Preparation method of magnetic molecularly imprinted polymer for adsorbing various microcystin homologs

Abstract

The invention discloses a preparation method of a magnetic molecularly imprinted polymer for adsorbing various microcystin homologs, and belongs to the technical field of water environment research. The preparation method comprises the following steps: synthesizing Fe3O4 by adopting a chemical coprecipitation method, modifying the surfaces of Fe3O4 magnetic nanoparticles with SiO2 and phosphate groups, and polymerizing to generate a polymer by utilizing a directional surface molecular imprinting strategy and adopting metformin with guanidyl similar to that of algal toxin as a virtual template molecule, dopamine as a functional monomer and a cross-linking agent and ammonium persulfate as an initiator. And eluting the template with ethanol, and drying to obtain the magnetic molecularly imprinted polymer (MMIPs) with specific adsorption performance on various microcystic toxins. In the adsorption process, phosphate radicals in an imprinting cavity can be combined with guanidyl in template molecules to improve the adsorption capacity of the MMIPs to the template molecules. The adsorption rate of the magnetic molecularly imprinted polymer to MC-LR reaches 90%-95%, the adsorption rate of the magnetic molecularly imprinted polymer to MC-RR reaches 75%-79%, the adsorption rate of the magnetic molecularly imprinted polymer to MC-YR reaches 85%-90%, and the method is high in sensitivity, good in selectivity and easy to operate and has wide market application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of water environment research, and particularly relates to a preparation method of a magnetic molecular imprinting polymer for adsorbing a plurality of microcystin homologues. Background Art

[0002] Cyanobacteria, also known as blue bacteria, are widely distributed in various environments in nature, including fresh water, sea water, humid and dry soil, even rock surfaces and other harsh environments. Cyanobacteria are simple in structure, without obvious nuclei, and their cell walls contain mucins. They are photoautotrophic Gram-negative microorganisms. The outbreak of cyanobacterial blooms will not only reduce the dissolved oxygen content in the water, inhibit the growth of other algae, interfere with the growth of fish, and cause an imbalance in the ecosystem, but also release a variety of toxic substances when cyanobacterial cells age, die, and rupture, causing water pollution and causing serious impacts on human daily life activities. The main types of cyanobacteria that cause blooms are Microcystis aeruginosa, Anabaena, Dinoflagellate, Noctiluca, Scintillans, Spiral Anabaena, and Foamy Globule. Among them, the microcystins (MCs) produced by Microcystis aeruginosa are the most common in water bodies.

[0003] Microcystins (MCs) are one of the most threatening algal toxins in cyanobacteria blooms. MCs are biologically active cyclic heptapeptides, and there are more than 90 homologues depending on the amino acids that make them up. On the one hand, microcystins are stable in physical and chemical properties, and the degradation of MCs in natural water bodies mainly depends on photodegradation and microbial degradation. They are not easily degraded and can exist stably in the water environment. On the other hand, microcystins can be transmitted through water bodies or food chains, and enter the human body through the digestive tract and other pathways, causing damage to the liver, kidneys, and nerves, posing a serious threat to human health. Therefore, it is of great practical significance to understand the types of algal toxins in water bodies, control their content, and study the enrichment and separation technology of microcystins. Among them, microcystin-LR (MC-LR), microcystin-RR (MC-RR), and microcystin-YR (MC-YR) have received extensive attention and research due to their widespread presence in water bodies, high content, and strong toxicity.

[0004] At present, conventional high-performance liquid chromatography often uses solid phase extraction columns (SPE) to pre-treat samples to enrich algae toxins before detecting algae toxins. Then, the chromatographic peaks and retention times of different algae toxin homologues in the sample are detected by ultraviolet detector to determine the composition and content of algae toxins in the sample. This method has good detection results for the content of algae toxins in water bodies. However, the C18-SPE extraction technology used in the pre-treatment process lacks selectivity. This method is prone to introduce impurities after enriching algae toxins, which interferes with the test results. Therefore, it is particularly important to develop a material with high selectivity for multiple algae toxins and specific adsorption properties. Summary of the invention

[0005] The present invention aims at the defects of the existing technology for detecting algae toxins, and invents a preparation method of a magnetic molecular imprinted polymer for adsorbing multiple microcystin homologues. The magnetic molecular imprinted polymer (MMIPs) includes a magnetic part and an imprinting layer part. MMIPs has high selectivity for the target and the characteristics of simple and rapid separation of magnetic materials, and realizes the specific adsorption of multiple phycocyanin homologues.

