CN105044273A - Method for detecting dopamine on basis of nanoparticle label oxidation-reduction cycle - Google Patents
Method for detecting dopamine on basis of nanoparticle label oxidation-reduction cycle Download PDFInfo
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- VYFYYTLLBUKUHU-UHFFFAOYSA-N dopamine Chemical compound NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 title claims abstract description 136
- 229960003638 dopamine Drugs 0.000 title claims abstract description 68
- 239000002105 nanoparticle Substances 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 title claims abstract description 24
- 230000033116 oxidation-reduction process Effects 0.000 title abstract 2
- 239000002131 composite material Substances 0.000 claims abstract description 29
- 239000000523 sample Substances 0.000 claims abstract description 26
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 25
- 229910021389 graphene Inorganic materials 0.000 claims abstract description 24
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 16
- 239000010931 gold Substances 0.000 claims abstract description 16
- 229910052737 gold Inorganic materials 0.000 claims abstract description 16
- 108020004414 DNA Proteins 0.000 claims abstract description 13
- 102000053602 DNA Human genes 0.000 claims abstract description 11
- 238000001514 detection method Methods 0.000 claims abstract description 9
- 230000000694 effects Effects 0.000 claims abstract description 6
- 239000012488 sample solution Substances 0.000 claims abstract description 6
- 230000000295 complement effect Effects 0.000 claims abstract description 4
- 108020004682 Single-Stranded DNA Proteins 0.000 claims abstract description 3
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 claims description 57
- 239000000243 solution Substances 0.000 claims description 47
- 239000002245 particle Substances 0.000 claims description 29
- 229910052697 platinum Inorganic materials 0.000 claims description 28
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- 238000002360 preparation method Methods 0.000 claims description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 230000008836 DNA modification Effects 0.000 claims description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims description 6
- 239000008367 deionised water Substances 0.000 claims description 6
- 229910021641 deionized water Inorganic materials 0.000 claims description 6
- 238000010438 heat treatment Methods 0.000 claims description 6
- 230000004044 response Effects 0.000 claims description 6
- NGNBDVOYPDDBFK-UHFFFAOYSA-N 2-[2,4-di(pentan-2-yl)phenoxy]acetyl chloride Chemical compound CCCC(C)C1=CC=C(OCC(Cl)=O)C(C(C)CCC)=C1 NGNBDVOYPDDBFK-UHFFFAOYSA-N 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 4
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 4
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- SJUCACGNNJFHLB-UHFFFAOYSA-N O=C1N[ClH](=O)NC2=C1NC(=O)N2 Chemical compound O=C1N[ClH](=O)NC2=C1NC(=O)N2 SJUCACGNNJFHLB-UHFFFAOYSA-N 0.000 claims description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 4
- 238000009835 boiling Methods 0.000 claims description 4
- 239000011734 sodium Substances 0.000 claims description 4
- 239000001509 sodium citrate Substances 0.000 claims description 4
- 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 claims description 4
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 4
- 239000001117 sulphuric acid Substances 0.000 claims description 4
- 235000011149 sulphuric acid Nutrition 0.000 claims description 4
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 4
- 239000012498 ultrapure water Substances 0.000 claims description 4
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 claims description 3
- PZBFGYYEXUXCOF-UHFFFAOYSA-N TCEP Chemical compound OC(=O)CCP(CCC(O)=O)CCC(O)=O PZBFGYYEXUXCOF-UHFFFAOYSA-N 0.000 claims description 3
- 230000003197 catalytic effect Effects 0.000 claims description 3
- GPRSOIDYHMXAGW-UHFFFAOYSA-N cyclopenta-1,3-diene cyclopentanecarboxylic acid iron Chemical compound [CH-]1[CH-][CH-][C-]([CH-]1)C(=O)O.[CH-]1C=CC=C1.[Fe] GPRSOIDYHMXAGW-UHFFFAOYSA-N 0.000 claims description 3
