Fluorescent probe, bicolor fluorescence immunochromatography test strip for simultaneously detecting AFB1 and T-2 toxins and preparation method of bicolor fluorescence immunochromatography test strip

Abstract

The invention belongs to the technical field of food safety detection, and relates to a fluorescent probe, a bicolor fluorescence immunochromatography test strip for simultaneously detecting AFB1 and T-2 toxins and a preparation method of the bicolor fluorescence immunochromatography test strip. The probe is ZGO nano particles coupled with a mycotoxin antibody, the ZGO nano particles comprise green fluorescence ZGO: Mn nano particles and blue fluorescence ZGO: Mo nano particles, the ZGO: Mn nano particles are Zn2GeO4: Mn nano particles, and the ZGO: Mo nano particles are Zn2GeO4: Mo nano particles. The test strip disclosed by the invention can be used for qualitatively and quantitatively detecting various fungaltoxins, meets the requirements of simultaneously detecting AFB1 and T-2 fungaltoxins, and is expected to provide a powerful tool for rapidly detecting co-pollution of various fungaltoxins in grains and feeds.

Description

Technical Field

[0001] The present invention belongs to the technical field of food safety detection, and in particular, relates to a fluorescent probe and a dual-color fluorescent immunochromatographic test strip for simultaneously detecting AFB1 and T-2 toxins, and a preparation method thereof. Background Art

[0002] Mycotoxins are toxic secondary metabolites produced mainly by filamentous fungi. Fungi that produce mycotoxins have the opportunity to contaminate food crops and feed before harvest, after harvest and during storage. Among them, aflatoxin B1 (AFB1) is the most dangerous mycotoxin discovered to date. It has strong carcinogenicity, teratogenicity and mutagenicity, and can cause acute poisoning and liver cancer. T-2 toxin is an immunotoxin that can cause apoptosis of blood lymphocytes, leukopenia and granulocytopenia, and inhibit bone marrow hematopoiesis. Cereals are often infected with multiple mycotoxins at the same time, and the synergistic toxicological effects produced by the coexistence of these mycotoxins will greatly increase their harm to human and animal health. This has led to a growing need for a cheap and rapid solution to simultaneously detect multiple mycotoxins in cereals. Summary of the invention

[0003] The purpose of the present invention is to provide a fluorescent probe and a dual-color fluorescent immunochromatographic test strip (abbreviated as ZGO-ICAs) for simultaneously detecting AFB1 and T-2 toxins and a preparation method thereof.

[0004] A first aspect of the present invention provides a mycotoxin fluorescent detection probe, wherein the probe is a ZGO nanoparticle coupled with a mycotoxin antibody, the ZGO nanoparticle comprises a green fluorescent ZGO:Mn nanoparticle and a blue fluorescent ZGO:Mo nanoparticle, the ZGO:Mn nanoparticle is a Zn2GeO4:Mn nanoparticle, and the ZGO:Mo nanoparticle is a Zn2GeO4:Mo nanoparticle.

[0005] In the present invention, the green fluorescent ZGO:Mn nanoparticles and the blue fluorescent ZGO:Mo nanoparticles are collectively referred to as dual-color fluorescent nanoparticles. For the convenience of description, ZGO is used to collectively refer to ZGO:Mn and ZGO:Mo.

[0006] According to a preferred embodiment of the present invention, the probe is prepared by a preparation method comprising the following steps:

[0007] (1) obtaining ZGO nanoparticles and coating them with a silica shell;

[0008] (2) ZGO nanoparticles coated with silica shells are amino- and carboxy-lated to obtain ZGO-COOH;

[0009] (3) ZGO-COOH was coupled to fungal toxin antibody to obtain the long afterglow nanofluorescence detection probe ZGO-mAb.

[0010] The fluorescent detection probe of the present invention can be coupled to the corresponding mycotoxin antibody according to the target toxin. According to a preferred embodiment of the present invention, the mycotoxin antibody is the antibody AFB1-mAb and T-2-mAb of the mycotoxin AFB1 and T-2.