[0006] In order to achieve the above technical objectives, the technical solution adopted by the present invention is:

[0007] A material synthesis capable of simultaneously adsorbing multiple microcystin homologues comprises the following steps:

[0008] Preparation of magnetic materials and preparation of magnetic molecular imprinted polymers;

[0009] The magnetic material is prepared by the following steps:

[0010] (1)Fe 3 O 4 Preparation: First weigh 1.35g of ferric chloride and 2.58g of sodium citrate into a 100mL beaker, add 80mL of distilled water, and sonicate to completely dissolve. Then add 1g of urea and 0.6g of sodium polyacrylate, place the beaker on a powerful stirrer for 30 minutes. After the end, transfer the solution to a reactor and heat it in a vacuum drying oven at 200°C for 12 hours. After cooling to room temperature, wash the product with water and ethanol three times each, and finally dry it for 12 hours and collect it for later use;

[0011] (2) Ferroferric oxide coated silica material (Fe 3 O 4 @SiO 2 Preparation of MMS: First weigh 0.5 g of prepared Fe 3 O 4 In a 100mL three-necked flask, add 0.6g urea and 1g hexadecyltrimethylammonium bromide, and add 30mL distilled water to form a suspension. Then add 1mL isopropanol and 30mL methane along the wall of the flask, and then add 120 microliters of tetraethyl orthosilicate every 6 minutes under vigorous mechanical stirring with a stirring paddle, a total of 1.2mL. Keep stirring and move the flask into a water bath for 16 hours. After the reaction is completed, pour off the supernatant, add 0.6g ammonium nitrate and 60mL ethanol and reflux it overnight. After the reaction is completed, wash three times with distilled water, freeze-dry, and collect for later use;

[0012] (3) Phosphate-functionalized Fe 3 O 4 @SiO 2 (Fe 3 O4 @SiO 2 -P, MMS-P) preparation: Diethylphosphoethyltriethoxysilane was placed in a 50mL three-necked flask, acetic acid was added and the mixture was refluxed in a water bath to pre-hydrolyze. After cooling to room temperature, 0.5g MMS and 25mL toluene were added and refluxed for two hours. After the reaction was completed, the mixture was washed with methanol and ethanol three times respectively, dried under vacuum at 70°C, and collected for later use;

[0013] The preparation of the molecular imprinted polymer (MMS-P@MIPs, MMIPs) comprises the following steps:

[0014] Weigh the prepared MMS-P, add dopamine, ammonium persulfate, virtual template and water in sequence, stir mechanically to react for 5-7 hours, wash with ethanol 2-3 times after the reaction, freeze-dry. Then, perform Soxhlet extraction, use ethanol as washing liquid to elute the metformin virtual template molecule, and finally freeze-dry to obtain magnetic molecular imprinting polymers (MMIPs).

[0015] Furthermore, in step (1), the drying condition after washing the product is 45°C-50°C.

[0016] Furthermore, in step (2), the water bath temperature is 70°C-80°C, and the overnight reflux temperature is 60°C.

[0017] Furthermore, in step (3), the water bath is at 80° C. and refluxed for 6-8 hours.

[0018] Furthermore, in step (3), the volume ratio of diethylphosphoethyltriethoxysilane to acetic acid is 0.4:0.275.

[0019] Furthermore, in the preparation of the magnetic molecular imprinted polymer, the virtual template is metformin.

[0020] Furthermore, in the preparation of the magnetic molecular imprinting polymer, the solid-to-liquid ratio of MMS-P, dopamine, ammonium persulfate, metformin and water is 0.1 g: 50 mg: 50 mg: 0.1 g: 25 mL.

[0021] Furthermore, in the preparation of the magnetic molecular imprinted polymer, the mechanical stirring reaction is performed for 5-7 hours, and the ethanol washing is performed 2-3 times.

[0022] A detection method capable of simultaneously adsorbing multiple microcystin homologues, the specific steps are as follows:

[0023] Several 3 mg portions of dried magnetic virtual template molecular imprinting polymer were placed in 5 mL centrifuge tubes, and MCs solutions of different concentrations were prepared using water as the solvent. Then, 1 mL of algae toxin solution of different concentrations was added to each centrifuge tube. After oscillation for 20 min, a magnet was placed at the bottom of the centrifuge tube for magnetic separation. The supernatant was then aspirated with a syringe, filtered through a 0.22 μm organic filter membrane, and analyzed by high performance liquid chromatography, and the changes in peak areas were recorded.

[0024] Furthermore, the multiple algal toxins are microcystins MC-RR, MC-LR and MC-YR.

[0025] Further, the working conditions of the high performance liquid chromatography are:

[0026] Mobile phase: methanol solution and 0.1% trifluoroacetic acid aqueous solution, ratio 65:35 (V / V). Flow rate: 1mL / min. Injection volume: 50μL. Detector: UV detector, wavelength 238nm. Column temperature: 30℃. Injection time: 10min. Liquid chromatography column: column length 250mm, inner diameter 4.6mm, filler particle size 5μm.

[0027] Beneficial Effects

[0028] The present invention adopts a specific surface-localized molecular imprinting method to construct a magnetic molecular imprinting polymer (MMIPs) on magnetic nanoparticles with metformin as a virtual template, dopamine as a functional monomer and cross-linking agent, and ammonium persulfate as an initiator. The polymer has a specific adsorption capacity for MCs.

