CN115267177A - Nano enzyme immunochromatography test strip, preparation method and application - Google Patents
Nano enzyme immunochromatography test strip, preparation method and application Download PDFInfo
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- CN115267177A CN115267177A CN202210844210.3A CN202210844210A CN115267177A CN 115267177 A CN115267177 A CN 115267177A CN 202210844210 A CN202210844210 A CN 202210844210A CN 115267177 A CN115267177 A CN 115267177A
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- 102000004190 Enzymes Human genes 0.000 title claims abstract description 65
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- 238000002360 preparation method Methods 0.000 title claims abstract description 25
- 238000003317 immunochromatography Methods 0.000 title claims abstract description 11
- 238000001514 detection method Methods 0.000 claims abstract description 94
- 239000002115 aflatoxin B1 Substances 0.000 claims abstract description 41
- 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 abstract description 37
- 229930020125 aflatoxin-B1 Natural products 0.000 claims abstract description 36
- UVBUBMSSQKOIBE-DSLOAKGESA-N fumonisin B1 Chemical compound OC(=O)C[C@@H](C(O)=O)CC(=O)O[C@H]([C@H](C)CCCC)[C@@H](OC(=O)C[C@@H](CC(O)=O)C(O)=O)C[C@@H](C)C[C@H](O)CCCC[C@@H](O)C[C@H](O)[C@H](C)N UVBUBMSSQKOIBE-DSLOAKGESA-N 0.000 claims abstract description 34
- QZIADBYRQILELJ-UHFFFAOYSA-N fumonisin B1 Natural products CCCCC(C)C(OC(=O)CC(CC(=O)O)C(=O)O)C(C)(CC(C)CC(O)CCCCC(O)CC(O)C(C)N)OC(=O)CC(CC(=O)O)C(=O)O QZIADBYRQILELJ-UHFFFAOYSA-N 0.000 claims abstract description 34
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- 238000006555 catalytic reaction Methods 0.000 claims abstract description 20
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- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 18
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- 239000000020 Nitrocellulose Substances 0.000 claims description 17
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 8
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- 229910052742 iron Inorganic materials 0.000 claims description 8
- ONDPHDOFVYQSGI-UHFFFAOYSA-N zinc nitrate Chemical compound [Zn+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O ONDPHDOFVYQSGI-UHFFFAOYSA-N 0.000 claims description 8
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- QNRATNLHPGXHMA-XZHTYLCXSA-N (r)-(6-ethoxyquinolin-4-yl)-[(2s,4s,5r)-5-ethyl-1-azabicyclo[2.2.2]octan-2-yl]methanol;hydrochloride Chemical compound Cl.C([C@H]([C@H](C1)CC)C2)CN1[C@@H]2[C@H](O)C1=CC=NC2=CC=C(OCC)C=C21 QNRATNLHPGXHMA-XZHTYLCXSA-N 0.000 claims description 4
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- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 3
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- MFLKDEMTKSVIBK-UHFFFAOYSA-N zinc;2-methylimidazol-3-ide Chemical compound [Zn+2].CC1=NC=C[N-]1.CC1=NC=C[N-]1 MFLKDEMTKSVIBK-UHFFFAOYSA-N 0.000 claims description 3
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- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
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- XWIYFDMXXLINPU-UHFFFAOYSA-N Aflatoxin G Chemical compound O=C1OCCC2=C1C(=O)OC1=C2C(OC)=CC2=C1C1C=COC1O2 XWIYFDMXXLINPU-UHFFFAOYSA-N 0.000 description 1
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- CKUAXEQHGKSLHN-UHFFFAOYSA-N [C].[N] Chemical compound [C].[N] CKUAXEQHGKSLHN-UHFFFAOYSA-N 0.000 description 1
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- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
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- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
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Images
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
- G01N33/54387—Immunochromatographic test strips
-
- 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
- G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
-
- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
-
- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
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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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
- G01N33/587—Nanoparticles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
- G01N2333/38—Assays involving biological materials from specific organisms or of a specific nature from fungi from Aspergillus
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- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
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- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
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- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Nanotechnology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a nano-enzyme immunochromatographic test strip, a preparation method and application. The immunochromatography test strip comprises an immunochromatography test strip, a catalytic reaction liquid and a reaction reagent, wherein the reaction reagent contains a nano enzyme-labeled monoclonal antibody for identifying a first object to be detected and a nano enzyme-labeled monoclonal antibody for identifying a second object to be detected, and the catalytic reaction liquid is a buffer solution containing TMB and hydrogen peroxide and having a pH value of 3-5. The method can be used for synchronous detection of aflatoxin B1 and fumonisin B1 in grain products, and has the characteristics of simple and rapid operation, high sensitivity and adjustable detection range.
Description
Technical Field
The invention belongs to the field of biological detection, and particularly relates to a nano-enzyme immunochromatographic test strip, a preparation method and application.
Background
The mycotoxin is a toxic secondary metabolite produced by infecting agricultural products and food with fungi, and the aflatoxin B in the food product1And fumonisin B1, has strong carcinogenic, teratogenic, mutagenic and other effects, and is greatly harmful to human health. And the aflatoxin B1 and the fumonisin B1 are often detected simultaneously by grain products, particularly samples such as wheat, corn and the like, so that the research and development of a method for synchronously detecting the two toxins are very necessary. Existing method for synchronously detecting aflatoxin B1And fumonisins B1 by liquid chromatography-mass spectrometry, immunochromatography, and the like. At present, most of immunochromatographic rapid detection methods for synchronously detecting aflatoxin B1 and fumonisin B1 mainly use gold-labeled test strips or fluorescence-labeled test strips, are high in cost, have only one detection range, once products beyond the detection range need to be diluted or enriched to adapt to the detection range and then re-detect, and are low in sensitivity.
