Nitrofuran metabolite magnetic surface molecularly imprinted polymer as well as preparation method and application thereof

Abstract

The invention provides a nitrofuran metabolite magnetic surface molecularly imprinted polymer as well as a preparation method and application thereof, and relates to the technical field of molecularly imprinted polymers. The preparation method comprises the following steps: by virtue of a surface imprinting polymerization method, taking CS-Fe3O4-coated HNTs as a carrier, and taking AHD and AMOZ as dual-template molecules; mAA is a functional monomer; a deep eutectic solvent synthesized by choline chloride and p-coumaric acid is used as an auxiliary functional monomer, AIBN is used as an initiator, and EGDMA is used as a cross-linking agent; the magnetic surface molecularly imprinted polymer is prepared by using a magnetic solid-phase extraction technology as a template and acetonitrile as a pore-foaming agent, and is successfully applied to enrichment and detection of nitrofuran metabolites in an actual sample by combining a magnetic solid-phase extraction technology and high performance liquid chromatography. According to the method, trace detection of nitrofuran metabolites such as AOZ, AMOZ, AHD and SEM can be achieved, the detection limit is as low as 0.25-0.93 ng / mL, and the adding standard recovery rate is 80-95% RSDlt; 3.5%; the zebra fish can also be applied to water environment restoration, the survival rate of zebra fish is remarkably increased, and it is proved that the zebra fish has toxicology relieving and ecological restoration functions.

Description

Technical Field

[0001] This invention relates to the field of molecularly imprinted polymer technology, and in particular to magnetic surface molecularly imprinted polymeric materials of nitrofuran metabolites, their preparation methods, and their applications in improving the survival rate of aquatic organisms. Background Technology

[0002] Nitrofurans (such as furazolidone, nitrofurazone, nitrofurantoin, and nitrofurantoin) were once widely used in livestock and aquaculture for infection control and growth promotion due to their broad-spectrum antibacterial activity. However, they are rapidly metabolized in animals to produce stable conjugated metabolites such as 3-amino-2-oxazolidinone (AOZ), 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), 1-aminohydantoin (AHD), and semicarbazine (SEM). These metabolites bind tightly to tissue proteins, have long half-lives, and have been shown to have potential carcinogenic, mutagenic, and teratogenic effects. Nevertheless, due to their historical use and environmental residues, these metabolites are still frequently detected in aquatic products and environmental water bodies, posing a serious threat to food safety and ecological health. Currently, various detection technologies have been developed. Among them, chromatography-mass spectrometry (LC-MS / MS, HPLC-MS) is widely used due to its sensitivity and quantitative accuracy. However, this method requires expensive instruments, is complex to operate, and has time-consuming sample pretreatment. Immunological methods such as ELISA and immunoaffinity column detection are simple to operate and have high throughput, but antibody preparation is complex, has poor stability, and is prone to cross-reaction. Molecularly imprinted polymers (MIPs) have gradually gained attention due to their structural stability, low cost, and artificial "specific binding sites," becoming a hot topic for the separation and enrichment of trace pollutants. However, existing molecular imprinting techniques still have several limitations in the detection of nitrofuran metabolites: First, the selection of templates is often inappropriate; most studies use the original drug as a template, making it difficult to accurately identify metabolites, or they only target a single metabolite (such as AOZ), lacking multi-target recognition capabilities. Second, the polymerization system is highly dependent on organic solvents, resulting in a significant environmental burden during preparation, and some imprinted sites lack specificity and selectivity. Third, the material forms are mostly powders or microspheres, requiring centrifugation and filtration for separation, which is cumbersome and makes it difficult to process large-volume samples rapidly, and also lacks magnetic response characteristics. Fourth, existing patents such as CN110361462A, which uses alternative templates and pipette tip polymerization, and CN103275272A, which prepares AOZ imprinted membranes on 96-well plates, fail to address higher-level needs such as multi-target recognition, green preparation, and environmental remediation. It is particularly noteworthy that most current technologies only focus on the "detection" level, without considering whether the materials can mitigate the ecotoxicity of pollutants. In fact, the residues of nitrofuran metabolites in the aquatic environment not only affect the test results, but also directly threaten the survival of aquatic organisms. Currently, there is a lack of functional materials that can simultaneously achieve selective removal and toxicity mitigation. Summary of the Invention

[0003] To address the aforementioned technical challenges, this invention proposes a magnetic surface molecularly imprinted polymer based on a deep eutectic solvent-assisted dual template. This polymer can efficiently, greenly, and repeatedly remove various nitrofuran metabolites from water. Furthermore, it has been verified in a zebrafish model that it can significantly reduce pollutant-induced mortality and improve the survival rate of aquatic organisms. This breaks through the limitations of traditional molecularly imprinted materials, which are confined to laboratory testing, and provides a novel technical approach for food safety testing and environmental remediation.