[0029] Magnetic molecular imprinted polymers (MMIPs) include a magnetic part and an imprinting layer part. The magnetic part is Fe 3 O 4 .Fe 3 O 4 Magnetic nanomaterials have the advantages of good stability and low toxicity, and can be completely separated from the adsorption system under the action of an external magnetic field. 3 O 4 Magnetic nanoparticles surface modified with SiO 2 and phosphate groups, which are provided by diethylphosphoethyltriethoxysilane. At the same time, phosphate groups have a positioning effect. During the adsorption process, the phosphate groups in the imprinted cavities will combine with the guanidine groups in the template molecules to enhance the adsorption capacity of MIPs on the template molecules. Metformin, which has a guanidine group similar to that of algae toxins, was used as a virtual template molecule. Since both the metformin simulation template and MCs contain guanidine groups, cavities similar to the MCs structure can be formed on the surface of silica microspheres through molecular imprinting and elution processes, thereby achieving the simultaneous adsorption of the three algae toxin homologues MC-LR, MC-RR, and MC-YR.

[0030] The invention has high sensitivity, good selectivity and simple operation, and can realize strong adsorption of various algae toxin homologues. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a schematic diagram of the synthesis of MMS-P@MIPs polymer in Example 1;

[0032] Figure 2 The transmission electron microscope (TEM) is used to examine the Fe 3 O 4 (A, B), morphology analysis of MMS (C, D) and scanning electron microscopy (SEM) image (E) and elemental analysis energy spectrum (F) of MMS-P@MIPs;

[0033] Figure 3 The infrared spectrum of Fe in Example 1 3 O 4 , MMS, and MMS-P were analyzed for their functional groups and chemical bonds;

[0034] Figure 4 Zeta potential for Fe in Example 1 3 O 4 , MMS, MMS-P, and MMS-P@MIP for stability analysis;

[0035] Figure 5 It is the fitting diagram of Langmuir and Freundlich isotherm adsorption model of the magnetic molecular imprinted polymer of Example 1 and Comparative Example 1 to microcystin-LR;

[0036] Figure 6 It is a fitting diagram of the kinetic adsorption model of the magnetic molecular imprinted polymer of Example 1 and Comparative Example 1 to microcystin-LR;

[0037] Figure 7 It is the fitting diagram of thermodynamic adsorption model of MMIPs in Example 1 and MNIPs in Comparative Example 1 to MC-LR;

[0038] Figure 8 The Langmuir and Freundlich isotherm adsorption model fitting diagrams of the magnetic molecular imprinted polymer of Example 1 and Comparative Example 1 for microcystin-RR;

[0039] Fig. 9 It is the fitting diagram of the kinetic adsorption model of the magnetic molecular imprinting polymer of Example 1 and Comparative Example 1 to microcystin-RR;

[0040] Fig.10 It is the fitting diagram of thermodynamic adsorption model of MMIPs in Example 1 and MNIPs in Comparative Example 1 for MC-RR;

[0041] Fig.11 It is the fitting diagram of the kinetic adsorption model of the magnetic molecular imprinted polymer of Example 1 and Comparative Example 1 to microcystin-YR;

[0042] Fig.12 Adsorption selectivity diagram of microcystins for MMIPs;

[0043] Fig.13 Reproducibility plot of adsorption of microcystins by MMIPs. DETAILED DESCRIPTION

[0044] The technical solution of the present invention is further described below in conjunction with specific embodiments, but is not limited thereto.

[0045] Example 1

[0046] A material synthesis capable of simultaneously adsorbing multiple microcystin homologues comprises the following steps:

[0047] Preparation of magnetic materials and preparation of magnetic molecular imprinted polymers;

[0048] The magnetic material is prepared by the following steps:

[0049] (1)Fe 3 O 4 Preparation: First weigh 1.35g of ferric chloride and 2.58g of sodium citrate into a 100mL beaker, add 80mL of distilled water, and sonicate to completely dissolve them. Then add 1g of urea and 0.6g of sodium polyacrylate, and place the beaker on a strong stirrer for 30 minutes. After the end, transfer the solution to a reactor and heat it in a vacuum drying oven at 200°C for 12 hours. After cooling to room temperature, wash the product with water and ethanol three times each, and finally dry it at 45°C for 12 hours and collect it for later use;

[0050] (2) Preparation of MMS: First weigh 0.5 g of prepared Fe 3 O 4 In a 100mL three-necked flask, add 0.6g urea and 1g hexadecyltrimethylammonium bromide, and add 30mL distilled water to form a suspension. Then add 1mL isopropanol and 30mL methane along the wall of the flask, and then add 120 microliters of tetraethyl orthosilicate every 6 minutes under vigorous mechanical stirring with a stirring paddle, a total of 1.2mL. Keep stirring and move the flask into a 70℃ water bath for 16 hours. After the reaction is completed, pour off the supernatant, add 0.6g ammonium nitrate and 60mL ethanol and reflux it at 60℃ overnight. After the reaction is completed, wash three times with distilled water, freeze-dry, and collect for later use;

[0051] (3) Preparation of MMS-P: 0.4 mL of diethylphosphoethyltriethoxysilane was placed in a 50 mL three-necked flask, 0.275 mL of acetic acid was added, and the mixture was refluxed in a water bath at 80°C for 6 hours for pre-hydrolysis. After cooling to room temperature, 0.5 g of MMS and 25 mL of toluene were added and refluxed for two hours. After the reaction was completed, the mixture was washed three times with methanol and ethanol respectively, dried under vacuum at 70°C, and collected for later use.

