Molecularly imprinted magnetic inverse opal photonic crystal microsphere capable of specifically recognizing zearalenone as well as preparation method and application of molecularly imprinted magnetic inverse opal photonic crystal microsphere

Abstract

The invention discloses a molecularly imprinted magnetic inverse opal photonic crystal microsphere capable of specifically recognizing zearalenone as well as a preparation method and application of the molecularly imprinted magnetic inverse opal photonic crystal microsphere. A false template similar to the zearalenone in structure is introduced into the microsphere, and the magnetic inverse opal photonic crystal microsphere is used as a substrate; a molecular imprinting layer capable of specifically recognizing zearalenone is modified on the surface of the microsphere. The microspheres prepared by the invention have very high imprinting factors on ZEN, and the enrichment specificity of ZEN is greatly improved. The novel magnetic molecular imprinting photonic crystal microsphere based on the inverse opal structure is synthesized through a false template strategy and a surface molecular imprinting technology, the novel magnetic molecular imprinting photonic crystal microsphere shows unique technical advantages in the field of ZEN separation and detection, and efficient enrichment of target toxin ZEN is achieved through the selective adsorption characteristic of a periodic pore channel structure; by combining the high separation efficiency of high performance liquid chromatography, an analysis system integrating separation and detection is constructed.

Description

Technical Field

[0001] The present invention belongs to the field of separation science, and in particular relates to a molecularly imprinted magnetic inverse opal photonic crystal microsphere capable of specifically identifying zearalenone, and a preparation method and application thereof. Background Art

[0002] Globally, zearalenone (ZEN) contamination has become a common problem affecting grain safety. Zearalenone, abbreviated as ZEN, also known as F-2 toxin, is a fungal secondary metabolite with estrogen-like activity. 18 H 22 O. Zearalenone, due to its molecular structure similar to 17β-estradiol, can interfere with the normal function of the endocrine system by competitively binding to estrogen receptors. It can compete with endogenous estrogens for receptor binding sites, activate the transcriptional activity of downstream estrogen response elements, and induce pathological changes in the reproductive system. Epidemiological studies have confirmed that ZEN exposure is significantly associated with a variety of estrogen-related diseases, including menstrual disorders in women, precocious puberty in children, and reproductive system diseases such as endometriosis. In order to ensure the safety of the entire grain product chain, it is necessary to establish a complete zearalenone (ZEN) prevention and control system to accurately detect ZEN in various links such as grain production, transportation, and sales. Therefore, it is necessary to establish a pretreatment technology with strong anti-interference and high selectivity to achieve the detection and analysis of the target object.

[0003] Currently, the main pretreatment technologies for ZEN in samples include liquid-liquid extraction, immunoaffinity columns, and solid-phase extraction. However, liquid-liquid extraction is often a tedious process that requires a large amount of organic solvents, while immunoaffinity columns are subject to certain limitations due to their high production costs, sensitivity to environmental factors, easy degradation and denaturation, and inability to be reused. Solid-phase extraction is further divided into non-selective solid-phase extraction and selective solid-phase extraction. The former has low selectivity and is difficult to be used efficiently for the extraction and enrichment of trace target analytes in complex samples. The latter can improve the effectiveness of the method by selecting different adsorption materials. Among them, antibody-based adsorption materials have strong specificity, but the acquisition of antibodies is time-consuming and expensive. Therefore, it is necessary to develop a solid-phase extraction pretreatment material that is simple to operate, highly specific, and low-cost for the separation and enrichment of ZEN in samples.

[0004] Molecular imprinting technology (MIP) simulates the interaction between antigens and antibodies. It forms a rigid polymer, known as a molecularly imprinted polymer, by combining target analyte template molecules with corresponding functional monomers in a porogen under the action of a crosslinker. After the template is removed by an eluent, the polymer remains highly crosslinked. Due to its advantages such as predictable structure, identification, structural stability, high enrichment capacity, and reusability, it is widely used in chromatographic separations, sensors, and solid-phase extraction. Summary of the Invention

[0005] Purpose of the invention: In response to the problems existing in the prior art, the present invention provides a molecularly imprinted magnetic inverse opal photonic crystal microsphere that can specifically identify zearalenone (ZEN). The imprinting factor of these microspheres for ZEN is very high, which greatly improves the enrichment specificity of ZEN. Compared with other materials for modifying biological antibodies, the stability of these microspheres is improved and the production cost is greatly reduced.

[0006] The present invention also provides a preparation method and application of the molecularly imprinted magnetic inverse opal photonic crystal microspheres capable of specifically identifying zearalenone (ZEN).