[0004] The present invention provides a magnetic surface molecularly imprinted polymer of nitrofuran metabolites, and the preparation method is as follows:

[0005] (1) Mix choline chloride and p-coumaric acid in a molar ratio of 3:1 and stir at 85-95℃ until a uniform and transparent liquid is formed, thus obtaining a clear and transparent deep eutectic solvent DES solution.

[0006] (2) Dissolve any two template molecules from AOZ, AMOZ, AHD, and SEM together with methacrylic acid (MAA) and the DES solution prepared in step (1) in acetonitrile, sonicate for a certain time, and then perform prepolymerization by shaking on a shaker.

[0007] (3) A certain amount of chitosan-modified magnetized halloysite (CS-Fe3O4@HNT) was dispersed in acetonitrile, and after sonication for a certain time, it was combined with the solution obtained in step (2) and transferred to a container. Ethylene glycol dimethacrylate (EGDMA) was added and sonicated to mix.

[0008] (4) Add a certain amount of azobisisobutyronitrile (AIBN), and under nitrogen protection, stir thoroughly in a room temperature water bath and heat to 60-70℃, and continue to react for a certain time.

[0009] (5) After the reaction is complete, the synthesized MIP material is separated by an external magnetic field, and the template molecules are eluted to obtain the molecularly imprinted polymer.

[0010] (6) The obtained molecularly imprinted polymer was vacuum dried to obtain a magnetic surface molecularly imprinted polymer: coumaric acid-MIP.

[0011] Preferably, in step (2), the molar ratio of the two template molecules to methacrylic acid is 1:1:4.

[0012] Preferably, in step (2), the template molecules are 1-aminohydantoin (AHD) and 5-methylmorpholino-3-amino-2-azolidinone (AMOZ).

[0013] Preferably, in step (3), the chitosan-modified magnetized halloysite (CS-Fe3O4@HNT) is prepared by the following steps:

[0014] Weigh a certain amount of halloysite powder into a three-necked flask, add sufficient deionized water and ultrasonically disperse, then add FeCl3·6H2O and FeSO4·7H2O, Fe 3+ and Fe 2+ The molar ratio was 1.2:1-2:1; the mixture was stirred thoroughly in a water bath at 55-65℃ under a nitrogen atmosphere, then concentrated ammonia was added until the pH reached 9-10, and the mixture was heated to 75-85℃ for a period of time; after the reaction was completed, the magnetic halloysite was separated by an external magnetic field, washed first with deionized water, then with anhydrous ethanol until neutral, and finally vacuum dried at 55-65℃ to obtain magnetized halloysite Fe3O4@HNT; the amount of halloysite added as a carrier was determined according to the magnetite loading of 30%-60%;

[0015] A certain amount of Fe3O4@HNT was weighed and dispersed in deionized water, and stirred in a water bath at 50°C to obtain an HNT suspension. Then, a 1% (w / w) chitosan solution was slowly added to the HNT suspension under stirring, and the reaction was continued for a period of time. The mass ratio of Fe3O4@HNT to chitosan was 1:1. The solvent of the chitosan solution was 0.25%-2% (w / w) acetic acid. After the reaction was completed, the mixture was separated by a magnet and washed with distilled water to remove unadsorbed chitosan and excess acetic acid. After freeze-drying, chitosan-modified magnetic HNT, namely CS-Fe3O4@HNT, was obtained.

[0016] Preferably, in step (3), the molar ratio of ethylene glycol dimethacrylate (EGDMA) to the template molecule is 20:1-35:1.

[0017] As a preferred option, in step (5), the method for eluting template molecules is as follows: the separated MIP material is subjected to Soxhlet extraction with methanol-acetic acid solution in a water bath at 75-85℃ until template molecules are no longer detectable. The volume ratio of methanol to acetic acid in the methanol-acetic acid solution is 9:1.

[0018] Working principle of the invention:

[0019] This invention utilizes surface-imprinted polymerization with chitosan-modified magnetic halloysite nanotubes (CS-Fe3O4@HNTs) as a carrier to impart high specific surface area, good dispersibility, and rapid magnetic separation capability to the material, significantly improving operational efficiency and reusability. 1-Aminohydantoin (AHD) and 5-methylmorpholino-3-amino-2-azolidinyl ketone (AMOZ) serve as dual-template molecules to construct multi-target recognition sites. Methacrylic acid (MAA) is a functional monomer that forms hydrogen bonds and electrostatic interactions with the template molecules through its carboxyl group. Deep eutectic solvent (DES), synthesized from choline chloride and p-coumaric acid, serves as an auxiliary functional monomer. During the polymerization process, additional hydrogen bonds and π–π interactions are provided to enhance the hydrogen bonding and electrostatic interactions between the template and monomer, thereby improving the specificity of the imprinted sites and the greenness of material preparation. Azobisisobutyronitrile (AIBN) is used as an initiator, which decomposes under nitrogen protection to generate free radicals and initiate the polymerization reaction. Ethylene glycol dimethacrylate (EGDMA) is used as a crosslinking agent to form a three-dimensional polymer framework and ensure the structural stability of the material. Acetonitrile is used as a porogen to prepare magnetic surface molecularly imprinted polymers. Combined with magnetic solid-phase extraction technology and high-performance liquid chromatography, it has been successfully applied to the enrichment and detection of nitrofuran metabolites in actual samples.