[0052] The molecularly imprinted polymer is prepared by the following steps:

[0053] Weigh 0.1g of the prepared MMS-P, add 50mg dopamine, 50mg ammonium persulfate, 0.1g metformin and 25mL water in sequence, stir mechanically to react for 5 hours, wash with ethanol three times after the reaction, and freeze-dry. Then, perform Soxhlet extraction, use ethanol as the washing liquid to elute the metformin virtual template molecule, and finally freeze-dry to obtain magnetic molecular imprinted polymers (MMIPs).

[0054] The Fe 3 O 4 (A, B), MMS (C, D) and MMS-P@MIPs were subjected to morphological analysis. Figure 2 As shown. Figure 2 A, 2B show that the synthesized Fe 3 O 4 The particles are spherical, uniform in size and shape, with an average diameter of about 100nm. As can be seen from Figures 2C and 2D, a layer of porous silica is successfully coated on the surface of the magnetic nanoparticles, and the thickness of the silicon layer is about 50nm. After coating the imprinting layer, the surface of MMS-P@MIPs presents a highly cross-linked morphology with an average diameter of 200nm (2E). The elemental distribution of MMS-P@MIPs was analyzed by energy spectrum (2F), which illustrates the distribution of Fe, Si, O and P elements on the surface of the nanoparticles, confirming the successful preparation of MMS-P@MIPs.

[0055] Using infrared spectroscopy, Fe 3 O 4 , MMS, and MMS-P were analyzed for their functional groups and chemical bonds. Figure 3 As shown, 588cm -1 The absorption peak near 1080cm is the stretching vibration absorption peak of Fe-O, indicating that the ferroferric oxide magnetic nanoparticles were successfully prepared in the first step of the polymer synthesis process; -1 The absorption peak at 1205cm is the asymmetric stretching vibration absorption peak of Si-O-Si, indicating that a layer of silicon dioxide is successfully coated on the surface of the ferroferric oxide magnetic nanoparticles. -1The absorption peak at is the stretching vibration absorption peak of PO, indicating that the phosphate groups are successfully modified on the surface of the ferroferric oxide magnetic nanomaterial.

[0056] Zeta potential of Fe 3 O 4 , MMS, MMS-P, MMS-P@MIP for stability analysis, such as Figure 4 Negative Zeta potential indicates that the material has a negative charge. Zeta potential is determined by Fe 3 O 4 To Fe 3 O 4 @SiO 2 -P@MIP becomes lower and lower during the process, indicating that the synthesized materials are becoming more and more stable.

[0057] Example 2

[0058] A material synthesis capable of simultaneously adsorbing multiple microcystin homologues comprises the following steps:

[0059] Preparation of magnetic materials and preparation of magnetic molecular imprinted polymers;

[0060] The magnetic material is prepared by the following steps:

[0061] (1)Fe 3 O 4 Preparation: First weigh 1.35g of ferric chloride and 2.58g of sodium citrate into a 100mL beaker, add 80mL of distilled water, and sonicate to completely dissolve them. Then add 1g of urea and 0.6g of sodium polyacrylate, and place the beaker on a strong stirrer for 30 minutes. After the end, transfer the solution to a reactor and heat it in a vacuum drying oven at 200°C for 12 hours. After cooling to room temperature, wash the product with water and ethanol three times each, and finally dry it at 50°C for 12 hours and collect it for later use;

[0062] (2) Preparation of MMS: First weigh 0.5 g of prepared Fe 3 O 4 In a 100mL three-necked flask, add 0.6g urea and 1g hexadecyltrimethylammonium bromide, and add 30mL distilled water to form a suspension. Then add 1mL isopropanol and 30mL methane along the wall of the flask, and then add 120 microliters of tetraethyl orthosilicate every 6 minutes under vigorous mechanical stirring with a stirring paddle, a total of 1.2mL. Keep stirring and move the flask into an 80℃ water bath for 16 hours. After the reaction is completed, pour off the supernatant, add 0.6g ammonium nitrate and 60mL ethanol and reflux it at 60℃ overnight. After the reaction is completed, wash three times with distilled water, freeze-dry, and collect for later use;

[0063] (3) Preparation of MMS-P: 0.4 mL of diethylphosphoethyltriethoxysilane was placed in a 50 mL three-necked flask, 0.275 mL of acetic acid was added, and the mixture was refluxed in a water bath at 80°C for 8 hours for pre-hydrolysis. After cooling to room temperature, 0.5 g of MMS and 25 mL of toluene were added and refluxed for two hours. After the reaction was completed, the mixture was washed three times with methanol and ethanol respectively, dried under vacuum at 70°C, and collected for later use.

