LU500224B1

LU500224B1 - Assay kit for detecting a variety of mycotoxins, its method and application

Info

Publication number: LU500224B1
Application number: LU500224A
Authority: LU
Inventors: Shuang Li; Jialei Bai; Dianpeng Han; Zhixian Gao; Tie Han; Yuan Peng
Original assignee: Inst Of Environmental And Operational Medicine Academy Of Military Medical Science Academy Of Milita
Priority date: 2021-03-30
Filing date: 2021-05-31
Publication date: 2021-11-30
Also published as: CN113092766A

Abstract

The present invention relates to the field of analysis and detection technology, and in particular to an assay kit for detecting a variety of mycotoxins, its method and application. The assay kit includes: upconversion nanoparticles separately coupled to the antigen of the mycotoxin to be tested, magnetic nanoparticles separately coupled to the antibody of the mycotoxin to be tested, and standard solution of the mycotoxin; the mycotoxin to be tested includes at least zearalenone and fumonisin B1. The assay kit and method according to the present invention have the advantages of high sensitivity and good specificity, which are of great practical significance for the ultrasensitive detection of a variety of mycotoxins and are of good guidance for realizing on-site rapid and ultrasensitive detection technology.

Description

Assay kit for detecting a variety of mycotoxins, its method and application

BACKGROUND OF THE INVENTION Field of the Invention

[01] The present invention relates to the field of analysis and detection technology, and in particular to an assay kit for detecting a variety of mycotoxins, its method and application. Description of the Related Art

[02] Food safety is a growing global concern. In particular, one of the most prevalent and harmful is mycotoxins. Mycotoxins are secondary metabolites and non-protein small molecule compounds produced by fungi under certain temperature and humidity conditions. They are widely distributed, diverse, toxic, difficult to detect, and a variety of toxins may be present in the same contaminated food. Fumonisin B1 (FB1) and zearalenone (ZEN) are two mycotoxins commonly found in cereals and dairy products such as corn, oats, and wheat. They often cause acute, chronic, teratogenic and carcinogenic to humans and animals, and inevitably and extensively continue to affect human health. Therefore, people are paying more and more attention to the contamination of mycotoxins in food and feed.

[03] The commonly used methods for detecting mycotoxins include chromatography, immunoassay, electrochemical detection, and Raman spectroscopy. Gas chromatography (GC), thin-layer chromatography (TLC), and high performance liquid chromatography (HPLC) are highly sensitive and specific, and are recognized as confirmatory methods in the field of analysis. However, they are time-consuming, expensive in equipment, cumbersome and rely on professionals for sample pre-treatment, and are difficult to apply to on-site testing. Other methods have average sensitivity and are generally more 1 complex to operate. With the rapid development of nanomaterials and nanotechnology, it offers great possibilities to further improve the sensitivity and convenience of Mycotoxin detection sensors and use them for field detection.

[04] Therefore, it is important to establish a rapid, efficient and sensitive Mycotoxin detection method for rapid food safety detection.

[05] In view of this, the present invention is proposed.

SUMMARY OF THE INVENTION

[06] The primary inventive object of the present invention is to provide an assay kit for detecting a variety of mycotoxins.

[07] The second inventive object of the present invention is to provide the method of preparing the assay kit for detecting a variety of mycotoxins.

[08] The third invention object of the present invention is to provide a method for detecting a variety of mycotoxins.

[09] The fourth invention object of the present invention is to provide an application of the above assay kit and method.

[10] In order to accomplish the inventive purpose of the present invention, the technical solutions used are:

[11] A first aspect of the present invention proposes an assay kit for detecting a variety of mycotoxins, the assay kit comprising the following components: upconversion nanoparticles separately coupled to an antigen of the mycotoxin to be tested; magnetic nanoparticles separately coupled to antibodies to the mycotoxins to be tested; a standard solution of the mycotoxin; the mycotoxin to be tested comprises at least zearalenone and fumonisin B1; the antibody is a monoclonal antibody.

[12] The second aspect of the present invention proposes a method for preparing an assay kit, wherein the method for preparing upconversion nanoparticles coupled to an 2 antigen of the mycotoxin to be tested comprises at least the steps of modifying the aminated upconversion nanoparticles with streptavidin using the glutaraldehyde method to obtain streptavidin-modified upconversion nanoparticles, incubating the streptavidin-modified upconversion nanoparticles with a biotinylated artificial antigen of the mycotoxin to be tested; preferably, the concentration of the upconverted nanoparticles is 2 mg/mL and the concentration of the biotinylated mycotoxin artificial antigen to be tested is 1.5 mg/mL; more preferably, the temperature of the incubation is 37°C and the time of incubation is 4 to 8 hours.

[13] The third aspect of the present invention proposes a method for detecting a variety of mycotoxins, using the reagents in the assay kit comprising at least the steps of S1. drawing a standard curve. mixing a series of concentration of the mycotoxin standard solution with the upconversion nanoparticles coupled with mycotoxin antigen, respectively, adding the magnetic nanoparticles coupled with mycotoxin monoclonal antibody for binding, magnetic separation, collecting the supernatant, detecting the fluorescence signals corresponding to different mycotoxins in the supernatant, and establishing a standard curve of mycotoxin standard concentration and fluorescence signal, S2. detecting the sample to be tested. mixing the sample to be tested with the mycotoxin standard to prepare a mixed sample to be tested; mixing said mixed sample to be tested with the upconversion nanoparticles coupled with mycotoxin antigen, adding the magnetic nanoparticles coupled with mycotoxin monoclonal antibody for binding, magnetic separation, collecting the supernatant, detecting the 3 fluorescence signals corresponding to different mycotoxins in the supernatant;.

S3, calculating the concentration of mycotoxins in the solution to be tested according to the standard curve.

[14] The present invention has at least the following beneficial effects: The present invention utilizes the unique luminescence characteristics of upconversion nanoparticles, the specificity of antibodies and the separation enrichment effect of magnetic nanoparticles. Not only can detect two mycotoxins at the same time, but also avoid the interference of autofluorescence in the immunoassay process, which effectively improves the accuracy and sensitivity of the analysis. Therefore, the assay kit and method according to the present invention have the advantages of high sensitivity, good specificity, etc., have important practical significance for the ultra-sensitive detection of a variety of mycotoxins, and have good guiding significance for realizing on-site rapid and ultra-sensitive detection technology.

