A mycotoxin detection probe and kit, and a mycotoxin detection method

Abstract

The present invention provides a mycotoxin detection probe and a kit, and a mycotoxin detection method, and belongs to the technical field of mycotoxin detection. The mycotoxin detection probe provided by the present invention includes a magnetic core multilayer quantum dot composite nanomaterial with a core-shell three-dimensional structure and a mycotoxin antibody loaded on the surface of the magnetic core multilayer quantum dot composite nanomaterial with a core-shell three-dimensional structure. The present invention utilizes Fe3O4 magnetic nanoparticles as the inner core, and its strong magnetic responsiveness to the external magnetic field can give the probe good magnetic separation characteristics. By utilizing magnetic separation technology, complex sample pretreatment methods can be omitted, and the concentration of the sample to be tested can be quickly realized to avoid sample loss, thereby improving detection sensitivity and reducing analysis time. The present invention wraps two layers of water-soluble polymers on the surface of the inner core, and each layer of the water-soluble polymer surface is respectively loaded with carboxylated quantum dots, which can make the probe have good stability and biocompatibility in complex samples.

Description

Technical Field

[0001] The present invention relates to the technical field of mycotoxin detection, and in particular to a mycotoxin detection probe and kit, and a mycotoxin detection method. Background Art

[0002] Mycotoxins, such as aflatoxin B1 (AFB1), fumonisin B1 (FB1), and ochratoxin A (OTA), are toxic secondary metabolites produced by filamentous fungi, posing a great threat to global agriculture and human health. Given their excellent chemical and thermal stability and low molecular weight (300-700 Da), these mycotoxins can be concentrated in plants and animals and easily enter the human body through the food chain (such as juice, meat, and milk), causing serious diseases including liver damage, kidney failure, and cancer. Worse still, the coexistence of multiple mycotoxins in contaminated foods is a common phenomenon because various fungi have the same ecological conditions for growth or toxin production. According to the Food and Agriculture Organization (FAO), more than 25% of food crops worldwide are contaminated with mycotoxins, and up to 91.4% of samples are co-contaminated. Therefore, there is a strong demand for a simple method for ultrasensitive simultaneous detection of multiple mycotoxins in real food.

[0003] Immunochromatographic testing has become a mature and powerful point-of-care testing tool due to its outstanding advantages such as rapidity, low cost, portability, and easy operation. In addition, immunochromatographic detection systems have also shown great potential in multiplex detection. Previous studies have shown that the construction of multiple parallel test lines or dot arrays on nitrocellulose membranes can simultaneously identify multiple targets in one ICA strip. The main obstacles of traditional immunochromatographic detection methods are low sensitivity and limited quantitative capabilities due to the use of colorimetric nanolabels (mainly gold nanoparticles) as signal labels. Recently, a class of fluorescent labels, such as fluorescent dyes, time-resolved fluorescent nanoparticles, upconversion nanomaterials, and quantum dots, have been integrated into immunochromatographic detection systems to improve their sensitivity and quantitative capabilities.

[0004] However, when detecting mycotoxins in food, complex food ingredients can easily affect the colloidal stability of fluorescent labels. At the same time, in order to avoid the matrix effect of real food, the sample needs to be diluted or subjected to complex pretreatment (such as extraction, purification, washing, centrifugation), which will lead to a decrease in detection sensitivity. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a mycotoxin detection probe and a kit, and a mycotoxin detection method. The mycotoxin detection probe provided by the present invention has high stability and high detection sensitivity when used for mycotoxin detection.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a mycotoxin detection probe, comprising a magnetic core multilayer quantum dot composite nanomaterial with a core-shell three-dimensional structure and a mycotoxin antibody loaded on the surface of the magnetic core multilayer quantum dot composite nanomaterial with a core-shell three-dimensional structure;

[0008] The mycotoxin antibody is one or more of aflatoxin B1 antibody, ochratoxin A antibody and fumonisin B1 antibody;

[0009] The core-shell three-dimensional structured magnetic core multilayer quantum dot composite nanomaterial comprises a Fe3O4 magnetic nanoparticle core and two layers of water-soluble polymers sequentially wrapped on the surface of the core, and the surface of each layer of the water-soluble polymer is loaded with carboxylated quantum dots.

[0010] Preferably, the water-soluble polymer is PEI, and the number average molecular weight of the PEI is 5,000 to 80,000.

[0011] Preferably, the carboxylated quantum dots are CdSe / ZnS quantum dots, and the surface of the CdSe / ZnS quantum dots is modified with 3-mercaptopropionic acid; the CdSe / ZnS quantum dots have CdSe as the core and ZnS as the shell.

[0012] Preferably, the particle size of the Fe3O4 magnetic nanoparticle core is 80 to 600 nm.

[0013] Preferably, the method for preparing the core-shell three-dimensional structured magnetic core multilayer quantum dot composite nanomaterial comprises the following steps:

[0014] Ultrasonic mixing of Fe3O4 magnetic nanoparticles and the first water-soluble polymer solution to obtain Fe3O4 magnetic nanoparticles coated with a single layer of water-soluble polymer;

[0015] The Fe3O4 magnetic nanoparticles wrapped by the single-layer water-soluble polymer are mixed with the first carboxylated quantum dots to obtain a magnetic core quantum dot composite nanomaterial loaded with a single-layer quantum dot;

[0016] Ultrasonic mixing the magnetic core quantum dot composite nanomaterial loaded with a single layer of quantum dots and a second water-soluble polymer solution to obtain a magnetic core quantum dot composite nanomaterial wrapped with two layers of water-soluble polymer;

[0017] The magnetic core quantum dot composite nanomaterial wrapped by the two layers of water-soluble polymer is mixed with the second carboxylation quantum dot to obtain a magnetic core multilayer quantum dot composite nanomaterial with a core-shell three-dimensional structure.