[0020] During the preparation process, template molecules (AHD, AMOZ), functional monomers (MAA), and deep eutectic solvent (DES) form stable pre-assembled complexes through hydrogen bonding, electrostatic interactions, and π–π interactions. Subsequently, under the action of crosslinking agent EGDMA and initiator AIBN, free radical polymerization occurs on the surface of magnetic halloysite nanotube carriers (CS-Fe3O4@HNTs), generating a crosslinked and dense polymer network. After polymerization, the template molecules are removed by elution with methanol-acetic acid solution, leaving "imprint recognition holes" on the polymer surface that highly match the shape, size, and functional group distribution of the template molecules. These recognition holes are the specific binding sites of the material, which can selectively bind to and remove corresponding nitrofuran metabolites in complex aquatic environments.

[0021] The present invention provides the application of a magnetic surface molecularly imprinted polymer for nitrofuran metabolites in the detection of nitrofuran metabolites:

[0022] The magnetic surface molecularly imprinted polymer material for nitrofuran metabolites provided by this invention is added to a water sample, and after magnetic separation, it is detected by HPLC or LC-MS / MS. This enables trace detection of nitrofuran metabolites such as AOZ, AMOZ, AHD, and SEM, with detection limits as low as 0.25–0.93 ng / mL, spiked recoveries of 80–95%, and RSD < 3.5%.

[0023] This invention provides an application of a nitrofuran metabolite magnetic surface molecularly imprinted polymer in aquatic environment remediation:

[0024] The magnetic surface molecularly imprinted polymer material for nitrofuran metabolites provided by this invention is introduced into water bodies polluted by nitrofuran metabolites. By adsorption, the concentration of nitrofuran metabolites in the water body is reduced, thereby reducing the toxic effects on aquatic organisms.

[0025] In zebrafish experiments, the polluted water samples treated with the magnetic surface molecularly imprinted polymer material of nitrofuran metabolites provided by this invention significantly improved the survival rate of zebrafish (the 24-hour survival rate increased from about 40% to 80%), demonstrating its toxicological mitigation and ecological restoration functions.

[0026] The beneficial effects of this invention are:

[0027] 1. Strong multi-target recognition capability

[0028] This invention employs a dual-template strategy of AHD and AMOZ, unlike existing technologies that use the original drug or a single metabolite as a template. By constructing recognition holes through dual templates, it is possible to simultaneously achieve efficient recognition and enrichment of multiple nitrofuran metabolites such as AOZ, AMOZ, AHD, and SEM, thus broadening the scope of application and avoiding the limitations of "single-target detection" in existing technologies.

[0029] 2. Green preparation and high selectivity coexist.

[0030] This invention introduces choline chloride-coumaric acid deep eutectic solvent (DES) as an auxiliary functional monomer, which can enhance the interaction between the template and the functional monomer through hydrogen bonding and π–π interaction, thereby improving the specificity and binding strength of the imprinted site. It also partially replaces organic solvents, making the preparation process more green and environmentally friendly, which is an advantage that traditional MIP preparation does not have.

[0031] 3. Magnetic separation is simple to operate.

[0032] This invention uses chitosan-modified magnetic halloysite nanotubes (CS-Fe3O4@HNTs) as a carrier. The material has a high specific surface area and good dispersibility. At the same time, it can achieve rapid separation under the action of an external magnetic field, avoiding the cumbersome process of centrifugation or filtration required by traditional powder or microsphere MIP, and greatly improving the operation efficiency and reusability.

[0033] 4. Excellent detection performance

[0034] Experimental results show that the magnetic surface molecularly imprinted polymer material of nitrofuran metabolites of the present invention has a spiked recovery rate of 80–95% in tap water samples, a relative standard deviation (RSD) of less than 3.5%, and a limit of detection (LOD) of 0.25–0.93 ng / mL, exhibiting excellent sensitivity and repeatability, which is significantly better than some existing MIP or immunological methods.

[0035] 5. Environmental remediation and toxicological mitigation functions

[0036] This invention is the first to verify the ecotoxicological effects of the material in a zebrafish model. Results showed that only about 40% of zebrafish survived within 24 hours in untreated water samples contaminated with nitrofuran metabolites, while the survival rate of zebrafish in water samples treated with the material of this invention increased to nearly 80%, and the material itself showed no significant toxicity. This result indicates that the invention can not only remove pollutants but also significantly alleviate the toxic effects of nitrofuran metabolites on aquatic organisms, achieving integrated application value of detection, treatment, and ecological restoration—a unique advantage never before seen in existing technologies. Attached Figure Description

[0037] Figure 1 Here are SEM images of HNT, Fe3O4@HNT, CS-Fe3O4@HNT and coumaric acid-MIP in the examples;

[0038] Figure 2 The images shown are TEM images of Fe3O4@HNT, CS-Fe3O4@HNT, and coumaric acid-MIP in the examples.

