CN109061144A - A kind of qualitative, quantitative immuno-chromatographic test paper strip and its preparation method and application for detecting zearalenone - Google Patents
A kind of qualitative, quantitative immuno-chromatographic test paper strip and its preparation method and application for detecting zearalenone Download PDFInfo
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- MBMQEIFVQACCCH-UHFFFAOYSA-N trans-Zearalenon Natural products O=C1OC(C)CCCC(=O)CCCC=CC2=CC(O)=CC(O)=C21 MBMQEIFVQACCCH-UHFFFAOYSA-N 0.000 title claims abstract description 120
- MBMQEIFVQACCCH-QBODLPLBSA-N zearalenone Chemical compound O=C1O[C@@H](C)CCCC(=O)CCC\C=C\C2=CC(O)=CC(O)=C21 MBMQEIFVQACCCH-QBODLPLBSA-N 0.000 title claims abstract description 118
- 238000012360 testing method Methods 0.000 title claims abstract description 60
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 238000001514 detection method Methods 0.000 claims abstract description 108
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 57
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- 102000036639 antigens Human genes 0.000 claims abstract description 40
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- KEOYKWIOAINZSQ-UHFFFAOYSA-N alpha-Zearalenol Natural products CC1CCCC(O)CCC=CCc2cc(O)cc(O)c2C(=O)O1 KEOYKWIOAINZSQ-UHFFFAOYSA-N 0.000 description 4
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- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 description 2
- FPQFYIAXQDXNOR-UHFFFAOYSA-N 7beta-trans-zearalenol Natural products O=C1OC(C)CCCC(O)CCCC=CC2=CC(O)=CC(O)=C21 FPQFYIAXQDXNOR-UHFFFAOYSA-N 0.000 description 2
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- FPQFYIAXQDXNOR-QDKLYSGJSA-N alpha-Zearalenol Chemical compound O=C1O[C@@H](C)CCC[C@H](O)CCC\C=C\C2=CC(O)=CC(O)=C21 FPQFYIAXQDXNOR-QDKLYSGJSA-N 0.000 description 2
- 150000008064 anhydrides Chemical class 0.000 description 2
- FPQFYIAXQDXNOR-PMRAARRBSA-N beta-Zearalenol Chemical compound O=C1O[C@@H](C)CCC[C@@H](O)CCC\C=C\C2=CC(O)=CC(O)=C21 FPQFYIAXQDXNOR-PMRAARRBSA-N 0.000 description 2
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- 239000001509 sodium citrate Substances 0.000 description 2
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- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 2
- 229940038773 trisodium citrate Drugs 0.000 description 2
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- 235000011293 Brassica napus Nutrition 0.000 description 1
- 235000004977 Brassica sinapistrum Nutrition 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 231100000678 Mycotoxin Toxicity 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 description 1
- 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 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 238000005411 Van der Waals force Methods 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
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- 238000000502 dialysis Methods 0.000 description 1
- 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 1
- 235000013601 eggs Nutrition 0.000 description 1
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- 239000005556 hormone Substances 0.000 description 1
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- 231100000915 pathological change Toxicity 0.000 description 1
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- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000002953 phosphate buffered saline Substances 0.000 description 1
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- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 235000015277 pork Nutrition 0.000 description 1
- TYJJADVDDVDEDZ-UHFFFAOYSA-M potassium hydrogencarbonate Chemical compound [K+].OC([O-])=O TYJJADVDDVDEDZ-UHFFFAOYSA-M 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
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- 235000014101 wine Nutrition 0.000 description 1
Classifications
-
- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- 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
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- 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
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- 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
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The present invention relates to technical field of food safety detection, in particular to a kind of qualitative, quantitative immuno-chromatographic test paper strip and its preparation method and application for detecting zearalenone.The qualitative, quantitative immuno-chromatographic test paper strip, successively overlap joint is provided with filter paper, sample pad, glass fibre membrane, nitrocellulose filter and blotting paper on bottom plate；The zearalenone monoclonal antibody of colloid gold label is attached on glass fibre membrane；It is provided with detection line and nature controlling line on nitrocellulose filter, the zearalenone artificial antigen of fluorescent microsphere label is coated in detection line, is coated with secondary antibody on nature controlling line.Spetrophotometry of the present invention realizes qualitative and quantitative detection to the zearalenone in detection sample simultaneously, and sample results obtain Dual system verifying in the detection, as a result relatively reliable, saves detection time.
Description
Technical Field
The invention relates to the technical field of food safety detection, in particular to a qualitative and quantitative immunochromatographic test strip for detecting zearalenone, and a preparation method and application thereof.
