CN111678962B - Preparation method and application of aflatoxin electrochemical sensor - Google Patents
Preparation method and application of aflatoxin electrochemical sensor Download PDFInfo
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- 229930195730 Aflatoxin Natural products 0.000 title claims abstract description 57
- XWIYFDMXXLINPU-UHFFFAOYSA-N Aflatoxin G Chemical compound O=C1OCCC2=C1C(=O)OC1=C2C(OC)=CC2=C1C1C=COC1O2 XWIYFDMXXLINPU-UHFFFAOYSA-N 0.000 title claims abstract description 57
- 239000005409 aflatoxin Substances 0.000 title claims abstract description 57
- 238000002360 preparation method Methods 0.000 title claims abstract description 35
- 238000001514 detection method Methods 0.000 claims abstract description 52
- 239000002105 nanoparticle Substances 0.000 claims abstract description 42
- 229910016411 CuxO Inorganic materials 0.000 claims abstract description 32
- 229910000422 cerium(IV) oxide Inorganic materials 0.000 claims abstract description 22
- 238000000034 method Methods 0.000 claims abstract description 17
- 238000011534 incubation Methods 0.000 claims abstract description 10
- 239000000243 solution Substances 0.000 claims description 88
- 239000012498 ultrapure water Substances 0.000 claims description 64
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 60
- 238000005406 washing Methods 0.000 claims description 29
- 238000001035 drying Methods 0.000 claims description 28
- 239000010949 copper Substances 0.000 claims description 21
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 20
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 20
- 238000004140 cleaning Methods 0.000 claims description 20
- 238000003756 stirring Methods 0.000 claims description 20
- HSJPMRKMPBAUAU-UHFFFAOYSA-N cerium(3+);trinitrate Chemical compound [Ce+3].[O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O HSJPMRKMPBAUAU-UHFFFAOYSA-N 0.000 claims description 16
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 15
- 239000002253 acid Substances 0.000 claims description 15
- ORTQZVOHEJQUHG-UHFFFAOYSA-L copper(II) chloride Chemical compound Cl[Cu]Cl ORTQZVOHEJQUHG-UHFFFAOYSA-L 0.000 claims description 15
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 claims description 15
- 230000012447 hatching Effects 0.000 claims description 14
- 229960005070 ascorbic acid Drugs 0.000 claims description 10
- 235000010323 ascorbic acid Nutrition 0.000 claims description 10
- 239000011668 ascorbic acid Substances 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 10
- 238000005498 polishing Methods 0.000 claims description 10
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 8
- 238000001291 vacuum drying Methods 0.000 claims description 8
- 238000012360 testing method Methods 0.000 claims description 7
- 229930063498 Aflatoxin G1 Natural products 0.000 claims description 6
- XWIYFDMXXLINPU-WNWIJWBNSA-N Aflatoxin G1 Chemical compound O=C1OCCC2=C1C(=O)OC1=C2C(OC)=CC2=C1[C@@H]1C=CO[C@@H]1O2 XWIYFDMXXLINPU-WNWIJWBNSA-N 0.000 claims description 6
- 239000002115 aflatoxin B1 Substances 0.000 claims description 6
- OQIQSTLJSLGHID-WNWIJWBNSA-N aflatoxin B1 Chemical compound C=1([C@@H]2C=CO[C@@H]2OC=1C=C(C1=2)OC)C=2OC(=O)C2=C1CCC2=O OQIQSTLJSLGHID-WNWIJWBNSA-N 0.000 claims description 6
- 239000002098 aflatoxin G1 Substances 0.000 claims description 6
- 229930020125 aflatoxin-B1 Natural products 0.000 claims description 6
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 5
- RVGRUAULSDPKGF-UHFFFAOYSA-N Poloxamer Chemical compound C1CO1.CC1CO1 RVGRUAULSDPKGF-UHFFFAOYSA-N 0.000 claims description 5
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 5
- 239000000427 antigen Substances 0.000 claims description 5
- 102000036639 antigens Human genes 0.000 claims description 5
- 108091007433 antigens Proteins 0.000 claims description 5
- 229940098773 bovine serum albumin Drugs 0.000 claims description 5
- 238000006243 chemical reaction Methods 0.000 claims description 5
- 229910021397 glassy carbon Inorganic materials 0.000 claims description 5
- 238000010438 heat treatment Methods 0.000 claims description 5
- 229960000502 poloxamer Drugs 0.000 claims description 5
- 229920001983 poloxamer Polymers 0.000 claims description 5
- 239000000843 powder Substances 0.000 claims description 5
- 239000001509 sodium citrate Substances 0.000 claims description 5
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 5
- 238000009210 therapy by ultrasound Methods 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 4
- 239000007853 buffer solution Substances 0.000 claims description 4
- 230000010355 oscillation Effects 0.000 claims description 4
- 239000008055 phosphate buffer solution Substances 0.000 claims description 4
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 3
- 230000008859 change Effects 0.000 claims description 3
- ZOMNIUBKTOKEHS-UHFFFAOYSA-L dimercury dichloride Chemical class Cl[Hg][Hg]Cl ZOMNIUBKTOKEHS-UHFFFAOYSA-L 0.000 claims description 3
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 claims description 3
- -1 potassium ferricyanide Chemical compound 0.000 claims description 3
- 238000005070 sampling Methods 0.000 claims description 3
- 239000000463 material Substances 0.000 abstract description 7
- 239000000758 substrate Substances 0.000 abstract description 6
- 230000003197 catalytic effect Effects 0.000 abstract description 5
- 239000003550 marker Substances 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 238000003018 immunoassay Methods 0.000 abstract description 3
