Preparation and Application of a Zearalenone Molecularly Imprinted Covalent Organic Framework

Abstract

The present application relates to the preparation of a molecularly imprinted covalent organic framework for zearalenone and its application in the detection of trace zearalenone residues in cereals by dispersed solid phase extraction, belonging to the technical field of organic functional material synthesis and sample pretreatment. Molecularly imprinted covalent organic framework nanoparticles were prepared by precipitation polymerization and used as adsorption materials for dispersed solid phase extraction. They were combined with HPLC-FLD to detect zearalenone in complex cereal samples. The prepared molecularly imprinted covalent organic framework has good selectivity and reusability for zearalenone, with an imprinting factor (IF) of up to 10.1 and an adsorption capacity of 177.2 mg g ‑1 , and after five cycles, it still maintained a recovery rate of over 92% for the target molecule. This overcomes the shortcomings of traditional adsorbents, such as poor selectivity, large matrix effects, and difficulty in recycling; it can meet the requirements for the detection of trace zearalenone in various matrices.

Description

Technical Field

[0001] The present application relates to the preparation of a zearalenone molecularly imprinted covalent organic skeleton and its dispersed solid phase extraction application, belonging to the technical field of organic functional material synthesis and sample pretreatment. Background Art

[0002] Zearalenone (ZEN), one of the most common Fusarium mycotoxins produced by Fusarium solani, may exhibit carcinogenic, hepatotoxic, genotoxic, and immunosuppressive effects. It is one of the most frequently detected mycotoxins in cereal samples. Due to its chemical and thermal stability, it is difficult to degrade or remove during food processing. Consequently, it poses significant risks to humans, livestock, and the environment. However, the complex matrix of cereal samples and the presence of interference from other contaminants often lead to a series of instabilities during detection. Therefore, the development of a highly selective pretreatment method for the extraction of ZEN is of great significance for food and environmental safety.

[0003] Covalent organic frameworks (COFs) are a class of porous crystalline polymers composed of light elements linked by covalent bonds. Covalent organic frameworks have been widely used in the fields of adsorption and sample pretreatment due to their low density, stable structure, uniform pores, and ease of post-modification. However, since the interaction between COFs and target compounds is relatively simple, they still cannot meet the requirements for selective extraction of target molecules. Molecular imprinting technology is a technology that uses molecularly imprinted polymers (MIPs) to mimic substrate-enzyme specific interactions for specific recognition of target molecules. However, the synthesis process of traditional molecularly imprinted polymers is relatively complex, and the flexible framework is prone to collapse and leakage, resulting in reduced selective extraction ability.

[0004] Currently, sample pretreatment methods for zearalenone toxins in food mainly include liquid-liquid extraction (LLE), solid-phase extraction (SPE), dispersive solid-phase extraction (DSPE), and immunoaffinity column extraction (IAC). Among these methods, immunoaffinity column extraction offers good selectivity, but it is expensive and cannot be recycled. Liquid-liquid extraction consumes a lot of solvent, is complex to operate, and lacks the ability to extract selectively. Dispersive solid-phase extraction is currently considered one of the most efficient pretreatment methods due to its low solvent consumption, simple operation, and recyclability; however, it also suffers from issues such as low adsorption capacity.

[0005] Therefore, it is necessary to develop a highly selective dispersed solid phase extraction adsorbent for the selective extraction and analysis of zearalenone in complex samples. Summary of the Invention

[0006] In order to solve the above problems, the present application provides a preparation of a molecularly imprinted covalent organic framework for zearalenone and a dispersed solid-phase extraction method thereof, combining COFs with MIPs to provide a selective recognition site for the selective enrichment and extraction of trace zearalenone in actual samples. First, a molecularly imprinted covalent organic framework (MICOF) was obtained by room temperature synthesis. The prepared MICOF has good selective adsorption capacity and a high imprinting factor for zearalenone; further, this MICOF was used as an adsorbent to develop a dispersed solid-phase extraction pretreatment method for the highly selective extraction of zearalenone in actual grain samples.