[0007] The present invention also provides a preparation method and application of the magnetic inverse opal structure-based colloidal crystal hydrogel microspheres.

[0008] Technical solution: In order to achieve the above-mentioned purpose, the present invention describes a molecularly imprinted magnetic inverse opal photonic crystal microsphere that can specifically identify zearalenone. The microsphere introduces a pseudo-template similar to the structure of zearalenone, uses magnetic inverse opal photonic crystal microspheres as a substrate, and modifies the surface of the microsphere with a molecular imprinting layer that can specifically identify zearalenone.

[0009] Wherein, the pseudo template having a structure similar to ZEN is curcumin, quercetin or 5,7-dimethylcoumarin.

[0010] Among them, the molecular imprinting layer is synthesized with a pseudo template molecule similar to the ZEN structure. After the template is removed by introducing functional monomers and reacting with cross-linkers, an imprinting cavity similar to the ZEN structure with specific recognition function is formed on the surface of the microsphere.

[0011] Among them, the functional monomer is 2,33-aminopropyltriethoxysilane, and the crosslinking agent is ethyl orthosilicate. Three functional monomers are used in the present invention, namely 3-aminopropyltriethoxysilane (APTES), methacrylic acid (MAA) and acrylamide (AM). The latter two cannot successfully prepare polymers.

[0012] The method for preparing molecularly imprinted magnetic inverse opal photonic crystal microspheres capable of specifically recognizing zearalenone according to the present invention comprises the following steps:

[0013] (1) Preparation of magnetic inverse opal photonic crystal microspheres and modification of hydroxyl groups: Microspheres were prepared by microfluidic self-assembly. Based on the water-in-oil principle, methyl silicone oil was used to cut the mixed emulsion into uniform small droplets. The desired microspheres were obtained after calcination. The microspheres were treated with a mixed solution of hydrogen peroxide and concentrated sulfuric acid to complete the modification of hydroxyl groups on the surface of the microspheres.

[0014] (2) Modification of the molecular imprinting layer of the magnetic inverse opal photonic crystal microspheres: taking the hydroxyl-modified photonic crystal microspheres prepared in step (1), adding a template molecule, a porogen, a functional monomer, and a cross-linking agent and reacting them, and removing the template after the reaction is completed, thus completing the modification of the molecular imprinting layer on the surface of the microspheres to obtain molecularly imprinted magnetic inverse opal photonic crystal microspheres that can specifically recognize zearalenone.

[0015] The mixed emulsion in step (1) is obtained by subjecting a polystyrene nanoparticle solution, a silicon dioxide nanoparticle solution and a ferroferric oxide nanoparticle solution to ultrasonic, vortex and centrifugal operations, adding double distilled water, and diluting the three solutions and then mixing them evenly.

[0016] Wherein, in step (1), the flow rate of methyl silicone oil is 8-10 mL / h, and the flow rate of the mixed emulsion is 5-8 mL / h; the calcination temperature is set to 700-800°C, and the calcination time is 3-4 hours; the modification time is 6-8 hours, the temperature is 25-30°C, and the rotation speed is 160-200 rpm.

[0017] Preferably, the flow rates of the two solutions in step (1) are: methyl silicone oil 8 mL / h, mixed emulsion 5 mL / h; the calcination temperature in step (1) is set to 700°C, and the calcination time is 3 hours; the modification time in step (1) is 6 hours, the temperature is 25°C, and the rotation speed is 160 rpm.

[0018] Wherein, in step (2), the porogens are ethanol and water, and the molar ratio of the template, the functional monomer and the cross-linking agent is 1:3-4:8-10.

[0019] Wherein, in step (2), the porogen is ethanol and water, the ratio of the two is 1:2, and the molar ratio of the template, the functional monomer and the cross-linking agent is 1:4:10.

[0020] Wherein, the reaction time in step (2) is 20-24 hours, the temperature is 25-30° C., and the rotation speed is 150-200 rpm.

[0021] Preferably, the reaction time in step (2) is 24 hours, the temperature is 25° C., and the rotation speed is 200 rpm.

[0022] Application of the magnetic inverse opal photonic crystal microspheres of the present invention in the specific adsorption of zearalenone.

[0023] The molecularly imprinted magnetic inverse opal photonic crystal microspheres prepared by the present invention have an imprinting factor on ZEN of up to 39.7.

[0024] Specifically, the preparation method of surface-modified molecularly imprinted magnetic inverse opal photonic crystal microspheres described in the present invention mainly includes two steps, namely, synthesizing a silica-imprinted polymer of zearalenone using a pseudo-template strategy and synthesizing magnetic inverse-structured photonic crystal microspheres (MIPCMs) with a three-dimensional ordered porous structure through a microfluidic self-assembly platform.

