CN110057892B - Preparation method and application of mHPBs-Hemin/rGO labeled electrochemical immunosensor - Google Patents

Preparation method and application of mHPBs-Hemin/rGO labeled electrochemical immunosensor Download PDF

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CN110057892B
CN110057892B CN201910333993.7A CN201910333993A CN110057892B CN 110057892 B CN110057892 B CN 110057892B CN 201910333993 A CN201910333993 A CN 201910333993A CN 110057892 B CN110057892 B CN 110057892B
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hemin
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rgo
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李月云
张春燕
禹晓东
张栓
贾翌雷
李新进
徐振
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Shandong University of Technology
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Abstract

The invention belongs to the technical field of novel nano materials, immunoassay and biosensing detection, and provides a preparation method and application of an mHPBs-Hemin/rGO labeled electrochemical immunosensor. According to the invention, the mesoporous hollow Prussian blue nano cubic spaced hemin functionalized graphene porous composite material is introduced into the construction of an electrochemical immunosensor as a current signal amplification mechanism for the first time, so that a current response signal is efficiently amplified, and quantitative and sensitive detection of aflatoxin is realized. The electrochemical immunosensor disclosed by the invention is simple and convenient to operate, low in cost, high in sensitivity, wide in detection range, and has important scientific significance and application value for quantitative and sensitive detection of aflatoxin.

Description

Preparation method and application of mHPBs-Hemin/rGO labeled electrochemical immunosensor
Technical Field
The invention belongs to the technical field of novel functional nano materials, immunoassay and biosensing detection, and provides a preparation method and application of an mHPBs-Hemin/rGO labeled electrochemical immunosensor.
Background
Aflatoxin is a very toxic mycotoxin, and as early as 1993, aflatoxin is defined as a carcinogen by the world health organization WHO, among the carcinogens known at present, is the first one. Aflatoxin is widely present in grain and oil crops such as corn, wheat and peanut, and various meat and dairy products, has strong destructiveness to human and animal liver tissues, is inevitably taken in production and life, and can cause cancer and even death in severe cases. According to the national food sanitation standard, the allowable maximum content of aflatoxin B1 in corn, peanut and peanut oil is 20 mug/kg, and the standard of countries of European Union and the like is 2 mug/kg, so that the sensitive and accurate detection of aflatoxin has important significance for the safety of food industry.
At present, the aflatoxin detection mainly comprises a thin film chromatography method and a liquid chromatography method, but the two methods have long detection period, complicated sample pretreatment and complex operation, and can not meet the requirements of the modern society. With the continuous development of nanotechnology and immunoassay technology, electrochemical immunosensors stand out and show good application prospects in the aspects of disease monitoring, environmental protection, food safety and the like. The electrochemical immunosensor for sensitively detecting aflatoxin is designed and prepared by taking acidified gold nanoparticles MUA-Au NPs as a substrate material and taking mesoporous hollow Prussian blue nano cubic spaced Hemin functionalized graphene mHPBs-Hemin/rGO as a marker for amplifying signals.
The gold nano-particle Au NPs have good conductivity and biocompatibility, and can not only efficiently transfer charges, but also provide stable binding sites for antibodies after being acidified by the sulfydryl undecanoic acid MUA, so that the stability of the construction of the working electrode sensing interface of the electrochemical immunosensor is improved. Hemin functionalized graphene pair H2O2The reduction reaction of (a) has a good catalytic effect,and effectively overcomes the defect that Hemin is easy to agglomerate and inactivate in the solution. The mesoporous hollow Prussian blue nano-cube with the framework structure has large specific surface area and has high specific surface area to H2O2The reduction has a certain catalytic action, mHPBs are used as a Hemin/rGO spacer to form a composite material with a complex pore structure, and a multi-layer porous structure of the composite material can form a large number of reaction active sites to efficiently catalyze H2O2The reduction effectively amplifies the electric response signal and improves the sensitivity of the electrochemical immunosensor.
The electrochemical immunosensor designed and constructed by the invention realizes quantitative and sensitive detection of aflatoxin, has low detection limit, wide detection range, good reproducibility and stability, and good application prospect in sensitive and rapid detection of aflatoxin.
Disclosure of Invention
The invention provides a preparation method and application of an mHPBs-Hemin/rGO labeled electrochemical immunosensor, and realizes accurate and sensitive detection of aflatoxin.
One of the purposes of the invention is to provide a preparation method of an mHPBs-Hemin/rGO labeled electrochemical immunosensor.
The second purpose of the invention is to apply the prepared mHPBs-Hemin/rGO labeled electrochemical immunosensor to the detection of aflatoxin.
The technical scheme of the invention comprises the following steps.
