Filter membrane with MOF (Metal Organic Framework) derived porous structure, preparation method, mycotoxin separation and extraction device and separation and extraction method

Abstract

The invention discloses a filter membrane with an MOF derived porous structure, a preparation method, a mycotoxin separation and extraction device and a separation and extraction method, and relates to the technical field of mycotoxin pretreatment. A porous functional layer is constructed on the surface of a filter membrane substrate and in pore channels through a controlled conversion process, the porous functional layer has a porous structure containing a Zr-O-C network, microporous channels are reserved, polar functional groups are remained on the surface, and the MOF-derived porous structure filter membrane which does not contain a complete MOF crystal structure is formed. The membrane is used in a circulation type working mode, a target analyte can quickly pass through the membrane layer along with fluid, and the matrix effect can be effectively reduced. The membrane material can realize rapid and simplified sample purification on the premise of ensuring high recovery rate of a target object. The method has the advantages of simple preparation process, stable structure, low flow resistance, wide applicable solvent range and the like, and is particularly suitable for rapid pretreatment analysis of trace micromolecule pollutants in complex food and agricultural product matrixes.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of mycotoxin pretreatment, in particular to a filter membrane with MOF derived porous structure, a preparation method, a mycotoxin separation and extraction device and a separation and extraction method. BACKGROUND

[0002] With the increasing attention of the global public to food safety issues, especially in key links such as food storage, transportation and processing, mycotoxin pollution has become one of the important factors restricting the quality and safety of agricultural products and the development of trade. Mycotoxins are a class of secondary metabolites produced by various molds under suitable temperature and humidity conditions, which have high toxicity, strong thermal stability and are not easy to degrade. They are widely present in food and its products, especially in corn, wheat, soybeans and related feed raw materials and processing by-products. Once they enter the food chain, they not only pose a potential threat to human and animal health, but also may cause serious economic losses and international trade barriers.

[0003] Studies have shown that typical mycotoxins such as aflatoxins (especially AFB1), ochratoxin, deoxynivalenol (DON) and zearalenone (ZEN) have strong carcinogenicity, mutagenicity, immunotoxicity and neurotoxicity, and long-term low-dose exposure may also cause chronic health hazards. In order to reduce the exposure risk of mycotoxins, countries around the world and international organizations have successively formulated relatively strict limit standards, and have made clear requirements for the content of mycotoxins in food and its deep processing products, feed and animal-derived food. Under this background, it is of great significance to establish sensitive, rapid and accurate mycotoxin detection technology for protecting public health, regulating food circulation and promoting international trade of agricultural products.

[0004] Although traditional analysis methods such as high performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS / MS) have been widely used for qualitative and quantitative detection of mycotoxins, these methods usually rely on expensive instruments and require high technical level of operators. In addition, the sample pretreatment steps are complicated, often involving multiple operations such as organic solvent extraction, purification, concentration, etc., which is time-consuming and high-cost, and is not conducive to rapid screening and daily supervision of large quantities of samples. Especially in the primary detection institutions, enterprise self-inspection laboratories and on-site rapid monitoring scenes, the complex pretreatment process seriously restricts the popularization and application of mycotoxin detection methods.

[0005] The existing MOF material is directly adsorbed with the toxin by means of the MOF material, so as to realize the separation of the toxin and other substances. The recovery rate of the direct filter paper to the mycotoxin is low, and the stability of the detection result is poor.

[0006] In view of this, the present application is proposed. SUMMARY

[0007] The application aims to provide a filter membrane with MOF derived porous structure, a preparation method, a mycotoxin separation and extraction device and a separation and extraction method to solve the problems of complex mycotoxin pretreatment steps, long time consumption, high cost, low sample recovery rate and poor stability of detection results.

[0008] The MOF derived porous structure is a porous structure without MOF structure, has a porous structure containing a Zr-O-C network, retains micropore channels and has residual polar functional groups on the surface. The derived porous membrane prepared by taking a Zr-BDC MOF as a precursor and performing heat treatment on the precursor in nitrogen atmosphere has a hierarchical pore structure combining micropores and mesopores, has high specific surface area and chemical stability and is suitable for sample pretreatment and target molecule adsorption.

[0009] The application is implemented in the following manner: In a first aspect, the application provides a filter membrane with MOF derived porous structure, which comprises a porous network structure formed on the filter membrane, and a preparation method of the filter membrane comprises the following steps: S1: mixing a zirconium source and an organic ligand in a solvent according to a molar ratio of 0.5-2:1 to obtain a precursor solution; S2: immersing a pretreated filter membrane substrate in the precursor solution, reacting at 100-120 ℃ for 6-12 h to obtain a filter membrane loaded with the precursor; S3: after the reaction, washing the filter membrane loaded with the precursor and then drying the filter membrane; S4: heating the dried filter membrane to 250-350 ℃ under nitrogen protection and keeping the temperature for 0.5-2 h.

