Preparation of IUCNPs and its application in mycotoxin immunochromatographic test strips

Abstract

The present invention discloses a preparation method of IUCNPs and its application in mycotoxin immunochromatographic test strips, comprising the following preparation steps: taking an appropriate amount of YC1 with a concentration of 1 mol / L 3 , YbC1 3 , ErCl3, LuCl 3 Add the salt solution to a three-necked flask, heat to 130°C, and remove water for 15 minutes without opening until the rare earth salt solution turns into a white solid; add 6 mL of oleic acid and 15 mL of octadecene with a pipette, heat to 150°C, and make the salt solution completely dissolved and turn orange-yellow. Cool the solution to below 60°C, and add the prepared NaOH and NH 4 F methanol solution, adjust the temperature to 120℃ to remove methanol and water; wash and centrifuge with anhydrous ethanol and cyclohexane, dissolve the prepared IUCNPs in cyclohexane solution, and store in a glass bottle at room temperature. Beneficial effects of the present invention: The fluorescence intensity and fluorescence lifetime of IUCNPs are significantly enhanced, the fluorescence performance is stronger, the specificity is strong, the accuracy is high, and the retesting of the test strip only takes 10 to 15 minutes, which has a faster detection speed.

Description

Technical Field

[0001] The present invention relates to the technical field of immunological diagnosis, and in particular to an immunochromatographic test strip which uses improved upconversion nanoparticles (IUCNPs) as fluorescent signal probes and can dually detect aflatoxin B1 (AFB1) and zearalenone (ZEN), and a preparation method and application thereof. Background Art

[0002] In recent years, multiple UCNPs-ICA has been successfully used for the qualitative or semi-quantitative detection of a single target analyte in a sample. However, some agricultural products such as corn can be contaminated by several fungi at the same time, resulting in widespread co-contamination of mycotoxins in grain and feed, which endangers the health of humans and livestock and causes serious economic losses. The presence of multiple mycotoxins co-contamination poses a challenge to the application of traditional single ICA test strips, and single ICA test strips also increase detection costs and time. Therefore, it is necessary to develop a simple and rapid ICA method for the simultaneous detection of multiple targets. Compared with ICA for single target detection, the multiple ICA method can simultaneously and specifically identify several different target mycotoxins, improve detection efficiency, and reduce detection time and cost. Therefore, the development of an immunochromatographic test strip based on modified UCNPs for dual detection of AFB and ZEN has great potential and broad application prospects in the rapid and efficient detection of trace AFB1 and ZEN residues in a variety of grains. Summary of the invention

[0003] To solve the above technical problems, the present invention aims to provide a method for preparing IUCNPs, an immunochromatographic test strip for dual detection of AFB1 and ZEN based on IUCNPs, and a method for preparing and use of an immunochromatographic test strip for dual detection of AFB1 and ZEN.

[0004] To achieve the above-mentioned purpose, the present invention provides the following technical scheme: a method for preparing IUCNPs, comprising the following preparation steps: taking an appropriate amount of YC13, YbC13, ErCl3, and LuCl3 salt solution with a concentration of 1 mol / L and adding it to a three-necked flask, heating it to 130°C, and removing water for 15 minutes without opening until the rare earth salt solution becomes a white solid; adding 6 mL of oleic acid and 15 mL of octadecene with a pipette, heating it to 150°C, so that the salt solution is completely dissolved and turns orange-yellow, and cooling the solution to 60°C Next, add the prepared NaOH and NH4F methanol solutions, adjust the temperature to 120°C to remove methanol and water; wait until all bubbles disappear, quickly evacuate with a vacuum pump, pass nitrogen, repeat the process of evacuating and passing nitrogen 3 times until the temperature reaches 150°C; heat to 300°C in a nitrogen atmosphere, maintain for 1.5 hours, and then cool to room temperature under nitrogen protection; finally, wash and centrifuge with anhydrous ethanol and cyclohexane, dissolve the prepared IUCNPs in cyclohexane solution, and store in a glass bottle at room temperature.

[0005] Preferably, the amounts of YC13, YbC13, ErCl3, and LuCl3 are 430 μL, 250 μL, 200 μL, and 300 μL, respectively. 3+ The doping ratio is 30%.

[0006] Preferably, the specific preparation of the NaOH and NH4F methanol solution includes the following steps: accurately weighing 0.1 g of NaOH and ultrasonically dissolving it in 5 mL of methanol solution, and then weighing 0.148 g of NH4F and ultrasonically dissolving it in the NaOH methanol solution.

[0007] Preferably, the average particle size of the IUCNPs is 60-70 nm.

