Probe for detecting t-2 toxin, kit and application thereof

Abstract

This invention discloses a probe, kit, and application for detecting T-2 toxin. The disclosed probe preparation method includes: adding a T-2 toxin monoclonal antibody to an aqueous solution of cerium vanadate nanomaterials and allowing it to stand at room temperature; then adding BSA for reaction; subsequently, centrifuging to collect the solid reaction product, and washing the product to obtain the T-2 toxin detection probe. The disclosed kit contains the probe of this invention. This invention significantly improves the sensitivity for T-2 detection and can be applied to the detection of related toxins in food.

Description

Technical Field

[0001] This invention designs a mycotoxin immunoassay technique, which relates to a probe, a kit for detecting T-2 toxin, and their applications. Background Technology

[0002] Mycotoxins can have serious adverse effects on human health, such as immunotoxicity, neurotoxicity, and even carcinogenic effects.

[0003] T-2 toxin is a type of mycotoxin produced by various Fusarium species. The main strains that produce T-2 toxin in nature are Fusarium fungi, such as Fusarium trifidum, Fusarium spp., Fusarium cladosporum, Fusarium oxysporum, Fusarium moniliforme, and Fusarium moniliforme. T-2 toxin is a common contaminant in grains, especially oats, corn, and wheat.

[0004] T-2 toxin can spread through the food chain and bioaccumulation, contaminating animal-derived foods and posing a potential threat to human health. T-2 toxin exhibits hepatotoxicity, digestive toxicity, neurotoxicity, immunotoxicity, and reproductive toxicity, causing symptoms in animals and humans such as hepatocellular edema, gastrointestinal ulcers, meningeal hemorrhage, lymphocyte necrosis, and decreased pregnancy rates.

[0005] Existing technologies for detecting T-2 toxin include ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS), enzyme-linked immunosorbent assays (ELISAs), multiplex polymerase chain reaction (mPCR), and electrochemically induced sensors. These analytical methods have high sensitivity and specificity, but they require expensive equipment and cumbersome sample preparation and cleaning procedures, making them unsuitable for rapid detection. Summary of the Invention

[0006] In view of the defects and deficiencies in the prior art, the present invention provides a probe for detecting T-2 toxin.

[0007] Therefore, the probe preparation method provided by the present invention includes: adding T-2 toxin monoclonal antibody to an aqueous solution of cerium vanadate nanomaterial and placing it at room temperature, then adding BSA to react; then centrifuging to collect the solid reaction product, and washing the reaction product to obtain a probe for T-2 toxin detection;

[0008] The method for preparing cerium vanadate nanomaterials includes: adding a second solution dropwise to a first solution and adjusting the pH value to 9.5–10.5; the first solution is an aqueous solution of cerium nitrate and ethylenediaminetetraacetic acid; the second solution is an aqueous solution of ammonium metavanadate; then reacting at 2 MPa–32 MPa and 170–190 °C; and cooling to room temperature after the reaction is complete to obtain cerium vanadate nanomaterials.

[0009] An alternative approach is to add the second solution dropwise to the first solution and then adjust the pH to 10.

[0010] The preferred embodiment is that the cerium vanadate nanomaterial has a rod-shaped structure with a particle size distribution range of 800–1350 nm and an average particle length of 1057.72 nm.

[0011] The present invention also provides a T-2 toxin detection kit. For this purpose, the kit contains an immunochromatographic test strip adsorbed with T-2 toxin antigen, and also contains a reagent bottle containing the aforementioned probe or an aqueous solution of the probe.

[0012] The probes and kits of this invention can be used to detect T-2 toxin in food.

[0013] The probe of this invention is a novel nanoprobe prepared by labeling antibodies with cerium vanadate as a signal marker, which greatly improves the intensity of the colorimetric signal, makes the color signal easier to identify, and greatly enhances the sensitivity of detection; and the sensitivity reaches a new level: the probe provided by this invention has a limit of detection of 0.079 ng / mL for T-2 toxin; the probe of this invention has high specificity for T-2 toxin and no specificity for other toxins. Attached Figure Description

[0014] The accompanying drawings are provided to further illustrate the present disclosure and form part of the specification. They are used together with the following detailed description to explain the present disclosure, but do not constitute a limitation thereof. In the drawings:

[0015] Figure 1 This is a schematic diagram illustrating the working principle of the reagent kit of the present invention.

