CN104614527A - Method for establishing electrochemical immunosensor for detecting carcino-embryonic antigen - Google Patents
Method for establishing electrochemical immunosensor for detecting carcino-embryonic antigen Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57473—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving carcinoembryonic antigen, i.e. CEA
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
- G01N33/5438—Electrodes
Abstract
The invention relates to a method for establishing an electrochemical immunosensor for detecting carcino-embryonic antigen, in particular to a method for establishing an electrochemical immunosensor for detecting carcino-embryonic antigen based on a graphite/bovine serum albumin stabilized silver nanoparticle (GR/Ag@BSA) composite material. The method is characterized by comprising the following steps: firstly, depositing a layer of gold nanoparticles on the surface of a glassy carbon electrode by using a potential deposition method, sequentially immobilizing primary antibodies and antigen on the surface of the electrode, and finally immobilizing a second antibody solution marked by GR/Ag@BSA through specific reaction of antigen antibodies. The gold nanoparticles are excellent in conductivity, the used GR/Ag@BSA composite material not only integrates excellent conductivity of GR and good biocompatibility of BSA, but also is high in catalytic activity to hydrogen peroxide reduction, and the electrochemical immunosensor which is established based on the material is relatively high in detection sensitivity on carcino-embryonic antigen and relatively in detection limit.
Description
Technical field
The invention belongs to electrochemical immunosensor technical field, be specifically related to a kind of construction method detecting the electrochemical immunosensor of carcinomebryonic antigen.
Background technology
Carcinomebryonic antigen (CEA) is a broad spectrum activity tumor markers, and it can reflect the existence of kinds of tumors to people, estimates it is a good tumor markers to the Outcome measure of colorectal cancer, breast cancer and lung cancer, PD, monitoring and prognosis.The immune analysis method of current detection CEA mainly contains radioimmunoassay, enzyme-linked immunosorbent assay, piezoelectric immuno analytic approach, fluoroimmunoassay etc.Although these methods are reliably sensitive, there are some shortcomings: have radiation hazard, analysis time is grown, need complicated electrical instrumentation etc.
In recent years, electrochemical immunosensor receives extensive concern, and this has the inherent advantages such as good portability, low cost, high detection sensitivity due to it.In electrochemical immunoanalytical process, enzyme is usually used to make signal probe, but the easy deactivation of enzyme, expensive, therefore, build the attention causing analysts without enzyme immunosensor.Nano silver grain is to also original high catalytic activity of hydrogen peroxide, and this catalysis characteristics makes Nano silver grain become the potential material built without enzyme immunosensor.But the bio-compatibility of simple silver nano material is bad, therefore needs to prepare and there is the become reconciled material of bio-compatibility of high catalytic activity build without enzyme sensor.The good biological that the stable Nano silver grain (Ag@BSA) of bovine serum albumin has gathered the catalytic activity of Nano silver grain and BSA is compatible, in the structure of sensor, have good application prospect.
Graphene (GR) has excellent electrochemical stability, high electric conductivity, large specific surface area, is the focus that people pay close attention to.In addition, the compound substance based on GR combines different materials characteristic separately, also receives the extensive concern of people.Therefore, the material that GR and Ag@BSA compound obtains not only has been had electric conductivity, high catalytic performance, good bio-compatibility, the fixing of antibody can be applied to better.
Gold nano is modified glassy carbon electrode surface by the method for the present invention's electro-deposition, then immobilized primary antibodie and antigen successively, resist with GR/Ag BSA compound substance mark two, prepared a kind of electrochemical immunosensor detecting CEA, this sensor has high sensitivity, selectivity and stability.
Summary of the invention
The object of the present invention is to provide a kind of construction method detecting the electrochemical immunosensor of CEA, especially based on the construction method of the electrochemical immunosensor of the detection CEA of GR/Ag@BSA compound substance.
For achieving the above object, the present invention adopts following technical matters:
The construction method of electrochemical immunosensor of the present invention comprises the steps:
(1) glass-carbon electrode of electrochemical deposition golden nanometer particle modification;
(2) preparation of diallyl dimethyl ammoniumchloride (PDDA) functionalization reduced graphene (GR);
(3) bovine serum albumin (BSA) stablizes the preparation of Nano silver grain;
(4) preparation of bovine serum albumin protection Nano silver grain and functionalization reduced graphene compound substance (GR/Ag@BSA);
(5) GR/Ag@BSA marks carcinomebryonic antigen second antibody (GR/Ag@BSA-Ab
2) preparation;
(6) Sandwich immunoassay is utilized to build electrochemical immunosensor.