[0018] The present invention provides a method for preparing the above-mentioned mycotoxin detection probe, comprising the following steps:

[0019] The core-shell three-dimensional structured magnetic core multilayer quantum dot composite nanomaterial is mixed with a carboxyl activator to perform an activation reaction to obtain an activated magnetic core multilayer quantum dot composite nanomaterial;

[0020] The activated magnetic core multilayer quantum dot composite nanomaterial is mixed with a fungal toxin antibody for loading, and bovine serum albumin is added after loading to obtain a fungal toxin detection probe.

[0021] The present invention provides a kit for detecting fungal toxins, comprising the above-mentioned fungal toxin detection probe of the immunochromatographic test strip;

[0022] The immunochromatographic test strip comprises a test strip matrix and a detection line and a control line located on the surface of the test strip matrix;

[0023] The components of the test line include mycotoxin binding antigens;

[0024] The fungal toxins detected by the kit are one or more of aflatoxin B1, ochratoxin A and fumonisin B1; when the kit detects multiple fungal toxins, each toxin corresponds to a detection line.

[0025] Preferably, the components of the control line include goat anti-mouse IgG antibody.

[0026] The present invention provides a method for detecting mycotoxins, wherein the mycotoxins are one or more of aflatoxin B1, ochratoxin A and fumonisin B1, and the detection method comprises the following steps:

[0027] The sample solution to be tested is mixed with the above-mentioned mycotoxin detection probe and then separated by magnetic adsorption, and the obtained solid is dispersed in a buffer solution to obtain a detection solution;

[0028] The detection liquid is added to one end of the immunochromatographic test strip, the fluorescence intensity of the detection line is tested, and the mycotoxin concentration of the detection liquid is obtained according to the fluorescence intensity and a predetermined standard curve; the standard curve is a fitting curve of the mycotoxin concentration and the fluorescence intensity.

[0029] Preferably, the minimum detection limit of aflatoxin B1 is 0.42 pg / mL, the minimum detection limit of ochratoxin A is 11.48 pg / mL, and the minimum detection limit of fumonisin B1 is 4.21 pg / mL.

[0030] The invention provides a mycotoxin detection probe, comprising a core-shell three-dimensional structure of a magnetic core multilayer quantum dot composite nanomaterial and a mycotoxin antibody loaded on the surface of the core-shell three-dimensional structure of the magnetic core multilayer quantum dot composite nanomaterial; the mycotoxin antibody is one or more of aflatoxin B1 antibody, ochratoxin A antibody and fumonisin B1 antibody; the core-shell three-dimensional structure of the magnetic core multilayer quantum dot composite nanomaterial comprises a Fe3O4 magnetic nanoparticle core and two layers of water-soluble polymers sequentially wrapped on the surface of the core, and each layer of the water-soluble polymer surface is respectively loaded with carboxylated quantum dots. The magnetic core multilayer quantum dot composite nanomaterial of the invention can be used as an enrichment tool for immunochromatographic detection and as an excellent fluorescent label. The composite nanomaterial uses Fe3O4 magnetic nanoparticles as the core, and its strong magnetic responsiveness to the external magnetic field can give the probe good magnetic separation characteristics. By using magnetic separation technology, complex sample pretreatment methods can be omitted, and the concentration of the sample to be tested can be quickly realized to avoid sample loss, thereby improving detection sensitivity and reducing analysis time. The present invention adopts a method of coating two layers of water-soluble polymer on the surface of the inner core, and each layer of water-soluble polymer is loaded with carboxylated quantum dots on the surface, which can make the probe have good stability and biocompatibility in complex samples. At the same time, the two layers of water-soluble polymer can increase the specific surface area of ​​the probe and improve the ability to capture target substances; the double-layer carboxylated quantum dot structure can also greatly improve the fluorescence signal of the material.

[0031] The present invention provides a kit for detecting mycotoxins, comprising an immunochromatographic test strip and the above-mentioned mycotoxins detection probe. The kit for detecting mycotoxins provided by the present invention has high detection sensitivity. In the present invention, the color development principle of the immunochromatographic test strip for detecting mycotoxins is based on the competitive reaction between the mycotoxins in the sample and the mycotoxins-BSA antigens fixed on the test line of the test strip. After the detection probe is incubated with the mycotoxins in the sample and magnetically enriched, the collected probe-mycotoxins immune complex is loaded onto the sample pad of the test strip. Under capillary action, the sample solution moves along the NC membrane toward the absorption pad. If there is no target mycotoxins in the sample solution, the probe-mycotoxins immune complex cannot be formed, so the free probe will recognize the corresponding capture antigens pre-fixed on the test line, making the test line fluorescent. When the sample is contaminated with a target mycotoxins, it will bind to the corresponding probe, hindering its immune binding reaction with the capture antigens on the test line. In this case, the corresponding test line binds less probes and shows a lower fluorescence intensity. When a high concentration of target true toxins is present in the sample, they will completely prevent the probe from binding to the test line; therefore, the fluorescent signal cannot be detected. Excess probe is bound by anti-mouse IgG immobilized in the control zone so that a bright control line should always be visible and this can be used to determine the effectiveness of the assay.