[0025] The specific steps are as follows:

[0026] (1) Synthesis of zearalenone-based silica-based molecularly imprinted polymers using a pseudo-template strategy: The pseudo-template molecule was placed in a beaker, and a porogen, functional monomer, cross-linker, and initiator were added in sequence. The mixture was placed on a magnetic stirrer for reaction. After the reaction, the mixture was centrifuged and the template molecule was washed with an eluent to obtain a molecularly imprinted polymer (MIP). The preparation of non-molecularly imprinted polymers (NIPs) was similar to that of MIPs, except that no pseudo-template molecule was added.

[0027] (2) Preparation and modification of magnetic inverse opal photonic crystal microspheres: Using a mixed emulsion and methyl silicone oil, uniform microspheres were formed through microfluidic self-assembly based on the water-in-oil principle. After curing, drying, and high-temperature calcination, magnetic inverse opal photonic crystal microspheres (MIPCMs) with a three-dimensional ordered porous structure were obtained. Piranha solution was added to the MIPCMs to modify the surface of the magnetic inverse opal photonic crystal microspheres with hydroxyl groups, facilitating subsequent modification of the molecular imprinting layer.

[0028] (3) Preparation of surface molecularly imprinted magnetic inverse structure photonic crystal microspheres: Take the MIPCMs modified with perhydroxyl groups in step (2), add pseudo-template molecules, porogens, functional monomers, and cross-linking agents in sequence, place them on a magnetic stirrer for sufficient reaction, discard the remaining solution after the reaction, and wash off the template molecules to obtain surface molecularly imprinted magnetic inverse opal photonic crystal microspheres.

[0029] Wherein, the pseudo-template molecule in step (1) is curcumin, the porogens are ethanol and water, the functional monomer is 3-aminopropyltriethoxysilane, the cross-linking agent is ethyl silicate, and the initiator is acetic acid or ammonia water.

[0030] Preferably, in step (1), the rotation speed of the magnetic stirrer is 800 rpm, the stirring temperature is 25° C., and the stirring time is 48 hours.

[0031] Preferably, when the template molecules are eluted by centrifugation in step (1), the eluent is ethanol.

[0032] Preferably, the porogen in step (1) and step (3) is a mixed solution of ethanol and water (volume ratio is 1:1).

[0033] The microspheres of the present invention are first synthesized by microfluidic self-assembly method into magnetic inverse opal photonic crystal microspheres, the surface of the microspheres is modified with hydroxyl groups, and then a molecular imprinting polymer imprinting layer capable of specifically recognizing ZEN is modified on the surface for the specific enrichment and separation of ZEN.

[0034] Design mechanism: The present invention adopts the self-assembly method to synthesize magnetic inverse structure stone photonic crystal microspheres, and combines the molecular imprinting technology to achieve the selective separation and enrichment of zearalenone. First, three pseudo-templates with similar structures to ZEN and easy to obtain are selected, namely curcumin, quercetin and 5,7-dimethylcoumarin. Secondly, functional monomers are added to form a stable pre-assembled system with the template molecules through directional intermolecular interactions. Finally, a cross-linking agent is added to fix the functional groups of the functional monomers within a stable range, and to combine with the template molecules through hydrogen bonds around the template molecules. It also ensures that the structure of the polymer does not change after the template is eluted, leaving an effective hole structure on the molecular imprinting surface. After the polymerization is completed, the template is eluted with an organic solvent, so that an imprinted cavity similar to ZEN will be formed on the surface of the polymer, which has the ability to selectively recognize ZEN.

[0035] This method uses a self-assembly method to synthesize magnetic inverse opal photonic crystal microspheres (MIPCMs). After modifying the MIPCM surface with hydroxyl groups, amino active sites are introduced onto the microsphere surface by adding functional monomers. Template molecules are then added to form a pre-bound complex with the functional monomers. A cross-linking agent is then introduced to construct a molecularly imprinted layer with specific recognition capabilities on the microsphere surface. After magnetic separation, the template molecules are removed using gradient elution, ultimately yielding a MIPCMs@MIP composite material that can specifically recognize ZEN.

[0036] This invention utilizes a pseudotemplate strategy and surface molecular imprinting technology to synthesize a novel magnetic molecularly imprinted photonic crystal microsphere based on an inverse opal structure, demonstrating unique technical advantages in the field of mycotoxin separation and detection. This material achieves efficient enrichment of target toxins through the selective adsorption properties of its periodic pore structure. Combined with the high separation efficiency of high-performance liquid chromatography, this material creates an integrated analytical system for separation and detection.