[0008] A method for preparing an immunochromatographic test strip for dual detection of AFB1 and ZEN comprises the following steps: S1: modifying the surface carboxyl groups of IUCNPs with polyacrylic acid, and coupling them with AFB1-mAb and ZEN-mAb respectively according to the carbodiamine method to prepare corresponding immunofluorescent probes; S2: pretreating a sample pad and a conjugate pad, and spraying the prepared two IUCNPs fluorescent probes onto the pretreated conjugate pad after mixing them in a ratio of 1:2 at a spraying speed of 8 μL / cm2, and drying them at 37°C in vacuum for 1 minute. Set aside after 5 minutes; S3: spray the test lines coated with AFB1-BSA and ZEN-BSA (referred to as T1 and T2 lines) and the control line of goat anti-mouse IgG antibody on the nitrocellulose membrane in horizontal order, and dry it in a 50°C forced air drying oven overnight; S4: assemble the NC membrane dried in S3, the sample pad treated with the treatment liquid, the binding pad dried in S2, and the absorbent pad in sequence on the PVC sticky bottom plate, cut them into 4 mm wide test strips, put them in a tin foil bag containing a desiccant, and seal them at room temperature for storage.

[0009] Preferably, the PAA carboxylation method in S1 comprises the following steps: taking IUCNPs dispersed in a cyclohexane solution, ultrasonically washing with anhydrous ethanol and a pH=3 HCl solution, and centrifuging at 10000 r / min for 10 min to obtain a precipitate; continuing to add anhydrous ethanol and HCl to the precipitate, repeating the washing for 3 times to obtain IUCNPs with good water solubility, and dissolving the precipitate in ultrapure water; preparing a 3% PAA solution, adjusting its pH to 9.5 with a 4 mol / L NaOH solution; adding the 3% PAA solution to the IUCNPs aqueous solution at a ratio of 1:2, detecting with a pH test paper to ensure that the reaction system always remains alkaline, ultrasonically washing the above-mentioned mixed solution for 1 h to obtain surface carboxylated IUCNPs, centrifuging at high speed to obtain a precipitate, dispersing it in ultrapure water and storing it in a refrigerator at 4°C;

[0010] The method for preparing the immunofluorescence probe comprises the following steps:

[0011] ① Take two tubes of 500μL carboxylated IUCNPs, centrifuge at 12000r / min for 10min, and take the precipitate. Under the same centrifugation conditions, wash the precipitate once with 500μL ultrapure water; ② Prepare MES solution with pH=6.5, and then use this solution to prepare 25mg / mL EDC and 37.5mg / mL NHS solutions; ③ Add 500μL MES 6.5 solution, 40μL EDC solution and 40μL NHS solution to the precipitate in step ①, and activate the mixed solution in a rotary mixer for 20min, and centrifuge at high speed to take the precipitate; ④ After re-dissolving and washing the precipitate in step ③ with MES 6.5 solution, re-dissolve the precipitate with 500μL MES 6.5 solution, add 20μg AFB1-mAb and 10μg ZEN-mAb to the two tubes of solution, and incubate for 2h. Then, 550 μL of 1% BSA prepared in MES 6.5 solution was taken for blocking for 0.5 h; ⑤ After the solution in step ④ was centrifuged at high speed to obtain the precipitate, it was washed with MES 6.5 solution and centrifuged at high speed to obtain the precipitate. Finally, the precipitate was resuspended in 100 μL of PBS preservation solution containing 0.5% Tween-20, 3% sucrose, 1% trehalose and 1% BSA to prepare the IUCNPs fluorescent probe stock solution labeled with AFB1-mAb and ZEN-mAb respectively.

[0012] Preferably, in S2, the sample pad and conjugate pad pretreatment method comprises the following steps: soaking the sample pad and conjugate pad in a PBS treatment solution containing 0.5% BSA, 1% Tween-20, 0.5% PVP-40, 3% sucrose and 1% trehalose for 30 minutes, and then drying at 37°C overnight.

[0013] Preferably, the concentrations of AFB1-BSA, ZEN-BSA and goat anti-mouse IgG antibody in S3 are 0.8 mg / mL, 0.8 mg / mL and 0.5 mg / mL respectively, and the spraying speed is 0.8 μL / cm.

[0014] An immunochromatographic test strip for dual detection of AFB1 and ZEN comprises the immunochromatographic test strip prepared as claimed in claims 5 to 8, wherein the immunochromatographic test strip comprises a sample pad, a conjugate pad, a nitrocellulose membrane (NC membrane), a PVC bottom plate, and a water absorbent pad.