[0016] Figure 2 Characterization of the cerium vanadate nanomaterials prepared in the embodiments of the present invention; Figure AF shows scanning electron microscope (SEM) and high-resolution transmission electron microscope (HRTEM) images of CR1 (A, D), CR2 (B, E) and CR3 (C, F); Figure GI shows the particle size distribution of CR1, CR2 and CR3 respectively; Figure J shows the EDS pattern of CR2; K is the X-ray diffraction (XRD) pattern of CR2; LN is the UV-Vis, FT-IR and zeta potential analysis of CR2 and CR2-ICA probe respectively.

[0017] Figure 3The detection sensitivities of nanomaterial probes of different sizes prepared in the embodiments of the present invention are shown below; numbers 1-14 correspond to the concentrations of T-2 in the standards: 0, 0.02, 0.06, 0.15, 1.15, 2.6, 3.9, 5.85, 8.77, 13.16, 19.7, 29.6, 66.7 and 100 ng / mL, respectively; Figures A, C, and D are detection photographs of CR1-ICA, CR2-ICA and CR3-ICA probes, respectively; Figures DF and F are the relationships between T-2 concentration and detection line intensity after detection of CR1-ICA, CR2-ICA and CR3-ICA, respectively, with the inner figures showing their respective linear relationships; Figure G is a comparison of the detection range and limit of detection (LOD) of CR1-ICA, CR2-ICA and CR3-ICA.

[0018] Figure 4 To demonstrate the specificity and reproducibility of the probe prepared in this invention, Figure A shows the intra-batch and inter-batch recoveries when the T-2 concentrations are 13 ng / mL and 0.15 ng / mL, respectively; Figure B shows the specificity analysis of the probe, with numbers 1-8 representing: blank, patulin, T-2 toxin, fumonisin B1, zearalenone, citric acid, deoxynivalenol, and aflatoxin B1, respectively; Figure C compares the limit of detection of the probe of this invention with the linear range of existing detection methods; Figure D shows the reproducibility analysis of the probe.

[0019] Figure 5 This invention demonstrates the practical application of the immunochromatographic test strips. Figures A and B show the detection of corn (A) and oats (B) by the immunochromatographic test strips. In Figure A, 1-14 correspond to T-2 toxin concentrations of 0, 0.03, 0.04, 0.06, 0.51, 1.15, 1.73, 3.9, 8.77, 13.16, 19.7, 29.6, 44.4, and 100 ng / mL, respectively. In Figure B, 1-13 correspond to concentrations of 0, 0.03, 0.04, 0.06, 0.51, 1.15, 1.73, 3.9, 8.77, 13.16, 19.7, and 29.6, respectively. The T-2 toxin concentration was 66.7 ng / mL; Figure C shows the sample recovery rate detected by the probe; Figure D shows the detection of T-2 toxin in corn and oat flake samples at different concentrations (29.6 and 1.15 ng / mL).

[0020] Figure 6 The results are from the gold nanoparticle immunochromatographic test strip in Example 3 of this invention; the numbers 1-15 represent the concentrations of the standard product T-2: 0, 0.02, 0.06, 0.15, 1.15, 2.6, 3.9, 5.85, 8.77, 19.7, 66.7, 100, 150 and 200 ng / mL, respectively. Detailed Implementation

[0021] Unless otherwise specified, the scientific and technical terms used in this article are intended for understanding by those skilled in the art.

[0022] The immunochromatographic test strip used in this invention includes a backing plate with a nitrocellulose membrane attached to it. One end of the nitrocellulose membrane is covered with an absorbent pad, and the other end is covered with a sample pad and a conjugate pad. A detection line (T line) and a control line (C line) are set transversely on the uncovered surface of the nitrocellulose membrane. The conjugate pad and sample pad are soaked in blocking solution and then dried overnight in an oven at 37°C for blocking treatment. The test strip of this invention can be prepared using existing technologies, such as the preparation method disclosed in Bai F, Bu T, Li R, et al. Rose petals-like Bi semimetal embedded on the zeolitic imidazolate frameworks based-immunochromatographic strip to sensitively detect acetamiprid.[J]. Journal of hazardous materials, 2022. The preparation method of the test strip used in the following examples is as follows:

[0023] The preparation method of nitrocellulose membrane includes: coating 1 mg / mL T-2 toxin-bovine serum albumin conjugate onto the detection line at a streak rate of 0.8 μL / cm to obtain the detection line, and coating 1 mg / mL goat anti-mouse immunoglobulin onto the control line at a streak rate of 1 μL / cm to obtain the control line; then drying at 37°C for later use.