[0032] The present invention provides a method for detecting mycotoxins. The present invention uses the above-mentioned mycotoxin detection probe as an enrichment tool for immunochromatographic detection, which can quickly achieve the concentration of the sample to be tested, avoid sample loss, thereby improving the detection sensitivity and reducing the analysis time. The method of the present invention has a minimum detection limit of 0.42 pg / mL for aflatoxin B1, a minimum detection limit of 11.48 pg / mL for ochratoxin A, and a minimum detection limit of 4.21 pg / mL for fumonisin B1. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The preparation flow chart of the core-shell three-dimensional structured magnetic core multilayer quantum dot composite nanomaterial MagQBD;

[0034] Figure 2 HRTEM test results of the product obtained in Example 1;

[0035] Figure 3 The characterization results of the magnetic core multilayer quantum dot composite nanomaterial MagQBD prepared in Example 1;

[0036] Figure 4 The stability test results of the magnetic core multilayer quantum dot shell composite nanomaterial prepared in Example 1;

[0037] Figure 5 Schematic diagram of the preparation process of mycotoxin detection probe;

[0038] Figure 6 This is the experimental principle diagram of the simultaneous detection of three fungal toxins by quantum dot fluorescence immunochromatography;

[0039] Figure 7 The photos are of test strips for detecting different concentrations of aflatoxin B1, ochratoxin A, and fumonisin B1 under ultraviolet light;

[0040] Figure 8 for Figure 7 Corresponding fluorescence intensity values ​​and standard curve;

[0041] Fig. 9 To detect different concentrations of ochratoxin A, fumonisin B1 and aflatoxin B1 by standard colloidal gold immunochromatographic colorimetric results;

[0042] Fig.10 The following are the fluorescence images and corresponding fluorescence intensity values ​​of the commercial quantum dot microsphere immunolayer detecting three target fungal toxins;

[0043] Fig.11 It is the test result of the immunochromatographic strip specificity experiment based on MagQBD;

[0044] Fig.12This is the result of the repeatability test of the MagQBD-based immunochromatographic strip. DETAILED DESCRIPTION

[0045] The present invention provides a mycotoxin detection probe, comprising a magnetic core multilayer quantum dot composite nanomaterial with a core-shell three-dimensional structure and a mycotoxin antibody loaded on the surface of the magnetic core multilayer quantum dot composite nanomaterial with a core-shell three-dimensional structure;

[0046] The mycotoxin antibody is one or more of aflatoxin B1 antibody, ochratoxin A antibody and fumonisin B1 antibody;

[0047] The core-shell three-dimensional structured magnetic core multilayer quantum dot composite nanomaterial comprises a Fe3O4 magnetic nanoparticle core and two layers of water-soluble polymers sequentially wrapped on the surface of the core, and the surface of each layer of the water-soluble polymer is loaded with carboxylated quantum dots.

[0048] In the present invention, the particle size of the Fe3O4 magnetic nanoparticle core is preferably 80 to 600 nm, more preferably 100 to 500 nm, and further preferably 200 to 300 nm.

[0049] The present invention has no special requirements on the source of the Fe3O4 magnetic nanoparticles. Commercially available Fe3O4 magnetic nanoparticles in the art can be used or self-prepared. When self-prepared, the preparation method preferably includes the following steps:

[0050] Soluble trivalent iron, a reducing agent, a surfactant and a solvent are mixed and subjected to a reduction reaction to obtain Fe3O4 magnetic nanoparticles.

[0051] In the present invention, the soluble trivalent iron is preferably ferric chloride hexahydrate; the reducing agent is preferably anhydrous sodium acetate; the surfactant is preferably PVP; and the solvent is preferably ethylene glycol or ethylene glycol ether.

[0052] In the present invention, the temperature of the reduction reaction is preferably 200° C., and the time is preferably 8 to 48 hours, more preferably 12 to 36 hours.

[0053] In the present invention, after the reduction reaction, the present invention preferably sequentially washes and dries the obtained reduction reaction liquid. In the present invention, the washing method is preferably washing twice with deionized water and ethanol respectively; in the present invention, the drying method is preferably vacuum drying, and the drying temperature is preferably 60°C.

[0054] In the present invention, the water-soluble polymer is preferably PEI, and the number average molecular weight of the PEI is preferably 5000 to 80000, more preferably 10000 to 50000. In the present invention, the thickness of the two layers of water-soluble polymer is preferably 10 to 100 nm, more preferably 30 nm.

[0055] In the present invention, the carboxylated quantum dots are preferably CdSe / ZnS quantum dots, and the surface of the CdSe / ZnS quantum dots is modified with 3-mercaptopropionic acid; the CdSe / ZnS quantum dots have CdSe as the core and ZnS as the shell. In the present invention, the source of the CdSe / ZnS quantum dots modified with 3-mercaptopropionic acid is preferably commercially available. As a specific embodiment of the present invention, the CdSe / ZnS quantum dots modified with 3-mercaptopropionic acid are purchased from Xingshuo Nano Technology Co., Ltd., model CdSe / ZnS-MPA-631.

[0056] In the present invention, the method for preparing the core-shell three-dimensional structured magnetic core multilayer quantum dot composite nanomaterial preferably comprises the following steps:

[0057] Ultrasonic mixing of Fe3O4 magnetic nanoparticles and the first water-soluble polymer solution to obtain Fe3O4 magnetic nanoparticles coated with a single layer of water-soluble polymer;

[0058] The Fe3O4 magnetic nanoparticles wrapped by the single-layer water-soluble polymer are mixed with the first carboxylated quantum dots to obtain a magnetic core quantum dot composite nanomaterial loaded with a single-layer quantum dot;

[0059] Ultrasonic mixing the magnetic core quantum dot composite nanomaterial loaded with a single layer of quantum dots and a second water-soluble polymer solution to obtain a magnetic core quantum dot composite nanomaterial wrapped with two layers of water-soluble polymer;

[0060] The magnetic core quantum dot composite nanomaterial wrapped by the two layers of water-soluble polymer is mixed with the second carboxylation quantum dot to obtain a magnetic core multilayer quantum dot composite nanomaterial with a core-shell three-dimensional structure.