[0007] Technical Solution

[0008] The first aspect of the present application provides a method for preparing a zearalenone molecularly imprinted covalent organic framework, comprising the following steps:

[0009] Step S11: dissolving tetrakis(4-aminophenyl)methane, aldehyde monomer and pseudo-template in a reaction solvent to obtain solution A;

[0010] Step S12: adding the catalyst dropwise to solution A to undergo polymerization reaction, eluting the pseudo-template, washing, and drying to obtain a molecularly imprinted covalent organic framework.

[0011] Furthermore, in step S11, the aldehyde monomer is selected from one of terephthalaldehyde, 4,4'-biphenyldicarboxaldehyde, 1,4-di(4-formaldehydephenyl)benzene and [1,1':4',1":4",1"'-quaterphenyl]-4,4"'-dicarboxaldehyde;

[0012] Furthermore, the structural formula of the aldehyde monomer is:

[0013]

[0014] Furthermore, the molar ratio of the aldehyde monomer to tetrakis(4-aminophenyl)methane is 2:3 to 6:1;

[0015] Furthermore, the molar ratio of the aldehyde monomer to tetrakis(4-aminophenyl)methane is 2:3 to 2:1, 2:1 to 6:1;

[0016] Furthermore, the pseudo template is selected from one of warfarin, quercetin and 2,4-dihydroxybenzoic acid cyclododecyl ester.

[0017] Furthermore, the molar ratio of the aldehyde monomer to the pseudo template is 10:1 to 4:1, 4:1 to 1:4.

[0018] In one embodiment, the pseudo template is warfarin;

[0019] Furthermore, the reaction solvent is selected from one of dioxane, acetonitrile, methanol and tetrahydrofuran;

[0020] In one embodiment, the reaction solvent is dioxane;

[0021] Furthermore, ultrasonic conditions were used to accelerate dissolution and form a transparent solution;

[0022] Furthermore, the catalyst in step S12 is selected from one of acetic acid and scandium trifluoromethanesulfonate.

[0023] In one embodiment, the catalyst is scandium trifluoromethanesulfonate;

[0024] Furthermore, the catalyst is 0.02-0.14 equivalents of the imine bond generated by the reaction.

[0025] In one embodiment, the catalyst is added dropwise to solution A under ultrasound;

[0026] Furthermore, the polymerization reaction time in step S12 is 3-48h;

[0027] Furthermore, in step S12: the washing solvent is methanol / acetic acid with a volume ratio of 9:1, and after the pseudo template is eluted, it is washed with methanol until neutral;

[0028] Furthermore, in step S12: drying conditions are vacuum drying at 40-80° C. to obtain MICOF;

[0029] The second aspect of the present application is to provide an application of a molecularly imprinted covalent organic framework prepared by the above-mentioned preparation method, which is applied in dispersed solid phase extraction for the selective extraction and analysis of zearalenone.

[0030] Furthermore, the sample pretreatment for the detection of zearalenone in food specifically includes:

[0031] Step S21: The grain sample is homogenized, ultrasonically extracted, dried with nitrogen, redissolved in acetonitrile, and diluted with water to obtain solution B;

[0032] Step S22: adding the molecularly imprinted covalent organic framework to solution B, and eluting with solvent after dispersive solid phase extraction;

[0033] Step S23: Detection and quantification.

[0034] Furthermore, step S23 uses HPLC-FLD method for quantitative detection.

[0035] Furthermore, the pH of solution B is 4-9.

[0036] Furthermore, the elution solvent in step S22 is selected from acetonitrile, methanol, ethanol, acetonitrile / acetic acid with a volume ratio of 9:1, methanol / acetic acid with a volume ratio of 9:1, or ethanol / acetic acid with a volume ratio of 9:1;

[0037] Preferably, the extraction time in step S22 is 5-30 min;

[0038] Preferably, the elution time in step S22 is 5-30 min.

[0039] In one embodiment, the amount of the aldehyde monomer is 0.05 mmol; the volume of the organic solvent in step S11 is 1-4 mL; and the amount of the catalyst in step S12 is 1-10 mg.

[0040] In one embodiment, 10 g of grain is taken, 18 mL of acetonitrile (ACN) and 2 mL of water are added, and ultrasonic extraction is performed for 20 minutes. The extract is then collected, dried on a nitrogen blower, redissolved with 1 mL of ACN, and diluted to 10 mL with water. Furthermore, in step S22, the amount of the molecularly imprinted covalent organic framework used is 2-14 mg. Furthermore, the volume of the elution solvent in step S22 is 0.5-3 mL.

