 1
- 241000255925 Diptera Species 0.000 description 1
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 238000007397 LAMP assay Methods 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108700011259 MicroRNAs Proteins 0.000 description 1
- WHNWPMSKXPGLAX-UHFFFAOYSA-N N-Vinyl-2-pyrrolidone Chemical compound C=CN1CCCC1=O WHNWPMSKXPGLAX-UHFFFAOYSA-N 0.000 description 1
- 101710147059 Nicking endonuclease Proteins 0.000 description 1
- VYLQGYLYRQKMFU-UHFFFAOYSA-N Ochratoxin A Natural products CC1Cc2c(Cl)cc(CNC(Cc3ccccc3)C(=O)O)cc2C(=O)O1 VYLQGYLYRQKMFU-UHFFFAOYSA-N 0.000 description 1
- 229920003081 Povidone K 30 Polymers 0.000 description 1
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
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- 230000005518 electrochemistry Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- FDWREHZXQUYJFJ-UHFFFAOYSA-M gold monochloride Chemical compound [Cl-].[Au+] FDWREHZXQUYJFJ-UHFFFAOYSA-M 0.000 description 1
- 244000052637 human pathogen Species 0.000 description 1
- 238000011901 isothermal amplification Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- RWQKHEORZBHNRI-BMIGLBTASA-N ochratoxin A Chemical compound C([C@H](NC(=O)C1=CC(Cl)=C2C[C@H](OC(=O)C2=C1O)C)C(O)=O)C1=CC=CC=C1 RWQKHEORZBHNRI-BMIGLBTASA-N 0.000 description 1
- DAEYIVCTQUFNTM-UHFFFAOYSA-N ochratoxin B Natural products OC1=C2C(=O)OC(C)CC2=CC=C1C(=O)NC(C(O)=O)CC1=CC=CC=C1 DAEYIVCTQUFNTM-UHFFFAOYSA-N 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- -1 potassium ferricyanide Chemical compound 0.000 description 1
- 239000000276 potassium ferrocyanide Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 239000011435 rock Substances 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
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- 230000005477 standard model Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- XOGGUFAVLNCTRS-UHFFFAOYSA-N tetrapotassium;iron(2+);hexacyanide Chemical compound [K+].[K+].[K+].[K+].[Fe+2].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] XOGGUFAVLNCTRS-UHFFFAOYSA-N 0.000 description 1
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
- 229940038773 trisodium citrate Drugs 0.000 description 1
Abstract
The invention relates to a preparation method and application of an electrochemical transducer with a signal amplification technology. The preparation method comprises the following steps: firstly, preparing a gold-platinum nano particle, fixing thionine as an electrochemical reagent on the surface of the gold-platinum nano particle, fixing probe DNA on the surface of the gold-platinum nano particle so as to obtain an electrochemical probe, and simultaneously, self-assembling a hairpin DNA on the surface of a gold electrode to obtain an electrochemical transducer. Target DNA and the hairpin DNA assembled on the surface of the gold electrode are partially complemented and paired so as to open a hairpin structure when the target DNA is added to the electrochemical transducer, then another hairpin DNA chain replaces a target DNA chain through a competitive effect, and thus the target DNA chain is released; the released target chain unceasingly opens other hairpin structured DNA of the surface of an electrode, thereby realizing the recycling of the target DNA chain under the cyclic action, and the recycling action is combined the prepared electrochemical probe to measure target DNA. The transducer provided by the invention is high in selectivity and detection sensitivity, the gold-platinum nano particle is utilized as a carrier to support thionine to form the probe, and the transducer has the advantage of electrochemical stability.
Description
Technical field
The invention belongs to analytical chemistry and technical field of electrochemistry, relate to a kind of signal amplification technique electrochemical sensor preparation method and application.In addition, the invention still further relates to the method that adopts described electrochemical sensor to detect DNA.
Background technology
Detect particular sequence DNA is very important in clinical diagnosis, gene therapy and other biological medical research.Therefore the method for, setting up the DNA of high sensitivity fast detecting particular sequence is very necessary.
