WO2023207166A1 - Five-linked fluorescence immunoassay quantitative test strip for simultaneously testing various mycotoxins and heavy metal cd in cereals - Google Patents

Abstract

A five-linked fluorescence immunoassay quantitative test strip for simultaneously testing various mycotoxins and heavy metal Cd in cereals. A preparation method for the five-linked fluorescence immunoassay quantitative test strip comprises the following steps: respectively coating 0.01-1.0 mg/mL of AFB1, FB1, OTA, and ZEN complete antigen solutions on a nitrocellulose (NC) membrane as test lines T1-T4, coating 0.1-1.0 mg/mL of a Cd complete antigen solution on the NC membrane as a test line T5, coating 0.05-1mg/mL of a goat anti-mouse secondary antibody solution on the NC membrane as a control line C, and drying; and then sequentially pasting a sample pad, the NC membrane containing the test lines and the control line, and absorbent paper on a PVC bottom plate to be assembled into the test strip. The five-linked fluorescence immunoassay quantitative test strip can be used for simultaneously testing various mycotoxins and heavy metal Cd in cereals on one test strip, and has the advantages of being high in sensitivity, low in cost, convenient to operate, rapid in quantitative test, and good in stability.

Description

A five-link fluorescent immunoquantitative test strip for simultaneous detection of multiple mycotoxins and heavy metal Cd in cereals Technical field

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.

Background technique

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. Seriously, the combined toxic effects of mycotoxins and heavy metals have great potential harm to the human body.

Aflatoxin (AFT) 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 (FBs) 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. It is classified by lARC It is a Class 2B carcinogen with nephrotoxicity, hepatotoxicity, embryotoxicity, teratogenicity, neurotoxicity, immunotoxicity, genotoxicity, carcinogenicity and intestinal toxicity. Zearalenone (ZEN) is mainly a secondary metabolite produced by Fusarium graminearum and Fusarium third line. It is classified as a Category 3 carcinogen by IARC and is a potential stimulator of human breast tumors. It also has Hepatotoxicity, hematological toxicity and immunotoxicity. The pollution of heavy metal ions such as Pb(II), Cd(II), and Hg(II) in soil has attracted worldwide attention. The accumulation of Cd(II) can cause a variety of acute and chronic diseases in the human body, and can lead to DNA damage and Enzyme activity is interfered with, ultimately leading to heart, lung, liver, and kidney failure.

At present, the detection methods of mycotoxins include instrumental methods, chemical analysis methods, immunoassay methods, etc., and the detection methods of heavy metals include instrumental methods, immunoassay methods, etc. There are no reports related to the simultaneous detection of two types of substances. The two-step detection requires the extraction of mycotoxins and heavy metals separately, and then detects them separately. The operation is cumbersome, requires high professional skills of the operators, and cannot achieve rapid quantitative detection on site.

At present, immunochromatography is very mature in the detection of single samples, but it cannot detect multiple analytes simultaneously. If multiple analytes need to be detected, more test strips will be needed, which will increase the cost and time of testing and greatly limit the efficiency of on-site screening of large batches of samples. There have been studies to detect two or more target substances on one test strip, but the strips will interfere with each other, which can easily lead to misjudgment of the results, and the sensitivity is also reduced, resulting in the inability to detect target substances with lower concentrations. . Most of the existing multi-linked immunochromatography test strips can only detect 2-3 analytes, such as Chinese patent CN 113552359A, and the more types of analytes, the greater the impact on the detection effect, making it difficult to detect five or more substances. The detection does not conform to the actual situation of the types of contamination contained in the actual samples. Therefore, it is urgent to establish a more sensitive, rapid, large-throughput, and simultaneous detection method for multiple mycotoxins and heavy metals in cereals.

Contents of the invention

[technical problem]

Conventional detection methods are to separately detect heavy metals, mycotoxins and organophosphorus pesticide residues in grains, and the procedures are cumbersome. For simultaneous detection with test strips, multiple operations are required, and the operation steps are cumbersome.

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. .

[Technical solutions]

In order to solve at least one of the above problems, 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. Use the competitive immunoassay method to use it as a fluorescent probe for immunochromatography. By taking photos with a camera tool, 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:

Spray the complete antigen solutions of AFB1, FB1, OTA, ZEN and Cd onto the nitrocellulose membrane (NC membrane) as five detection lines T1 to T5. Apply the goat anti-mouse secondary antibody solution to the nitrocellulose membrane (NC membrane). membrane) as the quality control line C, then 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.

