Preparation method and application of a magnetic molecularly imprinted inverse opal photonic crystal microsphere

Abstract

The present invention discloses a preparation method and application of magnetic molecularly imprinted inverse opal photonic crystal microspheres. The microspheres are prepared by first synthesizing core-shell type nanoscale magnetic molecularly imprinted nanoparticles capable of specifically recognizing AFB1, and then self-assembling the nanoparticles with polystyrene and silica nanoparticles into a novel molecularly imprinted inverse opal microsphere. The magnetic molecularly imprinted nanoparticles are prepared by using magnetite as a substrate and synthesizing a molecularly imprinted layer capable of specifically recognizing aflatoxin B1 through a surface molecular imprinting strategy using a bifunctional monomer. The magnetic molecularly imprinted photonic crystal microspheres prepared by the present invention have obvious porous structures and magnetic properties, can rapidly and selectively enrich aflatoxin B1 in samples. Compared with traditional enrichment materials such as immunoaffinity columns, the materials of the present invention are simple to prepare, low in cost, easy to store and reusable.

Description

Technical Field

[0001] The present invention belongs to the field of separation science, and particularly relates to a preparation method and application of magnetic molecularly imprinted inverse opal photonic crystal microspheres. Background Art

[0002] Food safety issues have always been the focus of people's attention. During the processing, transportation, storage, and sales of food, fungal contamination may occur due to irregular supervision and operation. Aflatoxin is one of the most common mycotoxins, which is a toxic secondary metabolite produced by Aspergillus flavus and Aspergillus parasiticus. It can be divided into aflatoxin B1, B2, G1, G2, etc. Among them, aflatoxin B1 has the highest toxicity and is also one of the main pathogenic factors of liver cancer. It widely exists in food agricultural products such as peanuts, corn, soybeans, and wheat. Due to its high solubility in fat, after the food is contaminated by AFB1, it can enter the human and animal bodies through the food chain, seriously endangering the health of humans and animals. Therefore, it has always received great attention internationally and was recognized as a "1A" carcinogen by the International Agency for Research on Cancer in 1987. Since the content of aflatoxin in food samples is low and there is matrix interference, high-efficiency and highly selective sample pretreatment technologies are required to achieve the extraction and analysis of target substances.

[0003] Currently, the pretreatment technologies for AFB1 in samples to be measured mainly include liquid-liquid extraction (LLE), immunoaffinity columns (IAC), and solid-phase extraction. However, liquid-liquid extraction is often a cumbersome process that requires a large amount of organic solvents. Immunoaffinity columns have high production costs, are sensitive to environmental factors, are easily degraded and denatured, and cannot be reused, which are subject to certain limitations. Solid-phase extraction can be divided into non-selective solid-phase extraction and selective solid-phase extraction. The former has low selectivity and is difficult to be efficiently used 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, the antibody-based adsorption material has strong specificity, but the acquisition of antibodies is time-consuming and expensive. Therefore, it is necessary to develop a solid-phase extraction pretreatment material with simple operation, high specificity, and low cost for separating and enriching AFB1 in actual samples.

[0004] In the prior art, a magnetic photonic crystal microsphere for enriching and separating aflatoxin B1, its preparation method and application are disclosed (CN2021109175482). The prepared core-shell type surface molecularly imprinted magnetic inverse opal photonic crystal microspheres can selectively extract aflatoxin B1 from samples. Compared with materials modified with biological antibodies, they can greatly improve the stability of the materials and reduce the material preparation cost. However, the core-shell type surface molecularly imprinted magnetic inverse opal photonic crystal microspheres are prepared by using a monofunctional monomer on the surface of the photonic crystal microspheres, so they can only provide a single recognition group, resulting in poor selectivity for target molecules. Moreover, the surface polymer has caused certain damage to the ordered porous structure of the inverse opal microspheres, affecting the mass transfer and thus the separation and enrichment time. Summary of the Invention

[0005] Object of the Invention: Aiming at some problems existing in the prior art and materials, the present invention provides a magnetic molecularly imprinted inverse opal photonic crystal microsphere. The magnetic molecularly imprinted inverse opal photonic crystal microsphere (MPCM@MIP) prepared by the present invention for separating and enriching aflatoxin B1 has a very high imprinting factor and a very fast enrichment speed, improving the specificity and enrichment speed.