[0010] The application provides a filter membrane based on MOF derived porous structure, a porous functional layer is constructed on the surface of a filter membrane substrate and inside pore channels through a controlled conversion process, the porous functional layer has a porous structure containing a Zr-O-C network, retains micropore channels and has residual polar functional groups on the surface, and forms a filter membrane with MOF derived porous structure without complete MOF crystal structure. The filter membrane is used as a core separation unit to assemble a mycotoxin separation and extraction device.

[0011] The prior art MOF membrane realizes capture of toxins through physical adsorption or chemical adsorption. The physical adsorption principle is that the MOF provides a high specific surface area and a channel network, and mycotoxin molecules (such as aflatoxin and ochratoxin) can enter the pore channels through pore size matching and are physically adsorbed. The chemical adsorption principle is that carboxylic acid, hydroxyl, amino and metal clusters on the MOF framework provide chemical adsorption sites, and toxins are fixed on the surface of the membrane or inside the pore channels through hydrogen bonding, coordination, π-π interaction or electrostatic interaction.

[0012] Unlike traditional MOF membrane or MOF powder filled functional layer, the metal-organic coordination structure in the MOF derived porous structure based filter membrane provided by the application only acts as a structure building precursor in the preparation process, and after thermal or chemical conversion, the original crystal structure is destroyed and rearranged to form a porous network structure with amorphous or low crystalline characteristics. The derived structure retains high porosity and good flow performance while the surface residual metal-oxygen bond, carboxyl residue and weak polar functional group, thus giving the membrane material moderate interface interaction ability. In the sample pretreatment process (i.e. mycotoxin extraction and separation process), the membrane is used in a flow-through mode, and the target analyte can pass through the membrane layer quickly, while the macromolecular substances, pigments, proteins, polysaccharides and other interfering components in the sample matrix are weakened or partially retained under the synergistic action of the porous structure and surface functional groups, thereby effectively reducing the matrix effect. Since the membrane material only produces weak and reversible interaction with the target analyte, the pretreatment process does not require additional elution steps, and can achieve rapid and simplified sample purification while ensuring high recovery of the target substance. It has the advantages of simple preparation process, stable structure, low flow resistance, wide solvent application range, etc., and is especially suitable for rapid pretreatment analysis of trace small molecule pollutants in complex food and agricultural product matrices.

[0013] The inventors found that when using a membrane material without MOF derived porous structure for mycotoxin separation and extraction, the recovery rate of mycotoxin in the sample was significantly reduced, and the detection result was unstable; while using a filter membrane based on MOF derived porous structure for mycotoxin separation and extraction, the high specific surface area, uniform and adjustable pore size structure and selective action on mycotoxin molecules were realized, which effectively separated and purified the sample matrix, significantly reduced the interference of non-target components on detection, and significantly improved the recovery rate and kept it stable within the methodological requirements.

[0014] In one embodiment, the pretreated filter membrane substrate is immersed in a precursor solution and reacted at 100°C, 101°C, 103°C, 103.4°C, 107.8°C, 110°C, 111°C, 112°C, 112.1°C, 112.5°C, 115°C, 115.6°C, 116°C, 118°C, 118.9°C, 119°C or 120°C for 6, 7, 8, 9, 10, 11 or 12 hours.