Disclosure of Invention
The invention aims to provide a nano enzyme immunochromatographic test strip, a preparation method and application. The nanoenzyme immunochromatographic test strip has the characteristics of simple and rapid operation, high sensitivity and low cost, can regulate and control the detection range according to requirements, and is used for synchronously detecting two objects to be detected in a sample.
In order to achieve the purpose, the invention adopts the following technical scheme:
the nano enzyme immunochromatographic test strip comprises an immunochromatographic test strip, a catalytic reaction liquid and a reaction reagent containing a nano enzyme-labeled monoclonal antibody for identifying a first object to be detected and a nano enzyme-labeled monoclonal antibody for identifying a second object to be detected, wherein: the immunochromatography test strip comprises a backing base plate, wherein a water absorption pad, a detection pad and a sample pad are sequentially adhered to one side of the backing base plate from top to bottom, the adjacent pads are connected at the joint in an overlapping manner, the detection pad takes a nitrocellulose membrane as a base pad, a transverse quality control line and two detection lines are arranged on the nitrocellulose membrane from top to bottom, the quality control line is coated with a goat anti-mouse polyclonal antibody, the two detection lines are positioned below the quality control line, the detection lines are respectively coated with a first to-be-detected object-bovine serum albumin conjugate and a second to-be-detected object-bovine serum albumin conjugate, and the catalytic reaction solution is a buffer solution containing TMB and hydrogen peroxide and having a pH value of 3-5.
According to the scheme, the substances to be detected are aflatoxin B1 and fumonisin B1 respectively.
According to the scheme, the IC50 of the anti-aflatoxin B1 monoclonal antibody is less than or equal to 1.6ng/mL; the IC50 of the monoclonal antibody of the fumonisin B1 is less than or equal to 0.32ng/mL.
According to the scheme, the regular rhombic dodecahedron particles with the particle size of 100-200nm of the nano enzyme are uniformly distributed with iron, carbon and nitrogen elements, and the iron elements exist on the carbon substrate in a single atom form.
According to the scheme, the nano enzyme is prepared by taking zinc nitrate and 2-methylimidazole as precursors of an organic metal framework ZIF-8 and heme as a precursor of monatomic iron through in-situ high-temperature carbonization, wherein the high-temperature carbonization temperature is 800-900 ℃, and the carbonization time is 1.5-2.5 hours.
According to the scheme, the mass percent of the heme is 5-10% of the mass percent of the zinc nitrate.
According to the scheme, the carbonization heating rate is 5-10 ℃/min.
According to the scheme, the length of the water absorption pad is 40-45mm, the length of the sample pad is 10-15mm, the length of the detection pad is 22-28mm, the distance between the quality control line on the detection pad and the upper edge of the nitrocellulose membrane is 3-4mm, the distance between every two adjacent detection lines is 1-2mm, and the distance between the detection line close to the quality control line and the quality control line is 5-7mm.
According to the above scheme, the sample pad can be selected from FUSION3 and FUSION5, preferably FUSION5, and the detection pad can be selected from IAB 135, IAB 120 and CN95, preferably CN 95.
According to the scheme, the coating amount of the aflatoxin-bovine serum albumin conjugate required by each centimeter of detection line on the detection pad is 0.12-0.24 mu g, the coating amount of the fumonisin B1-bovine serum albumin conjugate required by each centimeter of detection line is 0.3-0.6 mu g, and the coating amount of the goat anti-mouse polyclonal antibody required by each centimeter of quality control line is 0.12-0.24 mu g.
According to the scheme, the concentration of hydrogen peroxide in the catalytic reaction liquid is 0.1-0.2mM; TMB concentration is 0.05-0.1mM.
The preparation method of the nano enzyme immunochromatographic test strip comprises the following steps:
providing an immunochromatography test strip:
(1) Cutting the absorbent paper into absorbent pads;
(2) Preparation of a detection pad:
preparing coating liquid from a first to-be-detected object-bovine serum albumin conjugate and a second to-be-detected object-bovine serum albumin conjugate, coating the coating liquid on a nitrocellulose membrane at intervals in a membrane scratching mode to obtain two detection lines, and drying the two detection lines for 30-60 minutes at 37-40 ℃;
preparing a coating solution with the concentration of 0.2-0.4mg/mL by using the goat anti-mouse polyclonal antibody, transversely coating the coating solution on a nitrocellulose membrane in a membrane scratching mode, and drying the nitrocellulose membrane for 30-60 minutes at the temperature of 37-40 ℃;
providing a catalytic reaction solution;
providing a monoclonal antibody which is marked by the nano enzyme and is used for identifying the first detection object and a monoclonal antibody which is marked by the nano enzyme and is used for identifying the second detection object.
According to the scheme, the coating buffer solution used in the preparation of the coating solution of the substance to be detected-bovine serum albumin conjugate, the coating solution of the substance to be detected-bovine serum albumin conjugate and the coating solution of the goat anti-mouse polyclonal antibody is as follows: 2.9g of Na in 1L of water2HPO4·12H2O，0.2g KCl，0.2g KH2PO4And 8.0g NaCl.