[0006] The present invention also provides the magnetic molecularly imprinted inverse opal photonic crystal microsphere and its application as described above.

[0007] Technical Solution: To achieve the above object, the preparation method of a magnetic molecularly imprinted inverse opal photonic crystal microsphere according to the present invention includes the following steps:

[0008] (1) Surface modification of magnetic iron tetroxide: Adjust the solution of tetraethyl orthosilicate to be alkaline, and perform silica coating on the magnetic iron oxide nanoparticles through mechanical stirring in a water bath environment, and then use a silane coupling agent to form magnetic nanoparticles with surface grafted double bonds;

[0009] (2) Preparation of magnetic molecularly imprinted nanoparticles: Take the modified magnetic nanoparticles in step (1), add a template molecule, a bifunctional monomer, a crosslinking agent and a reaction solvent, and then carry out the reaction; subsequently, in an inert gas atmosphere, add an initiator, and after the reaction is completed, discard the remaining reaction liquid under the action of magnetic force. After the product is eluted with the template molecule, magnetic molecularly imprinted nanoparticles are obtained.

[0010] (3) Preparation of magnetic molecularly imprinted inverse opal photonic crystal microspheres: Take the magnetic molecularly imprinted nanoparticles prepared in step (2), mix them with pure water, and then add silica nanoparticles and polystyrene nanoparticle emulsion, and form uniform microspheres through microfluidic self-assembly. After curing and drying, the polystyrene nanoparticles are removed by high-temperature calcination to obtain inverse opal structure magnetic molecularly imprinted photonic crystal microspheres.

[0011] The microspheres of the present invention are prepared by first synthesizing nanoscale magnetic molecularly imprinted nanoparticles that can specifically recognize AFB1, and then self-assembling these nanoparticles with polystyrene and silica nanoparticles to form a novel molecularly imprinted inverse opal microsphere. Among them, the nanoscale magnetic molecularly imprinted nanoparticles are based on magnetite, and a molecularly imprinted layer that can specifically recognize aflatoxin B1 is synthesized using a bifunctional monomer through a surface molecular imprinting strategy.

[0012] Among them, in step (1), ammonia water is used to adjust the solution pH of tetraethyl orthosilicate to 8.0 - 9.0, the mechanical stirring speed is 400 - 750 r / min, the constant temperature water bath is 50 °C, and the silane coupling agent is 3-(methacryloyloxy)propyltrimethoxysilane.

[0013] Among them, in step (2), the template molecule is 5,7-dimethoxycoumarin, the bifunctional monomers are methacrylic acid and styrene respectively, the crosslinking agent is ethylene glycol dimethacrylate, the initiator is azobisisobutyronitrile, and the reaction solvent is acetonitrile or dimethyl sulfoxide or N,N-dimethylformamide.

[0014] Among them, in step (2), after taking the modified magnetic nanoparticles in step (1), adding the template molecule, bifunctional monomers, crosslinking agent and reaction solvent, the mixture is placed at 30 - 40 °C for reaction for 1 - 2 hours.

[0015] Preferably, in step (2), after taking the modified magnetic nanoparticles in step (1), adding the template molecule, bifunctional monomers, crosslinking agent and reaction solvent, the mixture is placed at 37 °C for reaction for 1 hour. Among them, in step (2), under a nitrogen atmosphere, the initiator is added, and the mixture is placed at 60 - 80 °C for reaction for 20 - 24 h.

[0016] Among them, in step (3), the concentration of the aqueous solution of the magnetic molecularly imprinted nanoparticles is 2 - 3%, the concentration of the silica nanoparticle emulsion is 15 - 25%, and the concentration of the polystyrene nanoparticle emulsion is 8 - 15%.

