CN111751432A - Preparation of electrochemical immunosensor based on PdCuPt - Google Patents
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Abstract
The invention belongs to the technical field of novel nano composite materials, immunoassay and biosensing, and provides a preparation method of an electrochemical immunosensor based on PdCuPt. The invention takes the ultra-small PdCuPt nanocube with a unique double-dodecahedron structure and the PdCuPt @ Fe-N-rGO formed by assembling Fe-N-rGO nano sheets as an electrochemical signal amplification platform, realizes the quantitative detection of the hepatitis B virus surface antigen HBs-Ag, has the advantages of strong specificity, high sensitivity, low detection limit and the like, and has important scientific significance and application value for the detection of hepatitis B.
Description
Technical Field
The invention belongs to the technical field of novel nano composite materials, immunoassay and biosensing, and provides preparation of an electrochemical immunosensor based on PdCuPt, which is applied to detection of hepatitis B virus surface antigen HBs-Ag.
Background
Malignant tumors have extremely high lethality, and in all diseases, the prevalence rate and the lethality rate of the tumors are in the front, which seriously threatens the life health of people in the world. Although the technology is rapidly developed at present, the cancer is still hard to cure at present, so that the early cancer is rapidly and accurately detected, and the method has great significance for curing the cancer.
Hepatitis b virus infection can cause cancer mortality, the second liver cancer, and is of particular concern. The reliable tumor marker for diagnosing and prognosing the diseases caused by hepatitis B virus infection is determined to be hepatitis B virus surface antigen HBs-Ag through research, so that the development of sensitive, rapid and accurate detection of the hepatitis B virus surface antigen HBs-Ag has important significance for the detection of hepatitis B virus.
The subminiature PdCuPt nanocube composed of the three metals has a unique biconvex structure, small particle size and large surface area, shows better conductivity and catalytic performance compared with single-component particles, and has good biocompatibility and is easy to combine with an antibody, so that sensitive and rapid electrochemical detection is realized; the Fe-N-rGO nanosheet has a large specific surface area, PdCuPt nanocubes can be effectively loaded, and Fe-N is inserted into the rGO so that the Fe-N-rGO nanosheet has better conductivity and catalytic performance; the PdCuPt @ Fe-N-rGO formed by assembling the PdCuPt nanocube and the Fe-N-rGO nano sheet can further realize signal amplification and improve the sensitivity of detecting HBs-Ag of the hepatitis B virus surface antigen.
The invention takes the PdCuPt @ Fe-N-rGO formed by assembling the subminiature PdCuPt nanocube with the unique dodecahedron structure and the Fe-N-rGO nano sheet as an electrochemical signal amplification platform, has the advantages of simple operation, strong specificity, high sensitivity, low detection limit and the like, has good reproducibility, stability and selectivity, and realizes the ultrasensitive detection of the HBs-Ag of the hepatitis B virus surface antigen.
Disclosure of Invention
The invention provides preparation of an electrochemical immunosensor based on PdCuPt, which realizes ultra-sensitive detection of hepatitis B virus surface antigen HBs-Ag.
The invention aims to provide a preparation method of an electrochemical immunosensor based on PdCuPt.
The other purpose of the invention is to use the prepared PdCuPt-based electrochemical immunosensor for detecting hepatitis B virus surface antigen HBs-Ag.
The technical scheme of the invention comprises the following steps:
1. a preparation method of a PdCuPt-based electrochemical immunosensor comprises the following steps:
(1) al for glassy carbon electrode with diameter of 3.0-5.0 mm2O3Polishing the polishing powder into a mirror surface, and ultrasonically cleaning the mirror surface in absolute ethyl alcohol;
(2) dropwise adding the 6.0 muL and 1.0-2.0 mg/mL PdCuPt @ Fe-N-rGO dispersed liquid to the surface of the electrode, washing with ultrapure water, and airing at room temperature;
(3) dropwise adding a hepatitis B virus surface antigen HBs-Ag antibody of 6.0 muL and 5-15 mug/mL to the surface of an electrode, washing the surface of the electrode by a phosphate buffer solution with pH =7.0, and drying in a refrigerator at 4.0 ℃;
(4) continuously dropwise adding bovine serum albumin solution of 3.0 muL and 1-2 mg/mL to the surface of the electrode to seal the non-specific active sites on the surface of the electrode, washing the surface of the electrode by phosphate buffer solution with pH =7.0, and airing in a refrigerator at 4.0 ℃;
(5) continuously dropwise adding a series of hepatitis B virus surface antigen HBs-Ag solutions with different concentrations, wherein the HBs-Ag solutions are 6.0 mu L and 10 fg/mL-100 ng/mL, washing with a phosphate buffer solution with the pH =7.0, and drying in a refrigerator at the temperature of 4.0 ℃ to obtain the PdCuPt-based electrochemical immunosensor.