- 230000003321 amplification Effects 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 241000228197 Aspergillus flavus Species 0.000 description 2
- 230000000711 cancerogenic effect Effects 0.000 description 2
- 231100000315 carcinogenic Toxicity 0.000 description 2
- 239000011258 core-shell material Substances 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 1
- 206010062016 Immunosuppression Diseases 0.000 description 1
- 206010067125 Liver injury Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010061481 Renal injury Diseases 0.000 description 1
- 208000031320 Teratogenesis Diseases 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000013043 chemical agent Substances 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- BERDEBHAJNAUOM-UHFFFAOYSA-N copper(I) oxide Inorganic materials [Cu]O[Cu] BERDEBHAJNAUOM-UHFFFAOYSA-N 0.000 description 1
- KRFJLUBVMFXRPN-UHFFFAOYSA-N cuprous oxide Chemical compound [O-2].[Cu+].[Cu+] KRFJLUBVMFXRPN-UHFFFAOYSA-N 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 208000037806 kidney injury Diseases 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 230000003071 parasitic effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
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- 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
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- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/308—Electrodes, e.g. test electrodes; Half-cells at least partially made of carbon
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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
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Abstract
The invention belongs to the technical field of immunoassay and biosensing, and provides a preparation method and application of an aflatoxin electrochemical sensor. According to the invention, AuPt nanoparticles are used as a substrate material, the excellent conductivity of the AuPt nanoparticles can effectively reduce background signals, and meanwhile, the AuPt nanoparticles have large surface area and can load sufficient antibodies; using Au/CuxO@CeO2Incubation with a detection antibody is used as a signal marker to further enhance the catalytic performance of the electrochemical sensor and amplify signals, so that quantitative detection of aflatoxin is realized, and the method has the advantages of low detection limit, high sensitivity, good repeatability, selectivity and stability and the like, and has important scientific significance and application value for detection of aflatoxin.
Description
Technical Field
The invention belongs to the technical field of immunoassay, nano materials and biosensing, and provides a preparation method and application of an aflatoxin electrochemical sensor.
Background
Aflatoxin has strong biological toxicity and carcinogenic capacity, and aflatoxin can be generated when the aflatoxin is polluted by aspergillus flavus and parasitic aspergillus flavus, and further pollutes animal-derived foods such as milk, eggs and meat through a food chain, and indirectly enters a human body, so that serious consequences such as liver and kidney injury, reproductive disorder, immunosuppression, carcinogenic teratogenesis and the like are finally caused.
The existing detection methods of aflatoxin comprise thin layer chromatography, liquid chromatography, radioimmunoassay, enzyme-linked immunosorbent assay, affinity chromatography, microcolumn screening, gold-labeled test paper method and the like. These methods have more or less disadvantages, such as tedious and complicated operation process, long time consumption and large labor capacity; the instrument and equipment are expensive, large in size and complex in operation, and the rapid field analysis is difficult to realize; sensitivity and reproducibility were poor. The electrochemical immunoassay developed in recent years is beginning to be widely applied to aflatoxin detection, and has the advantages of high selectivity, quick response, simple operation, convenient carrying, suitability for on-site detection and the like, so researchers are actively exploring and developing various novel electrochemical sensors for detecting aflatoxin.
The invention successfully constructs an aflatoxin electrochemical sensor, the substrate material of the sensor is AuPt nano particles, the bimetallic structure of the sensor has good conductivity, the electron transmission rate can be accelerated, the rugged surface morphology ensures that the sensor has larger specific surface area, and the antibody Ab can be better loaded1. Marker Au/CuxO@CeO2The multi-thorn core-shell structure is adopted, the surface appearance of the multi-thorn structure not only increases the specific surface area of the material, but also can better fix the antibody Ab2Meanwhile, the composite material has excellent catalytic activity, and can realize amplification of detection signals, so that the sensitivity of the immunosensor is increased. The invention adopts AuPt nano particles as substrate material, Au/CuxO@CeO2The electrochemical sensor constructed by the detection antibody marker realizes the detection of aflatoxin, has low detection limit, high sensitivity, acceptable repeatability, selectivity and stability, and has important scientific significance and application value for the detection of aflatoxin.
