CN102980925A - Sandwich type electrochemical immunosensor, preparation method and application thereof - Google Patents

Sandwich type electrochemical immunosensor, preparation method and application thereof Download PDF

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CN102980925A
CN102980925A CN2012104857503A CN201210485750A CN102980925A CN 102980925 A CN102980925 A CN 102980925A CN 2012104857503 A CN2012104857503 A CN 2012104857503A CN 201210485750 A CN201210485750 A CN 201210485750A CN 102980925 A CN102980925 A CN 102980925A
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preparation
electrochemical immunosensor
nano particle
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CN102980925B (en
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吴丹
张勇
魏琴
范海霞
杜斌
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University of Jinan
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Abstract

The invention relates to an electrochemical immunosensor, a preparation method and application thereof, in particular to a sandwich type electrochemical immunosensor constructed by a marker based on dumbbell type Pt-Fe3O4 nano particles serving as a second antibody, a preparation method thereof, and application of the electrochemical immunosensor prepared by using the method in measurement of tumor markers. The electrochemical immunosensor is high in accuracy, stability, reproducibility and sensitivity, can quickly and conveniently perform immunoassay detection and can be used for clinical analysis.

Description

A kind of sandwich type electrochemical immunosensor, Its Preparation Method And Use
Technical field
The present invention relates to a kind of electrochemical immunosensor, Its Preparation Method And Use, more specifically, the present invention relates to a kind of based on dumbbell shape Pt-Fe 3O 4Sandwich type electrochemical immunosensor, its preparation method that nano particle makes up as two anti-labels, and by the purposes of electrochemical immunosensor in measuring tumor markers of the method preparation.
Background technology
The existence of tumor markers or content can be indicated the character of tumour, help to understand tissue origin and the Cell Differentiation of tumour, thereby help pathological diagnosis, histologic classification, judging prognosis and the guiding clinical treatment of tumour.Its discovery and application become after diagnostic imaging and pathological diagnosis, one of method that current clinical detection tumour is commonly used, and be used for following up a case by regular visits to and the result for the treatment of observation of tumour.
The clinical detection method of tumor markers is a lot of at present, and radioimmunoassay, enzyme-linked immuno assay determination method and chemiluminscence immunoassay method etc. are arranged.But there is following deficiency in these detection methods.
(1) radioimmunoassay: though have easy and simple to handle, low cost and other advantages, this method radioactive contamination is serious, and sensitivity is low, detects limit for height, has therefore limited its application.
(2) enzyme-linked immuno assay determination method: the enzyme-linked immuno assay determination method is simple because of the label preparation, and the term of validity is long, and the characteristics such as environmentally safe have obtained popularizing rapidly and development.But the easy inactivation of the enzyme in the enzyme-linked immuno assay determination method causes its signal time short, has reduced sensitivity and the reappearance of the method.
(3) chemiluminscence immunoassay method: this method have sensitivity, fast, stable, selectivity is strong, favorable reproducibility, easy operating, the flexile advantage of method.But affect the many factors of the testing result of chemiluminescence analysis, so its less stable, and after chemical reaction occured, the luminous of sample can't be reproduced.
Electrochemical immunosensor, especially sandwich type electrochemical immunosensor owing to have the detection sensitivity height, selectivity is good and is easy to the advantage such as miniaturization, enjoy people's concern.
Graphene has fabulous crystallinity and outstanding electronics, thermodynamics and mechanical property, is the focus of people's extensive concern therefore.In the research and application of Graphene, in order to give full play to its advantageous property, must carry out functionalization to Graphene.Namely on grapheme material advantage basis, further material is carried out property modification and optimization, synthesized the nitrogen-doped graphene of biocompatibility excellence, thereby reach the better fixedly purpose of decorative material.
In order to realize the accurate detection to the early stage low abundance proteins mark of tumour, people adopt the nano material thing that serves as a mark usually, such as metal nanoparticle, quantum dot, carbon nano-tube etc., because they have specific surface area is large, the surfactivity site is many, good photoelectric characteristic and the stronger advantages such as bioaffinity, can accelerate the speed of response interface electronic conduction or catalysis electrode surface chemical reaction, therefore be widely used among the structure of electrochemical immunosensor.The sort signal amplification of nano material is so that the sensitivity of sensor has obtained greatly improving.Wherein, the dumbbell shape nano particle is a kind of ideal nano material with applications well prospect, and it is the nano particle of the dumbbell shape structure that forms of the nano particle close contact by two kinds of difference in functionalitys.Two independent nano particles in the dumbbell shape nano particle are by having obtained the not available special performance of single nano particle because the electronics at generation of interfaces shifts the interfacial interaction that causes.And dumbbell shape Pt-Fe 3O 4Nano particle there is not yet report for detection of the structure of Diagnostic Value of Several Serum Tumor Markers electrochemical immunosensor.
