CN105044273B - A kind of method that dopamine is detected based on nanometer particle to mark redox cycle - Google Patents
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Abstract
The invention belongs to electrochemical sensor field, it is related to a kind of method that dopamine is detected based on nanometer particle to mark redox cycle, connect dopamine fit complementary seriess single stranded DNA by the use of golden platinum composite nanoparticle as probe, add dopamine fit more within the probe, form the golden platinum composite nanoparticle that double-stranded DNA is modified, when adding sample solution containing dopamine in the golden platinum composite nanoparticle solution modified in double-stranded DNA, dopamine and the fit specific binding of dopamine, probe is released；Then, the probe being released using the gold electrode capture that golden nanometer particle and graphene oxide are modified, recycles the catalytic action of golden platinum composite nanoparticle to produce the detection that electrochemical signals realize dopamine.The inventive method have simply, the high advantage of sensitivity.
Description
Technical field
The invention belongs to electrochemical sensor field is and in particular to a kind of examined based on nanometer particle to mark redox cycle
The method surveying dopamine.
Background technology
Dopamine is to secrete, by hypothalamuses, the emotion information transmission being mainly responsible for brain, and in medical science, dopamine is usually used in controlling
Treat depression.In human body, DOPAMINE CONTENT IN RABBIT is not enough, body will muscle out of hand instinct, seriously lack can cause old silly
Slow-witted, schizophrenia, parkinson's syndrome etc..Adopting chromatograph of liquid to analyze the detection of dopamine more, Fluorometric assay of fluorescence-labeled is high
Effect liquid phase chromatogram mass spectrometry, the analysis method such as gas chromatography combined with mass spectrometry.Because these methods need large-scale instrument,
Have the shortcomings that operating cost costliness requires it is impossible to meet fast and convenient detection, need to set up simplicity, quickly detect DOPA
The new method of amine.Feng et al. utilizes 4- amino -3- diazanyl -5- sulfydryl -1,2,4- triazoles (AHMT) and golden nanometer particle
(AuNPs) link makes probe (AHMT-AuNPs), forms hydrogen bond using dopamine with AHMT and promotes AHMT-AuNPs probe to gather
Collection, causes the color change of colloidal solution to achieve the mensure to dopamine (Feng J J, Guo H, Li Y F, et
al.Single molecular functionalized gold nanoparticles for hydrogen-bonding
recognition and colorimetric detection of dopamine with high sensitivity and
selectivity[J].ACS applied materials&interfaces,2013,5(4):1226-1231).Zhao etc. will
Carbon nano tube modified to electrode surface it is achieved that to dopamine and ascorbic simultaneously detection (Zhao J, Zhang W,
Sherrell P,et al.Carbon nanotube nanoweb–bioelectrode for highly selective
dopamine sensing[J].ACS applied materials&interfaces,2011,4(1):44-48).Hsu et al.