[0011] According to a preferred embodiment of the present invention, ZGO nanoparticles are obtained by a hydrothermal synthesis method, comprising the following steps:

[0012] (i) under vigorous stirring, Zn(NO3)2, Mn precursor / Mo precursor and concentrated nitric acid are mixed, then Na2GeO3 is slowly added dropwise to the mixture, and then ammonium hydroxide is added to quickly adjust the pH to 9-10; wherein the Mn precursor is Mn(CH3COO)2 and the Mo precursor is Na2MoO4;

[0013] (ii) stirring the mixture obtained in step (i) at room temperature, then transferring it into an autoclave and maintaining it at 180-220° C. for 8-12 hours; collecting the obtained product ZGO:Mn or ZGO:Mo after washing it three times with ultrapure water, and drying it in an oven.

[0014] According to a preferred embodiment of the present invention, the step of coating the silica shell layer includes: dispersing ZGO:Mn or ZGO:Mo in ultrapure water and mixing with CTAB and sodium hydroxide solution; adding an ethanol solution of tetraethyl orthosilicate, stirring and reacting at 40-50°C, and then washing with ultrapure water to obtain silicon-coated ZGO:Mn or ZGO:Mo for subsequent modification.

[0015] According to the method of the present invention, before coupling with antibodies, the ZGO nanoparticles need to be surface modified. According to a preferred embodiment of the present invention, the steps of amination and carboxylation include:

[0016] ZGO nanoparticles coated with silica shell were modified with ATPES to obtain amino-modified ZGO-NH2, which was then carboxylated with diglycolic anhydride to obtain ZGO-COOH.

[0017] Further, the method of amination includes: dispersing 10 mg of ZGO wrapped with a silica shell in 3 mL of DMF, adding 400 μL of APTES, stirring at 80°C for 24 hours, and then washing with anhydrous ethanol and ultrapure water three times to obtain ZGO-NH2. The method of carboxylation includes: adding 12 mg of diglycolic anhydride and 10 mg of ZGO-NH2 to 4 mL of DMF, vigorously stirring overnight at room temperature, and then washing with anhydrous ethanol and ultrapure water three times to obtain ZGO-COOH.

[0018] According to a preferred embodiment of the present invention, the step of coupling ZGO-COOH to mycotoxin antibodies includes: activating the carboxyl groups on the surface of ZGO-COOH; then adding mycotoxin antibodies AFB1-mAb and T-2-mAb respectively, and reacting at room temperature; centrifuging the reaction mixture, adding BSA to block the non-specific sites of ZGO-mAb, and obtaining ZGO-COOH-coupled mycotoxin antibodies.

[0019] Further, the step of coupling ZGO-COOH with mycotoxin antibodies includes: mixing 1.68 mg ZGO:Mn-COOH or 2.8 mg ZGO:Mo-COOH with 0.5 mL MES (0.1 mol / L, pH 5.5), 100 μL EDC (50 mg / mL) and 100 uL NHS (50 mg / mL), reacting for 30 min, and activating the carboxyl groups on the surface of ZGO. In order to remove the unactivated carboxyl groups, the material was washed twice with 1 mL MES (0.1 mol / L, pH 5.5), and the product was resuspended in 0.5 mL MES (0.1 mol / L, pH 5.5), so that carboxyl-activated ZGO:Mn-COOH and ZGO:Mo-COOH were obtained. Then, 10 μg of AFB1-mAb and 6 μg of T-2-mAb were added to ZGO:Mn-COOH and ZGO:Mo-COOH, respectively, and reacted at room temperature for 2 h. Then, the mixture was centrifuged to remove the supernatant, and 500 μL of BSA (1%, w / v) was added and reacted for 0.5 h to block the nonspecific sites of ZGO-mAb. Finally, the ZGO-mAb probe was resuspended with 0.28 mL PBS (pH 7.4, containing 3% sucrose, 1% BSA and 1% trehalose, 1% Tween-20 (w / v)) and stored in a 4 °C refrigerator for subsequent use.

[0020] The second aspect of the present invention provides a dual-color fluorescent immunochromatographic test strip (abbreviated as ZGO-ICAs) for simultaneously detecting AFB1 and T-2 toxins, wherein the test strip comprises, in order along the sample flow direction: a sample pad, absorbent paper, and a pad fixed with a nitrocellulose membrane, wherein the sample pad and the absorbent paper are arranged on the pad and located on opposite sides of the pad; wherein:

[0021] The nitrocellulose membrane is provided with a T1 detection line, a T2 detection line and a C line in sequence along the sample flow direction; the T1 detection line is fixedly provided with AFB1-BSA specifically binding to AFB1-mAb; the T2 detection line is fixedly provided with T-2-BSA specifically binding to T-2-mAb; and the C line is fixedly provided with goat anti-mouse IgG.