Disclosure of Invention
The invention provides a preparation method and application of an aflatoxin electrochemical sensor,
the aflatoxin sensitive detection is realized.
One of the purposes of the invention is to provide a preparation method of an aflatoxin electrochemical sensor.
The second purpose of the invention is to apply the prepared aflatoxin electrochemical sensor to the high-sensitivity and specific detection of aflatoxin.
The technical scheme of the invention comprises the following steps.
1. A preparation method of an aflatoxin electrochemical sensor comprises the following steps:
(1) al for glassy carbon electrode with diameter of 4 mm2O3Polishing the polishing powder into a mirror surface, and cleaning with ultrapure water;
(2) dripping a 6 mu L of 1.0-3.0 mg/mL AuPt nano particle solution on the surface of the electrode, drying at room temperature, washing the surface of the electrode with ultrapure water, and drying;
(3) continuing to capture the aflatoxin capture antibody Ab of 6 muL and 8-12 mug/mL1Dropwise adding the solution to the surface of an electrode, washing with ultrapure water, and drying in a refrigerator at 4 ℃;
(4) continuously dropwise adding bovine serum albumin BSA solution with the mass fraction of 1% to the surface of the electrode by 3 muL, 0.5-1.5 mg/mL to seal non-specific active sites on the surface of the electrode, washing the surface of the electrode with ultrapure water, removing unbound BSA, and drying in a refrigerator at 4 ℃;
(5) continuously dropwise adding a series of aflatoxin antigen solutions with different concentrations, namely 6 muL and 10 pg/mL-60 ng/mL, washing the surface of the electrode with ultrapure water, and airing in a refrigerator at 4 ℃;
(6) continuously dropwise adding 6 mu L of detection antibody hatching Au/Cu of 1.5-3.5 mg/mLxO@CeO2-Ab2And putting the solution on the surface of an electrode, incubating for 40 min in a refrigerator at 4 ℃, washing with ultrapure water, and airing to obtain the aflatoxin electrochemical sensor.
2. The preparation method of the AuPt nano particle solution comprises the following steps:
(1) preparation of AuPt nanoparticles
Sequentially adding 50-150 mg of poloxamer F127 and 20-40 mg of potassium iodide into 1.0 mL of ultrapure water, and uniformly stirring; adding 0.8 mL, 10-30 mmol/L chloroplatinic acid, 2.5 mL and 10-30 mmol/L chloroauric acid, and uniformly stirring; adding 2.5 mL of ascorbic acid of 0.05-0.15 mol/L, and reacting for 15 min at 90 ℃; respectively centrifugally cleaning ultrapure water and ethanol for three times, and drying at 60 ℃ for 12 h to prepare AuPt nanoparticles;
(2) preparation of AuPt nanoparticle solution
5-15 mg of AuPt nanoparticles are dispersed in 5 mL of ultrapure water, and the solution is subjected to ultrasonic treatment for 10 min to prepare the AuPt nanoparticle solution.
3. Detection of antibody hatching Au/CuxO@CeO2-Ab2The solution was prepared by the following steps:
(1)Au/CuxO@CeO2preparation of
Putting 100 mL of 0.01 mol/L copper chloride solution in a water bath at 55 ℃; adding 10 mL of 2 mol/L sodium hydroxide solution, and reacting for 30 min; then 10 mL of ascorbic acid with the concentration of 0.6 mol/L is added for reaction for 3 hours; respectively centrifugally cleaning ultrapure water and ethanol for three times, and vacuum drying at 60 ℃ for 5 hours to obtain Cu2An O cube;
adding 20-40 mg of Cu into 30 mL of ultrapure water2Stirring the mixture evenly in an O cube, adding 500-700 mu L of cerium nitrate and 0.1 mol/L of cerium nitrate, and heating the mixture to 60 ℃; adding 3.0-4.0 mL of 0.15 mol/L ammonia water, and reacting for 3 h; adding 55-75 mu L of chloroauric acid of 20 mmol/L, and reacting for 15 min; adding 65 μ L, 20 mmol/L sodium citrate, and reacting for 30 min; centrifugally cleaning the solution for three times by ultrapure water, and drying the solution for 8 to 12 hours in vacuum at the temperature of 60 ℃ to obtain Au/CuxO@CeO2;
The copper chloride solution is prepared by adding 0.14g of copper chloride into 100 mL of ultrapure water and stirring for 5 min;
(2) detection of antibody hatching Au/CuxO@CeO2-Ab2Preparation of the solution
1.0-3.0 mL of Au/Cu with the concentration of 2 mg/mLxO@CeO2Adding the solution into 0.5-1.5 mL of aflatoxin detection antibody solution Ab with the concentration of 10 mu g/mL2Performing shake incubation for 12 h in a constant-temperature oscillation box at 4 ℃, centrifuging and washing, adding 1.0-3.0 mL of phosphoric acid buffer solution with pH = 7.38, and preparing the detection antibody incubation Au/CuxO@CeO2-Ab2The solution was stored at 4 ℃ until use.
