CN115267194A - Indirect competitive enzyme-linked immunosorbent assay method for detecting aflatoxin B1 - Google Patents
Indirect competitive enzyme-linked immunosorbent assay method for detecting aflatoxin B1 Download PDFInfo
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- CN115267194A CN115267194A CN202210854212.0A CN202210854212A CN115267194A CN 115267194 A CN115267194 A CN 115267194A CN 202210854212 A CN202210854212 A CN 202210854212A CN 115267194 A CN115267194 A CN 115267194A
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- 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 title claims abstract description 105
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Images
Classifications
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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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J23/00—Catalysts comprising metals or metal oxides or hydroxides, not provided for in group B01J21/00
- B01J23/70—Catalysts comprising metals or metal oxides or hydroxides, not provided for in group B01J21/00 of the iron group metals or copper
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- 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
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
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- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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- 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
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Abstract
The invention belongs to the technical field of toxin analysis and detection, and particularly relates to an indirect competitive enzyme-linked immunosorbent assay method for detecting aflatoxin B1. The detection method of the aflatoxin B1 adopts an indirect competition method, and firstly, a complete antigen AFB1-B is usedSA is fixed on the bottom of the plate, AFB1 monoclonal antibody and AFB1 sample to be detected are added simultaneously after being closed, and then Fe with the surface coupled with second antibody IgG is added3O4@SiO2The @ CuO nano-particles are combined by IgG and monoclonal antibodies to realize the fixation of the nano-particles on the plate bottom, so that TMB substrate color development is catalyzed, and the quantitative detection of AFB1 standard substances and actual samples is finally realized by establishing an AFB1 standard curve. The method disclosed by the invention is easy to operate, low in cost, rapid and sensitive in detection, capable of eliminating errors caused by instability of the natural peroxidase, accurate and reliable in detection result and high in specificity.
Description
Technical Field
The invention belongs to the technical field of toxin analysis and detection, and particularly relates to an indirect competitive enzyme-linked immunosorbent assay method for detecting aflatoxin B1.
Background
Aflatoxins (AFs) are secondary metabolites generated based on aspergillus flavus and aspergillus parasiticus, are one of mycotoxins with the greatest harm, have a wide existence range and a large pollution range, and bring great threat to the life health safety of people. Wherein, aflatoxin B1 (AFB 1) is one of aflatoxins with strong toxicity and high carcinogenicity, is very easy to pollute agricultural products under natural conditions, can cause hepatitis and liver cancer after being eaten by human bodies, seriously threatens the health of the human bodies, has the median lethal dose of 0.36mg/kg, and belongs to the extremely virulent poison range.
At present, the detection methods for AFB1 include thin layer chromatography, high performance liquid detection, capillary electrophoresis, fluorescence photometry, enzyme-linked immunosorbent assay and the like, wherein the enzyme-linked immunosorbent assay is widely used due to the advantages of high sensitivity, simple detection steps, low cost and few interference factors.
However, most of the existing traditional enzyme-linked immunosorbent technologies are researched by using natural peroxidase (such as natural horseradish peroxidase), but the natural peroxidase is expensive, greatly influenced by environmental factors, easy to denature and inactivate, and difficult to store. In addition, the conventional detection method at present lacks specificity, so that the detection result is easy to generate false positive. Therefore, the development of a more economical, simple and specific method for detecting the content of AFB1 in agricultural products is urgently needed.
Disclosure of Invention
In order to solve the above problems, the present invention aims to provide an indirect competitive enzyme-linked immunosorbent assay method for detecting aflatoxin B1, which is easy to operate, low in cost, rapid and sensitive in detection, capable of eliminating errors caused by instability of natural peroxidase, accurate and reliable in detection result, and high in specificity.
In order to realize the aim of the invention, the indirect competitive enzyme-linked immunosorbent assay method for detecting the aflatoxin B1 adopts the technical scheme that:
an indirect competitive enzyme-linked immunosorbent assay method for detecting aflatoxin B1, which comprises the following steps:
1) Extracting aflatoxin B1 in a sample to be detected to obtain a sample solution to be detected;
2) Coating the aflatoxin B1-BSA complete antigen on an enzyme label plate, then washing, patting dry and sealing, and simultaneously adding a sample solution to be detected and an aflatoxin B1 monoclonal antibody solution for a competitive reaction;
3) After the competition reaction, washing was carried out, and then Fe of which surface was conjugated with a secondary antibody IgG was added3O4@SiO2The @ CuO nano-particle dispersion liquid reacts in a heat-preservation and moisture-preservation environment to realize the fixation of nano-particles on the bottom of the ELISA plate;
4) Step 3) washing and drying after reaction, adding a buffer solution and a TMB color development solution for a light-shielding reaction, then stopping the reaction, measuring absorbance by using an enzyme-labeling instrument, and realizing quantitative detection of the aflatoxin B1 in the sample to be detected according to the measured absorbance and a standard working curve of the aflatoxin B1;
wherein, the concentration of the aflatoxin B1 monoclonal antibody solution is 2 mug/mL;fe surface conjugated with Secondary antibody IgG3O4@SiO2The concentration of the @ CuO nano-particle dispersion liquid is 0.5mg/mL; sample solution to be detected, aflatoxin B1 monoclonal antibody solution and Fe with surface coupled with second antibody IgG3O4@SiO2The volume ratio of the @ CuO nano-particle dispersion liquid is 1: 1; in step 3), fe is used3O4@SiO2The @ CuO nano particles are uniform spherical, and the particle size is 50-200 nm; fe surface conjugated with Secondary antibody IgG3O4@SiO2In the preparation of the @ CuO nano-particles, the coupling steps are as follows: firstly adopting APTES to Fe3O4@SiO2Performing amination modification on @ CuO nanoparticles, and then adopting a crosslinking agent sulfo-SMCC to realize that a second antibody IgG is in Fe3O4@SiO2Coupling of the surface of @ CuO nanoparticles.
In the method of the invention, heterogeneous bimetallic nanoparticles (Fe) with high peroxidase-like activity are adopted3O4@SiO2@ CuO) was used in place of native horseradish peroxidase (HRP). At the same time by adding Fe3O4@SiO2The @ CuO surface is coupled with a second antibody IgG to prepare a signal marker Fe3O4@SiO2@ CuO-IgG, which catalyzes the color development of the substrate 3, 5-Tetramethylbenzidine (TMB).