[0022] The third aspect of the present invention provides a method for preparing the above-mentioned dual-color fluorescent immunochromatographic test strip, comprising the following steps:

[0023] (1) AFB1-BSA, T-2-BSA, and goat anti-mouse IgG solutions were uniformly sprayed onto the nitrocellulose membrane as T1, T2 detection lines, and C line, respectively, and then placed in an oven for drying;

[0024] (2) Test strip assembly: Using a PVC plate as a backing, assemble the absorbent paper, nitrocellulose membrane, and sample pad in sequence to obtain a dual-channel fluorescent immunochromatographic test strip, which is then stored in a sealed, dry, and cool environment.

[0025] According to a preferred embodiment of the present invention, AFB1-BSA, T-2-BSA and goat anti-mouse IgG solutions are uniformly sprayed onto the nitrocellulose membrane at a speed of 0.8-1.2 μL / cm; the streaking concentrations of AFB1-BSA, T-2-BSA and goat anti-mouse IgG are 0.6 mg / mL, 0.6 mg / mL and 0.5 mg / mL, respectively; and the intervals between the T1 and T2 detection lines and the C line are 3-5 mm.

[0026] A fourth aspect of the present invention provides a kit for simultaneously detecting AFB1 and T-2 toxins, the kit comprising:

[0027] (a) the above-mentioned dual-color fluorescent immunochromatographic test strip;

[0028] (b) The above-mentioned fungal toxin fluorescent detection probe.

[0029] The present invention utilizes the fluorescence emission of the ZGO nanoparticles under 254nm ultraviolet light excitation and the principle of competitive immunoassay to achieve qualitative and quantitative detection of fungal toxins. The detection principle of the dual-color fluorescent immunochromatographic test strip of the present invention is as follows: Figure 4 As shown, the details are as follows:

[0030] When the target toxin does not exist in the sample or is below the detection limit, the target toxin antibody on the fluorescent detection probe will specifically bind to the target toxin antigen on the T line, the T line will emit fluorescence, and the test result will be negative; when the target toxin content in the sample is greater than or equal to the detection limit, the target toxin and the corresponding antigen on the T line will competitively bind to the antibody on the fluorescent detection probe, the antibody on the fluorescent detection probe will preferentially bind to the target toxin, the binding site on the antibody is closed and cannot bind to the target toxin antigen on the T line, the T line does not emit fluorescence, and the test result is positive; the target toxin content is inversely proportional to the fluorescence intensity of the T line, the higher the content, the lower the corresponding T line fluorescence intensity. The test strip of the present invention can achieve qualitative or quantitative detection of the target toxin.

[0031] The fifth aspect of the present invention provides a method for simultaneously detecting AFB1 and T-2 toxins, using the above-mentioned kit, and the detection method is selected from at least one of the following methods:

[0032] Method 1 (qualitative detection) includes the following steps:

[0033] (1) mixing a test solution with a fluorescent detection probe to obtain a mixed solution;

[0034] (2) Add the mixed solution dropwise to the sample pad, let it stand at room temperature, and then place it in a 254nm excitation UV dark box. After the fluorescence stabilizes, observe the T line and C line of the test strip to perform a qualitative test on whether the test solution contains AFB1 and T-2 toxins;

[0035] Method 2 (quantitative detection) includes the following steps:

[0036] (1) mixing a test solution with a fluorescent detection probe to obtain a mixed solution;

[0037] (2) Add the mixed solution to the sample pad, let it stand at room temperature, and then place it in a 254nm excitation UV dark box. After the fluorescence is stable, take a photo to record the T line and C line of the test strip, and use ImageJ software to obtain the grayscale result;

[0038] (3) Substituting the fluorescence intensity obtained by the detection into the standard working curve to calculate the fungal toxin content in the test solution; the standard working curve is a fitting curve based on the fungal toxin standard solutions with different concentration gradients and their corresponding fluorescence intensities.