4. Detecting aflatoxin by the following steps:
(1) testing by using an electrochemical workstation in a three-electrode system, taking a saturated calomel electrode as a reference electrode, taking a platinum wire electrode as an auxiliary electrode, taking the prepared sensor as a working electrode, and testing in 10 mL of potassium ferricyanide solution containing 10 mmol/L;
(2) detecting aflatoxin by using a time-lapse current method, wherein the input voltage is-0.4V, the sampling interval is 0.1 s, and the running time is 300 s;
(3) when the background current tended to be stable, 10 μ L of 5 mol/L hydrogen peroxide solution was injected into 10 mL of 50 mmol/L phosphate buffer solution with pH = 7.38 every 50 s, and the change in current was recorded.
The aflatoxin is selected from one of the following: aflatoxin B1, aflatoxin G1.
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 substrate material used by the method is AuPt nanoparticles, and has the advantages of simplicity in synthesis, good stability and the like, and the gully-shaped surface appearance enables the substrate material to have a larger surface area and can load a large amount of antibody Ab1Meanwhile, the bimetal structure also has good conductivity, and high-efficiency signal conduction can be realized. Au/CuxO@CeO2As a detection antibody marker, the sensitivity of the electrochemical sensor can be increased, the catalytic activity of the electrochemical sensor can be improved, and the amplification of a detection signal can be realized. Au/CuxO@CeO2Is a multi-spine core-shell structure, Cu2The O and CuO have good catalytic activity under the synergistic catalytic action, can realize the amplification of detection signals, and simultaneously endow the detection signals with larger specific surface area due to the multi-thorn morphology, and can effectively load an antibody Ab2. The invention adopts AuPt nano particles as substrate material, Au/CuxO@CeO2The electrochemical sensor constructed by the detection antibody marker realizes sensitive and rapid detection of aflatoxin.
(2) The aflatoxin electrochemical sensor realizes detection of aflatoxin, wherein the linear range of detection of aflatoxin B1 is 10 pg-60 ng/mL, the detection limit is 3.33pg/mL, the linear range of detection of aflatoxin G1 is 10 pg-60 ng/mL, and the detection limit is 3.33pg/mL, which shows that the aflatoxin electrochemical sensor based on Au/Cu is based on Au/CuxO@CeO2The electrochemical sensor can achieve the aim of accurate determination.
Detailed Description
The present invention will now be further illustrated by, but not limited to, specific embodiments thereof.
Embodiment 1 a method for preparing an aflatoxin electrochemical sensor, comprising the following steps:
(1) al for glassy carbon electrode with diameter of 4 mm2O3Polishing the polishing powder into a mirror surface, and cleaning with ultrapure water;
(2) dripping a 6 mu L and 1.0 mg/mL AuPt nano particle solution on the surface of the electrode, drying at room temperature, washing the surface of the electrode with ultrapure water, and drying;
(3) continuing to capture 6 muL and 8 mug/mL aflatoxin-captured antibody Ab1Dropwise adding the solution to the surface of an electrode, washing with ultrapure water, and drying in a refrigerator at 4 ℃;
(4) continuously dropwise adding bovine serum albumin BSA solution with the mass fraction of 1% to the surface of the electrode by 3 muL and 0.5 mg/mL to seal non-specific active sites on the surface of the electrode, washing the surface of the electrode with ultrapure water, removing unbound BSA, and drying in a refrigerator at 4 ℃;
(5) continuously dropwise adding a series of aflatoxin antigen solutions with different concentrations, namely 6 muL and 10 pg/mL-60 ng/mL, washing the surface of the electrode with ultrapure water, and airing in a refrigerator at 4 ℃;
(6) continuously dropwise adding detection antibody hatching Au/Cu of 6 mu L and 1.5 mg/mLxO@CeO2-Ab2And putting the solution on the surface of an electrode, incubating for 40 min in a refrigerator at 4 ℃, washing with ultrapure water, and airing to obtain the aflatoxin electrochemical sensor.