The invention is proved by catalytic activity experiments to compare with the nano-particle Fe3O4、Fe3O4@SiO2Fe of the invention3O4@SiO2The @ CuO nano-particles have stable structures and stronger peroxidase-like activity; meanwhile, compared with natural Horse Radish Peroxidase (HRP), the Fe of the invention3O4@SiO2The catalytic activity of the @ CuO nano-particles is kept stable in a wide pH range (from 3.0 to 5.0), and the nano-particles have higher catalytic activity within a reaction time of 10-60 min at a temperature of 30-50 ℃, while the natural HRP is sensitive to pH, the activity is obviously reduced at a temperature of above 30 ℃, and the catalytic activity is also obviously reduced along with the increase of the reaction time. It can be seen that the nanoparticles of the present invention are stronger than other nanoparticles or native HRP enzymeThe method has environmental adaptability and universality, and can greatly meet the quantitative detection requirements of aflatoxin B1 in different enzyme-linked immunosorbent assay systems.
Further, in order to ensure the enzyme-linked immunosorbent assay effect of the whole system and further improve the sensitivity and accuracy of detecting the aflatoxin B1, the invention is used for detecting the aflatoxin B1 monoclonal antibody and Fe coupled with the second antibody IgG3O4@SiO2The use amounts of the @ CuO nano-particles and the sample solution to be detected are strictly controlled, so that the color development is more obvious, the color development degree can be better linked with the content of the aflatoxin B1, and the high-sensitivity and accurate detection of the aflatoxin B1 becomes possible.
Furthermore, in the process of constructing the enzyme-linked immune system, the invention considers that AFB1 is a hapten, namely a small molecular antigen, only has one epitope and cannot be effectively fixed at the bottom of the ELISA plate, so the invention provides the following steps after experimental verification: firstly, fixing a complete antigen AFB1-BSA at the bottom of a plate, adding an AFB1 monoclonal antibody and an AFB1 sample to be detected for competitive reaction after sealing, and then adding Fe coupled with IgG3O4@SiO2The @ CuO nano-particles are combined by IgG and monoclonal antibodies to realize the fixation of the nano-particles on the plate bottom, so that TMB substrate color development is catalyzed, and an AFB1 standard curve is established, so that the problem of quantitative detection of AFB1 standard products and actual samples is finally realized.
In summary, the indirect competitive enzyme-linked immunosorbent assay method for detecting the aflatoxin B1 adopts Fe for the first time3O4@SiO2The @ CuO nano-particles are used as nano-material enzyme to replace natural enzyme, an indirect competitive enzyme-linked immunosorbent assay (ELISA) based on AFB1 antigen-antibody specific binding is constructed for detecting AFB1 in products, and a brand-new determination idea can be provided for detecting aflatoxin B1.
In addition, the detection method only needs one absorbance measuring instrument, has the characteristics of simple required instrument, easy operation, low cost, quick and sensitive detection, can especially eliminate errors caused by instability of the natural peroxidase, and has accurate and reliable detection results and high specificity.
In a preferred embodiment of the present invention, the sample to be tested is grain, including but not limited to peanut, corn, and wheat, as long as the test sample contains a certain amount of AFB1 and has the detection requirement of AFB1.
The invention does not specially limit the type of the extraction solvent for extracting the AFB1 in the sample to be detected, and only needs to realize the effective extraction of the aflatoxin B1. Preferably, in order to ensure the extraction effect of aflatoxin B1, and thus not affect subsequent determination, in step 1), when aflatoxin B1 in the sample to be detected is extracted, the extraction solvent adopted is 50% methanol-water (V/V), and each gram of sample to be detected corresponds to 5mL of extraction solvent. In order to further improve the extraction efficiency and the extraction effect, a pretreatment mode of adding a solvent, performing ultrasonic-assisted extraction and then centrifuging can be adopted. Such as may be: weighing a sample to be detected, grinding the sample to be detected into powder or small particles, adding a subsequent solvent, uniformly mixing, placing in an ultrasonic disperser for ultrasonic treatment, centrifuging, and taking supernatant for detection.
In the invention, aflatoxin B1-BSA complete antigen is marked as AFB1-BSA, and since AFB1 is a hapten per se and cannot be fixed at the bottom of an enzyme label plate, the complete antigen AFB1-BSA is coated at the bottom of the plate, after being sealed, AFB1 monoclonal antibody is added to compete with a sample to be detected for reaction, and then Fe coupled with IgG is added3O4@SiO2The @ CuO nanoparticle is used for realizing the immobilization of the nanoparticle on the plate bottom through the combination of IgG and monoclonal antibodies, thereby catalyzing the color development of a substrate. Preferably, the step of complete antigen coating is: AFB1-BSA was diluted to a concentration of 1. Mu.g/mL with carbonate coating buffer, and then plated on an ELISA plate at 100 ng/well, and the plate was left at 4 ℃ overnight. Further preferably, the blocking treatment of the complete antigen is: adding a sealing solution into the mixture at a rate of 200 mu L/hole, and preserving heat and moisture for 2h in an environment at 37 ℃. More preferably, the adopted confining liquid is bovine serum albumin solution with the concentration of 10mg/mL, and the preparation method comprises the following steps: to 1g of bovine serum albumin, 100mL of PBS was added.
The competitive reaction is carried out in the invention, AFB1-BSA is coated on an enzyme label plate, then a sample solution to be detected and an aflatoxin B1 monoclonal antibody solution are added simultaneously, at the moment, AFB1 in the sample solution to be detected and AFB1-BSA on the enzyme label plate compete to bind with the aflatoxin B1 monoclonal antibody, the higher the concentration of AFB1 in the sample solution to be detected is, the more aflatoxin B1 monoclonal antibody competitively binds, the less antibody is bound with AFB1-BSA on the bottom of the plate, and finally, the lighter the color development of a reaction hole is, so that the absorbance of the developing solution is substituted into a standard curve, and the purpose of quantitatively detecting the aflatoxin B1 can be achieved. Preferably, in step 2), the conditions of the competition reaction are: and (3) carrying out competitive reaction for 1h at 37 ℃ under the environment of heat preservation and moisture preservation.