Generally, detecting DNA all hybridizes to realize based on DNA.In order to read hybridization reaction, usual way is that the part complementary series of target dna is carried out to mark.The general part complementary series of the target-marking DNA such as fluorescence, electrochemiluminescence, chemiluminescent substance that adopts makes DNA probe, according to the concentration of the signal strength measuring target dna of DNA probe after hybridization reaction.Yet, because the concentration of target dna in living things system is extremely low, must adopt suitable detection method to improve the sensitivity of sensor.Therefore, some DNA detection methods of amplifying based on signal are developed: enzyme is cut cycle signal amplifying technique [Hun X., Liu F., Mei Z.H., et.al., Signal amplified strategy based on target-induced strand release couplingcleavage of nicking endonuclease for the ultrasensitive detection of ochratoxin A, Biosensors and Bioelectronics, 2013, 39, 145-151], roll ring amplifying technique [Wen Y.Q., Xu Y., Mao X.H., et.al., DNAzyme-Based Rolling-Circle Amplification DNA Machine for Ultrasensitive Analysis of MicroRNA in Drosophila Larva, Anal.Chem.2012, 84, 7664-7669], polymerase chain reaction [Hartman M.R., Yang D.Y., Tran T.N.N., et.al., Thermostable branched DNA nanostructures as modular primers for polymerase chain reaction, Angewandte Chemie International Edition, 2013, 52, 8699 – 8702], ring mediated isothermal amplification [Dhama K., Karthik K., Chakraborty S., et.al., Loop-mediated isothermal amplification of DNA (LAMP): A new diagnostic tool lights the world of diagnosis of animal and human pathogens:A review, Pakistan Journal of Biological Sciences, 2014, 17, 151-166] etc.These methods respectively have its advantage, can meet in various degree the testing requirement to DNA, but sensitivity are not high.So necessary development is a kind of highly sensitive, simple, stable detection method.
Summary of the invention
In view of the deficiencies in the prior art, the object of the present invention is to provide a kind of signal amplification technique electrochemical sensor preparation method and application.And the method that the electrochemical sensor detection DNA described in a kind of employing is provided.
The object of the present invention is achieved like this:
The present invention realizes by following measures: a kind of signal amplification technique electrochemical sensor preparation method, is characterized in that comprising the following steps:
(1) preparation of gold-nano platinum particle;
(2) preparation of electrochemical probe;
(3) preparation of electrochemical sensor;
The preparation of gold-nano platinum particle of the present invention comprises the following steps: preparation and reserve fund sol solution glass container (volumetric flask, brown wide-necked bottle, round-bottomed flask) used are steeped to 30min with chloroazotic acid, then with intermediate water, rinse dry for standby; The HAuCl that adds 50mL1mM in 1000mL round-bottomed flask
4, be heated with stirring to boiling, then add fast the sodium citrate (NaC of 5mL38.8mM
6h
5o
7), when solution becomes claret, in solution, add successively 5mL4.0 * 10
-3molL
-1hPtCl
6, 15mL10gL
-1pVP, continue heating 2h after being heated with stirring to boiling.Solution becomes sepia from claret, makes Au@PtNPs (gold-nano platinum particle), is transferred to brown bottle, and 4 ℃ store for future use.
The preparation of electrochemical probe of the present invention comprises the following steps: first, gets the centrifuge tube of 2mL, adds 10 μ L DNA1 and 10 μ L pH8.2, and the Tris-HCl buffer solution of 50mM, 10 μ L TCEP reaction 1h, in order to activate sulfydryl; Then, separately get 2mL centrifuge tube and add successively 1mL gold-nano platinum particle and 1000 μ L thionine solution reaction 0.5h, thionine is adsorbed on gold-nano platinum particle; Gold-the nano platinum particle that has adsorbed thionine is joined in the sulfydryl DNA1 having activated, put into shaking table jog reaction 16h.Sulfydryl DNA1 and gold-nano platinum particle are coupled together; Finally, under 15000 rotating speeds, centrifugal 30min, obtains red precipitate, and with 10mM, the phosphate buffer solution of pH8.0 washing three times, is dispersed in 10mM, obtains DNA1/Th/Au@PtNPs electrochemical probe in the phosphate buffer solution of pH8.0, and 4 ℃ keep in Dark Place.
The preparation of electrochemical sensor of the present invention comprises the following steps:
1. the pre-service of gold electrode
Gold electrode is through α-A1 of 0.05 μ m
2o
3after burnishing powder polishing, with redistilled water, rinse well, and in ultrasound bath ultrasonic 5min, with high pure nitrogen, dry up; Employing three-electrode system detects, and working electrode is gold electrode, to electrode, is platinum electrode, and contrast electrode is Ag/AgCl electrode, at K
3[Fe (CN)
6] in solution, it is 0～0.8V that voltage is set, and gold electrode is carried out to cyclic voltammetric (CV) scanning; If redox current peak in 0～1000mV, has illustrated that electrode surface is processed good.Otherwise again process, until meet the requirements; After having surveyed, with intermediate water, rinse electrode, dry up electrode surface, standby.