The glass-carbon electrode that described step (1) electrochemical deposition golden nanometer particle is modified, specifically comprises the following steps:
A uses the alundum (Al2O3) burnishing powder polishing glass-carbon electrode of 1.0,0.3 and 0.5 μm successively, then clean with ultrapure water, naturally dries;
It is in the chlorauric acid solution of 1% that the glass-carbon electrode that process is clean is immersed 3 mL massfractions by b, be working electrode with glass-carbon electrode, platinum electrode is to electrode, silver/silver chloride electrode is contrast electrode, at room temperature, prepare the glass-carbon electrode of golden nanometer particle modification with operating potential potentiostatic electrodeposition 30 s of-0.2 V, ultrapure water cleans up, and dries.
The preparation of described step (2) PDDA functionalization GR, specifically comprises the following steps:
The preparation of a graphene oxide: weighing 0.75 g dag, to join 100 mL volume ratios be in the phosphoric acid of 1:9 and the mixed solution of the concentrated sulphuric acid, and in water-bath, be heated to 30 DEG C, weighing 4.5 g potassium permanganate joins in above-mentioned mixed liquor, be warming up to 50 DEG C, after magnetic agitation reacts 12 h, sample is poured into and adds in 300 mL frozen water of 4 mL hydrogen peroxide in advance, the mixed solution obtained 10000 turns of centrifuging 15 min, with milli-Q water 7 times, in vacuum drying chamber, obtain solid oxidation Graphene after 60 DEG C of drying 12 h;
The preparation of b GR: take the obtained graphene oxide dissolution of solid of 25 mg step (2) a in 150 mL ultrapure waters, ultrasound wave process 30 min, 100 μ L hydrazine hydrates are added successively in solution, 560 μ L ammoniacal liquor, 10 mg polyvinylpyrrolidones, at 90 DEG C of reactions 1 h, the mixed solution obtained 10000 turns of centrifuging 15 min, with milli-Q water 5 times, in vacuum drying chamber, obtain solid GR after 60 DEG C of drying 12 h;
The preparation of c PDDA functionalization GR: the GR solid taking 2.5 mg step (2) b obtained is dissolved in 5 mL ultrapure waters, ultrasound wave process 30 min, in solution, add 10 mL massfractions is the PDDA of 1%, stirred at ambient temperature 2 h, the mixed solution obtained 10000 turns of centrifuging 10 min, again be dissolved in 5 mL ultrapure waters with after milli-Q water 3 times, successfully obtained PDDA-GR solution.
Described step (3) bovine serum albumin (BSA) stablizes the preparation of Nano silver grain, specifically comprises the following steps:
45 mg BSA are dissolved in 10 mL ultrapure waters, after stirred at ambient temperature 5 min, be heated to 60 DEG C, be rapidly in solution the silver nitrate adding 10 mL 8 mmol/L, continue stirring 45 min, the mixed solution obtained 10000 turns of centrifuging 5 min, with milli-Q water 5 times, obtain solid Ag BSA after drying under room temperature.
The preparation of described step (4) bovine serum albumin protection Nano silver grain and functionalization reduced graphene compound substance (GR/Ag@BSA), specifically comprises the following steps:
Taking Ag@BSA prepared by 1 mg step (3) is dissolved in 1 mL ultrapure water, the PDDA-GR solution mixing prepared with 1 mL step (2) after stirring 5 min, stirred at ambient temperature 4 h, by mixed liquor 1000 turns of centrifuging 5 min obtained, abandoning supernatant, is dissolved into obtained GR/Ag@BSA solution in the PBS buffer solution of 1 mL pH 7.4.
Described step (5) prepares the Ab that GR/Ag@BSA marks
2, specifically comprise the following steps:
The glutaraldehyde solution that the 1 mL GR/Ag@BSA solution that step (4) is prepared by a and 3 mL massfractions are 2.5% mixes, stirred at ambient temperature 2 h, the mixed liquor obtained 10000 turns of centrifuging 5 min, abandoning supernatant, is dissolved in the PBS buffer solution of 1 mL pH 7.4;
B is by the Ab of 1 mL 20 μ g/mL
2add in above-mentioned solution to stir and hatch 2 h, 10000 turns of centrifuging 5 min, abandoning supernatant, is again dissolved in the PBS buffer solution of 1 mL pH 7.4, obtains GR/Ag BSA-Ab
2solution.