BRIEF DESCRIPTION OF THE DRAWINGS

[15] Figure 1 shows a schematic diagram of the detection principle of the present invention;

[16] FIG. 2 shows transmission electron micrographs of two upconversion nanoparticles (UCNPs); [171 FIG. 3 shows XPS characterization diagram of two different luminescent UCNPs coupled with biotin-labeled FB1 and ZEN toxin antigens;

[18] Figure 4 shows SEM characterization image of the magnetic nanoparticles before coupling;

[19] Figure 5 shows SEM characterization image of magnetic nanoparticles after coupling FB1 toxin antibodies;

[20] Figure 6 shows the SEM characterization image of magnetic 4 nanoparticles after coupling ZEN toxin antibody;

[21] Figure 7 shows the XPS characterization diagram of magnetic nanoparticles after coupling with monoclonal antibodies FB1 and ZEN toxins;

[22] Figure 8 shows the standard curve plotted with the concentrations of FBI and ZEN standards as horizontal coordinates and the fluorescence intensity corresponding to each concentration as vertical coordinates;

[23] Figure 9 shows the specificity experiments of a highly sensitive immunofluorescence sensor based on magnetic separation and two-color upconversion for the simultaneous detection of fumonisin Bl and zearalenone;

[24] Figure 10 shows the results of volumetric screening experiments of upconversion nanoparticles coupled to the mycotoxin antigen to be tested;

[25] Figure 11 shows the results of the buffer type screening test for the reaction system;

[26] Figure 12 shows the results of the pH screening test of the reaction system;

[27] Figure 13 shows the results of the reaction competition time screening test.

DESCRIPTION OF THE EMBODIMENTS

[28] It should be noted that the following detailed descriptions are exemplary and are intended to provide further clarification of this application. Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which the present application belongs.

129] It is noted that the terms used herein are intended to describe specific embodiments only, and are not intended to limit exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form also includes the plural form, and it is 5 also to be understood that when the terms "comprising" and/or "including" are used in this description, they indicate the presence of features, steps, operations and/or components, and/or combinations thereof.

[30] The technical solutions of the present invention will be clearly and completely described below in connection with the embodiments, and it is clear that the described embodiments are a part of the embodiments of the present invention, and not all of them. Based on the embodiments in the present invention, all other embodiments obtained by a person skilled in the art without making creative labor fall within the scope of protection of the present invention.

[31] A first aspect of an embodiment of the present invention proposes an assay kit for detecting mycotoxins, which can detect a variety of mycotoxins simultaneously, comprising the following components:

[32] upconversion nanoparticles separately coupled to an antigen of the mycotoxin to be tested;

[33] magnetic nanoparticles separately coupled to antibodies to the mycotoxins to be tested, and the antibody preferably is a monoclonal antibody;

[34] a standard solution of the mycotoxin;

[35] the mycotoxin to be tested comprises at least zearalenone and fumonisin B1, but are not limited thereto; a variety of mycotoxins can be detected by replacing antigens and antibodies according to the inventive idea of the present invention. The assay kit and method according to the present invention have the advantages of high sensitivity and good specificity, etc., which are of great practical significance for the ultrasensitive detection of a variety of mycotoxins and are of good guidance for realizing on-site rapid and ultrasensitive detection technology. In addition, few methods have been reported for the simultaneous detection of both FB1 and ZEN, which are 6 co-occurring in many crops or feeds, so the establishment of a method capable of detecting both toxins is of high value from both the methodological and practical application perspectives.

[36] Optionally, the upconversion nanoparticles are selected from particles with a diameter of 25 to 30 nm; nanoparticles with a specific size can further improve the sensitivity of the detection. Preferably NaYF,: Yb’ Tm’ or NaYF,:Yb”*,Er”* . The conditions of the fluorescence signal at the time of assay kit use are related to the upconversion nanoparticles used, and for NaYF;:Yb,Tm and NaYF,:Yb,Er, the measurement conditions preferably include an excitation wavelength of 980 nm, a slit width of 10 nm, and emission wavelengths of 480 nm and 550 nm, respectively.

[371 Optionally, the upconversion nanoparticles and magnetic nanoparticles are preserved in 0.01 M PBS at a pH preferably of 7.0. It was found through screening experiments that the assay kit under this condition has the highest fluorescence intensity during detection, which is conducive to improving the sensitivity of the assay kit.

[38] Optionally, the concentration of upconverted nanoparticles coupled with fumonisin B1 antigen is 1 to 3 mg/mL, preferably 2 mg/mL.

[39] Optionally, the concentration of magnetic nanoparticles coupled with a fumonisin B1 monoclonal antibody is 1 to 3 mg/mL, preferably 2 mg/mL.

[40] Optionally, the concentration of the upconversion nanoparticles coupled with zearalenone antigen is 1 to 3 mg/mL, preferably 2 mg/mL.

[41] Optionally, the concentration of magnetic nanoparticles coupled with zearalenone monoclonal antibody is 1 to 3 mg/mL, preferably 2 mg/mL.

142] If the concentration of upconversion nanoparticles is too high, the upconversion nanoparticles are wasted, increasing the cost of detection, and it is also possible that the fluorescence is burst due to the high concentration instead; if the concentration of upconversion nanoparticles is too low, the 7 fluorescence intensity is too low, and the fluorescence signal is not detected.

[43] If the concentration of magnetic nanoparticles is too high, when the immunocompetitive reaction is formed, the antibodies coupled on the magnetic nanoparticles will be fully recognized with the antigens coupled on the upconversion nanoparticles, and the magnetic separation will lead to the precipitation of all the upconversion nanoparticles to the bottom, and no fluorescence signal will be detected in the supernatant. If the concentration of magnetic nanoparticles is too low, a good magnetic separation effect cannot be achieved.

[44] Optionally, the standard solution is a series of standard solutions in a concentration of 0.05 ~ 5 ng/mL, specifically 0.05 ng/mL, 0.1 ng/mL, 0.2 ng/mL, 0.5 ng/mL, 1 ng/mL, 2 ng/mL, 5 ng/mL.