To ensure Fe3O4@SiO2The peroxidase-like activity of the @ CuO nanoparticles to improve detection sensitivity and accuracy, preferably, in step 3), fe3O4@SiO2The preparation method of the @ CuO nano-particles comprises the following steps:
(1) under the protection of nitrogen, 20mL of deionized water and 7.5mL of FeCl acidified by hydrochloric acid3The solution is stirred and mixed for 0.5h, and then 5mL of Na is dropwise added at a constant speed2SO3The solution, when the color of the solution changes from reddish brown to yellow, begins to drop 200mL NH3·H2Stirring O solution vigorously, precipitating black solid, stirring for 40min, and vacuum drying to obtain Fe3O4A nanoparticle; wherein FeCl3Solution, na2SO3Solution, NH3·H2The concentration of the O solution is 2mol/L, 1mol/L and 0.85mol/L in sequence;
(2) 600. Mu.L of Fe with a concentration of 8.4mg/mL3O4The nanoparticle dispersion was added to a 4mL, 0.08mol/L aqueous solution of cetyltrimethylammonium bromide to obtain a dispersion, and the dispersion was added to a solution of 24mL of deionized water, 8mL of ethylene glycol, and 0.57mL of NH3·H2Heating in 70 deg.C water bath, stirring at 120rpm for 10min, adding 0.57mL n-decane, stirring for 40min, dropwise adding 0.14mL 1,3, 5-trimethylbenzene, stirring for 2h to homogenize, adding 366 μ L tetraethyl silicate, stirring for 3h, centrifuging, and precipitating to obtain Si-coated productO2Fe of the layer3O4Nanoparticles, which were then put in a solution containing 48mg of NH4NO348mL of ethanol, stirring at 60 ℃ for 2h, then repeating the extraction 3 times to remove the cetyltrimethylammonium bromide, centrifuging to collect the product, washing with ethanol and drying in vacuum to obtain Fe3O4@SiO2A nanoparticle;
(3) after 84mg of copper nitrate is fully dissolved in 18mL of deionized water, the pH value is adjusted to 10-11 by using 1mol/L NaOH solution, and 12mg of Fe is added3O4@SiO2Vibrating the nano particles for 10min to uniformly disperse the nano particles, then carrying out water bath on the mixed solution for 24h at the temperature of 80 ℃ to generate black precipitates, washing the black precipitates twice by using deionized water and absolute ethyl alcohol, and carrying out vacuum drying to obtain Fe3O4@SiO2@ CuO nanoparticles.
The invention adopts the steps to prepare the obtained Fe3O4@SiO2The @ CuO nano-particle is uniform and spherical, has the particle size of 50-200 nm, has a monoclinic crystal type stable structure, has better peroxidase-like enzyme catalytic activity than other nano-particles as proved by tests, and can be effectively coupled with a second antibody IgG after the surface of the nano-particle is aminated.
IgG and Fe based on improved secondary antibodies3O4@SiO2In consideration of coupling success rate and coupling effect of @ CuO nanoparticles, it is preferable that Fe of the second antibody IgG is surface-coupled in step 3)3O4@SiO2The preparation method of the @ CuO nano-particles comprises the following specific steps:
a) Mixing Fe3O4@SiO2Uniformly dispersing the @ CuO nano particles in absolute ethyl alcohol, then adding APTES, condensing and refluxing for 24h at 80 ℃, then washing and drying to obtain aminated Fe3O4@SiO2@ CuO nanoparticles; then taking aminated Fe3O4@SiO2@ CuO nano particles are washed, added with a cross-linking agent sulfo-SMCC and oscillated for 0.5h; wherein, fe3O4@SiO2The dosage ratio of the @ CuO nano particles to the APTES is 40 mg: 150 muL; aminated Fe3O4@SiO2@ CuO nanoparticlesThe dosage ratio of the granules to the sulfo-SMCC is 10 mg: 60 mu L; the concentration of the cross-linking agent sulfo-SMCC is 2mg/mL;
b) Diluting a second antibody IgG by PBS, adding a boric acid buffer solution containing EDTA and a 2-iminosulfane hydrochloride solution, and oscillating for reaction for 1h; wherein the volume dosage of the second antibody IgG, PBS, boric acid buffer solution containing EDTA and 2-iminothiolane hydrochloride solution is 1.5 muL, 20 muL, 500 muL and 200 muL; the concentration of the 2-iminothiolane hydrochloride solution is 2mg/mL;
c) Adding the reaction system obtained in the step b) into the reaction system obtained in the step a), and mixing and oscillating for 2h to obtain Fe with the surface coupled with a second antibody IgG3O4@SiO2@ CuO nanoparticles, noted Fe3O4@SiO2@CuO-IgG。
Further, the invention prepares Fe with surface coupled second antibody IgG3O4@SiO2After the @ CuO nano particles are washed, the nano particles are diluted into a dispersion liquid with required concentration by adopting a sodium acetate buffer solution with the pH value of 3.6.
The pH, temperature and reaction time all affect Fe3O4@SiO2@ CuO nanoparticles. For improving Fe3O4@SiO2The catalytic activity of @ CuO, preferably, in step 4), the buffer is sodium acetate buffer at pH 3.6; the temperature of the water bath light-proof reaction is 37 ℃, and the time is 10-15 min. Further, the termination reaction is: adding a stop solution, and standing at room temperature for 4-6 min to stop the reaction; the stop solution can be 2M sulfuric acid solution.
Preferably, in step 2) to step 4) of the present invention, the washing is: washing with washing solution for 3 times, each time for 3min; the wash was 0.1M PBST, pH 7.4.
In the invention, the absorbance value of the sample to be detected can be specifically measured by adopting an enzyme-labeling instrument. Preferably, in the step 4), the measurement wavelength is 450nm when the absorbance is measured.