2. golden nanometer particle modified gold electrode preparation
First get 10 μ L AuNPs (diameter 15nm) and be coated in gold electrode surfaces, obtain golden nanometer particle modified gold electrode (AuNPs/GE), be placed in lucifuge place and naturally dry.
3. the pre-service of hairpin dna
Get a certain amount of 10
-6the sulfydryl DNA4 (or DNA3) of M, in 2mL centrifuge tube, is placed in 95 ℃ of constant temperature water baths and heats 5min, is placed in immediately the cooling 1min of frozen water and obtains hairpin dna after taking-up.
4. the preparation of electrochemical sensor
Get 3 of the above-mentioned hairpin dnas of 10 μ L and be coated in AuNPs/GE surface, 4 ℃ of self assembly 24h; Then get 10 μ L10
-4the mercaptoethanol of M drips in electrode surface, and then reaction 30min rinses twice with the phosphate buffer solution of 10mM, obtains electrochemical sensor.
Utilize electrochemical sensor prepared by said method to detect a method for DNA content, comprise the steps:
(1) get again the electrode surface that 10 μ L target dnas 2 are added drop-wise to electrochemical sensor, hatch after 2h at 37 ℃, then get 10 μ L hairpin dnas 4 and be added drop-wise to electrode surface.At 37 ℃, hatch 4h, then use 10mM, the phosphate buffer solution of pH8.0 rinses twice.Finally, get electrochemical probe prepared by 10 μ L and be added drop-wise to electrode surface, continue to hatch 1h.
(2) electrode system is inserted to 0.1M, in the phosphate buffer solution of pH7.4, the above-mentioned gained gold electrode of take is working electrode, and Ag/AgC1 (saturated KCl) electrode is contrast electrode, and platinum electrode is that electrode is surveyed to electric signal (I
p/ A), carry out the mensuration of DNA
Invention has been investigated electrode surface and has been dripped after hairpin dna 4, the impact of reaction time (Time/h) on strength of current.As shown in Figure 2, first oxidation peak current increases along with the increase in reaction time, and when the time reaches 4h, peak current no longer increases, and presents the trend remaining unchanged.The peak optimization reaction time of selecting is 4h.
Also investigated electrochemical probe adsorption time impact on strength of current on electrode, as shown in Figure 3,10, in 60min, peak current increases along with the increase of adsorption time.When adsorption time continues to increase, peak current substantially no longer changes.Therefore, take 60min as optimum adsorption time.
Remarkable result of the present invention
The present invention has studied the relation between variable concentrations DNA2 and electrochemical signals intensity, has obtained detecting typical curve, the range of linearity and the linear equation of DNA2.
Under optimum condition, the concentration of target dna 2 is 3.0 * 10
-14～2.0 * 10
-12within the scope of M, be certain linear relationship with strength of current, its equation of linear regression is I
p=6.6 * 10
5c+1.0 * 10
-8(I
pelectrochemical signals intensity; C is the concentration of DNA2, n=9) (Fig. 4), linearly dependent coefficient R=0.9991, detectability is 0.9 * 10
-15m (3 σ).
By same 5 electrode pair concentration, be 2.0 * 10
-13m object DNA2 carries out respectively 17 error at measurments and is respectively 2.9%, 2.6%, 2.7%, 2.9%, 2.5%, and in mensuration process, electrode surface is all without obscission.Take golden nanometer particle as carrier loaded thionine is as probe is to 2.0 * 10
-13m object DNA2 carries out respectively 17 error at measurments and is respectively 9.3%, 7.9%, 7.7%, 3.2%, 5.7%, and in mensuration process, 4 electrode surfaces have obscission.Show take that gold-nano platinum particle has the feature that stability is high as carrier loaded thionine as probe.
Accompanying drawing explanation
Fig. 1. signal amplification technique is measured object schematic diagram.
Fig. 2. electrode surface drips after hairpin dna 4, the impact of reaction time on strength of current.
Fig. 3. electrochemical probe is the impact of adsorption time on strength of current on electrode.
Fig. 4. strength of current and object concentration relationship figure.
Embodiment
Example below will illustrate method of operating of the present invention, but can not be as limitation of the invention.
