Kit for detecting ochratoxin a and aflatoxin b1 and application thereof

Abstract

The present application relates to the technical field of chemical detection, and discloses a kit for detecting ochratoxin A and aflatoxin B1 and application thereof.The kit comprises a 96-well plate, photosensitive microspheres-ochratoxin A-BSA-Ag complex and / or photosensitive microspheres-aflatoxin B1-BSA-Ag complex, luminescent microspheres-ochratoxin A-Ab complex and / or luminescent microspheres-aflatoxin B1-Ab complex.The kit for detecting ochratoxin A and aflatoxin B1 has a short detection time of only 12 min, greatly reducing the detection time, and in actual sample detection, the recovery rate of OTA is greater than 94%, and the RSD is less than 4.7%;the recovery rate of AFB1 is greater than 90%, and the RSD is less than 4.2%, indicating that the method has good precision and good accuracy.

Description

Technical Field

[0001] This invention relates to the field of chemical detection technology, and in particular to kits for detecting ochratoxin A and aflatoxin B1 and their applications. Background Technology

[0002] Ochratoxin A (OTA) is a highly toxic fungal toxin produced by various Penicillium and Aspergillus molds. It affects humans by contaminating crops and animal feed; therefore, moldy feed, grains, and their products are major sources of OTA. Ochratoxin A primarily damages the kidneys and liver, exhibiting nephrotoxicity, hepatotoxicity, teratogenicity, carcinogenicity, and mutagenicity. Most importantly, OTA accumulates in the food chain; long-term consumption of food and water containing OTA can lead to serious health problems and pose a significant health risk. Similarly, aflatoxin B1 (AFB1) is a highly toxic fungal toxin widely present in nature. Due to its excellent heat and light stability, it is not easily decomposed under conventional cooking and heating methods, and has been classified as a Group 1 carcinogen by the International Agency for Research on Cancer. OTA and AFB1, as widely contaminating fungal toxins, can contaminate grains and foods, thus directly affecting the human body.

[0003] This invention is proposed to enable rapid on-site detection of ochratoxin A and aflatoxin B1. Summary of the Invention

[0004] The purpose of this invention is to provide a kit for detecting ochratoxin A and aflatoxin B1 and its application. The kit requires only 12 minutes for detection, which greatly reduces the detection time. In actual sample detection, the spiked recovery rate of OTA is greater than 94%, and the RSD is less than 4.7%; the spiked recovery rate of AFB1 is greater than 90%, and the RSD is less than 4.2%, indicating that the method has good precision and accuracy.

[0005] To achieve the above objectives, the present invention provides a kit for detecting ochratoxin A and aflatoxin B1, comprising a 96-well plate, a photosensitive microsphere-ochratoxin A-BSA-Ag complex and / or a photosensitive microsphere-aflatoxin B1-BSA-Ag complex, a luminescent microsphere-ochratoxin A-Ab complex and / or a luminescent microsphere-aflatoxin B1-Ab complex; The luminescent microsphere-ochratoxin A-Ab complex and the luminescent microsphere-aflatoxin B1-Ab complex were added to 96-well plates, respectively. The sample solution to be tested was added, and the plates were incubated for 6 min. Immediately afterwards, the photosensitive microsphere-ochratoxin A-BSA-Ag complex and the photosensitive microsphere-aflatoxin B1-BSA-Ag complex were added, respectively, and the plates were incubated for 6 min. After the incubation, the luminescence signal value was measured immediately.

[0006] Furthermore, the photosensitive microsphere-ochratoxin A-BSA-Ag complex is obtained by coupling photosensitive microspheres with ochratoxin A-BSA-Ag, and the photosensitive microsphere-aflatoxin B1-BSA-Ag complex is obtained by coupling photosensitive microspheres with aflatoxin B1-BSA-Ag. The luminescent microsphere-ochratoxin A-Ab complex was obtained by coupling luminescent microspheres with ochratoxin A-Ab, and the luminescent microsphere-aflatoxin B1-Ab complex was obtained by coupling luminescent microspheres with aflatoxin B1-Ab.