- 239000008363 phosphate buffer Substances 0.000 claims description 3
- 239000012286 potassium permanganate Substances 0.000 claims description 3
- 238000004064 recycling Methods 0.000 claims description 3
- 230000009870 specific binding Effects 0.000 claims description 3
- 238000013019 agitation Methods 0.000 claims description 2
- 235000010323 ascorbic acid Nutrition 0.000 claims description 2
- 229960005070 ascorbic acid Drugs 0.000 claims description 2
- 239000011668 ascorbic acid Substances 0.000 claims description 2
- 238000001816 cooling Methods 0.000 claims description 2
- 238000000502 dialysis Methods 0.000 claims description 2
- 238000004090 dissolution Methods 0.000 claims description 2
- 238000001704 evaporation Methods 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims description 2
- 239000008187 granular material Substances 0.000 claims description 2
- 238000000227 grinding Methods 0.000 claims description 2
- 239000005457 ice water Substances 0.000 claims description 2
- 238000009830 intercalation Methods 0.000 claims description 2
- 230000002687 intercalation Effects 0.000 claims description 2
- 238000003760 magnetic stirring Methods 0.000 claims description 2
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- 239000004317 sodium nitrate Substances 0.000 claims description 2
- 239000007787 solid Substances 0.000 claims description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 claims 1
- 239000012467 final product Substances 0.000 claims 1
- JUWSSMXCCAMYGX-UHFFFAOYSA-N gold platinum Chemical compound [Pt].[Au] JUWSSMXCCAMYGX-UHFFFAOYSA-N 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 150000002343 gold Chemical class 0.000 abstract description 3
- 230000008901 benefit Effects 0.000 abstract description 2
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- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 3
- 230000008859 change Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- QEDXSHCYPROEOK-UHFFFAOYSA-N 3-phosphanylpropanoic acid Chemical class OC(=O)CCP QEDXSHCYPROEOK-UHFFFAOYSA-N 0.000 description 1
- RDIMQHBOTMWMJA-UHFFFAOYSA-N 4-amino-3-hydrazinyl-1h-1,2,4-triazole-5-thione Chemical compound NNC1=NNC(=S)N1N RDIMQHBOTMWMJA-UHFFFAOYSA-N 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 239000004593 Epoxy Chemical group 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
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- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 1
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- 239000010452 phosphate Substances 0.000 description 1
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- KKEYFWRCBNTPAC-UHFFFAOYSA-L terephthalate(2-) Chemical compound [O-]C(=O)C1=CC=C(C([O-])=O)C=C1 KKEYFWRCBNTPAC-UHFFFAOYSA-L 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the field of electrochemical sensors and relates to a method for detecting dopamine on the basis of a nanoparticle label oxidation-reduction cycle. According to the method, gold platinum composite nanoparticles are connected to dopamine aptamer complementary sequence single-stranded DNA to form a probe, the dopamine aptamer is added into the probe, so that double-stranded DNA modified gold platinum composite nanoparticles are obtained, when a dopamine-containing sample solution is added into the double-stranded DNA modified gold platinum composite nanoparticles, dopamine is specifically bonded with the dopamine aptamer and the probe is released; the released probe is captured by gold nanoparticles and a graphene oxide modified gold electrode, and the catalysis effect of gold platinum composite nanoparticles is used to generate an electrochemical signal to realize detection of dopamine. The method provided by the invention has the advantages of being simple and high in sensitivity.
Description
Technical field
The invention belongs to electrochemical sensor field, be specifically related to a kind of method detecting dopamine based on nanometer particle to mark redox cycle.