[0015] The invention discloses an immunochromatographic test strip for dual detection of AFB1 and ZEN, which is used for simultaneously detecting AFB1 and ZEN in cereals. When the cereals are corn and wheat, the detection limits of AFB1 and ZEN in corn and wheat are 0.1 μg / kg and 0.25 μg / kg respectively.

[0016] Advantages of the present invention: First, the solvent thermal method is used to synthesize 30% Lu doped 3+ The fluorescence intensity and fluorescence lifetime of IUCNPs are significantly enhanced, and the fluorescence performance is stronger; second, IUCNPs are used as fluorescent markers in immunochromatography for the first time, and the detection limits of the obtained test strips for AFB1 and ZEN standard solutions are 0.025ng / mL and 0.1ng / mL, respectively, and the detection limits of AFB1 and ZEN in spiked corn and wheat are 0.1μg / kg and 0.25μg / kg, respectively, which are far below the national limit standards and have good detection sensitivity. In addition, the test card can be used in conjunction with immunofluorescence readers and other equipment to complete the rapid detection of various mycotoxins on site, reducing the detection cost and having broad application prospects; third, the test strips have good specificity and high sensitivity; fourth, the detection of two toxins, AFB1 and ZEN, is integrated on one test strip to form a dual-channel detection with strong specificity and high accuracy, and the test strips only take 10 to 15 minutes, and the detection is fast. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the preparation of IUCNPs fluorescent immunoprobe and the structure of the double detection fluorescent immunoassay strip in the present invention;

[0018] Figure 2 This is a scanning electron microscopy image of IUCNPs synthesized by the present invention;

[0019] Figure 3 It is a schematic diagram of the fluorescence spectrum of IUCNPs described in the present invention;

[0020] Figure 4 It is a schematic diagram of the specific results of the double detection fluorescent immunoassay strip of the present invention;

[0021] Figure 5 It is a schematic diagram of the detection sensitivity of the immune test strip of the present invention to wheat spiked samples;

[0022] Figure 6 This is a schematic diagram of the detection sensitivity of the dual-detection fluorescent immunoassay strip of the present invention to corn spiked samples. DETAILED DESCRIPTION

[0023] The specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. It should be noted that the experimental methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0024] If the specific conditions are not specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are used. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially. In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be clearly and completely described below.

[0025] Example 1

[0026] Reference Figures 1-2 The purpose of this embodiment is to provide an immunochromatographic test strip for dual rapid detection of AFB1 and ZEN based on improved UCNPs (i.e., IUCNPs). The test strip has high sensitivity to AFB1 and ZEN, good specificity, fast detection speed, and is simple and easy to operate, and can be used by non-professionals. It meets the requirements of grain storage and sales agencies, entry-exit quarantine, customs, production enterprises and supervision departments to quickly and accurately determine the content of AFB1 and ZEN toxins, and is convenient for grassroots promotion and application.

[0027] This embodiment also provides a method for preparing IUCNPs. The IUCNPs prepared by the solvothermal method have excellent fluorescence properties, regular shapes and suitable sizes, and have good application prospects in the field of rapid immunochromatographic detection as immunofluorescence probes.

[0028] The present embodiment also aims to provide a method for preparing the above-mentioned fluorescent immunochromatographic test strip, integrating the detection of two toxins on one test strip to form a dual-channel detection, which can detect two toxins at the same time with only one operation, reducing the loss of manpower and material resources caused by using a single detection method to detect two toxins, improving efficiency, and eliminating mutual interference between the two T lines. Figure 1 Figure a is a schematic diagram of the preparation of the IUCNPs fluorescent immunoprobe in the present invention. Figure 1 Figure b is a schematic diagram of the structure of the dual detection fluorescent immunoassay strip of the present invention.

[0029] More specifically, this embodiment provides a method for preparing IUCNPs, comprising the following preparation steps: taking an appropriate amount of 1 mol / L YC13, YbC13, ErCl3, and LuCl3 salt solution in a three-necked flask, heating to 130°C, and removing water for 15 minutes without opening until the rare earth salt solution becomes a white solid. Use a pipette to add 6mL OA and 15mL ODE, and heat to 150°C to completely dissolve the salt solution in an orange-yellow color. Cool the above solution to below 60°C, add the prepared NaOH and NH4F methanol solutions, and adjust the temperature to 120°C to remove methanol and water. When all bubbles disappear, quickly evacuate with a vacuum pump, pass nitrogen, and repeat the process of evacuating and passing nitrogen 3 times until the temperature rises to 150°C. Heat to 300°C in a nitrogen atmosphere, maintain for 1.5h, and then cool to room temperature under nitrogen protection. Finally, the mixture was washed and centrifuged with anhydrous ethanol and cyclohexane, and the prepared IUCNPs were dissolved in cyclohexane solution and stored in a glass bottle at room temperature.