[0039] Figure 3 Infrared images of HNT, Fe3O4@HNT, CS-Fe3O4@HNT, coumaric acid-MIP, and NIP in the examples;

[0040] Figure 4 The image shows a magnetometer reading of the vibrating sample of coumaric acid-MIP in the examples.

[0041] Figure 5 The figure shows the test results of the isothermal adsorption experiment in the examples;

[0042] Figure 6 The figure shows the test results of the kinetic adsorption experiment in the example;

[0043] Figure 7 This is a graph showing the test results selectively examined in the embodiments;

[0044] Figure 8 This is a diagram showing the optimization results of the optimal material amount in the examples;

[0045] Figure 9 This is a schematic diagram illustrating the concentration-dependent developmental toxicity of nitrofuran metabolites in zebrafish embryos in the examples.

[0046] Figure 10 This is a schematic diagram illustrating the developmental toxicity assessment of coumaric acid-MIP in zebrafish embryos in the examples;

[0047] Figure 11The bar chart shows the mortality rate of zebrafish in this example. Detailed Implementation

[0048] Example 1

[0049] This embodiment provides a magnetic surface molecularly imprinted polymer of nitrofuran metabolites, prepared by the following method:

[0050] (1) Synthesis of eutectic solvent

[0051] Choline chloride was selected as HBA and p-coumaric acid as HBD. 0.03 mol and 0.01 mol of each were taken at a molar ratio of 3:1, and the mixture was heated and stirred at 90 °C for 2 h until a homogeneous and transparent liquid was formed, yielding a clear and transparent deep eutectic solvent solution of DES. The prepared DES was used as an auxiliary monomer in the synthesis of coumaric acid-MIP.

[0052] (2) Synthesis of CS-Fe3O4@HNT

[0053] Accurately weigh 2.5g of halloysite powder into a three-necked flask, add 150ml of deionized water, and ultrasonically disperse for 15min. Add 5.8g of FeCl3·6H2O and 4.8g of FeSO4·7H2O. Stir the mixture in a 60℃ water bath under a nitrogen atmosphere for 10min, then add concentrated ammonia to adjust the pH to 9-10, and continue heating to 80℃ for 2 hours. After the reaction is complete, separate the magnetic halloysite using an external magnetic field. Wash three times with deionized water, then wash with anhydrous ethanol until neutral, and finally dry under vacuum at 60℃ for 12 hours to obtain magnetized halloysite Fe3O4@HNT.

[0054] 1 g of Fe3O4@HNT was accurately weighed and dispersed in 100 ml of deionized water. The mixture was stirred and refluxed in a 50°C water bath for 15 min to obtain an HNT suspension. Then, 100 ml of a 1% (w / w) chitosan solution (using 1% (w / w) acetic acid as the solvent) was slowly added to the HNT suspension under stirring, and the reaction continued for 12 h. After completion, the mixture was separated using a magnet and washed three times with distilled water to remove unadsorbed chitosan and excess acetic acid. After freeze-drying, chitosan-modified magnetic HNTs, namely CS-Fe3O4@HNT, were obtained.

[0055] (3) Preparation of magnetic surface molecularly imprinted polymers of nitrofuran metabolites

[0056] Take 0.2 mmol of AHD and AMOZ as dual template molecules, and dissolve them together with 0.8 mmol of methacrylic acid (MAA) and 2 ml of DES solution prepared in step (1) in 45 ml of acetonitrile. After sonication for 30 min, prepolymerize by shaking on a shaker at 25 °C for 12 h. All the above steps must be performed in the dark.

[0057] Take another 0.3g-1g of CS-Fe3O4@HNT and disperse it in 25ml of acetonitrile (0.5g in this example). After sonicating for 20min, combine it with the solution obtained in step (3) and transfer it to a three-necked flask. Add 14mmol of ethylene glycol dimethacrylate (EGDMA) and sonicate in a sealed container for 30min.

[0058] Add 0.1g of azobisisobutyronitrile (AIBN) and 40ml of acetonitrile, stir in a water bath at room temperature for 15min, then gradually raise the temperature to 60℃ and continue the reaction for 12h.

[0059] After the reaction was completed, the synthesized MIP material was separated by an external magnetic field and subjected to Soxhlet extraction in an 80°C water bath with about 500 ml of methanol:acetic acid (volume ratio = 9:1) to elute the template molecules until no template molecules could be detected, thus obtaining a magnetic surface molecularly imprinted polymer.

[0060] The obtained magnetic surface molecularly imprinted polymer was vacuum dried at 60 °C for 12 h to obtain coumaric acid-MIP, which was then stored in a desiccator in a light-proof and airtight container.