2. Preparation of a PdCuPt-based electrochemical immunosensor, and preparation of related materials, wherein the preparation steps are as follows:
(1) preparation of PdCuPt bisdodecahedron nano-cube
At room temperature, 10.0-30.0 mg of Pd (acac)25.0-15.0 mg of Pt (acac)210.0-20.0 mg of Cu (acac)2100.0 to 110.0mg of decaalkyltrimethylAdding ammonium bromide and 100.0-110.0 mg of sodium citrate into a 50mL round-bottom flask containing 6.0-10.0 mL of a mixed solvent (oleylamine (3.0-5.0 mL) and N, N-dimethylformamide (3.0-5.0 mL)), stirring the mixture for 30 min, heating the round-bottom flask in an oil bath at 180 ℃ for 24h, washing with a mixed solution of toluene and ethanol (1: 3) and centrifuging (8000 rpm, 15 min) for five times to collect a synthetic product, and storing the collected product in an ethanol solution;
(2) preparation of GO nanosheets
Placing 5-15 g of graphite and 3.5-10.5 g of sodium nitrate into a flask, adding 350-1050 mL of sulfuric acid under ice-bath stirring, adding 10-30 g of potassium permanganate under vigorous stirring, keeping the flask at room temperature for 6 days, then adding sulfuric acid (200-600 mL, 5 wt%) into the mixture, keeping the mixture at 98 ℃ for 2 hours, adding hydrogen peroxide (10-30 mL, 30 wt%) when the temperature is reduced to 50 ℃, stopping the reaction, centrifugally washing, collecting the precipitate, and freeze-drying for 2 days;
(3) preparation of Fe-N-rGO nanosheets
Fe-N-rGO is synthesized by taking the prepared GO nano-sheets as raw materials, GO is dissolved in deionized water (2 mg/mL), ultrasonic treatment is carried out for 3 hours, and 0.5-1.5 mL of FeCl is added3·6H2Adding O (3 mg/mL) water solution into 50mL GO suspension, performing ultrasonic treatment for 3min, freezing with liquid nitrogen, freeze-drying at-60 deg.C for 72 h, and adding the dried sample into NH3Carrying out heat treatment for 1 h at 750 ℃ in the atmosphere to obtain Fe-N-rGO;
(4) preparation of PdCuPt @ Fe-N-rGO
To 10.0mg of prepared Fe-N-rGO was added 5.0-15.0 mL of PdCuPt bisdodecahedral nanocube dispersion (1.0 mg/mL), then the mixture was sonicated for 1 h, after centrifugation, the black precipitate was dried at room temperature, then 20.0mg of black powder was redispersed in 5.0mL of buffer solution (pH = 7.4).
3. A preparation method of an electrochemical immunosensor based on PdCuPt is used for detecting hepatitis B virus surface antigen HBs-Ag, and comprises the following steps:
(1) testing in a three-electrode system by using an electrochemical workstation, taking a saturated calomel electrode as a reference electrode, a platinum wire electrode as a counter electrode and the prepared immunosensor as a working electrode, and testing in 10 mL of phosphate buffer solution with the pH of 5.0-8.5 and containing 5.0 mmol/L of hydrogen peroxide solution;
(2) detecting the analyte by a time-lapse current method, wherein the input voltage is-0.4V, the sampling interval is 0.1 s, and the running time is 400 s;
(3) when the background current tends to be stable, 10 muL and 5 mol/L hydrogen peroxide solution is injected into 10 mL and 50mmol/L phosphate buffer solution with pH =7.0 every 50 s, and the change of the current is recorded;
(3) recording current peak values corresponding to hepatitis B virus surface antigens HBs-Ag under different concentrations;
(4) and obtaining the concentration of the hepatitis B virus surface antigen HBs-Ag in the sample to be detected by using a working curve method.