Background
Zearalenone (Zearalenone, abbreviated as ZEN) is a hormone mycotoxin produced by fusarium, Zearalenone has various derivatives with similar structures, mainly comprises α -zearalenol, β -zearalenol, α -zearalenol, β -zearalenol, Zearalenone and the like, the Zearalenone can be formed by metabolic transformation of Zearalenone in animals and plants, and can also be produced by fusarium, the Zearalenone and the metabolites thereof have estrogen effect and are commonly found in grains and animal and plant tissues, various cereal pathological changes can be caused, and the Zearalenone and the metabolites thereof have estrogen-like effect and can cause overhigh level of animal estrogen and serious reproduction and sterility problems.
In view of serious harm of the zearalenone, the national regulation specifies that the limit standard of the zearalenone in wheat and wheat flour is 60 mu g/kg and the limit standard of corn and corn flour (dregs and flakes) is 60 mu g/kg for grains and products thereof.
The first method for measuring zearalenone in the current national standard food for food safety is liquid chromatography, and is suitable for measuring zearalenone in grains and grain products, wines, soy sauce, vinegar, sauce and sauce products, soybeans, rapeseeds and edible vegetable oil. The second method is a fluorescence photometry, which is suitable for measuring zearalenone in soybean, rapeseed and edible vegetable oil. The third method is a solid phase extraction column purification liquid chromatography-mass spectrometry method, and is suitable for the determination of zearalenone in beef, pork, beef liver, milk and eggs. In addition, there are some detection methods commonly used, such as immunoassay, thin layer chromatography, gas chromatography-mass spectrometry, and the like.
The immunochromatography technology is a unique immunoassay mode appearing in the early 80 s, and generally takes a strip-shaped fiber chromatography material as a solid phase, enables a sample solution to swim on a chromatography strip through capillary action, enriches or retains immune complexes in a certain area (detection line) of the chromatography material in the chromatography process through an immune reaction principle of antigen-antibody combination, and obtains an intuitive experimental result (for example, the detection line has strips with different colors) through enzyme reaction or direct application of a visual marker (for example, colloidal gold); the free label crosses the detection line, so as to achieve the purpose of automatically separating from the bound label. Common visual labeling carriers of the immunochromatography technology include colloidal gold, latex, colloidal selenium and the like, wherein the most successful label is the colloidal gold. However, the colloidal gold immunochromatographic test strip has the following defects:
⑴ the general test paper strips are qualitatively analyzed by naked eye observation results, and accurate quantitative detection cannot be realized.
⑵ the effect of different material matrixes is obvious, the background interference of the sample is large, and false positive results are easy to generate.
⑶, the positive result can not be preserved, and the result is not accurate and reliable after the judgment time is usually exceeded.
In order to meet the requirement of quantitative detection on samples in the market, various quantitative detection test strips based on different markers are also diversified, and common quantitative detection test strips are characterized in that after a single marker is used for marking an antibody, a developing signal value is read through a small instrument, and then quantitative detection is performed through drawing a standard curve. At present, fluorescence immunochromatography methods based on different element markers have appeared and high sensitivity is obtained, but the quantitative detection method also has the following defects:
⑴, in actual detection, the proportion of negative results is high, but all test strips of the method need to be read by an instrument, otherwise, the results cannot be obtained, and the time is long when a large number of samples are detected.
⑵ in quantitative determination, the detection department still needs a threshold to distinguish between negative and positive, so the data processing amount is large.
Disclosure of Invention
In view of the above, the invention provides a qualitative and quantitative immunochromatographic test strip for detecting zearalenone, and a preparation method and application thereof. The qualitative and quantitative immunochromatographic test strip is high in sensitivity, short in detection time and simple and convenient to operate, and can be used for qualitatively and quantitatively detecting zearalenone in a detection sample.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a qualitative and quantitative immunochromatographic test strip for detecting zearalenone, wherein a base plate is sequentially overlapped with filter paper, a sample pad, a glass fiber membrane, a nitrocellulose membrane and absorbent paper;
a zearalenone monoclonal antibody marked by colloidal gold is attached to the glass fiber membrane;
the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is coated with a zearalenone artificial antigen marked by fluorescent microspheres, and the quality control line is coated with a second antibody.
In the invention, the zearalenone artificial antigen marked by the fluorescent microspheres is zearalenone coupled with macromolecular protein and the fluorescent microspheres.
Preferably, the macromolecular protein is bovine serum albumin or ovalbumin.