[0039] The beneficial effects of the present invention are:

[0040] (1) The present invention provides a mycotoxin fluorescent detection probe and a kit for simultaneously detecting AFB1 and T-2 toxins, which meets the needs of simultaneously detecting AFB1 and T-2 mycotoxins and is expected to provide a powerful tool for the rapid detection of multiple mycotoxins coexisting in food.

[0041] (2) The present invention proposes a fluorescent immunochromatographic test strip detection method, which can achieve the purpose of qualitative and quantitative detection of a variety of fungal toxins, has the advantages of simple and rapid operation, high sensitivity, and intuitive results, and is of great significance in maintaining food safety and consumer rights.

[0042] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The above and other objects, features and advantages of the present invention will become more apparent through a more detailed description of exemplary embodiments of the present invention with reference to the accompanying drawings.

[0044] Figure 1 TEM image and particle size distribution diagram of ZGO nanoparticles prepared according to one embodiment of the present invention.

[0045] Figure 2 XRD diagram and fluorescence detection diagram of ZGO nanoparticles prepared according to one embodiment of the present invention.

[0046] Figure 3 This is a UV-visible absorption spectrum of ZGO nanoparticles prepared according to one embodiment of the present invention.

[0047] Figure 4 The figure is a schematic diagram of the working principle of a dual-color fluorescent immunochromatographic test strip prepared according to one embodiment of the present invention.

[0048] Figure 5 The sensitivity and standard curve results of a dual-color fluorescent immunochromatographic test strip prepared according to one embodiment of the present invention.

[0049] Figure 6 The specific results of the dual-color fluorescent immunochromatographic test strip prepared in one embodiment of the present invention. DETAILED DESCRIPTION

[0050] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0051] Example 1

[0052] This example is used to illustrate the preparation of the fluorescent probe ZGO-mAb. The specific method is as follows:

[0053] (1) Preparation of dual-color ZGO nanoparticles Zn2GeO4:Mn and Zn2GeO4:Mo (abbreviated as ZGO:Mn and ZGO:Mo). ZGO:Mn was synthesized by direct hydrothermal method. Specifically, 4 mL of Zn(NO3)2 (2 M), 250 μL of Mn(CH3COO)2 (80 mM) and 1.2 mL of concentrated nitric acid were added under vigorous stirring. Subsequently, 10 mL of Na2GeO3 (0.4 M) was slowly introduced into the above mixture. The pH was quickly adjusted to 9.5 by adding ammonium hydroxide (28 wt%). The resulting mixture was stirred at room temperature for 1 h and then transferred to a Teflon autoclave and reacted at 200 °C for 10 h. Finally, the resulting product (ZGO:Mn) was collected after washing three times with ultrapure water and dried in an oven at 60 °C for 12 h. ZGO:Mo was successfully prepared by the same steps using Na2MoO4 instead of Mn(CH3COO)2 as a precursor.

[0054] (2) The method for coating ZGO nanoparticles with silicon comprises the following steps: the synthesized ZGO:Mn or ZGO:Mo (10 mg) is dispersed in ultrapure water (10 mL), and mixed with CTAB (100 mL, 0.1 M) and sodium hydroxide solution (100 mL, 0.1 m). A 20 vol% tetraethyl orthosilicate (TEOS) ethanol solution (50 μL) is added. The reaction is stirred at 45°C for 2 h, and then washed with ultrapure water 3 times to obtain silicon-coated ZGO:Mn and ZGO:Mo for subsequent surface modification.

[0055] (3) Surface modification of ZGO nanoparticles, comprising the following steps:

[0056] Amination: 10 mg of silicon-coated ZGO nanoparticles were dispersed in 3 mL of DMF, and 400 μL of APTES was added, and stirred at 80 °C for 24 h to obtain aminated ZGO nanoparticles ZGO-NH2, which were then washed three times with anhydrous ethanol and ultrapure water.

[0057] Carboxylation: 12 mg of diglycolic anhydride and 10 mg of ZGO-NH2 were dissolved in 4 mL of DMF and stirred vigorously at room temperature overnight to obtain ZGO-COOH, which was then washed with anhydrous ethanol and ultrapure water three times.