The nitrogen-doped graphene that the present invention disperses shitosan drips and is coated onto the glass-carbon electrode surface, makes crosslinking chemical with glutaraldehyde and is used for fixedly primary antibodie and antigen, dumbbell shape Pt-Fe 3O 4Nano particle is as two anti-labels, prepared the sandwich type electrochemical immunosensor that detects Diagnostic Value of Several Serum Tumor Markers, test result shows that the sensitivity of the electrochemical immunosensor of said method preparation, selectivity and stability are high, based on above-mentioned discovery, the inventor has finished the present invention.
Summary of the invention
An object of the present invention is to provide a kind of sandwich type electrochemical immunosensor, described electrochemical immunosensor comprises: working electrode, contrast electrode and to electrode, the basal electrode of described working electrode is glass-carbon electrode, and its surface is nitrogen-doped graphene, glutaraldehyde, primary antibodie, bovine serum albumin, tumor markers antigen and the dumbbell shape Pt-Fe of beautify chitosan dispersion successively 3O 4Two of nano particle hatching resists.
A further object of the present invention is to provide a kind of preparation method of sandwich type electrochemical immunosensor, transducer sensitivity height, the favorable reproducibility, easy and simple to handle of described method preparation.
Another object of the present invention provides the purposes of described sandwich type electrochemical immunosensor in measuring tumor markers.
Described tumor markers is the tumor markers of cervical carcinoma, cancer of the stomach, liver cancer and oophoroma.
In order to solve the problems of the technologies described above, the present invention realizes by following measures.
Sandwich type electrochemical immunosensor of the present invention comprises: working electrode, contrast electrode and to electrode, the basal electrode of described working electrode is glass-carbon electrode, and its surface is nitrogen-doped graphene, glutaraldehyde, primary antibodie, bovine serum albumin, tumor markers antigen and the dumbbell shape Pt-Fe of beautify chitosan dispersion successively 3O 4Two of nano particle hatching resists.Described working electrode and contrast electrode, electrode is formed the sandwich type electrochemical immunosensor detect Diagnostic Value of Several Serum Tumor Markers antigen.Described contrast electrode is saturated calomel electrode, is platinum electrode to electrode.
The preparation method of sandwich type electrochemical immunosensor of the present invention may further comprise the steps:
The Graphene that a, synthetic nitrogen mix;
B, preparation dumbbell shape Pt-Fe 3O 4Two anti-(Ab of nano particle hatching 2) solution;
C, preparation electrochemical immunosensor working electrode;
D, making electrochemical immunosensor working curve.
Wherein, the Graphene that the synthetic nitrogen of step a mixes specifically may further comprise the steps:
1. graphene oxide is synthetic: graphite and potassium permanganate are mixed (mass ratio 1:9), put into the there-necked flask with magneton, then add the concentrated sulphuric acid and phosphoric acid mixed liquor (volume ratio 9:1), there-necked flask is put into oil bath, be heated to 50 ℃, react after 12 h, sample is poured on ice, add hydrogen peroxide, magnetic agitation 0.5 h.After reaction finished, the potpourri that obtains is centrifugal, washing, abandoning supernatant obtained brown color pressed powder graphene oxide after drying;
2. nitrogen-doped graphene is synthetic: take by weighing the graphene oxide that 1. above-mentioned steps prepares, after water-soluble, regulate its pH=10.0 with 30% ammoniacal liquor, add hydrazine hydrate (being 1:35 with graphene oxide liquor capacity ratio), room temperature magnetic agitation 10 min, be transferred to autoclave, under 120 ℃ of conditions, react 12 h.The mixed liquor that obtains, centrifuge washing is washed neutrality, and the room temperature vacuum drying obtains nitrogen-doped graphene.