Preferably, the fluorescent microsphere is prepared by taking rare earth ions with the diameter of 0.01-10 mu m as a marker to wrap the fluorescent material.
Preferably, the fluorescent substance is an organic or inorganic fluorescent substance, or a dopant of a plurality of fluorescent substances, or a quantum dot.
Preferably, the colloidal gold particles are gold particles having a diameter of 10 to 100 nm.
Preferably, the colloidal gold particles are gold particles with a diameter of 10-100 nm, which are prepared by reducing chloroauric acid with trisodium citrate, and the surfaces of the gold particles have negative charges and can be coupled with proteins.
The invention also provides a preparation method of the qualitative and quantitative immunochromatographic test strip, which comprises the following steps:
coupling zearalenone with macromolecules to obtain a zearalenone artificial antigen, and then marking the zearalenone artificial antigen by adopting fluorescent microspheres to obtain the zearalenone artificial antigen marked by the fluorescent microspheres;
coating a zearalenone artificial antigen marked by fluorescent microspheres at a detection line position of a nitrocellulose membrane, and coating a second antibody at a quality control line position of the nitrocellulose membrane;
adopting colloidal gold particles to mark the zearalenone monoclonal antibody to obtain a colloidal gold-marked zearalenone monoclonal antibody, and attaching the colloidal gold-marked zearalenone monoclonal antibody to a glass fiber membrane;
and sequentially overlapping and sticking filter paper, a sample pad, a glass fiber membrane, a nitrocellulose membrane and absorbent paper on the bottom plate to obtain the qualitative and quantitative immunochromatographic test strip for detecting zearalenone.
Preferably, the spraying amount of the colloidal gold labeled zearalenone monoclonal antibody is 7-9 muL/cm.
Preferably, the spraying amount of the colloidal gold-labeled zearalenone monoclonal antibody is 8 μ L/cm.
Preferably, the coating concentration of the fluorescent microsphere labeled zearalenone artificial antigen is 1.0-5.0 mg/mL, and the spraying amount is 0.7-0.8 muL/cm; the coating concentration of the second antibody is 0.5-5.0 mg/mL, and the spraying amount is 0.7-0.8 muL/cm.
Preferably, the coating concentration of the fluorescent microsphere labeled zearalenone artificial antigen is 1.0-5.0 mg/mL, and the spraying amount is 0.74 mu L/cm; the coating concentration of the second antibody is 0.5-5.0 mg/mL, and the spraying amount is 0.74 mu L/cm.
Preferably, the preparation method of the colloidal gold labeled zearalenone monoclonal antibody comprises the following steps:
taking 40mL of colloidal gold solution, adjusting the pH value to 7.0 by 0.02M potassium carbonate, adding a zearalenone monoclonal antibody to enable the final concentration to reach 10-60 mu g/mL, fully mixing, stirring at room temperature for reaction for 1-4 h, then adding 10% bovine serum albumin, reacting at room temperature for 0.5-1 h, centrifuging at (2000-5000) × g for 5-20 min, re-dissolving the precipitate by 0.02M phosphate buffer solution with the pH value of 7.0, wherein the re-dissolving volume is one tenth of the initial volume, and storing at 4 ℃.
In the specific embodiment provided by the invention, the preparation method of the colloidal gold labeled zearalenone monoclonal antibody is as follows:
taking 40mL of colloidal gold solution, adjusting the pH value to 7.0 by using 0.02M potassium carbonate, adding 400 mu g of zearalenone monoclonal antibody, fully mixing, stirring and reacting at room temperature for 1h, then adding 10% bovine serum albumin, reacting at room temperature for 0.5h, centrifuging at 5000 Xg for 20min, re-dissolving the precipitate by using 0.02M phosphate buffer solution with the pH value of 7.0, wherein the re-dissolving volume is one tenth of the initial volume, and storing at 4 ℃.
In the invention, the attachment of the colloidal gold labeled zearalenone monoclonal antibody on the glass fiber membrane specifically comprises the following steps: spraying the composite solution on a glass fiber membrane, and drying in vacuum at 25 ℃ for 1-2 h, wherein the spraying amount is 2-4 mu L/cm.
Preferably, the coating concentration of the fluorescent microsphere labeled zearalenone artificial antigen is 1.0-5.0 mg/mL, and the spraying amount is 0.7-0.8 muL/cm; the coating concentration of the second antibody is 0.5-5.0 mg/mL, and the spraying amount is 0.7-0.8 muL/cm.