The beneficial effects of the invention are as follows:
in the process of the invention, use is made of compounds having peroxidase catalytic activityNanoparticles (Fe)3O4@SiO2@ CuO), conjugated secondary antibodies (IgG), instead of natural horseradish peroxidase (HRP) -labeled secondary antibodies in traditional enzyme-linked immunoassay methods, to perform indirect competitive enzyme-linked immunoassay for detection of AFB1. Compared with other methods for detecting AFB1, the method provided by the invention has the advantages that the nanoparticles are used for replacing HRP in the traditional indirect competitive enzyme-linked immunosorbent assay, so that errors caused by instability of the HRP can be effectively eliminated, and the detection result is more accurate. Meanwhile, the AFB1 monoclonal antibody can be specifically combined with the antigen in the sample, so that the detection result has high specificity. In addition, the method has the advantages of simple required instrument, easy operation, low cost and quick and sensitive detection.
Furthermore, the detection range of the method of the invention on AFB1 is 0.06-61.9 ng/mL, the detection limit is 0.0037ng/mL, and the detection limit is significantly lower than the maximum limit of AFB1 in grain agricultural products specified by national standards (the national standard [ GB 2761-2017 ] specifies that the AFB1 limit of peanut and peanut products is 2 mug/kg, the AFB1 limit of corn and corn products is 20 mug/kg, and the AFB1 limit of wheat and wheat products is 5 mug/kg). Meanwhile, the linear relation between the absorbance and the logarithmic value of the concentration of the standard substance is good: y = -0.38903lg (x) +1.109863, correlation coefficient R2=0.98953, and the average standard recovery rates of the peanut, corn and wheat samples are 105.3%, 96.15% and 88.15%, respectively, so that the method can be successfully applied to detection of AFB1 in actual samples, has good accuracy and repeatability, and has high practical application and popularization value.
Drawings
FIG. 1 is a schematic diagram of the detection process of the indirect competitive ELISA method of the present invention;
FIG. 2 is a standard working curve of the indirect competition method enzyme-linked immunoassay AFB1 of the present invention;
FIG. 3 is a linear relationship between the logarithmic value of the AFB1 standard concentration and the absorbance in the present invention;
FIG. 4 shows Fe prepared by the present invention3O4@SiO2Transmission Electron Microscope (TEM) images of @ CuO nanoparticles;
FIG. 5 shows the present inventionMing prepared Fe3O4@SiO2An elemental map of @ CuO nanoparticles;
FIG. 6 shows Fe prepared by the present invention3O4@SiO2Infrared spectroscopy (FT-IR) profile of @ CuO nanoparticles;
FIG. 7 shows Fe prepared by the present invention3O4@SiO2X-ray diffraction (XRD) pattern of @ CuO nanoparticles;
FIG. 8 shows Fe prepared by the present invention3O4@SiO2Fluorescence analysis of @ CuO-IgG;
FIG. 9 shows Fe prepared by the present invention3O4、Fe3O4@SiO2And Fe3O4@SiO2Color change plot of @ CuO nanoparticle catalyzed reaction;
FIG. 10 shows Fe prepared by the present invention3O4、Fe3O4@SiO2And Fe3O4@SiO2The ultraviolet absorption spectrogram of the @ CuO nano-particle catalytic reaction solution;
FIG. 11 shows reaction system for Fe at different pH3O4@SiO2The effect of the catalytic activity of @ CuO nanoparticles;
FIG. 12 shows different reaction temperatures vs. Fe3O4@SiO2The effect of the catalytic activity of @ CuO nanoparticles;
FIG. 13 shows different reaction times vs. Fe3O4@SiO2The effect of the catalytic activity of @ CuO nanoparticles;
FIG. 14 is a graph comparing the indirect competitive enzyme-linked immunoassay for various mycotoxins with AFB1.
Detailed Description
The indirect competitive ELISA method for detecting aflatoxin B1 of the present invention is specifically described below with reference to specific examples, but the present invention is not limited to the technical scheme of the present invention.
In the following examples, reference is made to Fe3O4@SiO2The @ CuO nano-particles are prepared by adopting the following preparation method:
(1) in general has N2In a three-necked flask, 20mL of deionized water was added, followed by 7.5mL of FeCl acidified with hydrochloric acid3The solution (2 mol/L) is stirred for 0.5h and then 5mL of Na is dropwise added at a constant speed2SO3(1 mol/L), when the color of the solution changes from reddish brown to yellow, 200mL of NH with the concentration of 0.85mol/L is slowly added3·H2Stirring O solution vigorously, precipitating black solid by reaction, and vacuum drying at 45 deg.C to obtain Fe3O4And (3) nanoparticles.
(2) 600 μ L of Fe-containing solution3O4The chloroform solution of nanoparticles (8.4 mg/mL) was added to 4mL of a cetyltrimethylammonium bromide (CTAB) aqueous solution (0.08 mol/L) to obtain a dispersion, and the dispersion was added to a solution prepared from 24mL of deionized water, 8mL of ethylene glycol, and 0.57mL of NH3·H2O, heating in water bath at 70 deg.C, stirring at 120rpm for 10min, adding 0.57mL n-decane, stirring for 40min, dropwise adding 0.14mL 1,3, 5-trimethylbenzene, stirring for 2h for homogenization, adding 366 μ L tetraethyl silicate (TEDS), stirring for 3h, centrifuging, and precipitating to obtain SiO-coated product2Fe of the layer3O4Nanoparticles, which were then put in a solution containing 48mg of NH4NO348mL of ethanol and stirred at 60 ℃ for 2h. Extracting for 3 times to remove surfactant CTAB, centrifuging to collect product, washing with ethanol, and vacuum drying at 45 deg.C to obtain Fe3O4@SiO2And (3) nanoparticles.
(3) After 84mg of copper nitrate is fully dissolved in 18mL of deionized water, 1mol/L NaOH is used for adjusting the pH value of the solution to between 10 and 11, and 12mg of Fe is added3O4@SiO2And shaking the nano particles for 10min to uniformly disperse the nano particles. The mixed solution is bathed for 24h at 80 ℃ to generate black precipitate, washed twice by deionized water and absolute ethyl alcohol, and dried in vacuum at 45 ℃ to obtain Fe3O4@SiO2@ CuO nanoparticles.