Described step (6) builds electrochemical immunosensor, specifically comprises the following steps:
The glassy carbon electrode surface that golden nanometer particle prepared by CEA first antibody to the above-mentioned steps (1) that a drips painting 5 μ L 1 mg/mL is modified, the PBS buffer solution of incubated at room temperature 1 h, pH 7.4 cleans 3 times, dries;
B above-mentioned electrode surface drip 5 μ L massfractions be the BSA solution of 1% with the nonspecific activity site on enclosed-electrode surface, the PBS buffer solution of pH 7.4 cleans 3 times, dries;
The CEA antigenic solution of 5 μ L 0.005-150 ng/mL drips and is coated onto Different electrodes surface by c respectively, and the PBS buffer solution of incubated at room temperature 40 min, pH 7.4 cleans 3 times, dries;
D is by 5 μ L GR/Ag@BSA-Ab
2drip to electrode surface, the PBS buffer solution of incubated at room temperature 40 min, pH 7.4 cleans 3 times, dries, and namely obtains the working electrode of electrochemical immunosensor;
Silver/silver chloride reference electrode, the working electrode of platinum filament to electrode and above-mentioned preparation are connected on electrochemical workstation by e;
The PBS buffer solution of f pH 7.4 as end liquid, by the response of chronoamperometry testing electrode pair hydrogen peroxide, according to the current value of gained and the logarithm of CEA concentration linear, drawing curve.
Accompanying drawing explanation
Fig. 1 is the working curve that electrochemical immunosensor of the present invention measures CEA.
Embodiment
Below by way of specific embodiment, technical scheme of the present invention is described, but protection scope of the present invention is not limited thereto.
Embodiment 1 prepares the electrochemical immunosensor detecting CEA standard model.
Step 1. prepares the GR of PDDA functionalization
The preparation of a graphene oxide: be that phosphoric acid and the concentrated sulphuric acid mixed solution of 1:9 joins in there-necked flask by 100 mL volume ratios, then taking 0.75 g dag joins in above-mentioned solution, be heated to 30 DEG C, add 4.5 g potassium permanganate in solution after, regulate water-bath temperature to 50 DEG C, 12 h are reacted under magnetic agitation condition, after reaction stops, sample is poured into and adds in 300 mL frozen water of 4 mL hydrogen peroxide in advance, abundant stirring, the mixed liquor obtained is through centrifuging, with milli-Q water 7 times, 60 DEG C of dryings in vacuum drying chamber subsequently, brown color graphene oxide solid is obtained after drying,
The preparation of b GR: graphene oxide prepared by 25 mg above-mentioned steps a is dissolved in 150 mL ultrapure waters, ultrasound wave process 30 min, 100 μ L hydrazine hydrates are added successively, 560 μ L ammoniacal liquor, 10 mg polyvinylpyrrolidones in solution, 90 DEG C of reaction 1 h in water-bath, the mixed liquor centrifuging obtained, with milli-Q water 5 times, abandoning supernatant, 60 DEG C of dryings in vacuum drying chamber, obtain black GR solid after drying;
The preparation of c PDDA functionalization GR: take GR prepared by 2.5 mg above-mentioned steps b and join in 5 mL ultrapure waters, ultrasound wave process 30 min, the PDDA being 1% by the 10 mL massfractions prepared in advance joins in above-mentioned solution, stirred at ambient temperature 2 h, the mixed liquor obtained through centrifuging, with milli-Q water 3 times, abandoning supernatant, again be dissolved in 5 mL ultrapure waters, successfully obtained PDDA-GR solution.
The preparation of step 2. Ag@BSA: 45 mg BSA pressed powders are dissolved in 10 mL ultrapure waters, stirred at ambient temperature 5 min, 60 DEG C are heated in water-bath, add rapidly the liquor argenti nitratis ophthalmicus of 10 mL 8 mmol/L, continue to stir 45 min at 60 DEG C, the mixed solution obtained is through centrifuging, with milli-Q water 5 times, abandoning supernatant, at room temperature dry, obtain solid Ag@BSA.