Preferably, the coating concentration of the fluorescent microsphere labeled zearalenone artificial antigen is 1.0-5.0 mg/mL, and the spraying amount is 0.74 mu L/cm; the coating concentration of the second antibody is 0.5-5.0 mg/mL, and the spraying amount is 0.74 mu L/cm.
In the embodiment provided by the invention, the coating concentration of the zearalenone artificial antigen marked by the fluorescent microspheres is 2mg/mL, and the spraying amount is 0.74 mu L/cm; the coating concentration of the secondary antibody was 1mg/mL, and the sprayed amount was 0.74. mu.L/cm.
Preferably, the fluorescent microsphere labeled zearalenone artificial antigen and the second antibody are coated on the nitrocellulose membrane in a spraying mode, and the spraying steps of the detection line and the quality control line on the nitrocellulose membrane are as follows:
adjusting the concentration of the zearalenone artificial antigen marked by the fluorescent microspheres to be 1-5 mg/mL by using 0.01-0.5M phosphate buffer solution with the pH value of 7.0, and spraying the zearalenone artificial antigen at the position of a detection line, wherein the spraying amount is 0.74 mu L/cm;
adjusting the concentration of the second antibody to be 1-5 mg/mL by using 0.01-0.5M phosphate buffer solution with the pH value of 7.0, and spraying the second antibody at the position of a quality control line, wherein the spraying amount is 0.74 mu L/cm;
and drying the cellulose nitrate membrane at 37 ℃ for 8-12 h.
Preferably, the concentration of the zearalenone artificial antigen labeled with fluorescent microspheres is adjusted to 2mg/mL by using 0.02M phosphate buffer solution with pH 7.0.
Preferably, the second antibody concentration is adjusted to 1.0mg/mL using 0.02M phosphate buffer pH 7.0.
Preferably, the detection line and the quality control line are separated by 5-7 mm.
In one embodiment of the present invention, the detection line and the quality control line are separated by 5 mm.
In the invention, the top edge of the nitrocellulose membrane lapped with the glass fiber membrane is 10-12 mm away from the detection line.
Preferably, the top edge of the nitrocellulose membrane at the end that overlaps the glass fiber membrane is 10mm from the test line.
In the invention, the top edge of the nitrocellulose membrane lapped with the glass fiber membrane is 15-19 mm away from the quality control line.
Preferably, the top edge of the nitrocellulose membrane at the end that overlaps the glass fiber membrane is 15mm from the control line.
In particular embodiments provided herein, the second antibody is a goat anti-mouse antibody.
The invention also provides a method for qualitatively and quantitatively detecting zearalenone, which comprises the following steps:
(1) qualitative detection
A detection sample is added on a sample pad of the qualitative and quantitative immunochromatographic test strip provided by the invention, and after reaction for 10-12 min, the red strip conditions of a test strip detection line and a quality control line are observed:
the detection line and the quality control line both show red strips, and the content of zearalenone in the detection sample is less than 60 mug/kg, and the qualitative judgment is negative;
if the detection line does not display a red strip and the quality control line displays a red strip, the content of zearalenone in the detection sample is more than or equal to 60 mu g/kg, and the detection sample is qualitatively judged to be positive;
if a weak light red strip appears on the detection line in the judgment of the qualitative detection result, which indicates that the sample may contain zearalenone but the concentration is lower than 60 mug/kg, the quantitative method can be adopted for detection.
(2) Quantitative detection
And the quantitative detection is to obtain the content of the zearalenone in the detection sample according to the standard curve.
In the present invention, the detection sample is coix seed.
The invention provides a qualitative and quantitative immunochromatographic test strip for detecting zearalenone, and a preparation method and application thereof. The qualitative and quantitative immunochromatographic test strip is characterized in that filter paper, a sample pad, a glass fiber membrane, a nitrocellulose membrane and absorbent paper are sequentially overlapped on a bottom plate; a zearalenone monoclonal antibody marked by colloidal gold is attached to the glass fiber membrane; the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is coated with a zearalenone artificial antigen marked by fluorescent microspheres, and the quality control line is coated with a second antibody. The invention has the beneficial effects that:
1. the double-color development system realizes qualitative and quantitative detection of zearalenone in a detection sample at the same time: the method comprises the step of carrying out qualitative detection on one test strip by using a colloidal gold color development system to visually detect the existence of a detection line and a quality control line red strip. And if the colloidal gold shows a positive sample, reading the fluorescent color development system by a fluorescent microsphere reader to obtain the accurate concentration of the positive sample, thereby realizing quantitative detection. Two detection functions are simultaneously realized by one test strip, and the result of the sample is verified in a binary system in the detection, so that the result is more reliable.
