WO2023207166A1 - 一种同时检测谷物中多种真菌毒素和重金属Cd的五联荧光免疫定量试纸条 - Google Patents

一种同时检测谷物中多种真菌毒素和重金属Cd的五联荧光免疫定量试纸条 Download PDF

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WO2023207166A1
WO2023207166A1 PCT/CN2022/141458 CN2022141458W WO2023207166A1 WO 2023207166 A1 WO2023207166 A1 WO 2023207166A1 CN 2022141458 W CN2022141458 W CN 2022141458W WO 2023207166 A1 WO2023207166 A1 WO 2023207166A1
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solution
afb1
zen
ota
complete antigen
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French (fr)
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孙秀兰
孙嘉笛
张银志
汤璐瑶
王良哲
鲍琦
朱志远
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Jiangnan University
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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
    • G01N33/558—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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies

Definitions

  • the invention relates to a five-link fluorescent immunoquantitative test strip for simultaneously detecting multiple mycotoxins and heavy metal Cd in grains, and belongs to the technical field of rapid immunoassay detection.
  • Cereal foods and their products are rich in a variety of nutrients needed by humans. Global food production continues to increase. In 2021, global food production will exceed 2.8 billion tons. However, food is increasingly contaminated by a variety of mycotoxins and heavy metals at the same time. Furthermore, the combined toxic effects of mycotoxins and heavy metals have great potential harm to the human body.
  • Aflatoxin is mainly a metabolite of Aspergillus flavus and Aspergillus parasiticus. More than 20 species have been discovered so far, among which aflatoxin B1 (Aflatoxin B1, AFB1) ranks first in terms of toxicity, carcinogenicity and pollution frequency. It is a Class 1A strong carcinogen classified by IARC. Its toxicity is 68 times and 10 times that of arsenic and potassium cyanide respectively. It has teratogenicity, carcinogenicity, mutagenicity, neurotoxicity and nephrotoxicity, and can produce harmful effects on humans and animals. varying degrees of harm.
  • Fumonisins are mainly produced by Fusarium moniliforme and Fusarium multisporum. 11 structures have been discovered, among which fumonisin B1 (Fumonisin B1, FB1) has the highest content and the strongest toxicity. It mainly acts on the liver, Kidney, has been classified as a Category 2B carcinogen by IARC. Ochratoxin is a type of secondary metabolite produced by Aspergillus and Penicillium. Among them, Ochratoxin A (OTA) has the highest content, widest distribution and strongest toxicity.
  • OTA Ochratoxin A
  • ZEN Zearalenone
  • the detection methods of mycotoxins include instrumental methods, chemical analysis methods, immunoassay methods, etc.
  • the detection methods of heavy metals include instrumental methods, immunoassay methods, etc.
  • Single-type test strips cannot detect two or more substances simultaneously, quickly and at low cost; at the same time, existing immunochromatographic methods for detecting multiple toxins or multiple heavy metals can only achieve qualitative and semi-quantitative detection, or have low detection sensitivity. .
  • the present invention uses fluorescent microspheres to replace traditional colloidal gold, uses fluorescent microspheres to label the antibody complexes of AFB1, FB1, OTA, ZEN and Cd-EDTA, and adopts a method of spraying micro-detection points to form an array detection.
  • Image J software performs RGB analysis on the pictures to obtain the fluorescence values of dot matrix strips with different brightness on the detection line. , which can perform simultaneous and rapid quantitative analysis of AFB1, FB1, OTA, ZEN and Cd in the sample.
  • the first object of the present invention is to provide a method for preparing a five-link fluorescent immunoquantitative test strip for simultaneous detection of AFB1, FB1, OTA, ZEN and Cd, including the following steps:
  • the final concentration of the AFB1 complete antigen solution, FBl complete antigen solution, OTA complete antigen solution and ZEN complete antigen solution is 0.01-1.0 mg/mL
  • the final concentration of the Cd complete antigen solution is 0.01-1.0 mg/mL.
  • the concentration is 0.1-1.0mg/mL.
  • the final concentration of the goat anti-mouse secondary antibody solution is 0.05-1 mg/mL.
  • 0.01-1.0 mg/mL AFB1 complete antigen solution is applied to the nitrocellulose membrane (NC membrane) as the detection line T1
  • 0.01-1.0 mg/mL FB1 complete antigen solution is applied to On the nitrocellulose membrane (NC membrane) as the detection line T2
  • 0.01-1.0mg/mL ZEN complete antigen solution The antigen solution is applied to the nitrocellulose membrane (NC membrane) as the detection line T4.
  • the 0.1-1.0mg/mL Cd complete antigen solution is applied to the nitrocellulose membrane (NC membrane) as the detection line T5.
  • the 0.05-1mg/mL Cd complete antigen solution is applied to the nitrocellulose membrane (NC membrane) as the detection line T5.
  • mL of goat anti-mouse secondary antibody solution was applied to the nitrocellulose membrane (NC membrane) as quality control line C.
  • the distance between the detection lines T1, T2, T3, T4, and T5 is 1-3 mm
  • the distance between the detection line T1 and the quality control line C is 1-3 mm
  • the distance between the detection line T2- T5 and quality control line C are on both sides of the test line T1.
  • one end of the sample pad and the nitrocellulose membrane containing the detection line and the quality control line overlap each other, and the length of the overlapping area is 2- 4mm; the absorbent paper and the other end of the nitrocellulose membrane containing the detection line and the quality control line overlap each other, and the length of the overlapping area is 2-4mm; the absorbent paper, the sample pad and the nitric acid membrane containing the detection line and the quality control line
  • the overlapping portion of the cellulose membrane is located above the nitrocellulose membrane containing the detection line and quality control line.
  • the distance between the detection line T5 and the sample pad is 4-6 mm, and the distance between the quality control line C and the absorbent paper is 4-6 mm.
  • the widths of the detection lines T1, T2, T3, T4, T5 and the quality control line C are 1.5-2.5 mm.
  • the spraying method of AFB1 complete antigen solution, FB1 complete antigen solution, OTA complete antigen solution, ZEN complete antigen solution, Cd complete antigen solution and goat anti-mouse secondary antibody solution is to form an array of trace detection points detection method.
  • the spraying amounts of the detection line width per centimeter of the nitrocellulose membrane AFB1 complete antigen solution, FB1 complete antigen solution, OTA complete antigen solution, ZEN complete antigen solution and Cd complete antigen solution are respectively 10-40nL; when the width of the nitrocellulose membrane is 0.4cm, the spraying volume is 5-30nL respectively.
  • the spraying amount per centimeter of the quality control line width on the nitrocellulose membrane is 1-3 ⁇ L/cm; when the width of the nitrocellulose membrane is 0.4cm, the spraying amount is 0.4-1.2 ⁇ L.
  • the drying is to place the coated nitrocellulose membrane in a 37°C vacuum drying oven for drying before use.
  • the complete antigen solution of AFB1, FBl, OTA, ZEN, Cd and goat anti-mouse secondary antibody is diluted with 0.02mol/L pH 7.0 phosphate buffer solution (PBS).
  • the second object of the present invention is to prepare a five-link fluorescent immunoquantitative test strip for simultaneously detecting AFB1, FBl, OTA, ZEN and Cd prepared by the method of the present invention.
  • the detection concentration of AFB1 is 0.05-10ng/mL
  • the detection concentration of FB1 is 1-12ng/mL
  • the detection concentration of OTA is 0.5-60ng/mL
  • the detection concentration of ZEN is 0.1-40ng/mL
  • the detection concentration of Cd is 1-40ng/mL.
  • the width of the five-link fluorescent immunoquantitative test strip is 3-5 mm.
  • the third object of the present invention is to provide a method for simultaneously detecting AFB1, FBl, OTA and ZEN and heavy metal Cd in cereals.
  • the method includes the following steps:
  • step (2) Combine the test solution of step (1), PBS buffer, monoclonal antibody to AFB1 labeled with Eu 3+ -fluorescent microspheres, monoclonal antibody to FB1 labeled with Eu 3+ -fluorescent microspheres, and Eu 3+ -fluorescent microsphere labeled OTA monoclonal antibody, Eu 3+ -fluorescent microsphere labeled ZEN monoclonal antibody, Eu 3+ -fluorescent microsphere labeled Cd monoclonal antibody were mixed evenly to obtain pre- The solution to be tested after treatment;
  • step (3) Add the solution to be tested in step (2) to the sample pad of the five-link fluorescent immunoquantitative test strip of the present invention for chromatography, and then take photos with a camera tool, and Image J software performs RGB analysis on the pictures. Obtain the corresponding fluorescence intensity value C value of the sample at the detection line T1, T2, T3, T4, T5 and quality control line C, the T1 value of AFB1, the T2 value of FB1, the T3 value of OTA, the T4 value of ZEN, and the Cd T5 value;
  • the extraction in step (1) specifically includes the following steps:
  • the solution to be tested in step (2) the monoclonal antibody of AFB1 labeled with Eu 3+ -fluorescent microspheres, the monoclonal antibody of FBl labeled with Eu 3+ -fluorescent microspheres, Eu 3+ -fluorescent microsphere labeled OTA monoclonal antibody, Eu 3+ -fluorescent microsphere labeled ZEN monoclonal antibody, Eu 3+ -fluorescent microsphere labeled Cd monoclonal antibody and the volume of PBS buffer
  • the ratio is (3-5):(3-5):(3-5):(3-5):(3-5):(5-10):(85-95), more preferably 5:3 :3:3:3:5:87.
  • the uniform mixing in step (2) involves incubating at 20-37°C for 10-20 minutes to fully compete for the reaction.
  • the amount of the pretreated solution to be tested in step (3) is 30-50 ⁇ L/cm 2 .
  • the imaging tool in step (3) includes a mobile phone and a camera.
  • the method for constructing the standard curve described in step (4) includes the following steps;
  • Negative grain samples were used to prepare AFB1/FB1/OTA/ZEN/Cd-EDTA at concentrations of 0.05/1/0.5/0.1/1ng/mL, 0.25/2/1/0.5/5ng/mL, and 0.5/4/2/ respectively. 1/10ng/mL, 1/6/4/5/15ng/mL, 2/8/8/10/20ng/mL, 5/10/30/20/30ng/mL and 10/12/60/40/
  • the 40ng/mL standard solution is used for five-link fluorescence immunoassay strip detection, in which the standard solution is a 0.01M pH 7.0 PBS solution containing 5% methanol (v/v);
  • AFB1 monoclonal antibody (AFB1 fluorescent probe), FB1 monoclonal antibody (FB1 fluorescent probe), OTA monoclonal antibody (OTA fluorescent probe), ZEN monoclonal antibody (ZEN fluorescent probe) Probe), heavy metal Cd monoclonal antibody (Cd-EDTA fluorescent probe) as fluorescent probe, 1 ⁇ L AFB1 fluorescent probe, 2 ⁇ L FB1 fluorescent probe, 1 ⁇ L OTA fluorescent probe, 1 ⁇ L ZEN fluorescent probe, 0.5 ⁇ L Cd -EDTA fluorescent probe, 89.5 ⁇ L of PBS buffer and 5 ⁇ L of standard solution were homogenized and incubated at room temperature for 10 min, then slowly dripped into the sample pad of the five-link fluorescent immunoquantitative test strip, with a sample volume of 50 ⁇ L/cm 2 and chromatography at 37°C.