In one embodiment of the present invention, 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, and the final concentration of the Cd complete antigen solution is 0.01-1.0 mg/mL. The concentration is 0.1-1.0mg/mL.

In one embodiment of the present invention, the final concentration of the goat anti-mouse secondary antibody solution is 0.05-1 mg/mL.

In one embodiment of the invention, 0.01-1.0 mg/mL AFB1 complete antigen solution is applied to the nitrocellulose membrane (NC membrane) as the detection line T1, and 0.01-1.0 mg/mL FB1 complete antigen solution is applied to On the nitrocellulose membrane (NC membrane) as the detection line T2, apply 0.01-1.0mg/mL OTA complete antigen solution to the nitrocellulose membrane (NC membrane) as the detection line T3, and 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.

In one embodiment of the present invention, 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, and the distance between the detection line T2- T5 and quality control line C are on both sides of the test line T1.

In one embodiment of the present invention, in the five-link fluorescent immunoquantitative test strip, 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.

In one embodiment of the present invention, 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.

In one embodiment of the present invention, the widths of the detection lines T1, T2, T3, T4, T5 and the quality control line C are 1.5-2.5 mm.

In one embodiment of the present invention, 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.

In one embodiment of the present invention, 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.

In one embodiment of the present invention, the spraying amount per centimeter of the quality control line width on the nitrocellulose membrane is 1-3 μL/cm; when the width of the nitrocellulose membrane is 0.4cm, the spraying amount is 0.4-1.2 μL.

In one embodiment of the present invention, the drying is to place the coated nitrocellulose membrane in a 37°C vacuum drying oven for drying before use.

In one embodiment of the invention, 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.

In one embodiment 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, and the detection concentration of ZEN is 0.1-40ng/mL, the detection concentration of Cd is 1-40ng/mL.

In one embodiment of the present invention, 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:

(1) Extract AFB1, FB1, OTA, ZEN and heavy metal Cd from the grains to obtain the solution to be tested;

(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;

(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;

(4) Substitute the obtained C value, T1 value, T2 value, T3 value, T4 value and T5 value into the standard curve of AFB1, FB1, OTA, ZEN and Cd to obtain the AFB1, FB1, OTA, ZEN and Cd values in the sample to be tested. Cd concentration.

In one embodiment of the present invention, the extraction in step (1) specifically includes the following steps:

Crush and sieve the grains to obtain crushed grains; prepare an extract according to a volume ratio of methanol and nitric acid solution of 85:15; then add the extract to the crushed grains for extraction and centrifugation to obtain a solution containing AFB1, FB1, The solution of OTA, ZEN and Cd; the sieving is through an 80 mesh sieve, the concentration of the nitric acid solution is 1.2mol/L, the ratio of the crushed grains and the extracting liquid is 3:1 in mL/g, and the extraction is at 25 Ultrasonic extraction was performed at ℃ for 25 min, and centrifugation was performed at 4000 rpm for 5 min.

In one embodiment of the present invention, 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.

In one embodiment of the present invention, the uniform mixing in step (2) involves incubating at 20-37°C for 10-20 minutes to fully compete for the reaction.

In one embodiment of the present invention, the amount of the pretreated solution to be tested in step (3) is 30-50 μL/cm ² .

In one embodiment of the present invention, the imaging tool in step (3) includes a mobile phone and a camera.

In one embodiment of the present invention, 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);

Eu ³⁺ -fluorescent microsphere-labeled 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, 1μL AFB1 fluorescent probe, 2μL FB1 fluorescent probe, 1μL OTA fluorescent probe, 1μL ZEN fluorescent probe, 0.5μL Cd -EDTA fluorescent probe, 89.5 μL of PBS buffer and 5 μL 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 μL/cm ² and chromatography at 37°C. After 15 minutes, take a picture with a camera tool, perform RGB analysis on the picture with Image J software, and record the fluorescence values of T1, T2, T3, T4, and T5 of the detection lines of the five-link fluorescent immunoquantitative test strip; each concentration measures six In parallel, set the fluorescence value of the T line of the standard solution with a concentration of 0 ppb as T0, and the fluorescence values of the detection lines T1, T2, T3, T4, and T5 of other spiked concentrations as T1, T2, T3, T4, and T5. According to 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 logarithmic value of each Cd standard concentration is used as the abscissa, and T5/T0 × 100 (%) is used as the ordinate to draw a standard curve. The competition inhibition rate is set to (1-T5/T0)×100%.