[45] The second aspect of the present invention relates to a method for preparing the assay kit, specifically as shown in Figure 1, Figure a. The aminated upconversion nanoparticles are obtained, after which the two aminated upconversion nanoparticles are coupled to two toxin artificial antigens, Biotin-FB1-BSA and Biotin-ZEN-BSA, respectively, by the classical glutaraldehyde method using the interaction of streptavidin and biotin. Two different upconverted fluorescent probes, Biotin-FB1-BSA-UCNPs and Biotin-ZEN-BSA-UCNPs, are formed; the magnetic nanoparticles are coupled with FB1 monoclonal antibody and ZEN monoclonal antibody, respectively, to obtain two different capture probes, McAbs-FB1-MNPs and McAbs-ZEN-MNPs.

[46] The present invention proposes a way to prepare amino-modified UCNPs by synthesizing UCNPs of NaYF,:Yb”*,Tm** and NaYF,:Yb”*,Er”* using a solvothermal method, comprising at least the following steps:

[47] © Heating 1 mmol RECI;-6H,0 (Y: Yb: Tm=78:20:2), 8-10 mL oleic acid and 12-20 mL 1-octadecene in a homogeneous solution to 120 °C and 8 degassing under nitrogen or argon protection.

[48] (@ Stirring the mixed solution continuously for 20-40 min and then cooled; Adding 10 mL of methanol solution containing 2.5 mmol NaOH and 4 mmol NH4F and stirring at 45-55°C for 20-40 min to consume all the fluoride.

[49] © Heating the solution to 115 ~ 120 °C until the full evaporation of methanol, raising the temperature of the solution to 310 ~ 320 °C and maintain it for 1.5 h, and keep it ventilated with nitrogen or argon gas.

[50] @ Cooling and washing the precipitate repeatedly with deionized water and ethanol three times to obtain OA-UCNPs.

[51] © Dissolving 300 mg of PEI in 5 mL of ultrapure water and then injecting with cyclohexane solution containing 10 mg of OA-UCNPs and stirring vigorously and magnetically at room temperature for 18 to 36 h.

[52] © After the reaction, the PEI-UCNPs are washed several times with ultrapure water and ethanol to remove the excess PEI; the resulting products had amino modifications on the surface and were dispersed in ultrapure water for use.

[53] The obtained amino-modified UCNPs prepared by the embodiments of the present invention have the technical advantages of uniform particle size and high luminescence intensity.

[54] Wherein: the method of preparing upconversion nanoparticles coupled to an antigen of the mycotoxin to be tested comprises at least the steps of:

[55] modifying the aminated upconversion nanoparticles with streptavidin using the glutaraldehyde method to obtain streptavidin-modified upconversion nanoparticles;

[56] incubating the streptavidin-modified upconversion nanoparticles with a biotinylated artificial antigen of the mycotoxin to be tested;

[57] preferably, the concentration of the upconverted nanoparticles is 1 to 3 9 mg/mL, preferably 2 mg/ml and the concentration of the biotinylated mycotoxin artificial antigen to be tested is 1 to 3 mg/mL, preferably 1.5 mg/mL.

[581 More preferably, the temperature of the incubation is 37°C and the time of incubation is 4 to 8 hours.

[59] Wherein, the upconversion nanoparticles are preferably NaYF,:Yb,Tm and NaYF,:Yb,Er. The upconversion nanoparticles can be prepared by conventional methods in the art, for example, by classical solvothermal synthesis.

[60] According to the similar method described above, the present invention can detect a variety of mycotoxins, for example, three different luminescent UCNPs can be used: NaYF,,Yb”*,Er”* (green light) NaYF, Yb”*,Tm”* (blue light) NaYF,, Yb”*,Ho”* (red light) to detect three kinds of mycotoxins. The upconversion fluorescent probes are first prepared by coupling three different luminescent upconversion nanoparticles to three toxin antigens; and the bead capture probes are prepared by coupling magnetic beads to three toxin antibodies. The specific experimental protocol is to mix a certain volume of the toxin standard with a fixed concentration of the upconverted fluorescent probe, when there will be no immune recognition reaction. When the magnetic bead capture probe is added to the above mixture, according to the principle of competitive immunoassay, the toxin small molecule and toxin antigen will compete to bind to the toxin monoclonal antibody, making part of the magnetic bead capture probe bind to the small molecule and another part of the capture probe bind to the toxin antigen, finally forming three magnetic bead-upconversion particle complexes. When the reaction is finished, the solution is separated using an external magnetic field, and what remained in the supernatant solution are three different luminescent upconverted fluorescent signal probes. The changes in fluorescence intensity are recorded 10 by 980 nm NIR excitation of the supernatant solution with the emission peaks of UCNPs at 480 nm (blue light), 550 nm (green light) and 650 nm (red light) as monitoring signals. The intensity change of the fluorescence signal is linearly related to the concentration of the added target, whereby the quantitative detection of the three targets is achieved.

[61] A third aspect of the present invention relates to the detection method using the assay kit, the principle of which is shown in Fig. 1, Fig. b. A certain amount of FBI and ZEN is mixed with fixed concentrations of two upconversion fluorescent = probes, Biotin-FB1-BSA-UCNPs and Biotin-ZEN-BSA-UCNPs, at which time the two toxins will not react with the toxin artificial antigen for recognition. When two different capture probes, McAbs-FB1-MNPs and McAbs-ZEN-MNPs, are added to the above mixture, according to the principle of competitive immunoassay, the toxin small molecule and the toxin artificial antigen will compete for immune recognition with the toxin monoclonal antibody, making part of the magnetic capture probe bind to the toxin standard and the other part of the capture probe bind to the toxin artificial antigen, finally forming two magnetic bead-upconversion particle complexes. After completing the above process, the solution was separated using an external magnetic field, and the remaining in the supernatant solution are Biotin-FB1-BSA-UCNPs and Biotin-ZEN-BSA-UCNPs. The supernatant solution obtained by magnetic separation is light-excited by a 980 nm laser, and the change in fluorescence intensity produced by the upconverted particles is observed. The emission peak of UCNPs at 480 nm is used as the FB1 concentration monitoring signal, and the emission peak of UCNPs at 550 nm is used as the ZEN concentration monitoring signal. The intensity of the fluorescence signal of UCNPs iss positively correlated with the concentration of added FB1 and ZEN, which could be quantitatively detected and a standard curve is made. The sample to 11 be tested is determined by the same method and the measured fluorescence signal is substituted into the standard curve to obtain the concentration of FB1 and ZEN therein. The present invention takes advantage of the unique luminescence properties of upconverted nanoparticles, the specificity of antibodies and the separation enrichment effect of magnetic nanoparticles.