  • each AFB1 standard The logarithmic value of the product concentration is the abscissa, T1/T0 ⁇ 100 (%) is the ordinate to draw a standard curve, and the competition inhibition rate is set to (1-T1/T0) ⁇ 100%; the logarithm value of each FB1 standard concentration is The abscissa, T2/T0 ⁇ 100 (%) is used as the ordinate to draw a standard curve, and the competition inhibition rate is set to (1-T2/T0) ⁇ 100%; the logarithmic value of each OTA standard concentration is used as the abscissa, T3/T0 ⁇ 100 (%) is the ordinate to draw a standard curve, and the competition inhibition rate is set to (1-T3/T0) ⁇ 100%; taking the logarithmic value of each ZEN standard concentration as the abscissa, T4/T0 ⁇ 100 (%) is The ordinate is used to draw a standard curve, and the competition inhibition rate is set to (1-T4/T0) ⁇ 100%; the
  • the concentration range of AFB1 in the solution to be tested in step (1) is 0-30ng/mL
  • the concentration range of FB1 is 0-60ng/mL
  • the concentration range of OTA is 0-10ng/mL.
  • the concentration range of mL and ZEN is 0-50ng/mL
  • the concentration range of Cd is 0-40ng/mL. If the concentration is too high and exceeds the linear detection range of the test strip, the detected results will have no use value and need to be treated with PBS.
  • the buffer is used as a solvent for dilution; if the concentration is too low and below the national standard, there is no need for testing.
  • the fourth object of the present invention is the application of the five-link fluorescent immunoquantitative test strip of the present invention in the field of food testing.
  • the fifth object of the present invention is the application of the method of the present invention for simultaneously detecting AFB1, FBl, OTA, ZEN and heavy metal Cd in cereals in the field of food testing.
  • the operation is simple and the conditions are mild. It provides technical support for the simultaneous detection of multiple mycotoxins (AFB1, FB1, OTA, ZEN) and Cd, and simplifies the operation process.
  • the method of the present invention can achieve rapid quantitative and simultaneous detection of the contents of mycotoxins (AFB1, FB1, OTA, ZEN) and heavy metal Cd in cereal samples, with strong specificity and high sensitivity.
  • concentration of AFB1 is 0.05- At 10ng/mL
  • the logarithmic value of its concentration has a linear relationship with T2/T0.
  • the method of the present invention uses AFB1, FB1, OTA, ZEN, and Cd monoclonal antibodies as recognition targets, and uses time-resolved fluorescent microspheres as the signal source.
  • the volume of the fluorescent microspheres is much larger than the volume of the fluorescent dye molecules. Not only can it effectively eliminate non-specific fluorescence interference, but the particle size is uniform, as shown in Figure 1, the surface functional groups are stable and controllable, the experimental repeatability is good, the detection specificity is strong, and rapid quantitative detection can be achieved with high sensitivity and small error. , providing great convenience for instant detection.
  • the five-link fluorescent immunoquantitative test strip of the present invention has the characteristics of high sensitivity, low cost, easy operation, rapid quantitative detection, good stability, and is suitable for industrial and commercial departments, third-party testing institutions, government regulatory departments at all levels, and dairy products enterprises. It has broad market prospects and is easy to promote and use.
  • Figure 1 Transmission electron microscope image of Eu 3+ -fluorescent microspheres.
  • Figure 2 Structural diagram of five-link fluorescent immunoquantitative test strips for various mycotoxins and heavy metal Cd.
  • Figure 3 Standard curve of labeled fluorescent microsphere immunochromatography for detection of AFB1 (A), FB1 standard curve (B), OTA standard curve (C), ZEN standard curve (D), heavy metal Cd standard curve ( E) and the five-link fluorescent immunoquantitative test strip diagram under ultraviolet light (F).
  • Figure 4 Optimization of complete antigen concentration in the detection line (T1 line) of AFB1 immunochromatography (A), optimization of complete antigen concentration in the detection line (T2 line) of FB1 immunochromatography (B), OTA immunochromatography Optimization of the complete antigen concentration of the detection line (T3 line) (C), optimization of the complete antigen concentration of the ZEN immunochromatography detection line (T3 line) (D), complete optimization of the detection line (T5 line) of the Cd immunochromatography method Optimization of antigen concentration (E).
  • Figure 5 Effect of loading amount of fluorescent probe on fluorescence intensity of detection line.
  • Figure 6 The effect of the incubation time of the fluorescent probe and the test solution (A) or the chromatography time after adding the test strip (B) on the fluorescence intensity and T/T0.
  • Figure 7 AFB1, FB1, OTA, ZEN and Cd specificity test results.
  • the AFB1 monoclonal antibody, FB1 monoclonal antibody, OTA monoclonal antibody, ZEN monoclonal antibody, Cd monoclonal antibody and goat anti-mouse secondary antibody used in the examples were all purchased from Beijing Kaiyuan Technology Co., Ltd.
  • the preparation method includes the following steps:
  • the complete antigen solution is sprayed onto the nitrocellulose membrane (NC membrane) using a micro-detection point formation array detection method, and the 0.1 mg/mL AFB1 complete antigen solution (AFB1-OVA) is sprayed onto the nitrocellulose membrane (NC membrane) As the detection line T1, spray 0.1 mg/mL FB1 complete antigen solution (FB1-OVA) onto the nitrocellulose membrane (NC membrane) as the detection line T2, and spray 0.1 mg/mL OTA complete antigen solution (OTA-OVA) onto the nitrocellulose membrane (NC membrane).
  • NC membrane On the nitrocellulose membrane (NC membrane) as the detection line T3, spray 0.1mg/mL ZEN complete antigen solution (ZEN-OVA) onto the nitrocellulose membrane (NC membrane) as the detection line T4, and 0.3mg/mL Cd complete
  • ZEN-OVA ZEN complete antigen solution
  • Cd-EDTA-OVA The antigen solution
  • Cd-EDTA-OVA was applied to the nitrocellulose membrane (NC membrane) as the detection line T5
  • the 0.5 mg/mL goat anti-mouse secondary antibody solution was applied to the nitrocellulose membrane (NC membrane) as the quality control.
  • test line T1, T2, T3, T4, and T5 is all 1.2mm
  • the distance between test line T1 and quality control line C is 1.2mm
  • test line T2 and quality control line C are on both sides of test line T1
  • the width of the test lines T1, T2, T3, T4, T5 and the quality control line C depends on the diameter of the film spraying instrument pipe, which is 2mm, and dried at 37°C for 2 hours;
  • the sample pad, the nitrocellulose membrane containing the detection line and the quality control line, and the absorbent paper are sequentially pasted on the PVC bottom plate to assemble a test strip; the sample pad is stacked on the nitrocellulose membrane containing the detection line and the quality control line. The two overlap by 3mm.
  • the absorbent paper is stacked on the nitrocellulose membrane containing the detection line and the quality control line, and the two overlap by 3mm; the distance between the detection line T5 and the sample pad is 5mm, and the distance between the quality control line C and the absorbent paper The distance is 5mm;
  • the preparation method includes the following steps:
  • Activation buffer 2-(N-morpholine)ethanesulfonic acid (MES, C 6 H 13 NO 4 S ⁇ H 2 O) solution with pH 4.5-6.50.05M;
  • Coupling buffer Phosphate buffer (PBS) pH 7.0-8.00.01M (avoid using solvents with free amines);
  • Activator 1 mg/mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide nitrate (EDC, C 8 H 17 N 3 ⁇ HCl) solution and 1 mg/mL N-hydroxyl Succinimide (NHS, C 4 H 5 NO 3 ) solution;
  • Blocking buffer pH 6.0-7.00.01M phosphate buffer (PBS) containing 1% BSA, 0.05% Tween-20;
  • Antibody protection solution Tris-HCl with pH 6.0-7.00.05M containing 5% trehalose, 0.1% PVPK-30, 0.5% BSA, 0.1% TritonX-100;
  • Labeling wash solution Tris-HCl pH 6.0-7.00.05M containing 0.1% Tween-20;
  • Microsphere complex solution/reaction sustained release solution Tris-HCl containing 5% sucrose, 1% bovine serum albumin, 1% Tween-20, pH 6.0-7.00.05M;
  • the preparation method of ZEN monoclonal antibody (Eu-ZEN-mAb) and Eu 3+ -fluorescent microsphere-labeled Cd monoclonal antibody (Eu-Cd-EDTA-mAb) is to combine the Eu-AFB1-monoclonal antibody preparation method with The AFB1 monoclonal antibodies were replaced with FB1 monoclonal antibodies, OTA monoclonal antibodies, ZEN monoclonal antibodies, and Cd monoclonal antibodies to obtain Eu 3+ -fluorescent microsphere-labeled FB1 monoclonal antibodies, OTA monoclonal antibodies, and ZEN monoclonal antibodies. Clonal antibodies and Cd monoclonal antibodies should be stored at 4°C for later
  • the standard curve is constructed by:
  • Negative grain samples were used to prepare AFB1/FB1/OTA/ZEN/Cd-EDTA at concentrations of 0.05/1/0.5/0.1/1ng/mL, 0.25/2/1/0.5/5ng/mL, and 0.5/4/2/ respectively. 1/10ng/mL, 1/6/4/5/15ng/mL, 2/8/8/10/20ng/mL, 5/10/30/20/30ng/mL and 10/12/60/40/
  • the 40ng/mL standard solution is used for five-link fluorescence immunoassay strip detection, in which the standard solution is a 0.01M pH 7.0 PBS solution containing 5% methanol (v/v).
  • AFB1 fluorescent probe The Eu 3+ -fluorescent microsphere-labeled AFB1 monoclonal antibody (AFB1 fluorescent probe), FB1 monoclonal antibody (FB1 fluorescent probe), OTA monoclonal antibody (OTA fluorescent probe) prepared in Example 2, ZEN Monoclonal antibody (ZEN fluorescent probe) and heavy metal Cd monoclonal antibody (Cd-EDTA fluorescent probe) are used as fluorescent probes.
  • AFB1 fluorescent probe 2 ⁇ L FB1 fluorescent probe, 1 ⁇ L OTA fluorescent probe, and 1 ⁇ L ZEN fluorescent probe.
  • the probe 0.5 ⁇ L Cd-EDTA fluorescent probe, 89.5 ⁇ L PBS buffer and 5 ⁇ L standard solutions of different concentrations were homogenized and incubated at room temperature for 10 min, and then slowly dripped into the sample pad of the five-link fluorescent immunoquantitative test strip of Example 1.
  • the sample volume is 50 ⁇ L/cm 2.
  • Image J software performs RGB analysis on the image and records the detection lines T1, T2, T3, T4, and T5 of the five-link fluorescent immunoquantitative test strip. Fluorescence value; each concentration is measured in six parallels.
  • the fluorescence value of the T line of the standard solution with a concentration of 0 ppb is set to T0, and the fluorescence values of the detection lines T1, T2, T3, T4, and T5 of other spiked concentrations are T1, T2, and T3.
  • T4, T5 draw the standard curve with the logarithm value of each AFB1 standard concentration as the abscissa and T1/T0 ⁇ 100 (%) as the ordinate, and the competition inhibition rate is set as (1-T1/T0) ⁇ 100%;
  • the logarithmic value of each FB1 standard concentration is the abscissa, and T2/T0 ⁇ 100 (%) is the ordinate to draw a standard curve.