[0058] (4) Preparation of ZGO fluorescent probe ZGO-mAb, including the following steps: 1.68 mg ZGO:Mn-COOH or 2.8 mg ZGO:Mo-COOH, 0.5 mL MES (0.1 mol / L, pH 5.5), 100 μL EDC (50 mg / mL) and 100 μL NHS (50 mg / mL) were mixed and reacted for 30 min to activate the carboxyl groups on the surface of ZGO. In order to remove the unactivated carboxyl groups, the activated nanoparticles were washed twice with 1 mL MES (0.1 mol / L, pH 5.5). The product was resuspended in 0.5 mL MES (0.1 mol / L, pH 5.5), and carboxyl-activated ZGO:Mn-COOH and ZGO:Mo-COOH were obtained. Then, 10 μg of AFB1-mAb and 6 μg of T-2-mAb were added to ZGO:Mn-COOH and ZGO:Mo-COOH, respectively, and reacted at room temperature for 2 h. Then, the mixture was centrifuged to remove the supernatant, and 500 μL of BSA (1%, w / v) was added and reacted for 0.5 h to block the nonspecific sites of ZGO-mAb. Finally, the ZGO-mAb probe was resuspended with 0.28 mL PBS (pH 7.4, containing 3% sucrose, 1% BSA and 1% trehalose, 1% Tween-20 (w / v)) and stored in a 4 ° C refrigerator for subsequent use. That is, two fluorescent detection probes ZGO:Mn-AFB1-mAb and ZGO:Mo-T-2-mAb coupled with different antibodies were obtained.

[0059] (5) Performance characterization

[0060] The morphology, structure and size of ZGO nanoparticles were characterized by TEM and XRD. Figure 1 and Figure 2 . Figure 1 A and B are TEM images of ZGO:Mn and ZGO:Mo respectively. Figure 1 Figures C and D are the particle size distribution diagrams of ZGO:Mn and ZGO:Mo, respectively. The TEM images show that the ZGO nanoparticles have good dispersibility, regular shape, and rod-like structure. The particle size of ZGO:Mn is about 64nm, and the particle size of ZGO:Mo increases to about 63nm. Figure 2 A and B are the XRD patterns and fluorescence detection patterns of ZGO:Mn and ZGO:Mo, respectively. The XRD spectrum shows that the diffraction peak positions of ZGO:Mn and ZGO:Mo nanoparticles are consistent with the ZGO standard card (JCPDS No.11-0687), indicating that they are all pure hexagonal phase crystals. The fluorescence detection pattern shows that both ZGO:Mn and ZGO:Mo nanoparticles have fluorescence.

[0061] refer to Figure 3, UV absorption spectrum of ZGO after connecting with antibody. After coupling with monoclonal antibody, the characteristic peak of monoclonal antibody at 278nm shifts to 282nm, while there is no obvious absorption peak when the antibody is not connected, indicating that the antibody is successfully connected to the ZGO surface.

[0062] Example 2

[0063] This example is used to illustrate the preparation of a dual-channel fluorescent immunochromatographic test strip for simultaneously detecting AFB1 and T-2 toxins. Figure 4 The dual-channel fluorescent immunochromatographic test strip includes, in sequence along the sample flow direction: a sample pad, absorbent paper, and a pad fixed with a nitrocellulose membrane, wherein the sample pad and the absorbent paper are arranged on the pad and located on two opposite sides of the pad;

[0064] The nitrocellulose membrane is provided with a T1 detection line, a T2 detection line and a C line in sequence along the sample flow direction; the T1 detection line is fixedly provided with AFB1-BSA specifically binding to AFB1-mAb; the T2 detection line is fixedly provided with T-2-BSA specifically binding to T-2-mAb; and the C line is fixedly provided with goat anti-mouse IgG.

[0065] The preparation method of the dual-channel fluorescent immunochromatographic test strip comprises the following steps:

[0066] Preparation of nitrocellulose membrane: AFB1-BSA, T-2-BSA, and goat anti-mouse IgG solution were uniformly sprayed onto the nitrocellulose membrane at a speed of 1.0 μL / cm, respectively as T1, T2 detection lines and C line, with a spacing of 4 mm between T1, T2, and C lines, and dried in a 50°C oven for 12 h. Test strip assembly: Using PVC board as a liner, assemble absorbent paper, nitrocellulose membrane, and sample pad in turn, cut into 4.0 mm wide single strips, and obtain ZGO immunochromatographic test strips for dual-channel detection of fungal toxins, which are sealed, dried, and stored in a cool place.