[0017] Preferably, the concentration of the aqueous solution of the magnetic molecularly imprinted nanoparticles is 2.5%, the concentration of the silica nanoparticle emulsion is 20%, and the concentration of the polystyrene nanoparticle emulsion is 10%. The above concentrations are all mass fractions.

[0018] Among them, in step (3), the curing and drying temperature is 60 - 100 °C, the time is 16 - 24 hours, the high-temperature calcination temperature is 500 - 600 °C, and the time is 2 - 3 hours.

[0019] Preferably, the curing and drying temperature is 60 °C, the time is 16 hours, the high-temperature calcination temperature is 550 °C, and the time is 3 hours.

[0020] Preferably, in step (3), Soxhlet extraction method is used to elute the template. When performing Soxhlet reflux, the eluent is a mixed solution of methanol and acetic acid (volume ratio 9:1).

[0021] Preferably, the particle size of the magnetite is 10 - 50 nm, the particle size of the silica nanoparticles is 5 - 10 nm, and the particle size of the polystyrene nanoparticles is 250 - 300 nm.

[0022] The magnetic molecularly imprinted inverse opal photonic crystal microspheres prepared by the preparation method of the present invention. In the synthesis process of the magnetic molecularly imprinted inverse opal photonic crystal microspheres of the present invention, only 2 mg of magnetic molecularly imprinted nanoparticles with specific recognition function needs to be added to synthesize 60 mg of inverse opal microspheres. The polymer dosage is small (1 / 30), the adsorption capacity is up to 5.2 μg / mg, and the selectivity coefficient is between 2.5 and 4.0.

[0023] Application of the magnetic molecularly imprinted inverse opal photonic crystal microspheres of the present invention in the selective and efficient separation and enrichment of aflatoxin B1.

[0024] Among them, the microspheres can complete the separation and enrichment of the target toxin aflatoxin B1 with high specificity in a short time through an external magnetic field, and the time does not exceed 20 minutes, and the imprinting factor is up to 8.0.

[0025] The present invention first coats magnetite nanoparticles with silica, then modifies polymerizable functional groups such as allyl groups on its surface, and finally reacts it with a molecular prepolymer solution to obtain magnetic molecularly imprinted nanoparticles capable of specifically recognizing AFB1. Then, it is mixed with silica nanoparticles and polystyrene nanoparticle emulsion and assembled into magnetic molecularly imprinted inverse opal microspheres through a microfluidic system for the separation and enrichment of AFB1 in actual samples. Among them, the core-shell magnetic molecularly imprinted nanoparticles synthesize a molecularly imprinted layer capable of specifically recognizing AFB1 through a surface molecular imprinting strategy using a bifunctional monomer. Compared with traditional enrichment materials such as antibodies and immunoaffinity columns, the materials of the present invention are simple to prepare, low in cost, easy to store and reusable.

[0026] Specifically, the present invention mixes magnetite coated with silica on the surface and modified with allyl functional groups with a molecular imprinting prepolymer solution, and obtains magnetic molecularly imprinted nanoparticles capable of specifically recognizing AFB1 through thermal initiation polymerization, and then self-assembles them into magnetic molecularly imprinted inverse opal microspheres through a microfluidic system. Among them, the SiO2 nanoparticles used in the microspheres have a particle size of 5 - 10 nm, the PS nanoparticles have a particle size of 250 - 300 nm, and the magnetite has a particle size of 10 - 50 nm.

[0027] Design mechanism: The selective recognition principle of the magnetic molecularly imprinted inverse opal photonic crystal microspheres of the present invention: After tetraethyl orthosilicate coats silica on the surface of magnetic nanoparticles and modifies allyl functional groups, using it as a carrier, with the structural analogs DMC, MUAC or MDAC of AFB1 as template molecules, an interaction will occur between them and the bifunctional monomer to form a template-monomer complex. Then, the crosslinking agent and the bifunctional monomer will form a dense cross-linked polymer film on the surface of the magnetic nanoparticles under the catalysis of the initiator, and fix the template in it. After the polymerization is completed, the template molecules are eluted by Soxhlet extraction, thus leaving cavities on the surface of the magnetic nanoparticles that match the spatial configuration and binding sites of AFB1. These cavities have the ability to selectively recognize the structural analog AFB1 of the template molecule. Then, it is self-assembled with SiO2 nanoparticles and PS nanoemulsion into inverse opal structure microspheres. Its unique porous structure is conducive to improving the adsorption capacity and mass transfer, and can efficiently and selectively separate and enrich aflatoxin B1.