Background technology
Dopamine is the emotion information transmission of being secreted primary responsibility brain by hypothalamus, and in medical science, dopamine is usually used in Cure of depression.In human body, DOPAMINE CONTENT IN RABBIT is not enough, and body will the instinct of muscle out of hand, seriously lacks and can cause senile dementia, schizophrenia, parkinson's syndrome etc.The detection of dopamine adopts liquid chromatograph analysis, Fluorometric assay of fluorescence-labeled more, high performance liquid chromatography-mass spectrometry, the analytical approachs such as gas chromatography-mass spectrometry.Because these methods need large-scale instrument, there is the shortcomings such as operation cost is expensive, fast and convenient testing requirement can not be met, need to set up easy, to detect dopamine fast new method.The people such as Feng utilize 4-amino-3-diazanyl-5-sulfydryl-1, 2, 4-triazole (AHMT) links with golden nanometer particle (AuNPs) and makes probe (AHMT-AuNPs), utilizing dopamine and AHMT to form hydrogen bond impels AHMT-AuNPs probe to assemble, the color of colloidal solution is caused to change the mensuration (FengJJ achieved dopamine, GuoH, LiYF, etal.Singlemolecularfunctionalizedgoldnanoparticlesforhy drogen-bondingrecognitionandcolorimetricdetectionofdopam inewithhighsensitivityandselectivity [J] .ACSappliedmaterials & interfaces, 2013, 5 (4): 1226-1231).Zhao etc. arrive electrode surface by carbon nano tube modified, achieve dopamine and the ascorbic (ZhaoJ of detection simultaneously, ZhangW, SherrellP, etal.Carbonnanotubenanoweb – bioelectrodeforhighlyselectivedopaminesensing [J] .ACSappliedmaterials & interfaces, 2011,4 (1): 44-48).Detection (the HsuMS that plastic film covering carries out dopamine on electrode made by the terephthalate polyester that nanowires of gold is modified softness by the people such as Hsu, ChenYL, LeeCY, etal.Goldnanostructuresonflexiblesubstratesaselectrochem icaldopaminesensors [J] .ACSappliedmaterials & interfaces, 2012,4 (10): 5570-5575).Huang etc. Au-Pt Nanoparticle Modified to carbon nano-fiber will detect the content (HuangY of dopamine, MiaoYE, JiS, etal.ElectrospuncarbonnanofibersdecoratedwithAg – Ptbimetallicnanoparticlesforselectivedetectionofdopamine [J] .ACSappliedmaterials & interfaces, 2014,6 (15): 12449-12456).Graphene oxide because of containing functional groups such as carboxyl, hydroxyl, epoxy radicals, and has the features such as dirigibility, printing opacity are strong, suitable extensive preparation, is widely used in recent years in biology sensor.Borini group utilizes the characteristic of graphene oxide to prepare to can be used for the biology sensor (BoriniS detecting humidity and temperature, WhiteR, WeiD, etal.Ultrafastgrapheneoxidehumiditysensors [J] .ACSnano, 2013,7 (12): 11166-11173).The sensor that Li group then utilizes AC dielectric electrophoresis to make based on graphene oxide can highly sensitive detection gas containing nitrogen oxide (LiW, GengX, GuoY, etal.Reducedgrapheneoxideelectricallycontactedgraphenese nsorforhighlysensitivenitricoxidedetection [J] .ACSnano, 2011,5 (9): 6955-6961).In the present invention, first, dopamine is fit complementary series and the effect of golden platinum composite nanoparticle form probe, and it is fit then to add dopamine, forms the golden platinum composite nanoparticle that double-stranded DNA is modified.When adding containing dopamine sample solution in the golden platinum composite nanoparticle solution modified at double-stranded DNA, dopamine and the fit specific binding of dopamine, probe is released.Then, the gold electrode utilizing golden nanometer particle and graphene oxide to modify catches the probe released, and the catalytic action recycling golden platinum composite nanoparticle produces the detection that electrochemical signals realizes dopamine.
Summary of the invention
The present invention is intended to the method for inventing the mensuration dopamine that a kind of method is simple, cost is low, highly sensitive, selectivity is high.
Realizing goal of the invention technical scheme is:
A kind of method detecting dopamine based on nanometer particle to mark redox cycle.Its principle utilizes the fit complementary series single stranded DNA of golden platinum composite nano-granule sub-connection dopamine as probe.In above-mentioned probe, add dopamine more fit, form the golden platinum composite nanoparticle that double-stranded DNA is modified.When adding the sample solution containing dopamine in the golden platinum composite nanoparticle solution modified at double-stranded DNA, dopamine and the fit specific binding of dopamine, probe is released.Then, the gold electrode utilizing golden nanometer particle and graphene oxide to modify catches the probe released, and the catalytic action recycling golden platinum composite nanoparticle produces the detection that electrochemical signals realizes dopamine.
Determination step is:
(1) preparation of nano particle
First, by platinum acid chloride solution and sodium citrate solution 0.22 μm of micro-pore-film filtration.