1. A preparation method of mHPBs-Hemin/rGO labeled electrochemical immunosensor comprises the following steps:
(1) polishing a glassy carbon electrode with the diameter of 4.0 mm by using alumina powder to form a mirror surface, and ultrasonically cleaning the mirror surface in ultrapure water;
(2) dropwise adding 6.0 muA-Au NPs to the surface of the electrode, drying at room temperature, cleaning the surface of the electrode with ultrapure water, and drying;
(3) continuously capturing the aflatoxin capture antibody Ab of 6.0 muL and 10.0-20.0 mug/mL1Dropwise adding the solution to the surface of an electrode, and placing the electrode in a refrigerator at 4.0 ℃ for airing;
(4) dropwise adding a bovine serum albumin BSA solution of 3.0 muL and 0.5-1.5% to the surface of the electrode to seal non-specific active sites on the surface of the electrode, washing the surface of the electrode by using a phosphate buffer solution with pH =7.2, and airing in a refrigerator of 4.0 ℃;
(5) dropwise adding a series of aflatoxin antigen solutions with different concentrations, namely 6.0 muL and 1.0 pg/mL-100.0 ng/mL, placing in a 4.0 ℃ refrigerator for 30.0-40.0 min, washing with a phosphate buffer solution with pH =7.2, and placing in a 4.0 ℃ refrigerator for airing;
(6) dropwise adding 6.0 muL and 0.5-2.5 mg/mL mHPBs-Hemin/rGO labeled aflatoxin antibody mHPBs-Hemin/rGO-Ab2And (3) placing the mixture in a refrigerator at 4.0 ℃ for 30.0-40.0 min, washing the mixture by using a phosphate buffer solution with the pH =7.2, and placing the mixture in the refrigerator at 4.0 ℃ for airing to obtain the mHPBs-Hemin/rGO labeled electrochemical immunosensor.
2. The preparation of MUA-Au NPs comprises the following steps:
adding 99.0 mL of ultrapure water into a 500 mL three-neck flask, continuously adding 0.5-1.5 mL of chloroauric acid solution with the mass fraction of 1.0%, heating in an oil bath at 100 ℃ for 15.0-25.0 min under magnetic stirring, adding 1.5-3.5 mL of sodium citrate solution with the mass fraction of 1.0%, continuously heating in an oil bath at 100 ℃ for 10.0-20.0 min, and cooling to room temperature to obtain Au NPs;
centrifuging 1.0-2.0 mL of Au NPs at 12000 rpm for 20 min, adding 1.0-2.0 mL of ultrapure water into the precipitate, and performing ultrasonic treatment for 5.0-10.0 min to obtain an Au NPs dispersion liquid A; adding 0.05 mL of 15.0-25.0 mmol/L of 11-mercaptoundecanoic acid MUA ethanol solution into 0.5-1.0 mL of ultrapure water, and carrying out ultrasonic treatment for 60 min to obtain a solution B; and slowly adding the solution B into the Au NPs dispersion liquid A, oscillating for 2.0-4.0 h at room temperature, and centrifuging the obtained solution at 9000 rpm for 30 min to obtain MUA-Au NPs.
3. mHPBs-Hemin/rGO-Ab2The preparation method comprises the following steps:
(1) preparation of mHPBs
Adding 1.0-5.0 g of polyvinylpyrrolidone and 100.0-160.0 mg of potassium ferricyanide into 20.0-60.0 mL of 0.1 mol/L hydrochloric acid solution under magnetic stirring, continuing to magnetically stir for 20.0-40.0 min, reacting the obtained clear solution at 80.0 ℃ for 16.0-24.0 h, centrifuging at 10000 rpm for 5 min, respectively washing the obtained precipitate with absolute ethyl alcohol and ultrapure water for three times, and drying at room temperature for 12.0 h to obtain mesoporous Prussian blue seeds;
adding 1.0-5.0 g of polyvinylpyrrolidone, 100.0-160.0 mg of potassium ferricyanide and 5.0-15.0 mg of mesoporous Prussian blue seeds into 20.0-60.0 mL of 0.01 mol/L hydrochloric acid solution under magnetic stirring, continuing to magnetically stir for 20.0-40.0 min, reacting the mixed liquid at 80 ℃ for 10.0-14.0 h, centrifuging at 10000 rpm for 5 min, respectively washing the obtained precipitate with absolute ethyl alcohol and ultrapure water for three times, drying at room temperature for 12.0 h, and obtaining Prussian blue mPBs through mesopores;
weighing 50.0-150.0 mg of polyvinylpyrrolidone and 15.0-25.0 mg of mPBS, adding the mixture into 15.0-25.0 mL of 1.0 mol/L hydrochloric acid solution under magnetic stirring, continuing to magnetically stir for 1.0-3.0 h, transferring the mixed liquid into a polytetrafluoroethylene high-pressure reaction kettle, preserving heat at 140 ℃ for 3.0-5.0 h, centrifuging at 10000 rpm for 5 min, and respectively washing the obtained precipitate with absolute ethyl alcohol and ultrapure water for three times to prepare mesoporous hollow Prussian blue nano cubic mHPBs;
(2) preparation of mHPBs-Hemin/rGO
Dissolving 5.0-10.0 mg Hemin and 5.0-10.0 g diaminopyridine in 50.0-100.0 mL of graphene oxide GO solution of 1.0 mg/mL, carrying out ultrasonic dispersion for 20.0-40.0 min, continuously adding 10.0 mL of mHPBs solution of 5.0-15.0 mg/mL, carrying out magnetic stirring for 2.0 h, transferring the mixed liquid into a polytetrafluoroethylene high-pressure reaction kettle, carrying out heat preservation for 3.0-5.0 h at 130 ℃, centrifuging for 10 min at 8000 rpm, transferring the precipitate into a 50 mL culture dish, freezing for 12 h in a refrigerator at-20.0 ℃, putting into a freeze dryer, and carrying out freeze drying at-80.0 ℃ to obtain mHPBs-Hemin/rGO;
（3）mHPBs-Hemin/rGO-Ab2preparation of
Adding 2.0-12.0 mg of mHPBs-Hemin/rGO into 2.0 mL of phosphate buffer solution with pH =7.2, performing ultrasonic treatment for 10.0-20.0 min, and adding the mixed liquid into 2.0 mL of 20.0 microgram/mL of aflatoxinPlacing the antibody solution in a constant temperature oscillator at 4.0 ℃ for oscillation for 8.0-12.0 h to prepare mHPBs-Hemin/rGO-Ab2And storing in a refrigerator at 4.0 ℃ for later use.