[0030] Furthermore, the amounts of YC13, YbC13, ErCl3, and LuCl3 are 430 μL, 250 μL, 200 μL, and 300 μL, respectively. 3+ The doping ratio is 30%.

[0031] The specific preparation method of the NaOH and NH4F methanol solution is: accurately weigh 0.1g NaOH and ultrasonically dissolve it in 5mL methanol solution, and then weigh 0.148g NH4F and ultrasonically dissolve it in the above NaOH methanol solution. The average particle size of the IUCNPs is 60-80nm.

[0032] This embodiment also provides a method for preparing an immunochromatographic test strip for simultaneously detecting AFB1 and ZEN based on IUCNPs, comprising the following preparation steps:

[0033] (1) The surface carboxyl groups of IUCNPs were modified by PAA and conjugated with AFB1-mAb and ZEN-mAb according to the carbodiamine method to prepare the corresponding immunofluorescence probes.

[0034] (2) Pre-treating the sample pad and the conjugate pad, and evenly mixing the two IUCNPs fluorescent probes in a ratio of 1:2, and then spraying them onto the pre-treated conjugate pad at a spraying speed of 8 μL / cm, and vacuum drying them at 37°C for 15 min before use.

[0035] (3) The test lines (denoted as T1 and T2 lines) coated with AFB1-BSA and ZEN-BSA and the control line of goat anti-mouse IgG antibody were sprayed on the nitrocellulose membrane in horizontal order, and dried in a forced air drying oven at 50° C. overnight.

[0036] (4) The NC membrane dried in step (3), the sample pad treated with the treatment solution, the binding pad dried in step (2), and the absorbent pad are assembled in sequence on a PVC adhesive base plate, cut into 4 mm wide test strips, placed in a tin foil bag containing a desiccant, and sealed and stored at room temperature for later use.

[0037] In step (1), the main purpose of PAA carboxylation is to replace the carboxyl groups in the oleic acid molecules coated on the surface of IUCNPs, so that the free carboxyl groups on the particle surface are modified, so as to be coupled with the free amino groups on the toxin monoclonal antibody in the subsequent step, thereby preparing an immunofluorescent probe. The method comprises: taking IUCNPs dispersed in a cyclohexane solution, ultrasonically washing them with anhydrous ethanol and a pH=3 HCl solution, and centrifuging them at a high speed of 10000r / min for 10min to obtain a precipitate. Continue to add anhydrous ethanol and HCl to the precipitate, repeat washing 3 times, obtain IUCNPs with good water solubility, and dissolve them in ultrapure water. Prepare a 3% PAA solution, adjust its pH to 9.5 with a 4mol / L NaOH solution. Add the 3% PAA solution to the IUCNPs aqueous solution at a ratio of 1:2, and detect it with a pH test paper to ensure that the reaction system always remains alkaline. Ultrasonicate the above-mentioned mixed solution for 1h to obtain surface carboxylated IUCNPs, centrifuge the precipitate at a high speed, disperse it in ultrapure water and store it in a refrigerator at 4°C.

[0038] The preparation method of the immunofluorescence probe comprises:

[0039] ① Take 500 μL of carboxylated IUCNPs from two tubes respectively, centrifuge at 12000 r / min for 10 min, and take the precipitate. Wash the precipitate once with 500 μL of ultrapure water under the same centrifugal conditions.

[0040] ② Prepare MES solution with pH = 6.5, and then use this solution to prepare EDC and NHS solutions with concentrations of 25 mg / mL and 37.5 mg / mL respectively.

[0041] ③ Add 500 μL of MES 6.5 solution, 40 μL of EDC solution and 40 μL of NHS solution to the precipitate in step ①, activate the mixture in a rotary mixer for 20 min, and centrifuge at high speed to obtain the precipitate.

[0042] ④ After re-dissolving the precipitate in step ③ with MES 6.5 solution, re-dissolve the precipitate with 500 μL MES 6.5 solution, add 20 μg AFB1-mAb and 10 μg ZEN-mAb to the two tubes respectively, incubate in a rotating mixer for 2 hours, and then take 550 μL of 1% BSA prepared with MES 6.5 solution to block for 0.5 hours.

[0043] ⑤ After the solution in step ④ was centrifuged at high speed to obtain a precipitate, it was washed with MES 6.5 solution and centrifuged at high speed to obtain a precipitate. Finally, the precipitate was resuspended in 100 μL of PBS preservation solution containing 0.5% Tween-20, 3% sucrose, 1% trehalose and 1% BSA to prepare the IUCNPs fluorescent probe stock solution labeled with AFB1-mAb and ZEN-mAb, respectively.