  • the competitive inhibition rate is set to (1-T2/T0) ⁇ 100%; the concentration of each OTA standard is the The log value is the abscissa, T3/T0 ⁇ 100 (%) is the ordinate to draw a standard curve, the competition inhibition rate is set to (1-T3/T0) ⁇ 100%; the log value of each ZEN standard concentration is the abscissa, T4/T0 ⁇ 100 (%) is used as the ordinate to draw a standard curve, and the competitive inhibition rate is set as (1-T4/T0) ⁇ 100%; taking the logarithmic value of each Cd standard concentration as the abscissa, T5/T0 ⁇ 100 ( %) is the ordinate to draw a standard curve, and the competition inhibition rate is set as (1-T5/T0) ⁇ 100%.
  • Example 3 Please refer to Example 3 for the method of constructing the standard curve of the complete antigen solution directly streaked and sprayed on the test strip.
  • the detection limit of AFB1 is 0.524ng/mL
  • the detection limit of FB1 is 0.498ng/mL
  • the detection limit of OTA is 0.619ng/mL
  • the detection limit of ZEN The detection limit of Cd is 0.549ng/mL
  • the detection limit of Cd is 0.536ng/mL, both of which are higher than the detection limit of dot matrix test paper strips.
  • a method for simultaneously detecting mycotoxins (AFB1, FBl, OTA, ZEN) and heavy metal Cd in cereals includes the following steps:
  • step (2) Combine the test solution obtained in step (1), the monoclonal antibody of AFB1 labeled with Eu 3+ -fluorescent microspheres, the monoclonal antibody of FB1 labeled with Eu 3+ -fluorescent microspheres, and Eu 3+ -fluorescent microspheres.
  • the volume ratio of the microsphere-labeled OTA monoclonal antibody, Eu 3+ -fluorescent microsphere-labeled ZEN monoclonal antibody, Eu 3+ -fluorescent microsphere-labeled Cd monoclonal antibody and PBS buffer is 5:3. :3:3:3:5:87, incubate at 37°C for 15 minutes to obtain the test solution after pretreatment;
  • step (3) Add the solution to be tested obtained in step (2) to the sample pad of the five-link fluorescent immunoquantitative test strip of Example 1, perform chromatography at 37°C for 15 minutes, and add a sample volume of 50 ⁇ L/cm 2 ; then take pictures with a mobile phone , Image J software performs RGB analysis on the picture, and obtains the corresponding fluorescence intensity values of the sample at the detection lines T1, T2, T3, T4, T5 and quality control line C: T1 value of AFB1, T2 value of FB1, and T3 value of OTA , T4 value of ZEN, T5 value and C value of Cd;
  • the concentrations of AFB1-OVA, FB1-OVA, OTA-OVA, ZEN-OVA and Cd-EDTA-OVA were diluted to 0.1mg/mL and 0.3mg/mL respectively. mL, 0.5mg/mL, 0.7mg/mL and 0.9mg/mL.
  • Biodot's array spray spotter to spray onto the NC film, and pass through the negative control group and the positive experimental group (positive sample: AFB1 is 0.05ng/mL, FB1 is 1ng/mL, OTA is 0.25ng/mL, ZEN is 2ng/mL and Cd-EDTA (0.1ng/mL) for analysis, choose to observe the brightness of each band under ultraviolet light, and then select the antigen concentration with the best inhibition rate as the optimal spray point concentration.
  • Example 5 To evaluate the effects of different concentrations of AFB1-OVA, FB1-OVA, OTA-OVA, ZEN-OVA, and Cd-EDTA-OVA on the detection results of the five-linked immunoquantitative test strips, refer to Example 5 for the detection method.
  • the fluorescence intensity on the detection line is relatively strong, and the competitive inhibition rate is the largest. Therefore, it is best to choose an AFB1 antigen concentration of 0.3 mg/mL as the detection line T1.
  • Spraying concentration when the FB1-OVA concentration is 0.3mg/mL, the fluorescence intensity on the detection line is relatively strong, and the competitive inhibition rate is the largest, so the FB1 antigen concentration of 0.3mg/mL is selected as the optimal spraying concentration for detection line T2;
  • the OTA-OVA concentration is 0.5mg/mL, the fluorescence intensity on the detection line is relatively strong, and the competitive inhibition rate is the largest.
  • the OTA antigen concentration of 0.5mg/mL is selected as the optimal spraying concentration for detection line T3; when ZEN-OVA When the concentration is 0.5mg/mL, the fluorescence intensity on the detection line is relatively strong, and the competitive inhibition rate is the largest. Therefore, the ZEN antigen concentration of 0.5mg/mL is selected as the optimal spraying concentration for detection line T4; when the Cd-EDTA-OVA concentration When it is 0.3 mg/mL, the fluorescence intensity on the detection line is relatively strong, and the competitive inhibition rate is the highest. Therefore, the Cd-EDTA antigen concentration of 0.3 mg/mL is selected as the optimal spray concentration for detection line T5.
  • Eu-AFB1-mAb Eu-FB1-mAb
  • Eu-OTA-mAb Eu-ZEN-mAb
  • Five types of Eu-Cd-EDTA-mAb probes were optimized for loading. First, 0.1 ⁇ L, 0.5 ⁇ L, 1 ⁇ L, 1.5 ⁇ L and 2 ⁇ L probes were used to load the probes using five-link array immunochromatography test strips. Optimization.
  • Example 5 To evaluate the effects of different concentrations of AFB1-OVA, FB1-OVA, OTA-OVA, ZEN-OVA, and Cd-EDTA-OVA on the detection results of the five-link immunoquantitative test strip, refer to Example 5 for the method.
  • the optimal pre-incubation time is determined based on the fluorescence intensity on each detection line of the test strip under different pre-incubation times of the antigen and antibody. From Figure 6(A), we can see the T/ The ratio of T0 is the highest, and as time goes by, the value of T/T0 continues to decrease. When the pre-incubation time is 10 minutes, the ratio basically tends to balance and does not change, indicating that the antigen-antibody binding reaction has been completed after 10 minutes. Therefore, 10 min was selected as the pre-reaction time before the sample was added to the test strip immunochromatography.
  • Antibody affinity is used to measure the specific binding ability between an antigen and an antibody. It is determined by the binding strength of the binding cluster and the determinant. Its essence is a non-covalent force, including the attraction between amino acids, hydrogen bonds, Hydrophobic force, etc., reflects the ability of an antibody molecule to react with a hapten molecule or a determinant of an antigen molecule. Identification of monoclonal antibody affinity by indirect non-competitive ELISA method.
  • the main principle is to fix the coating material at the bottom of a 96-well plate, add the test substance and monoclonal antibody, the coating material at the bottom of the plate combines with the antibody, the excess antibody will be washed away, and the antigen-antibody complex trapped at the bottom of the plate will Combine the substance with the enzyme-labeled secondary antibody, and add chromogenic solution and stop solution.
  • the specific steps are as follows:
  • step (2) After washing according to step (2), dilute the 1 mg/mL AFB1 monoclonal antibody into 1 mg/mL, 0.33 mg/mL, 0.11 mg/mL, 0.037 mg/mL, 0.012 mg/mL, There are 8 concentrations of 0.004mg/mL, 0.001mg/mL, and 0.0003mg/mL. Add 100 ⁇ L to each well and incubate at 37°C for 1 hour;
  • step (2) wash three times according to step (2) and pat dry, add 100 ⁇ L enzyme-labeled secondary antibody to each well (dilute 1 ⁇ L of horseradish peroxidase-goat anti-mouse secondary antibody with 5mL 0.01M PBS) ;
  • Color development Spin the liquid in the wells dry, wash the plate three times and pat dry according to step (2), add 100 ⁇ L of color development solution (color development solution A) to each well, and accurately weigh 13.6g sodium acetate and 1.6g citric acid. , add 0.3mL of 30% hydrogen peroxide after ultrasonic dissolution, adjust the volume to 500mL with ultrapure water, and store at 4°C for later use; for color development B solution, weigh 0.95g of citric acid, 0.2g of ethylenediaminetetraacetic acid, and measure 50mL of glycerol.
  • Termination Add 50 ⁇ L of stop solution (2mol/L H 2 SO 4 ) to each well and detect immediately;
  • Reading Use a multifunctional microplate reader to read the absorbance values of AFB1 monoclonal antibodies at different coating concentrations at 450 nm.
  • [Ab'] t and [Ab] t are respectively the molar concentration of the antibody at OD max /2 corresponding to two different concentrations of antigen, and [Ab'] t is the lower coating concentration, [Ab] t is the higher coating concentration; n is the ratio of two different concentrations (n>1), and K a is the affinity constant of the monoclonal antibody.
  • OTA monoclonal antibody For the affinity determination of FB1 monoclonal antibody, OTA monoclonal antibody, ZEN monoclonal antibody, and Cd-EDTA monoclonal antibody, refer to the procedures for AFB1 monoclonal antibody.
  • the affinity between the antibody and the antigen is an important parameter that affects the detection sensitivity of the test strip.
  • the value of the constant K a is generally used to indicate the size of the affinity. If K a ⁇ 10 7 L/mol, then the antibody affinity is low and the specificity is poor; if K a >10 7 L/mol, the antibody affinity is high. The specificity is better.
  • Table 2 to Table 6 show the affinity experimental results of AFB1, FB1, OTA, ZEN, and Cd-EDTA monoclonal antibodies. From Table 2 to Table 6, it can be seen that the average K a of AFB1, FB1, OTA, ZEN and Cd-EDTA are 1.842 ⁇ 10 9 L/mol, 2.041 ⁇ 10 9 L/mol, and 2.395 ⁇ 10 9 L respectively. /mol, 2.082 ⁇ 10 9 L/mol and 7.020 ⁇ 10 8 L/mol, indicating that AFB1, FB1, OTA, ZEN and Cd-EDTA monoclonal antibodies are high-affinity antibodies and can be used for the specific detection needs of this study.
  • test strips from the same batch to detect positive samples (AFB1 is 0.05ng/mL, FB1 is 1ng/mL, OTA is 0.25ng/mL, ZEN is 2ng/mL and Cd-EDTA is 0.1ng/mL) and negative samples respectively.
  • Samples were analyzed for intra-batch differences in test strips; positive samples were detected by using test strips from different batches (AFB1 was 0.05ng/mL, FB1 was 1ng/mL, OTA was 0.25ng/mL, and ZEN was 2ng/mL).
  • the intra-batch variation coefficients of T0, T and T/T0 (%) obtained through the analysis of the coefficient of variation calculation formula are all less than 8.97%, indicating that the intra-batch and inter-batch variation coefficients of the five-link immune quantitative test strips are small. It has high precision and good accuracy, and basically meets the requirements of quantitative detection test strips.
  • Example 5 In order to further evaluate the detection effect of the five-link array immunochromatography test strip in actual samples, rice, corn and wheat samples were purchased from the supermarket, and the sample extracts were obtained according to the method described in Example 5 for AFB1 and Cd double linkage. Immunochromatographic test strip detection. UPLC-MS/MS was used to verify whether the samples contained AFB1, FB1, OTA and ZEN, and ICP-MS was used to verify whether the three samples contained Cd.
  • the negative rice, corn and wheat samples were added to perform spike recovery tests respectively.