4. Detecting aflatoxin by the following steps:
(1) an electrochemical workstation is used, a saturated calomel electrode is used as a reference electrode, a platinum wire electrode is used as a counter electrode, and the prepared sensor and the working electrode form a three-electrode system, and the three-electrode system is tested in 10.0 mL phosphate buffer solution with the pH = 5.91-7.73;
(2) using a time-current method to carry out the steps of setting the initial potential to be-0.4V, the sampling interval to be 0.1 s and the running time to be 400.0 s;
(3) after the background current stabilized, 10.0 μ L, 5.0 mol/L of H was injected into 10.0 mL of pH =7.2 phosphate buffer solution2O2After the current is stabilized again, recording the change of the current;
(4) measuring by adopting aflatoxin antigens with different concentrations, and recording current value changes corresponding to the aflatoxin antigens with different concentrations;
(5) and obtaining the concentration of the aflatoxin antigen in the sample to be detected by using a working curve method.
The aflatoxin antigen mentioned above is AFB 1.
The raw materials used in the present invention are all available from chemical agents companies or biopharmaceutical companies.
Advantageous results of the invention
(1) The MUA-Au NPs are used as the substrate material of the working electrode of the electrochemical immunosensor, so that the charges can be efficiently transferred, and the aflatoxin antibody can be stably combined, so that the detection sensitivity of the working electrode of the electrochemical immunosensor and the stability of the construction of a sensing interface are improved.
(2) The invention applies mHPBs-Hemin/rGO as a signal amplification mechanism to an electrochemical immunosensor for the first time. The graphene has a very large specific surface area, and the graphene is taken as a supporting material to load Hemin, so that the characteristic that Hemin is easy to agglomerate and inactivate in an oxidation medium is reduced, and the graphene has peroxidase-like HeminGood catalytic ability. Prussian blue is a typical frame structure substance, forms a mesoporous hollow structure after being etched, has increased specific surface area, and is specific to H2O2The reduction reaction of (2) has a certain catalytic action. The mesoporous hollow Prussian blue nano-cubic mHPBs are inserted into the graphene sheet layer as the spacer, so that the irreversible stacking phenomenon of graphene can be effectively reduced, the composite material forms a complex porous structure, the specific surface area of the composite material is increased, more reactive active sites are exposed, and H in the electrolyte is catalyzed more efficiently2O2The reduction reaction of the electrochemical immunosensor can effectively amplify response signals and improve the sensitivity of the electrochemical immunosensor.
(3) The linear range of the electrochemical immunosensor marked by the mHPBs-Hemin/rGO for detecting the aflatoxin AFB1 is 1.0 pg/mL-100.0 ng/mL, and the detection limit is 0.33 pg/mL, so that the mHPBs-Hemin/rGO electrochemical immunosensor can achieve the purpose of accurate quantitative detection, and has good application prospects for high-sensitivity and high-selectivity quantitative detection of the aflatoxin.
Detailed Description
The present invention will now be further described in detail by way of specific embodiments thereof, but not limited thereto.
Example 1 a method for preparing mhbps-Hemin/rGO labeled electrochemical immunosensor, comprising the steps of:
(1) polishing a glassy carbon electrode with the diameter of 4.0 mm by using alumina powder to form a mirror surface, and ultrasonically cleaning the mirror surface in ultrapure water;
(2) dropwise adding 6.0 muA-Au NPs to the surface of the electrode, drying at room temperature, cleaning the surface of the electrode with ultrapure water, and drying;
(3) continuing to capture the aflatoxin capture antibody Ab at 6.0 muL and 10.0 mug/mL1Dropwise adding the solution to the surface of an electrode, and placing the electrode in a refrigerator at 4.0 ℃ for airing;
(4) dropwise adding a bovine serum albumin BSA solution of 3.0 muL and 0.5% to the surface of the electrode to seal non-specific active sites on the surface of the electrode, washing the surface of the electrode by using a phosphate buffer solution with pH =7.2, and airing in a refrigerator of 4.0 ℃;
(5) dropwise adding a series of aflatoxin antigen solutions with different concentrations, namely 6.0 muL and 1.0 pg/mL-100.0 ng/mL, placing in a 4.0 ℃ refrigerator for 30.0 min, washing with a phosphate buffer solution with pH =7.2, and placing in a 4.0 ℃ refrigerator for airing;
(6) dropwise adding 6.0 muL and 0.5 mg/mL mHPBs-Hemin/rGO marked aflatoxin antibody mHPBs-Hemin/rGO-Ab2After 30.0 min in a refrigerator at 4.0 ℃, washing with phosphate buffer solution with pH =7.2, and airing in the refrigerator at 4.0 ℃ to obtain the mHPBs-Hemin/rGO labeled electrochemical immunosensor.