[0007] Furthermore, the concentration of photosensitive microspheres was 1 mg / mL, the biological concentration of luminescent microspheres was 1 mg / mL; the concentration of aflatoxin B1-BSA-Ag was 11 mg / mL, the concentration of aflatoxin B1-Ab was 8.2 mg / mL; the concentration of ochratoxin A-BSA-Ag was 10.5 mg / mL, and the concentration of ochratoxin A-Ab was 7.5 mg / mL. In the photosensitive microsphere-ochratoxin A-BSA-Ag complex, the ratio of photosensitive microspheres to ochratoxin A-BSA-Ag when coupled is 1 mL: 2 μL. In the photosensitive microsphere-aflatoxin B1-BSA-Ag complex, the ratio of photosensitive microspheres to aflatoxin B1-BSA-Ag when coupled is 1 mL: 2 μL. In the luminescent microsphere-ochratoxin A-Ab complex, the ratio of luminescent microspheres to ochratoxin A-Ab during coupling is luminescent microspheres: ochratoxin A-Ab = 1 mL: 2 μL; In the luminescent microsphere-aflatoxin B1-Ab complex, the ratio of luminescent microspheres to aflatoxin B1-Ab during coupling is luminescent microspheres:aflatoxin B1-Ab = 1 mL: 2 μL.

[0008] Furthermore, the method for preparing the photosensitive microspheres is as follows: Step 1, Preparation of dihydroxysilylphthalocyanine SiPc(OH)2: Dichlorosilylphthalocyanine was placed in an ice-water bath, and 98% sulfuric acid was slowly added while maintaining the ice-water bath. The mixture was stirred until completely dissolved. The solution was then filtered into an Erlenmeyer flask filled with ice water. The Erlenmeyer flask was wrapped with aluminum foil and placed in a refrigerator at 4°C. Crystals were allowed to precipitate by standing. The precipitate was then filtered and washed until neutral. The precipitate was dried in a vacuum drying oven and the powder was collected for later use. Step 2, Preparation of functionalized silicon phthalocyanine derivatives with flexible unsaturated side chains: Weigh out dihydroxysilyl phthalocyanine, and under nitrogen protection, first add DMF and stir for 10 min. Then add 0.08 mL of a mixture of 3-(trimethoxysilyl)propyl methacrylate (MPS) and 1 mL of N,N-dimethylformamide (DMF), and heat and stir continuously. Pour the reaction mixture into ice water, place it in a refrigerator at 4°C, and let it precipitate overnight. Filter, wash the precipitate, and collect it in a desiccator after vacuum drying. Step 3, Preparation of photosensitive microspheres: Weigh the functionalized phthalocyanine derivative, add 1 mL of benzyl alcohol and sonicate to dissolve. Mix the solution with ethylene glycol and add it dropwise to a vial containing carboxylated polystyrene microspheres. Stir while adding the solution at 60°C. Then place the vial in an oil bath and stir magnetically for 10-15 min. Centrifuge the solution under standard conditions for 20 min. Wash the precipitate several times with anhydrous ethanol and then wash it with ultrapure water. Finally, resuspend the precipitate in ultrapure water and store it in a refrigerator at 4°C in the dark for later use.

[0009] Furthermore, the method for preparing the luminescent microspheres is as follows: Mix the europium ternary complex Eu(TTA)3phen solution, thioxene solution, and 9,10-bis(phenylethynyl)BPEA solution. Add ethylene glycol and benzyl alcohol to the mixed solution. Add the solution dropwise to a vial containing 200 µL of carboxylated polystyrene microspheres while stirring. Then place the vial in an oil bath and stir magnetically. Centrifuge the solution for 20 min. Wash the precipitate several times with anhydrous ethanol. Finally, resuspend the precipitate in ultrapure water and store it in a refrigerator at 4°C in the dark for later use.

[0010] Furthermore, the concentration of Eu(TTA)3phen solution was 10 mg / mL, the concentration of Thioxene solution was 10 mg / mL, and the concentration of BPEA solution was 2 mg / mL; the volume ratio of Eu(TTA)3phen solution, Thioxene solution, BPEA solution, ethylene glycol, and benzyl alcohol was 400 µL: 60 µL: 38 µL: 1162 µL: 140 µL, with a total volume of 1.8 mL.

[0011] This invention also provides a method for using the above-mentioned reagent kit, including the following steps: The luminescent microsphere-ochratoxin A-Ab complex and the luminescent microsphere-aflatoxin B1-Ab complex were added to 96-well plates, respectively. The sample solution to be tested was added, and the plates were incubated for 6 min. Immediately afterwards, the photosensitive microsphere-ochratoxin A-BSA-Ag complex and the photosensitive microsphere-aflatoxin B1-BSA-Ag complex were added, respectively, and the plates were incubated for 6 min. After the incubation, the luminescence signal value was measured immediately.

[0012] The present invention also provides the application of the above-mentioned kit in the detection of ochratoxin A and aflatoxin B1, wherein the application is not for the purpose of diagnosis or treatment of disease.