The preparation dumbbell shape Pt-Fe of step b 3O 4Two anti-solution of nano particle hatching specifically may further comprise the steps:
1. in the ar gas environment that constantly flows, with diacetone platinum (II), oleic acid, oleyl amine and n-octadecane mix (ratio of amount of substance is 1:3:3:30);
2. above-mentioned mixed solution is heated to 120 ℃ with constant speed.Then add Fe (CO) 5(with the ratio of the amount of substance of diacetone platinum (II) be 4:1), temperature is increased to 280 ℃ and keep 20 min;
3. resulting mixed solution is precipitated with excess ethyl alcohol, centrifugal 10 min then, the sediment with preparation is dissolved in the normal hexane again, and with ethanol cyclic washing, centrifugal;
4. product obtained above is dissolved in the toluene, then vacuum drying can obtain dumbbell shape Pt-Fe 3O 4The nano particle solid;
The above-mentioned Pt-Fe that 5. will take by weighing 3O 4It is 1:18 that nano particle joins the CTAB(mass ratio) in the solution, ultrasonic 0.5 h.Add Ab behind centrifugal 15 min 2With the PBS buffer solution of pH=7.4, at 4 ℃ of concussion 24 h, obtain dumbbell shape Pt-Fe 3O 4Two anti-solution of nano particle hatching.
The method for preparing the electrochemical immunosensor working electrode of step c specifically may further comprise the steps:
1. polishing is carried out on the glass-carbon electrode surface and made its any surface finish;
2. the chitosan solution with massfraction 0.5% is that the solvent compound concentration is the nitrogen-doped graphene solution of 1 mg/mL.Then nitrogen-doped graphene solution is dripped on the electrode, room temperature is dried;
3. glutaraldehyde is dripped to electrode surface, and keep when electrode is half-dried, dripping the primary antibodie solution of 1 mg/mL about moistening placement 1 h, dry;
4. be that 1% BSA solution is to eliminate the non-specific avtive spot of electrode at the electrode face finish massfraction again.Be that 7.4 PBS solution cleans electrode with pH, after drying, drip the tumor markers antigenic solution of upper a series of variable concentrations;
5. with Pt-Fe 3O 4The two anti-solution that in advance hatching is good are modified on the electrode, dry 4 ℃ of preservations, namely obtain the working electrode of electrochemical immunosensor.
The making electrochemical immunosensor working curve of steps d, step is as follows:
1. with contrast electrode-saturated calomel electrode, to the working electrode exact connect ion of electrode-platinum electrode and above-mentioned preparation on electrochemical workstation;
2. the PBS buffer solution of pH 7.4 (containing 0.1 mol/LKCl as supporting electrolyte) detects the good working electrode of modification to the response of hydrogen peroxide as end liquid by chronoamperometry; According to the relation of gained current-responsive and tumor markers antigen concentration of standard solution, drawing curve.
Another aspect of the present invention provides the purposes of electrochemical immunosensor in measuring Diagnostic Value of Several Serum Tumor Markers of said method preparation.Described tumour is cervical carcinoma, cancer of the stomach, liver cancer and oophoroma, described Cervical Tumor mark is SCC, CA12-5 and CEA, described stomach neoplasms tumor markers is CA72-4, CEA and CA19-9, described liver cancer tumor markers is AFP and CEA, and described oophoroma tumor marker is CA12-5, AFP, HCG and CEA.
Useful achievement of the present invention:
(1) the present inventor is first with dumbbell shape Pt-Fe 3O 4Nano particle is incorporated into as two anti-labels in the middle of the preparation of electrochemical immunosensor of tumor markers, utilizes Pt nano particle and Fe 3O 4The synergy of nano particle makes the electrode of made that higher sensitivity and wider sensing range be arranged.
(2) in preparation method of the present invention, because the introducing of nitrogen-atoms can be adjusted band structure and the physical chemistry form of Graphene, so that the sensitivity that the Graphene that nitrogen mixes has further improved sensor.
(3) use identical nano material and method of modifying, utilize the specific binding of antigen and antibody, only need to change highly sensitive, the specific detection that the tumor-marker species can realize Diagnostic Value of Several Serum Tumor Markers, the method is simple to operate, detection speed is fast, can realize at short notice a large amount of sample tests, be conducive to the commercialization of tumor markers sensor.
(4) with dumbbell shape Pt-Fe 3O 4Nano particle and tumor markers two anti-directly hatchings, utilize its excellent biocompatibility and high catalytic performance, in two labels that resist, needn't use enzyme, avoided because of the inactivation of enzyme and leaked the detection error that causes, simplify the making step of two anti-labels, significantly improved reappearance and the stability of electrochemical immunosensor.