According to the scheme, the monoclonal antibody containing the nano enzyme label and used for identifying the object to be detected is obtained by mixing a nano enzyme solution and a monoclonal antibody solution for identifying the object to be detected, stirring at room temperature and centrifuging.
According to the scheme, the substances to be detected are aflatoxin B1 and fumonisin B1 respectively;
the preparation method of the anti-aflatoxin B1 monoclonal antibody containing the nano-enzyme label comprises the following steps: mixing and shaking the nano enzyme material and the solution of the monoclonal antibody for resisting the aflatoxin B1, wherein: nano-enzyme: anti-aflatoxin B1The mass ratio of the monoclonal antibody is (2.5) - (1) to (1), so that the nano enzyme material and the monoclonal antibody against aflatoxin B1 are fully combined, then redundant active sites are sealed, and the monoclonal antibody is obtained by post-treatment;
the monoclonal antibody containing nano enzyme labeled anti-fumonisin B1 is composed of nano enzyme and anti-fumonisin B1The monoclonal antibody is prepared by electrostatic force adsorption and sealing, and the preparation method comprises the following steps: mixing and shaking a nano enzyme material and a solution of a monoclonal antibody against fumonisin B1, wherein: nano enzyme and fumonisin B1The mass ratio of the monoclonal antibody is 4.
According to the scheme, the aflatoxin B1-bovine serum albumin conjugate and the fumonisin B1-bovine serum albumin conjugate are respectively prepared into coating solutions with the concentrations of 0.2-0.4 and 0.5-1.0 mg/mL.
According to the scheme, the preparation method of the catalytic reaction liquid comprises the following steps: adding TMB solution and hydrogen peroxide solution into buffer solution with pH of 3-5.
The application of the nano enzyme immunochromatographic test strip provides a sample solution to be detected, provides a nano enzyme-labeled monoclonal antibody solution for identifying a first object to be detected and a nano enzyme-labeled monoclonal antibody solution for identifying a second object to be detected, dilutes and uniformly mixes the nano enzyme-labeled monoclonal antibody solution for identifying the first object to be detected and the nano enzyme-labeled monoclonal antibody solution for identifying the second object to be detected with a detection buffer solution, adds the sample solution to be detected into the diluted nano enzyme-labeled monoclonal antibody solution for identifying the object to be detected, and incubates for a period of time; inserting a sample pad of the immunochromatographic test strip into the mixed solution, adding a catalytic reaction solution into the detection line after a black strip of the quality control line appears, measuring and obtaining the RGB value of the detection line after a period of time, and carrying out quantitative analysis on the content of the substance to be detected based on the RGB value-substance concentration standard curve.
According to the above protocol, the detection buffer is PBS buffer containing 2% (v/v) tween-20,0.1% (w/v) sucrose and 0.5% (w/v) BSA.
According to the scheme, the sample is agricultural products such as corn or wheat. The pretreatment method of the sample comprises adding methanol water solution into the sample to be detected, shaking for extraction, centrifuging to obtain supernatant, and filtering to obtain sample extract.
According to the scheme, the quantitative equation of the RGB color values is as follows: the intensity =0.3R +0.59G +0.11B, wherein R, G and B are color intensity values of red, yellow and blue of the detection line respectively and can be measured by photographing through a color meter or a camera;
according to the scheme, the method for obtaining the RGB value-to-be-detected substance concentration standard curve comprises the following steps:
preparing a series of standard solutions with gradient concentration;
and (3) respectively taking the standard solutions with the gradient concentrations as the solutions to be detected for detection, adding the catalytic reaction solution for secondary detection after a black strip of a quality control line appears, obtaining the RGD value of the detection line corresponding to each concentration standard solution, and obtaining an RGB value-concentration standard curve of the object to be detected based on the RGD value and the concentration of the standard solution.
According to the scheme, the substances to be detected are fumonisin B1 and aflatoxin B1 respectively, and the concentration of the fumonisin B1 in the standard curve preparation is 0.02-150ng-1(ii) a The concentration of aflatoxin B1 is 0.005-200ng-1。
According to the scheme, based on RGB value quantitative analysis, the detection limit of aflatoxin B1 (AFB 1) signals after secondary amplification is 0.0028ng/mL; the detection limit of fumonisins B1 (FB 1) after signal secondary amplification is 0.0139ng/mL.
The adopted nano enzyme labeling material is regular rhombic dodecahedron particles with the particle size of 100-200nm, iron, carbon and nitrogen elements are uniformly distributed, and the nano enzyme labeling material has a porous structure and is easy to combine with antibodiesThe anti-fumonisin B1 monoclonal antibody and the aflatoxin B1 monoclonal antibody can be combined, and the color development degree is obvious and the price is low compared with that of a nano-gold material; the iron element exists on the carbon-nitrogen substrate in a monoatomic form, has excellent artificial peroxidase activity and is based on H 202The medium catalyzes TMB color development, can enlarge the detection range and obviously improve the sensitivity.
The invention has the beneficial effects that:
(1) And (3) rapidly and synchronously detecting aflatoxin B1 and fumonisin B1. The nanoenzyme immunochromatographic test strip provided by the invention can realize synchronous and rapid detection of aflatoxin B1 and fumonisin B1 fungaltoxin on one test strip, and the used antibodies are monoclonal antibodies, so that the nanoenzyme immunochromatographic test strip has the advantages of good specificity, high sensitivity, no interference between the aflatoxin B1 and the fumonisin B1 fungaltoxin, simplicity and rapidness.