  • the added concentration of each sample was set up with three different sets of high, medium and low spike concentrations.
  • Each set of concentration gradients was set up with three sets of parallel tests.
  • the spiked recovery rate is used as the accuracy evaluation index, and the relative standard deviation (RSD%) of the detection results of repeated measurements of a certain concentration sample is used as the precision evaluation index.
  • the calculation formulas for spiked recovery and relative standard deviation are as follows (2) and (3):
  • AFB1/FB1/OTA/ZEN/Cd-EDTA were used to evaluate the specificity of the test strips.
  • AFM1, AFG1, AFG2 and AFB2 were used to evaluate the specificity of AFB1.
  • the final concentration of various substances is 10ng/mL; use FB2 and FB3 to specifically detect FB1, and the final concentration of various substances is 10ng/mL; OTA/ZEN uses OTB, DON, T-2 and The above-mentioned common mycotoxins were specifically analyzed, and the final concentration of each substance was 10ng/mL; Cd used Pb, Hg, and As for specific analysis of heavy metals, and the final concentration was 20ng/mL.

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Abstract

一种同时检测谷物中多种真菌毒素和重金属Cd的五联荧光免疫定量试纸条及其制备方法。五联荧光免疫定量试纸条的制备方法包括如下步骤：分别将0.01-1.0mg/mL AFB1、FB1、OTA、ZEN完全抗原溶液涂到NC膜上作为检测线T1～T4，将0.1-1.0mg/mL Cd完全抗原溶液涂到NC膜上作为检测线T5，将0.05-1mg/mL羊抗鼠二抗溶液涂到硝酸纤维素膜上作为质控线C，干燥；之后将样品垫、含有检测线和质控线的硝酸纤维素膜、吸水纸依次粘贴在PVC底板上组装成试纸条。五联荧光免疫定量试纸条可以实现在一张试纸条上同时检测谷物中多种真菌毒素和重金属Cd，并且具备灵敏度高、成本低廉、操作简便、快速定量检测、稳定性好的优点。

Description

一种同时检测谷物中多种真菌毒素和重金属Cd的五联荧光免疫定量试纸条 技术领域
本发明涉及一种同时检测谷物中多种真菌毒素和重金属Cd的五联荧光免疫定量试纸条，属于免疫分析快速检测技术领域。
背景技术
谷物类食品及其制品富含人类所需的多种营养物质，全球粮食的产量不断增加，2021年全球粮食产量已超过28亿吨，然而，粮食同时被多种真菌毒素和重金属污染现象愈发严重，真菌毒素和重金属的联合毒性作用对人体的潜在危害巨大。
黄曲霉毒素(Aflatoxin，AFT)主要是黄曲霉和寄生曲霉菌的代谢产物，目前已发现20余种，其中黄曲霉毒素B1(Aflatoxin B1，AFB1)的毒性、致癌性、污染频率均居于首位，是IARC划分的1A类强致癌物，其毒性分别为砒霜和氰化钾的68倍和10倍，具有致畸形、致癌性、致突变性、神经毒性和肾脏毒性，对人体和动物均会产生不同程度的危害。伏马毒素(Fumonisins，FBs)主要由串珠镰刀菌和多育镰孢菌产生，已发现11种结构，其中伏马毒素B1(Fumonisin B1，FB1)含量最高、毒性最强，主要作用于肝脏、肾脏，已被IARC列为2B类致癌物。赭曲霉毒素(Ochratoxin)是由曲霉属和青霉属产生的一类次级代谢产物，其中以赭曲霉毒素A(Ochratoxin A，OTA)含量最高、分布最广且毒性最强，lARC将其划为2B类致癌物，具有肾毒性、肝毒性、胚胎毒性、致畸性、神经毒性、免疫毒性、遗传毒性、致癌性以及肠道毒性等。玉米赤霉烯酮(Zearalenone，ZEN)主要是由禾谷镰刀菌和三线镰刀菌产生的次级代谢产物，被IARC归类为3类致癌物，是人类乳腺肿瘤发生的潜在刺激因子，还具有肝毒性、血液毒性和免疫毒性。土壤中重金属离子如Pb(II)、Cd(II)、Hg(II)的污染已经引起了全世界的关注，Cd(II)的蓄积会引起人体多种急性和慢性疾病，可导致DNA损坏和酶活性受到干扰，最终导致心脏、肺、肝脏和肾脏衰竭。
目前真菌毒素的检测方法包括仪器法、化学分析法和免疫分析法等，重金属的检测方法包括仪器法、免疫分析法等，未见报道同时检测两类物质的相关方法。两步检测需要对真菌毒素和重金属分别进行提取，然后分别进行检测，操作繁琐，对操作人员专业技能要求较高，无法实现现场快速定量检测。
目前，免疫色谱法在单一样品检测方面已经很成熟，但不能实现同时检测多种分析物。如果需要检测多种分析物则需要更多的试纸条，这样会增加检测成本和时间，极大地限制了大批量样本的现场筛选效率。已有研究实现在一个试纸条上检测两种或多种目标物质，但条带之间会产生相互干扰，易造成结果误判，灵敏度也有所降低，导致较低浓度的目标物无法检出。现有的多联免疫层析试纸条大多只能检测2-3种分析物，例如中国专利CN 113552359A，且分析物种类越多对检测效果的影响越大，难以实现对五种及以上物质的检测，也不符合实际样品中含有污染种类的实际情况。因此，建立一种更加灵敏、快速、可实现大通量、同时检测谷物中多种真菌毒素和重金属的方法成为了当务之急。
发明内容
[技术问题]
常规的检测方法都是单独检测谷物中的重金属、真菌毒素和有机磷农药残留，工序繁琐；用于试纸条同时检测，需要分多次操作，操作步骤繁琐。
单型试纸条无法满足同时、快速、低成本检测两种或多种物质；同时已有的检测多种毒素或多种重金属的免疫色谱法只能实现定性和半定量检测，或者检测灵敏度低。
[技术方案]
为了解决上述至少一个问题，本发明采用荧光微球替代传统胶体金，用荧光微球标记AFB1、FB1、OTA、ZEN及Cd-EDTA的抗体复合物，采用喷涂微量检测点形成阵列检测的方法，利用竞争免疫法，将其作为荧光探针用于免疫层析，通过通过摄像工具拍取照片，Image J软件对图片进行RGB分析，得到检测线上不同亮度的点阵条带荧光条带荧光值，可以对样品中的AFB1、FB1、OTA、ZEN及Cd进行同时快速定量分析。
本发明的第一个目的是提供一种制备同时检测AFB1、FB1、OTA、ZEN及Cd的五联荧光免疫定量试纸条的方法，包括如下步骤：
将AFB1、FB1、OTA、ZEN和Cd的完全抗原溶液分别喷涂到硝酸纤维素膜(NC膜)上作为5条检测 线T1～T5，将羊抗鼠二抗溶液涂到硝酸纤维素膜(NC膜)上作为质控线C，之后将样品垫、含有检测线和质控线的硝酸纤维素膜、吸水纸依次粘贴在PVC底板上组装成试纸条。
在本发明的一种实施方式中，所述AFB1完全抗原溶液、FB1完全抗原溶液、OTA完全抗原溶液和ZEN完全抗原溶液的终浓度为0.01-1.0mg/mL，所述Cd完全抗原溶液的终浓度为0.1-1.0mg/mL。
在本发明的一种实施方式中，所述羊抗鼠二抗溶液的终浓度为0.05-1mg/mL。
在本发明的一种实施方式中，将0.01-1.0mg/mL AFB1完全抗原溶液涂到硝酸纤维素膜(NC膜)上作为检测线T1，将0.01-1.0mg/mL FB1完全抗原溶液涂到硝酸纤维素膜(NC膜)上作为检测线T2，将0.01-1.0mg/mL OTA完全抗原溶液涂到硝酸纤维素膜(NC膜)上作为检测线T3，将0.01-1.0mg/mL ZEN完全抗原溶液涂到硝酸纤维素膜(NC膜)上作为检测线T4，将0.1-1.0mg/mL Cd完全抗原溶液涂到硝酸纤维素膜(NC膜)上作为检测线T5，将0.05-1mg/mL羊抗鼠二抗溶液涂到硝酸纤维素膜(NC膜)上作为质控线C。
在本发明的一种实施方式中，所述检测线T1、T2、T3、T4、T5的间距均为1-3mm，检测线T1和质控线C的距离为1-3mm，检测线T2-T5和质控线C在检测线T1的两边。
在本发明的一种实施方式中，所述五联荧光免疫定量试纸条中，样品垫和含有检测线、质控线的硝酸纤维素膜的一端部分相互重叠，重叠区域的长度为2-4mm；吸水纸和含有检测线、质控线的硝酸纤维素膜的另一端部分相互重叠，重叠区域的长度为2-4mm；所述吸水纸、样品垫与含有检测线、质控线的硝酸纤维素膜相互重叠的部分位于含有检测线、质控线的硝酸纤维素膜的上方。
在本发明的一种实施方式中，检测线T5距样品垫的距离为4-6mm，质控线C距吸水纸的距离为4-6mm。
在本发明的一种实施方式中，所述的检测线T1、T2、T3、T4、T5和质控线C的宽度为1.5-2.5mm。
在本发明的一种实施方式中，AFB1完全抗原溶液、FB1完全抗原溶液、OTA完全抗原溶液、ZEN完全抗原溶液、Cd完全抗原溶液及羊抗鼠二抗溶液的喷涂方式为微量检测点形成阵列检测的方式。
在本发明的一种实施方式中，每厘米的硝酸纤维素膜上检测线宽度AFB1完全抗原溶液、FB1完全抗原溶液、OTA完全抗原溶液、ZEN完全抗原溶液及Cd完全抗原溶液的喷涂量分别为10-40nL；当硝酸纤维素膜宽度为0.4cm时，喷涂量分别为5-30nL。
在本发明的一种实施方式中，每厘米的硝酸纤维素膜上质控线宽度喷涂量为1-3μL/cm；当硝酸纤维素膜宽度为0.4cm时，喷涂量为0.4-1.2μL。
在本发明的一种实施方式中，所述的干燥是将涂好的硝酸纤维素膜置于37℃真空干燥箱中烘干备用。