Nanowires of gold is modified and thin film is made on soft terephthalate polyester and is covered in carry out dopamine on electrode
Detection (Hsu M S, Chen Y L, Lee C Y, et al.Gold nanostructures on flexible
substrates as electrochemical dopamine sensors[J].ACS applied materials&
interfaces,2012,4(10):5570-5575).Huang etc. to examine on Au-Pt Nanoparticle Modified to carbon nano-fiber
Survey content (Huang Y, the Miao Y E, Ji S, et al.Electrospun carbon nanofibers of dopamine
decorated with Ag–Pt bimetallic nanoparticles for selective detection of
dopamine[J].ACS applied materials&interfaces,2014,6(15):12449-12456).Graphite oxide
Alkene because containing functional groups such as carboxyl, hydroxyl, epoxy radicals, and have motility, printing opacity is strong, the features such as suitably prepare on a large scale,
It is widely used in terms of biosensor in recent years.Borini group is prepared for can be used for examining using the characteristic of graphene oxide
Biosensor (Borini S, the White R, Wei D, et al.Ultrafast graphene of measuring moisture and temperature
oxide humidity sensors[J].ACS nano,2013,7(12):11166-11173).Li group is then situated between using exchange
Electrophoresis made sensor based on graphene oxide can highly sensitive detection gas containing nitrogen oxide (Li W, Geng X,
Guo Y,et al.Reduced graphene oxide electrically contacted graphene sensor for
highly sensitive nitric oxide detection[J].ACS nano,2011,5(9):6955-6961).At this
In invention, first, the fit complementary seriess of dopamine and golden platinum composite nanoparticle effect form probe, are subsequently adding dopamine and fit
Body, forms the golden platinum composite nanoparticle that double-stranded DNA is modified.When in the golden platinum composite nanoparticle solution modified in double-stranded DNA
When adding sample solution containing dopamine, dopamine and the fit specific binding of dopamine, probe is released.Then, utilize
The probe that the gold electrode capture of golden nanometer particle and graphene oxide modification releases, recycles golden platinum composite nanoparticle
Catalytic action produces the detection that electrochemical signals realize dopamine.
Content of the invention
It is contemplated that inventing a kind of side of the mensure dopamine that method is simple, low cost, sensitivity height, selectivity are high
Method.
Realizing goal of the invention technical scheme is：
A kind of method that dopamine is detected based on nanometer particle to mark redox cycle.Its principle is to be combined using golden platinum
Nanoparticle connects dopamine fit complementary seriess single stranded DNA as probe.Add dopamine fit again in above-mentioned probe, shape
Become the golden platinum composite nanoparticle that double-stranded DNA is modified.Contain when adding in the golden platinum composite nanoparticle solution modified in double-stranded DNA
During the sample solution of dopamine, dopamine and the fit specific binding of dopamine, probe is released.Then, using Jenner
The probe that the gold electrode capture of rice corpuscles and graphene oxide modification releases, recycles the catalysis of golden platinum composite nanoparticle
Effect produces the detection that electrochemical signals realize dopamine.
Determination step is：
(1) preparation of nanoparticle
First, by platinum acid chloride solution and sodium citrate solution with 0.22 μm of micro-pore-film filtration.
The preparation of golden nanometer particle：The sodium citrate solution 1.0mL that mass fraction is 1.0% is added rapidly to boiling
Concentration is in the chlorauric acid solution of 100.0mL of 0.3mmol/L, keeps stirring 10min under fluidized state in solution, when solution becomes
When becoming claret, stop heating, be cooled to room temperature, obtain golden nanometer particle.
The preparation of golden platinum composite nanoparticle：First, the chlorauric acid solution of the 1.0mL for 0.03mol/L, 0.1g by concentration
PVP and the solution of gold nanoparticles of 10mL of preparation be placed in the beaker being contained with 50mL water, and mix homogeneously, then by it
Transfer to heating in a 250mL flask.Mixed solution is heated to after seething with excitement and keep fluidized state 10min, in stirring
Add the platinum acid chloride solution that 7.0mL mass fraction is 1.0% while heating, be subsequently slow added into the Vitamin C of certain volume
Acid solution, when solution becomes dark brown rear stopping heating.Then more continuously stirred lower solution is cooled to room temperature, obtain golden platinum multiple
Close nanoparticle.Golden platinum composite nanoparticle prepared by 1mL is centrifuged 10min under conditions of 14000 revs/min, and uses phosphorus
Acid buffering solution washs three times, and is scattered in phosphate buffer solution.