(2) The sensitivity is high. The nano enzyme immunochromatographic test strip provided by the invention has the lowest detection limit of 0.0028ng/mL for aflatoxin B1 in a detection solution, and has the lowest detection limit of 0.0139ng/mL for fumonisin B1.
(3) The detection range can be regulated and controlled by combining the color development condition of the test strip and matching with the use of the catalytic reaction solution, and 5 dynamic detection sections exist.
Drawings
Fig. 1 is a schematic structural diagram of a nano-enzyme immunochromatographic test strip for synchronously detecting aflatoxin B1 and fumonisin B1 provided by the invention.
In the figure: the test method comprises the following steps of 1 water absorption pad, 2 test pads, 3 sample pads, 4 quality control lines, 5 aflatoxin test lines and 6 fumonisin test lines.
FIG. 2 is an SEM image of nanoenzymes.
FIG. 3 is a transmission electron microscope image and elemental distribution map of a high angle annular dark field scanning.
FIG. 4 is a transmission electron micrograph of nanoenzyme.
FIG. 5 nanometer BET plot.
Detailed Description
The nanometer enzyme immunochromatographic test strip for synchronously detecting aflatoxin B1 and fumonisin B1, the preparation method and the application thereof comprise the following steps:
1. the preparation of the nano enzyme immunochromatographic test strip comprises the following steps:
(1) Preparation of absorbent pad
Cutting the absorbent paper into pieces with the length of 43mm and the width of 4mm to obtain the absorbent pad;
(2) Preparation of detection pad
And (3) sticking a CN95 nitrocellulose membrane with the specification length of 25mm and the width of 4mm to a corresponding position of a paperboard, and then respectively spraying 2 detection lines and 1 quality control line.
Coating of detection lines:
accurately weighing 2.9g of Na2HPO4·12H2O，0.2g KCl，0.2g KH2PO4Adding 8.0g NaCl into ultrapure water, diluting to constant volume with a 1L volumetric flask, mixing, filtering with a 0.22 μm filter membrane, and making into 0.01mol/L Phosphate Buffer Solution (PBS).
Weighing aflatoxin B1Conjugates of bovine serum Albumin (AFB)1-BSA) 2.0mg, preparing a coating solution with a concentration of 0.2mg/mL with 10mL Phosphate Buffered Saline (PBS), coating the sample pad at a position 6mm away from the sample pad transversely on a nitrocellulose membrane by wire spraying to obtain a detection line, and spraying AFB on each millimeter of the detection line112ng BSA, weighing fumonisin B1Conjugates of bovine serum albumin (FB)1-BSA) 5.0mg, which is prepared into a coating solution with a concentration of 0.5mg/mL with 10mL of Phosphate Buffered Saline (PBS), and which is coated on the nitrocellulose membrane laterally at a position 4mm from the sample pad by a line spray method to obtain a detection line, wherein FB1-BSA 30ng is sprayed on each millimeter of the detection line, and then dried at 37 ℃ for 1 hour;
coating of quality control line:
weighing 2mg of goat anti-mouse polyclonal antibody, dissolving in 10mL of PBS solution to prepare coating solution with the concentration of 0.2 mg/mL; transversely coating the nitrocellulose membrane at a position which is 4mm away from the upper edge of the nitrocellulose membrane by a line spraying mode to obtain a quality control line, spraying 12ng of rabbit anti-mouse polyclonal antibody on each millimeter of the quality control line, and drying for 1h at 37 ℃;
(3) Preparation of sample pad
The melt 5 glass fiber membrane was cut into a size of 12mm long by 4mm wide to obtain a sample pad.
(4) Assembly of test strips
Sequentially adhering a water absorption pad, a detection pad and a sample pad on one surface of a paperboard from top to bottom, and overlapping and connecting adjacent pads at the joint, wherein the overlapping length is 2mm, thus obtaining the chromatographic test strip, which is shown in figure 1;
2. preparing reaction reagents of the nano enzyme-labeled anti-aflatoxin B1 monoclonal antibody and the nano enzyme-labeled anti-fumonisin B1 monoclonal antibody:
the monoclonal antibody against fumonisin B1 can be a monoclonal antibody secreted by a hybridoma cell strain Fm7A11 with the preservation number of CCTCC NO. C201636, and is specifically prepared according to a method reported in a patent with the patent application number of CN 201710131166.0.
The anti-aflatoxin B1 monoclonal antibody can be a monoclonal antibody secreted by a hybridoma cell line 3G1 with the preservation number of CCTCC NO. C201014, and is specifically prepared according to a method reported in a patent with the patent application number of CN 201210117614.9.
(1) Preparation of nanoenzyme
1.07g of Zn (NO) was weighed3)2·6H2O was dissolved in 40mL of methanol, mixed rapidly, another 40mL of methanol containing 2.35g of 2-methylimidazole and 0.0535g of hemoglobin was added rapidly, stirred vigorously at room temperature for 24h, the gray product was collected by centrifugation, rinsed 5 times with methanol, and dried under vacuum at 70 ℃ overnight. Transferring the obtained grey product powder into a porcelain boat at N2Heating from room temperature to 900 ℃ at a heating rate of 5 ℃/min for 2h under the condition, then naturally cooling the obtained material, grinding, and weighing to prepare 1.0mg/mL nano enzyme solution by using 10mM PBS buffer solution for later use. The SEM of nanoenzymes is shown in fig. 2, fig. 2 showing: the prepared nano enzyme particles have uniform size, are about 200nm and are in a regular rhombic dodecahedron shape.