[0044] In step (2), the sample pad and conjugate pad are pretreated by soaking the sample pad and conjugate pad in a PBS treatment solution containing 0.5% BSA, 1% Tween-20, 0.5% PVP-40, 3% sucrose and 1% trehalose for 30 minutes, and then drying them in a 37°C forced air drying oven overnight.

[0045] In step (3), the concentrations of AFB1-BSA, ZEN-BSA and goat anti-mouse IgG antibody are 0.8 mg / mL, 0.8 mg / mL and 0.5 mg / mL respectively, and the spraying speed is 0.8 μL / cm.

[0046] This embodiment also provides a sample determination method based on IUCNPs dual detection of AFB1 and ZEN immunochromatographic test strips, comprising the following detection steps:

[0047] (1) Crush the sample and pass it through a 20-mesh sieve, then mix thoroughly;

[0048] (2) Weigh 5.0 g of sample and add 25 mL of 60% methanol extract, and shake and mix for 5 min;

[0049] (3) Qualitative filtration with filter paper or centrifugation at 4000 rpm for 5 min;

[0050] (4) Take 100 μL of the filtrate, add 400 μL of PBS solution containing 0.5% Tween-20 to dilute, take 100 μL of the above dilution solution and drop it into the sample hole of the immunochromatographic test strip, and read the strip test results using an immunofluorescence analyzer after 10 minutes. The blank control strip T / C value -2.5SD obtained from the test is recorded as the cutoff value. When the test strip T / C value is less than the cutoff value, the result is recorded as positive, otherwise it is negative. Wherein: Figure 5 As shown, the detection limit of the AFB1 standard solution is 0.025 ng / mL, and the detection limit of the ZEN standard solution is 0.1 ng / mL.

[0051] like Figure 6 As shown, the detection limits of the fluorescent immunochromatographic test strip for simultaneously detecting AFB1 and ZEN in corn and wheat are 0.1 μg / kg and 0.25 μg / kg, respectively.

[0052] Compared with the prior art, the beneficial effects of this embodiment are:

[0053] 1.Reference Figure 2 Schematic diagram of IUCNPs scanning electron microscopy. In this example, the solvothermal method was used to synthesize 30% Lu-doped 3+ The average size of IUCNPs is 60-80nm, showing a regular hexagonal phase morphology. Compared with colloidal gold nanoparticles, IUCNPs have the advantages of cleaner synthesis route, low cost, and no matrix interference. Compared with basic UCNPs, the fluorescence intensity and fluorescence lifetime of IUCNPs are significantly enhanced, and the fluorescence performance is stronger. Therefore, IUCNPs can be used as a good biomarker in immunochromatography and have strong practical value in the field of rapid detection. It should be noted that, referring to Figure 3 Schematic diagram, 30% Lu 3+ The UCNPs have the best microscopic morphology and fluorescence properties.

[0054] 2. This example uses IUCNPs as a fluorescent marker for immunochromatography for the first time, and proposes a new type of immune test strip that can dually detect AFB1 and ZEN. my country's "GB 2761-2017 Limits of Fungal Toxins in Food" stipulates that the limit range of aflatoxin B1 in food is 5.0-20μg / kg; "GB2761-2011 Limits of Fungal Toxins in Food" supplements the requirement that the content of zearalenone in cereals and their products should be less than 60μg / kg. The test strip developed in this example has a detection limit of 0.025ng / mL and 0.1ng / mL for AFB1 and ZEN standard solutions, respectively, and a detection limit of 0.1μg / kg and 0.25μg / kg for AFB1 and ZEN in spiked corn and wheat, respectively, which is far lower than the national limit standard and has good detection sensitivity. In addition, the test card can be used in conjunction with equipment such as an immunofluorescence reader to complete the rapid detection of multiple mycotoxins on site, reducing the detection cost and having a wide range of application prospects.

[0055] 3. The test strip of this embodiment has good specificity and high sensitivity. When the test strip performs dual detection of AFB1 and ZEN, each antigen detection line has no cross reaction with other antibodies, and has no reaction with common fungal toxins such as ochratoxin A, vomitoxin, fumonisin, and T-2 toxin.