[0024] Preparation of sample pads: Cut glass fiber membranes into 15 mm long and 3 mm wide sizes, immerse them in a blocking solution (4% BSA), dry them at 37°C for 8 h to obtain sample pads, and then store them in a refrigerator at 4°C.

[0025] Preparation of the conjugate pad: The glass fiber membrane was cut into a size of 8 mm in length and 3 mm in width, immersed in the sealing solution (4% BSA), removed and dried at 37°C for 8 h to obtain the conjugate pad, and then stored in a refrigerator at 4°C.

[0026] Cut absorbent paper to a length of 18 mm and a width of 3 mm to obtain an absorbent pad;

[0027] Assembly of the test strip: First, attach the nitrocellulose membrane to the backing plate. Then, press the sample pad 2 mm onto the conjugation pad, press the conjugation pad 2 mm onto the nitrocellulose membrane, and press the absorbent pad 2 mm onto the nitrocellulose membrane in sequence. This gives you the immunochromatographic test strip for rapid detection of T-2 toxin.

[0028] See Figure 1 As shown. When using the kit of this invention, the sample pad of the test strip is first immersed in a mixed solution containing a CeVO4-mAb probe and the sample to be tested. The bound CeVO4-mAb-T-2 immune complex migrates to the test area of ​​the test strip via capillary action. On the nitrocellulose membrane, for the positive group, the T-2 toxin competes with the mAb for binding sites on the antigen, and the probe cannot be trapped on the test line. Therefore, the color intensity on the test line is inversely proportional to the concentration of T-2 toxin. For the negative group without T-2 toxin, the antigen and mAb specifically bind, so color is present on the test line. Based on this phenomenon, the amount of T-2 toxin in the sample solution affects the intensity of the test line color. The test results can be digitally evaluated using a test strip detector or visually evaluated with the naked eye.

[0029] The present invention will be further explained in conjunction with the following embodiments. All experimental reagents and testing instruments used in the following embodiments are commercially available.

[0030] Example 1:

[0031] 1.6 mmol of cerium nitrate hexahydrate and 2.0 mmol of ethylenediaminetetraacetic acid (EDTA) were dissolved in 15 mL of ultrapure water and mixed to obtain the first solution; 1.6 mmol of ammonium metavanadate (NH4VO3) was dissolved in 15 mL of ultrapure water at 80 °C to obtain the second solution;

[0032] Then the second solution was added dropwise to the first solution, and the pH of the solution was adjusted to 9, 10 and 12 respectively using 2M NaOH;

[0033] The three reactants were then sealed in a Teflon-lined stainless steel autoclave and reacted at 14 MPa - 17 MPa and 175 - 185 °C for 14 hours. After cooling to room temperature, they were washed twice with ultrapure water and ethanol to obtain CeVO4 nanorods.

[0034] Finally, the precipitate was resuspended in water, and the concentration of nanorods in each suspension after resuspension was 1 mg / mL.

[0035] CeVO4 nanorods of different sizes and shapes were named CR1 (pH=9), CR2 (pH=10), and CR3 (pH=12), respectively.

[0036] The materials synthesized in this embodiment were characterized as follows:

[0037] High-resolution transmission electron microscopy (HRTEM): See Figure 2 As shown in AF, SEM and TEM images indicate that, see Figure 2 As shown in GI, the prepared CR1, CR2, and CR3 have different shapes and sizes: CR1 is mainly slender rod-shaped with a particle size (diameter) distribution range of 500–900 nm and an average length (calculated using nanomeasuar software) of 710.10 nm; CR2 mainly appears as skeletal rods with wide wings and a thinner middle, radiating outwards at the edges, with a particle size distribution range of 800–1350 nm and an average length of 1057.72 nm; CR3 is a shorter rod-shaped material that tends to aggregate, with a particle size distribution range of 50–400 nm and an average length of 230.46 nm.