The transmission electron microscope picture, the element distribution picture and the transmission electron microscope picture of the high-angle annular dark field scanning of the nano enzyme are shown in figures 3 and 4, and figures 3-4 show that: and iron, carbon, nitrogen, and oxygen are uniformly distributed, and iron atoms are present in a monoatomic form on the carbonized ZIF-8 substrate.
BET diagram is shown in FIG. 5The BET test result shows that the specific surface area of the material is 592.2876m2Per g, pore volume of 0.387086cm3The/g and the average pore diameter is 2.61417nm, which indicates that the nano enzyme is a porous material.
(2) Preparing reaction reagents of the nano enzyme-labeled anti-aflatoxin B1 monoclonal antibody and the nano enzyme-labeled anti-fumonisin B1 monoclonal antibody:
weighing 1mg of the monoclonal antibody resisting AFB1, adding 5mL of PBS buffer solution to prepare 0.2mg/mL of monoclonal antibody solution resisting AFB 1. 1mg of the anti-FB 1 monoclonal antibody is weighed and added into 4mL PBS buffer solution to prepare 0.25mg/mL anti-FB 1 monoclonal antibody solution for later use.
Bovine serum albumin BSA 100mg was dissolved in 10mL of 10mM PBS buffer solution, and prepared into 1% (m/v) BSA-containing PBS buffer solution for use.
The nano enzyme-labeled anti-aflatoxin B1 monoclonal antibody is prepared according to the following method: to 800 μ L of 10mM PBS buffer solution (pH = 7.4), 100 μ L of 10mM PBS buffer solution containing 0.1mg nanoenzyme was added, and then 100 μ L of 10mM PBS buffer solution containing 20 μ g monoclonal antibody against AFB1 was added dropwise. The mixed solution was gently stirred at room temperature for 4 hours, centrifuged at 10000rpm for 20min at 4 ℃, and the resulting precipitate was redissolved with 1mL of 10mM PBS buffer solution containing 1% (m/v) BSA, and left at 4 ℃ for further use.
The nano enzyme-labeled fumonisin B1 monoclonal antibody is prepared according to the following method: to 800. Mu.L of 10mM PBS buffer solution (pH = 7.4), 100. Mu.L of 10mM PBS buffer solution containing 0.1mg nanoenzyme was added, and then 100. Mu.L of 10mM PBS buffer solution containing 25. Mu.g of monoclonal antibody against FB1 was added dropwise. The mixed solution was gently stirred at room temperature for 4 hours, centrifuged at 10000rpm for 20min at 4 ℃, and the resulting precipitate was redissolved with 1mL of 10mM PBS buffer solution containing 1% (m/v) BSA, and left at 4 ℃ for further use.
3. Preparation of catalytic reaction liquid:
48mg of TMB was weighed and then diluted to 200mL with N, N-Dimethylformamide (DMF) solution to prepare 1mM TMB solution. Taking out of commercial 30%2O2Adding 10 mu L of water solution into water to reach 50mL to prepare2mMH2O2The aqueous solution is prepared for use.
32.8mg of sodium acetate was weighed and added to 120. Mu.L of pure acetic acid, and a volume of water was determined to be 100mL, and the mixture was mixed to prepare a sodium acetate buffer (25mM, pH = 4.0).
mu.L of 1mM TMB solution and 1. Mu.L of 2mM H2O2The aqueous solution was added to 10. Mu.L of 25mM sodium acetate buffer solution to obtain a catalytic reaction solution.
Sodium acetate buffer (100mM, pH = 4.0) containing 32.8mg sodium acetate and 120. Mu.L pure acetic acid per 25mL water; the 1mM TMB solution was 24mg TMB per 100mL N, N-Dimethylformamide (DMF), and the 2mM H2O2 solution was a commercially available 37% hydrogen peroxide solution diluted 5000-fold with water.
4. The application of the nano enzyme immunochromatographic test strip for synchronously detecting the aflatoxin B1 and the fumonisin B1 comprises the following steps:
0.1g of sucrose, 0.5g of BSA,2mL of Tween-20 were weighed and dissolved in 100mL of PBS buffer solution to prepare a detection buffer solution.
Weighing 5g of 1#, 2#, 3# corn samples to be detected respectively, adding 15mL of 70% (v/v) methanol aqueous solution, mixing uniformly, shaking for 20min, centrifuging at 4000rpm for 5min at room temperature, taking supernatant, filtering with 0.22 μm filter membrane, taking 10 μ L of nano enzyme-labeled anti-aflatoxin B1 monoclonal antibody solution and 10 μ L of nano enzyme-labeled anti-fumonisin B1 monoclonal antibody solution, diluting and mixing uniformly with 115 μ L of detection buffer solution, adding 45 μ L of sample solution to be detected, mixing uniformly, inserting into a nano enzyme immunochromatography test strip, reacting at 37 ℃ for 8 min, observing whether a black line appears on a quality control line with naked eyes, adding 2 μ L of catalytic reaction liquid at the detection line, standing for 30s, determining the RGB value of the detection line, and carrying out quantitative analysis on the content of the object to be detected based on an RGB value-object concentration standard curve.