[0067] The streaking concentrations of AFB1-BSA, T-2-BSA, and goat anti-mouse IgG were 0.6 mg / mL, 0.6 mg / mL, and 0.5 mg / mL, respectively.

[0068] Example 3

[0069] This example is used to illustrate a method of the present invention for simultaneously detecting AFB1 and T-2 toxins, using the test strip prepared in Example 2 and the probe in Example 1, and includes the following steps:

[0070] (1) The test solution is mixed with the fluorescent detection probe to obtain a mixed solution; the dosage of the two fluorescent detection probes ZGO-mAb is: ZGO: Mn-mAb dosage is 54 μg, and ZGO: Mo-mAb dosage is 90 μg.

[0071] (2) Add 45 μL of the mixed solution to the sample pad, let it stand at room temperature for 15 minutes, then place it in a UV dark box instrument with an excitation wavelength of 254 nm. After the fluorescence is stable, take photos and record them, and use ImageJ software to obtain the fluorescence intensity of the T line and C line of the test strip. Calculate the fungal toxin content in the test solution based on the fluorescence intensity into the standard working curve. The standard working curve is a fitting curve based on different concentration gradient fungal toxin standard solutions and their corresponding fluorescence intensities.

[0072] The diluent for the detection of standard and actual samples is PBST solution. Preparation of PBST solution: 100 μL PBS (pH 7.4) containing 3% sucrose, 1% BSA (w / v), 1% trehalose (w / v) and 1% Tween-20.

[0073] Example 4

[0074] This example is used to illustrate the sensitivity and specificity of the dual-channel fluorescent immunochromatographic test strip for simultaneous detection of AFB1 and T-2 toxins. Detection was performed according to the method of Example 3.

[0075] Sensitivity test:

[0076] In the present invention, taking AFB1 and T-2 toxins as examples, the standard working curve drawing method includes the following steps:

[0077] AFB1 and T-2 standard solutions were prepared according to various concentration gradients; the AFB1 and T-2 standard solutions with concentration gradients were added dropwise to the immunochromatographic test strips, and the fluorescence signal results at the corresponding concentrations were obtained after 15 minutes; a standard curve was drawn with the concentrations of the AFB1 and T-2 standard solutions as the abscissa and B / B0 (inhibition rate) as the ordinate.

[0078] In the present invention, the cut-off threshold is defined as the mean of 10 blank sample results minus 3 times the standard deviation (SD), i.e., Mean-3SD. When a sample of a certain concentration is detected, if its T / C value is less than the cut-off threshold, it is positive, and vice versa. The lowest concentration when the T / C value of a positive sample is less than the cut-off threshold when compared with a blank sample is selected as the sensitivity of the test strip.

[0079] As a specific embodiment of the present invention, the concentration gradient of AFB1, T-2 standard solution and its B / B0, the specific standard curve is as follows Figure 5As shown in A and B, as the concentration of the fungal toxin standard increases, the corresponding fluorescence intensity decreases. After calculation, the cutoff threshold of AFB1 is defined at 0.442, and the cutoff threshold of T-2 is defined at 0.782. Figure 5 As shown in C and D.

[0080] When the concentration of AFB1 standard is in the range of 0.05 ng / mL to 20 ng / mL, there is a good linear relationship between B / B0 and AFB1 concentration. The standard curve equation is y=-0.08173x+0.88643(R 2 =0.9969), the detection limit is 0.01ng / mL; when the concentration of T-2 standard is in the range of 0.01ng / mL to 50ng / mL, there is a good linear relationship between B / B0 and T-2 concentration, and the standard curve equation is y=-0.02041x+0.89817(R 2 =0.9933), and the detection limit was 0.02 ng / mL. Compared with some previously reported methods for detecting AFB1, T-2 alone or dual-channel detection, the method of the present invention has considerable sensitivity and a wider linear range.