[0061] The present invention ultrasonically mixes Fe3O4 magnetic nanoparticles with a first water-soluble polymer solution to obtain Fe3O4 magnetic nanoparticles wrapped by a single layer of water-soluble polymer. In the present invention, the first water-soluble polymer solution is preferably a PEI aqueous solution, and the mass concentration of the PEI aqueous solution is preferably 0.2 to 5 mg / mL, more preferably 1 mg / mL. In the present invention, the mass ratio of the Fe3O4 magnetic nanoparticles to the first water-soluble polymer is preferably 1:1 to 5, more preferably 1:1.

[0062] In the present invention, the power of the ultrasonic mixing is preferably 20 to 40 kHz, more preferably 40 kHz; the ultrasonic time is preferably 10 to 60 min, more preferably 30 min. The present invention uses the ultrasound to wrap the first water-soluble polymer on the surface of the Fe3O4 magnetic nanoparticles. After the ultrasound, the present invention preferably performs magnetic adsorption separation on the Fe3O4 magnetic nanoparticles wrapped with a single layer of water-soluble polymer, and redisperses the separated solid in water.

[0063] After obtaining the Fe3O4 magnetic nanoparticles wrapped by the single-layer water-soluble polymer, the present invention mixes the Fe3O4 magnetic nanoparticles wrapped by the single-layer water-soluble polymer with the first carboxylated quantum dots to obtain a magnetic core quantum dot composite nanomaterial loaded with a single-layer quantum dot. In the present invention, the first carboxylated quantum dots are preferably CdSe / ZnS quantum dots modified with 3-mercaptopropionic acid. In the present invention, the concentration of the first carboxylated quantum dots is preferably 20 nM.

[0064] After the mixing, the present invention preferably performs ultrasound on the obtained mixed solution, and the ultrasound time is preferably 30 minutes. After the ultrasound, the present invention preferably performs magnetic adsorption separation on the obtained magnetic core quantum dot composite nanomaterial loaded with a single layer of quantum dots, and the separated solid is redispersed in water.

[0065] After obtaining the magnetic core quantum dot composite nanomaterial loaded with a single layer of quantum dots, the present invention ultrasonically mixes the magnetic core quantum dot composite nanomaterial loaded with a single layer of quantum dots with a second water-soluble polymer solution to obtain a magnetic core quantum dot composite nanomaterial wrapped with two layers of water-soluble polymers. In the present invention, the second water-soluble polymer solution is preferably a PEI aqueous solution, and the mass concentration of the PEI aqueous solution is preferably 0.2 to 5 mg / mL, more preferably 1 mg / mL. In the present invention, the mass ratio of the Fe3O4 magnetic nanoparticles to the second water-soluble polymer is preferably 1:1 to 5, more preferably 1:1.

[0066] In the present invention, the power of the ultrasonic mixing is preferably 20 to 40 kHz, more preferably 40 kHz; the ultrasonic time is preferably 10 to 60 min, more preferably 30 min. After the ultrasonication, the present invention preferably performs magnetic adsorption separation on the magnetic core quantum dot composite nanomaterial wrapped by two layers of water-soluble polymer, and redisperses the separated solid in water.

[0067] After obtaining the two layers of water-soluble polymer-wrapped magnetic core quantum dot composite nanomaterial, the present invention mixes the two layers of water-soluble polymer-wrapped magnetic core quantum dot composite nanomaterial with the second carboxylated quantum dot to obtain a core-shell three-dimensional structure of a magnetic core multilayer quantum dot composite nanomaterial. In the present invention, the second carboxylated quantum dot is preferably a CdSe / ZnS quantum dot modified with 3-mercaptopropionic acid. In the present invention, the concentration of the second carboxylated quantum dot is preferably 20 nM.

[0068] In the present invention, the mycotoxin antibody is one or more of aflatoxin B1 antibody, ochratoxin A antibody and fumonisin B1 antibody. The present invention has no special requirements on the specific types and sources of the above antibodies, and the above antibodies commercially available in the art can be used.

[0069] In the present invention, the mass ratio of the Fe3O4 magnetic nanoparticles to the mycotoxin antibody is preferably 1 mg Fe3O4: 5-20 μg mycotoxin antibody.

[0070] The present invention provides a method for preparing the above-mentioned mycotoxin detection probe, comprising the following steps:

[0071] The core-shell three-dimensional structured magnetic core multilayer quantum dot composite nanomaterial is mixed with a carboxyl activator to perform an activation reaction to obtain an activated magnetic core multilayer quantum dot composite nanomaterial;

[0072] The activated magnetic core multilayer quantum dot composite nanomaterial is mixed with a fungal toxin antibody for loading, and bovine serum albumin is added after loading to obtain a fungal toxin detection probe.

[0073] The present invention mixes a core-shell three-dimensional structured magnetic core multilayer quantum dot composite nanomaterial with a carboxyl activator, performs an activation reaction, and obtains an activated magnetic core multilayer quantum dot composite nanomaterial. Before the activation reaction, the present invention preferably pretreats the core-shell three-dimensional structured magnetic core multilayer quantum dot composite nanomaterial, and the pretreatment preferably includes:

[0074] The core-shell three-dimensional structured magnetic core multilayer quantum dot composite nanomaterial is washed with phosphate buffer and dispersed in MES buffer after washing.