Embodiment 2 a method for preparing an aflatoxin electrochemical sensor, comprising the following steps:
(1) al for glassy carbon electrode with diameter of 4 mm2O3Polishing the polishing powder into a mirror surface, and cleaning with ultrapure water;
(2) dripping a 6 mu L and 2.0 mg/mL AuPt nano particle solution on the surface of the electrode, drying at room temperature, washing the surface of the electrode with ultrapure water, and drying;
(3) continuing to capture the aflatoxin capture antibody Ab at 6 muL and 10 mug/mL1Drop-on electrode meterWashing with ultrapure water, and drying in a refrigerator at 4 ℃;
(4) continuously dropwise adding bovine serum albumin BSA solution with the mass fraction of 1% to the surface of the electrode by 3 muL and 1.0 mg/mL to seal non-specific active sites on the surface of the electrode, washing the surface of the electrode with ultrapure water, removing unbound BSA, and drying in a refrigerator at 4 ℃;
(5) continuously dropwise adding a series of aflatoxin antigen solutions with different concentrations, namely 6 muL and 10 pg/mL-60 ng/mL, washing the surface of the electrode with ultrapure water, and airing in a refrigerator at 4 ℃;
(6) continuously dropwise adding detection antibody hatching Au/Cu of 6 mu L and 2.5 mg/mLxO@CeO2-Ab2And putting the solution on the surface of an electrode, incubating for 40 min in a refrigerator at 4 ℃, washing with ultrapure water, and airing to obtain the aflatoxin electrochemical sensor.
Embodiment 3 a method for preparing an aflatoxin electrochemical sensor, comprising the following steps:
(1) al for glassy carbon electrode with diameter of 4 mm2O3Polishing the polishing powder into a mirror surface, and cleaning with ultrapure water;
(2) dripping a 6 mu L and 3.0 mg/mL AuPt nano particle solution on the surface of the electrode, drying at room temperature, washing the surface of the electrode with ultrapure water, and drying;
(3) continuing to capture the aflatoxin capture antibody Ab at 6 muL and 12 mug/mL1Dropwise adding the solution to the surface of an electrode, washing with ultrapure water, and drying in a refrigerator at 4 ℃;
(4) continuously dropwise adding bovine serum albumin BSA solution with the mass fraction of 1% to the surface of the electrode by 3 muL and 1.5 mg/mL to seal non-specific active sites on the surface of the electrode, washing the surface of the electrode with ultrapure water, removing unbound BSA, and drying in a refrigerator at 4 ℃;
(5) continuously dropwise adding a series of aflatoxin antigen solutions with different concentrations, namely 6 muL and 10 pg/mL-60 ng/mL, washing the surface of the electrode with ultrapure water, and airing in a refrigerator at 4 ℃;
(6) continuously dropwise adding detection antibody hatching Au/Cu of 6 mu L and 3.5 mg/mLxO@CeO2-Ab2Solution on the surface of the electrodeAnd incubating in a refrigerator at 4 ℃ for 40 min, washing with ultrapure water, and airing to obtain the aflatoxin electrochemical sensor.
Example 4 preparation of AuPt nanoparticle solution, the procedure was as follows:
(1) preparation of AuPt nanoparticles
Sequentially adding 50 mg of poloxamer F127 and 20 mg of potassium iodide into 1.0 mL of ultrapure water, and uniformly stirring; adding 0.8 mL, 10 mmol/L chloroplatinic acid, 2.5 mL and 10 mmol/L chloroauric acid, and stirring uniformly; adding 2.5 mL of ascorbic acid of 0.05 mol/L, and reacting for 15 min at 90 ℃; respectively centrifugally cleaning ultrapure water and ethanol for three times, and drying at 60 ℃ for 12 h to prepare AuPt nanoparticles;
(2) preparation of AuPt nanoparticle solution
5 mg of AuPt nanoparticles are dispersed in 5 mL of ultrapure water, and the solution is subjected to ultrasonic treatment for 10 min to prepare the AuPt nanoparticle solution.
Example 5 preparation of AuPt nanoparticle solution, the procedure was as follows:
(1) preparation of AuPt nanoparticles
Sequentially adding 100 mg of poloxamer F127 and 30 mg of potassium iodide into 1.0 mL of ultrapure water, and uniformly stirring; adding 0.8 mL, 20 mmol/L chloroplatinic acid, 2.5 mL and 20 mmol/L chloroauric acid, and stirring uniformly; adding 2.5 mL of ascorbic acid of 0.10 mol/L, and reacting for 15 min at 90 ℃; respectively centrifugally cleaning ultrapure water and ethanol for three times, and drying at 60 ℃ for 12 h to prepare AuPt nanoparticles;
(2) preparation of AuPt nanoparticle solution
10 mg of AuPt nanoparticles are dispersed in 5 mL of ultrapure water and subjected to ultrasonic treatment for 10 min to prepare an AuPt nanoparticle solution.