In one embodiment of the present invention, the standard curve in step (4) is specifically: when the concentration of AFB1 is 0.05-10ng/mL, the logarithm of the AFB1 concentration has a linear relationship with T/T0, and the linear equation is Y =-21.718Logx+47.003, x is the concentration of AFB1, Y is T1/T0, R ² =0.9896, the detection limit can reach 0.304ng/mL; when the concentration of FB1 is 1-60ng/mL, the logarithm of the concentration of FB1 and T/T0 has a linear relationship, the linear equation is Y=-36.733Logx+79.041, x is the concentration of FB1, Y is T2/T0, R ² =0.9774, the detection limit can reach 0.417ng/mL; when the concentration of OTA is 0.5- At 10ng/mL, the logarithm of OTA concentration has a linear relationship with T/T0. The linear equation is Y=-30.692Logx+51.728, x is the OTA concentration, Y is T3/T0, R ² =0.9645, and the detection limit can reach 0.308 ng/mL; when the concentration of ZEN is 0.1-50ng/mL, the logarithm of ZEN concentration has a linear relationship with T/T0. The linear equation is Y=-62.827Logx+86.945, x is the ZEN concentration, and Y is T4/T0 , R ² =0.9972, the detection limit can reach 0.370ng/mL; when the concentration of Cd-EDTA is 1-40ng/mL, the logarithm of the Cd-EDTA concentration has a linear relationship with T/T0, and the linear equation is Y=- 26.412Logx+67.024, x is Cd concentration, Y is T5/T0, R ² =0.9966, and the detection limit can reach 0.401ng/mL.

In one embodiment of the present invention, 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, and the concentration range of OTA is 0-10ng/mL. The concentration range of mL and ZEN is 0-50ng/mL, and 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.

[beneficial effect]

(1) The method of the present invention for extracting multiple mycotoxins (AFB1, FB1, OTA, ZEN) and heavy metal Cd from grains in one step, with short time (within 1 hour) and high recovery rate (90-110%), 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.

(2) 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. When the concentration of AFB1 is 0.05- At 10ng/mL, the logarithmic value of its concentration has a linear relationship with T2/T0. The linear equation is Y=-21.718Logx+47.003, R ² =0.9896, and the detection limit can reach 0.304ng/mL; when the concentration of FB1 is 1- At 60ng/mL, the logarithm of FB1 concentration has a linear relationship with T/T0. The linear equation is Y=-36.733Logx+79.041, x is the FB1 concentration, Y is T2/T0, R ² =0.9774, and the detection limit can reach 0.417 ng/mL; when the concentration of OTA is 0.5-10ng/mL, the logarithm of the OTA concentration has a linear relationship with T/T0. The linear equation is Y=-30.692Logx+51.728, x is the OTA concentration, and Y is T3/T0 , R ² =0.9645, the detection limit can reach 0.308ng/mL; when the concentration of ZEN is 0.1-50ng/mL, the logarithm of ZEN concentration has a linear relationship with T/T0, and the linear equation is Y=-62.827Logx+86.945 , x is the ZEN concentration, Y is T4/T0, R ² =0.9972, the detection limit can reach 0.370ng/mL; when the concentration of Cd-EDTA is 1-40ng/mL, the logarithm of the Cd-EDTA concentration is related to T/ T0 forms a linear relationship, the linear equation is Y=-26.412Logx+67.024, x is the Cd concentration, Y is T5/T0, R ² =0.9966, and the detection limit can reach 0.401ng/mL.

(3) 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.

(4) 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.

Description of drawings

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.

Detailed ways

Preferred embodiments of the present invention are described below. It should be understood that the embodiments are for the purpose of better explaining the present invention and are not intended to limit the present invention.

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.