Obtaining a standard curve based on RGB value-FB 1 concentration:
preparing standard solution with FB1 content of 0.08ng/mL, 0.4ng/mL, 2ng/mL, 8ng/mL, 40ng/mL, 200ng/mL, 600ng/mL, 800ng/mL, 1000ng/mL; the content of AFB1 is 0.02, 0.08, 0.4, 2, 40, 200, 800 and 2000ng/mL, after the reaction is carried out on the test paper strip in the same way as the method, the mobile phone recognizes RGB values, the final concentration of the fumonisin standard substance in the reaction container is respectively used as an abscissa, the intensity of a detection line corresponding to the standard substance solution with each concentration (the intensity is =0.3R +0.59G +0.11B, wherein R, G and B are respectively the color intensity values of red, yellow and blue of the detection line) is used as an ordinate, and a relation curve and a standard curve are obtained through fitting.
Obtaining a standard curve based on RGB value-AFB 1 concentration: y =4.18X +232.37 before amplification, Y =12.67X +174.59 after amplification, Y is an RGB value, and X is AFB1 concentration in ng/ml.
Obtaining of FB1 concentration standard curve: y =7.11X +228.32 before amplification and Y =16.84X +168.27 after amplification, Y is an RGB value, and X is an FB1 concentration in ng/ml.
The test strip can be fitted before and after amplification, and the RGB values of the test detection line before and after amplification are substituted into corresponding standard curves, so that the accurate content in the sample can be calculated.
Based on the quantitative analysis of the RGB values, we can obtain: the detection limit of aflatoxin B1 (AFB 1) signals after secondary amplification is 0.0028ng/mL; the detection limit of fumonisins B1 (FB 1) after signal secondary amplification is 0.0139ng/mL.
And (3) detection results:
the quality control line of the No. 1 test strip shows a black line, the 2 test lines show black and are close to the color of the negative control group, TMB color development liquid is dripped, the test lines turn blue and are close to the color of the negative control group, the RGB value is measured by mobile phone photographing and is substituted into the amplified standard curve according to a formula, the content of the sample AFB1 is calculated to be 0.78ng/g, and FB1 is not detected. Through detection of liquid chromatography-mass spectrometry (LC-MS/MS), aflatoxin B1 in a 1# sample to be detected is 0.71ng/g, fumonisin B1 is not detected, and the detection result is consistent with that of a test strip.
The quality control line of the 2# test strip shows a black line, the 2 test lines show black and are lighter than the negative control group, the mobile phone photographs to determine the RGB value, and the RGB value is substituted into the standard curve before amplification to obtain the sample with the AFB1 content of 15.56ng/g and the FB1 content of 63.18ng/g. Through detection of liquid chromatography mass spectrometry (LC-MS/MS), the content of aflatoxin B1 in a No. 2 sample to be detected is 17.19ng/g, and the content of fumonisin B1 is 61.53ng/g.
The quality control line of the No. 3 test strip shows a black line, while 2 test lines are colorless, TMB color development liquid is dripped on the test lines, the test lines turn blue, then the RGB values are measured by mobile phone photography and substituted into amplified standard curve, and the AFB1 content of the sample is 146.53ng/g, and the FB1 content of the sample is 1658.23ng/g. Through liquid chromatography mass spectrometry (LC-MS/MS) detection, the content of aflatoxin B1 in a 3# sample to be detected is 147.23ng/g, and fumonisin is 1670.14ng/g.
Claims (10)
1. The nano enzyme immunochromatographic test strip is characterized in that: the kit comprises an immunochromatography test strip, catalytic reaction liquid and a reaction reagent containing a nano enzyme-labeled monoclonal antibody for identifying a first object to be detected and a nano enzyme-labeled monoclonal antibody for identifying a second object to be detected, wherein: the immunochromatography test strip comprises a backing base plate, wherein a water absorption pad, a detection pad and a sample pad are sequentially adhered to one surface of the backing base plate from top to bottom, the adjacent pads are connected in an overlapped mode at the connection position, the detection pad takes a nitrocellulose membrane as a base pad, a transverse quality control line and two detection lines are arranged on the nitrocellulose membrane from top to bottom, the quality control line is coated with goat anti-mouse polyclonal antibodies, the two detection lines are located below the quality control line, the detection lines are respectively coated with a first object to be detected, namely bovine serum albumin conjugate and a second object to be detected, namely bovine serum albumin conjugate, and the catalytic reaction solution is a buffer solution containing TMB and hydrogen peroxide and having the pH value of 3-5; the concentration of hydrogen peroxide in the catalytic reaction liquid is 0.1-0.2mM; TMB concentration is 0.05-0.1mM.
2. The nanoenzyme immunochromatographic test strip of claim 1, which is characterized in that: the regular rhombic dodecahedron particles with the particle size of 100-200nm of the nano enzyme are uniformly distributed with iron, carbon and nitrogen elements, and the iron elements exist on a carbon substrate in a single atom form.
3. The nanoenzyme immunochromatographic test strip of claim 1, which is characterized in that: the nano enzyme is prepared by taking zinc nitrate and 2-methylimidazole as precursors of an organic metal framework ZIF-8 and heme as a precursor of monatomic iron through in-situ high-temperature carbonization, wherein the high-temperature carbonization temperature is 800-900 ℃, the carbonization time is 1.5-2.5h, and the heme accounts for 5-10% of the zinc nitrate in percentage by mass.