[0064] The magnetic molecular imprinted polymer is prepared by the following steps:

[0065] Weigh 0.1g of the prepared MMS-P, add 50mg dopamine, 50mg ammonium persulfate, 0.1g metformin and 25mL water in sequence, stir mechanically to react for 7 hours, wash twice with ethanol after the reaction, freeze-dry. Then, perform Soxhlet extraction, use ethanol as the washing liquid to elute the metformin virtual template molecule, and finally freeze-dry to obtain magnetic molecular imprinting polymers (MMIPs).

[0066] Comparative Example 1

[0067] Preparation of magnetic non-molecularly imprinted polymers (MNIPs). Compared with Example 1, this comparative example is the same as the preparation process of MMIPs in Example 1 except that metformin is not added during the preparation of magnetic molecularly imprinted polymers.

[0068] Performance Testing

[0069] Effect of phosphate location on material adsorption

[0070] Taking MC-LR as an example, the changes in the adsorption amount of the material before and after phosphate positioning were explored.

[0071] Table 1 Effect of phosphate positioning on the adsorption performance of materials

[0072]

[0073] As shown in Table 1, the initial concentration of MC-LR is 1.00 μg / mL. After adsorption of the MMS@MIP material synthesized by the positioning strategy without diethylphosphoethyltriethoxysilane to provide phosphate groups, the residual concentration of MC-LR is 0.82 μg / mL, and the material adsorption amount is about 61.33 μg / g. After adsorption of the MMS-P@MIP material prepared in Example 1 synthesized by the positioning imprinting strategy, the residual concentration of MC-LR is only 0.05 μg / mL, and the material adsorption amount can be as high as 317.12 μg / g. It shows that the adsorption performance of the material can be significantly improved by virtue of the combination of phosphate-guanidine groups.

[0074] Study on the Adsorption Performance of Microcystin-LR by Magnetic Molecularly Imprinted Polymer

[0075] There are two mathematical models to characterize the adsorption performance of molecular imprinting polymers: adsorption thermodynamics and adsorption kinetics. There are two common mathematical models for adsorption thermodynamics: Langmuir and Freundlich. Langmuir mainly simulates the force of monolayer adsorption, while Freundlich can simulate the force of multilayer adsorption. There are two main models for adsorption kinetics: pseudo-first-order kinetics and pseudo-second-order kinetics. The pseudo-first-order kinetics model assumes that the adsorption rate is mainly affected by physical adsorption, and this physical adsorption is mainly due to diffusion. The pseudo-second-order kinetics model assumes that adsorption is affected by both physical adsorption and chemical adsorption, and the force between the adsorbent and the target molecule is mainly chemical adsorption.

[0076] (1) Static adsorption

[0077] Weigh several portions of 3 mg of the eluted MMIPs of Example 1 and MNIPs of Comparative Example 1, and add six portions of MMIPs to 1 mL of MC-LR standard solutions with concentrations of 1, 2, 3, 4, 5, and 6 μg / mL, respectively; add six portions of MNIPs to 1 mL of MC-LR standard solutions with concentrations of 0.8, 1, 1.5, 2, 2.5, and 3 μg / mL, respectively, and place in a constant temperature shaker at room temperature for 20 minutes of oscillation adsorption. After the adsorption is completed, the solvent is magnetically separated. Take a certain amount of the supernatant to detect the concentration of MC-LR by high performance liquid chromatography.

[0078] Table 2 Correlation coefficients of Langmuir and Freundlich adsorption equations

[0079]

[0080] Depend on Figure 5 It can be seen from Table 2 that at the same concentration, the adsorption amount of MMIPs is significantly higher than that of MNIPs. This is because during the experiment, the template molecule and MMIPs formed a cavity with matching size and structure, while MNIPs did not form a site with specific recognition ability for the template molecule because no virtual template molecule was added during synthesis. It can also be seen that the adsorption of magnetic molecular imprinted polymer on algae toxin-LR conforms to the Langmuir isotherm adsorption model, that is, the adsorption of the template molecule in the solvent by the imprinted polymer is more inclined to monolayer adsorption.

[0081] (2) Dynamic adsorption

[0082] Weigh 3 mg portions of the MMIPs of Example 1 and the MNIPs of Comparative Example 1 that have been completely eluted, add them to 1 mL of 2 μg / mL MC-LR standard solution, place them in a constant temperature shaker and shake at room temperature, and adsorb for 3, 5, 8, 10, 20, and 30 minutes, respectively. After the adsorption is completed, separate the solvent magnetically. Take a certain amount of the supernatant and use high performance liquid chromatography to detect the concentration of MC-LR.