The preparation of step 3. GR/Ag@BSA compound substance: the Ag@BSA that 1 mg step 2 is obtained is dissolved in 1 mL ultrapure water, abundant stirring 5 min, the PDDA-GR that 1 mL step 1 is obtained is added in solution, continue at room temperature to stir 4 h, the mixed liquor obtained is through centrifuging, discard supernatant liquor, the potpourri obtained is dissolved in the PBS buffer solution of 1 mL pH 7.4, obtains GR/Ag@BSA solution.
Step 4. GR/Ag@BSA marks the preparation of second antibody solution: the glutaraldehyde solution being 2.5% by 1 obtained for step 3 mL GR/Ag@BSA solution and 3 mL massfractions mixes, stirred at ambient temperature 2 h, the mixed solution centrifuging obtained, abandoning supernatant, is dissolved in the PBS buffer solution of 1 mL pH 7.4; By the Ab of 1 mL 20 μ g/mL
2add in above-mentioned solution, stirred at ambient temperature hatches 2 h, and centrifuging discards supernatant liquor, is again dissolved in the PBS buffer solution of 1 mL pH 7.4, namely obtains GR/Ag@BSA-Ab
2mixed solution.
The preparation of step 5. golden nanometer particle modified glassy carbon electrode: the alundum (Al2O3) burnishing powder polishing glass-carbon electrode using 1.0,0.3 and 0.5 μm successively, then uses ultrapure water, naturally dries; It is in the chlorauric acid solution of 1% that the glass-carbon electrode that process is clean is immersed 3 mL massfractions, be working electrode with glass-carbon electrode, platinum electrode is to electrode, silver/silver chloride electrode is contrast electrode, at room temperature, prepare the glass-carbon electrode of golden nanometer particle modification with operating potential potentiostatic electrodeposition 30 s of-0.2 V, ultrapure water cleans, and dries.
The glassy carbon electrode surface that the CEA first antibody that step 6. droplet is coated with 5 μ L 1 mg/mL is modified to the golden nanometer particle that step 5 is obtained, incubated at room temperature 1 h, ultrapure water cleans, and dries.
The BSA solution that 5 μ L massfractions are 1% drips and is applied to the obtained electrode surface of step 6 by step 7., for the nonspecific activity site of enclosed-electrode, with the PBS buffer solution solution of pH 7.4 to electrode clean, dries.
The CEA antigenic solution of 5 μ L 0.005-150 ng/mL is dripped the Different electrodes surface being coated onto BSA and sheltering by step 8. respectively, and the PBS buffer solution cleaning of incubated at room temperature 40 min, pH 7.4, dries.
Step 9. is by GR/Ag@BSA-Ab obtained for 5 μ L steps 4
2drip to the electrode surface that step 8 is obtained, the PBS buffer solution cleaning of incubated at room temperature 40 min, pH 7.4, dries, namely obtains the working electrode of electrochemical immunosensor.
Silver/silver chloride reference electrode, platinum filament are connected on electrochemical workstation to electrode with according to working electrode prepared by above-mentioned steps by step 10..
The PBS buffer solution of step 11. pH 7.4 as end liquid, by the response of chronoamperometry testing electrode pair hydrogen peroxide, according to the current value of gained and the logarithm of CEA concentration linear, drawing curve.
The linearity curve of " response current-CEA concentration " that step 12. obtains is Current (μ A)=40.20+15.26 lg [CEA] (ng/mL), linearly dependent coefficient is 0.9997, as shown in Figure 1, CEA is within the scope of 0.005-150 ng/mL, the logarithm of response current value and CEA concentration keeps good linear relationship, detects and is limited to 0.002 ng/mL.
Embodiment 2 prepares the electrochemical immunosensor of the blood serum sample detected containing CEA.