Example 1 Preparation of five-link fluorescent immunoquantitative test strips for simultaneous detection of AFB1, FB1, OTA, ZEN and Cd in cereals

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). 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 The antigen solution (Cd-EDTA-OVA) was applied to the nitrocellulose membrane (NC membrane) as the detection line T5, and the 0.5 mg/mL goat anti-mouse secondary antibody solution was applied to the nitrocellulose membrane (NC membrane) as the quality control. Line C, where the distance between test lines T1, T2, T3, T4, and T5 is all 1.2mm, the distance between test line T1 and quality control line C is 1.2mm, and 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;

Then, 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. Similarly, 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;

Finally, use a strip cutter to cut the pasted board into test paper strips about 4mm wide, and assemble them with a plastic base and card case to obtain a five-linked fluorescence immunoassay test paper that simultaneously detects AFB1, FB1, OTA, ZEN and Cd in grains. The strips should be sealed and stored at 4°C for later use; the structural diagram is shown in Figure 2. From left to right on the bottom plate are the sample pad, nitrocellulose membrane and absorbent paper.

Example 2 Preparation of fluorescent probes labeled with Eu ³⁺ -fluorescent microspheres for AFB1, FBl, OTA, ZEN and Cd monoclonal antibodies

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;

For the preparation method of Eu ³⁺ -fluorescent microsphere-labeled AFB1 monoclonal antibody, refer to patent CN 110988339A, the details are as follows:

(1) Take 10 μL (1% solid content) of fluorescent microspheres (purchased from Bainatech New Material Technology Co., Ltd., particle size 300nm) that are internally wrapped with Eu ³⁺ and surface modified with carboxyl functional groups and placed at 4°C, and ultrasonic Disperse, add 1000μL activation buffer, centrifuge at 10000rpm at 4℃ for 15min;

(2) Discard the supernatant, add 800 μL of activation buffer, resuspend by sonication, and repeat centrifugation and washing three times;

(3) Discard the supernatant, add 200 μL of activation buffer and resuspend by ultrasound, add 1 μL of 1 mg/mL EDC solution and 1 μL of 1 mg/mL NHS solution as activators, and shake at room temperature at 500 rpm in the dark for 40 min;

(4) After activation is completed, centrifuge, discard the supernatant, and add PBS buffer to wash 3 times;

(5) Discard the supernatant, add 400 μL coupling buffer and resuspend by sonication, then add 0.5 μg AFB1 monoclonal antibody, and label with shaking for 2 hours at room temperature in the dark;

(6) After labeling, add 10% (v/v) blocking buffer and 100 μL antibody protection solution, and shake at room temperature for 40 minutes in the dark;

(7) After blocking, centrifuge and discard the supernatant, and wash 2-3 times with 800-1000 μL of labeling washing solution;

(8) Discard the supernatant and add 400 μL of fluorescent microsphere reconstituted solution to obtain Eu ³⁺ -fluorescent microsphere-labeled AFB1 monoclonal antibody (Eu-AFB1-mAb). Store at 4°C for later use.

Eu ³⁺ -fluorescent microsphere labeled FB1 monoclonal antibody (Eu-FB1-mAb), Eu ³⁺ -fluorescent microsphere labeled OTA monoclonal antibody (Eu-OTA-mAb), Eu ³⁺ -fluorescent microsphere labeled 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 use.

Example 3 Construction of a standard curve for five-link fluorescent immunoquantitative test strips of AFB1, FB1, OTA, ZEN and Cd

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).

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. Mix 1 μL AFB1 fluorescent probe, 2 μL FB1 fluorescent probe, 1 μL OTA fluorescent probe, and 1 μL ZEN fluorescent probe. The probe, 0.5 μL Cd-EDTA fluorescent probe, 89.5 μL PBS buffer and 5 μL 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 μL/cm ^2. After chromatography at 37°C for 15 minutes, take a photo with a mobile phone. 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%.

As can be seen in Figure 3, as the concentrations of AFB1, FB1, OTA, ZEN, and Cd increase, the fluorescence on the T line of the test strip will become lighter and lighter, so T1/T0, T2/T0, T3/T0 , T4/T0, T5/T0 will become smaller and smaller. When the concentration of AFB1 is 0.05-10ng/mL, the logarithm of the AFB1 concentration has a linear relationship with T/T0. The linear equation is Y=-21.718Logx+47.003, x is the concentration of AFB1, Y is T1/T0, R ² =0.9896, the detection limit can reach 0.304ng/mL; when the concentration of FB1 is 1-60ng/mL, the logarithm of the concentration of FB1 has a linear relationship with T/T0, The linear equation is Y=-36.733Logx+79.041, x is the concentration of FB1, Y is T2/T0, R ² =0.9774, the detection limit can reach 0.417ng/mL; when the concentration of OTA is 0.5-10ng/mL, the OTA concentration The logarithm of has a linear relationship with T/T0. The linear equation is Y=-30.692Logx+51.728, x is the OTA concentration, Y is T3/T0, R ² =0.9645, and the detection limit can reach 0.308ng/mL; when ZEN When the concentration is 0.1-50ng/mL, the logarithm of ZEN concentration has a linear relationship with T/T0. The linear equation is Y=-62.827Logx+86.945, x is the ZEN concentration, Y is T4/T0, R ² =0.9972, detection The limit can reach 0.370ng/mL; when the concentration of Cd-EDTA is 1-40ng/mL, the logarithm of the Cd-EDTA concentration has a linear relationship with T/T0, and the linear equation is Y=-26.412Logx+67.024, x is For Cd concentration, Y is T5/T0, R ² =0.9966, and the detection limit can reach 0.401ng/mL.