[0061] Characterization and analysis of magnetic surface molecularly imprinted polymers of nitrofuran metabolites

[0062] (1) Scanning electron microscope, transmission electron microscope

[0063] like Figure 1 The image shown is a SEM image of HNT, Fe3O4@HNT, CS-Fe3O4@HNT, and coumaric acid-MIP. Figure 1 -a shows that HNT appears as a long, tubular shape; after magnetization, as... Figure 1 -b shows iron oxide particles adhering to its surface; after chitosan modification, it can be seen that... Figure 1 -c contains more attached particles; MIPs were synthesized using chitosan-modified magnetic halloysite as a carrier, and... Figure 1 -d indicates that there are unidentified imprinted vacancies on the polymer surface, indicating that the MIP synthesis was successful. Figure 2 These are TEM images of Fe3O4@HNT, CS-Fe3O4@HNT, and coumaric acid-MIP. Figure 2 -a shows that iron oxide was successfully loaded onto the halloysite surface. Figure 2 -b shows that chitosan is modified on magnetic halloysite. Figure 2 -c allows you to see the state of the polymer; the obvious pores indicate that the polymer material has been successfully synthesized.

[0064] (2) Infrared analysis

[0065] Figure 3The Fourier transform infrared spectra of HNT, Fe3O4@HNT, CS-Fe3O4@HNT, coumaric acid-MIP, and NIP are shown. NIP was prepared using the method described in Example 1 of this invention, but without the addition of a template molecule during the preparation process.

[0066] Coumaric acid-MIP and NIP both at 2950cm -1 and 1730cm -1 The peaks are observed at 610 cm⁻¹, representing the CH stretching vibration in p-coumaric acid and the C=O stretching vibration in MAA, respectively; except for halloysite, the other four peaks are all at 610 cm⁻¹. -1 The peak is located at a point that is characteristic of the stretching vibration of Fe-O.

[0067] (3) VSM

[0068] VSM is a method used to study the superparamagnetic properties of magnetic nanoparticles. For example... Figure 4 As shown, the residual amount and coercivity of both samples approached zero, exhibiting superparamagnetic properties. Furthermore, no hysteresis was observed in these samples. These results indicate that the aforementioned polymers can achieve rapid magnetic separation and uniform suspension. The saturation magnetization values ​​of CS-Fe3O4@HNT and coumaric acid-MIP were 38.20 and 29.46 emu / g, respectively. The excellent magnetic response ensures rapid switching of MIPs during separation and reuse, improving enrichment efficiency.

[0069] Adsorption properties of magnetic surface molecularly imprinted polymers of nitrofuran metabolites

[0070] (1) Isothermal adsorption

[0071] 8 mg of MAA-MIP material and coumaric acid-MIP material were taken separately. MAA-MIP material was prepared using the preparation method of Example 1 of this invention, but without the addition of DES as an auxiliary monomer. 4 ml of a mixed standard solution of four nitrofuran metabolites (0.1-1 μg / ml) was added. The mixture was placed in a shaker and shaken at 110 rpm for 25°C for 180 min. After the shake, an external magnetic field was applied to separate the materials. The material surface was rinsed with 500 μL of deionized water, and then eluted with 3.5 mL of methanol-acetic acid (95:5, v:v) eluent by sonication, 1750 μL each time, for 15 min, twice. All eluents were combined, and 40 μL of derivatizing agent was added. The mixture was derivatized in a shaker at 37°C for 16 h, dried under nitrogen at 45°C, and reconstituted with 500 μL of acetonitrile. After filtering through a 0.22 μm filter, the sample was injected. The peak area was determined by HPLC, and the adsorption capacity of the two materials at different concentrations was calculated. Figure 5 Static adsorption isotherms of MAA-MIP and coumaric acid-MIP on nitrofuran metabolites.

[0072] The binding degree of both MIP materials to nitrofuran metabolites gradually increased with increasing initial concentration. When the template molecule concentration was 1 mg / mL, saturation was essentially reached. The adsorption capacities of MAA-MIP for SEM, AHD, AMOZ, and AOZ were 0.17, 0.21, 0.22, and 0.12 mg / g, respectively; while those for coumaric acid-MIP were 0.17, 0.30, 0.33, and 0.18 mg / g, respectively. It can be seen that the adsorption capacity of MIP is stronger with the participation of DES. This is because the use of multifunctional monomers in the synthesis of MIP helps to improve selectivity. The addition of DES has a synergistic effect on the recognition of monomers and templates, increasing the number of imprinted recognition sites and thus enhancing selectivity.

[0073] (2) Dynamic adsorption

[0074] Weigh 8 mg of MAA-MIP material and coumaric acid-MIP material respectively, add 4 ml of a mixed standard solution of four nitrofuran metabolites at 0.5 μg / mL, place in a shaker, shake at 110 rpm and 25 °C for 180 min, magnetically separate the materials, rinse the material surface with 500 μL of deionized water, and then elute the material with 3.5 mL of methanol-acetic acid (95:5, v:v) eluent by sonication, 1750 μL each time, sonication for 15 min, twice, combine all eluents, add 40 μL of derivatizing agent and derivatize in a shaker at 37 °C for 16 h, blow dry with nitrogen at 45 °C, redissolve in 500 μL of acetonitrile, filter through a 0.22 μm filter membrane and inject, and determine the peak area by HPLC.