Example: signal amplification technique electrochemical sensor detects the method for DNA
1. experimental section
1.1 instruments and reagent
CHI660B electrochemical workstation (Shanghai Chen Hua instrument company); Anke-TGL-16C flies father-in-law's board supercentrifuge (Shanghai City An Ting scientific instrument factory); PHS-3D type acidometer (Shanghai Lei Ci instrument plant); Adopt three-electrode system: gold electrode and modified gold electrode are working electrode, and Ag/AgC1 (saturated KCl) electrode is contrast electrode, and platinum electrode is to electrode.
Na
2hPO
412H
2o, Tianjin red rock chemical reagent factory; Three (2-carboxyethyl) phosphonium salt hydrochlorate (TCEP, tris (2-carboxyethyl) phosphine hydrochloride), gold chloride (HAuCl
4), trisodium citrate (Na
3c
6h
5o
7) be all purchased from Tianjin Bo Di Chemical Co., Ltd.; The potassium ferricyanide: K
3[Fe (CN)
6], Tianjin Bo Di Chemical Co., Ltd.; Potassium ferrocyanide: K
4[Fe (CN)
6] 3H
2o, Tianjin Guang Cheng chemical reagent company limited; Alumina powder: α-A1
2o
3, thionine (Th) is purchased from Shanghai Aladdin reagent company; Polyvinylpyrrolidone (PVP K30) Tianjin Guang Cheng chemical reagent company limited.
DNA artificial sequence synthetic used (match Parkson, Beijing bioengineering company limited buys) is as follows.
Preferred DNA1 partial sequence is: 5`-TTT TTT ATT CGA TCC GGT GCT CTT ATG CCC-HS-3`
Preferred DNA2 partial sequence is: 5 '-CAA TAA CTA CCG GGC ATT ACT GGC CTT-3 '
Preferred DNA3 partial sequence is: 5 '-SH-C
6-AAA GCC AGT AAT GCC CGG TAG TTA TTC CAT CGT GTA CAA TAA CTA CCG GGC ATA AGA GCA CCC TTG TAC-3 '
Preferred DNA4 partial sequence is: 5 '-TAG TTA TTG TAC ACG ATG GAA TAA CTA CCG GGC ATC CAT CGT GTA C-3 '.
1.2 experimental procedure
1.2.1 the preparation of gold-nano platinum particle
Preparation and reserve fund sol solution glass container (volumetric flask, brown wide-necked bottle, round-bottomed flask) used are steeped to 30min with chloroazotic acid, then with intermediate water, rinse dry for standby.The HAuCl that adds 50mL1mM in 1000mL round-bottomed flask
4, be heated with stirring to boiling, then add fast the sodium citrate (NaC of 5mL38.8mM
6h
5o
7), when solution becomes claret, in solution, add successively 5mL4.0 * 10
-3molL-
1hPtCl
6, 15mL10gL
-1pVP, continue heating 2h after being heated with stirring to boiling.Solution becomes sepia from claret, makes Au@PtNPs, is transferred to brown bottle, and 4 ℃ store for future use.
1.2.2 the preparation of electrochemical probe
First, get the centrifuge tube of 2mL, add 10 μ L10
-5the DNA1 of M and 10 μ L pH8.2, the Tris-HCl buffer solution of 50mM, the TCEP reaction 1h of 10 μ L10mM, in order to activate sulfydryl.Then, separately get 2mL centrifuge tube and add successively 1mL Au@PtNPs and 1000 μ L10
-4the thionine of M, solution reaction 0.5h, is adsorbed on gold-nano platinum particle thionine.The Au@PtNPs that has adsorbed thionine is joined in the sulfydryl DNA1 having activated, put into shaking table jog reaction 16h.Sulfydryl DNA1 and Au@PtNPs are coupled together.Finally, under 15000 rotating speeds, centrifugal 30min, obtains red precipitate, and with 10mM, the phosphate buffer solution of pH8.0 washing three times, is dispersed in 10mM, obtains DNA1/Th/Au@PtNPs electrochemical probe in the phosphate buffer solution of pH8.0, and 4 ℃ keep in Dark Place.
1.2.3 the preparation of electrochemical sensor
The pre-service of gold electrode
Gold electrode is through α-A1 of 0.05 μ m
2o
3after burnishing powder polishing, with redistilled water, rinse well, and in ultrasound bath ultrasonic 5min, with high pure nitrogen, dry up.Employing three-electrode system detects, and working electrode is gold electrode, to electrode, is platinum electrode, and contrast electrode is Ag/AgCl electrode, at K
3[Fe (CN)
6] in solution, it is 0～0.8V that voltage is set, and gold electrode is carried out to cyclic voltammetric (CV) scanning.If redox current peak in 0～1000mV, has illustrated that electrode surface is processed good.Otherwise again process, until meet the requirements.After having surveyed, with intermediate water, rinse electrode, dry up electrode surface, standby.First get AuNPs prepared by 10 μ L and drip and be coated in gold electrode surfaces, obtain golden nanometer particle modified gold electrode (AuNPs/GE), be placed in lucifuge place and naturally dry.