[0013] The advantages and positive effects of the kit for detecting ochratoxin A and aflatoxin B1 described in this invention and its application are as follows: 1. The method of using the kit in this invention is as follows: Add the luminescent microsphere-ochratoxin A-Ab complex and the luminescent microsphere-aflatoxin B1-Ab complex to a 96-well plate, add the sample solution to be tested, and incubate for 6 minutes. Immediately afterwards, add the photosensitive microsphere-ochratoxin A-BSA-Ag complex and the photosensitive microsphere-aflatoxin B1-BSA-Ag complex, respectively, and incubate for 6 minutes. After incubation, immediately measure the luminescence signal value. It can be seen that the kit of this invention requires only 12 minutes (6 minutes + 6 minutes) for detection, greatly reducing the detection time.

[0014] 2. This invention uses a 96-well plate for detection, which has low detection cost. In actual sample detection, the spiked recovery rate of OTA is greater than 94%, and the RSD is less than 4.7%; the spiked recovery rate of AFB1 is greater than 90%, and the RSD is less than 4.2%, indicating that the method has good precision and accuracy.

[0015] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0016] Figure 1 In the embodiments of the present invention (SiPC, Osi (C9H) 17 Infrared spectrum of O4)(2); Figure 2 In the embodiments of the present invention (SiPC, Osi (C9H) 17 The ultraviolet spectrum of O4)(2); Figure 3 The excited-state relaxation path of the material is obtained based on DFT calculation in the embodiments of the present invention; Figure 4The standard curves for ochratoxin A and aflatoxin B1 in this embodiment of the invention are shown below. (A) represents the standard curves for different concentrations of ochratoxin A antigen; (B) represents the standard curves for different concentrations of aflatoxin B1 antigen. Figure 5 The repeatability results in the embodiments of the present invention are as follows: (A) represents the repeatability of the method for detecting ochratoxin A; (B) represents the repeatability of the method for detecting aflatoxin B1. Figure 6 The results in this embodiment of the invention are selective, wherein (A) is the selectivity of the method for detecting ochratoxin A; and (B) is the selectivity of the method for detecting aflatoxin B1. Figure 7 The results of anti-interference in the embodiments of the present invention are as follows: (A) represents the anti-interference of the method for detecting aflatoxin B1, and (B) represents the anti-interference of the method for detecting ochratoxin A. Detailed Implementation

[0017] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0018] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0019] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0020] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention.

[0021] MPS, whose Chinese name is 3-(trimethoxysilyl)propyl methacrylate, is sourced from Shenzhen Huaan Tianxia Technology Co., Ltd.; DMF, whose Chinese name is N,N-dimethylformamide, is sourced from Shanghai Maclean. Eu(TTA)3phen is a europium ternary complex; Thioxene, whose Chinese name is thiophene, is sourced from Anaiji Chemical Co., Ltd.; BPEA, whose Chinese name is 9,10-bis(phenylethynyl), is sourced from Xiya Chemical Co., Ltd.

[0022] This invention employs a competitive method to capture and detect OTA and AFB1.

[0023] Photocatalytic chemiluminescence (LICA) is a homogeneous, wash-free chemiluminescence technology. This technology utilizes two key types of microspheres: photosensitive microspheres and luminescent microspheres. The photosensitive microspheres contain photosensitive materials capable of generating high-energy reactive oxygen species, while the luminescent microspheres contain luminescent materials capable of accepting energy transferred from these high-energy reactive oxygen species.

[0024] This invention couples the BSA antigen of the target toxin (OTA / AFB1) to photosensitive microspheres, and then couples the antibody of the target toxin (OTA / AFB1) to luminescent microspheres. When the target antigen is not present in the system, the photosensitive microspheres with the target toxin (OTA / AFB1)-BSA antigen and the luminescent microspheres with the target toxin (OTA / AFB1) antibody are brought closer together due to the specific action of the antigen and antibody, thus forming a photosensitive microsphere-BSA-antigen-antibody-luminescent microsphere complex. At this time, the distance between the photosensitive microspheres and the luminescent microspheres is reduced to less than 200 nm. When the photosensitive microspheres are excited by 680 nm excitation light, excited oxygen is generated, which transfers energy to the luminescent microspheres, thereby generating light of about 610 nm, and the instrument detects the signal value. When the test antigen is present in the system, the test antigen competes with the antigen coupled on the photosensitive microspheres. The test antigen binds to the antibody on the luminescent microspheres, resulting in a distance of more than 200 nm between the photosensitive microspheres and the luminescent microspheres. As a result, reactive oxygen species cannot transfer energy to the luminescent microspheres and therefore do not emit light.