(2) preparation of graphene oxide
First, the concentrated sulphuric acid 11.5mL that mass fraction is 98% is taken to be placed in conical flask, cold under conditions of being then put in 4 DEG C
But 1h.It was put in processing the conical flask containing cooling concentrated sulphuric acid in ice-water bath, under magnetic stirring, rapidly joined at grinding
The graphite powder containing 0.5g managed and the mixture of 0.25g sodium nitrate.After reaction 5min, point 6 potassium permanganate by 1.5g altogether
Add in solution, in controlling reaction temperature under conditions of 30-45 DEG C, stirring reaction 2.5h, solution becomes blackish green.Then will
Temperature control, between 80-100 DEG C, then adds 5mL deionized water with Dropping funnel, is further continued for reacting 30min.Subsequently with dripping
The 10mL hydrogen peroxide that mass fraction is 5% is slowly added to by liquid funnel, continues stirring 1h, and solution gradually becomes khaki, then will
Solution is transferred in evaporating dish and is dried in an oven, then again deionized water by the solid dissolving of gained, the oxidation that will be obtained
Graphene is put in dialyzer and is dialysed, and till graphene oxide solution is in neutrality, and dries.
(3) preparation of DNA modification nanoparticle
By the Tris-HCl of the 50mmol/L of the pH 8.2 of 20 μ L, the TCEP of the 10mol/L of 10 μ L, 10 μ L 1.0 × 10- 4The mercaptoethylmaine of mol/L and the 1.0 × 10 of 20 μ L-6The DNA1 of mol/L is added in the centrifuge tube of 2mL, is made using vibrator
The solution of gained is sufficiently mixed, and is then put in shaking table, reacts a hour under the conditions of 37 DEG C.It is subsequently adding washed gold
Platinum composite nanoparticle, and 16h is incubated under the conditions of 37 DEG C on shaking table.Subsequently, by solution centrifugal and with pH's 7.4
The 0.1mol/L phosphate buffer solution of 1.0mL flushes three times, and obtains DNA1 and modifies golden platinum composite nanoparticle probe.
It is subsequently adding 1.0 × 10-6Mol/L DNA2, centrifugation after incubation 2h on shaking table under the conditions of 37 DEG C, it is used in combination
The 0.1mol/L phosphate buffer solution of the 1.0mL of pH 7.4 flushes three times, and is scattered in 1.0mL phosphate buffer solution, obtains DNA
Modify nanoparticle, store under the conditions of 4 DEG C.
(4) analysis measures
Electro chemical analysis measuring principle used in the present invention is as shown in Fig. 1.
First, by gold electrode surfaces through grinding process, the golden nanometer particle of 10 μ L, 10 μ L graphite oxides in Deca successively
The gold electrode that alkene, prepared golden nanometer particle and graphene oxide are modified.
The sample solution that 100 μ L are contained dopamine adds in the DNA modification nano-particle solution of 100 μ L, under the conditions of 37 DEG C
After reaction 0.5h, centrifugation, and with the conditions of 37 DEG C on shaking table incubation 16h, and be scattered in 50 μ L phosphate buffer solutions,
Obtain the golden platinum composite nanoparticle probe after dopamine effect.The gold electrode subsequently golden nanometer particle and graphene oxide modified
After insertion reaction 0.5h, rinse electrode surface three times with the 0.1mol/L phosphate buffer solution of the 1.0mL of pH 7.4, by gained
Electrode, as working electrode, is inserted into 2mL and contains 0.1～20mM paranitrophenol, 0.1～20NaBH4With 0.1～20mM's
In the phosphate buffer solution of pH 7.4 of ferrocenecarboxylic acid, electrochemical signals are measured using three-electrode system.Rung according to electrochemistry
The mensure of the relational implementation dopamine of content of induction signal and dopamine.
Described potassium permanganate, paranitrophenol is purchased from seamount Pu Chemical Co., Ltd..
Described mercaptoethylmaine, mercaptoethanol, gold chloride HAuCl4·3H2O, chloroplatinic acid H2PtCl6·6H2O be 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 Na3C6H5O7, ferrocenecarboxylic acid FCA is purchased from Chemical Reagent Co., Ltd., Sinopharm Group.
Described graphite powder, dopamine D A buys in Shanghai Aladdin Reagent Company.