[0028] Compared with the prior art CN2021109175482, it prepares the microspheres first and then modifies the surface with imprinted polymers. However, in the pretreatment before polymerization, the surface of the microspheres is not hydroxylated, which will affect the grafting of double bonds and thus affect the surface coating of the molecularly imprinted polymer. In the present invention, first, silica is used to coat the surface of magnetic nanoparticles, and after increasing the amount of surface hydroxyl groups, a silane coupling agent is used for double bond modification, thus effectively improving the grafting of the molecularly imprinted polymer. Moreover, a bifunctional monomer is used for polymerization during the polymer synthesis process, which is conducive to improving molecular recognition. In the whole synthesis process, the prior art prepares the microspheres first and then does molecular imprinting on the surface of the microspheres, while the present invention synthesizes magnetic molecularly imprinted nanoparticles first as the components for synthesizing magnetic molecularly imprinted photonic crystal microspheres, avoiding the destruction of the ordered porous structure caused by the coating of the polymer layer on the surface of the microspheres. On the contrary, using magnetic molecularly imprinted nanoparticles as the components for synthesizing molecularly imprinted photonic crystal microspheres, the unique ordered porous structure of the inverse opal microspheres is well preserved, which is conducive to accelerating mass transfer and shortening the separation and enrichment time.

[0029] The magnetic molecularly imprinted inverse opal photonic crystal of the present invention uses magnetic molecularly imprinted nanoparticles as an adsorption carrier, and the molecularly imprinted layer polymerized on its surface can selectively recognize and enrich and separate AFB1 in the actual sample. Then, it is self-assembled into inverse opal photonic crystal microspheres through a microfluidic system. Its porous and ordered structure can accelerate the mass transfer and increase the enrichment amount. In the prior art CN2021109175482, after surface modification with a molecularly imprinted polymer, the porous structure is blocked by the polymer layer, which causes certain damage to the ordered porous structure of the inverse opal microspheres, affects the mass transfer, and further affects the separation and enrichment time. The microspheres of the present invention use a surface imprinting strategy to prepare an AFB1 molecularly imprinted layer on the surface of magnetic nanoparticles, which is beneficial for the elution and re-adsorption of the template molecule (where elution refers to the elution of the template during the preparation of the material; re-adsorption refers to the adsorption and enrichment of the template molecule or target during the use of the material for testing or application). It overcomes the defect that the imprinting sites are buried too deep in the traditional imprinting method, resulting in a decrease in adsorption efficiency. Moreover, its unique magnetic properties endow the microspheres with magnetism, eliminating the need for filtration or centrifugation, and facilitating the rapid separation of target molecules in complex matrices. The magnetic molecularly imprinted inverse opal photonic crystal microspheres prepared by the present invention can rapidly and selectively enrich and separate AFB1 in the actual sample. Moreover, the operation is simple and fast, the cost is low, the stability is good, and it can be reused, making it an excellent substitute for AFB1 biological antibodies.