[0015] The dried filter membrane was heated under nitrogen protection to 250.0 ℃, 251.0 ℃, 252.0 ℃, 253.0 ℃, 254.0 ℃, 255.0 ℃, 256.0 ℃, 257.0 ℃, 258.0 ℃, 259.0 ℃, 260.0 ℃, 261.0 ℃, 262.0 ℃, 263.0 ℃, 264.0 ℃, 265.0 ℃, 266.0 ℃, 267.0 ℃, 268.0 ℃, 269.0 ℃, 270.0 ℃, 271.0 ℃, 272.0 ℃, 273.0 ℃, 274.0 ℃, 275.0 ℃, 276.0 ℃, 277.0 ℃, and 278.0 ℃. ℃, 279.0℃, 280.0℃, 281.0℃, 282.0℃, 283.0℃, 284.0℃, 285.0℃, 286.0℃, 287.0℃, 288.0℃, 289.0℃, 290.0℃, 291.0℃, 292.0℃, 293.0℃, 294.0℃, 295.0℃, 296.0℃, 297.0℃, 298.0℃, 299.0℃, 300.0℃, 301.0℃, 302.0℃, 303.0℃, 304.0℃, 305.0℃, 306.0℃, 307.0℃, 308.0℃, 309.0℃ ℃, 310.0 ℃, 311.0 ℃, 312.0 ℃, 313.0 ℃, 314.0 ℃, 315.0 ℃, 316.0 ℃, 317.0 ℃, 318.0 ℃, 319.0 ℃, 320.0 ℃, 321.0 ℃, 322.0 ℃, 323.0 ℃, 324.0 ℃, 325.0 ℃, 326.0 ℃, 327.0 ℃, 328.0 ℃, 329.0 ℃, 330.0 ℃, 331.0 ℃, 332.0 ℃, 333.0 ℃, 334.0 ℃, 335.0 ℃, 336.0 ℃, 337.0 ℃, 338.0 ℃, 339.0 ℃, 340.0 ℃ Temperatures of 341.0℃, 342.0℃, 343.0℃, 344.0℃, 345.0℃, 346.0℃, or 350℃, with holding time of 0.5, 1, 1.5, or 2 hours.

[0016] Pretreated filter membrane substrate refers to filter membrane substrate that has been ultrasonically cleaned with ethanol and deionized water and then dried or oven-dried.

[0017] In a preferred embodiment of the present invention, the organic ligand is selected from terephthalic acid or aminoterephthalic acid; the zirconium source is selected from at least one of ZrCl4, ZrOCl2, ZrO2, Zr(OH)4 and Zr(NO3)4.

[0018] In a preferred embodiment of the present invention, the zirconium source is selected from ZrCl4.

[0019] In a preferred embodiment of the present invention, the solvent is selected from at least one of DMF and glacial acetic acid.

[0020] In a preferred embodiment of the present invention, the filter membrane substrate is selected from cellulose filter paper, polytetrafluoroethylene membrane or polyvinylidene fluoride.

[0021] Preferably, the filter membrane substrate is cellulose filter paper.

[0022] Secondly, the present invention also provides a method for preparing a filter membrane with a MOF-derived porous structure, comprising the following steps: S1: A precursor solution is obtained by mixing a zirconium source and an organic ligand in a solvent at a molar ratio of 0.5-2:1; S2: Immerse the pretreated filter membrane substrate in the precursor solution and react at 100–120 °C for 6–12 h to obtain a filter membrane loaded with the precursor. S3: After the reaction, the filter membrane loaded with the precursor is washed and then dried; S4: Heat the dried filter membrane to 250–350℃ under nitrogen protection and hold for 0.5–2 hours.

[0023] Thirdly, the present invention also provides the application of filter membranes with MOF-derived porous structures in the separation and extraction of mycotoxins.

[0024] In a preferred embodiment of the present invention, the mycotoxin is selected from at least one of aflatoxin B1, deoxynivalenol, zearalenone, aflatoxin B2, aflatoxin G1, aflatoxin G2, aflatoxin M1, aflatoxin M2, fumonisin B1, fumonisin B2, T.2 toxin, HT.2 toxin, ochratoxin, trichothecene toxins, citrinin, penicillic acid, beauveria bassiana, and patulin.

[0025] Fourthly, the present invention also provides a mycotoxin separation and extraction device, which includes: a sample container, a collection container and a syringe. The sample container is connected to the inlet of the syringe via a connecting pipe. A filter membrane with the above-mentioned MOF-derived porous structure is disposed between the sealing plug and the liquid outlet of the syringe. The inlet is located between the filter membrane and the sealing plug of the syringe, and the outlet of the syringe is connected to the collection container via a one-way valve. A one-way valve is also disposed on the connecting pipe.

[0026] The syringe can be a commercially available syringe. The syringe has an inlet on the side wall and an outlet at the bottom.

[0027] The syringe also has a movable plunger and a sealing plug connected to the end of the movable plunger, which ensures that the syringe chamber is isolated from the outside.

[0028] A one-way valve on the connecting tubing allows fluid to flow unidirectionally from the liquid outlet of the sample container to the inlet of the syringe. The syringe outlet is connected to a collection container via a one-way valve, which also allows fluid to flow unidirectionally from the outlet to the collection container.

[0029] The mycotoxin separation and extraction device of the present invention maintains the advantages of simple structure, convenient operation and no need for complicated instruments while ensuring recovery rate and detection accuracy. It can be combined with various detection methods such as time-resolved fluorescence detection, and is suitable for rapid on-site screening and routine monitoring of mycotoxins in grain, feed and other fields. It has good prospects for engineering application.