The reagent of all uses is all that chemistry is pure, and secondary deionized water ion configuration all used by all of solvent.
KH by 21mL 0.2mol/L2PO4, the Na of 78mL 0.2mol/L2HPO4·12H2O mixes, and obtains 0.1mol/L
PH is 7.4 phosphate buffered solution.
The Tris-HCl collocation method of the 50mmol/L of pH 8.2 is to weigh 2.423g trishydroxymethylaminomethane, uses
After the ultrapure water dissolution of 500mL, adjust PH to 8.2 with 0.1M hydrochloric acid, be finally diluted to 1000mL with ultra-pure water.
DNA obtains from Beijing SBS Genetech gene technology company limited.Their nucleotide sequence is as follows：
DNA1 5’-GTG TTC TCT GGC GCA CAC AGA GAC ACA GAA TGA GGC CC-(CH2)6-SH-
3’
DNA2 5’-GTC TCT GTG TGC GCC AGA GAA CAC TGG GGC AGA TAT GGG CCA GCA
CAG AAT GAG GCC C-3’
Electrochemical Detection adopts CHI820C electrochemical workstation (Shanghai Chen Hua instrument company).
TGL-16G type centrifuge (Town in Shanghai booth scientific instrument company limited) is used for centrifugation.
Brief description
Fig. 1 is based on nanometer particle to mark redox cycle and measures DA principle schematic.
The impact to electrochemical response signal for the action time of Fig. 2 probe and dopamine.
The impact to electrochemical signals for the incubation time of Fig. 3 electrode.
The advantage of invention and effect
Under optimal testing conditions, the concentration of the size of peak current and object dopamine is linear, and peak
The intensity of electric current increases with the increase of object dopamine concentration, illustrates that can pass through proposed method detects DOPA
The content of amine.With object dopamine concentration from 1.0 × 10-9To 1.0 × 10-6Mol/L increases, and peak current increases.Linearly
Regression equation is Ip=0.04347C+0.1289, and R is 0.9983 (C, nmol/L；Ip, μ A), test limit is 5.0 × 10-10mol/
L.
Specific embodiment
Further illustrate the present invention with reference to specific embodiment, but do not constitute the restriction further to invention.
The impact to electrochemical signals for the action time of embodiment 1 probe and dopamine
Experiment finds that probe is suitable for action time fit with dopamine, action time be not more grow or more short more
Good.As shown in Fig. 2 wherein a-f represents, 1 hour, 1.5 hours, 2.0 hours, 2.5 hours and 3.0 hours electric respectively 0.5 hour
Chemical signal curve, it can be seen that electrochemical signals reach higher value when action time is for 2.5h, then increases effect
The change of time electrochemical signals is inconspicuous, therefore choosing probe action time fit with dopamine is 2.5h.
The impact to electrochemical response signal for the incubation time of embodiment 2 electrode
The gold electrode of golden nanometer particle and graphene oxide modification is visited with the golden platinum composite nanoparticle after dopamine effect
Pin incubation time has important impact to the size of electrochemical signals.As shown in figure 3, a-h represents that incubation time is respectively
Electrochemical signals curve when 0.5h, 1.0h, 1.5h, 2.0h, 2.5h, 3.0h, 3.5h and 4.0h.It can be seen that with
Time increases from 0.5h to 3.0h, and response signal can gradually strengthen.But when incubation time continues to increase from 3h, response signal is anti-
And reduce, so take the incubation time of 3.0h.
The remolding sensitivity of embodiment 3 method is relatively
Replace the gold electrode that golden nanometer particle and graphene oxide are modified with graphene oxide modified gold electrode, to dopamine
By the step that above-mentioned (4) analysis measures, dopamine is detected.Obtaining the range of linearity is 7.0 × 10-8Mol/L～1.0 × 10- 6Mol/L, detection is limited to 3.0 × 10-8mol/L.With test limit as a comparison, the gold modified of golden nanometer particle and graphene oxide
The sensitivity of electrode is 60 times of graphene oxide modified gold electrode, in the presence of golden nanometer particle is described, drastically increases inspection
The sensitivity surveyed, the detection to dopamine has obvious potentiation.