4. The nanoenzyme immunochromatographic test strip of claim 1, which is characterized in that: the water absorption pad is 40-45mm long, the sample pad is 10-15mm long, the detection pad is 22-28mm long, the distance between a quality control line on the detection pad and the upper edge of the nitrocellulose membrane is 3-4mm, the distance between every two adjacent detection lines is 1-2mm, and the distance between the detection line close to the quality control line and the quality control line is 5-7mm;
the coating amount of the aflatoxin B1-bovine serum albumin conjugate required by each centimeter of detection line on the detection pad is 0.12-0.24 mug, the coating amount of the fumonisin B1-bovine serum albumin conjugate required by each centimeter of detection line is 0.3-0.6 mug, and the coating amount of the goat anti-mouse polyclonal antibody required by each centimeter of quality control line is 0.12-0.24 mug;
the substances to be detected are aflatoxin B1 and fumonisin B1 respectively, and the IC50 of the anti-aflatoxin B1 monoclonal antibody is less than or equal to 1.6ng/mL; the IC50 of the monoclonal antibody of the fumonisin B1 is less than or equal to 0.32ng/mL.
5. The preparation method of the nanoenzyme immunochromatographic test strip of claim 1, which is characterized by comprising the following steps:
preparing the immunochromatography test strip:
(1) Cutting the absorbent paper into absorbent pads;
(2) Preparation of a detection pad:
preparing coating liquid from a first to-be-detected object-bovine serum albumin conjugate and a second to-be-detected object-bovine serum albumin conjugate, coating the coating liquid on a nitrocellulose membrane at intervals in a membrane scratching mode to obtain two detection lines, and drying the two detection lines for 30-60 minutes at 37-40 ℃;
preparing a coating solution with the concentration of 0.2-0.4mg/mL by using the goat anti-mouse polyclonal antibody, transversely coating the coating solution on a nitrocellulose membrane in a membrane scratching mode, and then drying the nitrocellulose membrane for 30-60 minutes at 37-40 ℃;
providing a catalytic reaction solution;
a nanoenzyme-labeled monoclonal antibody recognizing a first detection substance and a nanoenzyme-labeled monoclonal antibody recognizing a second detection substance are provided.
6. The method for preparing the nanoenzyme immunochromatographic test strip of claim 5, which is characterized in that:
the preparation method of the catalytic reaction liquid comprises the following steps: adding TMB solution and hydrogen peroxide solution into buffer solution with pH of 3-5 to prepare;
the monoclonal antibody containing the nano enzyme label and identifying the detection object is obtained by mixing a nano enzyme solution and a monoclonal antibody solution for identifying the detection object, stirring at room temperature and centrifuging;
the substances to be detected are aflatoxin B1 and fumonisin B1 respectively;
the preparation method of the aflatoxin B1-resistant monoclonal antibody containing the nano-enzyme label comprises the following steps: mixing and shaking the nano enzyme material and the solution of the monoclonal antibody against aflatoxin B1, wherein: nano-enzyme: anti-aflatoxin B1The mass ratio of the monoclonal antibody is (2.5) - (1) to (1), so that the nano enzyme material and the monoclonal antibody against aflatoxin B1 are fully combined, then redundant active sites are sealed, and the monoclonal antibody is obtained by post-treatment;
the monoclonal antibody containing nano enzyme labeled anti-fumonisin B1 is composed of nano enzyme and anti-fumonisin B1The monoclonal antibody is prepared by electrostatic force adsorption and sealing, and the preparation method comprises the following steps: mixing and shaking a nano enzyme material and a solution of a monoclonal antibody against fumonisin B1, wherein: nano enzyme and fumonisin B resistance1The mass ratio of the monoclonal antibody is 4;
the aflatoxin B1-bovine serum albumin conjugate and the fumonisin B1-bovine serum albumin conjugate are respectively prepared into coating solutions with the concentrations of 0.2-0.4 and 0.5-1.0 mg/mL.
7. The application of the nanoenzyme immunochromatographic test strip of claim 1, which is characterized in that: providing a sample solution to be detected, providing a monoclonal antibody solution which is labeled by a nano enzyme and used for identifying a first object to be detected and a monoclonal antibody solution which is labeled by the nano enzyme and used for identifying a second object to be detected, diluting the monoclonal antibody solution which is labeled by the nano enzyme and used for identifying the first object to be detected and the monoclonal antibody solution which is labeled by the nano enzyme and used for identifying the second object to be detected by the nano enzyme with a detection buffer solution, uniformly mixing, adding the sample solution to be detected into the diluted monoclonal antibody solution which is labeled by the nano enzyme and used for identifying the object to be detected, and incubating for a period of time; inserting a sample pad of the immunochromatographic test strip into the mixed solution, adding a catalytic reaction solution into the detection line after a black strip of the quality control line appears, measuring and obtaining the RGB value of the detection line after a period of time, and carrying out quantitative analysis on the content of the substance to be detected based on the RGB value-substance concentration standard curve.