Advantageous results of the invention
(1) The subminiature trimetal PdCuPt nanocube prepared by the invention has a unique biconodecane structure, small particle size and large surface area, shows better conductivity and catalytic performance compared with single-component particles, has good biocompatibility, is easy to combine with an antibody, and realizes sensitive and rapid electrochemical detection; the Fe-N-rGO nanosheet has a large specific surface area, PdCuPt nanocubes can be effectively loaded, and Fe-N is inserted into the rGO so that the Fe-N-rGO nanosheet has better conductivity and catalytic performance; the PdCuPt @ Fe-N-rGO formed by assembling PdCuPt nanocubes and Fe-N-rGO nano sheets can further realize signal amplification, and the detection limit of the sensor is reduced through the synergistic effect and the multiple amplification effect, so that high-sensitivity and rapid electrochemical detection is realized;
(2) the electrochemical immunosensor based on PdCuPt is used for detecting hepatitis B virus surface antigen HBs-Ag, the linear detection range of the electrochemical immunosensor for the hepatitis B virus surface antigen HBs-Ag is 10 fg/mL-100 ng/mL, and the lowest detection lower limit is 3.3 fg/mL; the electrochemical immunosensor based on PdCuPt can accurately and quantitatively detect the hepatitis B virus surface antigen HBs-Ag.
Detailed Description
The present invention will now be further illustrated by, but not limited to, specific embodiments thereof.
Embodiment 1 preparation method of electrochemical immunosensor based on PdCuPt
(1) Al for glassy carbon electrode with diameter of 3.0 mm2O3Polishing the polishing powder into a mirror surface, and ultrasonically cleaning the mirror surface in absolute ethyl alcohol;
(2) dropwise adding 6.0 muL and 1.0 mg/mL of PdCuPt @ Fe-N-rGO dispersion liquid to the surface of the electrode, washing with ultrapure water, and airing at room temperature;
(3) dropwise adding the HBs-Ag antibody of the hepatitis B virus surface antigen of 6.0 muL and 5 mug/mL to the surface of the electrode, washing the surface of the electrode by phosphate buffer solution with pH =7.0, and drying in a refrigerator at 4.0 ℃;
(4) continuously dropwise adding bovine serum albumin solution of 3.0 muL and 1 mg/mL to the surface of the electrode to seal the non-specific active sites on the surface of the electrode, washing the surface of the electrode by phosphate buffer solution with pH =7.0, and airing in a refrigerator at 4.0 ℃;
(5) continuously dropwise adding a series of hepatitis B virus surface antigen HBs-Ag solutions with different concentrations, wherein the HBs-Ag solutions are 6.0 mu L and 10 fg/mL-100 ng/mL, washing with a phosphate buffer solution with the pH =7.0, and drying in a refrigerator at the temperature of 4.0 ℃ to obtain the PdCuPt-based electrochemical immunosensor.
Embodiment 2 preparation method of electrochemical immunosensor based on PdCuPt
(1) Al for glassy carbon electrode with diameter of 4.0 mm2O3Polishing the polishing powder into a mirror surface, and ultrasonically cleaning the mirror surface in absolute ethyl alcohol;
(2) dropwise adding 6.0 muL and 1.5 mg/mL of PdCuPt @ Fe-N-rGO dispersion liquid to the surface of the electrode, washing with ultrapure water, and airing at room temperature;
(3) dropwise adding the HBs-Ag antibody of the hepatitis B virus surface antigen of 6.0 muL and 10 mug/mL to the surface of the electrode, washing the surface of the electrode by phosphate buffer solution with pH =7.0, and drying in a refrigerator at 4.0 ℃;
(4) continuously dropwise adding bovine serum albumin solution of 3.0 muL and 1.5 mg/mL to the surface of the electrode to seal the non-specific active sites on the surface of the electrode, washing the surface of the electrode by phosphate buffer solution with pH =7.0, and airing in a refrigerator at 4.0 ℃;
(5) continuously dropwise adding a series of hepatitis B virus surface antigen HBs-Ag solutions with different concentrations, wherein the HBs-Ag solutions are 6.0 mu L and 10 fg/mL-100 ng/mL, washing with a phosphate buffer solution with the pH =7.0, and drying in a refrigerator at the temperature of 4.0 ℃ to obtain the PdCuPt-based electrochemical immunosensor.