Example 4 Comparison of membrane drawing methods using complete antigen solutions

Spray the complete antigen solution onto the nitrocellulose membrane (NC membrane) by streak spraying, and refer to Example 1 for the remaining steps.

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.

As shown in Table 1, when the complete antigen solution is directly sprayed by streaking, 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, and the detection limit of ZEN The detection limit of Cd is 0.549ng/mL, and the detection limit of Cd is 0.536ng/mL, both of which are higher than the detection limit of dot matrix test paper strips.

Table 1 Comparison results of detection limits (ng/Ml) of test strips with different marking methods

Scratching method	AFB1	FB1	OTA	ZEN	cd
Direct spraying	0.524	0.498	0.619	0.549	0.536
Dot matrix spraying	0.304	0.417	0.308	0.370	0.401

Example 5 Preliminary establishment of detection method

A method for simultaneously detecting mycotoxins (AFB1, FBl, OTA, ZEN) and heavy metal Cd in cereals, the method includes the following steps:

(1) Extract mycotoxins (AFB1, FB1, OTA, ZEN) and heavy metal Cd from grains:

Crush the negative sample rice and pass it through an 80-mesh sieve to obtain crushed rice; add 1.2 mL of concentrated nitric acid (14.4 mol/L) to 13.2 mL of ultrapure water, shake it thoroughly to make a 1.2 mol/L nitric acid solution; Then prepare the extract according to a volume ratio of methanol: nitric acid solution of 85:15; mix 1g of crushed rice, 500ppb Cd (cadmium standard), 200ppb AFB1, 200ppb FB1, 200ppb OTA, 200ppb ZEN (fungus All toxins were dissolved in methanol) and mixed evenly, then mixed with 1 mL of extraction solution and added to the solid-phase extraction column, ultrasonic extraction at 25°C for 25 min, and centrifugation at 4000 rpm for 5 min to remove insoluble substances to obtain AFB1, FB1, OTA, Solutions to be tested for ZEN and Cd;

(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;

(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 μL/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;

(4) Substitute the obtained T1 value, T2 value, T3 value, T4 value, T5 value and C value into the standard curve of AFB1, FB1, OTA, ZEN and Cd in Example 3 to obtain AFB1, FB1, Concentrations of OTA, ZEN and Cd.

Example 6 Condition Optimization

(1) Effect of the dosage of AFB1/FB1/OTA/ZEN/Cd complete antigen

The specific experimental investigation process is as follows:

In order to prepare five-linked array immunochromatography test strips, in this experiment, 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. Use 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.

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.

As shown in Figure 4, when the AFB1-OVA concentration is 0.3 mg/mL, 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; When 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. Therefore, 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.

(2) Optimization of fluorescent probe loading amount

In order to explore the effect of the amount of probe on the fluorescence intensity of each point of the five-link array immunochromatography test strip, Eu-AFB1-mAb, Eu-FB1-mAb, Eu-OTA-mAb, Eu-ZEN-mAb and Five types of Eu-Cd-EDTA-mAb probes were optimized for loading. First, 0.1 μL, 0.5 μL, 1 μL, 1.5 μL and 2 μL probes were used to load the probes using five-link array immunochromatography test strips. Optimization.

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.

After obtaining the optimal loading amount of Eu-AFB1-mAb, on this basis, optimize the loading amount of Eu-FB1-mAb, Eu-OTA-mAb, Eu-ZEN-mAb and Eu-Cd-EDTA-mAb in sequence. The optimized dosages of various probes are 0.1μL, 0.5μL, 1μL, 1.5μL and 2μL. All the above test results are taken by mobile phones and analyzed by Image J through RGB.