[0041] Beneficial effects

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] (1) COFs were combined with MIPs to prepare molecularly imprinted covalent organic frameworks (MIFs) using precipitation polymerization. These frameworks were then used to selectively extract, separate, and enrich zearalenone from cereals. The results showed good selectivity, with an imprinting factor (IF) of 10.1 and an adsorption capacity of 177.2 mg g -1 ;

[0044] (2) The obtained molecularly imprinted covalent organic framework is used as an adsorbent for pretreatment of dispersed solid phase extraction, overcoming the defects of traditional adsorbents such as poor selectivity, large matrix effect and difficulty in recycling;

[0045] (3) It improves the accuracy and precision of analyzing trace mycotoxins in complex matrices and provides a selective recognition site for the selective enrichment and extraction of trace zearalenone in actual samples;

[0046] (4) MICOF still retains a recovery rate of more than 92% after being recycled for 5 times, showing a good reusability. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is a schematic diagram of the preparation of molecularly imprinted covalent organic framework and the principle of dispersed solid phase extraction;

[0048] Figure 2 The infrared spectra of MICOF and MNCOF prepared in Example 1;

[0049] Figure 3 N2 adsorption-desorption isotherms and pore size distributions of MICOF and MNCOF prepared in Example 1;

[0050] Figure 4 SEM images of MICOF and MNCOF prepared in Example 1;

[0051] Figure 5 The adsorption kinetics and adsorption isotherms of zearalenone on MICOF and MNCOF prepared in Example 1 are shown;

[0052] Figure 6 Comparison of the adsorption capacity of four different mycotoxins by MICOF and MNCOF prepared in Example 1;

[0053] Figure 7 This is a test chart of the number of MICOF cycles and the recovery rate of zearalenone. DETAILED DESCRIPTION

[0054] The technical solutions of the present invention are further described in detail below with reference to specific embodiments. The embodiments described herein are only used to illustrate and explain the present invention, but are not used to limit the invention.

[0055] Unless otherwise specified, the sources of the reagents used in the examples of the present invention can be purchased commercially. Among them, the standard product of zearalenone was purchased from Tianjin Chemical Industry Development Co., Ltd. (TCIShanghai), and tetrakis(4-aminophenyl)methane, aldehyde monomer, pseudo template, catalyst and solvent were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0056] The present invention provides drawings of the test results of some embodiments. Other embodiments can be characterized or tested using the same test method. Those skilled in the art can directly and unambiguously determine the content of the embodiments of the present invention through the method provided by the present invention.

[0057] Example 1: Preparation of Molecularly Imprinted Covalent Organic Framework (MICOF) and Non-Imprinted Covalent Organic Framework (MNCOF)

[0058] Tetrakis(4-aminophenyl)methane (19.0 mg, 0.05 mmol), terephthalaldehyde (13.4 mg, 0.1 mmol) and pseudo-template warfarin (7.7 mg, 0.025 mmol) were dissolved in 5 mL of dioxane and sonicated to form a clear solution;

[0059] Scandium trifluoromethanesulfonate (6 mg / 200 μL) solution was slowly added dropwise to the mixed solution under ultrasound, and the polymerization reaction was carried out at room temperature for 24 hours; a yellow precipitate was obtained;

[0060] The yellow precipitate was eluted with tetrahydrofuran, methanol / acetic acid (9:1 v / v) and methanol in sequence to remove the template molecule, and dried in a vacuum oven at 60° C. overnight to obtain a yellow solid.

[0061] The preparation steps of non-imprinted covalent organic framework (MNCOF) are the same as those of MICOF except that no template molecules are added.

[0062] Figure 2 The infrared spectra of MICOF and MNCOF are as follows: Figure 2 As shown, the amino monomer is at 3325 cm -1 The amino peak at 1693 cm and the aldehyde monomer at 1693 cm -1 The disappearance of the aldehyde peak at 1621 cm -1 The emergence of , proved that the imine-bonded MICOF was successfully prepared.