[0030] Fifthly, the present invention also provides a method for extracting and separating mycotoxins, comprising the following steps: mixing the extract and the sample to be extracted into the sample container, and then pulling the movable plunger of the syringe to allow the liquid in the sample container to enter the chamber of the syringe through the connecting tube; and then pushing the movable plunger of the syringe to collect the separated mycotoxin in the collection container.

[0031] After extracting and separating mycotoxins from the test sample using the method provided by this invention, the detection requirements of methods such as test strips or high-performance liquid chromatography can be directly met.

[0032] In a preferred embodiment of the present invention, the extraction solvent is 50% ethanol; the mixing ratio of the extraction solvent to the sample to be extracted is 20-25 mL: 5 g.

[0033] Compared to other extraction solvents (water or other concentrations of ethanol), 50% ethanol, as the extraction solvent, consistently maintained spiked recoveries within the methodological requirements, demonstrating good accuracy and repeatability. This extraction system ensures simultaneous and efficient extraction of multiple mycotoxins while also considering sample matrix adaptability and compatibility with subsequent detection, showcasing significant comprehensive technical advantages.

[0034] The present invention has the following beneficial effects: This invention provides a filter membrane based on a MOF-derived porous structure. A porous functional layer is constructed on the surface and inside the pores of the filter membrane substrate through a controlled transformation process. This porous functional layer has a porous structure containing a Zr–O–C network, retains micropore channels, and has residual polar functional groups on its surface, forming a MOF-derived porous filter membrane without a complete MOF crystal structure. This membrane is used as the core separation unit to assemble a mycotoxin extraction and separation device.

[0035] Unlike traditional MOF membranes or MOF powder-filled functional layers, the metal-organic coordination structure in the MOF-derived porous membrane provided by this invention only serves as a precursor in the preparation process. After thermal or chemical transformation, its original crystal structure is destroyed and rearranged, forming a porous network structure with amorphous or low-crystallinity characteristics. This derived structure maintains high porosity and good flow performance while retaining metal-oxygen bonds, carboxyl residues, and weakly polar functional groups on its surface, thus endowing the membrane material with appropriate interfacial interaction capabilities. During the extraction and separation of mycotoxins from samples, the membrane operates in a flow-through mode. The target analyte can rapidly pass through the membrane layer with the fluid, while interfering components such as macromolecules, pigments, proteins, and polysaccharides in the sample matrix are weakened or partially retained through the synergistic effect of the porous structure and surface functional groups, effectively reducing the matrix effect. Since the membrane material only produces weak and reversible interactions with the target analyte, this pretreatment process requires no additional elution step, achieving rapid and simplified sample purification while ensuring high recovery rates of the target analyte. It has the advantages of simple preparation process, stable structure, low flow resistance and wide range of applicable solvents, and is especially suitable for rapid pretreatment analysis of trace small molecule pollutants in complex food and agricultural product matrices.

[0036] After using a filter membrane with a MOF-derived porous structure for mycotoxin separation and extraction, its high specific surface area, uniform and tunable pore size structure, and selective action on mycotoxin molecules enabled effective separation and purification of the sample matrix. This significantly reduced the interference of non-target components on detection, and the recovery rate was significantly improved and remained stable within the methodological requirements. Attached Figure Description

[0037] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0038] Figure 1 This is a schematic diagram of a mycotoxin separation and extraction device. Figure 2 The standard working curves of logC-T / T0 for three mycotoxins in grain matrix are shown. Figure 3 The image shows the test results of three mycotoxins in a grain matrix. Figure 4 The standard working curves for logC-T / T0 of three mycotoxins in processing by-products; Figure 5The image shows the test results of three mycotoxins in processing by-products. Figure 6 The standard working curve of logC-T / T0 for three mycotoxins in feed; Figure 7 The image shows the test results of three mycotoxins in feed. Figure 8 Comparison of AFB1, ZEN, and DON detection values ​​with HPLC values ​​in various matrices.

[0039] Figure labels: 1-sample vial; 2-dilution vial; 3-syringe; 4-filter membrane; 5-one-way valve; 6-inlet; 7-outlet; 8-sealing plug. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0041] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0042] Example 1 This embodiment provides a cellulose filter membrane with an MOF-derived porous structure.

[0043] 1. Materials and Reagents Membrane substrate material: cellulose filter paper; Zirconium tetrachloride (ZrCl4), terephthalic acid (BDC), N,N-dimethylformamide (DMF), ethanol, formic acid, nitrogen (N2), deionized water, and ethanol.