The pre-service of hairpin dna
Get a certain amount of 10
-6the sulfydryl DNA4 (or DNA3) of M, in 2mL centrifuge tube, is placed in 95 ℃ of constant temperature water baths and heats 5min, is placed in immediately the cooling 1min of frozen water and obtains hairpin dna after taking-up.
The preparation of electrochemical sensor
Get 3 of the above-mentioned hairpin dnas of 10 μ L and be coated in AuNPs/GE surface, 4 ℃ of self assembly 24h, then get 10 μ L10
-4the mercaptoethanol sealing of M, the phosphate buffer solution with 10mM after electrode is dry rinses twice, obtains electrochemical sensor.
Electrochemical gaging target dna
Design the target dna 2 of one group of concentration gradient, be added drop-wise to respectively on electrochemical sensor, hatch after 2h at 37 ℃, get 10 μ L hairpin dnas 4 and be added drop-wise to electrode surface, hatch 4h at 37 ℃, then use 10mM, the phosphate buffer solution of pH8.0 rinses twice.Finally, get electrochemical probe prepared by 10 μ L and be added drop-wise to electrode surface, continue to hatch 1h.Then utilize cyclic voltammetry or DPV to record strength of current, according to target dna 2 concentration and the strength of current relation typical curve of mapping to obtain, and calculate equation of linear regression according to typical curve.
To be added on electrochemical sensor containing the sample drop of target dna 2, hatch after 2h at 37 ℃, get 10 μ L hairpin dnas 4 and be added drop-wise to electrode surface, hatch 4h at 37 ℃, then use 10mM, the phosphate buffer solution of pH8.0 rinses twice.Finally, get electrochemical probe prepared by 10 μ L and be added drop-wise to electrode surface, continue to hatch 1h.Then utilize cyclic voltammetry or DPV to record strength of current, according to strength of current, target dna 2 content in recycling equation of linear regression calculation sample.
And adopt standard addition method to evaluate method, and the sample determination recovery is 97.0-106.0%, and measurement result is in Table 1, and method of the present invention has the advantages that precision is high in target dna detects.
The present invention is the model electrochemical sensor that the mechanism that recycles of based target DNA has been developed a kind of highly sensitive detection DNA.The sensor of development has following advantage: gold-nano platinum particle is that carrier loaded thionine is that probe has overcome probe and easily comes off and the unsettled defect of electrochemical signals; The circulation of target dna is amplified signal, and the electrochemical sensor of design has high stability, high sensitivity feature.Therefore, designed electrochemical sensor embodies good development prospect in building the research method that detects DNA.
Table 1. is containing target dna 2 sample analysis measurement results
| Numbering | Content a,b | Standard model addition | The amount of recording |
| 1 | 7.6 | 5.0 | 12.5 |
| 2 | 9.2 | 10.0 | 19.8 |
| 3 | 12.4 | 10.0 | 22.7 |
| 4 | 15.6 | 15.0 | 30.2 |
| 5 | 23.9 | 25.0 | 50.6 |
| 6 | 49.0 | 50.0 | 98.2 |
a7 measurement results
bunit: 10
-14m
[0001] sequence table
SEQUENCE LISTING
<110> Qingdao University of Science and Technology
<120> signal amplification technique electrochemical sensor detects the method for DNA
<160>4
<170>PatentIn version3.3
<210>1
<211>39
<212>DNA
<213> is series manually
<400>1
ttttttattc gatccggtgc tcttatgccc 39
<210>2
<211>27
<212>DNA
<213> is series manually
<400>2
caataactac cgggcattac tggcctt 27
<210>3
<211>69
<212>DNA
<213> is series manually
<400>3
aaagccagta atgcccggta gttattccat cgtgtacaat aactaccggg cataagagca cccttgtac 69
<210>4
<211>46
<212>DNA
<213> is series manually
<400>4
tagttattgt acacgatgga ataactaccg ggcatccatc gtgtac 46
Claims (3)
1. signal amplification technique electrochemical sensor detects a method of DNA, comprises the following steps:
(1) electrochemical probe preparation method is characterized in that:
(a) get the centrifuge tube of 2mL, add Tris-HCl buffer solution and the 10 μ L TCEP reaction 1h of the 50mM of 2～30 μ L DNA1 and 2～30 μ L pH8.2, in order to activate sulfydryl; Then, separately get 2mL centrifuge tube and add successively 1mL gold-nano platinum particle and 100～2000 μ L thionine solution reaction 0.5h, thionine is adsorbed on gold-nano platinum particle;
(b) gold-nano platinum particle that has adsorbed thionine is joined in the sulfydryl DNA1 having activated, put into shaking table jog reaction 16h, sulfydryl DNA1 and gold-nano platinum particle are coupled together; Finally, under 15000 rotating speeds, centrifugal 30min, obtains red precipitate, and with 10mM, and the phosphate buffer solution of pH8.0 washing three times is dispersed in the phosphate buffer solution of pH8.0 of 1mL10mM and obtains DNA1/Th/Au@PtNPs electrochemical probe;
(2) pre-service of gold electrode: gold electrode is through 0.2 μ m, 0.03 μ m, α-A1 of 0.05 μ m
2o
3after burnishing powder polishing, with redistilled water, rinse well, and in ultrasound bath ultrasonic 5min, with high pure nitrogen, dry up standby;
(3) golden nanometer particle modified gold electrode preparation: first get 2～30 μ L AuNPs solution and drip and be coated in gold electrode surfaces, obtain golden nanometer particle modified gold electrode (AuNPs/GE), be placed in lucifuge place and naturally dry;
(4) pre-service of hairpin dna: get a certain amount of 1.0 * 10
-4m～1.0 * 10
-7the sulfydryl DNA4 (or DNA3) of M, in 2mL centrifuge tube, is placed in 95 ℃ of constant temperature water baths and heats 5min, is placed in immediately the cooling 1min of frozen water and obtains hairpin dna after taking-up;
(5) preparation of electrochemical sensor: get 3 of the above-mentioned pretreated hairpin dnas of 2～30 μ L and be coated in AuNPs/GE surface, 4 ℃ of self assembly 24h; Then get 10 μ L10
-4the mercaptoethanol of M drips in electrode surface, and then reaction 30min rinses twice with the phosphate buffer solution of 10mM, obtains electrochemical sensor;
(6) drafting of typical curve: get the target dna 2 of one group of concentration gradient, be added drop-wise to respectively on electrochemical sensor, hatch after 2h at 37 ℃, get 10 μ L hairpin dnas 4 and be added drop-wise to electrode surface, at 37 ℃, hatch 4h, then use 10mM, the phosphate buffer solution of pH8.0 rinses twice; Finally, get electrochemical probe prepared by 10 μ L and be added drop-wise to electrode surface, continue to hatch 1h; Then utilize cyclic voltammetry or DPV to record strength of current, according to the relation of object concentration and the strength of current typical curve of mapping to obtain, and calculate equation of linear regression according to typical curve;
(7) sample determination: will be added on electrochemical sensor containing the sample drop of target dna 2, hatch after 2h at 37 ℃, get 10 μ L hairpin dnas 4 and be added drop-wise to electrode surface, hatch 4h at 37 ℃, then use 10mM, the phosphate buffer solution of pH8.0 rinses twice; Finally, get electrochemical probe prepared by 10 μ L and be added drop-wise to electrode surface, continue to hatch 1h; Then utilize cyclic voltammetry or DPV to record strength of current, according to strength of current, target dna 2 content in recycling equation of linear regression calculation sample.
2. according to the method for the mensuration target dna of claim 1, it is characterized in that described DNA sequence dna is as follows:
The partial sequence of described DNA1 is: 5`-TTT TTT ATT CGA TCC GGT GCT CTT ATGCCC-HS-3`;
The partial sequence of described DNA2 is: 5 '-CAA TAA CTA CCG GGC ATT ACT GGC CTT-3 ';
The partial sequence of described DNA3 is: 5 '-SH-C
6-AAA GCC AGT AAT GCC CGG TAG TTA TTCCAT CGT GTA CAA TA A CTA CCG GGC ATA AGA GCA CCC TTG TAC-3 ';
The partial sequence of described DNA4 is: 5 '-TAG TTA TTG TAC ACG ATG GAA TAA CTA CCG GGCATC CAT CGT GTA C-3 ';
3. the application of electrochemical sensor according to claim 1 in detecting DNA content.
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