[0025] The following examples provide a detailed description.

[0026] Example 1 Experimental Method Taking OTA detection as an example (the detection process for AFB1 is the same as that for OTA): by coupling photosensitive microspheres with OTA-BSA-Ag, a DMSs-OTA-BSA-Ag complex is obtained; by coupling luminescent microspheres with OTA-Ab, an RMSs-OTA-Ab complex is obtained; and then OTA is detected based on a photo-induced chemiluminescence detection platform.

[0027] 1. Preparation of dihydroxysilylphthalocyanine (SiPc(OH)2): First, weigh 250 mg of dichlorosilylphthalocyanine into a brown container. Place the container in an ice-water bath and slowly add 4.0 mL of sulfuric acid (98% by mass). Maintain the ice-water bath and stir for 2 hours until completely dissolved. Then, filter the solution into a 250 mL Erlenmeyer flask filled with ice water. After filtration, wrap the Erlenmeyer flask with aluminum foil and place it in a refrigerator at 4°C. After standing for 48 hours, crystals will precipitate. Filter and wash until neutral. Place the precipitate in a vacuum drying oven and dry at 60°C for 6 hours. Collect the powder and store it in a desiccator for later use. It is particularly important to note that the temperature must be controlled during the ice-water bath process, ensuring it remains below 5°C. The reaction process must be conducted in the dark.

[0028] 2. Functionalized silicon phthalocyanine derivatives (SiPC, Osi(C9H)) with flexible unsaturated side chains 17 Preparation of O4)2): Weigh 40 mg of dihydroxysilylphthalocyanine. Under nitrogen protection, first add 7 mL of DMF and stir for 10 min. Then add a mixture of (0.08 mL MPS + 1 mL DMF), heat to 40 °C, and stir continuously for 8 h. Pour the reaction mixture into an equal volume of ice water, place in a refrigerator at 4 °C overnight to precipitate a solid. Filter, wash, and vacuum dry the precipitate, then collect it in a desiccator for storage. The reaction process is carried out in the dark.

[0029] 3. Preparation of photosensitive microspheres: Weigh 10 mg of (SiPC, Osi (C9H) 17 O4) 2) Dissolve 1 mL of benzyl alcohol in a brown EP tube by sonication. Mix 0.8 mL of the above solution with 1 mL of ethylene glycol, and then add dropwise to a vial containing 200 µL of carboxylated polystyrene microspheres (CPMSs) (10% solid content). Stir continuously while adding the solution at 60 °C. Then, place the vial in an oil bath at 110 °C and stir magnetically for 10-15 min. Centrifuge the solution under standard conditions for 20 min. Wash the precipitate several times with anhydrous ethanol until the supernatant is colorless. Finally, wash once with ultrapure water. Resuspend the precipitate in 1 mL of ultrapure water and store in the dark at 4 °C.

[0030] 4. Preparation of luminescent microspheres: Mix 400 µL of a 10 mg / mL Eu(TTA)3phen solution, 60 µL of a 10 mg / mL Thioxene solution, and 38 µL of a 2 mg / mL BPEA solution. Add 1162 µL of ethylene glycol and 140 µL of benzyl alcohol to the mixture, bringing the total volume to 1.8 mL. Add the solution dropwise to a vial containing 200 µL of carboxylated polystyrene microspheres (CPMSs) (10% solids content) while stirring. Place the vial in an oil bath at 110 °C and stir magnetically for 10–15 min. Centrifuge the solution under standard conditions for 20 min. Wash the precipitate repeatedly with anhydrous ethanol until the supernatant shows no fluorescence when irradiated with a UV lamp. Finally, resuspend the precipitate in 1 mL of ultrapure water and store in the dark at 4 °C.

[0031] 5. Microsphere-conjugated antibody / BSA-antigen: 1) Take 20 μL of photosensitive microspheres (the same steps for coupling luminescent microspheres are used below) and add them to a 2 mL transparent centrifuge tube containing 1 mL of activation buffer (pH 7.4, 0.05 M MES). Mix well by sonication, then centrifuge at 11000 rpm and 4℃ for 35 min and discard the supernatant.

[0032] 2) Add 1 mL of activation buffer to the precipitate and sonicate to mix. Then, immediately add 6 μL of EDC (10 μg / μL) to the dispersed microspheres, vortex to mix, and then add 60 μL of NHS (10 μg / μL), vortex to mix, seal with sealing film, and place in a constant temperature shaking instrument at 70 r / min and 25℃ for 20 min to activate.