[0075] Figure 6 The adsorption curves show that the adsorption capacity of MMIPs gradually increases over time from 0 to 180 min, indicating the presence of a porous and uniform molecularly imprinted polymer layer on the MIP surface. After 180 min, the adsorption rate gradually slows down, eventually reaching adsorption equilibrium. Comparison reveals that coumaric acid-MIP has a significantly stronger adsorption capacity than MIP, further confirming that DES can enhance the adsorption capacity of MIP.

[0076] (3) Adsorption selectivity

[0077] To verify the specificity and selective recognition capabilities of the two MIPs, secnidazole and ornidazole, which have structures similar to nitrofuran metabolites, were selected as potential interfering agents. Ornidazole and secnidazole were used as competing compounds, and a 1 μg / mL mixed standard solution (containing four nitrofuran metabolites and two competing compounds) was prepared. 8 mg of each MIP material was weighed for adsorption, and the adsorption was performed by shaking at 110 rpm for 180 min at 25°C. After adsorption, the material was separated by magnetization, rinsed with 500 μL of deionized water, and then eluted by sonication with 3.5 mL of methanol and acetic acid (95:5, v:v) eluent, 1750 μL each time, for 15 min, twice. All eluents were combined, and 40 μL of derivatizing agent was added for derivatization in a shaker at 37°C for 16 h. The eluent was then dried under nitrogen at 45°C, redissolved in 500 μL of acetonitrile, filtered through a 0.22 μm filter, and injected. The peak area was determined by HPLC.

[0078] like Figure 7 As shown, the adsorption capacities of MIP for SEM, AHD, AMOZ, AOZ, secnidazole, and ornidazole were 0.40, 0.22, 0.19, 0.09, 0.06, and 0.03 mg / g, respectively; while those of coumaric acid-MIP were 0.37, 0.27, 0.29, 0.13, 0.05, and 0.02 mg / g. It can be seen that both MIPs have weak adsorption capacity for the competing drugs secnidazole and ornidazole, with the coumaric acid-MIP showing a more pronounced effect. This indicates that the addition of DES helps to improve adsorption selectivity.

[0079] (4) Adsorption temperature

[0080] Approximately 4 mg of coumaric acid-MIP material was used to prepare three 4 mL aliquots of a 0.5 μg / mL nitrofuran metabolite mixed standard solution. Adsorption was performed at 25°C, 35°C, and 45°C, respectively, by shaking at 110 rpm for 2 h. After adsorption, the material was magnetically separated, and the surface was simply rinsed with 500 μL of deionized water. Then, the material was ultrasonically eluted with 3.5 mL of methanol and acetic acid (95:5, v:v) eluent, 875 μL each time, for 10 min, for four times. All eluents were combined, and 40 μL of derivatizing agent was added. The mixture was derivatized in a shaker at 37°C for 16 h, dried under nitrogen at 45°C, and reconstituted with 500 μL of acetonitrile. The solution was filtered through a 0.22 μm membrane and injected. Peak area was determined by HPLC, and the recovery rates at different temperatures were compared.

[0081] Experimental results show that the adsorption capacity is the largest at a temperature of 25 °C, therefore 25 °C was chosen as the optimal adsorption temperature.

[0082] (5) Adsorption frequency

[0083] Take approximately 4 mg of coumaric acid-MIP material and prepare three 4 mL aliquots of a 0.5 μg / mL nitrofuran metabolite mixed standard solution. Adsorption was performed by shaking at 25°C for 2 h at frequencies of 80 rpm, 110 rpm, and 150 rpm, respectively. After adsorption, the material was magnetically separated, rinsed with 500 μL of deionized water, and then eluted by sonication with 3.5 mL of methanol and acetic acid (95:5, v:v) eluent, 875 μL each time, for 10 min, for 4 times. All eluents were combined, and 40 μL of derivatizing agent was added for derivatization on a shaker at 37°C for 16 h. The eluent was then dried under nitrogen at 45°C, redissolved in 500 μL of acetonitrile, filtered through a 0.22 μm membrane, and injected. Peak area was determined by HPLC, and the recoveries at different frequencies were compared.

[0084] Experimental results show that the adsorption capacity is the largest at a frequency of 110 rpm, therefore 110 rpm was selected as the optimal adsorption frequency.