[0038] Example 2:

[0039] Furthermore, probes were prepared using the novel CeVO4 nanomaterials prepared in Example 1. The preparation method used CeVO4 nanomaterials (CR1, CR2, and CR3) as signal markers, and T-2 toxin monoclonal antibody was added for adsorption. A comparison of this novel nanoprobe with traditional colloidal gold probes was also provided, including the following steps:

[0040] T-2 toxin monoclonal antibody (10 μL, purchased from Luoyang Baitong Experimental Materials Center) was added to 1 mL of CeVO4 nanosignal marker, incubated at room temperature for 30 min, and then BSA was added to a final concentration of 1%. The mixture was vortexed for 30 min to block uncoupled sites.

[0041] Centrifuge and wash with water (6000 rpm, 5 min) to remove non-attached BSA and monoclonal antibodies, and resuspend in 1 ml of ultrapure water to obtain three probes CR1-ICA, CR2-ICA and CR3-ICA.

[0042] Example 3: Sensitivity determination of a probe for rapid detection of T-2 toxin

[0043] Detection process: The T-2 toxin standard was dissolved in ultrapure water and continuously diluted to produce test solutions with concentrations ranging from 0 to 100 ng / mL. Ultrapure water served as a blank control. At the same time, the same samples were tested using gold nanoparticle test strips.

[0044] Each probe prepared in Example 2 was mixed with 100 μL of each T-2 toxin standard solution to obtain a test solution. The test strip was then immersed in 100 μL of the test solution. After the reaction, the colorimetric signal intensity of the test strip was observed with the naked eye under ambient light. The T line and C line were quantitatively detected by a test strip detector (ZF-1, Hangzhou Qiwei Instrument Co., Ltd.).

[0045] The minimum concentration of T-2 toxin observed by the naked eye to be significantly lighter than the negative control bar is defined as the visual detection limit (vLOD); the minimum concentration at which the T line completely disappears is taken as the threshold concentration. Competitive inhibition rate IC50 10 Defined as the visual detection limit (LOD).

[0046] Test results: see Figure 3 As the concentration of T-2 toxin increases, the colorimetric signal intensity of the T line on the test strip becomes increasingly faint until it disappears. In the colorimetric test strip, probes CR1-ICA, CR2-ICA, and CR3-ICA showed linear dynamic ranges of 0.51-8.77 ng / mL, 1.15-29.6 ng / mL, and 2.60-13.16 ng / mL, respectively; the T line intensity (Y) showed a good correlation with Lg[T-2 toxin concentration](X), with equations f as follows: Y = -337.93X + 421.25, (R... 2 = 0.990); Y = -335.69X +562.09, (R 2 = 0.984) and Y = -674.38X + 918.07, (R 2 = 0.981).

[0047] The threshold concentrations of T-2 toxin for the three sample probes were 13.16 ng / mL, 5.85 ng / mL, and 8.77 ng / mL, respectively, with LOD values ​​of 0.39 ng / mL, 0.079 ng / mL, and 0.09 ng / mL, respectively. Overall, the probe using CR2 as a signal marker was superior to the other two, exhibiting a lower detection limit and a wider detection range. Furthermore, the detection limit of this probe was 19 times that of the traditional gold nanoparticle test paper sensor (1.494 ng / mL). (See [reference needed]). Figure 6 As shown.

[0048] Further characterization of CR2 and CR2-ICA probes:

[0049] (1) EDS plot: Figure 2 The EDS plot results of JCR2 show that there are several elements, including Ce, V and O, in CR2.

[0050] (2) X-ray diffraction (XRD) pattern: Figure 2 In K, all characteristic peaks correspond to standard main peaks. Meanwhile, the pure CeVO4 phase can be associated with the diffraction peaks of the nanorods, and the absence of peaks belonging to extra phases indicates the high purity of the material. Furthermore, the diffraction peaks show significant crystallinity of the sample. Figure 2 K).

[0051] (3) FT-IR spectroscopy analysis: The tensile vibrations of Ce-O and VO were observed at 445 and 798 cm⁻¹. -1 There are two unique peaks at each location, and simultaneously, a peak at 2960 cm⁻¹ is present in the FT-IR spectrum of the CR2-ICA probe. -1 The characteristic peaks of CeVO4 nanorods, which are absent in CeVO4, demonstrate the successful coupling of CeVO4 nanorods and monoclonal antibodies. Figure 2 M).