[0037] The present invention proposes the preparation of high-zearalenone molecular imprinted polymers. Compared with other toxins, the adsorption imprinting factor for ZEN is generally lower. Therefore, the present invention explores the effect of molecular imprinted polymers synthesized under different conditions on the adsorption of ZEN, screens the optimal conditions, and modifies them onto magnetic inverse opal photonic crystal microspheres under these conditions. The present invention proves that the microspheres prepared based on this method have a significantly higher imprinting factor for ZEN adsorption than other inventions. Currently, existing research on the detection of zearalenone molecular imprinting technology has bottlenecks such as the adsorption effect being far lower than that of other toxins. The present invention increases the imprinting factor for ZEN adsorption to 39.7, significantly improving the adsorption effect.

[0038] Compared with liquid-liquid extraction, the material prepared by the present invention requires less organic solvent and a less complicated process for ZEN adsorption; compared with immunoaffinity columns, it has low production costs, low environmental requirements, and can be reused; compared with solid-phase extraction, it has high selectivity; compared with antibody-based adsorption materials, it has significantly lower costs; and compared with other molecular imprinting methods, it has better specific adsorption effects and significantly improved imprinting factors.

[0039] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0040] (1) Structural analogs of zearalenone were selected as pseudo-template molecules to prepare ZEN molecularly imprinted polymers, which reduced the cost while reducing the harm of toxins to the human body.

[0041] (2) The molecular imprinting polymers of ZEN were prepared by molecular imprinting technology, which can achieve the selective enrichment and separation of ZEN.

[0042] (3) The microsphere synthesis process is simple, the production cost is low, the production process is safe and harmless, and it can be used in large-scale industrial production.

[0043] (4) The prepared magnetic inverse opal photonic crystal microspheres have stable performance and can be stored and transported at room temperature without the need for special storage methods.

[0044] (5) The imprinting factor of the prepared microspherical magnetic inverse opal photonic crystal microspheres for ZEN adsorption is significantly improved compared with the existing reports, greatly improving the enrichment efficiency of ZEN. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Scanning electron microscopy characterization of MIPs and NIPs prepared using curcumin as a pseudo-template;

[0046] Figure 2 Comparison of the adsorption properties of different template molecules to their own template molecules;

[0047] Figure 3 The effect of porogen ratio on the adsorption performance of MIP and NIP;

[0048] Figure 4 The effects of different ratios of template, functional monomer and cross-linker on the adsorption properties of MIP and NIP;

[0049] Figure 5 The effect of the adsorption solvent ratio on the adsorption performance of MIP and NIP;

[0050] Figure 6 is the effect of initiator on the adsorption performance of MIP and NIP;

[0051] Figure 7 Scanning electron microscopy and metallographic microscopy characterization of magnetic inverse opal photonic crystal microspheres;

[0052] Figure 8 Scanning electron microscopy and metallographic microscopy characterization of MIPCMs surface modified with molecularly imprinted polymers and non-molecularly imprinted polymers;

[0053] Figure 9 Static adsorption experiments of ZEN by MIPCMs@MIP and MIPCMs@NIP;

[0054] Figure 10 Dynamic adsorption experiments of ZEN on MIPCMs@MIP and MIPCMs@NIP. DETAILED DESCRIPTION

[0055] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0056] The experimental methods described in the examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0057] The particle size of the Fe3SO4 dispersion was 10-50 nm and was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0058] Silica (SiO2) nanoparticles with a particle size of 5 nm were purchased from Sigma-Aldrich Trading Co., Ltd.;

[0059] Polystyrene nanospheres (PS) were purchased from Nanjing Caina Biotechnology Co., Ltd., CAS9006-53-6.

[0060] Example 1

[0061] (1) Synthesis of molecularly imprinted polymers (MIPs)

[0062] Place 0.1 mmol of curcumin in a beaker, add 4 mL of ethanol and 8 mL of water, sonicate for 10 minutes, then place the beaker on an 800 rpm magnetic stirrer and stir at room temperature for 10 minutes. After stirring, add 0.4 mmol of APTES to the beaker, sonicate for 10 minutes, and continue stirring for 30 minutes. Finally, add 1 mmol of TEOS, sonicate for 10 minutes, and continue stirring for 48 hours. If an initiator is needed, add 4 mmol / L acetic acid or ammonia 10 minutes after adding the crosslinker.

[0063] The preparation process of non-molecularly imprinted polymer (NIP) was the same as above, except that no template molecules were added.