[62] Based on the above principles, magnetic nanoparticles and monoclonal antibodies can be provided separately, used after coupling according to the method of embodiments of the present invention, or together in the form of magnetic nanoparticle-monoclonal antibody couples. The magnetic nanoparticles are epoxy modified to react with the amino group of the monoclonal antibody to achieve coupling.

[63] Specifically, at least the following steps are included: S1. drawing a standard curve. mixing a series of concentration of said mycotoxin standard solution with the upconversion nanoparticles coupled with mycotoxin antigen, respectively, adding the magnetic nanoparticles coupled with mycotoxin monoclonal antibody for binding, magnetic separation, collecting the supernatant, detecting the fluorescence signals corresponding to different mycotoxins in the supernatant, and establishing a standard curve of mycotoxin standard concentration and fluorescence signal, S2. detecting the sample to be tested. mixing the sample to be tested with the mycotoxin standard to prepare a mixed sample to be tested; mixing said mixed sample to be tested with said upconversion nanoparticles coupled with mycotoxin antigen, adding said magnetic nanoparticles coupled with mycotoxin monoclonal antibody for binding, magnetic separation, collecting the supernatant, detecting the fluorescence signals corresponding to different mycotoxins in the supernatant;. 12

[64] S3. calculating the concentration of mycotoxins in the solution to be tested according to the standard curve.

[65] Specifically, in S1, taking 50 uL each of mycotoxin standard solution and adding 160 ul, of upconversion nanoparticles coupled with FBI antigen and 180 pL of upconversion nanoparticles Biotin-ZEN-BSA-UCNPs coupled with ZEN antigen; adding 10 ul of magnetic nanoparticles coupled with FB1 monoclonal antibody and 10 uL of magnetic nanoparticles coupled with ZEN monoclonal antibody for competition;

[66] The competition is preferably performed under conditions of shaking reaction at room temperature for 30 minutes to 2 hours, preferably 1 hour.

[67] Specifically, in S2, mixing the sample to be tested with the standard solution of the standard mycotoxin to obtain the mixed sample to be tested, so that the final concentrations of the standard mycotoxin are 0.1 ng/mL, 0.5 ng/mL and 2 ng/mL, respectively, and taking 50 pL each of the mixed sample to be tested solution, adding 160 uL of upconversion nanoparticles coupled with FBI antigen and 180 ul of upconversion nanoparticles Biotin-ZEN-BSA-UCNPs coupled with ZEN antigen; adding 10 pl of magnetic nanoparticles coupled with FB1 monoclonal antibody and 10 uL of magnetic nanoparticles coupled with ZEN monoclonal antibody for competition;

[68] The conditions of the competition are preferably a shaking reaction at room temperature for 30 minutes to 2 hours, preferably 1 hour.

[69] Specifically, the above shock is a low-speed shock, specifically 650 rpm.

[70] Specifically, the detection method further includes the step of extracting the sample to be tested, preferably using an aqueous solution of methanol to extract the sample to be tested.

[71] The present invention also relates to the application of the above-mentioned assay kit or method for detecting mycotoxins in food and 13 feed. Wherein, the food includes solid food and liquid food, solid food includes various food crops, etc., and liquid food includes milk, etc.

[72] In the embodiments, biotin-labeled FBI and ZEN toxin artificial antigens, FB1 standards and ZEN standards are purchased from Shandong Lundu Biotechnology Co. Rare earth metals and other chemical reagents are purchased from Sigma, and the M270 epoxy-based magnetic bead kit is purchased from Thermo Fisher. The F97pro fluorescence spectrophotometer is purchased from Shanghai Lengguang Technology. Where specific conditions are not indicated in the Embodiments, conventional conditions or those recommended by the manufacturer were followed. The reagents or instruments used, where the manufacturer is not indicated, are conventional products that can be obtained through commercially available purchases. Embodiment 1

[73] Establishment of a highly sensitive immunofluorescent sensor based on magnetic separation and two-color upconversion for the simultaneous detection of fumonisin B1 and zearalenone.

[74] 1) Preparation of amino-modified UCNPs

[75] Synthesis of UCNPs of NaYF,:Yb”*,Tm** and NaYF,:Yb”*,Er”* by the solvothermal method.

[761 D Adding 1 mmol RECI3-6H20 (Y: Yb: Tm=78:20:2), 9 mL oleic acid and 15 mL 1-octadecene to a 100 mL three-necked flask. Heating and degassing the homogeneous solution to 120 °C and degassing under a nitrogen or argon protected environment.

235 In © Stirring the mixed solution continuously for 30 min, and then cooled naturally to room temperature. Slowly adding 10 mL methanol solution containing 2.5 mmol NaOH and 4 mmol NH4F drop by drop to the flask, and then stirring at 50 °C for 30 min to consume all the fluoride.

14

[78] @ Heating the solution to 120 °C and keep it for a long enough time to ensure sufficient evaporation of methanol. Warming the solution up to 320 °C and kept for 1.5 h. Passing Nitrogen or argon gas through the solution at regular intervals.

[79] @ Turning off the heating switch and allow the solution to cool naturally to room temperature. Repeatedly washing the precipitate with deionized water and ethanol three times to obtain OA-UCNPs.

[80] © Dissolving 300 mg of PEI in 5 mL of ultrapure water, then injecting cyclohexane solution containing 10 mg of OA-UCNPs and stirring vigorously and magnetically for 24 h at room temperature.

[81] © After the reaction, washing the PEI-UCNPs several times with ultrapure water and ethanol to remove the excess PEL The resulting product had amino modification on the surface and is dispersed in ultrapure water for use. The transmission electron micrographs of the obtained UCNPs are shown in Figure 2.