Embodiment 2 a method for preparing mhbps-Hemin/rGO labeled electrochemical immunosensor, comprising the steps of:
(1) polishing a glassy carbon electrode with the diameter of 4.0 mm by using alumina powder to form a mirror surface, and ultrasonically cleaning the mirror surface in ultrapure water;
(2) dropwise adding 6.0 muA-Au NPs to the surface of the electrode, drying at room temperature, cleaning the surface of the electrode with ultrapure water, and drying;
(3) continuing to capture the aflatoxin capture antibody Ab at 6.0 muL and 15.0 mug/mL1Dropwise adding the solution to the surface of an electrode, and placing the electrode in a refrigerator at 4.0 ℃ for airing;
(4) dropwise adding a bovine serum albumin BSA solution of 3.0 muL and 1.0% to the surface of the electrode to seal non-specific active sites on the surface of the electrode, washing the surface of the electrode by using a phosphate buffer solution with pH =7.2, and airing in a refrigerator of 4.0 ℃;
(5) dropwise adding a series of aflatoxin antigen solutions with different concentrations, namely 6.0 muL and 1.0 pg/mL-100.0 ng/mL, placing in a refrigerator at 4.0 ℃ for 35.0 min, washing with a phosphate buffer solution with pH =7.2, and placing in a refrigerator at 4.0 ℃ for airing;
(6) dropwise adding 6.0 muL and 1.5 mg/mL mHPBs-Hemin/rGO marked aflatoxin antibody mHPBs-Hemin/rGO-Ab2After 35.0 min in a refrigerator at 4.0 ℃, washing with phosphate buffer solution with pH =7.2, and airing in the refrigerator at 4.0 ℃ to obtain the mHPBs-Hemin/rGO labeled electrochemical immunosensor.
Example 3 a method for preparing mHPBs-Hemin/rGO labeled electrochemical immunosensor includes the following steps:
(1) polishing a glassy carbon electrode with the diameter of 4.0 mm by using alumina powder to form a mirror surface, and ultrasonically cleaning the mirror surface in ultrapure water;
(2) dropwise adding 6.0 muA-Au NPs to the surface of the electrode, drying at room temperature, cleaning the surface of the electrode with ultrapure water, and drying;
(3) continuing to capture the aflatoxin capture antibody Ab at 6.0 muL and 20.0 mug/mL1Dropwise adding the solution to the surface of an electrode, and placing the electrode in a refrigerator at 4.0 ℃ for airing;
(4) dropwise adding a bovine serum albumin BSA solution of 3.0 muL and 1.5% to the surface of the electrode to seal non-specific active sites on the surface of the electrode, washing the surface of the electrode by using a phosphate buffer solution with pH =7.2, and airing in a refrigerator of 4.0 ℃;
(5) dropwise adding a series of aflatoxin antigen solutions with different concentrations, namely 6.0 muL and 1.0 pg/mL-100.0 ng/mL, placing in a 4.0 ℃ refrigerator for 40.0 min, washing with a phosphate buffer solution with pH =7.2, and placing in a 4.0 ℃ refrigerator for airing;
(6) dropwise adding 6.0 muL and 2.5 mg/mL mHPBs-Hemin/rGO marked aflatoxin antibody mHPBs-Hemin/rGO-Ab2After 40.0 min in a 4.0 ℃ refrigerator, washing with phosphate buffer solution with pH =7.2, and airing in the 4.0 ℃ refrigerator to obtain the mHPBs-Hemin/rGO labeled electrochemical immunosensor.
Example 4 preparation of MUA-Au NPs, the procedure was as follows:
adding 99.0 mL of ultrapure water into a 500 mL three-neck flask, continuously adding 0.5 mL of chloroauric acid solution with the mass fraction of 1.0%, heating for 15.0 min in a 100 ℃ oil bath under magnetic stirring, adding 1.5 mL of sodium citrate solution with the mass fraction of 1.0%, continuously heating for 10.0 min in a 100 ℃ oil bath, and cooling to room temperature to obtain Au NPs;
centrifuging 1.0 mL of Au NPs at 12000 rpm for 20 min, adding 1.0 mL of ultrapure water into the precipitate, and performing ultrasonic treatment for 5.0 min to obtain an Au NPs dispersion liquid A; adding 0.05 mL of 15.0 mmol/L of 11-mercaptoundecanoic acid MUA ethanol solution into 0.5 mL of ultrapure water, and carrying out ultrasonic treatment for 60 min to obtain a solution B; and slowly adding the solution B into the Au NPs dispersion liquid A, shaking for 2.0 h at room temperature, and centrifuging the obtained solution at 9000 rpm for 30 min to obtain MUA-Au NPs.