Embodiment 3 preparation method of electrochemical immunosensor based on PdCuPt
(1) Al for glassy carbon electrode with diameter of 5.0 mm2O3Polishing the polishing powder into a mirror surface, and ultrasonically cleaning the mirror surface in absolute ethyl alcohol;
(2) dropwise adding the 6.0 muL and 2.0 mg/mL PdCuPt @ Fe-N-rGO dispersed liquid to the surface of the electrode, washing with ultrapure water, and airing at room temperature;
(3) dropwise adding the HBs-Ag antibody of the hepatitis B virus surface antigen of 6.0 muL and 15 mug/mL to the surface of the electrode, washing the surface of the electrode by phosphate buffer solution with pH =7.0, and drying in a refrigerator at 4.0 ℃;
(4) continuously dropwise adding bovine serum albumin solution of 3.0 muL and 2 mg/mL to the surface of the electrode to seal the non-specific active sites on the surface of the electrode, washing the surface of the electrode by phosphate buffer solution with pH =7.0, and airing in a refrigerator at 4.0 ℃;
(5) continuously dropwise adding a series of hepatitis B virus surface antigen HBs-Ag solutions with different concentrations, wherein the HBs-Ag solutions are 6.0 mu L and 10 fg/mL-100 ng/mL, washing with a phosphate buffer solution with the pH =7.0, and drying in a refrigerator at the temperature of 4.0 ℃ to obtain the PdCuPt-based electrochemical immunosensor.
Preparation of PdCuPt bisdodecahedral nanocubes as described in example 4
At room temperature, 10.0mg of Pd (acac)25.0mg of Pt (acac)210.0mg of Cu (acac)2100.0mg of decaalkyltrimethylammonium bromide, 100.0mg of sodium citrate were added to a 50mL round-bottomed flask containing 6.0 mL of a mixed solvent (oleylamine (3.0 mL) and N, N-dimethylformamide (3.0 mL)), the mixture was stirred for 30 min, and then the round-bottomed flask was heated in an oil bath at 180 ℃ for 24h, with toluene and N, N-dimethylformamide (3.0 mL)The ethanol (1: 3) mixed solution was washed and centrifuged (8000 rpm, 15 min) for five cycles to collect the synthesized product, and the collected product was stored in the ethanol solution.
Preparation of PdCuPt bisdodecahedral nanocubes as described in example 5
20.0mg of Pd (acac)210.0mg of Pt (acac)215.0 mg of Cu (acac)2105.0mg of decaalkyltrimethylammonium bromide and 105.0mg of sodium citrate were added to a 50mL round-bottom flask containing 8.0 mL of a mixed solvent (oleylamine (4.0 mL) and N, N-dimethylformamide (4.0 mL)), the mixture was stirred for 30 min, then the round-bottom flask was heated in an oil bath at 180 ℃ for 24h, and the synthesized product was collected by washing with a mixed solution of toluene and ethanol (1: 3) and centrifuging (8000 rpm, 15 min) for five cycles, and the collected product was stored in an ethanol solution.
Preparation of PdCuPt bisdodecahedral nanocubes as described in example 6
At room temperature, 30.0 mg of Pd (acac)215.0 mg of Pt (acac)220.0mg of Cu (acac)2110.0mg of decaalkyltrimethylammonium bromide and 110.0mg of sodium citrate were added to a 50mL round-bottomed flask containing 10.0 mL of a mixed solvent (oleylamine (5.0 mL) and N, N-dimethylformamide (5.0 mL)), the mixture was stirred for 30 min, then the round-bottomed flask was heated in an oil bath at 180 ℃ for 24h, and the synthesized product was collected by washing with a mixed solution of toluene and ethanol (1: 3) and centrifuging (8000 rpm, 15 min) for five cycles, and the collected product was stored in an ethanol solution.