The preparation of golden nanometer particle: by massfraction be 1.0% the sodium citrate solution 1.0mL concentration that joins boiling be fast in the chlorauric acid solution of the 100.0mL of 0.3mmol/L, 10min is stirred under solution keeps fluidized state, when solution becomes claret, stop heating, be cooled to room temperature, obtain golden nanometer particle.
The preparation of gold platinum composite nanoparticle: first, be the chlorauric acid solution of 1.0mL of 0.03mol/L by concentration, the solution of gold nanoparticles of the 10mL of the PVP of 0.1g and preparation is placed in the beaker being contained with 50mL water, and mix, then transferred in a 250mL flask and heated.Mixed solution is heated to boiling and after keeping fluidized state 10min, the platinum acid chloride solution that 7.0mL massfraction is 1.0% is added while agitating heating, slowly add the ascorbic acid solution of certain volume subsequently again, solution becomes dark brown rear stopping heating by the time.And then under Keep agitation, solution is cooled to room temperature, obtain golden platinum composite nanoparticle.Golden platinum composite nanoparticle prepared by 1mL centrifugal 10min under the condition of 14000 revs/min, and wash three times with phosphate buffer solution, and be scattered in phosphate buffer solution.
(2) preparation of graphene oxide
First, get massfraction be 98% concentrated sulphuric acid 11.5mL be placed in conical flask, cool 1h under being then put in the condition of 4 DEG C.The conical flask containing the cooling concentrated sulphuric acid processed is put in ice-water bath, under magnetic stirring, adds the potpourri containing 0.5g dag and 0.25g sodium nitrate that milled processed is crossed fast.After reaction 5min, divide 6 times and add in solution by the potassium permanganate amounting to 1.5g, in control temperature of reaction under the condition of 30-45 DEG C, stirring reaction 2.5h, solution becomes blackish green.Then temperature is controlled between 80-100 DEG C, then add 5mL deionized water with tap funnel, then continue reaction 30min.Slowly add with the 10mL hydrogen peroxide that massfraction is 5% by tap funnel subsequently, continue to stir 1h, solution becomes khaki gradually, again solution is transferred in evaporating dish and dry in an oven, and then with the dissolution of solid of deionized water by gained, obtained graphene oxide is put in dialysis membrane and dialyses, until graphene oxide solution is neutrality, and dry.
(3) preparation of DNA modification nano particle
By the TCEP of the 10mol/L of the Tris-HCl of the 50mmol/L of the pH8.2 of 20 μ L, 10 μ L, 1.0 × 10 of 10 μ L
-4the mercaptoethylmaine of mol/L and 1.0 × 10 of 20 μ L
-6the DNA1 of mol/L joins in the centrifuge tube of 2mL, uses Vib. that the solution of gained is fully mixed, is then put in shaking table, react one hour under 37 DEG C of conditions.Then add washed golden platinum composite nanoparticle, and on shaking table, hatch 16h under 37 DEG C of conditions.Subsequently, solution centrifugal is also rinsed three times with the 0.1mol/L phosphate buffer solution of the 1.0mL of pH7.4, obtains DNA1 and modify golden platinum composite nanoparticle probe.
Then 1.0 × 10 are added
-6mol/LDNA2, centrifuging hatch 2h on shaking table under 37 DEG C of conditions after, and rinse three times with the 0.1mol/L phosphate buffer solution of the 1.0mL of pH7.4, and be scattered in 1.0mL phosphate buffer solution, obtain DNA modification nano particle, store under 4 DEG C of conditions.
(4) mensuration is analyzed
Electrochemical analysis measuring principle used in the present invention is as shown in Fig. 1.
First, by gold electrode surfaces through grinding process, drip golden nanometer particle, the 10 μ L graphene oxides of upper 10 μ L successively, the gold electrode that obtained golden nanometer particle and graphene oxide are modified.