[0083] Table 3 Kinetic fitting parameters

[0084]

[0085] Depend on Figure 6 As can be seen from Table 3, the adsorption of MC-LR by MMIPs and MNIPs increased rapidly within 5 minutes and then tended to be flat, indicating that in the initial stage of adsorption, there were a large number of specific recognition holes on the polymer surface, which made the adsorption rate faster. As time went on, the specific recognition sites gradually decreased and the adsorption rate decreased. At the same time, the adsorption rate of MC-LR by MNIPs was lower than that of MMIPs because the adsorption of template molecules by MNIPs relied on the nonspecific adsorption of polydopamine and had no specific binding sites for MMIPs. Finally, from the results in the chart, it can be seen that the adsorption of MC-LR by magnetic molecular imprinting polymer is more consistent with the quasi-second-order kinetic equation, that is, the adsorption of MC-LR molecular imprinting is affected by both the diffusion of physical adsorption and the interaction between the chemical adsorption adsorbent and the template molecule.

[0086] (3) Thermodynamic adsorption

[0087] Weigh multiple portions of 3 mg of eluted MMIPs of Example 1 and MNIPs of Comparative Example 1, and divide them into three groups. In one group, six portions of MMIPs were added to 1 mL of MC-LR standard solution with concentrations of 1, 2, 3, 4, 5, and 6 μg / mL, and six portions of MNIPs were added to 1 mL of MC-LR standard solution with concentrations of 0.8, 1, 1.5, 2, 2.5, and 3 μg / mL, respectively, and placed in a constant temperature shaker at 15°C for oscillation adsorption for 20 minutes; the concentrations of the algae toxin standard solutions of the other two groups were the same as those of the previous group, and were placed in a constant temperature shaker at 25°C and 35°C for 20 minutes for oscillation adsorption. After the adsorption was completed, the solvent was magnetically separated. A certain amount of the supernatant was taken to detect the concentration of MC-LR by high performance liquid chromatography.

[0088] Table 4 MC-LR adsorption thermodynamic parameters

[0089]

[0090] Depend on Figure 7 It can be seen that the thermodynamic adsorption of magnetic molecular imprinted polymer on MC-LR conforms to the Langmuir isotherm adsorption model, that is, the adsorption of the template molecule in the solvent by the imprinted polymer is more inclined to monolayer adsorption. Table 4 shows the thermodynamic parameters of the adsorption of MC-LR by magnetic molecular imprinted polymer, indicating that the adsorption reaction can proceed spontaneously.

[0091] Study on the Adsorption Performance of Microcystin-RR by Magnetic Molecularly Imprinted Polymer

[0092] (1) Static adsorption

[0093] Weigh several portions of 3 mg of the eluted MMIPs of Example 1 and MNIPs of Comparative Example 1, and add six portions of MMIPs to 1 mL of MC-RR standard solutions with concentrations of 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 μg / mL, respectively; add six portions of MNIPs to 1 mL of MC-RR standard solutions with concentrations of 0.1, 0.2, 0.25, 0.3, 0.35, and 0.4 μg / mL, respectively, and place in a constant temperature shaker at room temperature for 20 minutes of oscillation adsorption. After the adsorption is completed, the solvent is magnetically separated. Take a certain amount of the supernatant to detect the concentration of MC-RR by high performance liquid chromatography.

[0094] Table 5 Correlation coefficients of Langmuir and Freundlich adsorption equations

[0095]

[0096] Depend on Figure 8 As can be seen from Table 5, when the concentration is the same, the adsorption amount of MMIPs is significantly higher than that of MNIPs. It can also be seen that the adsorption of algae toxin-RR by magnetic molecular imprinting polymers conforms to the Langmuir isotherm adsorption model, that is, the adsorption of template molecules in the solvent by the imprinted polymer is more inclined to monolayer adsorption.

[0097] (2) Dynamic adsorption

[0098] Weigh 3 mg portions of the MMIPs of Example 1 and the MNIPs of Comparative Example 1 that have been completely eluted, add them to 1 mL of 1 μg / mL MC-RR standard solution, place them in a constant temperature shaker and shake at room temperature, and adsorb for 3, 5, 8, 10, 20, and 30 minutes, respectively. After the adsorption is completed, separate the solvent magnetically. Take a certain amount of the supernatant and use high performance liquid chromatography to detect the concentration of MC-RR.

[0099] Table 6 Kinetic fitting parameters

[0100]

[0101] Depend on Fig. 9 From Table 6, it can be seen that the adsorption of MMIPs and MNIPs on MC-RR increases rapidly within 10 min and then tends to be flat. At the same time, the adsorption rate of MNIPs on MC-RR is lower than that of MMIPs. Finally, from the results in the chart, it can be seen that the adsorption of MC-RR by magnetic molecular imprinting polymer is more consistent with the quasi-second-order kinetic equation, that is, the adsorption of MC-RR molecular imprinting is affected by both the diffusion of physical adsorption and the interaction force between the adsorbent and the template molecule of chemical adsorption.