2. In practical application, the occupation ratio of negative samples is the largest, when a large number of samples are detected, the negative results are firstly eliminated through visual inspection, the detection time can be greatly saved, the positive results can be accurately quantified again, and the data and convenient storage of the results are realized.
Drawings
FIG. 1 is a schematic structural diagram of a qualitative and quantitative immunochromatographic test strip for zearalenone; the device comprises filter paper 1, a sample pad 2, a glass fiber membrane (colloidal gold antibody compound pad) 3, a nitrocellulose membrane (NC membrane) 4, a detection line 5, a quality control line 6, absorbent paper 7 and a PVC bottom plate 8, wherein the filter paper is made of a transparent material;
FIG. 2 is a schematic diagram of a qualitative and quantitative immunochromatographic test strip for the detection of zearalenone; wherein,zearalenone (ZEN),showing a colloidal gold-ZEN antibody complex,fluorescent microsphere-ZEN coupled antigen,anti-murine secondary antibody is shown.
Detailed Description
The invention discloses a qualitative and quantitative immunochromatographic test strip for detecting zearalenone, and a preparation method and application thereof. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The invention provides a qualitative and quantitative immunochromatographic test strip for detecting zearalenone. The test paper comprises a bottom plate, filter paper, a sample pad, a glass fiber pad, a nitrocellulose membrane and absorbent paper, wherein the filter paper, the sample pad, the glass fiber pad, the nitrocellulose membrane and the absorbent paper are sequentially pasted in a lap joint manner, a zearalenone monoclonal antibody compound marked by colloidal gold is sprayed on the glass fiber pad, and the nitrocellulose membrane is coated with a zearalenone artificial antigen marked by fluorescent microspheres as a detection line and a mouse-resistant antibody as a quality control line.
The fluorescent microspheres are special microspheres which are prepared by taking rare earth ions with longer fluorescence half-life period as markers and have the diameter of 0.01-10 mu m and are wrapped with fluorescent materials, and active groups are connected to the surfaces of the special microspheres; the fluorescent substance includes an organic or inorganic fluorescent substance or a dopant of a plurality of fluorescent substances and quantum dots.
The colloidal gold particles are gold particles with the diameter of 10-100 nm and prepared by reducing chloroauric acid with trisodium citrate, and the surfaces of the gold particles have negative charges and can be coupled with protein.
The invention adopts another technical scheme that a method for preparing the test strip is provided, which comprises the following steps:
⑴ preparation of nitrocellulose membranes
① preparing artificial antigen of zearalenone;
in order to prepare the nitrocellulose membrane, firstly, a zearalenone standard substance and protein macromolecules are subjected to covalent coupling to prepare an artificial antigen required by a quality control region, wherein the coupling method comprises the following steps: the mixed anhydride method. The coupling protein can be selected from: bovine serum albumin and ovalbumin are coupled with macromolecular protein, and then the fluorescent microspheres and the whole antigen are marked by an EDC-NHS oximation method.
② preparation of detection line and quality control line.
Respectively coating the zearalenone artificial antigen and the anti-mouse antibody marked by the fluorescent microspheres on a nitrocellulose membrane to prepare a detection line and a quality control line. Adjusting the concentration of the coating to be 1-5 mg/mL and the film spraying amount to be 0.74 mu L/cm respectively by using 0.1-0.5M PBS (phosphate buffer solution) with pH of 7.5, spraying zearalenone artificial antigen on a detection line, spraying antimouse antibody on a quality control line, and separating the two areas by 5 mm; after drying at 37 ℃ overnight, the mixture is stored in a room-temperature dry environment for later use.
⑵ preparation of colloidal gold antibody composite pad
① the colloidal gold particles are labeled with zearalenone monoclonal antibody by charge action and van der waals force, taking colloidal gold solution, adjusting pH to 7.0 with 0.02M potassium carbonate, adding 10-60 μ g zearalenone monoclonal antibody, fully mixing, stirring at room temperature for reaction for 1-4 hr, then adding 1% bovine serum albumin, reacting at room temperature for 1hr, centrifuging at 2000 Xg for 5-15 min, re-dissolving the precipitate with 0.02M phosphate buffer solution with pH of 7.0, the re-dissolved volume is one tenth of the initial volume, and storing at 4 ℃ for later use.
③ the gold-labeled antibody complex is sprayed on the glass fiber membrane by a BIODOT Dispensing System, dried in vacuum at 25 ℃ for 1-2 hr, and placed in a room temperature drying environment for standby.