在本发明的一种实施方式中，所述AFB1、FB1、OTA、ZEN、Cd和羊抗鼠二抗的完全抗原溶液用0.02mol/L pH 7.0的磷酸缓冲液(PBS)稀释。
本发明的第二个目的是本发明所述的方法制备得到的同时检测AFB1、FB1、OTA、ZEN和Cd的五联荧光免疫定量试纸条。
在本发明的一种实施方式中，所述AFB1的检测浓度为0.05-10ng/mL，FB1的检测浓度为1-12ng/mL，OTA的检测浓度为0.5-60ng/mL，ZEN的检测浓度为0.1-40ng/mL，Cd的检测浓度为1-40ng/mL。
在本发明的一种实施方式中，所述五联荧光免疫定量试纸条的宽度为3-5mm。
本发明的第三个目的是提供一种同时检测谷物中AFB1、FB1、OTA和ZEN和重金属Cd的方法，所述方法包括如下步骤：
(1)提取谷物中AFB1、FB1、OTA和ZEN和重金属Cd，得到待测溶液；
(2)将步骤(1)的待测溶液、PBS缓冲液、经Eu 3+-荧光微球标记的AFB1的单克隆抗体、经Eu 3+-荧光微球标记的FB1的单克隆抗体、经Eu 3+-荧光微球标记的OTA的单克隆抗体、经Eu 3+-荧光微球标记的ZEN的单克隆抗体、Eu 3+-荧光微球标记的Cd的单克隆抗体混合均匀，得到预处理之后的待测溶液；
(3)将步骤(2)的待测溶液加入本发明所述的五联荧光免疫定量试纸条的样品垫上进行层析，之后通过摄像工具拍取照片，Image J软件对图片进行RGB分析，得到样品在检测线T1、T2、T3、T4、T5和质控线C处相应的荧光强度值C值、AFB1的T1值、FB1的T2值、OTA的T3值、ZEN的T4值、Cd的T5值；
(4)将得到的C值、T1值、T2值、T3值、T4值、T5值代入AFB1、FB1、OTA、ZEN和Cd的标准曲线，得到待测样品中AFB1、FB1、OTA、ZEN和Cd的浓度。
在本发明的一种实施方式中，步骤(1)所述提取具体包括如下步骤：
将谷物粉碎过筛，得到粉碎后的谷物；按照甲醇和硝酸溶液体积比为85:15，配制得到提取液；之后在粉碎后的谷物中添加提取液进行提取、离心，得到含有AFB1、FB1、OTA和ZEN和Cd的溶液；其中过筛是过80目筛，硝酸溶液的浓度为1.2mol/L，粉碎后的谷物和提取液的比例以mL/g计为3:1，提取是在25℃下超声提取25min，离心是在4000rpm离心5min。
在本发明的一种实施方式中，步骤(2)所述待测溶液、Eu 3+-荧光微球标记的AFB1的单克隆抗体、Eu 3+-荧光微球标记的FB1的单克隆抗体、Eu 3+-荧光微球标记的OTA的单克隆抗体、Eu 3+-荧光微球标记的ZEN的单克隆抗体、Eu 3+-荧光微球标记的Cd的单克隆抗体和PBS缓冲液的体积比为(3-5):(3-5):(3-5):(3-5):(3-5):(5-10):(85-95)，进一步优选为5:3:3:3:3:5:87。
在本发明的一种实施方式中，步骤(2)所述混合均匀是在20-37℃孵育10-20min，使其充分竞争反应。
在本发明的一种实施方式中，步骤(3)所述预处理的待测溶液的用量为30-50μL/cm 2。
在本发明的一种实施方式中，步骤(3)所述摄像工具包括手机和相机。
在本发明的一种实施方式中，步骤(4)所述标准曲线的构建方法包括如下步骤；
用阴性谷物样品配制成AFB1/FB1/OTA/ZEN/Cd-EDTA浓度分别为0.05/1/0.5/0.1/1ng/mL、0.25/2/1/0.5/5ng/mL、0.5/4/2/1/10ng/mL、1/6/4/5/15ng/mL、2/8/8/10/20ng/mL、5/10/30/20/30ng/mL和10/12/60/40/40ng/mL的标准溶液用于五联荧光免疫定量试纸条检测，其中标准品释液为含5％甲醇(v/v)的0.01M pH 7.0的PBS溶液；
将Eu 3+-荧光微球标记的AFB1单克隆抗体(AFB1荧光探针)、FB1单克隆抗体(FB1荧光探针)、OTA单克隆抗体(OTA荧光探针)、ZEN单克隆抗体(ZEN荧光探针)、重金属Cd单克隆抗体(Cd-EDTA荧光探针)作为荧光探针，将1μL AFB1荧光探针、2μL FB1荧光探针、1μL OTA荧光探针、1μL ZEN荧光探针、0.5μL Cd-EDTA荧光探针、89.5μL的PBS缓冲液和5μL标准溶液均匀后室温孵育10min，缓慢滴入五联荧光免疫定量试纸条的样品垫，加样量为50μL/cm 2，37℃层析15min后，用通过摄像工具拍取照片，Image J软件对图片进行RGB分析，记录五联荧光免疫定量试纸条的检测线T1、T2、T3、T4、T5荧光值；每个浓度测定六个平行，设定浓度为0ppb标准液的T线荧光值为T0，其他加标浓度的检测线T1、T2、T3、T4、T5荧光值为T1、T2、T3、T4、T5，以各个AFB1标准品浓度的对数值为横坐标，T1/T0×100(％)为纵坐标绘制标准曲线，竞争抑制率设为(1-T1/T0)×100％；以各个FB1标准品浓度的对数值为横坐标，T2/T0×100(％)为纵坐标绘制标准曲线，竞争抑制率设为(1-T2/T0)×100％；以各个OTA标准品浓度的对数值为横坐标，T3/T0×100(％)为纵坐标绘制标准曲线，竞争抑制率设为(1-T3/T0)×100％；以各个ZEN标准品浓度的对数值为横坐标，T4/T0×100(％)为纵坐标绘制标准曲线，竞争抑制率设为(1-T4/T0)×100％；以各个Cd标准品浓度的对数值为横坐标，T5/T0×100(％)为纵坐标绘制标准曲线，竞争抑制率设为(1-T5/T0)×100％。
在本发明的一种实施方式中，步骤(4)所述标准曲线具体为：当AFB1的浓度为0.05-10ng/mL时，AFB1浓度的对数与T/T0成线性关系，线性方程为Y＝-21.718Logx+47.003，x为AFB1浓度，Y为T1/T0，R 2＝0.9896，检测限可达到0.304ng/mL；当FB1的浓度为1-60ng/mL时，FB1浓度的对数与T/T0成线性关系，线性方程为Y＝-36.733Logx+79.041，x为FB1浓度，Y为T2/T0，R 2＝0.9774，检测限可达到0.417ng/mL；当OTA的浓度为0.5-10ng/mL时，OTA浓度的对数与T/T0成线性关系，线性方程为Y＝-30.692Logx+51.728，x为OTA浓度，Y为T3/T0，R 2＝0.9645，检测限可达到0.308ng/mL；当ZEN的浓度为0.1-50ng/mL时，ZEN浓度的对数与T/T0成线性关系，线性方程为Y＝-62.827Logx+86.945，x为ZEN浓度，Y为T4/T0，R 2＝0.9972，检测限可达到0.370ng/mL；当Cd-EDTA的浓度为1-40ng/mL时，Cd-EDTA浓度的对数与T/T0成线性关系，线性方程为Y＝-26.412Logx+67.024，x为Cd浓度，Y为T5/T0，R 2＝0.9966，检测限可达到0.401ng/mL。
在本发明的一种实施方式中，步骤(1)的待测溶液中AFB1的浓度范围为0-30ng/mL、FB1的浓度范围为0-60ng/mL、OTA的浓度范围为0-10ng/mL、ZEN的浓度范围为0-50ng/mL、Cd的浓度范围为0-40ng/mL，如果浓度过高，超出试纸条的线性检测范围，则检测出来的结果无使用价值，需要以PBS缓冲液为溶剂进行稀释；浓度太低，低于国标，也就不需要进行检测了。
本发明的第四个目的是本发明所述的五联荧光免疫定量试纸条在食品检测领域的应用。
本发明的第五个目的是本发明所述的一种同时检测谷物中AFB1、FB1、OTA、ZEN和重金属Cd的方法在食品检测领域的应用。
[有益效果]
(1)本发明所述的从取谷物中一步提取多种真菌毒素(AFB1、FB1、OTA、ZEN)和重金属Cd的方法，时间短(1h以内)，回收率高(90-110％)，操作简单，条件温和，为同时检测多种真菌毒素(AFB1、FB1、OTA、ZEN)和Cd、简化操作流程提供了技术支持。
(2)本发明所述的方法能够实现快速定量同时检测谷物样品中真菌毒素(AFB1、FB1、OTA、ZEN)和重金属Cd的含量，特异性强、灵敏度高，其中当AFB1的浓度为0.05-10ng/mL时，其浓度的对数值与T2/T0成线性关系，线性方程为Y＝-21.718Logx+47.003，R 2＝0.9896，检测限可达到0.304ng/mL；当FB1的浓度为1-60ng/mL时，FB1浓度的对数与T/T0成线性关系，线性方程为Y＝-36.733Logx+79.041，x为FB1浓度，Y为T2/T0，R 2＝0.9774，检测限可达到0.417ng/mL；当OTA的浓度为0.5-10ng/mL时，OTA浓度的对数与T/T0成线性关系，线性方程为Y＝-30.692Logx+51.728，x为OTA浓度，Y为T3/T0，R 2＝0.9645，检测限可达到0.308ng/mL；当ZEN的浓度为0.1-50ng/mL时，ZEN浓度的对数与T/T0成线性关系，线性方程为Y＝-62.827Logx+86.945，x为ZEN浓度，Y为T4/T0，R 2＝0.9972，检测限可达到0.370ng/mL；当Cd-EDTA的浓度为1-40ng/mL时，Cd-EDTA浓度的对数与T/T0成线性关系，线性方程为Y＝-26.412Logx+67.024，x为Cd浓度，Y为T5/T0，R 2＝0.9966，检测限可达到0.401ng/mL。
(3)本发明所述的方法以AFB1、FB1、OTA、ZEN、Cd单克隆抗体作为识别靶点，以时间分辨荧光微球为信号源，荧光微球的体积远大于荧光染料分子的体积，不但可以有效消除非特异性荧光干扰，粒径均一，如图1所示，表面功能基团稳定可控，实验重复性较好，检测特异性强，可实现快速定量检测，且灵敏度高、误差小，为即时检测提供了极大的便捷。
(4)本发明的五联荧光免疫定量试纸条具有灵敏度高、成本低廉、操作简便、快速定量检测、稳定性好、适合工商部门、第三方检测机构、各级政府监管部门、乳制品企业等使用，市场前景广阔，易推广使用。
附图说明
图1：Eu 3+-荧光微球透射电镜图。
图2：多种真菌毒素和重金属Cd的五联荧光免疫定量试纸条结构图。
图3：标记荧光微球免疫层析法检测AFB1的标准曲线(A)、FB1的标准曲线(B)、OTA的标准曲线(C)、ZEN的标准曲线(D)、重金属Cd的标准曲线(E)及紫外灯下的五联荧光免疫定量试纸条图(F)。
图4：AFB1免疫层析法的检测线(T1线)完全抗原浓度的优化(A)、FB1免疫层析法的检测线(T2线)完全抗原浓度的优化(B)、OTA免疫层析法的检测线(T3线)完全抗原浓度的优化(C)、ZEN免疫层析法的检测线(T3线)完全抗原浓度的优化(D)、Cd免疫层析法的检测线(T5线)完全抗原浓度的优化(E)。