Electrochemical immunosensor of the present invention has shown good accuracy, stability, reappearance and high sensitivity, and immunoassay detects rapidly, convenient, can be used for clinical analysis.
Description of drawings
Below in conjunction with description of drawings and specific embodiment the present invention is done and to describe in further detail.
Fig. 1 is dumbbell shape Pt-Fe of the present invention 3O 4The TEM photo of nano particle.
Fig. 2 is (A) of the present invention Pt, (B) Fe 3O 4, (C) Pt-Fe 3O 4To H 2O 2Cyclic voltammogram.
Fig. 3 is the working curve that immunosensor of the present invention is measured SCC.
Embodiment
SCC, CA12-5, CEA, AFP, HCG, CA72-4 and the CA19-9 that the present invention uses all gives birth to worker's bioengineering company limited available from Shanghai, diacetone platinum (98%) is available from lark prestige company, oleyl amine (80%-90%), n-octadecane are available from Aladdin reagent company limited, and absolute ethyl alcohol (analyzing pure) is available from Tianjin Fu Ning Fine Chemical Co., Ltd; Normal hexane, oleic acid, dag and the potassium ferricyanide all are purchased from Chemical Reagent Co., Ltd., Sinopharm Group, glutaraldehyde, bovine serum albumin(BSA) (96%-99%) are available from Sigma-Aldrich company, hydrogen peroxide (30%), potassium dihydrogen phosphate, sodium hydrogen phosphate all are purchased from Tianjin and extensively become chemical reagent company limited, except glass-carbon electrode cleaned with ultrapure water, what whole experimentation was used all was intermediate waters.
The CHI760D electrochemical workstation is available from Shanghai occasion China Instr Ltd..
Embodiment 1 preparation sandwich type electrochemical immunosensor
(1) Graphene of synthetic nitrogen doping
Synthesizing of graphene oxide: graphite and the 1.8 g potassium permanganate of 0.3 g being mixed, put into the there-necked flask with magneton, is that the concentrated sulphuric acid and phosphoric acid mixed liquor 40 mL that 9:1 mixes join in the above there-necked flask with volume ratio.There-necked flask is put into oil bath, be heated to 50 ℃, react 12 h, after reaction finishes, sample is poured into about 40 mL on ice, magneton is put on the mixture of ice and water of toppling over magnetic agitation slowly simultaneously, add 30 % hydrogen peroxidase 10 .3 mL, ice melts gradually, magnetic agitation 0.5 h.After reaction finishes, with potpourri centrifugal 0.5 h under the rotating speed of 8000 R that obtains, then distinguish centrifuge washing three times with water, the hydrochloric acid of 20 mL 30%, the 20 mL ethanol of 20 mL respectively, abandoning supernatant, use at last 20 mL ether centrifuge washings, abandoning supernatant, the solid sample that obtains at last are put into 35 ℃ of dryings of vacuum drying chamber, obtain brown color pressed powder graphene oxide after drying is complete;
Synthesizing of nitrogen-doped graphene: take by weighing the graphene oxide of 140 mg above-mentioned steps preparation, add 70 mL water, add 30% ammoniacal liquor, regulate pH=10.0, add 2 mL hydrazine hydrates, room temperature magnetic agitation 10 min, be transferred to autoclave, under 80 ℃ of conditions, react 12 h.The mixed liquor that obtains is washed with water to neutrality after the centrifuging, the room temperature vacuum drying obtains nitrogen-doped graphene.