[0056] 4. This embodiment integrates the detection of two toxins, AFB1 and ZEN, into one test strip, forming a dual-channel detection with strong specificity and high accuracy. Compared with the 30 minutes required for the single-channel test strip to detect AFB1 and ZEN respectively, the dual-detection test strip only takes 10 to 15 minutes, which has a faster detection speed. In addition, the test strip is easy to operate, and professionals who do not need training can use the method of this embodiment for detection, which meets the requirements of grain storage and sales agencies, entry-exit quarantine, customs, production enterprises, supervision departments, etc. to quickly and accurately determine the content of AFB1 and ZEN toxins, and is convenient for grassroots promotion and application.

[0057] Example 2

[0058] The preparation of IUCNPs in this embodiment is as follows:

[0059] Take 430μL, 250μL, 200μL, 300μL of 1mol / L YC13, YbC13, ErCl3, LuCl3 salt solution respectively and add them to a three-necked flask, heat to 130℃, and remove water for 15min in an open state until the rare earth salt solution becomes a white solid. Use a pipette to add 6mLOA and 15mL ODE, heat to 150℃, and make the salt solution completely dissolved and orange-yellow. Accurately weigh 0.1g NaOH and ultrasonically dissolve it in 5mL methanol solution, and then weigh 0.148g NH4F and ultrasonically dissolve it in the above NaOH methanol solution. Cool the above salt solution to below 60℃, add the prepared NaOH and NH4F methanol solution, adjust the temperature to 120℃ to remove methanol and water. When all bubbles disappear, quickly evacuate with a vacuum pump, pass nitrogen, and repeat the process of evacuating and passing nitrogen 3 times until the temperature reaches 150℃. The temperature was raised to 300°C in a nitrogen atmosphere, maintained for 1.5 h, and then cooled to room temperature under nitrogen protection. Finally, the prepared IUCNPs were washed and centrifuged with anhydrous ethanol and cyclohexane, and dissolved in cyclohexane solution and stored in a glass bottle at room temperature.

[0060] 1. Surface carboxyl modification of IUCNPs. Take the IUCNPs dispersed in the cyclohexane solution, use anhydrous ethanol and pH = 3 HCl solution to ultrasonically wash them, and centrifuge at 10000r / min for 10min to obtain the precipitate. Continue to add anhydrous ethanol and HCl to the precipitate, repeat the washing 3 times, obtain IUCNPs with good water solubility, and dissolve it in ultrapure water. Prepare a 3% PAA solution and adjust its pH to 9.5 with a 4mol / L NaOH solution. Add the 3% PAA solution to the IUCNPs aqueous solution in a ratio of 1:2, and use pH test paper to detect to ensure that the reaction system always remains alkaline. Ultrasonicate the above mixed solution for 1h to obtain surface carboxylated IUCNPs, centrifuge at high speed to obtain the precipitate, disperse it in ultrapure water, and store it at 4°C.

[0061] 2. Preparation of two IUCNPs fluorescent immunoprobes. Take two tubes of 500μL carboxylated IUCNPs and centrifuge at 12000r / min for 10min to obtain the precipitate. Add 500μL ultrapure water to wash and centrifuge to obtain the precipitate. Prepare MES solution with a pH of 6.5, and then prepare EDC and NHS solutions with concentrations of 25mg / mL and 37.5mg / mL respectively. Add 500μL MES solution, 40μLEDC solution, and 40μL NHS solution to the precipitate. Rotate and activate the above mixture for 20min and centrifuge to obtain the precipitate. Redissolve and wash the above precipitate with MES 6.5 buffer and centrifuge to obtain the precipitate. Redissolve the above precipitate with 500μL MES 6.5 buffer, add 20μg AFB1-mAb and 10μg ZEN-mAb to the two tubes respectively, and place them in a rotating mixer for incubation for 2h. Then block with 1% BSA for 0.5h. After the blocked solution was centrifuged at high speed to obtain the precipitate, it was washed with MES 6.5 solution. Finally, the precipitate was resuspended in 100 μL of PBS preservation solution containing 0.5% Tween-20, 3% sucrose, 1% trehalose and 1% BSA to prepare the IUCNPs fluorescent probe stock solution labeled with AFB1-mAb and ZEN-mAb respectively.

[0062] Furthermore, an immunochromatographic test strip for dual detection of AFB1 and ZEN based on IUCNPs was prepared as follows:

[0063] 1. Spray the fluorescent probe onto the conjugate pad. Soak the conjugate pad in a PBS treatment solution containing 0.5% BSA, 1% Tween-20, 0.5% PVP-40, 3% sucrose and 1% trehalose for 30 minutes, and then place it in a 37°C air drying oven to dry overnight. Then mix the prepared AFB1 fluorescent probe and ZEN fluorescent probe in a volume ratio of 1:2 and spray them onto the conjugate pad. The spraying amount is 8μL / cm, and vacuum dry at 37°C for more than 15 minutes.