[82] 2) Upconversion nanoparticles coupled to toxin artificial antigens:

[83] Streptavidin is used to modify the aminated UCNPs with streptavidin prior to coupling to biotin-labeled complete antigen following the classical glutaraldehyde method.

[84] © Sonicating the amino-functionalized UCNPs for 20 min, measuring 5 mL of 2 mg/mL amino-functionalized UCNPs accurately, adding 1.25 mL of 25% glutaraldehyde, and shaking the mixed solution gently for 2 h at room temperature on a thermostatic shaker.

[85] @ Separating the resulting product by centrifugation (14,000 rpm, 10 min, 4°C) and washing three times with PBS to remove excess glutaraldehyde.

[86] © Redispering the above obtained product in 5 mL of 0.01 M PBS, and then adding 100 uL of 1.0 mg/mL streptavidin mix, and carrying out a 15 reaction at room temperature for 12 h (650 rpm). After the reaction, performing a centrifugal wash and redispersing in 5 mL of 0.01 M PBS.

[87] @ Mixing 50 pL, concentration 1.5 mg/mL of biotinylated ZEN-BSA (or FB1-BSA) with 5 mL of the above streptavidin-labeled UCNPs dispersion and incubating for 6h at 37°C.

[88] © Finally, the resulting product was centrifuged, washed and dispersed in 5 mL of PBS buffer containing 0.1% BSA.

[89] The XPS characterization image of two different luminescent UCNPs coupled with biotin-labeled toxin antigens is shown in Figure 3.

[90] As seen in Figure 3, the binding energies at 167.81 eV and 167.44 eV belong to S2p, where the presence of the S element indicates the successful coupling of the two biotin-labeled antigens.

[91] 3) Coupling of magnetic nanoparticles and monoclonal antibodies (prepared by M270 epoxy magnetic bead kit):

[92] G)Accurately weighing 5mg of M270 magnetic bead powder into a centrifuge tube, washing the magnetic beads with ImL C1 solution and shake;

[93] @ Putting the centrifuge tube on the magnetic stand for 1 min, let the magnetic beads gather on the tube wall, and discarding the supernatant.

[94] B®) Adding 50 pL of FBI (or ZEN) toxin antibody (concentration 1.5 mg/mL), then adding 200 pL. of CI solution (total amount is 250 pL), then adding 250 uL of C2 solution, and mix and shake slowly;

[95] DIncubating overnight (18 hours) in a vertical suspension at 37°C. Make sure that the liquid in the tube is well mixed.

[96] © Putting the centrifuge tube on the magnetic stand for 1 min, let the magnetic beads gather on the tube wall, and collecting the supernatant (unbound toxin antibody);

[97] © Adding 800 pL HB, shake and mixing well, separating on a magnetic stand for 1 min, discarding the supernatant; 16

[98] (DAdding 800 pL LB, shake and mixing well, separating on a magnetic stand for 1 min, discarding the supernatant;

[99] @Fast SB elution: adding 800 pL of SB, mixing for 3 min, separating on a magnetic stand for 1 min, discarding the supernatant, and repeat once;

[100] @Long-term SB elution: adding 800 pL of SB, mixing for 15 minutes, separating on a magnetic stand for 1 minute, discarding the supernatant, and repeat twice;

[101] (Adding 500 pL of SB (100 pL per mg magnetic beads) and storing at 4°C. The final concentration of the magnetic beads after the antibody is coupled to the magnetic beads is 10 mg/mL.

[102] Figure 4 is the SEM characterization image of the magnetic nanoparticles before coupling: Figure 5 is the SEM characterization image of the magnetic nanoparticles after coupling the FB1 toxin antibody; Figure 6 is the SEM characterization image of the magnetic nanoparticles after coupling the ZEN toxin antibody;

[103] As shown in Figures 4 to 6, before the toxin monoclonal antibody is coupled, the magnetic nanoparticles have uniform particle size and good dispersibility; after incubating with the toxin monoclonal antibody, adhesion appears and attachments appear on the surface. This proves that the antibody coupling is successful.

[104] As can be seen from Figure 7, the toxin antibodies were successfully coupled to the magnetic nanoparticles. The antigen antibodies belong to proteins, they contain elements such as sulfur (S) and phosphorus (P), while the materials coupled with them do not contain S and P inside. XPS elemental analysis was performed after coupling, and the binding energy at 163.18 eV and 163.29 eV belonged to S2p, and the element S appeared, indicating that the antibody coupling is successful and the coupling effect was good and the probe is stable.

17

[105] 4) First adding 50 uL of FBI and ZEN standard solution, and mixing with two different upconverted fluorescent probes, 160 pL of Biotin-FB1-BSA-UCNPs (concentration 2 mg/mL) and 180 pl of Biotin-ZEN-BSA-UCNPs (concentration 2 mg/mL).

[106] 5) Then adding 10 uL each of the two different magnetic complexes, anti-FB1-MNPs and anti-ZEN-MNPs, to the above mixed solution, diluting the concentration to 2 mg/mL, and shaking at room temperature for 1 h.

[107] 6) Separating the solution with an external magnetic field, collecting the supernatant, and then using an F97pro fluorescence spectrophotometer (excitation wavelength 980 nm, slit width 10 nm, voltage 900V) to detect the fluorescence signal at room temperature. The emission wavelengths are 480 nm and 550 nm.

[108] 7) Detecting different concentrations of FB1 and ZEN standards (0.05 ng/mL, 0.1 ng/mL, 0.2 ng/mL, 0.5 ng/mL, 1 ng/mL, 2 ng/mL, 5 ng/mL), and obtain the fluorescence spectrum. The standard curves calculated by software are Fæp=451.9611gX+819.265 (R*=0.9965), the detection range is 0.05-5 ng/mL, and the detection limit is 0.016 ng/mL (S/N = 3); Fizew=

612.031gX+1016.270 (R°=0.9976), the detection range is 0.05-5 ng/mL, and the detection limit is 0.012 ng/mL (S/N = 3). Drawing a standard curve with the fluorescence signal as the ordinate and the concentration of FB1 and ZEN standards as the abscissa, as shown in Figure 8. Embodiment 2

[109] A method for simultaneously detecting fumonisin B1 and zearalenone in com flour with a high-sensitivity immunofluorescence sensor based on magnetic separation and dual-color up-conversion.