[0033] 3) Once activation is complete, immediately place the centrifuge tube in a centrifuge and centrifuge at 11,000 rpm and 4°C for 35 minutes, then discard the supernatant.

[0034] 4) Add activation buffer to the precipitate again, centrifuge again under the same conditions, and discard the supernatant.

[0035] 5) Then add ultrapure water to the precipitate, ultrasonically clean it, and then centrifuge it again under the same conditions, discarding the supernatant.

[0036] 6) First, add 1 mL of microsphere coupling solution to the washed microspheres and disperse by sonication. Then, carefully add the following separately: Photosensitive microspheres coupled with ochratoxin A-BSA (OTA-BSA-Ag) were added to 2 μL to form a DMSs-OTA-BSA-Ag complex; 2 μL of luminescent microspheres conjugated with ochratoxin A antibody (OTA-Ab) were added to form an RMSs-OTA-Ab complex. 7) After carefully mixing, seal with sealing film and place in a constant temperature shaking instrument to avoid light. Incubate at 70 r / min, 25℃ for 120 min.

[0037] 8) After incubation, add 100 μL of Quick Block microsphere blocking solution to each microsphere. TM After shaking to mix the Western blocking solution, continue incubation for 120 min.

[0038] 9) After incubation, place the centrifuge tube in a centrifuge and centrifuge at 13500 rpm and 4℃ for 35 min. Discard the supernatant. Add 1 mL of 0.15% Tween-20 to the precipitate, sonicate to mix, centrifuge again, and finally add 1 mL of homogeneous luminescent diluent. Store at 4℃.

[0039] 6. Photosensitive materials (SiPC, Osi (C9H) 17 Characterization and theoretical calculation of O4)(2): The successful synthesis of the photosensitive material was confirmed by infrared and ultraviolet characterization, and the structure-property relationship was established through theoretical calculations to analyze the mechanism of action of the photosensitive material.

[0040] 7. Linear: Ochratoxin A standards of 78.1 ng / mL, 39.2 ng / mL, 9.8 ng / mL, 1.2 ng / mL, 0.3 ng / mL, 0.07 ng / mL, and 0.009 ng / mL were prepared in 96-well plates. First, the luminescent microsphere-conjugate antibody complex was added to the standards and mixed thoroughly. The mixture was incubated at 37°C for 6 min. Then, the photosensitive microsphere-conjugate BSA-antigen was added and mixed thoroughly, followed by incubation for another 6 min. After incubation, the luminescence signal value was immediately measured.

[0041] 8. Repeatability: Take 20 µL of the luminescent microsphere-conjugated antibody complex (RMSs-OTA-Ab complex) into each well of a 96-well test plate, add 20 µL of ochratoxin A standard, and incubate in an incubator with shaking for 6 min. Immediately afterward, add 20 µL of the photosensitive microsphere-conjugated BSA-antigen complex (DMSs-OTA-BSA-Ag complex), and incubate in an incubator with shaking for 6 min. After shaking, immediately measure the luminescence signal value.

[0042] 9. Selectivity: Selective determination of other toxins that may interfere with the determination of mycotoxins was performed. These toxins included zearalenone (ZEN), fumonisin (FB1), aflatoxin B1 (AFB1), T-2 toxin, vomitoxin (DON), patulin, and aflatoxin M1 (AFM1). The photoluminescence values ​​were determined according to the above detection steps.

[0043] 10. Anti-interference capability: Select some interfering substances that may interfere with the detection, and add zearalenone (ZEN), fumonisin (FB1), aflatoxin B1 (AFB1), T-2 toxin, vomitoxin (DON), patulin, and aflatoxin M1 (AFM1) respectively. Mix them with 10 µL of ochratoxin A at a concentration of 1 ng / mL, and determine their photo-induced chemiluminescence values ​​according to the above detection steps, and calculate the recovery rate.

[0044] 11. Testing of quality control samples: Quality control samples were purchased from the market for testing to determine the accuracy and reliability of this method in practical applications. To more accurately determine sample concentration, homogenized corn flour samples were used. Ochratoxin A was extracted from the samples according to the instructions, and the baseline concentration was determined. The recovery rate was then determined by adding ochratoxin A standards to the samples, thus confirming the accuracy and reliability of this method in actual sample testing.