[0064] 2. Antigen coating on NC membrane: AFB1-BSA, ZEN-BSA and 0.5 mg / mL goat anti-mouse IgG antibody were sprayed on the same nitrocellulose membrane in horizontal order, with a spacing of 3.5-4 mm between each test line and control line, and the spraying amount was 0.8 μL / cm, and vacuum dried at 37°C overnight.

[0065] 3. Assemble the test strips. Assemble the dried NC membrane, the sample pad treated with the treatment solution, the dried binding pad, and the absorbent pad in order on the PVC adhesive base plate, cut them into 4mm wide test strips, put them in a tin foil bag containing a desiccant, and seal them at room temperature for later use.

[0066] Example 3

[0067] Sample testing and result analysis

[0068] The sample was crushed and passed through a 20-mesh sieve. After thorough mixing, 5.0 g of sample powder was weighed and added to 25 mL of 60% methanol extract, and the mixture was shaken for 5 minutes. The sample was filtered using qualitative filter paper or centrifuged at 4000 r / min for 5 minutes, 100 μL of the filtrate was taken and diluted with 400 μL of PBS solution containing 0.5% Tween-20, and 100 μL of the above dilution was added dropwise to the sample well of the immunochromatographic test strip. After 10 minutes, the strip test results were read using an immunofluorescence analyzer. Figure 4 As shown in the figure, the detection limits of the fluorescent immunochromatographic test strip prepared in this study for AFB1 and ZEN in corn and wheat were 0.1μg / kg and 0.25μg / kg, respectively. Compared with the detection of AFB1 and ZEN standards, its detection limit was slightly reduced, but still far less than the national limit standard, indicating that the test strip has good anti-matrix interference ability and has broad application prospects in the field of real grain sample detection.

[0069] Example 4

[0070] Specificity analysis experiments

[0071] According to the method in Example 3, ochratoxin A, vomitoxin, T-2 toxin, fumonisin, and aflatoxin M1 were used in the detection of the fluorescent immunochromatographic test strip in Example 2. Figure 2 As shown, the T lines all showed strong signals, indicating that the test strips had good specificity and had no cross-reaction with the above-mentioned mycotoxins.

[0072] It should be understood that the present invention is described through implementation methods, and the embodiments are only clear and complete descriptions of the technical solutions proposed in the claims of the present invention, that is, explanations of the claims. Therefore, when judging whether the technical solutions recorded in the specification of the present invention are sufficiently disclosed, full consideration should be given to the core essence of the solutions defined by the claims. In the specification, there must be other technical problems that are not related to the core technical problems proposed in this embodiment. The corresponding technical features and technical solutions do not belong to the essence of this embodiment and are non-essential technical features. Therefore, they can be referred to as implicit disclosures. Those skilled in the art can fully implement them in combination with existing technologies and common knowledge. Therefore, there is no need to describe them in detail.

[0073] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for preparing IUCNPs, characterized in that: The method comprises the following preparation steps: Take an appropriate amount of 1 mol / L YC13, YbC13, ErCl3, and LuCl3 salt solutions into a three-necked flask, heat to 130°C, and remove water for 15 min without opening until the rare earth salt solution becomes a white solid. The amounts of YC13, YbC13, ErCl3, and LuCl3 are 430 μL, 250 μL, 200 μL, and 300 μL, respectively. 3+ The doping ratio is 30%; Use a pipette to add 6 mL of oleic acid and 15 mL of octadecene, heat to 150°C, and completely dissolve the salt solution to turn orange-yellow. Cool the solution to below 60°C, accurately weigh 0.1 g of NaOH and ultrasonically dissolve it in 5 mL of methanol solution, then weigh 0.148 g of NH4F and ultrasonically dissolve it in the methanol solution of NaOH, add the prepared NaOH and NH4F methanol solution, and adjust the temperature to 120°C to remove methanol and water; When all bubbles disappear, quickly evacuate with a vacuum pump and introduce nitrogen. Repeat the process of evacuating and introducing nitrogen three times until the temperature reaches 150°C. The temperature was raised to 300 °C in a nitrogen atmosphere, maintained for 1.5 h, and then cooled to room temperature under nitrogen protection; Finally, the mixture was washed and centrifuged with anhydrous ethanol and cyclohexane, and the prepared IUCNPs were dissolved in a cyclohexane solution and stored in a glass bottle at room temperature. The average particle size of the IUCNPs was 60-70 nm.