[0044] Sample extraction solution: ethanol, sodium chloride.

[0045] Sample dilution solution: Add 0.1 mL Tween-20, 0.1 g BSA and 10 μL to 500 mL PBS buffer.

[0046] Proclin 300, store at 4°C.

[0047] Preparation of sample pretreatment membranes based on MOF-derived porous structures 2. Membrane substrate pretreatment The selected membrane substrate was cut into circular pieces with a diameter of 2 cm; Ultrasonic cleaning with ethanol and deionized water for 10 min each; Dry at room temperature or at 50°C for later use.

[0048] 3. Construction of MOF precursors on membrane substrates (1) ZrCl4 and organic ligand BDC were dissolved in DMF at a molar ratio of 1:1 to obtain a precursor solution; (2) Immerse the pretreated membrane substrate completely in the precursor solution; (3) Transfer the system to a closed reaction vessel and react at 120 °C for 8 h; (3) After the reaction is complete, remove the membrane and wash it several times alternately with DMF and ethanol; (4) Dry at 60 °C to obtain a composite membrane supported on MOF precursor.

[0049] 4. Formation of MOF-derived porous structures: (1) Place the above composite membrane in a tube furnace; (2) Heating to 260 °C under nitrogen protection; (3) Keep warm for 1 hour; (4) Allow to cool naturally to room temperature.

[0050] Example 2 This embodiment provides a mycotoxin separation and extraction device. Figure 1 As shown, it includes: a sample vial 1, a dilution bottle 2, and a syringe 3. The syringe 3 has an inlet 6 on its side wall for connection to the sample vial 1 via a connecting tube. A filter membrane 4 is installed inside the syringe 3, located before the sealing plug 8 and the outlet 7. The inlet 6 is located between the filter membrane 4 and the sealing plug 8 of the syringe 3.

[0051] The outlet 7 of the syringe 3 is connected to the dilution bottle 2 via a one-way valve 5, and a one-way valve 5 is also installed on the connecting pipeline.

[0052] A one-way valve 5 on the connecting tubing allows fluid to flow unidirectionally from the liquid outlet of sample vial 1 to the inlet 6 of syringe 3. The outlet 7 of syringe 3 is connected to dilution vial 2 via one-way valve 5, which allows fluid to flow unidirectionally from outlet 7 to dilution vial 2.

[0053] The functions of each component are as follows: Syringe 3: By pulling up and pushing down, syringe 3 can draw up and push liquid samples, precisely controlling the amount of liquid and ensuring the accuracy of experimental results.

[0054] One-way valve 5: This valve controls the flow direction of the liquid, prevents backflow, and ensures the directionality of the liquid during operation. The left one-way valve 5 is for inflow only and outflow only, while the right one-way valve 5 is for outflow only and outflow only, thereby ensuring the separation of mycotoxins from the liquid sample.

[0055] Filter membrane 4: When liquid passes through the membrane filtration device, larger particles are intercepted by filter membrane 4, and only smaller molecules (such as fungal toxins) pass through the membrane and enter the downstream dilution bottle 2.

[0056] Sample vial 1: Used to store sample liquids, facilitating sample processing and subsequent analysis.

[0057] Dilution bottle 2: Used to receive the filtered sample and to bring the sample to a suitable concentration for detection by adding diluent.

[0058] The method of using this extraction and separation device is as follows: The extract and the sample to be extracted are mixed into the sample bottle 1. Then, by pulling the movable plunger of the syringe 3, the liquid in the sample bottle 1 enters the chamber of the syringe 3 through the connecting tube. Then, the movable plunger of the syringe 3 is pushed to collect the mycotoxin separation liquid in the dilution bottle 2.

[0059] Example 3 This embodiment provides a method for extracting and separating mycotoxins from corn.

[0060] 1. The experimental method includes the following steps: (1) Grind the dried corn in a small grinder for 1 minute and pass it through a 20-mesh sieve to remove larger particles.

[0061] (2) Weigh 5.00 ± 0.03 g of the sieved sample and put it into a 50 mL centrifuge tube pre-filled with 25 mL of sample extract solution. Shake well to ensure complete extraction of mycotoxins.

[0062] (3) Let stand for 10 minutes to allow the sample extract and solid impurities to completely separate into layers.

[0063] (4) Connect sample bottle 1 to extraction device, pull up the plunger of syringe 3 to allow the mixture to enter syringe 3.