Example 6 preparation of AuPt nanoparticle solution, the procedure was as follows:
(1) preparation of AuPt nanoparticles
Sequentially adding 150 mg of poloxamer F127 and 40 mg of potassium iodide into 1.0 mL of ultrapure water, and uniformly stirring; adding 0.8 mL, 30 mmol/L chloroplatinic acid, 2.5 mL and 30 mmol/L chloroauric acid, and stirring uniformly; adding 2.5 mL of ascorbic acid of 0.15 mol/L, and reacting for 15 min at 90 ℃; respectively centrifugally cleaning ultrapure water and ethanol for three times, and drying at 60 ℃ for 12 hours to prepare AuPt nanoparticles;
(2) preparation of AuPt nanoparticle solution
15 mg of AuPt nanoparticles are dispersed in 5 mL of ultrapure water and subjected to ultrasonic treatment for 10 min to prepare an AuPt nanoparticle solution.
Example 7 detection of antibody hatching Au/CuxO@CeO2-Ab2The solution was prepared by the following steps:
(1)Au/CuxO@CeO2preparation of
Putting 100 mL of 0.01 mol/L copper chloride solution in a water bath at 55 ℃; adding 10 mL of 2 mol/L sodium hydroxide solution, and reacting for 30 min; then 10 mL of ascorbic acid with the concentration of 0.6 mol/L is added for reaction for 3 hours; respectively centrifugally cleaning ultrapure water and ethanol for three times, and vacuum drying at 60 ℃ for 5 hours to obtain Cu2O cube;
20 mg of Cu was added to 30 mL of ultrapure water2Stirring the mixture evenly, adding 500 mu L of cerium nitrate with the concentration of 0.1 mol/L, and heating the mixture to 60 ℃; adding 3.0 mL of 0.15 mol/L ammonia water, and reacting for 3 h; adding 55 μ L of 20 mmol/L chloroauric acid, and reacting for 15 min; adding 65 μ L, 20 mmol/L sodium citrate, and reacting for 30 min; centrifugally cleaning with ultrapure water for three times, and vacuum drying at 60 ℃ for 8 h to obtain Au/CuxO@CeO2;
The copper chloride solution is prepared by adding 0.14g of copper chloride into 100 mL of ultrapure water and stirring for 5 min;
(2) detection of antibody hatching Au/CuxO@CeO2-Ab2Preparation of the solution
1.0 mL of Au/Cu with a concentration of 2 mg/mLxO@CeO2The solution was added to 0.5 mL of aflatoxin-detection antibody solution Ab at a concentration of 10. mu.g/mL2Performing shake incubation for 12 h in a constant temperature oscillation box at 4 ℃, centrifuging, washing, adding into 1.0 mL of phosphoric acid buffer solution with pH = 7.38, and preparing the detection antibody incubation Au/CuxO@CeO2-Ab2The solution was stored at 4 ℃ until use.
EXAMPLE 8 detection of antibody hatching Au/CuxO@CeO2-Ab2The solution was prepared by the following steps:
(1)Au/CuxO@CeO2preparation of
Putting 100 mL of 0.01 mol/L copper chloride solution in a water bath at 55 ℃; adding 10 mL of 2 mol/L sodium hydroxide solution, and reacting for 30 min; then 10 mL of ascorbic acid with the concentration of 0.6 mol/L is added for reaction for 3 hours; respectively centrifugally cleaning ultrapure water and ethanol for three times, and vacuum drying at 60 ℃ for 5 hours to obtain Cu2An O cube;
30 mg of Cu was added to 30 mL of ultrapure water2Stirring the mixture evenly, adding 600 mu L of cerium nitrate with the concentration of 0.1 mol/L, and heating the mixture to 60 ℃; adding 3.5 mL of 0.15 mol/L ammonia water, and reacting for 3 h; adding 65 μ L of 20 mmol/L chloroauric acid, and reacting for 15 min; adding 65 mu L of sodium citrate with the concentration of 20 mmol/L, and reacting for 30 min; centrifugally cleaning with ultrapure water for three times, and vacuum drying at 60 ℃ for 10 hours to obtain Au/CuxO@CeO2;
The copper chloride solution is prepared by adding 0.14g of copper chloride into 100 mL of ultrapure water and stirring for 5 min;
(2) detection of antibody hatching Au/CuxO@CeO2-Ab2Preparation of the solution
2.0 mL of Au/Cu at a concentration of 2 mg/mLxO@CeO2The solution was added to 1.0 mL Aflatoxin detection antibody solution Ab at a concentration of 10. mu.g/mL2Performing shake incubation for 12 h in a constant temperature oscillation box at 4 ℃, centrifuging, washing, adding into 2.0 mL of phosphoric acid buffer solution with pH = 7.38, and preparing the detection antibody incubation Au/CuxO@CeO2-Ab2The solution was stored at 4 ℃ until use.