The sample solution that 100 μ L contain dopamine is added in the DNA modification nano-particle solution of 100 μ L, after reacting 0.5h under 37 DEG C of conditions, centrifuging, and on shaking table, hatch 16h with under 37 DEG C of conditions, and be scattered in 50 μ L phosphate buffer solutions, obtain the golden platinum composite nanoparticle probe after dopamine effect.After the gold electrode intercalation reaction 0.5h subsequently golden nanometer particle and graphene oxide modified, electrode surface is rinsed three times with the 0.1mol/L phosphate buffer solution of the 1.0mL of pH7.4, using the electrode of gained as working electrode, be inserted into 2mL and contain 0.1 ~ 20mM p-nitrophenol, 0.1 ~ 20NaBH
4with in the phosphate buffer solution of the pH7.4 of the ferrocenecarboxylic acid of 0.1 ~ 20mM, three-electrode system is adopted to measure electrochemical signals.According to the mensuration of the relational implementation dopamine of the content of electrochemical response signal and dopamine.
Described potassium permanganate, p-nitrophenol is purchased from seamount Pu Chemical Co., Ltd..
Described mercaptoethylmaine, mercaptoethanol, gold chloride HAuCl
43H
2o, chloroplatinic acid H
2ptCl
66H
2o is all purchased from Shanghai Pu Guang reagent company limited.
Described three (2-carboxyethyl) phosphonium salt hydrochlorate TCEP is purchased from Tianjin red rock chemical reagent factory.
Described citrate three sodium Na
3c
6h
5o
7, ferrocenecarboxylic acid FCA is purchased from Chemical Reagent Co., Ltd., Sinopharm Group.
Described dag, dopamine D A buys in Shanghai Aladdin Reagent Company.
The reagent of all uses is all chemical pure, and secondary deionized water ion configuration all used by all solvents.
By the KH of 21mL0.2mol/L
2pO
4, 78mL0.2mol/L Na
2hPO
412H
2o mixes, and obtains the phosphate buffered solution that 0.1mol/LpH is 7.4.
The Tris-HCl collocation method of the 50mmol/L of pH8.2 takes 2.423g trishydroxymethylaminomethane, after dissolving, adjusts PH to 8.2, be finally diluted to 1000mL with ultrapure water with 0.1M hydrochloric acid with 500mL ultrapure water.
DNA obtains from match Parkson, Beijing gene technology company limited.Their nucleotide sequence is as follows:
DNA15’-GTGTTCTCTGGCGCACACAGAGACACAGAATGAGGCCC-(CH
2)
6-SH-3’
DNA25’-GTCTCTGTGTGCGCCAGAGAACACTGGGGCAGATATGGGCCAGCACAGAATGAGGCCC-3’
Electrochemical Detection adopts CHI820C electrochemical workstation (Shanghai Chen Hua instrument company).
TGL-16G type hydro-extractor (Town in Shanghai booth scientific instrument company limited) is for centrifuging.
Accompanying drawing explanation
Fig. 1 measures DA principle schematic based on nanometer particle to mark redox cycle.
The action time of Fig. 2 probe and dopamine is on the impact of electrochemical response signal.
The incubation time of Fig. 3 electrode is on the impact of electrochemical signals.
The advantage of invention and effect
Under the testing conditions of the best, the size of peak current and the concentration of object dopamine linear, and the intensity of peak current increases along with the increase of object dopamine concentration, illustrates and can pass through the content that proposed method detects dopamine.Along with the concentration of object dopamine is from 1.0 × 10
-9to 1.0 × 10
-6mol/L increases, and peak current increases.Equation of linear regression is Ip=0.04347C+0.1289, R is 0.9983 (C, nmol/L; Ip, μ A), detectability is 5.0 × 10
-10mol/L.
Embodiment
Further illustrate the present invention below in conjunction with specific embodiment, but do not form the further restriction to invention.
The action time of embodiment 1 probe and dopamine is on the impact of electrochemical signals
Experiment finds that the action time that probe and dopamine are fit wants suitable, and action time is not more to grow or more short better.As shown in Figure 2, wherein a-f represents 0.5 hour respectively, 1 hour, 1.5 hours, 2.0 hours, 2.5 hours and 3.0 hours electrochemical signals curves, as can be seen from the figure, when action time is 2.5h, electrochemical signals reaches higher value, then it is not obvious to increase electrochemical signals change action time, therefore chooses probe and dopamine fit action time is 2.5h.