[110] (1) Weighing 20g of crushed corn flour sample, adding 50mL of 70% methanol solution prepared in advance, then putting it into a magnetic stirrer 18 and stirring at high speed for 2min, filtering it with quantitative filter paper, measuring 10mL of filtrate, diluting it 10 times with double distilled water (to avoid the effect on fluorescence detection, it is necessary to ensure that the filtrate has no color), and filtering it with 0.45pm filter membrane.

[111] 2) Adding FB1 and ZEN standard substances to the filtrate of corn flour sample at three different concentrations, so that the final concentrations of FB1 and ZEN are 0.1 ng/mL, 0.5 ng/mL and 2 ng/mL, respectively, to obtain three different concentrations Samples of corn flour to be tested.

[112] 3) Adding three different concentrations of corn flour samples to be tested in two different upconversion fluorescent probes containing 160 uL of Biotin-FB1-BSA-UCNPs (concentration of 2 mg/mL) and 180 uL of Biotin-ZEN-BSA-UCNPs (concentration of 2 mg/mL).

[113] 4) Then adding 10 pL of anti-ZEN-MNPs and anti-FB1-MNPs to the above mixed solution, diluting to a concentration of 2 mg/mL, shaking slowly and reacting at room temperature for 60 minutes, and magnetically separating after the reaction and collecting the supernatant and then detecting the fluorescence signal by F97pro fluorescence spectrophotometer (excitation wavelength 980 nm, slit width 10 nm, voltage 900V) at room temperature with emission wavelengths of 480 nm and 550 nm.

[114] 5) The detected concentration calculated by the standard curve is compared with the actual added concentration. The experimental results are shown in Table 1: Table 1 Addition Measured Recovery Actual sample Target substance concentration AF concentration RSD(%) rate (%) *(ng mL”) (ng mL”)

0.1 392.48 0.11 113.69 3.7 FB, 0.5 679.11 0.49 97.93 2.2 corn flour 2 954.78 1.99 99.73 1.2 ZEN 01 3840 009 9074 43 — 19

0.5 802.49 0.45 89.48 1.1 2 1226.50 2.21 110.27 1.0 *n=3, each additive concentration is obtained by three parallel experiments

[115] According to the data in Table 1, the recovery rates of FB1 and ZEN are in the ranges of 97.9% ~ 113.7%, 89.5% ~ 110.3%, and the RSD ranges are

1.2% ~ 3.7% and 1.0% ~ 4.3%, respectively. The results show that the detection method can be applied to the actual corn flour sample detection, and the pre-processing is simple. Embodiment 3

[116] A method for simultaneously detecting fumonisin B1 and zearalenone in milk with a high-sensitivity immunofluorescence sensor based on magnetic separation and dual-color up-conversion.

[117] 1) Taking 1 mL of skim milk and diluting it to 9 mL, then adding three different concentrations of FB1 and ZEN standards so that the final concentrations of FB1 and ZEN are 0.1 ng/mL, 0.5 ng/mL and 2 ng/mL, respectively, to obtain three concentrations of the samples to be tested;

[118] 2) Adding three different concentrations of Milk samples to be tested in two different upconversion fluorescent probes containing 160 uL of Biotin-FB1-BSA-UCNPs (concentration of 2 mg/mL) and 180 uL of Biotin-ZEN-BSA-UCNPs (concentration of 2 mg/mL).

[119] 3) Then adding 10 uL of anti-ZEN-MNPs and anti-FB1-MNPs to the above mixed solution, diluting to a concentration of 2 mg/mL, shaking slowly and reacting at room temperature for 60 minutes, and magnetically separating after the reaction and collecting the supernatant and then detecting the fluorescence signal by F97pro fluorescence spectrophotometer (excitation wavelength 980 nm, slit width 10 nm, voltage 900V) at room temperature with emission wavelengths of 480 nm and 550 nm.

[120] 4) The detected concentration calculated by the standard curve is 20 compared with the actual added concentration. The experimental results are shown in Table 2: Table 2 Addition Measured Recovery Actual sample Target substance concentration AF concentration RSD(%) ] rate (%) *(ng mL") (ng mL")

0.1 392.71 0.11 113.82 1.7 FB, 0.5 672.38 0.47 94.63 3.0 2 962.45 2.07 103.70 1.9

0.1 364.07 0.09 85.97 1.6 ZEN 0.5 803.10 0.45 89.69 2.1 2 1213.80 2.10 105.13 2.7 *n=3, each additive concentration is obtained by three parallel experiments

[121] According to the data in Table 2, the recovery rates of FB1 and ZEN are in the range of 94.6%~113.8%, 86.0%~105.1%, and the RSD range are

1.7%~3.0% and 1.6%~2.7%, respectively. The results show that the detection method can be applied to the actual milk sample detection, and the pre-processing is simple.

Embodiment 4

[122] Experiments on the specificity of the method for simultaneously detecting fumonisin B1 and zearalenone in corn flour with a high-sensitivity immunofluorescence sensor based on magnetic separation and dual-color up-conversion.

[123] Adding a certain concentration of the standard to be tested (100 pL) in two different upconversion fluorescent probes containing 160 uL of Biotin-FB1-BSA-UCNPs (concentration of 2 mg/mL) and 180 uL of Biotin-ZEN-BSA-UCNPs (concentration of 2 mg/mL).

[124] 2) Then adding 10 uL of anti-ZEN-MNPs and anti-FB1-MNPs to the above mixed solution, diluting to a concentration of 2 mg/mL, shaking slowly 21 and reacting at room temperature for 60 minutes, and magnetically separating after the reaction and collect the supernatant.

[125] 3) And then detecting the fluorescence signal by F97pro fluorescence spectrophotometer (excitation wavelength 980 nm, slit width 10 nm, voltage 900V) at room temperature with emission wavelengths of 480 nm and 550 nm.

[126] In order to verify the specificity of the detection method according to the present invention, T-2 (trichothecenes, TS), OTA (Ochratoxin A, ochratoxin A), AFB1 (aflatoxin B1, aflatoxin B1), AFM1 (aflatoxin M1, aflatoxin A) were selected. M1) and AFM2 (aflatoxin M2, aflatoxin M2) and tested. It can be seen from Fig. 9 that the detection method of the present invention has good specificity, and the fluorescence intensity when the substances to be tested are FB1 and ZEN is significantly higher than the fluorescence intensity of other mycotoxins.