(2) preparation dumbbell shape Pt-Fe 3O 4Two anti-solution of nano particle hatching
1. in the ar gas environment that constantly flows, with 2 mmol diacetone platinum (II), the oleic acid of 6 mmol, the n-octadecane of 6 mmol oleyl amines and 20 mL mixes;
2. above-mentioned mixed solution is heated to 120 ℃ with the constant rate of speed of 3 ℃/min.After reaching this temperature, add the Fe (CO) of 8 mmol 5, temperature is increased to 280 ℃ and keep 20 min;
3. resulting mixed solution is precipitated with excess ethyl alcohol, then with centrifugal 10 min of 9000 R.Again the sediment that obtains is dissolved in the 20 mL normal hexanes, and uses the ethanol cyclic washing.Still adopt centrifugal 10 min of rotating speed of 9000 R in this process;
4. product obtained above is dissolved in the toluene, then puts into inherent 35 ℃ of vacuum drying chamber and carry out drying, can obtain dumbbell shape Pt-Fe 3O 4The nano particle solid; Fig. 1 is the dumbbell shape Pt-Fe that makes 3O 4The TEM photo of nano particle solid, as known in the figure, synthetic Pt-Fe 3O 4Nano particle is dumb-bell shape really, and particle diameter is approximately about 5 nm;
5. in order to show dumbbell shape Pt-Fe 3O 4The superior function of nano particle is being inquired into Pt-Fe 3O 4Before two anti-labels, at first to Pt-Fe 3O 4Nano particle and Pt nano particle thereof, Fe 3O 4Nano particle is to H 2O 2Catalytic capability carried out the contrast experiment.Pt-Fe as shown in Figure 2 3O 4Nano particle is to H 2O 2Catalytic capability more than Pt nano particle and Fe 3O 4Nano particle is to H 2O 2Catalytic capability, mainly be because Pt-Fe 3O 4Synergy caused it to H 2O 2Catalytic capability obviously strengthens.Therefore, dumbbell shape Pt-Fe is adopted in experiment 3O 4Nano particle is as two anti-labels;
6. take by weighing the aqueous solution that 0.018 g CTAB is made into 1 mL, then take by weighing the Pt-Fe of 1 mg 3O 4Nano particle joins in the CTAB solution that has prepared, ultrasonic 0.5 h.With this solution with centrifugal 15 min of the rotating speed of 9000 R.Then the Ab that adds 25 μ L, 60 mg/mL 2With 475 μ L pH be 7.4 PBS buffer solution, at 4 ℃ of concussion 24 h, obtain dumbbell shape Pt-Fe 3O 4Two anti-solution of nano particle hatching.
(3) preparation electrochemical immunosensor working electrode
1. be the glass-carbon electrode of 4 mm with diameter with the alundum (Al2O3) burnishing powder polishing of 1.0,0.3,0.05 mm, the ethanol ultrasonic cleaning, rinse well with ultrapure water again, then electrode is placed 0.05 mol/L potassium ferricyanide solution, scan at-0.2 ~ 0.6 V, make spike potential poor less than 110 mV, with ultrapure water cleaning electrode surface, dry up;
2. the chitosan solution with massfraction 0.5% is that the solvent compound concentration is the nitrogen-doped graphene solution of 1 mg/mL.Then the nitrogen-doped graphene solution of getting 6 μ L drips on the electrode, and room temperature is dried;
3. the glutaraldehyde with 6 μ L drips to electrode surface, and keeps about moistening placement 1 h, and the primary antibodie solution (shown in table 1, table 2, table 3 and table 4) dripping 6 μ L, 1 mg/mL when electrode is half-dried dries;
4. be that 1% BSA solution is to eliminate the non-specific avtive spot of electrode at electrode face finish 6 μ L massfractions again.Be that 7.4 PBS solution cleans electrode with pH, after drying, drip the tumor markers antigenic solution of upper a series of 6 μ L variable concentrations;
5. with 6 μ L Pt-Fe 3O 4The two anti-solution that hatching is good are modified on the electrode, dry 4 ℃ of preservations, namely obtain the working electrode of electrochemical immunosensor.
(4) make the electrochemical immunosensor working curve
Open electrochemical workstation, with contrast electrode-saturated calomel electrode, to the working electrode exact connect ion of electrode-platinum electrode and above-mentioned preparation on electrochemical workstation;
The PBS buffer solution of 10 mL pH 7.4 detects the good working electrode of modification to the response of hydrogen peroxide as end liquid by chronoamperometry; According to the concentration relationship of gained current-responsive and tumor markers antigen standard solution, drawing curve.
The detection of embodiment 2 Cervical Tumor marks: SCC, CA12-5 or CEA
Prepare electrochemical immunosensor according to embodiment 1 described step, adopt the Cervical Tumor mark shown in this electrochemical immunosensor detection table 1, its detection technique index sees Table 1.
The detection technique index of table 1 Cervical Tumor mark
By table 1 detection technique index result as can be known, the electrochemical immunosensor of the present invention's preparation is used for the detection of Cervical Tumor mark, and its range of linearity is wide, and detectability is low, and method is highly sensitive.