[0081] Specificity test:

[0082] AFB2, AFG1, FB1, OTA, and ZEN toxins were used as competitors, and the concentration of the above standard products was prepared to be 200 ng / mL; the detection effects of the dual-channel fluorescent immunochromatographic test strips on AFB1, T-2, AFB1+T-2, AFB2, AFG1, FB1, OTA, ZEN, DON and other toxins were tested respectively. Figure 6 As shown, ZEN, FB1, OTA, and DON will not interfere with the fluorescent signal detected by the dual-channel test strip, and there is no cross reaction, indicating that the dual-color fluorescent immunochromatographic test strip of the invention has high specificity; AFB2 and AFG1 toxin detection will interfere with the AFB1 content corresponding to the T2 line, because the two are homologues and have similar structures, but the concentrations of AFB2 and AFG1 are much greater than the concentration of AFB1, which will not affect the detection of cereal mycotoxins.

[0083] Example 5

[0084] The present invention is used to illustrate the actual sample detection of a dual-channel fluorescent immunochromatographic test strip for simultaneously detecting AFB1 and T-2 toxins.

[0085] Weigh 1g of sample into a 5mL polypropylene centrifuge tube, add a certain amount of toxin standard solution, add 2mL acetonitrile / water (84:16, V:V), ultrasonicate for 25min at room temperature and then shake for 25min, centrifuge at 6000r / min for 20min, and collect the supernatant. Dilute the supernatant 12.8 times with the above PBST buffer. Take 45μL of the diluted extract into the sample well of the test strip, let it stand for 15min and read the result. All experiments were repeated three times.

[0086] As shown in Table 1, AFB1 and T-2 were detected simultaneously on actual corn flour samples. Standard solutions of AFB1 / T-2 were added to negative corn samples at three concentrations (5.0 / 40μg / kg, 25 / 60μg / kg and 50 / 100μg / kg). The accuracy of the ZGO-ICAs of the present invention was verified with the mature HPLC-MS / MS method. The recovery rate of AFB1 was 90.84% ​​to 95.10%, and the maximum CV was 0.44-1.52%; the recovery rate of T-2 was 113.87% to 115.83%, and the maximum CV was 0.22-0.49%. This shows that the detection results of the ZGO-ICAs developed by the present invention are consistent with the detection results of HPLC-MS / MS, indicating that compared with the instrumental method, ZGO-ICAs have significant reliability in the quantitative detection of AFB1 and T-2.

[0087] Table 1 Comparison of the spiked results of AFB1 and T-2 detected by ZGO-ICAs and HPLC-MS / MS

[0088]

[0089] a Standard deviation (n=3)

[0090] b Recovery rate (RC)

[0091] c Coefficient of variation (CV)

[0092] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A fluorescent probe, characterized in that The probe is a ZGO nanoparticle coupled with a fungal toxin antibody, the ZGO nanoparticle includes a green fluorescent ZGO:Mn nanoparticle and a blue fluorescent ZGO:Mo nanoparticle, the ZGO:Mn nanoparticle is a Zn2GeO4:Mn nanoparticle, and the ZGO:Mo nanoparticle is a Zn2GeO4:Mo nanoparticle.

2. The fluorescent probe according to claim 1, characterized in that The probe is prepared by a preparation method comprising the following steps: (1) obtaining ZGO nanoparticles and coating them with a silica shell; (2) ZGO nanoparticles coated with silica shells are amino- and carboxy-lated to obtain ZGO-COOH; (3) coupling ZGO-COOH with fungal toxin antibody to obtain the long afterglow nanofluorescence detection probe ZGO-mAb; Preferably, the mycotoxin antibodies are antibodies to mycotoxins AFB1 and T-2.

3. The fluorescent probe according to claim 2, characterized in that The ZGO nanoparticles are obtained by a hydrothermal synthesis method, which includes the following steps: (i) under vigorous stirring, Zn(NO3)2, Mn precursor / Mo precursor and concentrated nitric acid are mixed, then Na2GeO3 is slowly added dropwise to the mixture, and then ammonium hydroxide is added to quickly adjust the pH to 9-10; wherein the Mn precursor is Mn(CH3COO)2 and the Mo precursor is Na2MoO4; (ii) stirring the mixture obtained in step (i) at room temperature, then transferring it into an autoclave and maintaining it at 180-220° C. for 8-12 hours; collecting the obtained product ZGO:Mn or ZGO:Mo after washing it three times with ultrapure water, and drying it in an oven.