[0064] (5) Push the syringe 3 lever to allow the mixture to pass through the filter membrane 4 into the dilution bottle 2 on the right side, which is pre-filled with 5 mL of sample diluent. The mixture is about 20 drops (1 mL).

[0065] (6) Remove dilution bottle 2, shake, and mix well.

[0066] (7) Add the mixture to the sample well of the mycotoxin multiplex test strip (FADZ31 from Nanjing Microtest Biotechnology Co., Ltd.), incubate at 37°C for 8 min to allow the sample to fully bind. After the reaction is complete, analyze the results using a fluorescence detector.

[0067] 2. In this embodiment, the sample extract was first screened, thus providing a technical basis for subsequent mycotoxin extraction and detection.

[0068] Using corn flour as a blank matrix for typical grain samples, a systematic methodology validation experiment was conducted. Mycotoxin standards were accurately added to the corn flour matrix, with spiked levels of aflatoxin B1 (AFB1), deoxynivalenol (DON), and zearalenone (ZEN) at 20 μg / kg, 500 μg / kg, and 200 μg / kg, respectively, to simulate the contamination state of mycotoxins in actual samples. Sterile water, 25% ethanol aqueous solution, 50% ethanol aqueous solution, 75% ethanol aqueous solution, and anhydrous ethanol were used as sample extraction solutions to compare and evaluate the extraction efficiency of the target mycotoxins under different extraction systems. The recovery rate of the spiked samples was used as the main evaluation index to systematically investigate the influence of the extraction solution composition on the accuracy of the detection results. The relevant experimental results are listed in Tables 1 to 5.

[0069] Experimental results show that the volume fraction of ethanol in the extraction solution has a significant impact on the extraction efficiency of mycotoxins. Compared with other extraction solutions, when using a 50% ethanol aqueous solution as the sample extraction solution, the spiked recoveries of AFB1, DON, and ZEN are consistently within the methodological requirements, demonstrating good accuracy and repeatability. This extraction system ensures simultaneous and efficient extraction of multiple mycotoxins while also considering sample matrix adaptability and compatibility with subsequent detection, demonstrating significant comprehensive technical advantages. In summary, using a 50% ethanol aqueous solution as the extraction solution not only meets the requirements for recovery and stability in the detection of multi-component mycotoxins but also lays a solid technical foundation for the reliable implementation of the pretreatment method of this invention and its widespread application in grain and feed samples.

[0070] Table 1. Extraction reagents for mycotoxins in maize - sterile water

[0071] Table 2. Extraction reagents for mycotoxins in maize - 25% ethanol

[0072] Table 3. Extraction reagents for mycotoxins in maize - 50% ethanol

[0073] Table 4. Extraction reagent for mycotoxins in maize - 75% ethanol

[0074] Table 5. Extraction reagents for mycotoxins in corn - 100% ethanol

[0075] Example 4 This embodiment tests the recovery rate of the separation and extraction method created in Example 3 and evaluates the accuracy of the test strip in detecting mycotoxins.

[0076] Using corn flour as a blank matrix for typical grain samples, mycotoxin standard solutions were added to spike aflatoxin B1 (AFB1), deoxynivalenone (DON), and zearalenone (ZEN) at levels of 20 μg / kg, 500 μg / kg, and 200 μg / kg, respectively. Under these conditions, the pretreatment effects (i.e., extraction and separation effects) of different filter materials were compared. Traditional cellulose filter paper and the cellulose filter membrane based on the MOF-derived porous structure proposed in this invention were used, with the recovery rate of the spiked samples as the evaluation index. The effects of different filter materials on the accuracy of mycotoxin detection and matrix effect were systematically investigated. The experimental results are shown in Tables 6 and 7, respectively.

[0077] Table 6 Results of Recovery Rate Determination of Three Mycotoxins - Filter Paper

[0078] Table 7. Results of Recovery of Three Mycotoxins - Sample Pretreatment Membrane Based on MOF-Derived Porous Structure

[0079] Experimental results show that when using only traditional filter paper for filtration, complex matrix components in the sample cannot be effectively removed, and the target mycotoxins are easily affected by matrix interference during pretreatment, resulting in low spiked recoveries of AFB1, DON, and ZEN, which are difficult to simultaneously meet the methodological requirements. In contrast, when using the sample pretreatment membrane based on the MOF-derived porous structure described in this invention for filtration, the spiked recoveries of the three mycotoxins are all consistently within the acceptable range, and the overall recovery rate is significantly improved, approaching 100%.