[0064] (2) Removal of template molecules

[0065] The polymer prepared above was placed in a centrifuge and centrifuged at 10,000 rpm for 10 minutes. The resulting supernatant was discarded, and ethanol was added to the remaining solid. After ultrasonic dispersion, the mixture was evenly centrifuged at 10,000 rpm for 10 minutes. The supernatant was discarded, and the remaining solid was further washed with ethanol. This washing process was repeated 2-3 times, followed by another 2-3 washes with purified water. The supernatant after centrifugation was observed until the solution was colorless. The supernatant from the final wash was tested with a UV spectrophotometer. The absence of template molecules confirmed that the residual template molecules in the polymer had been washed clean.

[0066] Example 2

[0067] Based on the molecularly imprinted polymer synthesized in Example 1, the effects of varying synthesis conditions on polymer adsorption were investigated. Following the methods of steps 1 and 2 above, the effects of varying template molecules, the ratio of template molecules to crosslinkers and functional monomers, and the ratio of porogens to adsorption solvents on the adsorption capacity of the synthesized molecularly imprinted polymer were investigated.

[0068] The adsorption process is as follows: 1 mL of a 500 ng / mL template molecule standard solution is added to each MIP and NIP in a centrifuge tube. The tube is shaken at 200 rpm for 10 hours. After 10 hours, the polymer and template molecules are completely adsorbed. After adsorption is complete, the tube is centrifuged at 10,000 rpm for 10 minutes, and the supernatant is collected for measurement and calculation of its concentration.

[0069] The calculation formula of adsorption amount Q is as follows:

[0070] Where C0 is the initial concentration of the solution in μmol / L, C1 is the concentration in the supernatant after adsorption in μmol / L, V is the initial solution volume, and M is the weight of the polymer. Q is in nmol / mg.

[0071] The calculation formula of imprinting factor IF is as follows:

[0072] Among them, Q MIP is the adsorption of the target by MIP, the unit is nmol / mg, Q NIP is the adsorption of NIP to the target compound, and the unit is nmol / mg.

[0073] (1) Template molecule

[0074] 0.1 mmol of each of curcumin, quercetin, and coumarin was weighed into a beaker. 4 ml of ethanol and 8 ml of water were added. After sonication for 10 minutes, the beaker was placed on a magnetic stirrer at 800 rpm and stirred at room temperature for 10 minutes. After stirring, 0.2 mmol of APTES was added to the beaker. After sonication for 10 minutes, stirring was continued for another half hour. Finally, 1 mmol of TEOS was added. After sonication for 10 minutes, stirring was continued for 48 hours. Three MIPs were finally obtained.

[0075] The preparation process of NIP was the same as above, except that no template molecule was added.

[0076] The adsorption effects of the three prepared molecularly imprinted polymers on themselves are as follows Figure 1 As shown, the imprinting factors of the MIPs synthesized using curcumin, quercetin, and coumarin for their own template molecules were 1.64, 1.37, and 1.09, respectively (the imprinting factor is the ratio of the amount of template molecule adsorbed by the molecularly imprinted polymer to the amount of template molecule adsorbed by the non-molecularly imprinted polymer); the template utilization rates were 69%, 63%, and 48%, respectively, and the yields were 53.2, 42.1, and 15.3 mg, respectively. Overall, the molecularly imprinted polymers synthesized using curcumin as a pseudo-template molecule exhibited good adsorption performance and high yield, so curcumin was selected as the template molecule for subsequent experiments. The template molecule adsorption performance was tested as follows: the polymer and template molecule were mixed in an adsorption solvent and shaken at room temperature overnight. The supernatant was then collected to measure the template molecule concentration, i.e., the template molecule concentration in the solution after adsorption. The adsorption capacity of the polymer was calculated based on the difference in template molecule concentration before and after adsorption.

[0077] (2) Porogen ratio

[0078] During the polymerization process, the reaction solvent acts as a porogen. The choice of reaction solution has a very important influence on the generation of the polymerization reaction and the performance of the prepared polymer. The type and ratio of the reaction solvent will directly affect the density of the polymer structure, the formation of the surface pore structure, and the adsorption capacity of the polymer to the template molecule. According to the method in the above step (1), the porogen ratio of ethanol: water (volume ratio) was selected as 1:0, 2:1, 1:1, 1:2 and 0:1 respectively, and different molecular imprinting polymers were synthesized, and their adsorption properties were investigated. The results are as follows Figure 2 As shown in Figure 2, the imprinting factor is highest at 1.68 when the ethanol:water ratio is 1:2, and the MIP adsorption capacity is also high at this time. Therefore, a 1:2 ethanol:water (volume ratio) porogen was selected for subsequent experiments.