Step 1. prepares the GR of PDDA functionalization
The preparation of a graphene oxide: be that phosphoric acid and the concentrated sulphuric acid mixed solution of 1:9 joins in there-necked flask by 100 mL volume ratios, then taking 0.75 g dag joins in above-mentioned solution, be heated to 30 DEG C, add 4.5 g potassium permanganate in solution after, regulate water-bath temperature to 50 DEG C, 12 h are reacted under magnetic agitation condition, after reaction stops, sample is poured into and adds in 300 mL frozen water of 4 mL hydrogen peroxide in advance, abundant stirring, the mixed liquor obtained is through centrifuging, with milli-Q water 7 times, 60 DEG C of dryings in vacuum drying chamber subsequently, brown color graphene oxide solid is obtained after drying,
The preparation of b GR: graphene oxide prepared by 25 mg above-mentioned steps a is dissolved in 150 mL ultrapure waters, ultrasound wave process 30 min, 100 μ L hydrazine hydrates are added successively, 560 μ L ammoniacal liquor, 10 mg polyvinylpyrrolidones in solution, 90 DEG C of reaction 1 h in water-bath, the mixed liquor centrifuging obtained, with milli-Q water 5 times, abandoning supernatant, 60 DEG C of dryings in vacuum drying chamber, obtain black GR solid after drying;
The preparation of c PDDA functionalization GR: take GR prepared by 2.5 mg above-mentioned steps b and join in 5 mL ultrapure waters, ultrasound wave process 30 min, the PDDA being 1% by the 10 mL massfractions prepared in advance joins in above-mentioned solution, stirred at ambient temperature 2 h, the mixed liquor obtained through centrifuging, with milli-Q water 3 times, abandoning supernatant, again be dissolved in 5 mL ultrapure waters, successfully obtained PDDA-GR solution.
The preparation of step 2. Ag@BSA: 45 mg BSA pressed powders are dissolved in 10 mL ultrapure waters, stirred at ambient temperature 5 min, 60 DEG C are heated in water-bath, add rapidly the liquor argenti nitratis ophthalmicus of 10 mL 8 mmol/L, continue to stir 45 min at 60 DEG C, the mixed solution obtained is through centrifuging, with milli-Q water 5 times, abandoning supernatant, at room temperature dry, obtain solid Ag@BSA.
The preparation of step 3. GR/Ag@BSA compound substance: the Ag@BSA that 1 mg step 2 is obtained is dissolved in 1 mL ultrapure water, abundant stirring 5 min, the PDDA-GR that 1 mL step 1 is obtained is added in solution, continue at room temperature to stir 4 h, the mixed liquor obtained is through centrifuging, discard supernatant liquor, the potpourri obtained is dissolved in the PBS buffer solution of 1 mL pH 7.4, obtains GR/Ag@BSA solution.
Step 4. GR/Ag@BSA marks the preparation of second antibody solution: the glutaraldehyde solution being 2.5% by 1 obtained for step 3 mL GR/Ag@BSA solution and 3 mL massfractions mixes, stirred at ambient temperature 2 h, the mixed solution centrifuging obtained, abandoning supernatant, is dissolved in the PBS buffer solution of 1 mL pH 7.4; By the Ab of 1 mL 20 μ g/mL
2add in above-mentioned solution, stirred at ambient temperature hatches 2 h, and centrifuging discards supernatant liquor, is again dissolved in the PBS buffer solution of 1 mL pH 7.4, namely obtains GR/Ag@BSA-Ab
2mixed solution.
The preparation of step 5. golden nanometer particle modified glassy carbon electrode: the alundum (Al2O3) burnishing powder polishing glass-carbon electrode using 1.0,0.3 and 0.5 μm successively, then uses ultrapure water, naturally dries; It is in the chlorauric acid solution of 1% that the glass-carbon electrode that process is clean is immersed 3 mL massfractions, be working electrode with glass-carbon electrode, platinum electrode is to electrode, silver/silver chloride electrode is contrast electrode, at room temperature, prepare the glass-carbon electrode of golden nanometer particle modification with operating potential potentiostatic electrodeposition 30 s of-0.2 V, ultrapure water cleans, and dries.
The glassy carbon electrode surface that the CEA first antibody that step 6. droplet is coated with 5 μ L 1 mg/mL is modified to the golden nanometer particle that step 5 is obtained, incubated at room temperature 1 h, ultrapure water cleans, and dries.
The BSA solution that 5 μ L massfractions are 1% drips and is applied to the obtained electrode surface of step 6 by step 7., for the nonspecific activity site of enclosed-electrode, with the PBS buffer solution solution of pH 7.4 to electrode clean, dries.
The blood serum sample that 5 μ L contain CEA is dripped the electrode surface being coated onto BSA and sheltering by step 8., and the PBS buffer solution cleaning of incubated at room temperature 40 min, pH 7.4, dries.
Step 9. is by GR/Ag@BSA-Ab obtained for 5 μ L steps 4
2drip to the electrode surface that step 8 is obtained, the PBS buffer solution cleaning of incubated at room temperature 40 min, pH 7.4, dries, namely obtains the working electrode of electrochemical immunosensor.