Example 9 detection of antibody hatching Au/CuxO@CeO2-Ab2The solution was prepared by the following steps:
(1)Au/CuxO@CeO2preparation of
Putting 100 mL of 0.01 mol/L copper chloride solution in a water bath at 55 ℃; adding 10 mL of 2 mol/L sodium hydroxide solution, and reacting for 30 min; then 10 mL of ascorbic acid with the concentration of 0.6 mol/L is added for reaction for 3 hours; respectively centrifugally cleaning ultrapure water and ethanol for three times, and vacuum drying at 60 ℃ for 5 hours to obtain Cu2An O cube;
adding into 30 mL of ultrapure waterAdding 40 mg of Cu2Stirring the mixture evenly, adding 700 mu L of cerium nitrate and 0.1 mol/L of cerium nitrate, and heating the mixture to 60 ℃; adding 4.0 mL of 0.15 mol/L ammonia water, and reacting for 3 h; adding 75 mu L of 20 mmol/L chloroauric acid, and reacting for 15 min; adding 65 μ L, 20 mmol/L sodium citrate, and reacting for 30 min; centrifugally cleaning with ultrapure water for three times, and vacuum drying at 60 ℃ for 12 hours to obtain Au/CuxO@CeO2;
The copper chloride solution is prepared by adding 0.14g of copper chloride into 100 mL of ultrapure water and stirring for 5 min;
(2) detection of antibody hatching Au/CuxO@CeO2-Ab2Preparation of the solution
3.0 mL of Au/Cu at a concentration of 2 mg/mLxO@CeO2The solution was added to 1.5 mL of aflatoxin-detection antibody solution Ab at a concentration of 10. mu.g/mL2In the preparation method, shaking and incubating in a constant temperature shaking box at 4 ℃ for 12 h, centrifuging and washing, adding into 3.0 mL of phosphate buffer solution with pH = 7.38 to prepare the detection antibody incubation Au/CuxO@CeO2-Ab2The solution was stored at 4 ℃ until use.
Example 10 detection of aflatoxin B1, the procedure was as follows:
(1) testing by using an electrochemical workstation in a three-electrode system, taking a saturated calomel electrode as a reference electrode, taking a platinum wire electrode as an auxiliary electrode, taking the prepared sensor as a working electrode, and testing in 10 mL of potassium ferricyanide solution containing 10 mmol/L;
(2) detecting aflatoxin by using a time-lapse current method, wherein the input voltage is-0.4V, the sampling interval is 0.1 s, and the running time is 300 s;
(3) when the background current tends to be stable, 10 mu L of 5 mol/L hydrogen peroxide solution is injected into 10 mL of 50 mmol/L phosphate buffer solution with pH = 7.38 every 50 s, and the current change is recorded;
(4) and drawing a working curve according to the linear relation between the obtained current and the concentration of the aflatoxin B1, wherein the linear range is 10 pg-60 ng/mL, and the lowest detection limit is 3.33 pg/mL.
Example 11 detection of aflatoxin G1
The aflatoxin G1 in the sample is detected according to the method in the embodiment 10, the linear range is 10 pg-60 ng/mL, and the detection limit is 3.33 pg/mL.