⑶ assembling the test paper strip;
the following materials are sequentially adhered to the bottom plate in an overlapping manner:
① filter paper;
② sample pad;
③ glass fiber pad sprayed with colloidal gold-zearalenone monoclonal antibody complex;
④ a nitrocellulose membrane with zearalenone artificial antigen marked by fluorescent microspheres as a detection line and an anti-mouse antibody as a quality control line;
⑤ absorbent paper.
Namely assembling the immunochromatographic test strip of the invention.
⑷ a detection method;
the method for detecting zearalenone in the coix seed sample by using the immunochromatographic test strip comprises the following steps:
① weighing 2g of semen Coicis, adding 10mL of the extractive solution, shaking for 1 min, and collecting supernatant for detection;
② adding 110 μ L Coicis semen extract sample into the sample hole of the test paper, reacting for 10min, and observing red strip condition of test paper detection line and quality control line;
③ if the detection line and the quality control line show red strips, the content of zearalenone in the sample is less than 60 μ g/kg, the qualitative judgment is negative, otherwise, if the detection line does not develop color and only the quality control line develops color, the content of zearalenone in the sample is more than 60 μ g/kg, and the qualitative judgment is positive.
④ according to the result displayed by the red strip of the colloidal gold, if the sample is positive, the test paper is inserted into the detection window of the fluorescence reader, the intensity of the fluorescence of the detection line and the quality control line can be displayed on the display according to the value, and according to the standard curve recorded in the instrument, the content of the zearalenone in the sample can be calculated, thus realizing the quantitative detection of the positive sample (after the colloidal gold is developed, the fluorescence data read within 20 minutes is effective).
The qualitative and quantitative immunochromatographic test strip for detecting zearalenone provided by the invention, and the preparation method and the reagent or instrument used in the application thereof can be purchased from the market.
The invention is further illustrated by the following examples:
example 1: preparation of qualitative and quantitative immunochromatographic test strip for detecting zearalenone in coix seeds
Preparation process of immunochromatography test strip
1. Preparing a nitrocellulose membrane;
(1) preparing a fluorescent microsphere labeled zearalenone (ZEN-BSA) artificial antigen:
the coupling method with the protein is a mixed anhydride method, the coupling ratio is 1:10-1:100, and the ZEN-BSA is obtained after coupling and dialysis purification. The labeling method of the fluorescent microspheres is an EDC-NHS method, the coupling ratio of the fluorescent microspheres to the artificial antigen protein is 1:1 by calculating the concentration of the artificial antigen protein. After marking, ultrafiltration and centrifugal washing are carried out for 3 times to obtain the zearalenone artificial antigen marked by the fluorescent microspheres.
(2) Preparation of detection line and quality control line:
coating fluorescent microsphere-labeled ZEN-BSA conjugate and anti-mouse antibody on a nitrocellulose membrane: diluting the antigen conjugate to a concentration of 2mg/mL with 0.02M PBS (phosphate buffered saline) having a pH of 7.5, and spraying the resulting solution onto a membrane as a detection line; diluting the concentration of the anti-mouse antibody to 1mg/mL, spraying the obtained solution on a membrane as a quality control line, wherein the membrane spraying volumes of two lines are 0.74 mu L/cm, the interval between the detection line and the top edge of the membrane is 10mm, the interval between the two lines is 5mm, drying is carried out for 12 hours at 37 ℃, and the membrane is placed in a drying cabinet for storage and standby.
2. Preparing a colloidal gold composite pad:
preparing a colloidal gold labeled zearalenone monoclonal antibody: taking 40mL of colloidal gold (about 25nm in size), adjusting the pH of the colloidal gold to 7.0 by using a potassium carbonate solution, adding 400 mu g of zearalenone monoclonal antibody, fully mixing, stirring at room temperature for reaction for 1hr, adding 4mL of 10% bovine serum albumin, sealing for reaction for 30min, centrifuging at 5000 Xg for 20min, centrifuging and washing with ultrapure water for 3 times, redissolving and precipitating the precipitate to 1/10 of the initial volume by using 0.02M PBS (containing 5% of sucrose and 0.05% of Tween-20) with the pH of 7.0, spraying a binding pad onto a 30 X0.8 cm glass fiber membrane, drying at 25 ℃ in vacuum for 1-2 hr, and placing in a drying cabinet for later use. The semi-finished product is assembled with an NC film. The result of detecting the sample is as follows: the standard curve concentrations under this condition were: 0. 10, 20, 40, 80, 160ppb, R2Is 0.9943, IC50At 56ng/mL, the linear regression equation is: y-1024.1987 x + 1198.6738.