Silver/silver chloride reference electrode, platinum filament are connected on electrochemical workstation to electrode with according to working electrode prepared by above-mentioned steps by step 10..
The PBS buffer solution of step 11. pH 7.4 as end liquid, by the response of chronoamperometry testing electrode pair hydrogen peroxide.
Claims (7)
1. detect a construction method for the electrochemical immunosensor of carcinomebryonic antigen (CEA), it is characterized in that comprising the following steps:
(1) glass-carbon electrode of electrochemical deposition golden nanometer particle modification;
(2) preparation of diallyl dimethyl ammoniumchloride (PDDA) functionalization graphene (GR);
(3) bovine serum albumin stablizes the preparation of Nano silver grain (Ag@BSA);
(4) bovine serum albumin stablizes the preparation of Nano silver grain and functionalization reduced graphene compound substance (GR/Ag@BSA);
(5) GR/Ag@BSA marks carcinomebryonic antigen second antibody (GR/Ag@BSA-Ab
2) preparation;
(6) Sandwich immunoassay is utilized to build electrochemical immunosensor.
2. a kind of construction method detecting the electrochemical immunosensor of CEA according to claim 1, is characterized in that described step (1) is specially:
A uses the alundum (Al2O3) burnishing powder polishing glass-carbon electrode of 1.0,0.3 and 0.5 μm successively, then clean with ultrapure water, naturally dries;
It is in the chlorauric acid solution of 1% that the glass-carbon electrode that process is clean is immersed 3 mL massfractions by b, be working electrode with glass-carbon electrode, platinum electrode is to electrode, silver/silver chloride electrode is contrast electrode, at room temperature, prepare the glass-carbon electrode of golden nanometer particle modification with operating potential potentiostatic electrodeposition 30 s of-0.2 V, ultrapure water cleans up, and dries.
3. a kind of construction method detecting the electrochemical immunosensor of CEA according to claim 1, is characterized in that described step (2) is specially:
The preparation of a graphene oxide: weighing 0.75 g dag, to be dissolved into 100 mL volume ratios be in the phosphoric acid of 1:9 and the mixed solution of the concentrated sulphuric acid, and in water-bath, be heated to 30 DEG C, 4.5 g potassium permanganate are joined in above-mentioned mixed liquor, be warming up to 50 DEG C, after magnetic agitation reacts 12 h, sample is poured into and adds in 300 mL frozen water of 4 mL hydrogen peroxide in advance, the mixed solution obtained 10000 turns of centrifuging 15 min, with milli-Q water 7 times, in vacuum drying chamber, obtain solid oxidation Graphene after 60 DEG C of drying 12 h;
The preparation of b reduced graphene: the graphene oxide taking 25 mg step (2) a obtained is dissolved in 150 mL ultrapure waters, ultrasound wave process 30 min, 100 μ L hydrazine hydrates are added successively in solution, 560 μ L ammoniacal liquor, 10 mg polyvinylpyrrolidones, at 90 DEG C of reactions 1 h, the mixed solution obtained 10000 turns of centrifuging 15 min, with milli-Q water 5 times, in vacuum drying chamber, obtain solid GR after 60 DEG C of drying 12 h;
The preparation of c PDDA functionalization GR: take the obtained GR dissolution of solid of 2.5 mg step (2) b in 5 mL ultrapure waters, ultrasound wave process 30 min, in solution, add 10 mL massfractions is the PDDA of 1%, stirred at ambient temperature 2 h, the mixed solution obtained 10000 turns of centrifuging 10 min, again be dissolved in 5 mL ultrapure waters with after milli-Q water 3 times, so just successfully prepared PDDA-GR solution.
4. a kind of construction method detecting the electrochemical immunosensor of CEA according to claim 1, is characterized in that described step (3) is specially:
45 mg BSA are dissolved in 10 mL ultrapure waters, after stirred at ambient temperature 5 min, 60 DEG C are heated in water-bath, be rapidly in solution the silver nitrate adding 10 mL 8 mmol/L, continue stirring 45 min, the mixed solution obtained 10000 turns of centrifuging 5 min, with milli-Q water 5 times, obtain solid Ag BSA after drying under room temperature.