If the amount of fluorescent probe loaded is too small, the fluorescence intensity of the detection line on the test strip will be too weak and the sensitivity will be low. If the amount of fluorescent probe loaded is too much, the fluorescence intensity will be too strong and background noise will easily occur. As shown in Figure 5, by comparing the fluorescence intensities of detection lines T1, T2, T3, T4, and T5, Eu-AFB1-mAb, Eu-FB1-mAb, Eu-OTA-mAb, Eu-ZEN-mAb, and Eu- The optimal loading volumes of Cd-EDTA-mAb are 1μL, 2μL, 1μL, 1μL and 0.5μL respectively.

(3) The impact of immunity time on test results

Mix 1 μL Eu-AFB1-mAb, 2 μL Eu-FB1-mAb, 1 μL Eu-OTA-mAb, 1 μL Eu-ZEN-mAb, 0.5 μL Eu-Cd-EDTA-mAb, and 5 μL spiked sample solution (AFB1=0.05 μg/ Mix L, FB1=0.05μg/L, OTA=0.05μg/L, ZEN=0.05μg/L, Cd=0.5μg/L mixed solution) and 89.5μ PBS buffer, and incubate for 0min, 5min, respectively at 37°C. After 10min, 15min and 20min, add the 5 sets of mixed solutions dropwise to the sample pad of the five-linked immunoquantitative test strip respectively. The sample volume is 50μL/cm ² , chromatograph at 37°C for 15min, take pictures with your mobile phone, and pass the Image J software. RGB analysis was performed and T-line fluorescence intensity was recorded.

Mix 1 μL Eu-AFB1-mAb, 2 μL Eu-FB1-mAb, 1 μL Eu-OTA-mAb, 1 μL Eu-ZEN-mAb, 0.5 μL Eu-Cd-EDTA-mAb, and 5 μL spiked sample solution (AFB1=0.05 μg/ Mix L, FB1=0.05μg/L, OTA=0.05μg/L, ZEN=0.05μg/L, Cd=0.5μg/L mixed solution) and 89.5μ PBS buffer, incubate at 37°C for 10 minutes, slowly drip into the five-link For the sample pad of the immunoquantitative test strip, the sample volume is 50 μL/cm ² , chromatography is performed at 37°C, take a photo with a mobile phone, and use Image J software to analyze through RGB and record the change in T-line fluorescence intensity every minute.

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.

Using the time of the immune reaction as the abscissa and the fluorescence intensity of the detection line as the ordinate, draw an immune kinetics curve to determine the optimal reaction time. Through the analysis of the graph (B), it is found that at 0-8 minutes, the fluorescence intensity of the detection line increases rapidly, at 8-15 minutes, the fluorescence intensity of the detection line increases slowly, and beyond 15 minutes, the fluorescence intensity of the detection line tends to balance and no longer increases. Therefore, it is more appropriate to choose 18 minutes for chromatography time.

Example 7 Antibody affinity determination

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:

(1) Coating: Dilute AFB1-OVA with a concentration of 1 mg/mL by 3-fold gradient dilution to three concentrations: 1 mg/mL, 0.33 mg/mL, and 0.11 mg/mL. Add 100 μL to each well and incubate at 37°C for 2 hours. ;

(2) Washing: Shake out the antigen-coated solution in the microwells, pat dry, add 300 μL of PBST washing solution, shake gently to wash, spin dry, repeat three times;

(3) Blocking: Add 300 μL of 3% of the test solution containing AFB1, FB1, OTA, ZEN and Cd obtained in step (1) of Example 5 to each well, and place it at a constant temperature of 37°C to block in the dark for 1 hour;

(4) Add antibody: 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 100μL to each well and incubate at 37°C for 1 hour;

(5) Add enzyme-labeled secondary antibody: wash three times according to step (2) and pat dry, add 100μL enzyme-labeled secondary antibody to each well (dilute 1μL of horseradish peroxidase-goat anti-mouse secondary antibody with 5mL 0.01M PBS) ;

(6) Color development: Spin the liquid in the wells dry, wash the plate three times and pat dry according to step (2), add 100 μL 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. Weigh 0.15g of 3,3',5,5'-tetramethylbenzidine, dissolve it in 3mL of dimethyl sulfoxide, adjust the volume to 500mL, and store it in the dark at 4°C; mix liquid A and liquid B at a ratio of 1:1 Mix in equal volumes) and react in the dark at 37°C for 15 minutes;

(7) Termination: Add 50 μL of stop solution (2mol/L H ₂ SO ₄ ) to each well and detect immediately;

(8) Reading: Use a multifunctional microplate reader to read the absorbance values of AFB1 monoclonal antibodies at different coating concentrations at 450 nm.