[0075] In the present invention, the concentration of the phosphate buffer is preferably 0.01M, and the pH value is preferably 7.4. In the present invention, the concentration of the MES buffer is preferably 0.1M, and the pH value is preferably 5.5. In the present invention, the mass ratio of the core-shell three-dimensional structured magnetic core multilayer quantum dot composite nanomaterial to the volume ratio of the MES buffer is preferably 1mg:500μL.

[0076] In the present invention, the carboxyl activating agent is preferably EDC and NHS. The molar ratio of EDC to NHS is preferably 1:1-4, more preferably 1:2-3.

[0077] In the present invention, the activation reaction temperature is preferably room temperature, and the time is preferably 15 min. After the activation reaction, the present invention preferably performs magnetic adsorption separation on the obtained activation reaction solution, and the obtained magnetic adsorption separation product is redispersed in PBS buffer.

[0078] After obtaining the activated magnetic core multilayer quantum dot composite nanomaterial, the present invention mixes the activated magnetic core multilayer quantum dot composite nanomaterial with a mycotoxin antibody for loading, and then adds bovine serum albumin to obtain a mycotoxin detection probe. In the present invention, the loading temperature is preferably room temperature, and the loading time is preferably 0.5 to 3 hours, and more preferably 2 hours.

[0079] In the present invention, the mass concentration of the bovine serum albumin is preferably 10%. In the present invention, the time for adding the bovine serum albumin is preferably 30 minutes. In the present invention, the bovine serum albumin plays a blocking role.

[0080] In the present invention, after adding bovine serum albumin, the present invention preferably performs magnetic adsorption separation on the obtained product, and the obtained magnetic adsorption separation product is redispersed in a preservation solution. In the present invention, the preservation solution is preferably a preservation solution containing 10mM PBS (pH7.4), 0.02% NaN3 (w / v), 0.5% BSA (w / v) and 0.05% Tween 20 (v / v).

[0081] The present invention provides a kit for detecting fungal toxins, comprising an immunochromatographic test strip and the fungal toxin detection probe or the fungal toxin detection probe prepared by the preparation method;

[0082] The immunochromatographic test strip comprises a test strip matrix and a detection line (T line) and a control line (C line) located on the surface of the test strip matrix;

[0083] The components of the test line include mycotoxin binding antigens;

[0084] The fungal toxins detected by the kit are one or more of aflatoxin B1, ochratoxin A and fumonisin B1; when the kit detects multiple fungal toxins, each toxin corresponds to a detection line.

[0085] In the present invention, the test strip substrate is preferably a nitrocellulose membrane (NC membrane). In the present invention, the component of the detection line includes mycotoxin binding antigen, and the specific component of the detection limit line is AFB1-BSA, OTA-BSA or FB1-BSA.

[0086] In the present invention, the components of the control line preferably include goat anti-mouse IgG antibody.

[0087] In the present invention, the method for preparing the immunochromatographic test strip preferably comprises the following steps:

[0088] The PBS buffer solution of the fungal toxin-binding antigen and the control line solution were sprayed on the surface of the nitrocellulose membrane.

[0089] In the present invention, the mycotoxin binding antigen is AFB1-BSA, OTA-BSA or FB1-BSA. In the present invention, the concentration of AFB1-BSA is preferably 0.8 mg / mL, the concentration of OTA-BSA is preferably 0.6 mg / mL, and the concentration of FB1-BSA is preferably 0.06 mg / mL.

[0090] In the present invention, the concentration of the PBS buffer is preferably 10 mM, and the pH value is preferably 7.4.

[0091] In the present invention, the concentration of the control line solution is preferably 0.4 mg / mL.

[0092] The present invention has no special requirements for the spraying method, and any spraying method well known to those skilled in the art can be used.

[0093] The present invention provides a method for detecting mycotoxins, wherein the mycotoxins are one or more of aflatoxin B1, ochratoxin A and fumonisin B1, and the detection method comprises the following steps:

[0094] The sample solution to be tested is mixed with the above-mentioned mycotoxin detection probe and then separated by magnetic adsorption, and the obtained solid is dispersed in a buffer solution to obtain a detection solution;

[0095] The detection liquid is added to one end of the immunochromatographic test strip, the fluorescence intensity of the detection line is tested, and the fungal toxin concentration of the detection liquid is obtained according to the fluorescence intensity and a predetermined standard curve; the standard curve is a fitting curve of the fungal toxin concentration and the fluorescence intensity.

[0096] In the present invention, the sample solution to be tested is preferably a food solution. In the present invention, the method for preparing the sample solution to be tested preferably comprises the following steps:

[0097] When the sample to be tested is solid, the present invention grinds the sample to be tested and mixes it with methanol aqueous solution, and filters it to obtain a solution of the sample to be tested.

[0098] In the present invention, the volume ratio of methanol to water in the methanol aqueous solution is preferably 7:3; the volume ratio of the mass of the sample crushed material to the methanol aqueous solution is preferably 1 g:5 mL.

[0099] When the sample to be tested is liquid, the liquid sample to be tested is directly used as the sample to be tested.

[0100] The present invention mixes the sample solution to be tested with the above-mentioned fungal toxin detection probe and then separates them by magnetic adsorption. In the present invention, the fungal toxin detection probe is preferably added in the form of a PBS buffer dispersion, and the concentration of the fungal toxin detection probe is preferably 1-20 mg / mL, more preferably 10 mg / mL. In the present invention, the volume ratio of the mass of the fungal toxin detection probe to the sample solution to be tested is preferably 1-20 mg:1 mL, more preferably 10 mg:1 mL.