[0079] (3) Ratio of template, functional monomer and cross-linker

[0080] Template molecules, functional monomers and cross-linking agents are three essential elements for the preparation of molecularly imprinted polymers. By changing the ratio of the three, the effect of their ratio on the adsorption of the target molecule was explored. The adsorption performance of the polymer was investigated when the ratio of template, functional monomer and cross-linking agent was 1:1:10, 1:2:10, 1:4:10, 1:2:5 and 1:2:20. The results are as follows Figure 3 As shown in the figure, by comparing the three data sets with a ratio of 1:1:10, 1:2:10, and 1:4:10, it can be found that when the ratio of template to cross-linker remains unchanged, the higher the content of functional monomer, the greater the adsorption of curcumin by MIP and NIP, and the higher the imprinting factor. By comparing the three data sets with a ratio of 1:4:10, 1:4:5, and 1:4:20, it can be found that the higher the ratio of cross-linker, the lower the adsorption of polymer and the weaker the adsorption capacity. When the ratio of template molecule, functional monomer, and cross-linker is 1:4:10, MIP has a larger adsorption capacity for curcumin and the highest imprinting factor of 1.511. Therefore, the ratio of 1:4:10 was finally selected for subsequent experiments.

[0081] (4) Ratio of adsorption solvent

[0082] In order to ensure that the template molecules have good solubility in the solution, the adsorption solution was selected as an ethanol-water solution. Solutions with ethanol:water (v / v) ratios of 1:0, 6:1, 3:1, 2:1, 1:1, 1:2 and 0:1 were prepared respectively. A certain amount of curcumin was added to make the final solution concentration 50μmol / L. The results of the effect of the adsorption solvent ratio on the polymer adsorption amount are shown in the figure. Figure 4 As can be seen in the figure, the adsorption capacity of MIP and NIP generally decreases with decreasing ethanol content. This is because curcumin is insoluble in water but soluble in ethanol. Therefore, it can be completely dissolved and evenly dispersed in solvents with high ethanol content, facilitating site-specific binding of the target molecule to the imprinted polymer surface. The best imprinting solvents were ethanol:water (v / v) ratios of 6:1 and 3:1, with the 6:1 ratio producing the highest adsorption capacity. Therefore, ethanol:water (v / v) = 6:1 was selected as the adsorption solvent for subsequent experiments.

[0083] (5) Selection of initiator

[0084] Initiators not only alter the reaction rate but can also affect the yield and performance of the synthesized product. The present invention uses acetic acid and aqueous ammonia as initiators to create acidic and alkaline conditions, with a catalyst concentration of 4 mmol / L. As shown in the figure, the addition of the alkaline initiator slightly increases the amount of curcumin adsorbed by the MIP, while the addition of the acidic initiator results in little change in the amount of curcumin adsorbed by the MIP. Figure 5The results showed that the imprinting factor was much lower after adding initiator than without initiator, so no initiator was selected as the optimal condition for synthesizing molecularly imprinted polymers.

[0085] Example 3

[0086] The MIP and NIP prepared under the optimal conditions were characterized by scanning electron microscopy. Figure 6 As shown, the surface of the molecularly imprinted polymer (MIP) exhibits distinct porous morphology. This three-dimensional network structure confirms the successful removal of the template molecules, and the resulting specific cavities can effectively recognize the target molecules. In contrast, the surface structure of the non-imprinted polymer (NIP) is dense and flat, lacking specific binding sites. This morphological difference verifies the effectiveness of molecular imprinting technology at the microscale.