图5：荧光探针上样量对检测线荧光强度的影响。
图6：荧光探针和待测溶液的孵育时间(A)或加入试纸条后的层析时间(B)对于荧光强度和T/T0的影响。
图7：AFB1、FB1、OTA、ZEN和Cd特异性测试结果。
具体实施方式
以下对本发明的优选实施例进行说明，应当理解实施例是为了更好地解释本发明，不用于限制本发明。
实施例中采用的AFB1单克隆抗体、FB1单克隆抗体、OTA单克隆抗体、ZEN单克隆抗体、Cd单克隆抗体和羊抗鼠二抗均购自北京开园科技有限公司。
实施例1 同时检测谷物中AFB1、FB1、OTA、ZEN和Cd的五联荧光免疫定量试纸条的制备
制备方法包括如下步骤：
利用微量检测点形成阵列检测的方式将完全抗原溶液喷涂到硝酸纤维素膜上(NC膜)，将0.1mg/mL AFB1完全抗原溶液(AFB1-OVA)喷涂到硝酸纤维素膜(NC膜)上作为检测线T1，将0.1mg/mLFB1完全抗原溶液(FB1-OVA)喷涂到硝酸纤维素膜(NC膜)上作为检测线T2，将0.1mg/mL OTA完全抗原溶液(OTA-OVA)喷涂到硝酸纤维素膜(NC膜)上作为检测线T3，将0.1mg/mLZEN完全抗原溶液(ZEN-OVA)喷涂到硝酸纤维素膜(NC膜)上作为检测线T4，将0.3mg/mL Cd完全抗原溶液(Cd-EDTA-OVA)涂到硝 酸纤维素膜(NC膜)上作为检测线T5，将0.5mg/mL羊抗鼠二抗溶液涂到硝酸纤维素膜(NC膜)上作为质控线C，其中检测线T1、T2、T3、T4、T5的间距均为1.2mm，检测线T1和质控线C的距离为1.2mm，检测线T2和质控线C在检测线T1的两边，检测线T1、T2、T3、T4、T5和质控线C的宽度取决于喷膜仪管路的直径为2mm，37℃干燥2h；
之后将样品垫、含有检测线、质控线的硝酸纤维素膜、吸水纸依次粘贴在PVC底板上组装成试纸条；样品垫叠在含有检测线、质控线的硝酸纤维素膜上，二者重叠3mm，类似地，吸水纸叠在含有检测线、质控线的硝酸纤维素膜上，二者重叠3mm；检测线T5距离样品垫的距离为5mm，质控线C距离吸水纸的距离为5mm；
最后，用切条机将粘贴好的板切成约4mm宽的试纸条，用塑料底座和卡壳组装，得到同时检测谷物中AFB1、FB1、OTA、ZEN和Cd的五联荧光免疫定量试纸条，4℃密封保存备用；结构图如图2所示，底板上从左到右依次为样品垫、硝酸纤维素膜和吸水纸。
实施例2 Eu 3+-荧光微球标记AFB1、FB1、OTA、ZEN和Cd单克隆抗体的荧光探针的制备
制备方法包括如下步骤：
活化缓冲液：pH 4.5-6.50.05M的2-(N-吗啡啉)乙磺酸(MES，C 6H 13NO 4S·H 2O)溶液；
偶联缓冲液：pH 7.0-8.00.01M的磷酸缓冲液(PBS)(避免使用存在游离胺的溶剂)；
活化剂：1mg/mL的1-(3-二甲氨基丙基)-3-乙基碳二亚胺硝酸盐(EDC，C 8H 17N 3·HCl)溶液以及1mg/mL的N-羟基琥珀酰亚胺(NHS，C 4H 5NO 3)溶液；
封闭缓冲液：含1％BSA、0.05％Tween-20的pH 6.0-7.00.01M的磷酸缓冲液(PBS)；
抗体保护液：含5％海藻糖、0.1％PVPK-30、0.5％BSA、0.1％TritonX-100的pH 6.0-7.00.05M的Tris-HCl；
标记洗涤液：含0.1％Tween-20的pH 6.0-7.00.05M的Tris-HCl；
微球复溶液/反应缓释液：含有5％蔗糖，1％牛血清白蛋白，1％Tween-20的pH 6.0-7.00.05M的Tris-HCl；
Eu 3+-荧光微球标记的AFB1单克隆抗体的制备方法，参考专利CN 110988339A，具体如下：
(1)取4℃放置的内部包裹Eu 3+、表面修饰有羧基官能团的荧光微球(购于百纳泰科新材料科技有限公司，粒径300nm)10μL(1％固形物含量)，超声分散，加入1000μL活化缓冲液，10000rpm于4℃离心15min；
(2)弃上清，加入800μL活化缓冲液，超声重悬，重复离心清洗3次；
(3)弃上清，加入200μL活化缓冲液超声重悬，加入1μL 1mg/mL EDC溶液、1μL 1mg/mL的NHS溶液作为活化剂，室温摇床500rpm避光振荡活化40min；
(4)活化完成后离心，弃上清，加入PBS缓冲液清洗3次；
(5)弃上清，加入400μL偶联缓冲液超声重悬，再加入0.5μgAFB1单克隆抗体，室温避光振荡标记2h；
(6)标记结束后加入10％(v/v)的封闭缓冲液和100μL抗体保护液，室温避光振荡40min；
(7)封闭后离心弃上清，用800-1000μL的标记洗涤液清洗2-3次；
(8)弃上清，加入400μL荧光微球复溶液，得到Eu 3+-荧光微球标记的AFB1单克隆抗体(Eu-AFB1-mAb)，4℃保存备用。
Eu 3+-荧光微球标记的FB1单克隆抗体(Eu-FB1-mAb)、Eu 3+-荧光微球标记的OTA单克隆抗体(Eu-OTA-mAb)、Eu 3+-荧光微球标记的ZEN单克隆抗体(Eu-ZEN-mAb)、Eu 3+-荧光微球标记的Cd单克隆抗体(Eu-Cd-EDTA-mAb)的制备方法是将Eu-AFB1-单克隆抗体制备方法中的AFB1单克隆抗体分别替换为FB1单克隆抗体、OTA单克隆抗体、ZEN单克隆抗体、Cd单克隆抗体，得到Eu 3+-荧光微球标记的FB1单克隆抗体、OTA单克隆抗体、ZEN单克隆抗体、Cd单克隆抗体，4℃保存备用。
实施例3 AFB1、FB1、OTA、ZEN和Cd的五联荧光免疫定量试纸条的标准曲线的构建
标准曲线的构建方法是：
用阴性谷物样品配制成AFB1/FB1/OTA/ZEN/Cd-EDTA浓度分别为0.05/1/0.5/0.1/1ng/mL、0.25/2/1/0.5/5ng/mL、0.5/4/2/1/10ng/mL、1/6/4/5/15ng/mL、2/8/8/10/20ng/mL、5/10/30/20/30ng/mL和10/12/60/40/40ng/mL的标准溶液用于五联荧光免疫定量试纸条检测，其中标准品释液为含5％甲醇(v/v)的0.01M p H 7.0的PBS溶液。
将实施例2制得的Eu 3+-荧光微球标记的AFB1单克隆抗体(AFB1荧光探针)、FB1单克隆抗体(FB1荧光探针)、OTA单克隆抗体(OTA荧光探针)、ZEN单克隆抗体(ZEN荧光探针)、重金属Cd单克隆抗体(Cd-EDTA荧光探针)作为荧光探针，将1μL AFB1荧光探针、2μL FB1荧光探针、1μL OTA荧光探针、1μL ZEN荧光探针、0.5μL Cd-EDTA荧光探针、89.5μL的PBS缓冲液和5μL不同浓度的标准溶液均匀后室温孵育10min，缓慢滴入实施例1的五联荧光免疫定量试纸条的样品垫，加样量为50μL/cm 2，37℃层析15min后，用手机拍照，Image J软件对图片进行RGB分析，记录五联荧光免疫定量试纸条的检测线T1、T2、T3、T4、T5荧光值；每个浓度测定六个平行，设定浓度为0ppb标准液的T线荧光值为T0，其他加标浓度的检测线T1、T2、T3、T4、T5荧光值为T1、T2、T3、T4、T5，以各个AFB1标准品浓度的对数值为横坐标，T1/T0×100(％)为纵坐标绘制标准曲线，竞争抑制率设为(1-T1/T0)×100％；以各个FB1标准品浓度的对数值为横坐标，T2/T0×100(％)为纵坐标绘制标准曲线，竞争抑制率设为(1-T2/T0)×100％；以各个OTA标准品浓度的对数值为横坐标，T3/T0×100(％)为纵坐标绘制标准曲线，竞争抑制率设为(1-T3/T0)×100％；以各个ZEN标准品浓度的对数值为横坐标，T4/T0×100(％)为纵坐标绘制标准曲线，竞争抑制率设为(1-T4/T0)×100％；以各个Cd标准品浓度的对数值为横坐标，T5/T0×100(％)为纵坐标绘制标准曲线，竞争抑制率设为(1-T5/T0)×100％。
如图3可以看出，随着AFB1、FB1、OTA、ZEN、Cd浓度的增大，试纸条T线条带上的荧光会越来越浅，所以T1/T0、T2/T0、T3/T0、T4/T0、T5/T0会越来越小，当AFB1的浓度为0.05-10ng/mL时，AFB1浓度的对数与T/T0成线性关系，线性方程为Y＝-21.718Logx+47.003，x为AFB1浓度，Y为T1/T0，R 2＝0.9896，检测限可达到0.304ng/mL；当FB1的浓度为1-60ng/mL时，FB1浓度的对数与T/T0成线性关系，线性方程为Y＝-36.733Logx+79.041，x为FB1浓度，Y为T2/T0，R 2＝0.9774，检测限可达到0.417ng/mL；当OTA的浓度为0.5-10ng/mL时，OTA浓度的对数与T/T0成线性关系，线性方程为Y＝-30.692Logx+51.728，x为OTA浓度，Y为T3/T0，R 2＝0.9645，检测限可达到0.308ng/mL；当ZEN的浓度为0.1-50ng/mL时，ZEN浓度的对数与T/T0成线性关系，线性方程为Y＝-62.827Logx+86.945，x为ZEN浓度，Y为T4/T0，R 2＝0.9972，检测限可达到0.370ng/mL；当Cd-EDTA的浓度为1-40ng/mL时，Cd-EDTA浓度的对数与T/T0成线性关系，线性方程为Y＝-26.412Logx+67.024，x为Cd浓度，Y为T5/T0，R 2＝0.9966，检测限可达到0.401ng/mL。
实施例4 完全抗原溶液划膜方式比较
通过划线喷涂的方式将完全抗原溶液喷涂到硝酸纤维素膜上(NC膜)，其余步骤参考实施例1。
完全抗原溶液直接划线喷涂试纸条标准曲线的构建方法参考实施例3。
如表1所示，采用直接划线喷涂完全抗原溶液时，AFB1的检测限为0.524ng/mL，FB1的检测限为0.498ng/mL，OTA的检测限为0.619ng/mL，ZEN的检测限为0.549ng/mL，Cd的检测限为0.536ng/mL，均高于点阵试纸条的检测限。
表1 不同划膜方式试纸条的检测限(ng/Ml)比较结果
划膜方式 AFB1 FB1 OTA ZEN Cd
直接喷涂 0.524 0.498 0.619 0.549 0.536
点阵喷涂 0.304 0.417 0.308 0.370 0.401
实施例5 检测方法的初步建立
一种同时检测谷物中真菌毒素(AFB1、FB1、OTA、ZEN)和重金属Cd的方法，所述方法包括如下步骤：
(1)提取谷物中真菌毒素(AFB1、FB1、OTA、ZEN)和重金属Cd：