[0030] The present invention provides a large number of hydroxyl groups for the grafting of double bonds by coating silica on the surface of magnetic nanoparticles, which is beneficial for the preparation of a molecularly imprinted layer for the efficient extraction and separation of aflatoxin B1 using a bifunctional monomer. Different from a monofunctional monomer that can only provide one molecular recognition group, a bifunctional monomer can improve the specificity of the molecular polymer by providing different recognition groups. Then, it is self-assembled into an inverse opal structure photonic crystal microsphere through microfluidics. Different from the defect that the porous structure on the surface of the inverse opal photonic crystal microsphere is buried by the polymer layer due to surface molecular imprinting technology, the novel magnetic molecularly imprinted photonic crystal microspheres prepared by this method use magnetic molecularly imprinted nanoparticles to replace traditional iron oxide as the synthesis component of the molecularly imprinted inverse protein microspheres, thus effectively avoiding the clogging problem caused by subsequent surface modification, perfectly preserving the ordered porous structure of the inverse protein microspheres, facilitating the acceleration of mass transfer, greatly shortening the enrichment time, increasing the enrichment amount, and the magnetic nanoparticles can endow substances with magnetic properties, and the molecularly imprinted layer on them has a molecular sieve effect, which can specifically enrich and separate target molecules in complex samples.

[0031] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0032] (1) A novel molecularly imprinted polymer capable of specifically recognizing AFB1 was synthesized through bifunctional monomers, improving the enrichment performance of the target toxin.

[0033] (2) In the present invention, an imprinting layer is polymerized on the surface of magnetic nanoparticles. Due to its unique magnetic properties, it is easy to separate and enrich through an external magnetic field without operations such as filtration or centrifugation, saving time and effort.

[0034] (3) The overall particle size of the magnetic molecularly imprinted nanoparticles prepared in the present invention is nanoscale. They are self-assembled inside the photonic crystal to synthesize novel magnetic molecularly imprinted inverse opal photonic crystal microspheres.

[0035] (4) The magnetic molecularly imprinted inverse opal photonic crystal microspheres prepared in the present invention well preserve the ordered porous structure of the inverse opal microspheres, have a high imprinting factor, accelerate the mass transfer, and shorten the enrichment time.

[0036] (5) The magnetic molecularly imprinted inverse opal photonic crystal microspheres prepared in the present invention have the advantages of simple preparation, good stability, high enrichment and separation efficiency, good specificity, and low cost, and can be industrially produced.

[0037] (6) The magnetic molecularly imprinted inverse opal photonic crystal microspheres prepared through self-assembly technology can meet the requirements for magnetic properties in different environments by adjusting the applied magnetic field, and are easy to store, effectively replacing traditional solid-phase extraction materials. Description of the Drawings

[0038] Figure 1 is the preparation technical route diagram of the magnetic molecularly imprinted inverse opal photonic crystal microspheres;

[0039] Figure 2 is the infrared characterization after modification polymerization of the magnetic molecularly imprinted nanoparticles;

[0040] Figure 3 is the influence of different template molecules on the adsorption performance of the magnetic molecularly imprinted nanoparticles;

[0041] Figure 4 is the influence of different functional monomers on the adsorption performance of the magnetic molecularly imprinted nanoparticles;

[0042] Figure 5 is the influence of different applied magnetic fields on the adsorption performance of the magnetic molecularly imprinted nanoparticles;

[0043] Figure 6 is the morphological characterization of the magnetic molecularly imprinted inverse opal photonic crystal microspheres;

[0044] Figure 7 is the adsorption saturation curve of the magnetic molecularly imprinted inverse opal photonic crystal microspheres;

[0045] Figure 8 For the adsorption kinetics of magnetic molecularly imprinted inverse opal photonic crystal microspheres;

[0046] Figure 9 For the adsorption specificity test of magnetic molecularly imprinted inverse opal photonic crystal microspheres. Specific implementation mode

[0047] In the experimental methods described in the examples, unless otherwise specified, they are all conventional methods; the reagents and materials, unless otherwise specified, can be obtained from commercial channels. The overall technical route for the preparation of magnetic molecularly imprinted inverse opal photonic crystal microspheres is shown in Figure 1 .

[0048] Among them, the particle size of the iron oxide (Fe3O4) dispersion is 10 - 50 nm, and the particle size of the silica (SiO2) nanoparticles is 5 - 10 nm. Both are purchased from Sigma-Aldrich.