Example 5 preparation of MUA-Au NPs, the procedure was as follows:
adding 99.0 mL of ultrapure water into a 500 mL three-neck flask, continuously adding 1.0 mL of chloroauric acid solution with the mass fraction of 1.0%, heating in 100 ℃ oil bath for 20.0 min under magnetic stirring, adding 2.5 mL of sodium citrate solution with the mass fraction of 1.0%, continuously heating in 100 ℃ oil bath for 15.0 min, and cooling to room temperature to obtain Au NPs;
centrifuging 1.5 mL of Au NPs at 12000 rpm for 20 min, adding 1.5 mL of ultrapure water into the precipitate, and performing ultrasonic treatment for 7.5 min to obtain an Au NPs dispersion liquid A; adding 0.05 mL of 20.0 mmol/L of 11-mercaptoundecanoic acid MUA ethanol solution into 0.7 mL of ultrapure water, and carrying out ultrasonic treatment for 60 min to obtain a solution B; and slowly adding the solution B into the Au NPs dispersion liquid A, shaking for 3.0 h at room temperature, and centrifuging the obtained solution at 9000 rpm for 30 min to obtain MUA-Au NPs.
Example 6 preparation of MUA-Au NPs, the procedure was as follows:
adding 99.0 mL of ultrapure water into a 500 mL three-neck flask, continuously adding 1.5 mL of chloroauric acid solution with the mass fraction of 1.0%, heating in 100 ℃ oil bath for 25.0 min under magnetic stirring, adding 3.5 mL of sodium citrate solution with the mass fraction of 1.0%, continuously heating in 100 ℃ oil bath for 20.0 min, and cooling to room temperature to obtain Au NPs;
centrifuging 2.0 mL of Au NPs at 12000 rpm for 20 min, adding 2.0 mL of ultrapure water into the precipitate, and performing ultrasonic treatment for 10.0 min to obtain an Au NPs dispersion liquid A; adding 0.05 mL of 25.0 mmol/L of 11-mercaptoundecanoic acid MUA ethanol solution into 1.0 mL of ultrapure water, and carrying out ultrasonic treatment for 60 min to obtain a solution B; and slowly adding the solution B into the Au NPs dispersion liquid A, oscillating for 2.0-4.0 h at room temperature, and centrifuging the obtained solution at 9000 rpm for 30 min to obtain MUA-Au NPs.
Example 7 mHPBs-Hemin/rGO-Ab2The preparation method comprises the following steps:
(1) preparation of mHPBs
Adding 1.0 g of polyvinylpyrrolidone and 100.0 mg of potassium ferricyanide into 20.0 mL of 0.1 mol/L hydrochloric acid solution under magnetic stirring, continuing to carry out magnetic stirring for 20.0 min, reacting the obtained clear solution at 80.0 ℃ for 16.0 h, centrifuging at 10000 rpm for 5 min, washing the obtained precipitate with absolute ethyl alcohol and ultrapure water for three times, and drying at room temperature for 12.0 h to obtain mesoporous Prussian blue seeds;
taking 1.0 g of polyvinylpyrrolidone, 100.0 mg of potassium ferricyanide and 5.0 mg of mesoporous Prussian blue seeds, adding the polyvinylpyrrolidone, 100.0 mg of potassium ferricyanide and 5.0 mg of mesoporous Prussian blue seeds into 20.0 mL of 0.01 mol/L hydrochloric acid solution under magnetic stirring, continuing to magnetically stir for 20.0 min, reacting the mixed liquid at 80 ℃ for 10.0 h, centrifuging the mixed liquid for 5 min at 10000 rpm, washing the obtained precipitate with absolute ethyl alcohol and ultrapure water for three times, and drying the precipitate at room temperature for 12.0 h to prepare mesoporous Prussian blue mPBs;
weighing 50.0 mg of polyvinylpyrrolidone and 15.0 mg of mPBS, adding the mixture into 15.0 mL of 1.0 mol/L hydrochloric acid solution under magnetic stirring, continuing to magnetically stir for 1.0 h, transferring the mixed liquid into a polytetrafluoroethylene high-pressure reaction kettle, preserving heat at 140 ℃ for 3.0 h, centrifuging the mixture for 5 min at 10000 rpm, washing the obtained precipitate with absolute ethyl alcohol and ultrapure water for three times, and preparing mesoporous hollow Prussian blue nano-cubic mHPBs;
(2) preparation of mHPBs-Hemin/rGO
Dissolving 5.0 mg Hemin Hemin and 5.0 g diaminopyridine in 50.0 mL and 1.0 mg/mL graphene oxide GO solutions, performing ultrasonic dispersion for 20.0 min, continuously adding 10.0 mL and 5.0 mg/mL mHPBs solutions, performing magnetic stirring for 2.0 h, transferring the mixed liquid into a polytetrafluoroethylene high-pressure reaction kettle, preserving heat at 130 ℃ for 3.0 h, centrifuging at 8000 rpm for 10 min, transferring the precipitate into a 50 mL culture dish, freezing in a refrigerator at-20.0 ℃ for 12 h, and putting into a freeze dryer for freeze drying at-80.0 ℃ to obtain mHPBsHemin/rGO;
（3）mHPBs-Hemin/rGO-Ab2preparation of
Adding 2.0 mg of mHPBs-Hemin/rGO into 2.0 mL of phosphate buffer solution with pH =7.2, performing ultrasonic treatment for 10.0 min, adding the mixed liquid into 2.0 mL of 20.0 mu g/mL of aflatoxin antibody solution, and placing the aflatoxin antibody solution in a 4.0 ℃ constant temperature oscillator for oscillation for 8.0 h to obtain mHPBs-Hemin/rGO-Ab2And storing in a refrigerator at 4.0 ℃ for later use.