Taking the logarithmic value of the monoclonal antibody concentration (mol/L) as the abscissa and the absorption photometric value OD _450nm as the ordinate, fit the "S" curve, find the maximum OD ₄₅₀ nm of each curve, and set it as OD _max . Then the antibody molar concentration corresponding to OD _max /2 is calculated through the fitting equation, and the three obtained coating originals of different concentrations are divided into two groups to form 3 groups, and 3 are calculated according to the following formula (1) Monoclonal antibody affinity constant _Ka , the average of the three affinity constants is the affinity constant of the antibody.

Among them, [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.

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.

Table 2 AFB1 antibody affinity constant results

Table 3 FB1 antibody affinity constant results

Table 4 OTA antibody affinity constant results

Table 5 ZEN antibody affinity constant results

Table 6 Cd-EDTA antibody affinity constant results

Example 8 Performance test of five-link immune quantitative test strips

(1) Precision test

Use 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). mL and Cd-EDTA at 0.1ng/mL) and negative samples to analyze the inter-batch variation of the test strips. Analyze inter-batch and intra-batch variation data to determine the precision and accuracy of the test strip.

The experimental operation is the same as in Example 5.

The fluorescence intensity value (T0) at the detection line of the negative control group (5% methanol-PBS) and the positive test group (AFB1=0.05; FB1=1ng/mL ; OTA=0.25ng/mL; ZEN=2ng/mL Cd=0.5μg/L) Fluorescence intensity value (T) at the T line to analyze inter-batch differences.

As can be seen from Table 7, 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.

Table 7 Inter-batch and intra-batch precision experiments of T1, T2, T3, T4 and T5 detection lines

(2) Accuracy test

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):

The results are shown in Table 8. In the actual sample spike recovery experiment, the recovery rate of the five-link immunoquantitative test strip was between 68.95% and 93.45%, and the coefficient of variation was less than 9.876%. It is consistent with the UPLC-MS/MS method and ICP -MS method comparison, the results are consistent.

Table 8 Accuracy test results of five-link immune quantitative test strips

(3) Cross-reactivity test

In order to verify the specificity of mycotoxins and heavy metals, the structural analogs or homologues of five substances 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. For specific detection, 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.

Mix 1 μL Eu-AFB1-mAb, 2 μL Eu-FB1-mAb, 1 μL Eu-OTA-mAb, 1 μL Eu-ZEN-mAb, 0.5 μL Eu-Cd-EDTA-mAb, 89.5 μL PBS buffer and 5 μL of the above 16 kinds of After the single solution is homogeneous, incubate at room temperature for 10 minutes, then slowly drip it into the sample pad of the five-link fluorescent immunoquantitative test strip. The sample volume is 50 μL/cm ² . After chromatography at 37°C for 15 minutes, take a picture with your mobile phone. Image J software performs RGB on the picture. Analyze and record the fluorescence intensity of the T line on the five-link fluorescent immunoquantitative test strip.

As shown in Figure 7, when the concentration of AFB1, FB1, OTA, ZEN is 10ng/mL and the concentration of Cd is 20ng/mL, the detection lines corresponding to T1, T2, T3, T4 and T5 disappear, while when other cross substances are added There was almost no change in fluorescence intensity after the test, and there was no obvious weakening, indicating that the test strip has good specificity and stable performance, meets market demand, and has broad market prospects.

Although the present invention has been disclosed above in terms of preferred embodiments, they are not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, The protection scope of the present invention should be defined by the claims.