5. a kind of construction method detecting the electrochemical immunosensor of CEA according to claim 1, is characterized in that described step (4) is specially:
Taking Ag@BSA prepared by 1 mg step (3) is dissolved in 1 mL ultrapure water, the PDDA-GR solution prepared with 1 mL step (2) after stirring 5 min mixes, stirred at ambient temperature 4 h, by mixed liquor 10000 turns of centrifuging 5 min obtained, abandoning supernatant, be dissolved in the PBS buffer solution of 1 mL pH 7.4, obtained GR/Ag@BSA solution.
6. a kind of preparation method detecting the electrochemical immunosensor of CEA according to claim 1, is characterized in that described step (5) is specially:
The glutaraldehyde solution that the 1 mL GR/Ag@BSA solution that step (4) is prepared by a and 3 mL massfractions are 2.5% mixes, stirred at ambient temperature 2 h, the mixed liquor obtained 10000 turns of centrifuging 5 min, abandoning supernatant, is dissolved in the PBS buffer solution of 1 mL pH 7.4;
B is by the Ab of 1 mL 20 μ g/mL
2add in above-mentioned solution, stirred at ambient temperature hatches 2 h, 10000 turns of centrifuging 5 min, and abandoning supernatant, is again dissolved in the PBS buffer solution of 1 mL pH 7.4, obtains GR/Ag BSA-Ab
2solution.
7. a kind of preparation method detecting the electrochemical immunosensor of CEA according to claim 1, is characterized in that described step (6) is specially:
The glassy carbon electrode surface that golden nanometer particle prepared by CEA first antibody to the above-mentioned steps (1) that a drips painting 5 μ L 1 mg/mL is modified, the PBS buffer solution of incubated at room temperature 1 h, pH 7.4 cleans 3 times, dries;
B above-mentioned electrode surface drip 5 μ L massfractions be the BSA solution of 1% with the nonspecific activity site on enclosed-electrode surface, the PBS solution of pH 7.4 cleans 3 times, dries;
The CEA antigenic solution of 5 μ L 0.005-150 ng/mL drips and is coated onto Different electrodes surface by c respectively, and the PBS buffer solution of incubated at room temperature 40 min, pH 7.4 cleans 3 times, dries;
D is by 5 μ L GR/Ag@BSA-Ab
2drip to electrode surface, the PBS buffer solution of incubated at room temperature 40 min, pH 7.4 cleans 3 times, dries, and namely obtains the working electrode of electrochemical immunosensor;
Silver/silver chloride reference electrode, the working electrode of platinum filament to electrode and above-mentioned preparation are connected on electrochemical workstation by e;
The PBS buffer solution of f pH 7.4 as end liquid, by the response of chronoamperometry testing electrode pair hydrogen peroxide, according to the current value of gained and the logarithm of CEA concentration linear, drawing curve.
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| CN105675697A (en) * | 2016-01-19 | 2016-06-15 | 济南大学 | Construction method of nanoprobe C60 based electrochemical immunosensor for carcino-embryonic antigens |
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| CN105675697A (en) * | 2016-01-19 | 2016-06-15 | 济南大学 | Construction method of nanoprobe C60 based electrochemical immunosensor for carcino-embryonic antigens |
| CN105699458A (en) * | 2016-02-03 | 2016-06-22 | 洪国粦 | Novel immunosensor for NT-proBNP detection and preparation method thereof |
| CN105928997A (en) * | 2016-07-11 | 2016-09-07 | 山东理工大学 | Preparation method and application of immunosensor based on Au-GQD@PtPd |
| CN106556632A (en) * | 2016-11-10 | 2017-04-05 | 无锡市明盛强力风机有限公司 | A kind of preparation method of modified glassy carbon electrode |
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| CN108693231A (en) * | 2018-05-25 | 2018-10-23 | 江南大学 | A kind of electrochemica biological sensor and preparation method thereof of detection carcinomebryonic antigen |
| CN112730562A (en) * | 2020-12-22 | 2021-04-30 | 河南中泽生物工程有限公司 | Electrochemical immunosensor for detecting tiamulin antigen and preparation method thereof |
| CN112858420A (en) * | 2021-03-30 | 2021-05-28 | 济南大学 | Preparation method and application of sandwich type electrochemical sensor constructed based on vanadium selenide/gold nanoparticles |
| CN112986348A (en) * | 2021-03-30 | 2021-06-18 | 济南大学 | Preparation and application of dual-mode electrochemical biosensor based on transition metal sulfide |
| CN112986348B (en) * | 2021-03-30 | 2022-10-11 | 济南大学 | Preparation and application of dual-mode electrochemical biosensor based on transition metal sulfide |
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