Example 8 mHPBs-Hemin/rGO-Ab2The preparation method comprises the following steps:
(1) preparation of mHPBs
Adding 3.0 g of polyvinylpyrrolidone and 130.0 mg of potassium ferricyanide into 40.0 mL of 0.1 mol/L hydrochloric acid solution under magnetic stirring, continuing to magnetically stir for 30.0 min, reacting the obtained clear solution at 80.0 ℃ for 20.0 h, centrifuging at 10000 rpm for 5 min, washing the obtained precipitate with absolute ethyl alcohol and ultrapure water for three times, and drying at room temperature for 12.0 h to obtain mesoporous Prussian blue seeds;
adding 3.0 g of polyvinylpyrrolidone, 130.0 mg of potassium ferricyanide and 10.0 mg of mesoporous Prussian blue seeds into 40.0 mL of 0.01 mol/L hydrochloric acid solution under magnetic stirring, continuing to magnetically stir for 30.0 min, reacting the mixed liquid at 80 ℃ for 12.0 h, centrifuging at 10000 rpm for 5 min, washing the obtained precipitate with absolute ethyl alcohol and ultrapure water for three times, and drying at room temperature for 12.0 h to prepare mesoporous Prussian blue mPBs;
weighing 130.0 mg of polyvinylpyrrolidone and 20.0 mg of mPBS, adding the mixture into 20.0 mL of 1.0 mol/L hydrochloric acid solution under magnetic stirring, continuing to magnetically stir for 2.0 h, transferring the mixed liquid into a polytetrafluoroethylene high-pressure reaction kettle, preserving heat at 140 ℃ for 4.0 h, centrifuging at 10000 rpm for 5 min, washing the obtained precipitate with absolute ethyl alcohol and ultrapure water for three times, and preparing mesoporous hollow Prussian blue nano-cubic mHPBs;
(2) preparation of mHPBs-Hemin/rGO
Dissolving 7.0 mg Hemin Hemin and 7.0 g diaminopyridine in 70.0 mL and 1.0 mg/mL graphene oxide GO solutions, performing ultrasonic dispersion for 30.0 min, continuously adding 10.0 mL and 10.0 mg/mL mHPBs solutions, performing magnetic stirring for 2.0 h, transferring the mixed liquid into a polytetrafluoroethylene high-pressure reaction kettle, preserving heat at 130 ℃ for 4.0 h, centrifuging at 8000 rpm for 10 min, transferring the precipitate into a 50 mL culture dish, freezing in a refrigerator at-20.0 ℃ for 12 h, and putting into a freeze dryer for freeze drying at-80.0 ℃ to obtain mHPBsHemin/rGO;
（3）mHPBs-Hemin/rGO-Ab2preparation of
Adding 8.0 mg of mHPBs-Hemin/rGO into 2.0 mL of phosphate buffer solution with pH =7.2, performing ultrasonic treatment for 15.0 min, adding the mixed liquid into 2.0 mL of 20.0 mu g/mL of aflatoxin antibody solution, and placing the aflatoxin antibody solution in a 4.0 ℃ constant temperature oscillator for oscillation for 10.0 h to obtain mHPBs-Hemin/rGO-Ab2And storing in a refrigerator at 4.0 ℃ for later use.
Example 9 mHPBs-Hemin/rGO-Ab2The preparation method comprises the following steps:
(1) preparation of mHPBs
Adding 5.0 g of polyvinylpyrrolidone and 160.0 mg of potassium ferricyanide into 60.0 mL of 0.1 mol/L hydrochloric acid solution under magnetic stirring, continuing to magnetically stir for 40.0 min, reacting the obtained clear solution at 80.0 ℃ for 24.0 h, centrifuging for 5 min at 10000 rpm, washing the obtained precipitate with absolute ethyl alcohol and ultrapure water for three times, and drying at room temperature for 12.0 h to obtain mesoporous Prussian blue seeds;
taking 5.0 g of polyvinylpyrrolidone, 160.0 mg of potassium ferricyanide and 15.0 mg of mesoporous Prussian blue seeds, adding the polyvinylpyrrolidone, the 160.0 mg of potassium ferricyanide and the 15.0 mg of mesoporous Prussian blue seeds into 60.0 mL of 0.01 mol/L hydrochloric acid solution under magnetic stirring, continuing to magnetically stir for 40.0 min, reacting the mixed liquid at 80 ℃, centrifuging for 5 min at 10000 rpm, washing the obtained precipitate with absolute ethyl alcohol and ultrapure water for three times, and drying at room temperature for 12.0 h to prepare mesoporous Prussian blue mPBs;
weighing 150.0 mg of polyvinylpyrrolidone and 25.0 mg of mPBS, adding the mixture into 25.0 mL of 1.0 mol/L hydrochloric acid solution under magnetic stirring, continuing to magnetically stir for 3.0 h, transferring the mixed liquid into a polytetrafluoroethylene high-pressure reaction kettle, preserving heat at 140 ℃ for 5.0 h, centrifuging at 10000 rpm for 5 min, washing the obtained precipitate with absolute ethyl alcohol and ultrapure water for three times, and preparing mesoporous hollow Prussian blue nano-cubic mHPBs;
(2) preparation of mHPBs-Hemin/rGO
Dissolving 10.0 mg Hemin Hemin and 10.0 g diaminopyridine in 100.0 mL and 1.0 mg/mL graphene oxide GO solutions, performing ultrasonic dispersion for 40.0 min, continuously adding 10.0 mL and 15.0 mg/mL mHPBs solutions, performing magnetic stirring for 2.0 h, transferring the mixed liquid into a polytetrafluoroethylene high-pressure reaction kettle, preserving heat at 130 ℃ for 5.0 h, centrifuging at 8000 rpm for 10 min, transferring the precipitate into a 50 mL culture dish, freezing in a refrigerator at-20.0 ℃ for 12 h, and putting into a freeze dryer for freeze drying at-80.0 ℃ to obtain mHPBsHemin/rGO;
（3）mHPBs-Hemin/rGO-Ab2preparation of
Adding 12.0 mg of mHPBs-Hemin/rGO into 2.0 mL of phosphate buffer solution with pH =7.2, performing ultrasonic treatment for 20.0 min, adding the mixed liquid into 2.0 mL of 20.0 mu g/mL of aflatoxin antibody solution, and placing the aflatoxin antibody solution in a 4.0 ℃ constant temperature oscillator for oscillation for 12.0 h to obtain the mHPBs-Hemin/rGO-Ab2And storing in a refrigerator at 4.0 ℃ for later use.