[0102] (3) Thermodynamic adsorption

[0103] Weigh multiple portions of 3 mg of eluted MMIPs of Example 1 and MNIPs of Comparative Example 1, and divide them into three groups. In one group, six portions of MMIPs were added to 1 mL of MC-RR standard solutions with concentrations of 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 μg / mL, respectively, and six portions of MNIPs were added to 1 mL of MC-RR standard solutions with concentrations of 0.1, 0.2, 0.25, 0.3, 0.35, and 0.4 μg / mL, respectively, and placed in a constant temperature shaker at 15°C for oscillation adsorption for 20 minutes; the concentrations of the algae toxin standard solutions of the other two groups were the same as those of the previous group, and were placed in a constant temperature shaker at 25°C and 35°C for 20 minutes for oscillation adsorption. After the adsorption was completed, the solvent was magnetically separated. A certain amount of the supernatant was taken to detect the concentration of MC-RR by high performance liquid chromatography.

[0104] Table 7 MC-RR adsorption thermodynamic parameters

[0105]

[0106] Depend on Fig.10 It can be obtained that the thermodynamic adsorption of magnetic molecular imprinted polymer on algae toxin-RR conforms to the Langmuir isotherm adsorption model, that is, the adsorption of the imprinted polymer on the template molecule in the solvent is more inclined to monolayer adsorption. Table 7 shows the thermodynamic parameters of the adsorption of magnetic molecular imprinted polymer on MC-RR, indicating that the adsorption reaction can proceed spontaneously.

[0107] Study on the Adsorption Performance of Microcystin-YR by Magnetic Molecularly Imprinted Polymer

[0108] (1) Dynamic adsorption

[0109] Weigh 3 mg portions of the eluted MMIPs of Example 1 and the MNIPs of Comparative Example 1, respectively, and add them to 1 mL of a 1 μg / mL MC-YR standard solution, place them in a constant temperature shaker and shake at room temperature, and adsorb for 3, 5, 8, 10, 20, and 30 minutes, respectively. After the adsorption is completed, separate the solvent by magnetic separation. Take a certain amount of the supernatant and use high performance liquid chromatography to detect the concentration of MC-YR.

[0110] Table 8 Kinetic fitting parameters

[0111]

[0112]

[0113] Depend on Fig.11It can be seen from Table 8 that the adsorption of MMIPs and MNIPs on MC-YR increases rapidly within 10 min and then tends to be flat. At the same time, the adsorption rate of MNIPs on MC-YR is lower than that of MMIPs. Finally, it can be seen from the results in the chart that the adsorption of MC-YR by magnetic molecular imprinting polymer is more in line with the pseudo-second-order kinetic equation, that is, the adsorption of MC-YR molecular imprinting is affected by both the diffusion of physical adsorption and the interaction between the adsorbent and the template molecule of chemical adsorption.

[0114] (2) According to the results of static adsorption and thermodynamic adsorption studies on algae toxin-LR and algae toxin-RR, it can be seen that both algae toxins are monolayer adsorbed and the adsorption reaction can proceed spontaneously. Algae toxin-YR is a homologue of algae toxin-LR and algae toxin-RR, so algae toxin-YR also conforms to a similar adsorption principle.

[0115] Study on adsorption selectivity and reproducibility of MMIPs

[0116] The most prominent feature of MMIPs is their ability to specifically select and recognize target molecules, which is an important indicator for evaluating the performance of imprinted materials. Taking MC-LR as an example, the interference selectivity of MMIPs was explored by adding common ions and substances such as aflatoxin and domoic acid to the target solution to be tested. Weigh 3 mg of the completely eluted Example 1 MMIPs and add them to 1 mL of MC-LR standard solution with a concentration of 1 μg / mL. At the same time, add interfering ions of different concentration multiples and place them in a constant temperature shaker to oscillate at room temperature. After the adsorption is completed, magnetically separate the solvent and take a certain amount of supernatant to detect the concentration of MC-LR by high performance liquid chromatography. Fig.12 As shown in the figure, after adding these interfering ions and interfering substances, there was no obvious signal change when MMIPs interacted with MC-LR, indicating that MMIPs have good anti-interference ability. This study also explored the reproducibility of MMIPs ( Fig.13 ). Adsorption tests were conducted on five different batches of materials, and it was found that the adsorption amount of MC-LR did not vary much, indicating that the synthesis process of MMIPs has good reproducibility.

[0117] Microcystin adsorption determination of real samples

[0118] Take a certain volume of lake water sample and centrifuge it, then filter it with a 0.22μm organic filter membrane to remove impurities in the lake water sample. Use the treated actual water sample as solvent to prepare 300μg / L MC-LR solution, 300μg / L MC-RR solution, 300μg / L MC-YR solution and a mixed solution of three algae toxins at a concentration of 300μg / L, then weigh four portions of 3mg MMIPs in Example 1, take 1mL of each of the above four solutions and oscillate and adsorb them for 20min, magnetically separate after adsorption, and take the supernatant. Finally, use a liquid chromatograph to detect the actual water sample, 300μg / L MC-LR solution, 300μg / L MC-RR solution, 300μg / L MC-YR solution, 300μg / L mixed solution of three algae toxins, and the supernatant after adsorption of the above four solutions. Each group of experiments was repeated 4 times, and the results were averaged. The adsorption results are shown in the following table.