The incubation time of embodiment 2 electrode is on the impact of electrochemical response signal
The size of golden platinum composite nanoparticle probe incubation time on electrochemical signals after the gold electrode that golden nanometer particle and graphene oxide are modified and dopamine effect has important impact.As shown in Figure 3, electrochemical signals curve when a-h represents that incubation time is 0.5h, 1.0h, 1.5h, 2.0h, 2.5h, 3.0h, 3.5h and 4.0h respectively.As can be seen from the figure, along with the time increases from 0.5h to 3.0h, response signal can strengthen gradually.But when incubation time continues to increase from 3h, response signal reduces on the contrary, so take the incubation time of 3.0h.
The remolding sensitivity of embodiment 3 method comparatively
With the gold electrode that graphene oxide modified gold electrode replaces golden nanometer particle and graphene oxide to modify, by above-mentioned (4), the step measured is analyzed to dopamine dopamine is detected.Obtaining the range of linearity is 7.0 × 10
-8mol/L ~ 1.0 × 10
-6mol/L, detects and is limited to 3.0 × 10
-8mol/L.Using detectability as comparing, the sensitivity of the gold electrode that golden nanometer particle and graphene oxide are modified is 60 times of graphene oxide modified gold electrode, under illustrating that golden nanometer particle exists, drastically increase the sensitivity of detection, to the detection of dopamine, there is obvious humidification.
Claims (7)
1. one kind is detected the method for dopamine based on nanometer particle to mark redox cycle, it is characterized in that utilizing the fit complementary series single stranded DNA of golden platinum composite nano-granule sub-connection dopamine as probe, add dopamine more within the probe fit, form the golden platinum composite nanoparticle that double-stranded DNA is modified, when adding containing dopamine sample solution in the golden platinum composite nanoparticle solution modified at double-stranded DNA, dopamine and the fit specific binding of dopamine, probe is released; Then, the gold electrode utilizing golden nanometer particle and graphene oxide to modify catches the probe released, and the catalytic action recycling golden platinum composite nanoparticle produces the detection that electrochemical signals realizes dopamine, and determination step is as follows:
(1) preparation of nano particle:
A, by platinum acid chloride solution and sodium citrate solution 0.22 μm of micro-pore-film filtration;
The preparation of b, golden nanometer particle: by massfraction be 1.0% the sodium citrate solution 1.0mL concentration that joins boiling be fast in the chlorauric acid solution of the 100.0mL of 0.3mmol/L, 10min is stirred under solution keeps fluidized state, when solution becomes claret, stop heating, be cooled to room temperature, obtain golden nanometer particle;
The preparation of c, golden platinum composite nanoparticle: first, be the chlorauric acid solution of 1.0mL of 0.03mol/L by concentration, the solution of gold nanoparticles of the 10mL of the PVP of 0.1g and preparation is placed in the beaker being contained with 50mL water, and mix, then transferred in a 250mL flask and heated; Mixed solution is heated to boiling and after keeping fluidized state 10min, the platinum acid chloride solution that 7.0mL massfraction is 1.0% is added while agitating heating, slowly add the ascorbic acid solution of certain volume subsequently again, solution becomes dark brown rear stopping heating by the time; And then under Keep agitation, solution is cooled to room temperature, obtain golden platinum composite nanoparticle; Golden platinum composite nanoparticle prepared by 1mL centrifugal 10min under the condition of 14000 revs/min, and wash three times with phosphate buffer solution, and be scattered in phosphate buffer solution;
(2) preparation of graphene oxide: first, get massfraction be 98% concentrated sulphuric acid 11.5mL be placed in conical flask, cool 1h under being then put in the condition of 4 DEG C; The conical flask containing the cooling concentrated sulphuric acid processed is put in ice-water bath, under magnetic stirring, adds the potpourri containing 0.5g dag and 0.25g sodium nitrate that milled processed is crossed fast; After reaction 5min, divide 6 times and the potassium permanganate amounting to 1.5g is added in solution, in control temperature of reaction under the condition of 30-45 DEG C, stirring reaction 2.5h, solution becomes blackish green; Then temperature is controlled between 80-100 DEG C, then add 5mL deionized water with tap funnel, then continue reaction 30min; Slowly add with the 10mL hydrogen peroxide that massfraction is 5% by tap funnel subsequently, continue to stir 1h, solution becomes khaki gradually, again solution is transferred in evaporating dish and dry in an oven, and then with the dissolution of solid of deionized water by gained, obtained graphene oxide is put in dialysis membrane and dialyses, until graphene oxide solution is neutrality, and dry;