2. A method for preparing an immunochromatographic test strip for dual detection of AFB1 and ZEN, characterized in that: The following steps are included: S1: using polyacrylic acid (PAA) to modify the surface carboxyl groups of the IUCNPs prepared in claim 1, and coupling them with AFB1-mAb and ZEN-mAb respectively according to the carbodiamine method to prepare the corresponding immunofluorescence probes; The PAA carboxylation method comprises the following steps: The IUCNPs dispersed in the cyclohexane solution were ultrasonically washed with anhydrous ethanol and pH=3 HCl solution, and then centrifuged at 10000 r / min for 10 min to obtain the precipitate; Anhydrous ethanol and HCl were continuously added to the precipitate, and the washing was repeated three times to obtain IUCNPs with good water solubility, which were then dissolved in ultrapure water; Prepare 3% PAA solution and adjust its pH to 9.5 with 4 mol / L NaOH solution; 3% PAA solution was added to the IUCNPs aqueous solution at a ratio of 1:2, and pH test paper was used to ensure that the reaction system always remained alkaline. The mixture was sonicated for 1 h to obtain surface carboxylated IUCNPs, and the precipitate was dispersed in ultrapure water and stored in a refrigerator at 4°C. The preparation method of the immunofluorescent probe comprises the following steps: ① taking two tubes of 500 μL of carboxylated IUCNPs respectively, centrifuging at 12000 r / min for 10 min, taking the precipitate, and washing the precipitate once with 500 μL of ultrapure water under the same centrifugal condition; ② preparing a MES solution with a pH of 6.5, and then using the solution to prepare EDC and NHS solutions with concentrations of 25 mg / mL and 37.5 mg / mL respectively; ③ adding 500 μL of MES 6.5 solution, 40 μL of EDC solution and 40 μL of NHS solution prepared in step ① to the precipitate in step ②, activating the mixed solution in a rotary mixer for 20 min, and taking the precipitate by high-speed centrifugation; ④ using MES6.5 solution to re-dissolve and wash the precipitate in step ③, and then re-dissolve the precipitate with 500 μL of MES 6.5 solution, adding 20 μg of AFB1-mAb and 10 μg of ZEN-mAb to the two tubes of solution respectively, incubating for 2 hours, and then taking 550 μL of MES ⑤ The solution in step ④ was centrifuged at high speed to obtain a precipitate, which was then washed with MES 6.5 solution and centrifuged at high speed to obtain a precipitate. Finally, the precipitate was resuspended in 100 μL of PBS containing 0.5% Tween-20, 3% sucrose, 1% trehalose and 1% BSA to prepare the IUCNPs fluorescent probe stock solutions labeled with AFB1-mAb and ZEN-mAb respectively; S2: Pre-treat the sample pad and conjugate pad, and evenly mix the two prepared IUCNPs fluorescent probes in a ratio of 1:2 and spray them onto the pre-treated conjugate pad at a spraying speed of 8 μL / cm. Vacuum dry them at 37°C for 15 min and set aside for use. Soak the sample pad and conjugate pad in a PBS treatment solution containing 0.5% BSA, 1% Tween-20, 0.5% PVP-40, 3% sucrose and 1% trehalose for 30 min, and then dry them at 37°C overnight. S3: spraying the test lines coated with AFB1-BSA and ZEN-BSA on the nitrocellulose membrane in horizontal order, which are recorded as T1 and T2 lines and the control line of goat anti-mouse IgG antibody, and drying them in a 50°C forced air drying oven overnight; the concentrations of AFB1-BSA, ZEN-BSA and goat anti-mouse IgG antibody are 0.8 mg / mL, 0.8 mg / mL and 0.5 mg / mL, respectively, and the spraying speed is 0.8 μL / cm; S4: Assemble the NC membrane dried in S3, the sample pad treated with the treatment solution, the binding pad dried in S2, and the absorbent pad in sequence on the PVC adhesive base plate, cut them into 4 mm wide test strips, put them in a tin foil bag containing a desiccant, and seal them at room temperature for later use.

3. An immunochromatographic test strip for dual detection of AFB1 and ZEN, characterized in that: The invention comprises the immunochromatographic test strip prepared as claimed in claim 2, wherein the immunochromatographic test strip comprises a sample pad, a conjugate pad, a nitrocellulose membrane (NC membrane), a PVC bottom plate, and a water-absorbing pad.

4. An application of the immunochromatographic test strip for dual detection of AFB1 and ZEN according to claim 3, characterized in that: The method is applied to dual detection of AFB1 and ZEN in cereals, when the cereals are corn and wheat, the detection limits of AFB1 and ZEN in corn and wheat are 0.1 μg / kg and 0.25 μg / kg, respectively.

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