[0119] Table 9 Determination results of MC-LR, MC-YY and MC-YR contents in water samples

[0120]

[0121]

[0122] The test results are shown in Table 9. The adsorption rate of the magnetic molecular imprinted polymer of the present invention to MC-LR is as high as 92.55%, the adsorption rate to MC-RR is as high as 79.20%, and the adsorption rate to MC-RR is as high as 86.82%. In addition, in the whole algae toxin adsorption determination process, the adsorption rate of the magnetic imprinted polymer of the present invention to MC-LR is 90-95%, the adsorption rate to MC-RR is 75-79%, and the adsorption rate to MC-YR is 85-90%. The above data show that the magnetic molecular imprinted polymer of the present invention has high selectivity for a variety of algae toxins and high adsorption performance.

[0123] It should be noted that the above embodiments are only partial embodiments of the preferred methods of implementing the present invention, rather than all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

Claims

1. A method for preparing a magnetic molecular imprinted polymer for adsorbing multiple microcystin homologues, characterized in that: The following steps are involved: Preparation of magnetic materials and preparation of magnetic molecular imprinted polymers; The preparation of the magnetic material comprises the following steps: (1) Preparation of Fe3O4: First, weigh 1.35g of ferric chloride and 2.58g of sodium citrate into a 100mL beaker, add 80mL of distilled water, and sonicate to completely dissolve them. Then add 1g of urea and 0.6g of sodium polyacrylate, and place the beaker on a powerful stirrer for 30 minutes. After the end, transfer the solution to a reactor and place it in a vacuum drying oven. Heat at 200°C for 12 hours. After cooling to room temperature, wash the product with water and ethanol three times each, and finally dry for 12 hours and collect for later use; (2) Preparation of ferroferric oxide-coated silica material - MMS: First weigh 0.5g of the prepared Fe3O4 into a 100mL three-necked flask, add 0.6g of urea and 1g of hexadecyltrimethylammonium bromide, and add 30mL of distilled water to form a suspension. Then add 1mL of isopropanol and 30mL of methane along the wall of the flask, and then add 120 microliters of tetraethyl orthosilicate every 6 minutes under vigorous mechanical stirring with a stirring paddle, a total of 1.2mL. Keep stirring and move the flask into a water bath for 16 hours. After the reaction is completed, pour off the supernatant, add 0.6g of ammonium nitrate and 60mL of ethanol and reflux it overnight. After the reaction is completed, wash three times with distilled water, freeze-dry, and collect for later use; (3) Preparation of phosphoric acid functionalized Fe3O4@SiO2-MMS-P: Diethylphosphoethyltriethoxysilane was placed in a 50 mL three-necked flask, acetic acid was added and the flask was refluxed in a water bath to pre-hydrolyze. After cooling to room temperature, 0.5 g MMS and 25 mL toluene were refluxed for two hours. After the reaction, they were washed with methanol and ethanol three times respectively, dried under vacuum at 70 °C, and collected for later use; The preparation of the magnetic molecular imprinted polymer-MMIPs comprises the following steps: The prepared MMS-P was weighed, dopamine, ammonium persulfate, virtual template and water were added in sequence, mechanically stirred, washed with ethanol after the reaction was completed, and freeze-dried. Then, the metformin virtual template molecules were eluted by Soxhlet extraction with ethanol as the washing liquid, and finally freeze-dried to obtain the magnetic molecular imprinting polymer.

2. The method for preparing a magnetic molecular imprinted polymer for adsorbing multiple microcystin homologues according to claim 1, characterized in that: In the step (1), the drying condition of the product after washing is 45°C-50°C.

3. The method for preparing a magnetic molecular imprinted polymer for adsorbing multiple microcystin homologues according to claim 1, characterized in that: In step (2), the water bath temperature is 70°C-80°C, and the overnight reflux temperature is 60°C.

4. The method for preparing a magnetic molecular imprinted polymer for adsorbing multiple microcystin homologues according to claim 1, characterized in that: In step (3), the water bath is at 80° C. and refluxed for 6-8 hours.

5. The method for preparing a magnetic molecular imprinted polymer for adsorbing multiple microcystin homologues according to claim 1, characterized in that: In the step (3), the volume ratio of diethylphosphoethyltriethoxysilane to acetic acid is 0.4:0.

275.

6. The method for preparing a magnetic molecular imprinted polymer for adsorbing multiple microcystin homologues according to claim 1, characterized in that: In the preparation of the magnetic molecular imprinted polymer, the reaction is carried out by mechanical stirring for 5-7 hours, and the ethanol washing is performed 2-3 times.

7. The method for preparing a magnetic molecular imprinted polymer for adsorbing multiple microcystin homologues according to claim 1, characterized in that: The virtual template in the preparation of the magnetic molecular imprinting polymer is metformin.

8. The method for preparing a magnetic molecular imprinted polymer for adsorbing multiple microcystin homologues according to claim 1, characterized in that: In the preparation of the magnetic molecular imprinting polymer, the solid-liquid ratio of MMS-P, dopamine, ammonium persulfate, metformin and water is 0.1 g: 50 mg: 50 mg: 0.1 g: 25 mL.

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