[0052] (4) Ultraviolet-Vis Spectroscopy (UV-Vis) Analysis: Pure CeVO4 exhibits a distinct absorption band at 311 nm in the UV-Vis spectrum, with a broad absorption peak between 200 nm and 600 nm. After the addition of protein, a significant blue shift and a strong absorption band at 298 nm were observed, indicating successful binding of the material and the monoclonal antibody. Figure 2 L).

[0053] (5) Zeta potential analysis: It can be seen that after CeVO4 is conjugated with the antibody, the zeta potential changes from -11.87 eV to -5.68 eV, proving the successful conjugation of the two. Figure 2 N).

[0054] Example 4: Specificity determination of a probe for rapid detection of T-2 toxin

[0055] Detection procedure: Aflatoxin B1 (AFB1), fumonisin B1 (FB1), patulin (PAT), zearalenone (ZEN), penicillin, and deoxynivalenol (DON) were diluted with ultrapure water to 100 ng / mL. 100 μL of each solution was taken as the test solution and mixed with 2 μL of CR2-ICA probe for incubation to obtain the test solution. The sample pad of the test strip was then immersed in 100 μL of the test solution. At the same time, 100 μL of ultrapure water was taken as the blank control solution. After reacting for a period of time, the intensity of the test line (T line) and control line (C line) was scanned using a test strip detector. The values ​​were used for specificity analysis.

[0056] See results Figure 4B. This study verified the specificity of the CR2-ICA probe in detecting T-2 toxin. When the sample concentration of T-2 toxin was 50 ng / mL, the color on the T-line was suppressed, while for other common toxins, a distinct brown color was observed on the T-line. This indicates that the invention can specifically identify T-2 toxin and has good specificity.

[0057] See Figure 4 Ten test strips from different batches were used to detect T-2 toxin at concentrations of 0.15 ng / mL and 13 ng / mL. The detection process was the same as the sensitivity detection process. The colorimetric signal intensity of the test strips was observed visually under ambient light, and the T-line and C-line were quantitatively detected using a test strip detector (ZF-1, Hangzhou Qiwei Instrument Co., Ltd.). The obtained T-line value was substituted into the sensitivity fitting curve to calculate the corresponding T-2 toxin concentration, such as... Figure 4 As shown in Figure D, the calculated T-2 toxin concentration is within the acceptable deviation of the spiked concentration, indicating good repeatability. The recovery rate is obtained by comparing the calculated concentration with the spiked concentration, as shown in Figure D. Figure 4 As shown in Figure A, it indicates that its repeatability is good.

[0058] The LOD of the CR2-ICA probe of this invention (0.079 ng / mL) was compared with the linear concentration of the T-2 toxin detection methods in existing literatures S1-S13. Figure 4 The results show that the probe of this invention has a wide linear range and the LOD is within the ideal range.

[0059] S1:Li, Y.;Xu, L.;Fu,

[0060] S2: Zhao, X.; Wang, Y.; Li, J.; Huo, B.; Huang, H.; Bai, J.; Peng, Y.; Li, S.; Han, D.; Ren, S.; Wang, J.; Gao, Z., A fluorescence aptasensor for the sensitive detection of T-2 toxin based on FRET by adjusting the surface electric potentials of UCNPs and MIL-101. Anal Chim Acta 2021, 1160, 338450.

[0061] S3: Zhao, X.; Wang, Y.; Li, J.; Huo, B.; Qin, Y.; Zhang, J.; Chen, M.; Peng, Y.; Bai, J.; Li, S.; Gao, Z., A fluorescence aptasensor based on controlled zirconium-based MOFs for the highly sensitive detection of T-2 toxin. Spectrochim Acta A Mol Biomol Spectrosc 2021, 259, 119893.

[0062] S4: Zhang, W.; Wang, Y.; Nan, M.; Li, Y.; Yun, J.; Wang, Y.; Bi, Y., Novel colorimetric aptasensor based on unmodified gold nanoparticle and ssDNA for rapid and sensitive detection of T-2 toxin. Food Chem 2021, 348, 129128.