The detection of embodiment 3 stomach neoplasms tumor markers: CA72-4, CEA or CA19-9
Prepare electrochemical immunosensor according to embodiment 1 described step, adopt the stomach neoplasms tumor markers shown in this electrochemical immunosensor detection table 2, its detection technique index sees Table 2.
The detection technique index of table 2 stomach neoplasms tumor markers
Figure 587913DEST_PATH_IMAGE002
By table 2 detection technique index result as can be known, this electrochemical immunosensor is used for the detection of stomach neoplasms tumor markers, and its range of linearity is wide, and detectability is low, and method is highly sensitive.
The detection of embodiment 4 liver cancer tumor markerses: AFP or CEA
Prepare electrochemical immunosensor according to embodiment 1 described step, adopt the liver cancer tumor markers shown in this electrochemical immunosensor detection table 3, its detection technique index sees Table 3.
The detection technique index of table 3 liver cancer tumor markers
Figure 928502DEST_PATH_IMAGE003
By table 3 detection technique index result as can be known, the electrochemical immunosensor of the present invention's preparation is used for the detection of liver cancer tumor markers, and its range of linearity is wide, and detectability is low, and method is highly sensitive.
The detection of embodiment 5 oophoroma tumor markers: CA12-5, AFP, HCG or CEA
Prepare electrochemical immunosensor according to embodiment 1 described step, adopt the oophoroma tumor marker shown in this electrochemical immunosensor detection table 4, its detection technique index sees Table 4.
The detection technique index of table 4 oophoroma tumor marker
Figure 323712DEST_PATH_IMAGE004
By table 4 detection technique index result as can be known, the electrochemical immunosensor of the present invention's preparation is used for the detection of oophoroma tumor marker, and its range of linearity is wide, and detectability is low, and method is highly sensitive.
The detection of tumor markers in embodiment 6 human serums
The concentration of preparation SCC antigen is followed successively by 0.05,0.2, and 0.5,4,6,8,10,12,14,16,18 ng/mL, 1 drafting obtains working curve according to embodiment, as shown in Figure 3, is used for the detection of human serum SCC.Get the fresh serum sample, get supernatant after the centrifuging, with the content that determines SCC in the human serum after the dilution of PBS buffer solution.Then add certain density SCC standard solution in the human serum, carry out recovery testu, the alluvial that obtains by mensuration and the radiometer of addition are calculated the average recovery rate of SCC in the sample.The detection type of other tumor markers is similar to SCC in the human serum, and testing result sees Table 5.
The testing result of Diagnostic Value of Several Serum Tumor Markers in table 5 human serum
Figure 138084DEST_PATH_IMAGE005
In the upper table nNumber of times for replicate determination
By table 5 testing result as can be known, result's relative standard deviation (RSD) is less than 5.0%, and average recovery rate is 95.8 ~ 103%, shows that the present invention is used for the detection of human serum Diagnostic Value of Several Serum Tumor Markers, and the precision of method is high, and the result accurately and reliably.

Claims (9)

1. sandwich type electrochemical immunosensor, described electrochemical immunosensor comprises: working electrode, contrast electrode and to electrode, the basal electrode of described working electrode is glass-carbon electrode, and its surface is nitrogen-doped graphene, glutaraldehyde, primary antibodie, bovine serum albumin, tumor markers antigen and the dumbbell shape Pt-Fe of beautify chitosan dispersion successively 3O 4Two of nano particle hatching resists, and described contrast electrode is saturated calomel electrode, and described is platinum electrode to electrode.
2. the preparation method of a sandwich type electrochemical immunosensor claimed in claim 1, described method comprises the steps:
The Graphene that a, synthetic nitrogen mix;
B, preparation dumbbell shape Pt-Fe 3O 4Two anti-(Ab of nano particle hatching 2) solution;
C, preparation electrochemical immunosensor working electrode;
D, making electrochemical immunosensor working curve.