3. Assembling the test strip:
⑴ Filter paper and sample pad size is 1X 30 cm;
⑵ spraying glass fiber coated with colloidal gold antibody compound, with specification of 0.8 × 30 cm;
⑶ spraying nitrocellulose membrane with detection line and quality control line, and specification of 2.5 × 30 cm;
⑷ absorbent paper with specification of 1.2 × 30 cm;
⑸ PVC soleplate, 5.5X 30 cm.
The materials are sequentially pasted according to the positions of all components in a test strip structural schematic diagram, the test strip with the size of 4 multiplied by 55mm is cut by a cutter after being assembled, the test strip is put into a plastic card shell, is compressed and put into an aluminum foil bag, and is sealed and stored after a drying agent is added, wherein the shelf life of the test strip in a room temperature environment is 6 months.
As shown in fig. 1, the immunochromatographic test strip is composed of: the NC film 4 sprayed with the detection line 5 and the quality control line 6, the gold pad 3 sprayed with the colloidal gold antibody compound, the sample pad 1, the filter paper 2 and the absorbent paper 7 are sequentially overlapped and stuck on the PVC bottom plate 8, and after being stuck, the test paper is cut into test paper strips with the size of 4 multiplied by 55mm by a slitter and is put into a plastic card shell, thus obtaining the complete test paper strip.
As shown in fig. 2, the detection principle is as follows:
after the sample is added into the test strip, if the sample is negative (the content of the zearalenone is less than 60 mug/kg), the sample is subjected to chromatography along the chromatography direction, a colloidal gold antibody compound on a binding pad surges to the position of a detection line, and the colloidal gold antibody compound and a zearalenone coupling antigen marked by fluorescent microspheres on the detection line undergo an immunological reaction based on antigen-antibody binding to form an antigen-antibody compound, and the colloidal gold is gathered on the detection line and shows that a red strip appears; part of the colloidal gold antibody compound which is not combined with the coupling antigen surges to the position of the quality control line, and the anti-mouse secondary antibody can be combined with the mouse-derived monoclonal antibody so that the quality control line also displays a red strip, and the negative result can be directly judged by naked eyes. If the sample is positive, the zearalenone in the sample is firstly combined with the colloidal gold antibody compound, and the colloidal gold antibody compound combined with the zearalenone can not be combined with the zearalenone coupling antigen marked by the fluorescent microspheres on the detection line any more, so that the colloidal gold of the detection line is colorless, and the sample is judged to be positive by naked eyes. Because the fluorescence microspheres on the detection line are subjected to light quenching due to the color development of the colloidal gold, the negative result of the color development is low in fluorescence value, and the color development is a positive result, and the fluorescence value is strong due to no quenching of the colloidal gold, and the fluorescence value of the fluorescence microspheres is positively correlated with the zearalenone in the sample, so that the fluorescence intensity of the fluorescence microspheres on the detection line is read by a fluorescence microsphere reader, and the specific concentration of the zearalenone in the coix seed sample is obtained through data analysis. In the detection system, a colloidal gold color development system is used for qualitative detection, and a fluorescent microsphere color development system is used for quantitative detection.
Qualitative and quantitative detection of zearalenone in sample
1. Crushing a coix seed sample, sieving with a 18-mesh sieve, accurately weighing 1g, extracting with 80% methanol solution, oscillating for 3min, centrifuging at 4 ℃ for 10min at 4000r/min, taking supernatant, diluting with 0.01MPBS (pH7.4) solution until the final concentration of methanol is 20%, sieving with a 0.22 μm organic filter, dropping 110 μ L of sample on a test strip sample pad, and reacting for 10 min; according to the instruction, the coix seeds are subjected to sample pretreatment.
2. The red strip condition of the test strip detection line and the quality control line is visually observed;
3. if the detection line and the quality control line both show red strips, the content of the zearalenone in the coix seed is less than 60 mug/kg, and the qualitative judgment is negative, otherwise, if the detection line does not show color, and only the quality control line shows the red strips, the content of the zearalenone in the sample is more than 60 mug/kg, and the qualitative judgment is positive.
4. According to the result displayed by the colloidal gold red strip, if the sample is positive, the test strip is inserted into a detection window of a fluorescence reader, the intensity of fluorescence of the detection line and the quality control line can be displayed on a display according to the magnitude of the numerical value, and the content of zearalenone in the sample can be calculated according to a standard curve recorded in the instrument, so that the quantitative detection of the positive sample is realized (after the colloidal gold is developed, the fluorescence data read within 20 minutes is effective).