[0063] S5:Guo, T.;Wang, C.;Zhou, H.;Zhang, Y.;Ma, L.; Wang, S., A facileaptasensor based on polydopamine nanospheres for high-sensitivity sensing ofT-2 toxin. Anal Methods 2021, 13 (24), 2654-2658.

[0064] S6:He, D.;Wu, Z.;Cui, B.;Xu, E.; Jin, Z., Building a FluorescentAptasensor Based on Exonuclease-Assisted Target Recycling Strategy for One-Step Detection of T-2 Toxin. Food Analytical Methods 2018, 12 (2), 625-632.

[0065] S7:Gao, L.;Sun, J.;Wang, L.;Fan, Q.;Zhu, G.;Guo, H.; Sun, X., Highlysensitive real-time detection of intracellular oxidative stress andapplication in mycotoxin toxicity evaluation based on living single-cellelectrochemical sensors. Analyst 2021, 146 (4), 1444-1454.

[0066] S8:Tan, X.;Yu, W.;Wang, Y.;Song, P.;Xu, Q.;Ming, D.; Yang, Y., Aswitchable and signal-amplified aptasensor based on metal organic frameworksas the quencher for turn-on detection of T-2 mycotoxin. Anal Bioanal Chem2021, 413 (26), 6595-6603.

[0067] S9:Qie, Z.;Yan, W.;Gao, Z.;Meng, W.;Xiao, R.; Wang, S., Ovalbuminantibody-based fluorometric immunochromatographic lateral flow assay usingCdSe / ZnS quantum dot beads as label for determination of T-2 toxin. MikrochimActa 2019, 186 (12), 816.

[0068] S10:Urusov, A. E.;Petrakova, A. V.;Bartosh, A. V.;Gubaydullina, M.K.;Zherdev, A. V.; Dzantiev, B. B., Immunochromatographic assay of T-2 toxinusing labeled anti-species antibodies. Applied Biochemistry and Microbiology2017, 53 (5), 594-599.

[0069] S11:Qie, Z.;Shi, J.;Yan, W.;Gao, Z.;Meng, W.;Xiao, R.; Wang, S.,Immunochromatographic assay for T-2 toxin based on luminescent quantum dotbeads. RSC Adv 2019, 9 (66), 38697-38702.

[0070] S12:Zhang, Z.;Wang, D.;Li, J.;Zhang, Q.; Li, P., Monoclonal antibody–europium conjugate-based lateral flow time-resolved fluoroimmunoassay forquantitative determination of T-2 toxin in cereals and feed. AnalyticalMethods 2015, 7 (6), 2822-2829.

[0071] S13: Zhang, X.;Ding, M.;Mao, Y.;Huang, X.;Xie, Actuators B: Chemical 2022, 359.

[0072] Example 5: Application of probes for rapid detection of T-2 toxin

[0073] Detection procedure: Corn and oats were pretreated with T-2 toxin by spiked solution. 1 gram of corn flour (or oat flakes) was added to 5 mL of a mixture of methanol and water (methanol to water volume ratio of 8:2), and the mixture was continuously vortexed for 3 minutes. After ultrasonic extraction for 20 minutes, the mixture was centrifuged at 8000 rpm for 10 minutes. After a 10-fold dilution, the supernatant was added to the detection solution for further analysis.

[0074] The prepared sample solution was diluted to different ratios, and 100 μL of each solution was used as the detection solution. It was mixed with 2 μL of CR2-ICA probe and incubated. The test strip was then immersed in 100 μL of the test solution. At the same time, 100 μL of ultrapure water was used as the blank control solution. The test strip detector was used to scan the detection line (T line) and control line for specificity analysis.

[0075] See results Figure 5 For CR2-ICA probes A and 5B, the intensity of the T-line gradually decreased with increasing T-2 toxin concentration; the critical values ​​(i.e., the concentration of T-2 toxin when the color of the T-line is lighter than that of the C-line) for oat and corn samples were 8.77 ng / mL and 19.7 ng / mL, respectively, and the visual detection limits (vLOD) for oat and corn were approximately 0.04 ng / mL and 0.06 ng / mL, respectively.