将阴性样本大米粉碎过80目筛，得到粉碎后的大米；取1.2mL浓硝酸(14.4mol/L)加入到13.2mL的超纯水中，充分震荡，制成1.2mol/L的硝酸溶液；之后按照体积比甲醇：硝酸溶液为85:15，配制得到提取液；将1g粉碎后的大米、500ppb Cd(镉标品)、200ppb的AFB1、200ppb的FB1、200ppb的OTA、200ppb的ZEN(真菌毒素均用甲醇溶解)混合均匀，之后与1mL提取液混合后加入到固相萃取柱中，在25℃下超声提取25min，提取结束后4000rpm离心5min去除不溶性物质，得到含有AFB1、FB1、OTA、ZEN 和Cd的待测溶液；
(2)将步骤(1)得到的待测溶液、经Eu 3+-荧光微球标记的AFB1的单克隆抗体、Eu 3+-荧光微球标记的FB1的单克隆抗体、Eu 3+-荧光微球标记的OTA的单克隆抗体、Eu 3+-荧光微球标记的ZEN的单克隆抗体、Eu 3+-荧光微球标记的Cd的单克隆抗体和PBS缓冲液按照体积比为5:3:3:3:3:5:87，在37℃孵育15min，得到预处理之后的待测溶液；
(3)将步骤(2)得到的待测溶液加入实施例1的五联荧光免疫定量试纸条的样品垫上，37℃层析15min，加样量为50μL/cm 2；之后利用用手机拍照，Image J软件对图片进行RGB分析，得到样品在检测线T1、T2、T3、T4、T5和质控线C处相应的荧光强度值：AFB1的T1值、FB1的T2值、OTA的T3值、ZEN的T4值、Cd的T5值和C值；
(4)将得到的T1值、T2值、T3值、T4值、T5值和C值代入实施例3的AFB1、FB1、OTA、ZEN和Cd的标准曲线，得到待测样品中AFB1、FB1、OTA、ZEN和Cd的浓度。
实施例6 条件优化
(1)AFB1/FB1/OTA/ZEN/Cd完全抗原的用量的影响
具体的实验探究过程如下：
为了制备五联点阵免疫层析试纸条，本实验将AFB1-OVA、FB1-OVA、OTA-OVA、ZEN-OVA和Cd-EDTA-OVA的浓度分别稀释成0.1mg/mL、0.3mg/mL、0.5mg/mL、0.7mg/mL和0.9mg/mL。利用Biodot的阵列喷点仪喷涂到NC膜上，通过阴性对照组和阳性实验组(阳性样本：AFB1为0.05ng/mL、FB1为1ng/mL、OTA为0.25ng/mL、ZEN为2ng/mL和Cd-EDTA为0.1ng/mL)进行分析，选择紫外灯下观察各个条带的亮度，然后选择抑制率最好的抗原浓度作为最佳的喷点浓度。
评价不同浓度的AFB1-OVA、FB1-OVA、OTA-OVA、ZEN-OVA、Cd-EDTA-OVA对五联免疫定量试纸条检测结果的影响，检测方法参考实施例5。
如图4所示，当AFB1-OVA浓度为0.3mg/mL时，检测线上的荧光强度相对较强，且竞争抑制率最大，因此选择AFB1抗原浓度为0.3mg/mL作为检测线T1最佳喷涂浓度；当FB1-OVA浓度为0.3mg/mL时，检测线上的荧光强度相对较强，且竞争抑制率最大，因此选择FB1抗原浓度为0.3mg/mL作为检测线T2最佳喷涂浓度；OTA-OVA浓度为0.5mg/mL时，检测线上的荧光强度相对较强，且竞争抑制率最大，因此选择OTA抗原浓度为0.5mg/mL作为检测线T3最佳喷涂浓度；当ZEN-OVA浓度为0.5mg/mL时，检测线上的荧光强度相对较强，且竞争抑制率最大，因此选择ZEN抗原浓度为0.5mg/mL作为检测线T4最佳喷涂浓度；当Cd-EDTA-OVA浓度为0.3mg/mL时，检测线上的荧光强度相对较强，且竞争抑制率最大，因此选择Cd-EDTA抗原浓度为0.3mg/mL作为检测线T5最佳喷涂浓度。
(2)荧光探针上样量的优化
为了探究探针的用量对五联点阵免疫层析试纸条的各点荧光强度的影响，对Eu-AFB1-mAb、Eu-FB1-mAb、Eu-OTA-mAb、Eu-ZEN-mAb和Eu-Cd-EDTA-mAb五种探针进行上样优化，首先取0.1μL、0.5μL、1μL、1.5μL和2μL的探针采用五联点阵免疫层析试纸条进行探针上样量的优化。
评价不同浓度的AFB1-OVA、FB1-OVA、OTA-OVA、ZEN-OVA、Cd-EDTA-OVA对五联免疫定量试纸条检测结果的影响，方法参考实施例5。
得到Eu-AFB1-mAb最佳上样量后，在此基础上依次进行Eu-FB1-mAb、Eu-OTA-mAb、Eu-ZEN-mAb和Eu-Cd-EDTA-mAb的上样量优化，各种探针的优化取用量均为0.1μL、0.5μL、1μL、1.5μL和2μL，以上所有的检测结果均采用手机拍照，Image J通过RGB进行分析所得。
荧光探针的上样量太少，导致试纸条上的检测线荧光强度太弱，灵敏度低，荧光探针的上样量太多容易导致荧光强度太强，容易产生背景噪音。如图5所示，通过比较检测线T1、T2、T3、T4、T5的荧光强度，得到Eu-AFB1-mAb、Eu-FB1-mAb、Eu-OTA-mAb、Eu-ZEN-mAb和Eu-Cd-EDTA-mAb最佳上样量分别为1μL、2μL、1μL、1μL和0.5μL。
(3)免疫时间对检测结果的影响
将1μL Eu-AFB1-mAb、2μL Eu-FB1-mAb、1μL Eu-OTA-mAb、1μL Eu-ZEN-mAb、0.5μL Eu-Cd-EDTA-mAb、5μL加标样品溶液(AFB1＝0.05μg/L、FB1＝0.05μg/L、OTA＝0.05μg/L、ZEN＝0.05μg/L、Cd＝0.5μg/L混合溶液)和89.5μLPBS缓冲液的混合，37℃条件下分别孵育0min、5min、10min、15min和20min后，将5组混合溶液分别滴加到五联免疫定量试纸条的样品垫，加样量为50μL/cm 2，37℃层析15min，用手机拍照，Image J软件通过RGB进行分析，记录T线荧光强度。
将1μL Eu-AFB1-mAb、2μL Eu-FB1-mAb、1μL Eu-OTA-mAb、1μL Eu-ZEN-mAb、0.5μL Eu-Cd-EDTA-mAb、5μL加标样品溶液(AFB1＝0.05μg/L、FB1＝0.05μg/L、OTA＝0.05μg/L、ZEN＝0.05μg/L、Cd＝0.5μg/L混合溶液)和89.5μLPBS缓冲液的混合，37℃孵育10min，缓慢滴入五联免疫定量试纸条的样品垫，加样量为50μL/cm 2，37℃层析，用手机拍照，Image J软件通过RGB进行分析，记录每分钟的T线荧光强度变化。
根据抗原抗体的不同的预孵育时间下试纸条各条检测线上的荧光强度来确定最佳的预孵育时间，从图6(A)中可以看到没有经过预孵育的对照组的T/T0的比值最高，而随着时间的不算增加，T/T0的数值不断下降，当预孵育时间为10min时，比值基本趋于平衡不在发生变化，说明10min之后抗原抗体结合反应已经全部完成，因此选择10min作为样品加入试纸条免疫层析之前的预反应时间。
以免疫反应的时间为横坐标，以检测线的荧光强度为纵坐标，绘制免疫动力学曲线以确定最佳的反应时间。通过分析图发(B)现，在0-8min时，检测线的荧光强度快速增加，8-15min时检测线的荧光强度缓慢增加，超过15min检测线的荧光强度趋于平衡，不再增加，因此层析时间选择18min较为合适。
实施例7 抗体亲和力测定
抗体亲和力用于测定抗原与抗体之间特异性结合的能力，由结合簇与决定簇的结合强度决定，其本质是一种非共价作用力，包括对氨基酸之间的吸引力、氢键、疏水性作用力等，体现了一个抗体分子和一个半抗原分子或抗原分子的一个决定簇起反应的能力。通过间接非竞争ELISA方法鉴定单克隆抗体亲和力。其原理主要是将包被原固定在96孔板底部，加入待测物和单抗，板底部的包被原与抗体结合，多余的抗体会被洗掉，截留在板底部的抗原-抗体复合物与酶标二抗相结合，加入显色液和终止液。具体操作步骤如下：
(1)包被：将浓度为1mg/mL的AFB1-OVA，3倍梯度稀释即1mg/mL、0.33mg/mL、0.11mg/mL稀释三个浓度，每孔加入100μL，37℃恒温孵育2h；
(2)洗涤：将微孔中的包被抗原的溶液甩出，拍干，加入300μL的PBST洗涤液，轻晃洗涤，甩干，重复三次；
(3)封闭：每孔加入300μL 3％的实施例5步骤(1)得到的含有AFB1、FB1、OTA、ZEN及Cd的待测溶液，放在37℃恒温避光封闭1h；；
(4)加抗体：按照步骤(2)进行洗涤后，将1mg/mL的AFB1单克隆抗体稀释成1mg/mL、0.33mg/mL、0.11mg/mL、0.037mg/mL、0.012mg/mL、0.004mg/mL、0.001mg/mL、0.0003mg/mL 8个浓度，每孔加入100μL，放在37℃下恒温孵育1h；
(5)加入酶标二抗：按照步骤(2)洗涤三次并拍干，每孔加入100μL酶标二抗(用5mL 0.01M PBS稀释1μL的辣根过氧化物酶-羊抗鼠二抗)；
(6)显色：甩干孔内液体，按照步骤(2)洗板三次拍干，每孔内加入100μL的显色液(显色A液，准确称取13.6g醋酸钠、1.6g柠檬酸，超声溶解后加入0.3mL 30％的双氧水，超纯水定容至500mL，4℃储存备用；显色B液，称取柠檬酸0.95g，乙二胺四乙酸0.2g，量取50mL甘油，称取3,3',5,5'-四甲基联苯胺0.15g，用3mL的二甲基亚砜溶解，定容至500mL，4℃避光保存；将A液和B液1:1等体积混合)，37℃条件下避光反应15min；
(7)终止：每孔加入50μL终止液(2mol/L H 2SO 4)，立即检测；
(8)读数：用多功能酶标仪读取不同包被浓度下不同浓度AFB1单克隆抗体在450nm条件下的吸光度值。
以单克隆抗体浓度(mol/L)的对数值为横坐标，吸收光度值OD 450nm为纵坐标，拟合出“S”曲线，找出每条曲线的最大OD 450nm，设为OD max，然后通过拟合方程计算出OD max/2所对应的抗体摩尔浓度，将所得到的3个不同浓度的包被原两两一组共组成3组，根据下式(1)分别计算出3个单克隆抗体亲和力常数K a，3个亲和力常数的平均值即为抗体的亲和力常数。
Figure PCTCN2022141458-appb-000001
其中，[Ab’] t、[Ab] t分别为两个不同浓度的抗原所对应的在OD max/2时的抗体的摩尔浓度，且，[Ab’] t为较低的包被浓度、[Ab] t为较高的包被浓度；n为两个不同浓度的比值(n>1)，K a为单克隆抗体的亲和力常 数。
FB1单克隆抗体、OTA单克隆抗体、ZEN单克隆抗体、Cd-EDTA单克隆抗体的亲和力测定参照AFB1单克隆抗体的操作步骤。