In the following examples, the surface-conjugated secondary antibody IgG referred to is Fe3O4@SiO2@ CuO nanoparticles, the specific coupling steps being:
mixing 40mg of Fe3O4@SiO2The @ CuO nano particles are suspended in 20mL of absolute ethyl alcohol, added with 150 mu L of APTES after ultrasonic homogenization and condensed and refluxed for 24h in a water bath kettle at the temperature of 80 ℃. Washing with anhydrous ethanol, and vacuum drying at 45 deg.C to obtain aminated Fe3O4@SiO2@ CuO nanoparticles. Then, 1.5. Mu.L of the secondary antibody IgG (rabbit anti-mouse) was added to 20. Mu.L of PBS (0.01M), and then 500. Mu.L of EDTA-containing boric acid buffer and 200. Mu.L of 2-iminosulfane hydrochloride solution (Traut's Reagent,2 mg/mL) were added and reacted in a shaker for 1 hour. 10mg of aminated Fe were weighed3O4@SiO2@ CuO nanoparticles, washed three times with 0.1M PBS without EDTA, added 60. Mu.L of sulfo-SMCC (2 mg/mL), shaken for 0.5h. Finally, adding the obtained antibody solution into a solution containing nano particles, mixing and shaking for 2 hours to obtain Fe3O4@SiO2@ CuO-IgG. After washing with sterile 0.01M PBS three times, the mixture was diluted to a concentration of 0.5mg/mL with 0.02mol/L sodium acetate buffer (pH 3.6) and stored for further use.
Example 1
The indirect competitive enzyme-linked immunosorbent assay method for detecting aflatoxin B1 in the embodiment is schematically shown in figure 1, and comprises the following specific steps:
1) Extraction of AFB1 in samples: weighing 1g of sample (peanut, corn or wheat agricultural product), grinding into powder or small particles, adding 5mL of extracting solution (50% methanol water solution, V/V), uniformly mixing, placing in an ultrasonic disperser, performing ultrasonic treatment for 15min, centrifuging (4000 rpm,5 min), and taking 3mL of supernatant as sample solution to be detected for detection;
2) Coating: using a coating buffer solution (0.05 mol/L carbonate buffer solution, pH9.6) to dilute aflatoxin B1-BSA complete antigen (AFB 1-BSA) to a concentration of 1 mug/mL, spreading the aflatoxin B1-BSA complete antigen on an enzyme label plate at a speed of 100 ng/hole, and placing the enzyme label plate in an environment at 4 ℃ overnight;
3) And (3) sealing: washing with washing solution (0.1M PBST, pH7.4) for 3 times, 3min each time, beating to dry, adding blocking solution (bovine serum albumin solution, 10 mg/mL) at 200 μ L/hole, and keeping warm and moisture at 37 deg.C for 2 hr;
4) And (3) competitive reaction: washing with washing solution for 3 times, 3min each time, beating to dry, adding 50 μ L sample solution to be tested into each well, adding 50 μ L AFB1 monoclonal antibody solution (2 μ g/mL), and performing competitive reaction for 1h in 37 deg.C environment;
5) Adding enzyme-labeled secondary antibody: washing with washing solution for 3 times, 3min each time, and adding 50. Mu.L of Fe surface-conjugated secondary antibody IgG to each well3O4@SiO2@ CuO nanoparticles (Fe)3O4@SiO2@ CuO-IgG) (0.5 mg/mL) and reacting for 1h in a heat-preservation and moisture-preservation environment at 37 ℃;
6) And (3) color development reaction: washing with washing solution for 3 times, each for 3min, drying, adding 100 μ L sodium acetate buffer (pH3.6) into each well, adding 6 μ L in situ TMB color development solution, and reacting in 37 deg.C water bath under dark condition for 15min;
7) And (3) terminating the reaction: mu.L of stop solution (2M H) was added to each well simultaneously2SO4) Standing at room temperature for 5min to terminate the reaction;
8) Measurement of absorbance (OD value): measuring the OD value at the position with the wavelength of 450nm by using a microplate reader; and determining the content of the aflatoxin B1 in the sample according to the measured absorbance and the standard working curve of the aflatoxin B1.
In the above embodiment, the process of establishing the standard working curve of aflatoxin B1 is as follows: diluting AFB1 standard substance with 50% methanol water solution (V/V) 4 times to 15 gradients to make AFB1 standard substance concentration (0.93 × 10)-9) About 0.25mg/mL, and setting a blank control group at the same time, wherein 16 gradients are provided; and then carrying out the operations of the steps 2) to 8), changing the sample solution into standard solutions with different concentrations, and measuring the OD value at the position of 450nm of the wavelength by using a microplate reader after the reaction is finished.
And (3) taking a logarithmic value of the concentration of the AFB1 standard substance with the base of 10 as an abscissa and an OD value at 450nm as an ordinate, drawing a standard curve and calculating a linear regression equation. As shown in FIG. 2, the OD value at 450nm gradually decreases with the increase of the concentration of AFB1 standard substance, and the standard S-shaped is shown.
As shown in FIG. 3, the absorbance was in a linear relationship with the logarithm of the standard concentration in the range of 0.06 to 61.9 ng/mL: y = -0.38903lg (x) +1.109863, correlation coefficient R2=0.98953. The lowest concentration of AFB1 which differs from the OD at 450nm of the negative control by 0.1 or more is considered as the lowest concentration of AFB1 at which inhibition can occur, and this concentration is used as the detection limit, so the detection limit of the method of the present invention is 0.0037ng/mL.
Experimental example 1 Fe3O4@SiO2Characterization of @ CuO nanoparticles
Prepared Fe by transmission electron microscope3O4@SiO2The morphology of the @ CuO nanoparticles was characterized, and the results are shown in FIG. 4. The transmission electron microscope image shows that the nano-particles are in a uniform spherical shape, the particle size range is 50-200 nm, and the average particle size is about 100nm (see figure 4). Fe3O4@SiO2The elemental mapping (fig. 5) of @ CuO nanoparticles indicates that the particles contain Fe, si, cu and O elements, the Fe elements are more concentrated and are the cores of the nanoparticles, and the rest elements are more uniformly distributed.
Fe3O4@SiO2In the preparation process of the @ CuO nano-particles, fe prepared in each step3O4、Fe3O4@SiO2And Fe3O4@SiO2The characteristic absorption peak of the @ CuO nanoparticle characterization element is obtained by adopting a Fourier infrared spectrum, and the result of FIG. 6 can be observed. Nano Fe3O4FTIR spectrum of (5) at 575cm-1Has a strong absorption peak corresponding to the vibration of Fe-O bonds. Fe3O4@SiO2The spectrum of (A) is at 796 and 1080cm-1There are additional peaks due to symmetric and asymmetric Si-O-Si stretching vibrations. 500-600cm-1The nearby absorption peak is ascribed to the vibrational mode of Cu-O bond, and FIG. 6 shows that Fe was successfully produced3O4@SiO2@ CuO nanoparticles.