4. The fluorescent probe according to claim 2, characterized in that The steps of coating the silica shell layer include: dispersing ZGO:Mn or ZGO:Mo in ultrapure water and mixing with CTAB and sodium hydroxide solution; adding ethanol solution of tetraethyl orthosilicate, stirring and reacting at 40-50°C, and then washing with ultrapure water to obtain silicon-coated ZGO:Mn and ZGO:Mo.

5. The fluorescent probe according to claim 2, characterized in that The steps of amination and carboxylation include: ZGO nanoparticles coated with silica shell were modified with ATPES to obtain amino-modified ZGO-NH2, which was then carboxylated with diglycolic anhydride to obtain ZGO-COOH.

6. The fluorescent probe according to claim 2, characterized in that The steps of conjugating ZGO-COOH to mycotoxin antibodies include: The carboxyl groups on the surface of ZGO-COOH were activated; then mycotoxin antibodies AFB1-mAb and T-2-mAb were added respectively and reacted at room temperature; the reaction mixture was centrifuged, and BSA was added to block the non-specific sites of ZGO-mAb to obtain ZGO-COOH-coupled mycotoxin antibodies.

7. A dual-color fluorescent immunochromatographic test strip for simultaneously detecting AFB1 and T-2 toxins, characterized in that: The test strip includes: a sample pad, absorbent paper and a liner fixed with a nitrocellulose membrane in sequence along the sample flow direction, wherein the sample pad and the absorbent paper are arranged on the liner and located on two opposite sides of the liner; wherein, The nitrocellulose membrane is provided with a T1 detection line, a T2 detection line and a C line in sequence along the sample flow direction; the T1 detection line is fixedly provided with AFB1-BSA specifically binding to AFB1-mAb; the T2 detection line is fixedly provided with T-2-BSA specifically binding to T-2-mAb; and the C line is fixedly provided with goat anti-mouse IgG.

8. The method for preparing the dual-color fluorescent immunochromatographic test strip according to claim 7, comprising the following steps: (1) AFB1-BSA, T-2-BSA, and goat anti-mouse IgG solutions are uniformly sprayed onto a nitrocellulose membrane as T1, T2 detection lines, and C line, respectively, and then placed in an oven for drying; preferably, AFB1-BSA, T-2-BSA, and goat anti-mouse IgG solutions are uniformly sprayed onto the nitrocellulose membrane at a speed of 0.8 to 1.2 μL / cm; the line concentrations of AFB1-BSA, T-2-BSA, and goat anti-mouse IgG are 0.6 mg / mL, 0.6 mg / mL, and 0.5 mg / mL, respectively; the interval between the T1, T2 detection lines and the C line is 3 to 5 mm; (2) Test strip assembly: Using a PVC plate as a backing, assemble the absorbent paper, nitrocellulose membrane, and sample pad in sequence to obtain a dual-channel fluorescent immunochromatographic test strip, which is then stored in a sealed, dry, and cool environment.

9. A kit for simultaneously detecting AFB1 and T-2 toxins, characterized in that: The kit comprises: (a) The dual-color fluorescent immunochromatographic test strip according to claim 8; (b) The fungal toxin fluorescent detection probe according to any one of claims 1 to 6.

10. A method for simultaneously detecting AFB1 and T-2 toxins, characterized in that: Using the kit according to claim 9, the detection method is selected from at least one of the following methods: Method 1 includes the following steps: (1) mixing a test solution with a fluorescent detection probe to obtain a mixed solution; (2) Add the mixed solution dropwise to the sample pad, let it stand at room temperature, and then place it in a 254nm excitation UV dark box. After the fluorescence stabilizes, observe the T line and C line of the test strip to perform a qualitative test on whether the test solution contains AFB1 and T-2 toxins; Method 2 includes the following steps: (1) mixing a test solution with a fluorescent detection probe to obtain a mixed solution; (2) Add the mixed solution to the sample pad, let it stand at room temperature, and then place it in a 254nm excitation UV dark box. After the fluorescence is stable, take a photo to record the T line and C line of the test strip, and use ImageJ software to obtain the grayscale result; (3) Substituting the fluorescence intensity obtained by the detection into the standard working curve to calculate the fungal toxin content in the test solution; the standard working curve is a fitting curve based on the fungal toxin standard solutions with different concentration gradients and their corresponding fluorescence intensities.

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