[0049] The particle size of the polystyrene nanospheres (PS) nanoparticles is about 300 nm, purchased from Nanjing Caina Biotechnology Co., Ltd., CAS9006-53-6.

[0050] Example 1

[0051] 1. Surface modification of magnetic nanoparticles:

[0052] (1) Silica coating: Take 1.5 g of Fe3O4 dispersion (375 mg of Fe3O4 in 1.5 g of Fe3O4 aqueous dispersion) in a 1000 mL three-necked flask, add 100 mL of 0.1 M HCL and ultrasonicate for 15 min, then wash with ultrapure water to neutrality under the action of an external magnetic field. Add 50 mL of ultrapure water and 200 mL of ethanol, ultrasonicate for 15 min to mix evenly, then stir in a water bath at 50 °C at a speed of 650 r / min for 10 min, and then add 2.5 mL of 25% ammonia water, stir for 30 min to make the liquid pH between 8.0 and 9.0. Add the mixed solvent of TEOS and ethanol (1.2 mL TEOS + 8.8 mL ethanol) 1 mL each time, add it in ten times at intervals of 5 min, and then stir at 50 °C for 20 h. After the reaction is completed, wash alternately with ethanol and ultrapure water several times to neutrality, and dry in vacuum for standby.

[0053] (2) Surface grafting of double bonds: Take 400 mg of the dried magnetic particles in a 250 mL three-necked flask, add 100 mL of methanol solution and ultrasonicate for 15 min. After the magnetic nanoparticles are mixed evenly, introduce nitrogen gas. Under the nitrogen atmosphere, add 7.5 mL of 3-(methacryloyloxy)propyltrimethoxysilane and ultrasonicate for 10 min, then place it in a constant temperature water bath at 25 °C and stir vigorously mechanically for 24 h. After the reaction is completed, wash alternately with ethanol and ultrapure water several times to neutrality, and dry in vacuum for standby.

[0054] Example 2

[0055] Using the magnetic nanoparticles prepared in Example 1 as the substrate, magnetic molecularly imprinted nanoparticles were synthesized, and the specific steps are as follows:

[0056] 1. Molecular imprinting polymerization:

[0057] Using surface molecular imprinting technology, 30 mg of surface-modified magnetic nanoparticles were placed in a centrifuge tube, and the template molecule, bifunctional monomer, and cross-linking agent were added, and the volume was fixed to 6 mL with a porogen (i.e., the reaction solvent acetonitrile). The reaction was carried out on a shaker at 37 °C for 1 h at a rotation speed of 200 rpm. Subsequently, nitrogen was passed for 6 min, and then the initiator was added. After sealing the centrifuge tube with a sealing film, it was placed on a shaker at 60 °C and 220 rpm for shaking reaction for 24 h. After the reaction was completed, the reaction liquid was discarded under the action of a magnet to obtain magnetic molecularly imprinted nanoparticles (MMIPs) coated with an organic polymer layer.

[0058] 2. Removal of the template molecule:

[0059] Using Soxhlet extraction method, the nanoparticles in step 1 were wrapped with filter paper and placed in a siphon tube. 120 mL of a methanol-acetic acid (9:1, v / v) mixture was added to the flask. After Soxhlet refluxing and eluting at 80 °C for 12 h, it was replaced with 120 mL of pure methanol to wash away acetic acid, and then vacuum drying was carried out to obtain magnetic molecularly imprinted nanoparticles. The preparation steps of non-imprinted magnetic molecularly imprinted nanoparticles (MNIPs) were the same as steps 1 and 2 above, but the template molecule was not added.

[0060] 3. Optimization of synthesis conditions:

[0061] According to the methods of steps 1 and 2 above, different template molecules, functional monomers, and magnetic addition amounts were investigated.