The Fe prepared in each step is mixed3O4、Fe3O4@SiO2And Fe3O4@SiO2The @ CuO nanoparticles were further analyzed for their crystal structure with an X-ray diffractometer (see FIG. 7). FIG. 7 shows the results for Fe3O4The nanoparticles have six characteristic diffraction peaks (30.2 deg. and 35.5 deg. °)43.3 degrees, 53.7 degrees, 57.2 degrees and 62.9 degrees), and the positions of six diffraction peaks are basically consistent with those of the standard product, and the peak shape is sharp, which indicates that the invention successfully synthesizes Fe3O4Nanoparticles. Fe3O4@SiO2Spectrogram of nano-particles and nano-Fe3O4The same, but with a reduced peak intensity. This is due to SiO2In Fe3O4And stacking on the crystal nucleus. Fe3O4@SiO29 characteristic peaks corresponding to CuO standard substance are observed in X-ray diffraction pattern of @ CuO nano particles, and Fe is proved3O4@SiO2The monoclinic crystal structure of @ CuO. These results further demonstrate Fe3O4@SiO2The successful preparation of @ CuO nanoparticles.
Experimental example 2 Fe3O4@SiO2@ CuO conjugated antibody Fe3O4@SiO2Activity assay of @ CuO-IgG
Fe analysis by the Indirect fluorescence immunoassay3O4@SiO2@ CuO was successfully coupled to IgG. The fluorescent dye Alexa flour 488 (goat anti-rabbit) can be specifically combined with secondary IgG (rabbit anti-mouse), and green fluorescence is displayed under the irradiation of blue excitation light of a fluorescence microscope. Taking 100 mu L of Fe3O4@SiO2@ CuO-IgG solution, 5 uL Alexa flour 488 fluorescent dye (goat anti-rabbit) is added, and the reaction is carried out for 12h in a dark oscillation way; washed 2-3 times with sterile PBS, 10 μ L was put on a slide and observed under a fluorescence microscope. Fe assayed as unconjugated IgG3O4@SiO2@ CuO is a control, and the results are shown in FIG. 8.
The results in FIG. 8 show that: fe3O4@SiO2@ CuO-IgG had significant green fluorescence, whereas the control group was non-fluorescent, indicating IgG plus Fe3O4@SiO2@ CuO was successfully coupled.
Experimental example 3 verification of Fe3O4@SiO2Peroxidase-like activity of @ CuO nanoparticles and determination of optimal reaction conditions
(1) Verification of Fe3O4@SiO2Peroxide-like of @ CuO nanoparticlesEnzyme activity: with TMB as a chromogenic substrate, in H2O2The catalytic oxidation reaction was carried out in the presence of oxygen, and the Fe prepared by the present invention was examined3O4、Fe3O4@SiO2And Fe3O4@SiO2@ CuO nanoparticles. 50 μ L of native Horse Radish Peroxidase (HRP) (25 ng/mL) was used as a positive control, and 50 μ L of LFe was added3O4、Fe3O4@SiO2And Fe3O4@SiO2The @ CuO nano mimic enzyme (1 mg/mL, solvent is ultrapure water) is added into the reaction solution containing 20 muL H2O2After incubation at 37 ℃ for 10 minutes in 500. Mu.L of sodium acetate buffer (0.2M, pH 3.6) (30%) and 10. Mu.L of TMB (10 mg/mL, solvent DMSO), the absorbance of the supernatant at 652nm was measured using a UV-visible spectrophotometer.
As shown in FIG. 9, contains Fe3O4(a1-a4)、Fe3O4@SiO2(b 1-b 4) and Fe3O4@SiO2The solution of @ CuO (c 1-c 4) nanoparticles was catalyzed to blue, and 50. Mu.L of H was added2SO4After (0.5 mol/L) the reaction was terminated, the solution turned from blue to yellow, the color depth being consistent with that before termination.
Absorption spectra As shown in FIG. 10, negative control (TMB-H) without nanoparticles2O2) The absorption peak at 652nm is very weak (d), and contains Fe3O4@SiO2The reaction system of @ CuO nano particle has a strong absorption peak (c) at 652nm, and in addition, fe3O4@SiO2The catalytic activity of the particles (b) is slightly lower than that of bare Fe3O4Nanoparticles (a) due to SiO2Occupy Fe3O4Resulting in a reduction of catalytic centers. Fe loaded with CuO3O4@SiO2The catalytic activity of the @ CuO nanoparticles is significantly enhanced due to Fe3O4And CuO.
The above results indicate Fe3O4、Fe3O4@SiO2And Fe3O4@SiO2The @ CuO nanoparticles all have peroxidase-like activity, but the Fe of the present invention3O4@SiO2The @ CuO nanoparticles are most active.
(2) Determination of optimal reaction conditions: fe3O4@SiO2The catalytic activity of @ CuO is influenced by a combination of pH, temperature and reaction time. Therefore, fe was examined at different pH (2.8-7.6), different temperature (30-60 ℃), different reaction time (0-60 min)3O4@SiO2The results of the relative catalytic activity of @ CuO and the positive control of natural horseradish peroxidase (HRP) are shown in FIGS. 11 to 13, and Fe3O4@SiO2@ CuO is represented by FSC in the figure.
The results show that the catalytic activity of the nanoparticles remains stable over a wide pH range (from 3.0 to 5.0) (fig. 11), is less sensitive to pH than native HRP, has a higher catalytic activity between 30 ℃ and 50 ℃, and the activity of native HRP decreases significantly above 30 ℃ (fig. 12). In addition, the activity of the nanoparticles remained stable for 10-60 min of reaction time, while the catalytic activity of HRP decreased with increasing reaction time (fig. 13). Therefore, the optimum reaction conditions are pH3.6, 37 ℃ and 10min of reaction time, in this case Fe3O4@SiO2The @ CuO nanoparticles have the strongest catalytic activity.