Claims (4)
1. A preparation method of an aflatoxin electrochemical sensor is characterized by comprising the following steps:
(1) al for glassy carbon electrode with diameter of 4 mm2O3Polishing the polishing powder into a mirror surface, and cleaning with ultrapure water;
(2) dripping a 6 mu L of 1.0-3.0 mg/mL AuPt nano particle solution on the surface of the electrode, drying at room temperature, washing the surface of the electrode with ultrapure water, and drying;
(3) continuing to capture the aflatoxin capture antibody Ab of 6 muL and 8-12 mug/mL1Dropwise adding the solution to the surface of an electrode, washing with ultrapure water, and drying in a refrigerator at 4 ℃;
(4) continuously dropwise adding bovine serum albumin BSA solution with the mass fraction of 1% to the surface of the electrode by 3 muL, 0.5-1.5 mg/mL to seal non-specific active sites on the surface of the electrode, washing the surface of the electrode with ultrapure water, removing unbound BSA, and drying in a refrigerator at 4 ℃;
(5) continuously dropwise adding a series of aflatoxin antigen solutions with different concentrations, namely 6 muL and 10 pg/mL-60 ng/mL, washing the surface of the electrode with ultrapure water, and airing in a refrigerator at 4 ℃;
(6) continuously dropwise adding detection antibody hatching Au/Cu of 6 muL and 1.5-3.5 mg/mLxO@CeO2-Ab2Placing the solution on the surface of an electrode, incubating for 40 min in a refrigerator at 4 ℃, washing with ultrapure water, and drying in the air to obtain the aflatoxin electrochemical sensor;
the preparation method of the AuPt nanoparticle solution comprises the following steps:
(1) preparation of AuPt nanoparticles
Sequentially adding 50-150 mg of poloxamer F127 and 20-40 mg of potassium iodide into 1.0 mL of ultrapure water, and uniformly stirring; adding 0.8 mL, 10-30 mmol/L chloroplatinic acid, 2.5 mL and 10-30 mmol/L chloroauric acid, and uniformly stirring; adding 2.5 mL of ascorbic acid of 0.05-0.15 mol/L, and reacting for 15 min at 90 ℃; respectively centrifugally cleaning ultrapure water and ethanol for three times, and drying at 60 ℃ for 12 h to prepare AuPt nanoparticles;
(2) preparation of AuPt nanoparticle solution
Dispersing 5-15 mg of AuPt nanoparticles into 5 mL of ultrapure water, and carrying out ultrasonic treatment for 10 min to prepare an AuPt nanoparticle solution;
the detection antibody hatches Au/CuxO@CeO2-Ab2The solution was prepared by the following steps:
(1)Au/CuxO@CeO2preparation of
Putting 100 mL of 0.01 mol/L copper chloride solution in a water bath at 55 ℃; adding 10 mL of 2 mol/L sodium hydroxide solution, and reacting for 30 min; then 10 mL of ascorbic acid with the concentration of 0.6 mol/L is added for reaction for 3 hours; respectively centrifugally cleaning ultrapure water and ethanol for three times, and vacuum drying at 60 ℃ for 5 hours to obtain Cu2An O cube;
adding 20-40 mg of Cu into 30 mL of ultrapure water2Stirring the mixture evenly, adding 500-700 mu L of cerium nitrate and 0.1 mol/L of cerium nitrate, and heating the mixture to 60 ℃; adding 3.0-4.0 mL of 0.15 mol/L ammonia water, and reacting for 3 h; adding 55-75 mu L of chloroauric acid of 20 mmol/L, and reacting for 15 min; adding 65 μ L, 20 mmol/L sodium citrate, and reacting for 30 min; centrifugally cleaning the solution for three times by ultrapure water, and drying the solution for 8 to 12 hours in vacuum at the temperature of 60 ℃ to obtain Au/CuxO@CeO2;
The copper chloride solution is prepared by adding 0.14g of copper chloride into 100 mL of ultrapure water and stirring for 5 min;
(2) detection of antibody hatching Au/CuxO@CeO2-Ab2Preparation of the solution
1.0-3.0 mL of Au/Cu with the concentration of 2 mg/mLxO@CeO2Adding the solution into 0.5-1.5 mL of aflatoxin detection antibody solution Ab with the concentration of 10 mu g/mL2Performing shake incubation for 12 h in a constant-temperature oscillation box at 4 ℃, centrifuging and washing, adding 1.0-3.0 mL of phosphoric acid buffer solution with pH = 7.38, and preparing the detection antibody incubation Au/CuxO@CeO2-Ab2The solution was stored at 4 ℃ until use.
2. The method for preparing an aflatoxin electrochemical sensor of claim 1, wherein the aflatoxin is selected from one of the following: aflatoxin B1, aflatoxin G1.
3. The aflatoxin electrochemical sensor prepared by the preparation method of claim 1, wherein the electrochemical sensor is used for detecting aflatoxin, and the steps are as follows:
(1) testing by using an electrochemical workstation in a three-electrode system, taking a saturated calomel electrode as a reference electrode, taking a platinum wire electrode as an auxiliary electrode, taking the prepared sensor as a working electrode, and testing in 10 mL of potassium ferricyanide solution containing 10 mmol/L;
(2) detecting aflatoxin by using a time-lapse current method, wherein the input voltage is-0.4V, the sampling interval is 0.1 s, and the running time is 300 s;
(3) when the background current tended to stabilize, 10 μ L of 5 mol/L hydrogen peroxide solution was injected into 10 mL of 50 mmol/L phosphate buffer solution with pH = 7.38 every 50 s, and the change in current was recorded.
4. An aflatoxin electrochemical sensor according to claim 3 wherein the aflatoxin is selected from one of the following: aflatoxin B1, aflatoxin G1.
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