[0085] (6) Amount of adsorbent material

[0086] Five 4 mL aliquots of 0.5 μg / mL nitrofuran metabolite mixed standard solutions were prepared using coumaric acid-MIP material at concentrations of 2, 4, 6, 8, and 10 mg. The solutions were then shaken at 110 rpm for 2 hours at 25°C for adsorption. After adsorption, the material was magnetically separated, rinsed briefly with 500 μL of deionized water, and then eluted using 3.5 mL of methanol and acetic acid (95:5, v:v) eluent (875 μL each time, 10 min each time, 4 times). All eluents were combined, and 40 μL of derivatizing agent was added. The eluent was then derivatized in a shaker at 37°C for 16 hours, dried under nitrogen at 45°C, redissolved in 500 μL of acetonitrile, filtered through a 0.22 μm membrane, and injected. The peak area was determined by HPLC, and the recovery rates after adsorption with different material amounts were compared.

[0087] like Figure 8 As shown, the adsorption capacity is maximized when the material amount is 8 mg; therefore, 8 mg is selected as the optimal adsorption material amount. Based on the above results, the optimized adsorption conditions of 25℃, 110 rpm, and 8 mg of material were selected to re-bind nitrofuran metabolites.

[0088] (7) Optimize elution conditions

[0089] Different elution times (5, 10, 15 min), eluent ratios (methanol:acetic acid = 9:1, 8:2, 95:5), eluent volumes (1.5, 3.5, 7.5 ml), and elution cycles (2, 3, 4) were used to elute coumaric acid-MIP. A four-factor, three-level orthogonal experimental design (L9(3)) was employed. 4To explore the optimal combination of factors, as shown in Table 1, we selected a methanol to acetic acid solvent ratio of 95:5, an elution solvent volume of 3.5 mL, an elution time of 15 min, and elution cycles of 2 as the theoretically optimized elution conditions.

[0090] Table 1. Results of Orthogonal Design and Experiment

[0091]

[0092] Application of nitrofuran metabolite magnetic surface molecularly imprinted polymers in real samples

[0093] (1) Determination of residual nitrofuran metabolites in water:

[0094] Take 500 ml of natural water and add 16 mg of coumaric acid-MIP material for enrichment. Shake at 110 rpm for 180 min at 25°C. After adsorption, separate the material using an external magnetic field. Wash the material with 500 μL of deionized water and then elute with 3.5 ml of methanol-acetic acid (95:5 v / v) for 15 min by sonication. Repeat twice, 1750 μL each time. Combine the eluents and add 60 μL of derivatizing agent. Derivatize in a 37°C water bath for 16 h. Blow dry under nitrogen. Redissolve in 500 μL of acetonitrile and filter through a 0.22 μm filter membrane for HPLC determination.

[0095] (2) Determination of residual nitrofuran metabolites in pork:

[0096] Take 0.1g of pork, add 4ml of acetonitrile, vortex, centrifuge at 8000r for 15min, and separate the supernatant. Extract with n-hexane and collect the lower layer. Add 8mg of material for adsorption, and adsorb at 25℃ water bath, 110r, for 180min with shaking. After adsorption, separate the material with a magnet, wash with 500ul of deionized water, and then elute with 3.5ml of methanol-acetic acid (95:5 v / v) sonicated for 15min, twice, 1750ul each time. Combine the eluents, add 60ul of derivatizing agent, derivatize at 37℃ water bath for 16h, blow dry under nitrogen, redissolve with 500ul of acetonitrile, filter through a 0.22um filter membrane, and perform HPLC analysis.

[0097] Application of magnetic surface molecularly imprinted polymers of nitrofuran metabolites in toxicological mitigation and ecological restoration

[0098] In zebrafish experiments, the effects of different concentrations of nitrofuran metabolites on zebrafish embryonic development were observed, such as... Figure 9 As shown. Abbreviations: PE, pericardial edema; YSE, yolk sac edema; OE, orbital edema; SC, scoliosis; RBL, shortened body length. Figure 10This study demonstrates the developmental toxicity assessment of the coumaric acid-MIP material of the present invention in zebrafish embryos. No significant malformations were observed in the coumaric acid-MIP treatment groups (5, 10, and 20 mg). OPP (o-phenylphenol) and DMSO served as positive and solvent controls, respectively. Abbreviations: PE, pericardial edema; YSE, yolk sac edema. Figure 11 The mortality rate of zebrafish larvae exposed to nitrofuran metabolites (50 and 80 μg / mL) was compared with and without coumaric acid-MIP (10 mg) treatment. Coumaric acid-MIP significantly reduced larval mortality compared with the metabolite treatment alone (p < 0.05).

[0099] In zebrafish experiments, the magnetic surface molecularly imprinted polymer material for nitrofuran metabolites provided by this invention significantly improved the survival rate of zebrafish after treatment, increasing it from approximately 40% to 80% after 24 hours, demonstrating its toxicological mitigation and ecological restoration functions. The significant improvement in zebrafish survival rate after treatment with this material indicates that it not only possesses selective removal capabilities but also toxicological mitigation and ecological restoration effects.