[0080] Therefore, sample pretreatment membranes based on MOF-derived porous structures, with their high specific surface area, regular and controllable pore structure, and designable surface chemical properties, can not only effectively retain interfering components that cause matrix effects in the sample during filtration, but also exhibit good permeability and protection against target mycotoxins, thus significantly reducing the impact of matrix effects on detection results. The application of this functionalized membrane material in mycotoxin pretreatment overcomes the technical limitations of traditional filter paper, which only provides mechanical separation and struggles to simultaneously achieve purification and recovery, demonstrating significant technological advantages and irreplaceability.

[0081] Experimental Example 1 To evaluate the applicability and reliability of the developed mycotoxin pretreatment device in different matrices, this embodiment conducted methodological validation. The pretreatment device was used for the extraction and purification of mycotoxins from grains, processing by-products, and feed matrices. The pretreatment efficiency and supporting detection methods were comprehensively evaluated using the linear relationship between T / T0 and LogC.

[0082] 1. Standard working curves of logC-T / T0 for three mycotoxins in different matrices Three mycotoxins were added at different concentrations (AFB1: 2.5, 5, 10, 25, 50, and 75 ppb; DON: 250, 500, 1000, 2000, 3500, and 5000 ppb; ZEN: 25, 50, 100, 250, 500, and 1000 ppb) to three matrices: grains, processing by-products (corn husks), and feed. Mycotoxins were extracted using a rapid pretreatment device, and the results were analyzed and interpreted. A standard curve was plotted with the logarithm of the standard concentration on the x-axis and T / T0 on the y-axis, where T is the fluorescence intensity of the test line on the paper strip and T0 is the fluorescence intensity of the control line. The linear equation and R² were obtained.

[0083] Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 and Figure 7 The results showed that Figure 3 In the middle, the lanes from left to right are AFB1: 2.5, 5, 10, 25, 50 and 75 ppb; DON: 250, 500, 1000, 2000, 3500 and 5000 ppb; ZEN: 25, 50, 100, 250, 500 and 1000 ppb. Figure 5 In the middle, the lanes from left to right are AFB1: 2.5, 5, 10, 25, 50 and 75 ppb; DON: 250, 500, 1000, 2000, 3500 and 5000 ppb; ZEN: 25, 50, 100, 250, 500 and 1000 ppb. Figure 7In the swimming pool, the lanes from left to right are: AFB1: 2.5, 5, 10, 25, 50, and 75 ppb; DON: 250, 500, 1000, 2000, 3500, and 5000 ppb; ZEN: 25, 50, 100, 250, 500, and 1000 ppb. In the three matrices (grains, processing by-products, and feed), within the set concentration range, the T / T0 values ​​of the three mycotoxins gradually decreased with increasing standard concentration. A good linear relationship was observed between T / T0 and the logarithm of the standard concentration, and the correlation coefficient R² obtained from the linear equation fitting was high, meeting the requirements for quantitative analysis.

[0084] The results above indicate that the established rapid pretreatment device combined with time-resolved fluorescent test strips is suitable for the simultaneous detection of AFB1, DON, and ZEN in grains, processing by-products, and feed, providing a reliable technical means for rapid screening of multi-matrix mycotoxins.

[0085] Experiment Example 2 This experimental example demonstrates sensitivity testing.

[0086] Three mycotoxins were added at different concentrations (AFB1: 2.5, 5, 10, 25, 50, and 75 ppb; DON: 250, 500, 1000, 2000, 3500, and 5000 ppb; ZEN: 25, 50, 100, 250, 500, and 1000 ppb) to three matrices: grains, processing by-products (corn bran), and feed. Mycotoxins were extracted using a rapid pretreatment device, and the results were analyzed and interpreted. The lowest concentration at which an inhibition rate exceeded 25% was defined as the method sensitivity.

[0087] As shown in Table 8, the results indicate that the lowest detection concentration of AFB1 by this method is 2.5 ppb in grains and processing by-products, and 5 ppb in feed ingredients. The detection limit of ZEN in all matrices is 25 ppb, while the detection limit of DON in all matrices is 250 ppb, all of which are better than the national standard. At the same time, the above results confirm that this method can maintain good detection sensitivity in different matrices.

[0088] Table 8 Detection sensitivity in different matrices

[0089] Experimental Example 3 This experimental example is used for reliability testing.

[0090] To verify the accuracy and reliability of the method provided in this invention, high-performance liquid chromatography (HPLC) was used as a reference method for comparative analysis. HPLC is a standard technique commonly used for the quantitative analysis of chemical compounds, possessing high accuracy, good repeatability, and is widely applied in the determination of various mycotoxins in food and feed.