[0045] Result Analysis of Example 2 like Figure 1 As shown, it is located between 1550-1620cm. -1 The vibrations between these vibrations belong to the skeletal stretching vibrations of C=C and C=N. These are the core vibrations of the phthalocyanine macrocyclic conjugated Π system and are one of the most important hallmarks of phthalocyanine ring formation. The vibrations located at 1480-1520 cm⁻¹ are... -1 Between these points, the skeletal stretching vibrations belonging to the CC and CN rings also originate from the breathing and stretching vibrations of the macrorings. These vibrations are typically located between 1320 and 1390 cm. -1 The peak between these two points corresponds to the CN stretching vibration of the isoindole ring (the portion connecting the benzene ring and the central nitrogen atom). This peak is very characteristic and has a relatively high intensity, typically located between 1100 and 1225 cm⁻¹. -1 This is attributed to the in-plane bending vibration of CH (related to the benzene ring). 1000-1100 cm⁻¹ -1 Within this range, it belongs to the concerted in-plane bending vibration of two adjacent CH bonds on the benzene ring. It is typically located at 720-745 cm⁻¹. -1 The out-of-plane bending vibration of CH, belonging to the phthalocyanine macrocycle, is another very characteristic fingerprint peak. 580cm -1 The absorption peak is attributed to the bending vibration of —Si—O—Si—, 910 cm⁻¹ -1 The strong absorption peak at [value missing] is attributed to the vibration of the Si-N bond in the phthalocyanine molecule. It is located at ~980-1020 cm⁻¹. -1 The peak at 1720 cm⁻¹ belongs to the Si-O stretching vibration and is the most critical characteristic peak of this material. -1 The strong absorption peak at 1166 cm⁻¹ is attributed to the stretching vibration of —O—CO (the stretching of the α,β unsaturated ester group). -1 The peak value at 3084 cm⁻¹ is attributed to the stretching vibration of the CoC. -1 and 3034 cm -1 The absorption peak at that point is attributed to the stretching vibration of the C-H ring in the benzene ring.

[0046] Ultraviolet-Vis absorption spectrum (UV-Vis) such as Figure 2 As shown, two characteristic absorption bands appear in the 300-400nm and 600-700nm ranges, corresponding to the B band and Q band in phthalocyanine dyes, respectively, proving the successful synthesis of the photosensitive material.

[0047] To more comprehensively evaluate the energy transfer, storage, and release capabilities of materials in excited states, the singlet-triplet bandgap (S1-T1) was determined by calculating the energy difference between the lowest excited singlet state (S1) and the lowest excited triplet state (T1). The S1 energy was obtained from TD-DFT calculations, while the T1 energy was calculated using an optimized singlet ground-state geometry and an unrestricted density functional theory method at the B3LYP / 6-31G(d) level. Figure 3 As shown, the process of singlet oxygen generation is as follows: the excited-state relaxation pathways of the three materials are similar, all first absorbing a photon and transitioning from the ground state (S0) to a singlet excited state (S1, S2, S3, S4, etc.). The high-energy states (such as S2, S3, S4) relax to the lowest singlet excited state (S1) through extremely fast internal conversion (IC). The exciton in the S1 state can transition back to S0 via fluorescence radiation or enter the long-lived triplet excited state (T1) via intersystem crossing (ISC). The T1 state can generate singlet oxygen through phosphorescence or energy transfer. Furthermore, as can be seen from the figure, S1 and S2 are concentrated in the medium energy range, while S3, S4 and the key T3 are clustered in the high energy region. The energies of the three are close, indicating that the intersystem crossing from S3 / S4 to T3 has a small energy gap. Moreover, the energy level of T3 is below S4 but higher than T1 and T2, showing a clear energy downslope from the high-energy singlet state (S3 / S4) to T3, which meets the thermodynamic requirements of intersystem crossing. The figure visually verifies the feasibility of energy transfer, namely the "S→T" energy transfer path.

[0048] According to the data in Table 1, SiPC, OSi(C9H) 17 The S1 states of O4)2 are relatively independent, and the energy gap between the lowest singlet excited state (S1) and the lowest triplet excited state (T1) is the smallest, proving that SiPC,OSi(C9H) 17 O4)2 has a high singlet oxygen yield, making it a highly efficient photosensitizer.

[0049] Table 1 Energy level data of three photosensitive materials

[0050] Under optimal detection conditions, different concentrations of ochratoxin A antigen and different concentrations of aflatoxin B1 antigen were measured using a photo-induced chemiluminescence detection platform. Figure 4 As shown in A and B, the linear equation of OTA is obtained as follows: I =-0.1292 c +0.5392, R 2 =0.9985, the linear range was 0.009~78.1 ng / mL, and the limit of detection was 3.7 ng / L; the linear equation for AFB1 was... I =-0.1024 c+0.3752, R 2 =0.9943, the linear range was 0.009 ~78.1 ng / mL, and the limit of detection was 0.88 ng / L.