Claims (4)
1. a kind of method based on nanometer particle to mark redox cycle detection dopamine is it is characterised in that be combined using golden platinum
Nanoparticle connects dopamine fit complementary seriess single stranded DNA as probe, then adds dopamine fit within the probe, is formed double
The golden platinum composite nanoparticle that chain DNA is modified, contains DOPA when adding in the golden platinum composite nanoparticle solution modified in double-stranded DNA
During amine sample solution, dopamine and the fit specific binding of dopamine, probe is released；Then, using golden nanometer particle
The probe that the gold electrode capture modified with graphene oxide releases, recycles the catalytic action of golden platinum composite nanoparticle to produce
Raw electrochemical signals realize the detection of dopamine, and determination step is as follows：
(1) preparation of nanoparticle：
A, by platinum acid chloride solution and sodium citrate solution with 0.22 μm of micro-pore-film filtration；
B, the preparation of golden nanometer particle：The sodium citrate solution 1.0mL that mass fraction is 1.0% is added rapidly to the dense of boiling
Spend in the chlorauric acid solution of the 100.0mL for 0.3mmol/L, keep stirring 10min under fluidized state in solution, when solution becomes
During claret, stop heating, be cooled to room temperature, obtain golden nanometer particle；
C, the preparation of golden platinum composite nanoparticle：First, the chlorauric acid solution of the 1.0mL for 0.03mol/L, 0.1g by concentration
The solution of gold nanoparticles of the 10mL of PVP and preparation is placed in the beaker being contained with 50mL water, and mix homogeneously, then by its turn
Move on to heating in a 250mL flask；Mixed solution is heated to after seething with excitement and keep fluidized state 10min, adds in stirring
Add the platinum acid chloride solution that 7.0mL mass fraction is 1.0% while hot, be subsequently slow added into the ascorbic acid of certain volume
Solution, when solution becomes dark brown rear stopping heating；Then more continuously stirred lower solution is cooled to room temperature, obtain golden platinum be combined
Nanoparticle；Golden platinum composite nanoparticle prepared by 1mL is centrifuged 10min under conditions of 14000 revs/min, and uses phosphoric acid
Buffer solution washs three times, and is scattered in phosphate buffer solution；
(2) preparation of graphene oxide：First, the concentrated sulphuric acid 11.5mL that mass fraction is 98% is taken to be placed in conical flask, then
1h is cooled down under conditions of being put in 4 DEG C；It was put in processing the conical flask containing cooling concentrated sulphuric acid in ice-water bath, in magnetic agitation
Under, rapidly join the mixture of the graphite powder containing 0.5g that milled processed crosses and 0.25g sodium nitrate；After reaction 5min, divide and will be total to for 6 times
The potassium permanganate of meter 1.5g adds in solution, in controlling reaction temperature under conditions of 30-45 DEG C, stirring reaction 2.5h, and solution
Become blackish green；Then, by temperature control between 80-100 DEG C, then add 5mL deionized water with Dropping funnel, be further continued for
Reaction 30min；Subsequently with Dropping funnel, the 10mL hydrogen peroxide that mass fraction is 5% is slowly added to, continue stirring 1h, solution by
Crossfade into khaki, then transfer the solution in evaporating dish and dry in an oven, then again deionized water by the solid of gained
Dissolving, the graphene oxide being obtained is put in dialyzer and is dialysed, and till graphene oxide solution is in neutrality, and dries
Dry；
(3) preparation of DNA modification nanoparticle：10mol/L by the Tris-HCl of the 50mmol/L of the pH 8.2 of 20 μ L, 10 μ L