[0091] First, AFB1, ZEN, and DON were detected using HPLC. HPLC detection methods were performed according to the national standards GB5009.22-2016 for AFB1, GB 5009.111-2016 for DON, and GB 5009.209-2016 for ZEN, obtaining reference values. Subsequently, the rapid mycotoxin pretreatment device (i.e., separation and extraction device) provided in this invention was used for sample pretreatment. Time-resolved fluorescence multiplex test strips were used to simultaneously detect these three mycotoxins, and the corresponding measured values ​​were recorded. A scatter plot was generated to compare the reference values ​​and measured values, thereby evaluating the detection performance.

[0092] like Figure 8 As shown, the reference values ​​and measured values ​​of AFB1, ZEN, and DON exhibit a strong linear relationship, with R² values ​​of 0.9878, 0.9826, and 0.9916, respectively. These results demonstrate that the detection method established in this invention is stable and reliable.

[0093] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A filter membrane with a MOF-derived porous structure, characterized in that, It includes: The porous network structure formed on the filter membrane is prepared by the following steps: S1: A precursor solution is obtained by mixing the zirconium source and the organic ligand in a solvent at a molar ratio of 0.5–2.0:1; S2: Immerse the pretreated filter membrane substrate in the precursor solution and react at 100–120 °C for 6–12 h to obtain a filter membrane loaded with the precursor. S3: After the reaction, the filter membrane loaded with the precursor is washed and then dried; S4: Heat the dried filter membrane to 250–350℃ under nitrogen protection and hold for 0.5–2 hours.

2. The filter membrane with MOF-derived porous structure according to claim 1, characterized in that, The organic ligand is selected from terephthalic acid or aminoterephthalic acid; the zirconium source is selected from at least one of ZrCl4, ZrOCl2, ZrO2, Zr(OH)4 and Zr(NO3)4; Preferably, the zirconium source is selected from ZrCl4.

3. The filter membrane with MOF-derived porous structure according to claim 1, characterized in that, The solvent is selected from at least one of DMF and glacial acetic acid.

4. The filter membrane with MOF-derived porous structure according to claim 1, characterized in that, The filter membrane substrate is selected from cellulose filter paper, polytetrafluoroethylene membrane or polyvinylidene fluoride.

5. The method for preparing a filter membrane with a MOF-derived porous structure as described in any one of claims 1-4, characterized in that, It includes the following steps: S1: A precursor solution is obtained by mixing a zirconium source and an organic ligand in a solvent at a molar ratio of 0.5-2:1; S2: Immerse the pretreated filter membrane substrate in the precursor solution and react at 100–120 °C for 6–12 h to obtain a filter membrane loaded with the precursor. S3: After the reaction, the filter membrane loaded with the precursor is washed and then dried; S4: Heat the dried filter membrane to 250–350℃ under nitrogen protection and hold for 0.5–2 hours.

6. The application of the filter membrane with MOF-derived porous structure as described in any one of claims 1-4 in the separation and extraction of mycotoxins.

7. The application according to claim 6, characterized in that, The mycotoxin is selected from at least one of aflatoxin B1, deoxynivalenol, zearalenone, aflatoxin B2, aflatoxin G1, aflatoxin G2, aflatoxin M1, aflatoxin M2, fumonisin B1, fumonisin B2, T.2 toxin, HT.2 toxin, ochratoxin, trichothecene toxins, citrinin, penicillic acid, beauveria bassiana, and patulin.

8. A mycotoxin separation and extraction device, characterized in that, It includes: samples The sample container, the collection container, and the syringe are provided. The sample container is connected to the inlet of the syringe via a connecting pipe. A filter membrane with a MOF-derived porous structure as described in any one of claims 1-4 is disposed between the sealing plug and the liquid outlet of the syringe. The inlet is located between the filter membrane and the sealing plug of the syringe. The outlet of the syringe is connected to the collection container via a one-way valve. A one-way valve is also provided on the connecting pipe.

9. A method for extracting and separating mycotoxins, characterized in that, It includes the following steps: mixing the extract and the sample to be extracted into the sample container as described in claim 8, and then, by pulling the movable plunger of the syringe, causing the liquid in the sample container to enter the chamber of the syringe through the connecting tube; and then pushing the movable plunger of the syringe to collect the mycotoxin separation liquid in the collection container.

10. The method for extracting and separating mycotoxins according to claim 9, characterized in that, The extraction solvent is 50% ethanol; the mixing ratio of the extraction solvent to the sample to be extracted is 20-25 mL: 5 g.

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