[0051] Under optimal conditions, the repeatability of ochratoxin A and aflatoxin B1 was determined using a photo-induced chemiluminescence detection platform. Each test was repeated 10 times, and the relative standard deviation (RSD) was calculated. The results are as follows: Figure 5 As shown in Figure AB, the RSD is greater than 4.0%, indicating that the method has good repeatability.

[0052] Selectively determine several possible interfering substances. Figure 6 As shown in section A, the test results with other interfering substances added at concentrations as high as 2500 ng / mL showed almost no inhibitory signal compared to the addition of the target toxin alone (OTA = 1.22 ng / mL), and from... Figure 6 As shown in Figure B, the test results with other interfering substances added at a concentration as high as 2500 ng / mL showed almost no inhibitory signal compared to the addition of the target toxin alone (AFB1 = 0.61 ng / mL). The experimental results indicate that the corresponding detection reagent prepared by this method only responds to the target substance corresponding to the detection reagent. This demonstrates the good specificity of the method.

[0053] Several possible interfering agents were selected for anti-interference testing, and the results were determined by... Figure 7 It can be seen that both methods have good anti-interference ability, and the calculated recovery rate is greater than 95%, indicating that the methods have high accuracy.

[0054] To evaluate the practicality of the established AlphaLISA detection platform, corn flour quality control samples containing ochratoxin A / aflatoxin B1 were selected as the samples. Under optimal conditions, the established detection platform was used, and the supernatant of the quality control samples was mixed with 10X PBS (pH=7.4) buffer at a ratio of 1:4. Data acquisition was performed according to the AlphaLISA method described above, as shown in Table 2. The results showed that the recovery rate of OTA in the quality control samples was greater than 94%, and the RSD was less than 4.7%, indicating that the method has high precision and good accuracy.

[0055] Similarly, as shown in Table 3, the results show that the spiked recoveries of the quality control sample AFB1 are all greater than 90%, and the RSDs are all less than 4.2%, indicating that the method has high precision and good accuracy.

[0056] Table 2. OTA spike recovery in quality control samples (n=3)

[0057] Table 3. Recovery of AFB1 spiked in quality control samples (n=3)

[0058] The detection kit described in this invention has a wide detection limit when detecting ochratoxin A and aflatoxin B1. It is homogeneous and wash-free, easy to operate, has good specificity, good repeatability, and good anti-interference. It can be used for rapid detection of mycotoxin contaminants and has application value in mycotoxin detection.