Example 10 detection of aflatoxin AFB1, the procedure was as follows:
(1) an electrochemical workstation is used, a saturated calomel electrode is used as a reference electrode, a platinum wire electrode is used as a counter electrode, and the prepared sensor and the working electrode form a three-electrode system, and the three-electrode system is tested in 10.0 mL phosphate buffer solution with the pH = 5.91-7.73;
(2) using a time-current method to carry out the steps of setting the initial potential to be-0.4V, the sampling interval to be 0.1 s and the running time to be 400.0 s;
(3) after the background current stabilized, 10.0 μ L, 5.0 mol/L of H was injected into 10.0 mL of pH =7.2 phosphate buffer solution2O2After the current is stabilized again, recording the change of the current;
(4) measuring by adopting aflatoxin antigens with different concentrations, and recording current value changes corresponding to the aflatoxin antigens with different concentrations;
(5) by utilizing a working curve method, the linear range of aflatoxin AFB1 is 1.0 pg/mL-100.0 ng/mL, and the detection limit is 0.33 pg/mL.

Claims (4)

1. A preparation method of mHPBs-Hemin/rGO labeled electrochemical immunosensor is characterized by comprising the following steps:
(1) polishing a glassy carbon electrode with the diameter of 4.0 mm by using alumina powder to form a mirror surface, and ultrasonically cleaning the mirror surface in ultrapure water;
(2) dropwise adding 6.0 muA-Au NPs to the surface of the electrode, drying at room temperature, cleaning the surface of the electrode with ultrapure water, and drying;
(3) continuously capturing the aflatoxin capture antibody Ab of 6.0 muL and 10.0-20.0 mug/mL1Dropwise adding the solution to the surface of an electrode, and placing the electrode in a refrigerator at 4.0 ℃ for airing;
(4) dropwise adding a bovine serum albumin BSA solution of 3.0 muL and 0.5-1.5% to the surface of the electrode to seal non-specific active sites on the surface of the electrode, washing the surface of the electrode by using a phosphate buffer solution with pH =7.2, and airing in a refrigerator of 4.0 ℃;
(5) dropwise adding a series of aflatoxin antigen solutions with different concentrations, namely 6.0 muL and 1.0 pg/mL-100.0 ng/mL, placing in a 4.0 ℃ refrigerator for 30.0-40.0 min, washing with a phosphate buffer solution with pH =7.2, and placing in a 4.0 ℃ refrigerator for airing;
(6) dropwise adding 6.0 muL and 0.5-2.5 mg/mL mHPBs-Hemin/rGO labeled aflatoxin antibody mHPBs-Hemin/rGO-Ab2Placing the mixture in a refrigerator at 4.0 ℃ for 30.0-40.0 min, washing the mixture with phosphate buffer solution with pH =7.2, placing the mixture in the refrigerator at 4.0 ℃ for airing to obtain the mHPBs-Hemin/rGO labeled electrochemical immunosensor;
the MUA-Au NPs are all named as gold nanoparticles acidified by mercaptoundecanoic acid;
the mHPBs are fully called mesoporous hollow Prussian blue;
the preparation method of the MUA-Au NPs is characterized by comprising the following steps:
adding 99.0 mL of ultrapure water into a 500 mL three-neck flask, continuously adding 0.5-1.5 mL of chloroauric acid solution with the mass fraction of 1.0%, heating in an oil bath at 100 ℃ for 15.0-25.0 min under magnetic stirring, adding 1.5-3.5 mL of sodium citrate solution with the mass fraction of 1.0%, continuously heating in an oil bath at 100 ℃ for 10.0-20.0 min, and cooling to room temperature to obtain Au NPs;
centrifuging 1.0-2.0 mL of Au NPs at 12000 rpm for 20 min, adding 1.0-2.0 mL of ultrapure water into the precipitate, and performing ultrasonic treatment for 5.0-10.0 min to obtain an Au NPs dispersion liquid A; adding 0.05 mL of 15.0-25.0 mmol/L of 11-mercaptoundecanoic acid MUA ethanol solution into 0.5-1.0 mL of ultrapure water, and carrying out ultrasonic treatment for 60 min to obtain a solution B; slowly adding the solution B into the Au NPs dispersion liquid A, oscillating for 2.0-4.0 h at room temperature, and centrifuging the obtained solution at 9000 rpm for 30 min to obtain MUA-Au NPs;
the mHPBs-Hemin/rGO-Ab2The preparation method is characterized by comprising the following steps:
(1) preparation of mHPBs