[0063] Figure 3 The N2 adsorption-desorption isotherms and pore size distributions of MICOF and MNCOF indicate the successful binding of the pseudo-template molecule warfarin to the covalent organic framework. After removing the template molecule, the pore size of MICOF is 3.2 nm, significantly larger than that of MNCOF, demonstrating the successful preparation of the molecularly imprinted covalent organic framework.

[0064] Figure 4 The SEM images of MICOF and MNCOF are as follows: Figure 4 As shown, the as-prepared MICOFs displayed a spherical morphology with a diameter of approximately 0.5–1 μm.

[0065] Example 2: Selective Adsorption Performance of Molecularly Imprinted Covalent Organic Frameworks

[0066] In order to evaluate the selective adsorption performance of the material of the present invention, the adsorption capacity of three other fungal toxins was compared and compared with the adsorption performance of MNCOF.

[0067] Verification of adsorption kinetics, adsorption isotherms, and adsorption selectivity of MICOF and MNCOF; only the verification method of MICOF is described in detail in the examples. Unless otherwise specified, the verification method of MNCOF is the same.

[0068] Adsorption kinetics: 10 mg of MICOF prepared in Example 1 of the present invention was weighed into a test tube and 10 mL of 10 mg L -1The mixture was briefly sonicated and then placed on a shaker at 150 rpm. At specific intervals (5, 10, 20, 30, 40, 50, 60, 90, and 120 min), 0.2 mL of the solution was collected, filtered through a 0.22 μm filter, and transferred to a chromatographic vial for analysis by HPLC-FLD.

[0069] Adsorption isotherm: 2 mg of the product MICOF of the present invention was weighed into a test tube, and 2 mL of 10-500 mg L -1 After a brief sonication, the test tube was placed on a shaker at 150 rpm for 1 h of adsorption, filtered through a 0.22 μm filter membrane, placed in a chromatographic vial, and analyzed by HPLC-FLD injection.

[0070] Adsorption selectivity: Weigh 2 mg of the product MICOF of the present invention into a test tube, add 2 mL of 10 mg L -1 After brief sonication, the tubes were placed on a shaker at 150 rpm for 1 h, filtered through a 0.22 μm filter membrane, and placed into chromatographic vials for analysis by HPLC-FLD.

[0071] Figure 5 The adsorption kinetics and adsorption isotherms of ZEN on MICOF and MNCOF are shown in Figure 2. Figure 5 As shown in the figure, MICOF has faster adsorption kinetics (20 min to reach adsorption equilibrium), while MNCOF is 60 min. -1 MICOF has a larger adsorption capacity for the target molecule ZEN (177.2 mg g -1 ), the calculated imprinting factor (IF) was 10.1, indicating good selectivity for the target molecule ZEN.

[0072] Figure 6 Comparison of the adsorption capacity of four different mycotoxins by MICOF and MNCOF (initial concentration was 10 mg / L -1 ).like Figure 5 As shown in the figure, MICOF has the largest adsorption capacity for ZEN, and the adsorption capacity for other mycotoxins is lower and comparable to that of non-imprinted materials, demonstrating the highly selective adsorption performance of the imprinted covalent organic framework.

[0073] Example 3: Application of molecularly imprinted covalent organic framework as pretreatment adsorbent for dispersed solid phase extraction in cereal samples

[0074] Pre-extraction of actual samples: 10 g of each grain was placed in a 50 mL centrifuge tube. 18 mL of acetonitrile (ACN) and 2 mL of water were added. The mixture was homogenized using a high-speed homogenizer for 3 minutes and ultrasonically extracted for 20 minutes. The extract was then collected (centrifuged at 10,000 rpm for 10 minutes), dried under nitrogen evaporation, and reconstituted in 1 mL of ACN for later use.

[0075] Dispersive Solid-Phase Extraction: Dilute 1 mL of the above solution to 10 mL with water to prepare the DSPE working solution. Add 10 mg of MICOF to the working solution and briefly sonicate. Incubate the suspension in a shaker at 180 rpm for 20 minutes. Centrifuge at 10,000 rpm for 10 minutes to collect the MICOF, elute with 1 mL of ACN, and filter through a 0.22 μm syringe filter. The filtrate is analyzed by HPLC-FLD.