Experiment Example 1

[127] Adding 10 uL of McAbs-FB1-MNPs (concentration of 2 mg/mL), and then adding different volumes of Biotin-FB1-BSA-UCNPs (volumes of 140, 160, 180, 200, and 220 pL) (all adding PBS to ensure the total volume of the reaction system is consistent), shaking and reacting slowly for 1 hour at room temperature, magnetically separating after the reaction, collecting the supernatant, and then measuring the fluorescence with a F97pro fluorescence spectrophotometer, finding out the volume of the probe when the fluorescence intensity remains stable, and the optimal volume of Biotin-FB1-BSA-UCNPs is determined is 160 pL.

[128] Adding 10 uL of McAbs-ZEN-MNPs (concentration of 2 mg/mL), and then adding different volumes of Biotin-ZEN-BSA-UCNPs (volumes of 140, 160, 180, 200, and 220 pL) (all adding PBS to ensure the total volume of the 22 reaction system is consistent), shaking and reacting slowly for 1 hour at room temperature, magnetically separating after the reaction, collecting the supernatant, and then measuring the fluorescence with a F97pro fluorescence spectrophotometer, finding out the volume of the probe when the fluorescence intensity remains stable, and the optimal volume of Biotin-ZEN-BSA-UCNPs is determined is 180 pL.

[129] The experimental results are shown in Figure 10.

Experiment Example 2

[130] According to the present invention, the buffer type of the reaction system preferably includes the following steps:

[131] Dispersing 180uL of Biotin-ZEN-BSA-UCNPs and 160uL of Biotin-FB1-BSA-UCNPs into five different buffers (0.01M PBS, 0.01M Tris-Hcl, HEPES, BR and Hybrid buffer), adjusting both the concentration to 2 mg/mL, add 50 pL of ZEN and FB1 standard solution and mixing together. Subsequently, adding 10uL of McAbs-FB1-MNPs and McAbs-ZEN-MNPs to the above-mentioned mixed solution, shaking slowly and reacting at room temperature for 1h, and magnetically separating after the reaction and collecting the supernatant. Then using the F97pro fluorescence spectrophotometer to measure the fluorescence and compare the fluorescence intensity in different buffers. The experimental results are shown in Figure 11.

[132] According to Figure 11, the best buffer is 0.01M PBS.

Experiment Example 3

[133] The pH of the reaction system preferably includes the following steps:

[134] Dispersing 180uL of Biotin-ZEN-BSA-UCNPs (concentration of 2 mg/mL) and 160uL of Biotin-FB1-BSA-UCNPs (concentration of 2 mg/mL) into five different pHs (6.6, 7.0, 7.4, 7.8, 8.2), adding 50 uL of ZEN and FB1 23 standard solution and mixing together. Subsequently, adding 10 uL of McAbs-FB1-MNPs and McAbs-ZEN-MNPs to the above mixed solution, shaking slowly and reacting at room temperature for 1h, and magnetically separating after the reaction and collect the supernatant. Then using the F97pro fluorescence spectrophotometer to measure the fluorescence and compare the fluorescence intensity in different pH, the test result is shown in Figure 12.

[135] According to Figure 12, the optimal pH is 7.0. Experiment Example 4

[136] The reaction competition time preferably includes the following steps:

[137] Based on the above three best conditions, mixing 50 uL of ZEN and FB1 standard solution with 180 uL of Biotin-ZEN-BSA-UCNPs (concentration of 2 mg/mL) and 160 ul. of Biotin-FB1-BSA-UCNPs (concentration of 2 mg/ml) together. Subsequently, adding 10 pL of McAbs-FB1-MNPs and McAbs-ZEN-MNPs to the above mixed solution, shaking slowly and reacting at room temperature for 20, 40, 60, 80, 100 min, and magnetically separating after the reaction and collecting the supernatant. Then using the F97pro fluorescence spectrophotometer to measure the fluorescence and compare the fluorescence intensity at different times. The experimental results are shown in Figure 13.

[138] According to Figure 13, the optimal reaction competition time is determined to be 60 min. Experiment Example 5

[139] Detection limit determination:

[140] Measuring 10 blank values without adding the target substance (i.e. mycotoxins), and using LOD=3*blank standard deviation/standard curve 24 slope to calculate.

[141] Experimental steps:

[142] Under the preferred conditions of Examples 1, 2, 3, and 4, mixing 50 pL. of 0.01M PBS with 180 pL of Biotin-ZEN-BSA-UCNPs and 160 uL of Biotin-FB1-BSA-UCNPs. Subsequently, adding 10 ul of anti-ZEN-MNPs and anti-FB1-MNPs to the above mixed solution, shaking slowly and reacting at room temperature for 1h, and magnetically separating after the reaction and collect the supernatant. Then using 980 nm semiconductor laser to excite and measure the fluorescence intensity at 480 nm and 550 nm. The experimental data obtained is shown in Table 3: Table 3 Fluorescence Blank value SD LOD emission peak

199.9381 202.6619 202.4381 201.1333 202.3667 0.016 480 nm(FB1) 2.348612

199.7762 201.1333 196.4381 205.1048 198.1333 ng/mL 550 nm(ZEN) 401.8952 400.3095 400.8333 398.2429 405.9476 2.363442 0.012 ng/mL Experiment Example 6

[143] Purchase commercialized FB1 and ZEN ELISA kits (Wuhan Liborui Biotechnology Co., Ltd., FB1 ELISA kit item number: MM-9509401, ZEN ELISA kit item number: MM-3286201), and conduct methodological comparison., the data is shown in Table 4 and Table 5: Table 4 Addition Measured | | Recovery Actual sample Target substance concentration concentration RSD(%) - i rate (%) *(ngmL”) (ng mL”)

0.1 0.12 114.60 2.0 FB, 0.5 0.52 99.93 1.6 Milk 2 1.68 100.13 2.3 0 00 10090 20

ZEN

0.5 0.48 95.60 3.1

2 2.01 97.53 2.4 *n=3, each additive concentration is obtained by three parallel experiments Table 5 Addition Measured | | Recovery Actual sample Target substance concentration concentration te (%) RSD(%) gm’) gmLh

0.1 0.12 121.63 2.0 FB, 0.5 0.54 101.97 3.0 2 1.82 86.80 3.4 corn flour oo

0.1 0.12 111.27 2.3 ZEN 0.5 0.54 94.00 4.4 2 1.82 100.70 5.1 *n=3, each additive concentration is obtained by three parallel experiments

[144] It can be seen from the above comparison that the method for simultaneously detecting two toxins established by the present invention is consistent with the traditional ELISA method, and the detection results are consistent, and the present invention has obvious advantages in the simultaneous detection of multiple components; in addition, the present invention is successfully applied to the detection of FBI and ZEN in corn flour and milk samples, with good accuracy and stability, and provides a new idea for realizing high-sensitivity detection of multiple targets.