Adding 1.0-5.0 g of polyvinylpyrrolidone and 100.0-160.0 mg of potassium ferricyanide into 20.0-60.0 mL of 0.1 mol/L hydrochloric acid solution under magnetic stirring, continuing to magnetically stir for 20.0-40.0 min, reacting the obtained clear solution at 80.0 ℃ for 16.0-24.0 h, centrifuging at 10000 rpm for 5 min, respectively washing the obtained precipitate with absolute ethyl alcohol and ultrapure water for three times, and drying at room temperature for 12.0 h to obtain mesoporous Prussian blue seeds;
adding 1.0-5.0 g of polyvinylpyrrolidone, 100.0-160.0 mg of potassium ferricyanide and 5.0-15.0 mg of mesoporous Prussian blue seeds into 20.0-60.0 mL of 0.01 mol/L hydrochloric acid solution under magnetic stirring, continuing to magnetically stir for 20.0-40.0 min, reacting the mixed liquid at 80 ℃ for 10.0-14.0 h, centrifuging at 10000 rpm for 5 min, respectively washing the obtained precipitate with absolute ethyl alcohol and ultrapure water for three times, drying at room temperature for 12.0 h, and obtaining Prussian blue mPBs through mesopores;
weighing 50.0-150.0 mg of polyvinylpyrrolidone and 15.0-25.0 mg of mPBS, adding the mixture into 15.0-25.0 mL of 1.0 mol/L hydrochloric acid solution under magnetic stirring, continuing to magnetically stir for 1.0-3.0 h, transferring the mixed liquid into a polytetrafluoroethylene high-pressure reaction kettle, preserving heat at 140 ℃ for 3.0-5.0 h, centrifuging at 10000 rpm for 5 min, and respectively washing the obtained precipitate with absolute ethyl alcohol and ultrapure water for three times to prepare mesoporous hollow Prussian blue nano cubic mHPBs;
(2) preparation of mHPBs-Hemin/rGO
Dissolving 5.0-10.0 mg Hemin and 5.0-10.0 g diaminopyridine in 50.0-100.0 mL of graphene oxide GO solution of 1.0 mg/mL, carrying out ultrasonic dispersion for 20.0-40.0 min, continuously adding 10.0 mL of mHPBs solution of 5.0-15.0 mg/mL, carrying out magnetic stirring for 2.0 h, transferring the mixed liquid into a polytetrafluoroethylene high-pressure reaction kettle, carrying out heat preservation for 3.0-5.0 h at 130 ℃, centrifuging for 10 min at 8000 rpm, transferring the precipitate into a 50 mL culture dish, freezing for 12 h in a refrigerator at-20.0 ℃, putting into a freeze dryer, and carrying out freeze drying at-80.0 ℃ to obtain mHPBs-Hemin/rGO;
（3）mHPBs-Hemin/rGO-Ab2preparation of
Adding 2.0-12.0 mg of mHPBs-Hemin/rGO into 2.0 mL of phosphate buffer solution with pH =7.2, performing ultrasonic treatment for 10.0-20.0 min, adding the mixed liquid into 2.0 mL of 20.0 mug/mL aflatoxin antibody solution, and placing the aflatoxin antibody solution in a 4.0 ℃ constant temperature oscillator for oscillation for 8.0-12.0 h to obtain the mHPBs-Hemin/rGO-Ab2And storing in a refrigerator at 4.0 ℃ for later use.
2. The method of claim 1, wherein the aflatoxin is AFB 1.
3. The electrochemical immunosensor prepared by the preparation method according to claim 1, is applied to aflatoxin detection, and is characterized in that the detection comprises the following specific steps:
(1) an electrochemical workstation is used, a saturated calomel electrode is used as a reference electrode, a platinum wire electrode is used as a counter electrode, and the prepared sensor and the working electrode form a three-electrode system, and the three-electrode system is tested in 10.0 mL phosphate buffer solution with the pH = 5.91-7.73;
(2) using a time-current method to carry out the steps of setting the initial potential to be-0.4V, the sampling interval to be 0.1 s and the running time to be 400.0 s;
(3) after the background current stabilized, 10.0 μ L, 5.0 mol/L of H was injected into 10.0 mL of pH =7.2 phosphate buffer solution2O2After the current is stabilized again, recording the change of the current;
(4) measuring by adopting aflatoxin antigens with different concentrations, and recording current value changes corresponding to the aflatoxin antigens with different concentrations;
(5) and obtaining the concentration of the aflatoxin antigen in the sample to be detected by using a working curve method.
4. The electrochemical immunosensor according to claim 3, wherein the aflatoxin is AFB 1.
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