[0076] See Figure 5 C and 5D, such as Figure 5As shown in C and 5D, the recoveries of corn and oats (based on the results obtained from the above tests, the detection line intensity of the actual detected T-2 toxin was substituted into the previously detected sensitivity curve to calculate the corresponding T-2 toxin concentration on the curve, and the recovery rate was obtained by comparing the calculated concentration with the spiked concentration) were 81.27%-108.27% and 81.90%-115.44%, respectively, with RSDs of less than 9.16% and 5.66%, respectively; verifying the reliability of the results measured using this sensor.

[0077] The preferred embodiments of this disclosure have been described in detail above with reference to the accompanying drawings. However, this disclosure is not limited to the specific details of the above embodiments. Within the scope of the technical concept of this disclosure, various simple modifications can be made to the technical solutions of this disclosure, and these simple modifications all fall within the protection scope of this disclosure.

[0078] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this disclosure will not describe the various possible combinations separately.

[0079] Furthermore, various different embodiments of this disclosure can be combined in any way, as long as they do not violate the spirit of this disclosure, they should also be regarded as the content disclosed in this disclosure.

Claims

1. The application of the probe for detecting T-2 toxin in the preparation of a kit for detecting T-2 toxin in food, the kit comprising a box containing an immunochromatographic test strip adsorbed with T-2 toxin antigen, and a reagent bottle containing the probe or an aqueous solution of the probe. The immunochromatographic test strip includes a backing plate with a nitrocellulose membrane attached to it. One end of the nitrocellulose membrane is covered with an absorbent pad, and the other end of the nitrocellulose membrane is covered with a sample pad and a conjugation pad in sequence. Detection lines and control lines are set horizontally on the non-covered surface of the nitrocellulose membrane. The method for preparing the nitrocellulose membrane comprises: The test line was prepared by applying 1 mg / mL T-2 toxin-bovine serum albumin conjugate to the test line at a streak rate of 0.8 μL / cm, and the control line was prepared by applying 1 mg / mL goat anti-mouse immunoglobulin to the control line at a streak rate of 1 μL / cm. Then dry at 37°C for later use; The preparation method of the sample pad includes: cutting the glass fiber membrane into a size of 15 mm in length and 3 mm in width, immersing it in a sealing solution, drying it at 37°C for 8 h to obtain the sample pad, and then storing it in a refrigerator at 4°C. The method for preparing the conjugate pad includes: cutting a glass fiber membrane into a size of 8 mm in length and 3 mm in width, immersing it in a sealing liquid, removing it, drying it at 37°C for 8 h to obtain the conjugate pad, and then storing it in a refrigerator at 4°C. The absorbent pad is obtained by cutting absorbent paper to a length of 18 mm and a width of 3 mm; The preparation method of the probe includes: adding T-2 toxin monoclonal antibody to an aqueous solution of cerium vanadate nanomaterials and placing it at room temperature, then adding BSA to react; then centrifuging to collect the solid reaction product, and washing the reaction product to obtain the probe for T-2 toxin detection; The method for preparing cerium vanadate nanomaterials includes: adding a second solution dropwise to a first solution and adjusting the pH value to 9.5–10.5; the first solution is an aqueous solution of cerium nitrate and ethylenediaminetetraacetic acid; the second solution is an aqueous solution of ammonium metavanadate; then reacting at 2 MPa–32 MPa and 170–190 °C; after the reaction is completed, cooling to room temperature to obtain cerium vanadate nanomaterials; When using the kit, the sample pad of the test strip is first immersed in a mixed solution containing CeVO4-mAb probes and the sample to be tested. The bound CeVO4-mAb-T-2 immune complex migrates to the test area of ​​the test strip through capillary action. On the nitrocellulose membrane, for the positive group, T-2 toxin competes with mAb for binding sites on the antigen, and the probe cannot be trapped on the test line. Therefore, the color intensity on the test line is inversely proportional to the concentration of T-2 toxin. For the negative group without T-2 toxin, the antigen and mAb bind specifically, so there is color on the test line. Based on this phenomenon, the amount of T-2 toxin in the sample solution affects the intensity of the test line color.

2. Use according to claim 1, characterized in that, After adding the second solution dropwise to the first solution, adjust the pH value to 10.

3. Use according to claim 2, characterized in that, The cerium vanadate nanomaterial has a rod-like structure with a particle size distribution range of 800–1350 nm and an average particle length of 1057.72 nm.

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