[0101] In the present invention, the mixing method is preferably oscillating mixing, the oscillating mixing speed is preferably 800 rpm, and the time is preferably 15 min.

[0102] In the present invention, the solid obtained by magnetic adsorption separation is dispersed in a buffer solution to obtain a detection solution. In the present invention, the buffer solution is preferably a borate buffer solution, the concentration of the boric acid buffer solution is preferably 10 mM, and the boric acid buffer solution is preferably a 1% Tween 20 solution.

[0103] The present invention adds the detection liquid to one end of the immunochromatographic test strip, and when a fluorescent signal appears on the detection line, it indicates that the detection liquid contains the fungal toxin corresponding to the detection line.

[0104] In the present invention, the amount of the detection solution added is preferably 50 to 100 μL, more preferably 60 to 80 μL. In the present invention, the fluorescence signal detection result is preferably tested after 20 minutes.

[0105] The present invention tests the fluorescence intensity of the test line, and obtains the mycotoxin concentration of the test solution according to the fluorescence intensity and a predetermined standard curve; the standard curve is a fitting curve of the mycotoxin concentration and the fluorescence intensity. In the present invention, taking aflatoxin B1 as an example, the method for obtaining the standard curve preferably includes the following steps:

[0106] Provide gradient concentrations of aflatoxin B1 standard solution;

[0107] The gradient concentration aflatoxin B1 standard solution is added to one end of the immunochromatographic test strip to obtain the fluorescence intensity corresponding to the gradient concentration aflatoxin B1 standard solution, and a standard curve is drawn with the aflatoxin B1 standard solution as the abscissa and the fluorescence intensity as the ordinate.

[0108] As a specific embodiment of the present invention, the gradient concentrations of aflatoxin B1 standard solution and its corresponding fluorescence intensity, and the specific standard curve are shown in Table 1.

[0109] Table 1 Aflatoxin B1 concentration, fluorescence intensity and standard curve

[0110]

[0111] In the present invention, the method for obtaining the standard curves of ochratoxin A and fumonisin B1 is the same as the method for obtaining the standard curve of aflatoxin B1, which will not be described in detail herein.

[0112] As a specific embodiment of the present invention, the gradient concentrations of ochratoxin A standard solution and its corresponding fluorescence intensity, and the specific standard curve are shown in Table 2.

[0113] Table 2 Ochratoxin A concentration, fluorescence intensity and standard curve

[0114]

[0115] As a specific embodiment of the present invention, the gradient concentrations of the fumonisin B1 standard solution and its corresponding fluorescence intensity, and the specific standard curve are shown in Table 3.

[0116] Table 3 Fumonisin B1 concentration, fluorescence intensity and standard curve

[0117]

[0118] In the present invention, the minimum detection limit of aflatoxin B1 is 0.42 pg / mL, the minimum detection limit of ochratoxin A is 11.48 pg / mL, and the minimum detection limit of fumonisin B1 is 4.21 pg / mL.

[0119] In the present invention, the detection range of aflatoxin B1 is preferably 1 to 0.00046 ng / mL, the detection range of ochratoxin A is preferably 10 to 0.0046 ng / mL, and the detection range of fumonisin B1 is preferably 10 to 0.0046 ng / mL.

[0120] A mycotoxin detection probe and kit, and a mycotoxin detection method provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0121] Example 1

[0122] Preparation of core-shell three-dimensional structured magnetic core multilayer quantum dot composite nanomaterials

[0123] (1) Preparation of 160nm Fe3O4 core with good dispersion:

[0124] First, add 1.36g of ferric chloride hexahydrate to 40mg of ethylene glycol solution and stir magnetically for 30min. Then, add 1g of reducing agent polyethylene glycol 8000 and 2.7g of alkali source anhydrous sodium acetate to the above solution and stir evenly until completely dissolved. Then, transfer the above mixed solution to a high-pressure reactor (polytetrafluoroethylene liner) and place it in a 200℃ blast oven for reaction for 8 to 48h. Then, after the reaction is completed, wait for the material to cool to room temperature, collect the precipitate by magnetic enrichment and wash it twice with deionized water and ethanol respectively. Finally, place it in a vacuum drying oven at 60℃ to dry it to obtain the product Fe3O4 microspheres.

[0125] (2) Preparation of PEI-coated Fe3O4 magnetic nanoparticles Fe3O4@PEI

[0126] 10 mg of Fe3O4 particles were reacted with 50 mL of PEI aqueous solution (0.2 mg / mL) under intense ultrasound for 30 min, and the PEI-coated Fe3O4 particles (Fe3O4@PEI) were collected by magnetic enrichment, washed twice and dispersed in 10 mL of deionized water.

[0127] (3) Preparation of MagQB composite nanomaterials loaded with single-layer quantum dots

[0128] The prepared Fe3O4@PEI solution was added to the solution and then 100 μL of CdSe / ZnS MPA QD (20 nM) was added to the solution. After ultrasonic treatment for 30 min, it was washed with deionized water and then magnetically enriched, and finally resuspended in 10 mL of deionized water for use.

[0129] (4) Preparation of MagQB@PEI composite nanomaterials wrapped with two layers of water-soluble polymers

[0130] 10 mL of MagQB particles were reacted with 50 mL of PEI aqueous solution (0.2 mg / mL) under intense ultrasound for 30 min, and the PEI-coated MagQB particles (MagQB@PEI) were collected by magnetic enrichment, washed twice, and then dispersed in 10 mL of deionized water.