(3) preparation of DNA modification nano particle: by the TCEP of the 10mol/L of the Tris-HCl of the 50mmol/L of the pH8.2 of 20 μ L, 10 μ L, 1.0 × 10 of 10 μ L
-4the mercaptoethylmaine of mol/L and 1.0 × 10 of 20 μ L
-6the DNA1 of mol/L joins in the centrifuge tube of 2mL, uses Vib. that the solution of gained is fully mixed, is then put in shaking table, react one hour under 37 DEG C of conditions; Then add washed golden platinum composite nanoparticle, and on shaking table, hatch 16h under 37 DEG C of conditions; Subsequently, solution centrifugal is also rinsed three times with the 0.1mol/L phosphate buffer solution of the 1.0mL of pH7.4, obtains DNA1 and modify golden platinum composite nanoparticle probe;
Then 1.0 × 10 are added
-6mol/LDNA2, centrifuging hatch 2h on shaking table under 37 DEG C of conditions after, and rinse three times with the 0.1mol/L phosphate buffer solution of the 1.0mL of pH7.4, and be scattered in 1.0mL phosphate buffer solution, obtain DNA modification nano particle, store under 4 DEG C of conditions;
(4) analyze mensuration: first, by gold electrode surfaces through grinding process, drip golden nanometer particle, the 10 μ L graphene oxides of upper 10 μ L successively, the gold electrode that obtained golden nanometer particle and graphene oxide are modified;
The sample solution that 100 μ L contain dopamine is added in the DNA modification nano-particle solution of 100 μ L, after reacting 0.5h under 37 DEG C of conditions, centrifuging, and on shaking table, hatch 16h with under 37 DEG C of conditions, and be scattered in 50 μ L phosphate buffer solutions, obtain the golden platinum composite nanoparticle probe after dopamine effect; After the gold electrode intercalation reaction 0.5h subsequently golden nanometer particle and graphene oxide modified, electrode surface is rinsed three times with the 0.1mol/L phosphate buffer solution of the 1.0mL of pH7.4, using the electrode of gained as working electrode, be inserted into 2mL and contain 0.1 ~ 20mM p-nitrophenol, 0.1 ~ 20NaBH
4with in the phosphate buffer solution of the pH7.4 of the ferrocenecarboxylic acid of 0.1 ~ 20mM, three-electrode system is adopted to measure electrochemical signals; According to the mensuration of the relational implementation dopamine of the content of electrochemical response signal and dopamine.
2. a kind of method detecting dopamine based on nanometer particle to mark redox cycle according to claim 1, it is characterized in that described chemical reagent is analytical reagent, all solution all uses secondary deionized water ion configuration.
3. a kind of method detecting dopamine based on nanometer particle to mark redox cycle according to claim 1, is characterized in that described phosphate buffer is by the KH of 21mL0.2mol/L
2pO
4, 78mL0.2mol/L Na
2hPO
412H
2o mixes, and to obtain final product.
4. a kind of method detecting dopamine based on nanometer particle to mark redox cycle according to claim 1, the Tris-HCl buffer solution collocation method that it is characterized in that the 50mmol/L of described pH8.2 takes 2.423g trishydroxymethylaminomethane, after dissolving with 500mL ultrapure water, adjust PH to 8.2 with 0.1M hydrochloric acid, be finally diluted to 1000mL with ultrapure water.
5. a kind of method detecting dopamine based on nanometer particle to mark redox cycle according to claim 1, is characterized in that the partial sequence of described DNA is:
DNA15’-GTGTTCTCTGGCGCACACAGAGACACAGAATGAGGCCC-(CH
2)
6-SH-3’
DNA25’-GTCTCTGTGTGCGCCAGAGAACACTGGGGCAGATATGGGCCAGCACAGAATGAGGCCC-3’。
6. a kind of method detecting dopamine based on nanometer particle to mark redox cycle according to claim 1, is characterized in that described electrochemical workstation selects CHI820C electrochemical workstation.
7. a kind of method detecting dopamine based on nanometer particle to mark redox cycle according to claim 1, is characterized in that TGL-16G type hydro-extractor selected by described hydro-extractor.
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