8. Use according to claim 7, characterized in that: the detection buffer solution is PBS buffer solution containing 2% (v/v) tween-20,0.1% (w/v) sucrose and 0.5% (w/v) BSA;
adding methanol water solution into a sample to be detected, shaking for extraction, centrifuging to obtain supernatant, and filtering to obtain a sample extracting solution;
the quantitative equation of the RGB color values is as follows: the intensity =0.3R +0.59G +0.11B, wherein R, G, B are the color intensity values of red, yellow and blue of the detection line respectively, and can be measured by photographing with a color measuring instrument or a camera.
9. Use according to claim 7, characterized in that: the method for obtaining the RGB value-concentration standard curve of the object to be detected comprises the following steps:
preparing a series of standard solutions with gradient concentration;
respectively taking the standard solutions with the gradient concentrations as the solutions to be detected for detection, adding a catalytic reaction solution for secondary detection after a black strip of a quality control line appears, obtaining RGD values of detection lines corresponding to the standard solutions with various concentrations, and obtaining a standard curve of RGB values-concentration standard curves of the substances to be detected based on the RGD values and the concentrations of the standard solutions;
the concentration of fumonisin B1 to be detected in the standard curve preparation is 0.02-150ng-1(ii) a The concentration of aflatoxin B1 is 0.005-200ng-1。
10. Use according to claim 7, characterized in that: based on RGB value quantitative analysis, the detection limit of aflatoxin B1 (AFB 1) signals amplified for the second time in the method is 0.0028ng/mL; the detection limit of the fumonisin B1 (FB 1) signal after secondary amplification is 0.0139ng/mL.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109709322A (en) * | 2019-01-15 | 2019-05-03 | 湖北工业大学 | A kind of detection method detecting aflatoxin B1 |
| US20210132066A1 (en) * | 2017-03-07 | 2021-05-06 | Oil Crops Research Institute, Chinese Academy Of Agricultural Sciences | Time-resolved fluorescence immunochromatographic kit for simultaneous detection of mixed contamination of five mycotoxins such as aflatoxin b1 and application thereof |
| CN114252428A (en) * | 2021-12-27 | 2022-03-29 | 江南大学 | Surface-enhanced Raman detection method for mycotoxin based on catalytic reaction of cuprous oxide nano composite enzyme |
| CN114384243A (en) * | 2022-01-18 | 2022-04-22 | 北京易斯威特生物科技股份有限公司 | Test strip for mycotoxin joint detection, preparation method and application thereof |
-
2022
- 2022-07-18 CN CN202210844210.3A patent/CN115267177A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20210132066A1 (en) * | 2017-03-07 | 2021-05-06 | Oil Crops Research Institute, Chinese Academy Of Agricultural Sciences | Time-resolved fluorescence immunochromatographic kit for simultaneous detection of mixed contamination of five mycotoxins such as aflatoxin b1 and application thereof |
| CN109709322A (en) * | 2019-01-15 | 2019-05-03 | 湖北工业大学 | A kind of detection method detecting aflatoxin B1 |
| CN114252428A (en) * | 2021-12-27 | 2022-03-29 | 江南大学 | Surface-enhanced Raman detection method for mycotoxin based on catalytic reaction of cuprous oxide nano composite enzyme |
| CN114384243A (en) * | 2022-01-18 | 2022-04-22 | 北京易斯威特生物科技股份有限公司 | Test strip for mycotoxin joint detection, preparation method and application thereof |
Non-Patent Citations (9)
| Title |
|---|
| JIAOJIAO HAN等: "Nanozyme-based lateral flow assay for the sensitive detection of Escherichia coli O157:H7 in milk", 《JOURNAL OF DAIRY SCIENCE》, vol. 101, no. 7, 31 December 2018 (2018-12-31) * |
| NUANFEI ZHU等: "Biomimic Nanozymes with Tunable Peroxidase-like Activity Based on the Confinement Effect of Metal−Organic Frameworks (MOFs) for Biosensing", 《ANALYTICAL CHEMISTRY》, vol. 94, 9 March 2022 (2022-03-09) * |
| WENDONG LIU等: "Hemin-assisted synthesis of peroxidase-like Fe-N-C nanozymes for detection of ascorbic acid-generating bio-enzymes", 《CHEMICAL ENGINEERING JOURNAL》, vol. 415, 11 February 2021 (2021-02-11), pages 4 * |
| XINFA CAI等: "Fe-N-C single-atom nanozyme for ultrasensitive, on-site and multiplex detection of mycotoxins using lateral flow immunoassay", 《JOURNAL OF HAZARDOUS MATERIALS》, vol. 441, 1 September 2022 (2022-09-01) * |
| XINFA CAI等: "Nanozyme-strip based on MnO2 nanosheets as a catalytic label for multi-scale detection of aflatoxin B1 with an ultrabroad working range", 《FOOD CHEMISTRY》, vol. 377, 31 December 2021 (2021-12-31), pages 3 - 4 * |
| 冯跃跃: "《电视原理与数字电视》", 28 February 2013, 北京理工大学出版社, pages: 59 * |
| 陈曲波等: "《临床免疫检验标准化操作程序》", 31 August 2019, 上海科学技术出版社, pages: 138 * |
| 陈秋梦: "MOFs衍生的铁基碳纳米酶及其分析应用", 《中国博士学位论文全文数据库》, no. 01, 15 January 2022 (2022-01-15) * |
| 马理: "真菌毒素多残留胶体金免疫层析试纸条的研制", 《中国优秀硕士学位论文全文数据库》, no. 02, 15 February 2017 (2017-02-15), pages 2 - 2 * |
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