[0131] (5) Preparation of core-shell three-dimensional structured magnetic core multilayer quantum dot composite nanomaterials MagQBD

[0132] The prepared MagQB@PEI solution was added to the solution and then 100 μL of CdSe / ZnS MPA QD (20 nM) was added to the solution. After ultrasonic treatment for 30 min, the solution was washed with deionized water and then magnetically enriched, and finally resuspended in 10 mL of deionized water for later use.

[0133] The preparation process of the core-shell three-dimensional structured magnetic core multilayer quantum dot composite nanomaterial MagQBD is shown in the figure. Figure 1 shown.

[0134] Figure 2 The HRTEM test results of the product obtained in Example 1. Wherein a is the Fe3O4 core particles obtained in step (1) of this embodiment, b is the Fe3O4@PEI particles obtained in step (2), c is the MagQB particles obtained in step (3), and d is the high-definition transmission electron microscopy (HRTEM) of the MagQBD particles obtained in step (5). It can be seen from the above HRTEM results that CdSe / ZnS quantum dots are evenly distributed on the surface of typical nearly spherical 160nm Fe3O4 nanoparticles, and the second layer of the quantum dot shell is constructed by PEI-mediated electrostatic adsorption, so that MagQBD has a thicker shell than MagQB, thereby loading more quantum dots. The high loading amount of carboxylated quantum dots makes the product have excellent and stable fluorescence properties, and there are sufficient sites to facilitate the subsequent antibody coupling reaction.

[0135] The characterization results of the magnetic core multilayer quantum dot composite nanomaterial MagQBD prepared in Example 1 are as follows: Figure 3 shown. Figure 3 a is the zeta potential test result, b is the photo of each component under visible light and ultraviolet light excitation during the synthesis process, and c is the fluorescence spectrum of each component. Figure 3 As can be seen in Figure a, the change in the zeta potential of each component during the material synthesis process can monitor the process of PEI-mediated self-assembly reaction adsorption of quantum dots. Figure 3 As can be seen in Figure c, under UV-365nm excitation, MagQBD has a higher fluorescence intensity than MagQB and quantum dot solution, which can be attributed to the higher loading amount of the double-layer quantum dot shell.

[0136] The stability test results of the magnetic core multilayer quantum dot shell composite nanomaterial prepared in Example 1 are as follows: Figure 4 shown. Figure 4 a is a photo of MagQBD in aqueous solutions with different pH values, b is the effect of storage time of MagQBD in aqueous solution on its fluorescence intensity, and c is the magnetic properties of MagQBD in aqueous solutions with different pH values. These results show the high stability of MagQBD in complex environments and its potential as a high-performance fluorescent probe for on-site rapid immunochromatographic detection systems.

[0137] Example 2

[0138] Preparation of mycotoxin detection probes

[0139] First, 1 mL of MagQBD (10 mg / mL) was washed with PBS (0.01 M, pH 7.4) and resuspended in 500 microliters of 1 mL 2-(N-morpholine) ethanesulfonic acid buffer (0.1 M, pH 5.5). The solution was incubated with 5 μL of carbodiimide solution (0.1 mM) and 10 μL of N-hydroxysuccinimide solution (100 mM) for 15 min. The activated MagQBD in each tube was collected from the solution by magnetic enrichment and resuspended in 200 μL of PBS (0.01 M, pH 7.4). 8 μg of anti-aflatoxin B1 antibody, anti-ochratoxin A antibody, and anti-fumonisin B1 antibody were added to the activated MagQBD solution. The mixture was shaken at room temperature for 2 h and then blocked with 50 μL of 10% BSA (w / v) for 30 min. The three immuno-MagQBDs were collected by magnetic separation and resuspended in 0.5 mL of storage solution containing 10 mM PBS (pH 7.4), 0.02% NaN3 (w / v), 0.5% BSA (w / v) and 0.05% Tween 20 (v / v) for use as liquid immunoprobes for immunochromatography.

[0140] The schematic diagram of the preparation process of the fungal toxin detection probe is as follows Figure 5 shown.

[0141] Example 3

[0142] The magnetic core multilayer quantum dot shell composite nanomaterial proposed by the present invention has the ability of specific capture / detection after being modified with antibodies against three kinds of fungi (aflatoxin B1, ochratoxin A, and fumonisin B1), and can be directly used for detection in an immunochromatographic system. In this embodiment, quantum dot fluorescent labels modified with antibodies against aflatoxin B1, ochratoxin A, and fumonisin B1 are used as labels for the immunochromatographic system to detect different concentrations of aflatoxin B1, ochratoxin A, and fumonisin B1. Figure 6 This is a schematic diagram of the experimental principle of simultaneous detection of three fungal toxins based on the immunochromatography using quantum dot fluorescence as shown in this example. Figure 7 The following are photos of test strips for detecting different concentrations of aflatoxin B1, ochratoxin A, and fumonisin B1 under ultraviolet light. Figure 7 As can be seen in the figure, as the concentration of the target mycotoxins increases, the fluorescence intensity on the detection lines (T1, T2, T3) gradually decreases. The detection sensitivity of aflatoxin B1, ochratoxin A, and fumonisin B1 was determined to be 0.42, 11.48, and 4.21 pg / mL, respectively.

[0143] Figure 8 yes Figure 7 Corresponding fluorescence intensity values ​​and standard curve.

[0144] Fig. 9It is the standard colloidal gold immunochromatographic colorimetric result for detecting different concentrations of ochratoxin A, fumonisin B1 and aflatoxin B1. Fig. 9 It can be seen that its detection sensitivity is 1000 times lower than that of the detection method of the present invention.