3. preparation method as claimed in claim 2, wherein, the Graphene that step a synthetic nitrogen mixes may further comprise the steps:
1. graphene oxide is synthetic: graphite and the potassium permanganate of mass ratio 1:9 are mixed, put into the there-necked flask with magneton, then add the concentrated sulphuric acid and phosphoric acid mixed liquor that volume ratio is 9:1, there-necked flask is put into oil bath, be heated to 50 ℃, react after 12 h, sample is poured on ice, add hydrogen peroxide, magnetic agitation 0.5 h; After reaction finished, the potpourri that obtains is centrifugal, washing, abandoning supernatant obtained brown color pressed powder graphene oxide after drying;
2. nitrogen-doped graphene is synthetic: take by weighing graphene oxide that 1. above-mentioned steps prepare water-soluble after, regulate its pH=10.0 with 30% ammoniacal liquor, add hydrazine hydrate, wherein said hydrazine hydrate is 1:35 with graphene oxide liquor capacity ratio, room temperature magnetic agitation 10 min, be transferred to autoclave, reaction 12 h under 120 ℃ of conditions, the mixed liquor that obtains, centrifuge washing, wash neutrality, the room temperature vacuum drying obtains nitrogen-doped graphene.
4. preparation method as claimed in claim 2, wherein, step b prepares dumbbell shape Pt-Fe 3O 4Two anti-solution of nano particle hatching may further comprise the steps:
1. in the ar gas environment that constantly flows, be that diacetone platinum (II), oleic acid, oleyl amine and the n-octadecane of 1:3:3:30 mixes with the ratio of amount of substance;
2. above-mentioned mixed solution is heated to 120 ℃ with constant speed, then adds Fe (CO) 5, wherein, described Fe (CO) 5With the ratio of the amount of substance of diacetone platinum (II) be 4:1, temperature is increased to 280 ℃ and keep 20 min;
3. resulting mixed solution is precipitated with excess ethyl alcohol, centrifugal 10 min then, the sediment with preparation is dissolved in the normal hexane again, and with ethanol cyclic washing, centrifugal;
4. product obtained above is dissolved in the toluene, then vacuum drying can obtain dumbbell shape Pt-Fe 3O 4The nano particle solid;
The above-mentioned Pt-Fe that 5. will take by weighing 3O 4Nano particle joins in the CTAB solution, and the mass ratio of the two is 1:18, and ultrasonic 0.5 h adds Ab behind centrifugal 15 min 2With the PBS buffer solution of pH=7.4, at 4 ℃ of concussion 24 h, obtain dumbbell shape Pt-Fe 3O 4Two anti-solution of nano particle hatching.
5. preparation method as claimed in claim 2, wherein, the method that step c prepares the electrochemical immunosensor working electrode may further comprise the steps:
1. polishing is carried out on the glass-carbon electrode surface and made its any surface finish;
2. the chitosan solution with massfraction 0.5% is that the solvent compound concentration is the nitrogen-doped graphene solution of 1 mg/mL, then described nitrogen-doped graphene solution is dripped on the electrode, and room temperature is dried;
3. glutaraldehyde is dripped to electrode surface, and keep when electrode is half-dried, dripping the primary antibodie solution of 1 mg/mL about moistening placement 1 h, dry;
4. being 1% BSA solution eliminating the non-specific avtive spot of electrode at the electrode face finish massfraction again, is that 7.4 PBS solution cleans electrode with pH, after drying, drips the tumor markers antigenic solution of going up a series of variable concentrations;
5. with Pt-Fe 3O 4The two anti-solution that in advance hatching is good are modified on the electrode, dry 4 ℃ of preservations, namely obtain the working electrode of electrochemical immunosensor.
6. preparation method as claimed in claim 2, wherein, the step that steps d is made the electrochemical immunosensor working curve is as follows:
1. will as the saturated calomel electrode of contrast electrode, as to the working electrode exact connect ion of the platinum electrode of electrode and above-mentioned preparation on electrochemical workstation;
2. contain 0.1 mol/LKCl as the PBS buffer solution of the pH 7.4 of supporting electrolyte as end liquid, detect by chronoamperometry and to modify good working electrode to the response of hydrogen peroxide; According to the relation of gained current-responsive and tumor markers antigen concentration of standard solution, drawing curve.
7. the purposes of sandwich type electrochemical immunosensor as claimed in claim 1 in measuring tumor markers.
8. purposes claimed in claim 7, wherein, described tumour is cervical carcinoma, cancer of the stomach, liver cancer and oophoroma.
9. purposes claimed in claim 8, wherein, described Cervical Tumor mark is SCC, CA12-5 and CEA, described stomach neoplasms tumor markers is CA72-4, CEA and CA19-9, described liver cancer tumor markers is AFP and CEA, and described oophoroma tumor marker is CA12-5, AFP, HCG and CEA.
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