Preparation of PdCuPt @ Fe-N-rGO dispersion as described in example 7
(1) Preparation of GO nanosheets
Placing 5 g of graphite and 3.5 g of sodium nitrate into a flask, adding 350 mL of sulfuric acid under ice-bath stirring, adding 10 g of potassium permanganate under vigorous stirring, keeping the flask at room temperature for 6 days, then adding sulfuric acid (200 mL, 5 wt%) into the mixture, keeping the mixture at 98 ℃ for 2 hours, adding hydrogen peroxide (10 mL, 30 wt%) when the temperature is reduced to 50 ℃ to terminate the reaction, centrifugally washing, collecting the precipitate, and freeze-drying for 2 days;
(2) preparation of Fe-N-rGO nanosheets
Fe-N-rGO is synthesized by taking the prepared GO nano-sheets as raw materials, GO is dissolved in deionized water (2 mg/mL), ultrasonic treatment is carried out for 3 hours, and 0.5 mL of FeCl is added3·6H2Adding O (3 mg/mL) water solution into 50mL GO suspension, performing ultrasonic treatment for 3min, freezing with liquid nitrogen, freeze-drying at-60 deg.C for 72 h, and adding the dried sample into NH3Carrying out heat treatment for 1 h at 750 ℃ in the atmosphere to obtain Fe-N-rGO;
(3) preparation of PdCuPt @ Fe-N-rGO
To 10.0mg of prepared Fe-N-rGO was added 5.0mL of pdcutpt bisdodecahedral nanocube dispersion (1.0 mg/mL), then the mixture was sonicated for 1 h, after centrifugation, the black precipitate was dried at room temperature, then 20.0mg of black powder was redispersed in 5.0mL of buffer solution (pH = 7.4).
Preparation of PdCuPt @ Fe-N-rGO dispersion as described in example 8
(1) Preparation of GO nanosheets
Placing 10 g of graphite and 7 g of sodium nitrate into a flask, adding 700 mL of sulfuric acid under ice bath stirring, adding 20 g of potassium permanganate under vigorous stirring, keeping the flask at room temperature for 6 days, then adding sulfuric acid (400 mL, 5 wt%) into the mixture, keeping the mixture at 98 ℃ for 2 hours, adding hydrogen peroxide (20 mL, 30 wt%) when the temperature is reduced to 50 ℃ to terminate the reaction, centrifugally washing, collecting the precipitate, and freeze-drying for 2 days;
(2) preparation of Fe-N-rGO nanosheets
Fe-N-rGO is synthesized by taking the prepared GO nano-sheets as raw materials, GO is dissolved in deionized water (2 mg/mL), ultrasonic treatment is carried out for 3 hours, and 1.0 mL of FeCl is added3·6H2Adding O (3 mg/mL) water solution into 50mL GO suspension, performing ultrasonic treatment for 3min, freezing with liquid nitrogen, freeze-drying at-60 deg.C for 72 h, and adding the dried sample into NH3Carrying out heat treatment for 1 h at 750 ℃ in the atmosphere to obtain Fe-N-rGO;
(3) preparation of PdCuPt @ Fe-N-rGO
To 10.0mg of prepared Fe-N-rGO was added 10 mL of PdCuPt bisdodecahedral nanocube dispersion (1.0 mg/mL), then the mixture was sonicated for 1 h, after centrifugation, the black precipitate was dried at room temperature, then 20.0mg of black powder was redispersed in 5.0mL of buffer solution (pH = 7.4).