TCEP, the 1.0 × 10 of 10 μ L-4The mercaptoethylmaine of mol/L and the 1.0 × 10 of 20 μ L-6The DNA1 of mol/L is added to 2mL's
In centrifuge tube, it is sufficiently mixed using the solution that vibrator makes gained, is then put in shaking table, reaction one is little under the conditions of 37 DEG C
When；It is subsequently adding washed gold platinum composite nanoparticle, and 16h is incubated under the conditions of 37 DEG C on shaking table；Subsequently, will be molten
Liquid is centrifuged and is flushed three times with the 0.1mol/L phosphate buffer solution of the 1.0mL of pH 7.4, obtains DNA1 and modifies golden platinum composite Nano
Particle probe；
It is subsequently adding 1.0 × 10-6Mol/L DNA2, centrifugation after incubation 2h on shaking table under the conditions of 37 DEG C, and use pH7.4
The 0.1mol/L phosphate buffer solution of 1.0mL flush three times, and be scattered in 1.0mL phosphate buffer solution, obtain DNA modification and receive
Rice corpuscles, store under the conditions of 4 DEG C；
(4) analysis measures：First, by gold electrode surfaces through grinding process, the golden nanometer particle of 10 μ L, 10 μ L in Deca successively
The gold electrode that graphene oxide, prepared golden nanometer particle and graphene oxide are modified；
The sample solution that 100 μ L are contained dopamine adds in the DNA modification nano-particle solution of 100 μ L, reacts under the conditions of 37 DEG C
After 0.5h, centrifugation, and it is incubated 16h on shaking table under the conditions of 37 DEG C, and be scattered in 50 μ L phosphate buffer solutions, much
Golden platinum composite nanoparticle probe after the effect of bar amine；The gold electrode insertion subsequently golden nanometer particle and graphene oxide modified
After reaction 0.5h, rinse electrode surface three times with the 0.1mol/L phosphate buffer solution of the 1.0mL of pH 7.4, by the electrode of gained
As working electrode, it is inserted into 2mL and contains 0.1～20mM paranitrophenol, 0.1～20NaBH4Two cyclopentadienyls with 0.1～20mM
In the phosphate buffer solution of pH 7.4 of iron formate, electrochemical signals are measured using three-electrode system；Believed according to electrochemical response
Mensure number with the relational implementation dopamine of the content of dopamine.
2. a kind of method that dopamine is detected based on nanometer particle to mark redox cycle according to claim 1, its feature
It is that described phosphate buffer solution is by the KH of 21mL 0.2mol/L2PO4, the Na of 78mL 0.2mol/L2HPO4·12H2O mixes
Close, obtain final product.
3. a kind of method that dopamine is detected based on nanometer particle to mark redox cycle according to claim 1, its feature
The Tris-HCl buffer solution collocation method of the 50mmol/L of pH 8.2 described in being is to weigh 2.423g trihydroxy methyl amino
Methane, after the ultrapure water dissolution of 500mL, adjusts pH to 8.2 with 0.1M hydrochloric acid, is finally diluted to 1000mL with ultra-pure water.
4. a kind of method that dopamine is detected based on nanometer particle to mark redox cycle according to claim 1, its feature
The sequence being described DNA is：
DNA1 5’-GTG TTC TCT GGC GCA CAC AGA GAC ACA GAA TGA GGC CC-(CH2)6-SH-3’
DNA2 5’-GTC TCT GTG TGC GCC AGA GAA CAC TGG GGC AGA TAT GGG CCA GCA CAG
AAT GAG GCC C-3’.
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Effective date of registration: 20180517 Address after: 200120 room 304, 3 lane, 720 lane, Cai Lun Road, China (Shanghai) free trade zone. Patentee after: Kai Hui Sagi Biotechnology (Shanghai) Co., Ltd. Address before: 266000 Qingdao University of Science & Technology, 53 Zhengzhou Road, Shibei District, Qingdao, Shandong Patentee before: Qingdao University of Science & Technology |
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