[145] Although this application is disclosed as above in preferred embodiments, it is not used to limit the claims. Any person skilled in the art can make several possible changes and modifications without departing from the concept of this application. The scope of protection shall be subject to the scope defined by the claims of this application. 26

Claims

[146] WHAT IS CLAIMED IS:

1. An assay kit for detecting a variety of mycotoxins, characterized in that the assay kit comprises the following components: upconversion nanoparticles separately coupled to an antigen of the mycotoxin to be tested, magnetic nanoparticles separately coupled to antibodies to the mycotoxins to be tested, a standard solution of the mycotoxin, preferably, the mycotoxin to be tested comprises at least zearalenone and fumonisin B1, more preferably, the antibody is a monoclonal antibody.

2. The assay kit according to claim 1, characterized in that the upconversion nanoparticles are selected from particles with a diameter of 25 ~ 30 nm; preferably NaYF,:Yb”*,Tm°* or NaYF,:Yb”*,Er”* .

3. The assay kit according to claim 1, characterized in that the upconversion nanoparticles and the magnetic nanoparticles are preserved in

0.01 M PBS, pH preferably 7.0.

4. The assay kit according to claim 1, characterized in that the concentration of the upconverted nanoparticles coupled with the fumonisin B1 antigen is 2 mg/mL, and/or, the concentration of magnetic nanoparticles coupled with a fumonisin B1 monoclonal antibody is 2 mg/mL, and/or, the concentration of the upconversion nanoparticles coupled with zearalenone antigen is 2 mg/mL, and/or, the concentration of magnetic nanoparticles coupled with zearalenone monoclonal antibody is 2 mg/mL.

27

5. The assay kit according to claim 1, characterized in that the standard solution is a series of standard solutions with a concentration of 0.05 ~ Sng/mL.

6. The method for preparing the assay kit according to claims 1 to 5, characterized in that the method for preparing upconversion nanoparticles coupled to an antigen of the mycotoxin to be tested comprises at least the steps of modifying the aminated upconversion nanoparticles with streptavidin using the glutaraldehyde method to obtain streptavidin-modified upconversion nanoparticles; incubating the streptavidin-modified upconversion nanoparticles with a biotinylated artificial antigen of the mycotoxin to be tested; preferably, the concentration of the upconverted nanoparticles is 2 mg/mL and the concentration of the biotinylated mycotoxin artificial antigen to be tested is 1.5 mg/mL; more preferably, the temperature of the incubation is 37°C and the time of incubation is 4 to 8 hours.

7. À method for detecting of a variety of mycotoxins, using the reagents in the assay kit according to any one of claims 1 to 5, comprising at least the steps of S1. drawing a standard curve, mixing a series of concentration of the mycotoxin standard solution with the upconversion nanoparticles coupled with mycotoxin antigen, respectively, adding the magnetic nanoparticles coupled with mycotoxin monoclonal antibody for binding, magnetic separation, collecting the supernatant, detecting the fluorescence signals corresponding to different mycotoxins in the supernatant, and establishing a standard curve of mycotoxin standard concentration and fluorescence signal, 28

S2. detecting the sample to be tested, mixing the sample to be tested with the mycotoxin standard to prepare a mixed sample to be tested; mixing the mixed sample to be tested with the upconversion nanoparticles coupled with mycotoxin antigen, adding the magnetic nanoparticles coupled with mycotoxin monoclonal antibody for binding, magnetic separation, collecting the supernatant, detecting the fluorescence signals corresponding to different mycotoxins in the supernatant; S3. calculating the concentration of mycotoxins in the solution to be tested according to the standard curve.

8. The method according to claim 7, characterized in that in S1, taking 50 uL each of mycotoxin standard solution and adding 160 uL of upconversion nanoparticles coupled with FB1 antigen and 180 uL of upconversion nanoparticles Biotin-ZEN-BSA-UCNPs coupled with ZEN antigen; adding 10 pl. of magnetic nanoparticles coupled with FBI monoclonal antibody and 10 uL of magnetic nanoparticles coupled with ZEN monoclonal antibody for competition; the competition is preferably performed under conditions of shaking reaction at room temperature for 30 minutes to 2 hours, preferably 1 hour.

9. The method according to claim 7, characterized in that in S2, mixing the sample to be tested with the standard solution of the standard mycotoxin to obtain the mixed sample to be tested, so that the final concentrations of the standard mycotoxin are 0.1 ng/mL, 0.5 ng/mL and 2 ng/mL, respectively, and taking 50 pL each of the mixed sample to be tested solution, adding 160 pL. of upconversion nanoparticles coupled with FB1 antigen and 180 pL of upconversion nanoparticles Biotin-ZEN-BSA-UCNPs coupled with ZEN antigen; adding 10 uL of magnetic nanoparticles coupled with FB1 monoclonal antibody and 10 uL of magnetic nanoparticles coupled with ZEN monoclonal antibody for competition; 29 the conditions of the competition are preferably a shaking reaction at room temperature for 30 minutes to 2 hours, preferably 1 hour, and/or, the method further comprising the step of extracting the sample to be tested, preferably, an aqueous solution of methanol is used to extract the sample to be tested.

10. Application of the assay kit according to any one of claims 1 to 5 or the method according to any one of claims 7 to 9 in the detection of a variety of mycotoxins in food and feed.