Preparation of PdCuPt @ Fe-N-rGO dispersion as described in example 9
(1) Preparation of GO nanosheets
Placing 15 g of graphite and 10.5 g of sodium nitrate into a flask, adding 1050mL of sulfuric acid under ice bath stirring, adding 30 g of potassium permanganate under vigorous stirring, keeping the flask at room temperature for 6 days, then adding sulfuric acid (600 mL, 5 wt%) into the mixture, keeping the mixture at 98 ℃ for 2 hours, adding hydrogen peroxide (30 mL, 30 wt%) when the temperature is reduced to 50 ℃ to terminate the reaction, centrifugally washing, collecting the precipitate, and freeze-drying for 2 days;
(2) preparation of Fe-N-rGO nanosheets
Fe-N-rGO is synthesized by taking the prepared GO nano-sheets as raw materials, GO is dissolved in deionized water (2 mg/mL), ultrasonic treatment is carried out for 3 hours, and 1.5 mL of FeCl is added3·6H2Adding O (3 mg/mL) water solution into 50mL GO suspension, performing ultrasonic treatment for 3min, freezing with liquid nitrogen, freeze-drying at-60 deg.C for 72 h, and adding the dried sample into NH3Carrying out heat treatment for 1 h at 750 ℃ in the atmosphere to obtain Fe-N-rGO;
(3) preparation of PdCuPt @ Fe-N-rGO
To 10.0mg of prepared Fe-N-rGO was added 15.0 mL of pdcutpt bisdodecahedral nanocube dispersion (1.0 mg/mL), then the mixture was sonicated for 1 h, after centrifugation, the black precipitate was dried at room temperature, then 20.0mg of black powder was redispersed in 5.0mL of buffer solution (pH = 7.4).
Detection of hepatitis B Virus surface antigen HBs-Ag by the PdCuPt-based electrochemical immunosensor described in example 10
(1) Testing in a three-electrode system by using an electrochemical workstation, taking a saturated calomel electrode as a reference electrode, a platinum wire electrode as a counter electrode and the prepared immunosensor as a working electrode, and testing in 10 mL of phosphate buffer solution with the pH of 5.0-8.5 and containing 5.0 mmol/L of hydrogen peroxide solution;
(2) detecting the analyte by a time-lapse current method, wherein the input voltage is-0.4V, the sampling interval is 0.1 s, and the running time is 400 s;
(3) when the background current tends to be stable, 10 muL and 5 mol/L hydrogen peroxide solution is injected into 10 mL and 50mmol/L phosphate buffer solution with pH =7.0 every 50 s, and the change of the current is recorded;
(3) recording current peak values corresponding to hepatitis B virus surface antigens HBs-Ag under different concentrations;
(4) the linear detection range of the hepatitis B virus surface antigen HBs-Ag in the sample to be detected is 10 fg/mL-100 ng/mL by using a working curve method, and the lowest detection lower limit is 3.3 fg/mL.
Claims (4)
1. The preparation method of the PdCuPt-based electrochemical immunosensor is characterized by comprising the following steps:
(1) preparation of PdCuPt @ Fe-N-rGO: combining a separately prepared subminiature PdCuPt bisdodecahedral nano cube with a Fe-N-rGO nano sheet;
(2) constructing an electrochemical immunosensor of unmarked PdCuPt @ Fe-N-rGO, measuring the content of HBs-Ag of the hepatitis B virus surface antigen, and drawing a working curve.
2. The electrochemical immunosensor for detecting hepatitis B virus surface antigen HBs-Ag according to claim 1, wherein the electrochemical immunosensor comprises: the PdCuPt @ Fe-N-rGO in the step (1) is prepared as follows:
(1) preparation of PdCuPt bisdodecahedron nano-cube
At room temperature, 10.0-30.0 mg of Pd (acac)25.0-15.0 mg of Pt (acac)210.0-20.0 mg of Cu (acac)2100.0 to 110.0mg of decaalkyltrimethylammonium bromide and 100.0 to 110.0mg of sodium citrate are added to a 50mL round bottom flask containing 6.0 to 10.0 mL of a mixed solvent (oleylamine (3.0 to 5.0 mL) and N, N-dimethylformamide (3.0 to 5.0 mL)), the mixture is stirred for 30 min, then the round bottom flask is heated in an oil bath at 180 ℃ for 24h, washed with a mixed solution of toluene and ethanol (1: 3) and centrifuged (8000 rpm,15 min) was circulated five times to collect the synthesis product, which was stored in ethanol solution;
(2) preparation of GO nanosheets
Placing 5-15 g of graphite and 3.5-10.5 g of sodium nitrate into a flask, adding 350-1050 mL of sulfuric acid under ice-bath stirring, adding 10-30 g of potassium permanganate under vigorous stirring, keeping the flask at room temperature for 6 days, then adding sulfuric acid (200-600 mL, 5 wt%) into the mixture, keeping the mixture at 98 ℃ for 2 hours, adding hydrogen peroxide (10-30 mL, 30 wt%) when the temperature is reduced to 50 ℃, stopping the reaction, centrifugally washing, collecting the precipitate, and freeze-drying for 2 days;
(3) preparation of Fe-N-rGO nanosheets
Fe-N-rGO is synthesized by taking the prepared GO nano-sheets as raw materials, GO is dissolved in deionized water (2 mg/mL), ultrasonic treatment is carried out for 3 hours, and 0.5-1.5 mL of FeCl is added3·6H2Adding O (3 mg/mL) water solution into 50mL GO suspension, performing ultrasonic treatment for 3min, freezing with liquid nitrogen, freeze-drying at-60 deg.C for 72 h, and adding the dried sample into NH3Carrying out heat treatment for 1 h at 750 ℃ in the atmosphere to obtain Fe-N-rGO;
(4) preparation of PdCuPt @ Fe-N-rGO
To 10.0mg of prepared Fe-N-rGO was added 5.0-15.0 mL of PdCuPt bisdodecahedral nanocube dispersion (1.0 mg/mL), then the mixture was sonicated for 1 h, after centrifugation, the black precipitate was dried at room temperature, then 20.0mg of black powder was redispersed in 5.0mL of buffer solution (pH = 7.4).