[0087] Example 4

[0088] Preparation of magnetic inverse opal photonic crystal microspheres: 3 mL of a 20% SiO₂ solution (mass fraction) was added to a centrifuge tube. 24 mL of a 10% PS emulsion (mass fraction) was then added. The centrifuge tube was sonicated to thoroughly mix the solution. After 2 hours, 1 mL of a 2.5% Fe₃O₄ solution was added to the centrifuge tube, and the tube was placed in an ultrasonic machine for 2 hours to obtain a mixed emulsion. The mixed emulsion was transferred to a syringe, and another syringe was filled with methyl silicone oil. The two syringes were fixed to a constant-current microfluidic pump and fitted with capillaries. The pump flow rate was adjusted to 8 mL / h for methyl silicone oil and 5 mL / h for the mixed emulsion. The syringes were slowly pushed. Utilizing the principle of water-in-oil, the methyl silicone oil flowing out of the capillary tube cut off the emulsion flowing out of the capillary tube. Once the flow rate was uniform, the cut-off portions formed into droplets of uniform size, resulting in the desired magnetic inverse opal photonic crystal microspheres. The resulting uniformly sized microspheres were placed on a plate and placed in an oven at 60°C overnight until the solvent in the plate evaporated. The microspheres were then transferred to a ceramic crucible and n-hexane was added to the crucible three times to completely remove any residual methyl silicone oil from the surface. Anhydrous ethanol was then added to the crucible to remove any residual n-hexane from the microspheres. Finally, the crucible was left at room temperature until the ethanol evaporated. The dried microspheres were placed in the crucible and placed in a tube furnace at 700°C for 3 hours. After 3 hours, the crucibles were removed and used for later use. This resulted in magnetic inverse-structured photonic crystal microspheres (MIPCMs) with a three-dimensional ordered porous structure. Next, 30% hydrogen peroxide and 70% concentrated sulfuric acid were mixed in a volume ratio of 3:7 to create a piranha solution. The piranha solution was added to the MIPCMs and modified at 25°C and 160 rpm for 6 hours. The microspheres were then centrifuged and dried to modify the microsphere surfaces with hydroxyl groups. The prepared MIPCMs were placed under a scanning electron microscope and a metallographic microscope for observation and characterization. Figure 7 Metallographic microscope images show that the microspheres are neat, regular, and full, with a bright spot in the center, indicating a complete surface structure. SEM images show that the prepared inverse-structured photonic crystal microspheres have a distinct, regular porous structure with a pore size of approximately 200 nm.

[0089] Example 5

[0090] Take 0.036g of curcumin and put it into a centrifuge tube, add 1mL of ethanol, and ultrasonicate to disperse it evenly. Take 10mg of the magnetic inverse opal photonic crystal microspheres prepared in Example 4 and put them into a centrifuge tube, add 11.75μL APTES and 2ml of water. Mix the two centrifuge tubes and react at a temperature of 25°C and a speed of 200rpm for 30 minutes. Then add 55.5μL of TEOS and react at room temperature and a speed of 150rpm for 20h. After centrifugation and drying, magnetic inverse opal photonic crystal microspheres modified with a molecular imprinting layer, namely MIPCMs@MIP, are obtained.

[0091] The preparation of MIPCMs@NIP was the same as above, except that curcumin was not added.

[0092] The prepared MIPCMs@MIP and MIPCMs@NIP were observed and characterized under a scanning electron microscope and a metallographic microscope. Figure 8 From the metallographic microscope images, it can be seen that the central light spots of MIPCMs@NIP and MIPCMs@MIP microspheres are significantly darker than those of the bare microspheres. Combined with the scanning electron microscope images, it can be observed that this is because a large amount of polymer is bound to the surface of the photonic crystal microspheres, resulting in a weakening of the microsphere structural color.

[0093] Example 6

[0094] The MIPCMs@MIP and MIPCMs@NIP prepared in Example 5 were used as materials to investigate and analyze their static adsorption properties.

[0095] 1 mg of MIPCMs@MIP and MIPCMs@NIP prepared in Example 5 was placed in a centrifuge tube, and 1 ml of ZEN solution with concentrations of 0.05 μg / mL, 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL, and 80 μg / mL was added, respectively (the solvent in step 4 of Example 2 was ethanol: water volume ratio of 6:1). The mixture was shaken at 200 rpm at room temperature for 4 hours, and the supernatant was taken for HPLC fluorescence detection after the end. The results are shown in FIG. Figure 9As shown, as the ZEN concentration continues to increase, the adsorption capacity of the target toxin by MIPCMs@MIP continues to increase, and saturation is not reached at the toxin concentration of 80μg / ml. At this time, the adsorption capacity of ZEN by MIPCMs@MIP is 16.2665μg / mg. The growth rate of adsorption is still very high at this time. To avoid waste of toxin, the ZEN concentration is no longer increased. In contrast, the adsorption capacity of ZEN by MIPCMs@NIP almost reaches equilibrium at 2μg / ml, with the adsorption capacity at this time being 0.41μg / mg. At this time, the imprinting factor (IF) of MIPCMs@MIP for ZEN is 39.7. This is because the microspheres have a large specific surface area, and after the molecularly imprinted polymer is modified on the surface, there are more binding sites, making it easier to specifically recognize and capture target molecules.

[0096] Example 7

[0097] The MIPCMs@MIP and MIPCMs@NIP prepared in Example 10 were used as materials to investigate and analyze their dynamic adsorption properties.