抗体与抗原之间的亲和力是影响试纸条检测灵敏度的重要参数。常数K a的值一般用来表示亲和力的大小，如果K a<10 7L/mol，那么抗体亲和力较低，特异性较差；如果K a>10 7L/mol，那么抗体亲和力较高，特异性较好。
表2-表6为AFB1、FB1、OTA、ZEN、Cd-EDTA单克隆抗体亲和力实验结果。从表2-表6可以看出：AFB1、FB1、OTA、ZEN和Cd-EDTA的K a的平均值分别为1.842×10 9L/mol、2.041×10 9L/mol、2.395×10 9L/mol、2.082×10 9L/mol和7.020×10 8L/mol，说明AFB1、FB1、OTA、ZEN和Cd-EDTA单克隆抗体均是高亲和力抗体，可用于本研究的特异性检测需求。
表2 AFB1抗体亲和力常数结果
Figure PCTCN2022141458-appb-000002
表3 FB1抗体亲和力常数结果
Figure PCTCN2022141458-appb-000003
表4 OTA抗体亲和力常数结果
Figure PCTCN2022141458-appb-000004
表5 ZEN抗体亲和力常数结果
Figure PCTCN2022141458-appb-000005
表6 Cd-EDTA抗体亲和力常数结果
Figure PCTCN2022141458-appb-000006
实施例8 五联免疫定量试纸条的性能测试
(1)精密度试验
采用同一批次的试纸条分别检测阳性样本(AFB1为0.05ng/mL、FB1为1ng/mL、OTA为0.25ng/mL、 ZEN为2ng/mL和Cd-EDTA为0.1ng/mL)和阴性样本对试纸条的批内差异进行分析；通过采用不同批次的试纸条分别检测阳性样本(AFB1为0.05ng/mL、FB1为1ng/mL、OTA为0.25ng/mL、ZEN为2ng/mL和Cd-EDTA为0.1ng/mL)和阴性样本对试纸条的批间差异进行分析。分析批间和批内差异的数据判断该试纸条的精密度和准确性。
实验操作同实施例5。
通过8个不同批次的五联免疫定量试纸条分别检测阴性对照组(5％甲醇-PBS)检测线处的荧光强度值(T0)和阳性试验组(AFB1＝0.05；FB1＝1ng/mL；OTA＝0.25ng/mL；ZEN＝2ng/mL Cd＝0.5μg/L)T线处的荧光强度值(T)分析批间差异。
由表7可知，通过变异系数计算公式分析得到T0、T和T/T0(％)的批内变异系数均小于8.97％，表明五联免疫定量试纸条的批内和批间变异系数小，精密度高，准确性好，基本符合定量检测试纸条的要求。
表7 T1、T2、T3、T4和T5检测线批间批内精密度实验
Figure PCTCN2022141458-appb-000007
Figure PCTCN2022141458-appb-000008
(2)准确度试验
为了进一步评估五联点阵免疫层析试纸条在实际样品中的检测效果，从超市购买大米、玉米和小麦样品，按照实施例5所述方法获得样品提取液，用于AFB1和Cd双联免疫层析试纸条检测。采用UPLC-MS/MS验证样品中是否含有有AFB1、FB1、OTA和ZEN，ICP-MS验证三种样品中是否含有Cd。
在阴性的大米、玉米和小麦样本中添加分别进行加标回收试验，每种样本的添加浓度均设置高、中、低三组不同的加标浓度，每组浓度梯度均设置三组平行试验。以加标回收率作为准确度评价指标，重复测定某一浓度样品的检测结果相对标准偏差(RSD％)作为精密度评价指标。加标回收率和相对标准偏差的计算公式如下式(2)、式(3)：
Figure PCTCN2022141458-appb-000009
Figure PCTCN2022141458-appb-000010
结果如表8所示，五联免疫定量试纸条在实际样品加标回收实验中，其回收率在68.95％-93.45％之间，变异系数小于9.876％，与UPLC-MS/MS法和ICP-MS法对比，结果一致性良好。
表8 五联免疫定量试纸条的准确度检测结果
Figure PCTCN2022141458-appb-000011
Figure PCTCN2022141458-appb-000012
Figure PCTCN2022141458-appb-000013
(3)交叉反应试验
为了验证真菌毒素和重金属的特异性，AFB1/FB1/OTA/ZEN/Cd-EDTA五种物质的结构类似物或同系物对试纸条进行特异性评价，用AFM1、AFG1、AFG2和AFB2对AFB1进行特异性检测，各类物质的终浓度为10ng/mL；用FB2和FB3对FB1进行特异性检测，各类物质的终浓度为10ng/mL；OTA/ZEN采用OTB、DON、T-2和上述几种常见的真菌毒素对其进行特异性分析，各类物质的终浓度为10ng/mL；Cd采用Pb、Hg、As进行重金属的特异性分析，终浓度为20ng/mL。
将1μL Eu-AFB1-mAb、2μL Eu-FB1-mAb、1μL Eu-OTA-mAb、1μL Eu-ZEN-mAb、0.5μL Eu-Cd-EDTA-mAb、89.5μL的PBS缓冲液和5μL上述16种单一溶液均匀后室温孵育10min，缓慢滴入五联荧光免疫定量试纸条的样品垫，加样量为50μL/cm 2，37℃层析15min后，用手机拍照，Image J软件对图片进行RGB分析，记录五联荧光免疫定量试纸条上T线的荧光强度。
如图7所示，浓度为10ng/mL的AFB1、FB1、OTA、ZEN和浓度为20ng/mL的Cd的时候对应T1、T2、T3、T4和T5的检测线消失，而在其他交叉物添加后荧光强度几乎没变化，没有明显的减弱，说明该试纸条的特异性良好性能稳定，符合市场需求，具有广阔的市场前景。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (12)

  1. 一种制备同时检测谷物中多种真菌毒素和重金属Cd的五联点阵免疫层析定量试纸条的方法，其特征在于，包括如下步骤：
    将AFB1、FB1、OTA、ZEN和Cd的完全抗原溶液分别喷涂到硝酸纤维素膜上作为检测线T1～T5，将羊抗鼠二抗溶液涂到硝酸纤维素膜上作为质控线C，之后将样品垫、含有检测线和质控线的硝酸纤维素膜、吸水纸依次粘贴在PVC底板上组装成试纸条。
  2. 根据权利要求1所述的方法，其特征在于，所述AFB1完全抗原溶液、FB1完全抗原溶液、OTA完全抗原溶液和ZEN完全抗原溶液的终浓度为0.01-1.0mg/mL，所述Cd完全抗原溶液的终浓度为0.1-1.0mg/mL；所述羊抗鼠二抗溶液的终浓度为0.05-1mg/mL。
  3. 根据权利要求1所述的方法，其特征在于，将AFB1完全抗原溶液涂到硝酸纤维素膜上作为检测线T1，将FB1完全抗原溶液涂到硝酸纤维素膜上作为检测线T2，将OTA完全抗原溶液涂到硝酸纤维素膜上作为检测线T3，ZEN完全抗原溶液涂到硝酸纤维素膜上作为检测线T4，将Cd完全抗原溶液涂到硝酸纤维素膜上作为检测线T5，将羊抗鼠二抗溶液涂到硝酸纤维素膜上作为质控线C。
  4. 根据权利要求3所述的方法，其特征在于，所述检测线T1、T2、T3、T4、T5的间距均为1-3mm，检测线T1和质控线C的距离为1-3mm，检测线T2-T5和质控线C在检测线T1的两边。
  5. 根据权利要求3所述的方法，其特征在于，检测线T5距样品垫的距离为4-6mm，质控线C距吸水纸的距离为4-6mm，所述的检测线T1、T2、T3、T4、T5和质控线C的宽度为1.5-2.5mm。
  6. 根据权利要求1所述的方法，其特征在于，所述AFB1完全抗原溶液、FB1完全抗原溶液、OTA完全抗原溶液、ZEN完全抗原溶液、Cd完全抗原溶液及羊抗鼠二抗溶液的喷涂方式为微量检测点形成阵列检测的方式。
  7. 根据权利要求6所述的方法，其特征在于，每厘米的硝酸纤维素膜上检测线宽度AFB1完全抗原溶液、FB1完全抗原溶液、OTA完全抗原溶液、ZEN完全抗原溶液及Cd完全抗原溶液的喷涂量分别为10-40n。
  8. 权利要求1-7任一项所述的方法制备得到的同时检测AFB1、FB1、OTA、ZEN和Cd的五联荧光免疫定量试纸条。
  9. 一种同时检测谷物中多种真菌毒素和重金属Cd的方法，其特征在于，所述方法包括如下步骤：
    (1)提取谷物中多种真菌毒素和重金属Cd，得到待测溶液；
    (2)将步骤(1)的待测溶液、PBS缓冲液、经Eu 3+-荧光微球标记的AFB1的单克隆抗体、经Eu 3+-荧光微球标记的FB1的单克隆抗体、经Eu 3+-荧光微球标记的OTA的单克隆抗体、经Eu 3+-荧光微球标记的ZEN的单克隆抗体、Eu 3+-荧光微球标记的Cd的单克隆抗体混合均匀，得到预处理之后的待测溶液；
    (3)将步骤(2)的的待测溶液加入权利要求4所述的五联荧光免疫定量试纸条的样品垫上进行层析，之后通过通过摄像工具拍取照片，Image J软件对图片进行RGB分析，得到样品在检测线T1、T2、T3、T4、T5和质控线C处相应的荧光强度值C值、AFB1的T1值、FB1的T2值、OTA的T3值、ZEN的T4值、Cd的T5值；
    (4)将得到的C值、T1值、T2值、T3值、T4值、T5值代入AFB1、FB1、OTA、ZEN、Cd的标准曲线，得到待测样品中AFB1、FB1、OTA、ZEN和Cd的浓度。
  10. 根据权利要求9所述的方法，其特征在于，步骤(1)所述提取具体包括如下步骤：
    将谷物粉碎过筛，得到粉碎后的谷物；按照甲醇和硝酸溶液体积比为85:15，配制得到提取液；之后在粉碎后的谷物中添加提取液进行提取、离心，得到含有AFB1、FB1、OTA、ZEN和Cd的溶液；其中过筛是过80目筛，硝酸溶液的浓度为1.2mol/L，粉碎后的谷物和提取液的比例以mL/g计为3:1，提取是在25℃下超声提取25min，离心是在4000rpm离心5min。
  11. 根据权利要求9所述的方法，其特征在于，步骤(2)所述待测溶液、Eu 3+-荧光微球标记的AFB1的单克隆抗体、Eu 3+-荧光微球标记的FB1的单克隆抗体、Eu 3+-荧光微球标记的OTA的单克隆抗体、Eu 3+-荧光微球标记的ZEN的单克隆抗体、Eu 3+-荧光微球标记的Cd的单克隆抗体和PBS缓冲液的体积比为(3-5):(3-5):(3-5):(3-5):(3-5):(5-10):(85-95)。
  12. 权利要求1～7任一项所述的方法或权利要求6所述的五联荧光免疫定量试纸条或权利要求8-11任一项所述的方法在食品检测领域的应用。
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