Experimental example 4 method specificity verification
In order to explore the detection specificity of the method for AFB1, three mycotoxins, namely Zearalenone (ZEN), deoxynivalenol (DON) and ochratoxin A (OTA), are selected as structural analogues of AFB1 to carry out detection similar to that of embodiment 1 of the invention, wherein the concentrations of ZEN, DON and OTA are 1ng/mL, the concentration of AFB1 is 0.2ng/mL, and a blank control group is arranged at the same time. The results are shown in FIG. 14.
As can be seen from fig. 14, even if the concentration of other mycotoxins was 5 times the concentration of AFB1, no competitive reaction occurred at all, and no change in absorbance was caused, indicating that the measurement method of the present invention has high specificity for the detection of AFB1.
Experimental example 5 method verification of accuracy and precision
In order to evaluate the accuracy and precision of the method, mildew-free peanut, corn and wheat samples are adopted, sample solutions are extracted, AFB1 standard solutions with different concentrations are added into the sample solutions to be used as standard adding samples for quantitative detection, and meanwhile, blank control is set. Spiked recovery and Relative Standard Deviation (RSD) were calculated and six replicates of each set of experiments were run. The results are shown in Table 1. The calculation method of the standard addition recovery rate comprises the following steps:
the RSD is calculated as: in the actual sample detection process, each sample detection is repeated in parallel for 6 times, 6 absorbance values are correspondingly obtained, and the standard deviation is calculated. The RSD calculation formula is as follows:
TABLE 1 spiked recovery of AFB1 in peanut, corn and wheat samples
The results in table 1 show that the average standard recovery rates of the peanut, corn and wheat samples are 105.3%, 96.15% and 88.15%, respectively, which indicates that the analysis method is suitable for detecting AFB1 in the peanut, corn and wheat samples and has good accuracy. RSD is less than 5.7%, which shows that the method has good repeatability.
In summary, the method of the invention uses nanoparticles (Fe) with peroxidase catalytic activity3O4@SiO2@ CuO), and a second antibody IgG is coupled to replace a natural horseradish peroxidase (HRP) labeled secondary antibody in the traditional enzyme-linked immunoassay method, so as to detect AFB1 by an indirect competitive enzyme-linked immunosorbent assay. Compared with other methods for detecting AFB1, the method disclosed by the invention can effectively eliminate errors caused by HRP instability, so that the method can effectively eliminate the errors caused by the HRP instabilityThe detection result is more accurate. Meanwhile, the AFB1 monoclonal antibody can be specifically combined with the antigen in the sample, so that the detection result has high specificity. In addition, the method has the advantages of simple required instrument, easy operation, low cost and quick and sensitive detection. Furthermore, the method is verified by methodology experiments, the detection range of the AFB1 is 0.06-61.9 ng/mL, the detection limit is 0.0037ng/mL, and the detection limit is obviously lower than the maximum limit of the AFB1 in the grain agricultural products specified by the national standard. Meanwhile, the linear relation between the absorbance and the logarithmic value of the concentration of the standard substance is good: y = -0.38903lg (x) +1.109863, correlation coefficient R2=0.98953, and the average standard recovery rates of the peanut, corn and wheat samples are 105.3%, 96.15% and 88.15%, respectively, so that the method can be successfully applied to detection of AFB1 in actual samples, has good accuracy and repeatability, and has high practical application and popularization value.
Claims (10)
1. An indirect competitive enzyme-linked immunosorbent assay method for detecting aflatoxin B1 is characterized by comprising the following steps:
1) Extracting aflatoxin B1 in a sample to be detected to obtain a sample solution to be detected;
2) Coating the aflatoxin B1-BSA complete antigen on an enzyme label plate, then washing, patting dry and sealing, and simultaneously adding a sample solution to be detected and an aflatoxin B1 monoclonal antibody solution for a competitive reaction;
3) After the competition reaction, washing was carried out, and then Fe of which surface was conjugated with a secondary antibody IgG was added3O4@SiO2The @ CuO nano-particle dispersion liquid reacts in a heat-preservation and moisture-preservation environment to realize the fixation of nano-particles on the bottom of the ELISA plate;
4) Step 3) washing and drying after reaction, adding a buffer solution and a TMB color development solution for a light-shielding reaction, then stopping the reaction, measuring absorbance by using an enzyme-labeling instrument, and realizing quantitative detection of the aflatoxin B1 in the sample to be detected according to the measured absorbance and a standard working curve of the aflatoxin B1;
wherein, the concentration of the aflatoxin B1 monoclonal antibody solution is 2 mug/mL; surface coupling of secondFe of antibody IgG3O4@SiO2The concentration of the @ CuO nanoparticle dispersion liquid is 0.5mg/mL; sample solution to be detected, aflatoxin B1 monoclonal antibody solution and Fe with surface coupled with second antibody IgG3O4@SiO2The volume ratio of the @ CuO nano-particle dispersion liquid is 1: 1; in step 3), fe is used3O4@SiO2The @ CuO nano-particles are uniform spherical, and the particle size is 50-200 nm; fe surface conjugated with Secondary antibody IgG3O4@SiO2In the preparation of the @ CuO nano-particle, the coupling steps are as follows: firstly adopting APTES to Fe3O4@SiO2Performing amination modification on @ CuO nanoparticles, and then adopting a crosslinking agent sulfo-SMCC to realize that a second antibody IgG is in Fe3O4@SiO2Coupling of the surface of @ CuO nanoparticles.
2. The indirect competitive ELISA method of detecting aflatoxin B1 of claim 1 wherein in step 1), the sample to be detected is grain; when aflatoxin B1 in a sample to be detected is extracted, the adopted extraction solvent is 50% methanol water (V/V), and each gram of sample to be detected corresponds to 5mL of extraction solvent.
3. The indirect competitive ELISA method for detecting aflatoxin B1 of claim 1 wherein in step 2), the aflatoxin B1-BSA complete antigen is denoted as AFB1-BSA, and the coating step is: AFB1-BSA was diluted to a concentration of 1. Mu.g/mL with carbonate coating buffer, and then plated on an ELISA plate at 100 ng/well, and the plate was left at 4 ℃ overnight.