Claims

1. A method for preparing a magnetic surface molecularly imprinted polymer of nitrofuran metabolites, characterized in that, Includes the following steps: (1) Mix choline chloride and p-coumaric acid in a molar ratio of 3:1 and stir at 85-95℃ until a uniform and transparent liquid is formed, thus obtaining a clear and transparent deep eutectic solvent DES solution. (2) Dissolve any two template molecules from AOZ, AMOZ, AHD, and SEM together with MAA methacrylic acid and the DES solution prepared in step (1) in acetonitrile, sonicate for a certain time, and then shake on a shaker for prepolymerization. (3) A certain amount of chitosan-modified magnetized halloysite CS-Fe3O4@HNT was dispersed in acetonitrile, sonicated for a certain time, and then combined with the solution obtained in step (2) and transferred to a container. Ethylene glycol dimethacrylate EGDMA was added and sonicated to mix. (4) Add a certain amount of azobisisobutyronitrile (AIBN), and under nitrogen protection, stir thoroughly in a room temperature water bath and heat to 60-70℃, and continue the reaction for a certain time. (5) After the reaction is complete, the synthesized MIP material is separated by an external magnetic field, and the template molecules are eluted to obtain the molecularly imprinted polymer. (6) The obtained molecularly imprinted polymer was vacuum dried to obtain a magnetic surface molecularly imprinted polymer: coumaric acid-MIP.

2. The method for preparing a nitrofuran metabolite magnetic surface molecularly imprinted polymer according to claim 1, characterized in that: In step (2), the molar ratio of the two template molecules to methacrylic acid is 1:1:

4.

3. The method for preparing a nitrofuran metabolite magnetic surface molecularly imprinted polymer according to claim 1, characterized in that: In step (2), the template molecules are 1-aminohydantoin AHD and 5-methylmorpholino-3-amino-2-azolidinone AMOZ.

4. The method for preparing a nitrofuran metabolite magnetic surface molecularly imprinted polymer according to claim 1, characterized in that: In step (3), the chitosan-modified magnetized halloysite CS-Fe3O4@HNT is prepared through the following steps: Weigh a certain amount of halloysite powder into a three-necked flask, add deionized water and ultrasonically disperse, then add FeCl3·6H2O and FeSO4·7H2O, Fe 3+ and Fe 2+ The molar ratio is 1.2:1-2:1; the mixture is stirred thoroughly in a water bath at 55-65℃ under a nitrogen atmosphere, then concentrated ammonia is added until the pH is 9-10, and the mixture is heated to 75-85℃ for a period of time. After the reaction was completed, the magnetic halloysite was separated by an external magnetic field. It was first washed with deionized water, then washed with anhydrous ethanol until neutral, and finally dried under vacuum at 55-65℃ to obtain magnetized halloysite Fe3O4@HNT. The amount of halloysite added as a carrier was determined according to the magnetite loading of 30%-60%. A certain amount of Fe3O4@HNT was weighed and dispersed in deionized water, and stirred in a water bath at 50°C to obtain an HNT suspension. Then, a 1% (w / w) chitosan solution was slowly added to the HNT suspension under stirring, and the reaction was continued for a period of time. The mass ratio of Fe3O4@HNT to chitosan was 1:

1. The solvent of the chitosan solution was 0.25%-2% (w / w) acetic acid. After the reaction was completed, the mixture was separated by a magnet and washed with distilled water to remove unadsorbed chitosan and excess acetic acid. After freeze-drying, chitosan-modified magnetic HNT, namely CS-Fe3O4@HNT, was obtained.

5. The method for preparing a nitrofuran metabolite magnetic surface molecularly imprinted polymer according to claim 1, characterized in that: In step (3), the molar ratio of ethylene glycol dimethacrylate (EGDMA) to the template molecule is 20:1-35:

1.

6. The method for preparing a nitrofuran metabolite magnetic surface molecularly imprinted polymer according to claim 1, characterized in that: In step (5), the template molecule elution method is as follows: the separated MIP material is subjected to Soxhlet extraction with methanol-acetic acid solution in a water bath at 75-85℃ until the template molecule is no longer detectable. The volume ratio of methanol to acetic acid in the methanol-acetic acid solution is 9:

1.

7. A magnetic surface molecularly imprinted polymer of nitrofuran metabolites, characterized in that: Prepared by the preparation method according to any one of claims 1-6.

8. The application of the nitrofuran metabolite magnetic surface molecularly imprinted polymer according to claim 7, characterized in that: This is used for the detection of nitrofuran metabolites, wherein the nitrofuran metabolites are at least one of AOZ, AMOZ, AHD, and SEM.

9. The application of the nitrofuran metabolite magnetic surface molecularly imprinted polymer according to claim 7, characterized in that: For water environment remediation, the magnetic surface molecularly imprinted polymer material of nitrofuran metabolites is introduced into water bodies polluted by nitrofuran metabolites, thereby reducing the concentration of nitrofuran metabolites in the water through adsorption.

10. The application of the nitrofuran metabolite magnetic surface molecularly imprinted polymer according to claim 7, characterized in that: It is used to improve the survival rate of zebrafish in water bodies polluted by nitrofuran metabolites.

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