[0098] 1 mg of MIPCMs@MIP and MIPCMs@NIP prepared in Example 5 was placed in a centrifuge tube, and 1 mL of a prepared 2 μg / mL ZEN standard solution was added to the centrifuge tube. The mixture was shaken at 200 rpm at room temperature for 1 minute, 5 minutes, 10 minutes, 30 minutes, 50 minutes, 70 minutes, 90 minutes, 120 minutes, 150 minutes, and 200 minutes. After the end, the supernatant was taken for HPLC-FLD analysis under the action of an external magnetic field. The results are shown in Figure 2. Figure 10 As shown in the figure, it can be clearly seen that the adsorption of ZEN by MIPCMs@MIP reaches equilibrium at around 90 minutes, with an adsorption capacity of 1.01 μg / mg, while the adsorption of ZEN by MIPCMs@NIP reaches equilibrium at around 120 minutes, with an adsorption capacity of 0.339 μg / mg. When the adsorption reaches equilibrium, the imprinting factor (IF) is 2.97. This is because the imprinting layer is more uniform after the molecularly imprinted polymer is modified on the surface of the microspheres. Due to the large specific surface area of ​​the microspheres, there are more binding sites on the surface, which can specifically adsorb more target molecules.

Claims

1. A molecularly imprinted magnetic inverse opal photonic crystal microsphere that can specifically recognize zearalenone, characterized in that: The microspheres introduce a pseudo template similar to the structure of zearalenone, use magnetic inverse opal photonic crystal microspheres as a substrate, and modify the surface of the microspheres with a molecular imprinting layer that can specifically recognize zearalenone.

2. The molecularly imprinted magnetic inverse opal photonic crystal microspheres capable of specifically recognizing zearalenone according to claim 1, characterized in that: The pseudo template having a structure similar to ZEN is curcumin, quercetin or 5,7-dimethylcoumarin.

3. The molecularly imprinted magnetic inverse opal photonic crystal microspheres capable of specifically recognizing zearalenone according to claim 1, characterized in that: The molecular imprinting layer is synthesized with a pseudo template molecule similar to the ZEN structure. After the functional monomers are introduced and the cross-linking agent reacts and the template is removed, an imprinting cavity similar to the ZEN structure with specific recognition function is formed on the surface of the microsphere.

4. The molecularly imprinted magnetic inverse opal photonic crystal microspheres capable of specifically recognizing zearalenone according to claim 3, characterized in that: The functional monomer is 2,33-aminopropyltriethoxysilane, and the crosslinking agent is ethyl orthosilicate.

5. A method for preparing molecularly imprinted magnetic inverse opal photonic crystal microspheres capable of specifically recognizing zearalenone according to claim 1, characterized in that: The following steps are involved: (1) Preparation of magnetic inverse opal photonic crystal microspheres and modification of hydroxyl groups: Microspheres were prepared by microfluidic self-assembly. Based on the water-in-oil principle, methyl silicone oil was used to cut the mixed emulsion into uniform small droplets. The desired microspheres were obtained after calcination. The microspheres were treated with a mixed solution of hydrogen peroxide and concentrated sulfuric acid to complete the modification of hydroxyl groups on the surface of the microspheres. (2) Modification of the molecular imprinting layer of the magnetic inverse opal photonic crystal microspheres: taking the hydroxyl-modified photonic crystal microspheres prepared in step (1), adding a template molecule, a porogen, a functional monomer, and a cross-linking agent and reacting them, and removing the template after the reaction is completed, thus completing the modification of the molecular imprinting layer on the surface of the microspheres to obtain molecularly imprinted magnetic inverse opal photonic crystal microspheres that can specifically recognize zearalenone.

6. The preparation method according to claim 5, characterized in that The mixed emulsion in step (1) is obtained by subjecting a polystyrene nanoparticle solution, a silicon dioxide nanoparticle solution and a ferroferric oxide nanoparticle solution to ultrasonic, vortex and centrifugal operations, adding double distilled water, and diluting the three solutions and then mixing them evenly.

7. The preparation method according to claim 5, characterized in that In step (1), the flow rate of methyl silicone oil is 8-10 mL / h, and the flow rate of the mixed emulsion is 5-8 mL / h; the calcination temperature is set to 700-800°C, and the calcination time is 3-4 hours; the modification time is 6-8 hours, the temperature is 25-30°C, and the rotation speed is 160-200 rpm.

8. The preparation method according to claim 5, characterized in that In step (2), the reaction solvent is ethanol and water, the initiator is acetic acid or ammonia water, and the molar ratio of the template, the functional monomer and the cross-linking agent is preferably 1:3-4:8-10.

9. The preparation method according to claim 5, characterized in that In step (2), the reaction time is 20-24 hours, the temperature is 25-30° C., and the rotation speed is 150-200 rpm.

10. Use of the magnetic inverse opal photonic crystal microspheres according to claim 1 for the specific adsorption of zearalenone.

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