[0145] Fig.10 The fluorescence images and corresponding fluorescence intensity values ​​of the commercial quantum dot microsphere immunolayer detecting three target fungal toxins. Fig.10 (a) is the fluorescence image and corresponding fluorescence intensity value of ochratoxin A; (b) is the fluorescence image and corresponding fluorescence intensity value of fumonisin B1; (c) is the fluorescence image and corresponding fluorescence intensity value of aflatoxin B1. Fig.10 It can be seen that its detection sensitivity is 50 times lower than that of the detection method of the present invention.

[0146] Fig.11 The results of the specificity test of the immunochromatographic strip based on MagQBD in Example 3 of the present invention are shown in the experimental results. The results show that the immunochromatographic strip based on MagQBD has good specificity and selectivity for the detection of three target mycotoxins (aflatoxin B1, ochratoxin A, and fumonisin B1).

[0147] Fig.12 This is the repeatability test result of the immunochromatographic strip based on MagQBD in Example 3 of the present invention, showing stable fluorescence signals of all test samples, reflecting the good reproducibility of the immunochromatographic strip based on MagQBD.

[0148] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A mycotoxin detection probe, comprising a core-shell three-dimensional structured magnetic core multilayer quantum dot composite nanomaterial and a mycotoxin antibody loaded on the surface of the core-shell three-dimensional structured magnetic core multilayer quantum dot composite nanomaterial; The mycotoxin antibody is one or more of aflatoxin B1 antibody, ochratoxin A antibody and fumonisin B1 antibody; The core-shell three-dimensional structured magnetic core multilayer quantum dot composite nanomaterial comprises Fe 3 O 4 A magnetic nanoparticle core and two layers of water-soluble polymers sequentially wrapped on the surface of the core, and the surface of each layer of the water-soluble polymer is respectively loaded with carboxylation quantum dots.

2. The fungal toxin detection probe according to claim 1, It is characterized in that The water-soluble polymer is PEI, and the number average molecular weight of the PEI is 5,000 to 80,000.

3. The fungal toxin detection probe according to claim 1 or 2, It is characterized in that The carboxylation quantum dots are CdSe / ZnS quantum dots, and the surfaces of the CdSe / ZnS quantum dots are modified with 3-mercaptopropionic acid; the CdSe / ZnS quantum dots have CdSe as the core and ZnS as the shell.

4. The fungal toxin detection probe according to claim 1, It is characterized in that The Fe 3 O 4 The particle size of the core of the magnetic nanoparticles is 80 to 600 nm.

5. The fungal toxin detection probe according to claim 1 or 2, It is characterized in that The method for preparing the magnetic core multilayer quantum dot composite nanomaterial with a core-shell three-dimensional structure comprises the following steps: Fe 3 O 4 The magnetic nanoparticles are ultrasonically mixed with the first water-soluble polymer solution to obtain a single layer of water-soluble polymer-wrapped Fe 3 O 4 Magnetic nanoparticles; The Fe 3 O 4 The magnetic nanoparticles are mixed with the first carboxylated quantum dots to obtain a magnetic core quantum dot composite nanomaterial loaded with a single layer of quantum dots; Ultrasonic mixing the magnetic core quantum dot composite nanomaterial loaded with a single layer of quantum dots and a second water-soluble polymer solution to obtain a magnetic core quantum dot composite nanomaterial wrapped with two layers of water-soluble polymer; The magnetic core quantum dot composite nanomaterial wrapped by the two layers of water-soluble polymer is mixed with the second carboxylation quantum dot to obtain a magnetic core multilayer quantum dot composite nanomaterial with a core-shell three-dimensional structure.

6. A method for preparing the mycotoxin detection probe according to any one of claims 1 to 5, The following steps are involved: The core-shell three-dimensional structured magnetic core multilayer quantum dot composite nanomaterial is mixed with a carboxyl activator to perform an activation reaction to obtain an activated magnetic core multilayer quantum dot composite nanomaterial; The activated magnetic core multilayer quantum dot composite nanomaterial is mixed with a fungal toxin antibody for loading, and bovine serum albumin is added after loading to obtain a fungal toxin detection probe.

7. A kit for detecting fungal toxins, comprising an immunochromatographic test strip and the fungal toxin detection probe according to any one of claims 1 to 5 or the fungal toxin detection probe prepared by the preparation method according to claim 6; The immunochromatographic test strip comprises a test strip matrix and a detection line and a control line located on the surface of the test strip matrix; The components of the test line include mycotoxin binding antigens; The fungal toxins detected by the kit are one or more of aflatoxin B1, ochratoxin A and fumonisin B1; when the kit detects multiple fungal toxins, each toxin corresponds to a detection line.

8. The fungal toxin detection kit according to claim 7, It is characterized in that The components of the control line included goat anti-mouse IgG antibody.

9. A method for detecting mycotoxins, wherein the mycotoxins are one or more of aflatoxin B1, ochratoxin A and fumonisin B1, and the detection method The following steps are involved: The sample solution to be tested is mixed with the mycotoxin detection probe according to any one of claims 1 to 5 or the mycotoxin detection probe prepared by the preparation method according to claim 6, and then separated by magnetic adsorption, and the obtained solid is dispersed in a buffer solution to obtain a test solution; The detection liquid is added to one end of the immunochromatographic test strip, the fluorescence intensity of the detection line is tested, and the fungal toxin concentration of the detection liquid is obtained according to the fluorescence intensity and a predetermined standard curve; the standard curve is a fitting curve of the fungal toxin concentration and the fluorescence intensity.

10. The detection method according to claim 9, It is characterized in that The minimum detection limit of aflatoxin B1 is 0.42 pg / mL, the minimum detection limit of ochratoxin A is 11.48 pg / mL, and the minimum detection limit of fumonisin B1 is 4.21 pg / mL.

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