5. Sample verification: in the experimental process, a series of known concentrations of a zearalenone standard substance are prepared, and the corresponding fluorescence intensity value is measured, so that a standard curve established according to the series of values and the corresponding concentrations is recorded into an instrument. The method is used for detecting 5 coix seed samples with known concentration (the known samples are quantified to be 0, 28, 49, 81 and 200ppb by a GC-MS method), and the detection result completely conforms to the result of a confirmed sample, and the specific data are shown in Table 1.
Table 1: comparison of the results between the detection method and the instrument confirmation method shows that n is 3
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A qualitative and quantitative immunochromatographic test strip for detecting zearalenone is characterized in that a base plate is sequentially overlapped with filter paper, a sample pad, a glass fiber membrane, a nitrocellulose membrane and absorbent paper;
the glass fiber membrane is attached with a zearalenone monoclonal antibody marked by colloidal gold;
the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is coated with a zearalenone artificial antigen marked by fluorescent microspheres, and the quality control line is coated with a second antibody.
2. The qualitative and quantitative immunochromatographic test strip according to claim 1, wherein the fluorescent microsphere labeled zearalenone artificial antigen is zearalenone coupled with a macromolecular protein and a fluorescent microsphere.
3. The qualitative and quantitative immunochromatographic test strip according to claim 2, wherein the macromolecular protein is bovine serum albumin or ovalbumin.
4. The qualitative and quantitative immunochromatographic test strip according to claim 2, wherein the fluorescent microspheres are microspheres coated with fluorescent substances prepared by using rare earth ions with diameters of 0.01-10 μm as markers.
5. The qualitative and quantitative immunochromatographic test strip according to claim 4, wherein the fluorescent substance is an organic or inorganic fluorescent substance, or a dopant of a plurality of fluorescent substances, or a quantum dot.
6. The qualitative and quantitative immunochromatographic test strip according to claim 1, wherein the colloidal gold particles are gold particles having a diameter of 10 to 100 nm.
7. The method for preparing the qualitative and quantitative immunochromatographic test strip of any one of claims 1 to 6, comprising the steps of:
coupling zearalenone with macromolecules to obtain a zearalenone artificial antigen, and then marking the zearalenone artificial antigen by adopting fluorescent microspheres to obtain the zearalenone artificial antigen marked by the fluorescent microspheres;
coating a zearalenone artificial antigen marked by fluorescent microspheres at a detection line position of a nitrocellulose membrane, and coating a second antibody at a quality control line position of the nitrocellulose membrane;
adopting colloidal gold particles to mark the zearalenone monoclonal antibody to obtain a colloidal gold-marked zearalenone monoclonal antibody, and attaching the colloidal gold-marked zearalenone monoclonal antibody to a glass fiber membrane;
and sequentially overlapping and sticking filter paper, a sample pad, a glass fiber membrane, a nitrocellulose membrane and absorbent paper on the bottom plate to obtain the qualitative and quantitative immunochromatographic test strip for detecting zearalenone.
8. The method according to claim 7, wherein the amount of the colloidal gold-labeled zearalenone monoclonal antibody sprayed is 7 to 9 μ L/cm.
9. The preparation method of claim 7, wherein the coating concentration of the fluorescent microsphere labeled zearalenone artificial antigen is 1.0-5.0 mg/mL, and the spraying amount is 0.7-0.8 μ L/cm; the coating concentration of the second antibody is 0.5-5.0 mg/mL, and the spraying amount is 0.7-0.8 muL/cm.
10. A method for qualitatively and quantitatively detecting zearalenone is characterized by comprising the following steps:
(1) qualitative detection
Adding a detection sample on a sample pad of the qualitative and quantitative immunochromatographic test strip of any one of claims 1 to 6, and observing red strips appearing in a detection line and a quality control line of the test strip after reacting for 10-12 min:
the detection line and the quality control line both show red strips, and the content of zearalenone in the detection sample is less than 60 mug/kg, and the qualitative judgment is negative;
if the detection line does not display a red strip and the quality control line displays a red strip, the content of zearalenone in the detection sample is more than or equal to 60 mu g/kg, and the detection sample is qualitatively judged to be positive;
(2) quantitative detection
And detecting the fluorescence intensity of the test strip which is qualitatively judged to be positive, and obtaining the content of the zearalenone in the detection sample according to the standard curve.
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