4. The indirect competitive ELISA method for detecting aflatoxin B1 of claim 1 wherein in step 2), the blocking treatment is: adding a sealing solution into the mixture at a rate of 200 mu L/hole, and preserving heat and moisture for 2h at 37 ℃.
5. The indirect competitive ELISA method for detecting aflatoxin B1 of claim 1 wherein in step 2), the conditions of the competition reaction are as follows: and (3) carrying out competitive reaction for 1h at 37 ℃ under the environment of heat preservation and moisture preservation.
6. The indirect competitive ELISA method of detecting aflatoxin B1 of claim 1 wherein in step 3), fe3O4@SiO2A preparation method of @ CuO nano-particles comprises the following steps:
(1) under the protection of nitrogen, 20mL of deionized water and 7.5mL of FeCl acidified by hydrochloric acid3The solution was stirred and mixed for 0.5h, then 5mL Na was added dropwise2SO3The solution, when the color of the solution changes from reddish brown to yellow, is started to drop 200mL of NH3·H2Stirring O solution vigorously, precipitating black solid, stirring for 40min, and vacuum drying to obtain Fe3O4A nanoparticle; wherein FeCl3Solution, na2SO3Solution, NH3·H2The mass concentration of the O solution is 2mol/L, 1mol/L and 0.85mol/L in sequence;
(2) 600. Mu.L of Fe with a concentration of 8.4mg/mL3O4The nanoparticle dispersion was added to a 4mL, 0.08mol/L aqueous solution of cetyltrimethylammonium bromide, which was then added to a solution consisting of 24mL deionized water, 8mL ethylene glycol, and 0.57mL NH3·H2Heating in 70 deg.C water bath, stirring at 120rpm for 10min, adding 0.57mL n-decane, stirring for 40min, dropwise adding 0.14mL 1,3, 5-trimethylbenzene, stirring for 2h to homogenize, adding 366 μ L tetraethyl silicate, stirring for 3h, centrifuging, and precipitating to obtain SiO-coated product2Fe of the layer3O4Nanoparticles, which were then put in a solution containing 48mg of NH4NO348mL of ethanol, stirring at 60 ℃ for 2h, then repeating the extraction 3 times to remove the cetyltrimethylammonium bromide, centrifuging to collect the product, washing with ethanol and drying in vacuum to obtain Fe3O4@SiO2A nanoparticle;
(3) after 84mg of copper nitrate is fully dissolved in 18mL of deionized water, 1mol/L NaOH solution is used for adjusting the pH value to 10-11, and 12mg of Fe is added3O4@SiO2Vibrating the nano particles for 10min to uniformly disperse the nano particles, then carrying out water bath on the mixed solution for 24h at the temperature of 80 ℃ to generate black precipitates, washing the black precipitates twice by using deionized water and absolute ethyl alcohol, and carrying out vacuum drying to obtain Fe3O4@SiO2@ CuO nanoparticles.
7. The indirect competitive ELISA method of detecting aflatoxin B1 of claim 1 or claim 6 wherein in step 3), the surface of the secondary antibody IgG is conjugated to Fe3O4@SiO2The preparation method of the @ CuO nano-particles comprises the following specific steps:
a) Mixing Fe3O4@SiO2Uniformly dispersing the @ CuO nano particles in absolute ethyl alcohol, then adding APTES, condensing and refluxing for 24h at 80 ℃, then washing and drying to obtain aminated Fe3O4@SiO2@ CuO nanoparticles; then taking aminated Fe3O4@SiO2@ CuO nano particles are washed, added with a cross-linking agent sulfo-SMCC and oscillated for 0.5h; wherein, fe3O4@SiO2The dosage ratio of the @ CuO nano particles to the APTES is 40 mg: 150 muL; aminated Fe3O4@SiO2The dosage ratio of the @ CuO nano particles to the sulfo-SMCC is 10 mg: 60 mu L; the concentration of the cross-linking agent sulfo-SMCC is 2mg/mL;
b) Diluting a second antibody IgG by PBS, adding a boric acid buffer solution containing EDTA and a 2-iminosulfane hydrochloride solution, and oscillating for reaction for 1h; wherein the volume dosages of the second antibody IgG, the PBS, the boric acid buffer solution containing EDTA and the 2-iminothiolane hydrochloride solution are 1.5 muL, 20 muL, 500 muL and 200 muL in sequence; the concentration of the 2-iminothiolane hydrochloride solution is 2mg/mL;
c) Adding the reaction system obtained in the step b) into the reaction system obtained in the step a), and mixing and oscillating for 2 hours to obtain Fe with the surface coupled with second antibody IgG3O4@SiO2@ CuO nanoparticles, noted Fe3O4@SiO2@CuO-IgG。
8. The indirect competitive ELISA method for detecting aflatoxin B1 as claimed in any one of claims 1 to 6 wherein in step 4), the buffer is sodium acetate buffer at pH 3.6; the temperature of the water bath light-proof reaction is 37 ℃, and the time is 10-15 min; the termination reaction is as follows: adding a stop solution, and standing for 4-6 min at room temperature to stop the reaction; the stop solution is a 2M sulfuric acid solution.
9. The indirect competitive ELISA method for detecting aflatoxin B1 of any one of claims 1-6, wherein in steps 2) -4), the washing is: washing with washing solution for 3 times, each for 3min; the wash was 0.1M PBST, pH 7.4.
10. The indirect competitive ELISA method for detecting aflatoxin B1 of any one of claims 1 to 6 wherein in step 4), the wavelength of measurement is 450nm when the absorbance is measured.
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| CN114457063A (en) * | 2022-03-11 | 2022-05-10 | 安徽黑娃食品科技有限公司 | Aspergillus flavus toxin B in degradation peanut1Preparation method of immobilized enzyme |
| JP7455442B1 (en) | 2023-06-16 | 2024-03-26 | 青▲島▼▲農▼▲業▼大学 | Aflatoxin B1 detection method based on NH2-MIL-53 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114457063A (en) * | 2022-03-11 | 2022-05-10 | 安徽黑娃食品科技有限公司 | Aspergillus flavus toxin B in degradation peanut1Preparation method of immobilized enzyme |
| JP7455442B1 (en) | 2023-06-16 | 2024-03-26 | 青▲島▼▲農▼▲業▼大学 | Aflatoxin B1 detection method based on NH2-MIL-53 |
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