[0076] Table 1 shows the validation of the MICOF DSPE-HPLC-FLD method. As shown in Table 1, the established method exhibits minimal matrix attenuation, good precision (intra-day and inter-day RSDs <5%), and a wide linear range. This method provides high data reliability and can be used to analyze samples at multiple concentration levels.

[0077] Table 2 shows the analysis results of the actual samples using the MICOF-DSPE-HPLC-FLD method. As shown in Table 2, the spike recovery test showed that the -1 A recovery rate of 95.1–103.1% can be achieved within the concentration range of 100 μg / mL, indicating that the method has high accuracy. The accuracy of the method was verified by quality control samples, further ensuring the reliability of the analytical method.

[0078] like Figure 7 As shown, the recycling frequency and recovery rate of MICOF were verified in the presence of zearalenone and zearalenone and other mycotoxins. MICOF retained a recovery rate of over 92% after five cycles, demonstrating the material's excellent reusability. Even in the presence of other mycotoxins, the recovery rate remained comparable to that of the blank control, demonstrating MICOF's high selectivity for zearalenone.

[0079] Table 1 MICOF DSPE-HPLC-FLD method validation

[0080]

[0081] Table 2 Analysis results of actual samples using MICOF-DSPE-HPLC-FLD method

[0082]

[0083]

[0084] 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 principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a zearalenone molecularly imprinted covalent organic framework, characterized in that: The following steps are involved: Step S11: dissolving tetrakis(4-aminophenyl)methane, aldehyde monomer and pseudo-template in a reaction solvent to obtain solution A; Step S12: adding the catalyst dropwise to solution A to undergo polymerization reaction, eluting the pseudo-template, washing, and drying to obtain a molecularly imprinted covalent organic framework; The pseudo template is selected from one of warfarin, quercetin and 2,4-dihydroxybenzoic acid cyclododecyl ester; The aldehyde monomer is selected from one of terephthalaldehyde, 4,4'-biphenyldicarboxaldehyde, 1,4-di(4-formaldehydephenyl)benzene and [1,1':4',1'':4'',1'''-quaterphenyl]-4,4'''-dicarboxaldehyde; The molar ratio of the aldehyde monomer to the pseudo template is 10:1 to 4:

1.

2. The method for preparing a molecularly imprinted covalent organic framework according to claim 1, wherein: The reaction solvent is selected from one of dioxane, acetonitrile, methanol and tetrahydrofuran; The polymerization reaction time in step S12 is 3-48 h; The catalyst in step S12 is selected from one of acetic acid and scandium trifluoromethanesulfonate.

3. The method for preparing a molecularly imprinted covalent organic framework according to claim 1, wherein: The catalyst is 0.02-0.14 equivalents of the imine bond generated by the reaction.

4. Use of the molecularly imprinted covalent organic framework prepared by the preparation method according to any one of claims 1 to 3, characterized in that: Applied in dispersive solid phase extraction for the selective extraction and analysis of zearalenone.

5. The use of the molecularly imprinted covalent organic framework according to claim 4, characterized in that: Sample pretreatment for the detection of zearalenone in cereals, including: Step S21: The grain sample is homogenized, ultrasonically extracted, dried with nitrogen, redissolved in acetonitrile, and diluted with water to obtain solution B; Step S22: adding the molecularly imprinted covalent organic framework to solution B, and eluting with solvent after dispersive solid phase extraction; Step S23: Detection and quantification.

6. The use of the molecularly imprinted covalent organic framework according to claim 5, characterized in that: In step S23, the HPLC-FLD method is used for quantitative detection.

7. The use of the molecularly imprinted covalent organic framework according to claim 5, characterized in that: The pH of solution B is 4-9.

8. The use of the molecularly imprinted covalent organic framework according to claim 5, characterized in that: In step S22, the elution solvent is selected from acetonitrile, methanol, ethanol, acetonitrile / acetic acid with a volume ratio of 9:1, methanol / acetic acid with a volume ratio of 9:1, or ethanol / acetic acid with a volume ratio of 9:1; The elution time in step S22 is 5-30 min.

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