3. The electrochemical immunosensor for detecting hepatitis B virus surface antigen HBs-Ag according to claim 1, wherein the electrochemical immunosensor comprises: the construction of the electrochemical immunosensor in the step (2) specifically comprises the following steps:
(1) al for glassy carbon electrode with diameter of 3.0-5.0 mm2O3Polishing the polishing powder into a mirror surface, and ultrasonically cleaning the mirror surface in absolute ethyl alcohol;
(2) dropwise adding the 6.0 muL and 1.0-2.0 mg/mL PdCuPt @ Fe-N-rGO dispersed liquid to the surface of the electrode, washing with ultrapure water, and airing at room temperature;
(3) dropwise adding a hepatitis B virus surface antigen HBs-Ag antibody of 6.0 muL and 5-15 mug/mL to the surface of an electrode, washing the surface of the electrode by a phosphate buffer solution with pH =7.0, and drying in a refrigerator at 4.0 ℃;
(4) continuously dropwise adding bovine serum albumin solution of 3.0 muL and 1-2 mg/mL to the surface of the electrode to seal the non-specific active sites on the surface of the electrode, washing the surface of the electrode by phosphate buffer solution with pH =7.0, and airing in a refrigerator at 4.0 ℃;
(5) continuously dropwise adding a series of hepatitis B virus surface antigen HBs-Ag solutions with different concentrations, wherein the HBs-Ag solutions are 6.0 mu L and 10 fg/mL-100 ng/mL, washing with a phosphate buffer solution with the pH =7.0, and drying in a refrigerator at the temperature of 4.0 ℃ to obtain the PdCuPt-based electrochemical immunosensor.
4. The electrochemical immunosensor for detecting hepatitis B virus surface antigen HBs-Ag according to claim 1, wherein the electrochemical immunosensor comprises: measuring the HBs-Ag on the surface of the hepatitis B virus in the step (2), and drawing a working curve, wherein the working curve is as follows:
(1) preparing hepatitis B virus surface antigen HBs-Ag solutions with different concentrations by adopting 10 mL of phosphate buffer solution with the pH value of 7.0 of 50mmol/L, respectively dripping 6 μ L of hepatitis B virus surface antigen HBs-Ag solutions with different concentrations on the surface of an electrode, reacting for 0.5-2 h, connecting the electrode to an electrochemical workstation after airing, respectively soaking the electrode in the phosphate buffer solution with the pH value of 7.0 to measure the redox current change of the electrode, injecting 10 μ L of hydrogen peroxide solution and 5 mol/L of hydrogen peroxide solution into the phosphate buffer solution with the pH value of 7.0 of 10 mL of 50mmol/L every 50 s when the background current tends to be stable, and recording the current change;
(2) drawing a working curve according to the linear relation between the obtained current difference and the concentration of the hepatitis B virus surface antigen HBs-Ag;
(3) and obtaining the concentration of the hepatitis B virus surface antigen HBs-Ag in the sample to be detected by using a working curve method.
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