[0059] Therefore, the present invention employs the above-mentioned kit for detecting ochratoxin A and aflatoxin B1 and its application. The detection time is short, requiring only 12 minutes, which greatly reduces the detection time. At the same time, in actual sample detection, the spiked recovery rate of OTA is greater than 94%, and the RSD is less than 4.7%; the spiked recovery rate of AFB1 is greater than 90%, and the RSD is less than 4.2%, indicating that the method has good precision and accuracy.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. The application of a kit for detecting ochratoxin A and aflatoxin B1 in the detection of ochratoxin A and aflatoxin B1, wherein the application is not for the purpose of disease diagnosis or treatment, characterized in that: The kit includes a 96-well plate, photosensitive microspheres-ochratoxin A-BSA-Ag complex and / or photosensitive microspheres-aflatoxin B1-BSA-Ag complex, luminescent microspheres-ochratoxin A-Ab complex and / or luminescent microspheres-aflatoxin B1-Ab complex; The photosensitive microsphere-ochratoxin A-BSA-Ag complex is obtained by coupling photosensitive microspheres with ochratoxin A-BSA-Ag, and the photosensitive microsphere-aflatoxin B1-BSA-Ag complex is obtained by coupling photosensitive microspheres with aflatoxin B1-BSA-Ag. The luminescent microsphere-ochratoxin A-Ab complex was obtained by coupling luminescent microspheres with ochratoxin A-Ab, and the luminescent microsphere-aflatoxin B1-Ab complex was obtained by coupling luminescent microspheres with aflatoxin B1-Ab. The detection limit for ochratoxin A was 3.7 ng / L, and the detection limit for aflatoxin B1 was up to 0.88 ng / L. The concentration of photosensitive microspheres was 1 mg / mL, the biological concentration of luminescent microspheres was 1 mg / mL; the concentration of aflatoxin B1-BSA-Ag was 11 mg / mL, the concentration of aflatoxin B1-Ab was 8.2 mg / mL; the concentration of ochratoxin A-BSA-Ag was 10.5 mg / mL, and the concentration of ochratoxin A-Ab was 7.5 mg / mL. In the photosensitive microsphere-ochratoxin A-BSA-Ag complex, the ratio of photosensitive microspheres to ochratoxin A-BSA-Ag when coupled is 1 mL: 2 μL. In the photosensitive microsphere-aflatoxin B1-BSA-Ag complex, the ratio of photosensitive microspheres to aflatoxin B1-BSA-Ag when coupled is 1 mL: 2 μL. In the luminescent microsphere-ochratoxin A-Ab complex, the ratio of luminescent microspheres to ochratoxin A-Ab during coupling is luminescent microspheres: ochratoxin A-Ab = 1 mL: 2 μL; In the luminescent microsphere-aflatoxin B1-Ab complex, the ratio of luminescent microspheres to aflatoxin B1-Ab during coupling is luminescent microspheres:aflatoxin B1-Ab = 1 mL: 2 μL. The method for preparing the photosensitive microspheres is as follows: Step 1, Preparation of dihydroxysilylphthalocyanine SiPc(OH)2: Weigh 250 mg of dichlorosilylphthalocyanine into a container, place the container in an ice-water bath, slowly add 4 mL of 98% sulfuric acid, keep in the ice-water bath, and stir until completely dissolved. Then, filter the solution into an Erlenmeyer flask filled with ice water, wrap the Erlenmeyer flask with aluminum foil and place it in a refrigerator at 4°C to allow crystals to precipitate. Then filter and wash until neutral, place the precipitate in a vacuum drying oven to dry, and collect the powder for later use. Step 2, Preparation of functionalized silicon phthalocyanine derivatives with flexible unsaturated side chains: Weigh 40 mg of dihydroxysilyl phthalocyanine, and under nitrogen protection, first add 7 mL of LDMF and stir for 10 min. Then add 0.08 mL of a mixture of 3-(trimethoxysilyl)propyl methacrylate (MPS) and 1 mL of N,N-dimethylformamide (DMF). Heat to 40 °C and stir continuously for 8 h. Pour the reaction mixture into ice water and place it in a refrigerator at 4 °C overnight to precipitate the solid. Filter, wash the precipitate, and vacuum dry it. Collect the precipitate and store it in a desiccator. Step 3, Preparation of photosensitive microspheres: Weigh 10 mg of functionalized phthalocyanine derivative, add 1 mL of benzyl alcohol and sonicate to dissolve. Take 0.8 mL of the mixed solution and mix it with 1 mL of ethylene glycol. Then add the mixture dropwise to a vial containing 200 μL of carboxylated polystyrene microspheres. Stir while adding the mixture at 60 °C. Then place the vial in an oil bath at 110 °C and stir magnetically for 10-15 min. Centrifuge the solution under standard conditions for 20 min. Wash the precipitate several times with anhydrous ethanol. Finally, wash the precipitate with ultrapure water. Finally, resuspend the precipitate in ultrapure water and store it in the dark at 4 °C for later use. The method for preparing the luminescent microspheres is as follows: Take 400 μL of a 10 mg / mL europium ternary complex Eu(TTA)3phen solution, 60 μL of a 10 mg / mL thioxene solution, and 38 μL of a 2 mg / mL 9,10-bis(phenylethynyl)BPEA solution and mix them. Add 1162 μL of ethylene glycol and 140 μL of benzyl alcohol to the mixed solution, for a total volume of 1.8 mL. Add the solution dropwise to a vial containing 200 μL of carboxylated polystyrene microspheres while stirring. Then place the vial in an oil bath at 110 °C and stir magnetically. Centrifuge the solution for 20 min, wash the precipitate several times with anhydrous ethanol, and finally resuspend the precipitate in ultrapure water. Store the solution in a refrigerator at 4 °C in the dark for later use.

2. The application according to claim 1, characterized in that, The application method includes the following steps: The luminescent microsphere-ochratoxin A-Ab complex and the luminescent microsphere-aflatoxin B1-Ab complex were added to 96-well plates, respectively. The sample solution to be tested was added, and the plates were incubated for 6 min. Immediately afterwards, the photosensitive microsphere-ochratoxin A-BSA-Ag complex and the photosensitive microsphere-aflatoxin B1-BSA-Ag complex were added, respectively, and the plates were incubated for 6 min. After the incubation, the luminescence signal value was measured immediately.

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