Claims (12)

1. A method for preparing a five-link array immunochromatography quantitative test strip for simultaneously detecting multiple mycotoxins and heavy metal Cd in cereals, which is characterized by including the following steps:

   Spray the complete antigen solutions of AFB1, FB1, OTA, ZEN and Cd onto the nitrocellulose membrane as detection lines T1 to T5, and apply the goat anti-mouse secondary antibody solution onto the nitrocellulose membrane as quality control line C. Paste the sample pad, nitrocellulose membrane containing the detection line and quality control line, and absorbent paper on the PVC bottom plate in sequence to assemble a test strip.
2. The method according to claim 1, wherein 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, and the Cd complete antigen The final concentration of the solution is 0.1-1.0 mg/mL; the final concentration of the goat anti-mouse secondary antibody solution is 0.05-1 mg/mL.
3. The method according to claim 1, characterized in that the AFB1 complete antigen solution is applied to the nitrocellulose membrane as the detection line T1, the FB1 complete antigen solution is applied to the nitrocellulose membrane as the detection line T2, and the OTA complete antigen solution is applied to the nitrocellulose membrane as the detection line T1. The antigen solution was applied to the nitrocellulose membrane as the detection line T3, the ZEN complete antigen solution was applied to the nitrocellulose membrane as the detection line T4, the Cd complete antigen solution was applied to the nitrocellulose membrane as the detection line T5, and the sheep anti- The mouse secondary antibody solution was applied to the nitrocellulose membrane as quality control line C.
4. The method according to claim 3, characterized in that the distance between the detection lines T1, T2, T3, T4 and T5 is 1-3mm, the distance between the detection line T1 and the quality control line C is 1-3mm, and the distance between the detection lines T1, T2, T3, T4 and T5 is 1-3mm. Lines T2-T5 and quality control line C are on both sides of the detection line T1.
5. The method according to claim 3, characterized in that the distance between the detection line T5 and the sample pad is 4-6mm, the distance between the quality control line C and the absorbent paper is 4-6mm, and the detection lines T1, T2, T3 , T4, T5 and quality control line C have a width of 1.5-2.5mm.
6. The method according to claim 1, characterized in that the spraying method of the AFB1 complete antigen solution, FBl complete antigen solution, OTA complete antigen solution, ZEN complete antigen solution, Cd complete antigen solution and goat anti-mouse secondary antibody solution is: Micro-detection points form an array detection method.
7. The method according to claim 6, characterized in that the detection line width per centimeter on the nitrocellulose membrane is sprayed with AFB1 complete antigen solution, FB1 complete antigen solution, OTA complete antigen solution, ZEN complete antigen solution and Cd complete antigen solution. The quantities are 10-40n respectively.
8. A five-link fluorescent immunoquantitative test strip for simultaneously detecting AFB1, FBl, OTA, ZEN and Cd prepared by the method described in any one of claims 1 to 7.
9. A method for simultaneously detecting multiple mycotoxins and heavy metal Cd in cereals, characterized in that the method includes the following steps:

   (1) Extract various mycotoxins and heavy metal Cd in grains to obtain the solution to be tested;

   (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;

   (3) Add the solution to be tested in step (2) to the sample pad of the five-link fluorescent immunoquantitative test strip described in claim 4 for chromatography, and then take photos with a camera tool, and use Image J software to process the pictures. RGB analysis, 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, and the T4 value of ZEN , T5 value of Cd;

   (4) Substitute the obtained C value, T1 value, T2 value, T3 value, T4 value, and T5 value into the standard curve of AFB1, FB1, OTA, ZEN, and Cd to obtain the AFB1, FB1, OTA, ZEN, and Cd values in the sample to be tested. Cd concentration.
10. The method according to claim 9, characterized in that the extraction in step (1) specifically includes the following steps:

    Crush and sieve the grains to obtain crushed grains; prepare an extract according to a volume ratio of methanol and nitric acid solution of 85:15; then add the extract to the crushed grains for extraction and centrifugation to obtain a solution containing AFB1, FB1, Solution of OTA, ZEN and Cd; the sieving is through an 80 mesh sieve, the concentration of the nitric acid solution is 1.2mol/L, the ratio of the crushed grains and the extracting liquid is 3:1 in mL/g, and the extraction is at 25 Ultrasonic extraction was performed at ℃ for 25 min, and centrifugation was performed at 4000 rpm for 5 min.
11. The method according to claim 9, characterized in that the test solution in step (2), a monoclonal antibody of Eu ³⁺ -fluorescent microsphere labeled AFB1, a monoclonal antibody of Eu ³⁺ -fluorescent microsphere labeled FBl Clonal antibodies, 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 PBS buffer The volume ratio of the liquid is (3-5):(3-5):(3-5):(3-5):(3-5):(5-10):(85-95).
12. Application of the method described in any one of claims 1 to 7 or the five-link fluorescent immunoquantitative test strip described in claim 6 or the method described in any one of claims 8-11 in the field of food testing.

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