[0062] (1) Template molecule

[0063] Take 30 mg of the magnetic nanoparticles with modified double bonds in Example 1 (the end product of Step 2 in Example 1) in a centrifuge tube. Add 0.03 mmol of template molecules (5,7-dimethoxycoumarin (DMC), 7-acetoxy-4-methylcoumarin (MUAC), or 7-diethylamino-4-methylcoumarin (MDAC)), 0.3 mmol of bifunctional monomer methacrylic acid MAA, 0.015 mmol of styrene ST, and 0.75 mmol of crosslinker ethylene glycol dimethacrylate EGDMA into the tube and make up the volume to 6 mL with acetonitrile. After reacting in a shaker at 37 °C for 1 h, the rotation speed is 200 rpm. Subsequently, pass nitrogen for 6 min, add 0.014 mmol of initiator azobisisobutyronitrile AIBN, seal the centrifuge tube with a sealing film, and finally place it in a shaker at 60 °C and shake at 220 rpm for 24 h. After the reaction is completed, remove the template molecules by Soxhlet extraction method, and finally three kinds of MMIPs can be obtained.

[0064] The adsorption properties of the three prepared magnetic molecularly imprinted nanoparticles for the corresponding template molecules are as Figure 3 shown. The imprinting factors of the MIPs synthesized with DMC, MUAC, and MDAC as templates for the corresponding templates are 1.44, 0.97, and 1.13 respectively (the imprinting factor is the ratio of the adsorption amount of the molecularly imprinted polymer for the template molecule to the adsorption amount of the non-molecularly imprinted polymer for the template molecule); the adsorption amounts are 1.53, 1.18, and 1.28 μg / mg respectively, indicating that the imprinted material synthesized with DMC as the template has good specificity and strong adsorption ability for AFB1. Therefore, DMC is selected as the template molecule for subsequent experiments. The adsorption performance / adsorption amount refers to Example 5, and the detection of the adsorption performance of the template molecule is as follows: Mix the polymer (three kinds of MMIPs) with the template molecule in the adsorption solvent, shake overnight at room temperature, take the supernatant to test the concentration of the template molecule in it, that is, the concentration of the template molecule in the solution after adsorption, and calculate the adsorption amount of the polymer according to the difference in the concentration of the template molecule before and after adsorption.

[0065] (2) Functional monomer

[0066] The strength of the interaction between the monomer and the template determines the strength of the selective recognition ability of the imprinted material for the target molecule. Therefore, it is crucial to select a suitable functional monomer. According to the method in the above step (1), methacrylic acid (MAA), 4-vinylpyridine (4-VP), styrene (ST), and acrylamide (AAm) are respectively selected as monomers to react with the template molecule DMC to prepare different magnetic molecularly imprinted nanoparticles, and adsorption experiments are carried out on their template molecules. The results are shown in Figure 4a), The adsorption amounts of MMIPs prepared with MAA, 4-VP, ST, and AAm as functional monomers for DMC were 1.53, 0.51, 3.34, and 0.16 μg / mg, respectively; the imprinting factors were 1.44, 1.02, 1.08, and 1. To improve the adsorption amount and selectivity, ST and MAA were used as bifunctional monomers, and their ratio was optimized. The results are as Figure 4 b, However, when ST:MAA = 1:2 (molar ratio), the adsorption amount of DMC was 2.06 μg / mg, and the imprinting factor was 1.62. The other imprinting factors were 1.34 (ST:MAA = 0:1), 1.09 (ST:MAA = 2:1), 1.21 (ST:MAA = 1:1), and 1.08 (ST:MAA = 1:0). Therefore, ST:MAA = 1:2 was selected as the bifunctional monomer.

[0067] In addition, using the method of Example 2, the effects of magnetic molecularly imprinted polymers prepared with template molecules acetyl-oxy-4-methylcoumarin (MUAC) or 7-diethylamino-4-methylcoumarin (MDAC) and monomers 4-vinylpyridine (4-VP) or styrene (ST) were also significantly inferior to those of the polymer prepared with DMC as the template molecule and ST:MAA = 1:2 as the bifunctional monomer.

[0068] (3) Magnetic amount

[0069] As the magnetic nanoparticles used as the substrate material, their content determines to a certain extent the uniformity of the surface molecular imprinting coating. Therefore, it is very necessary to select an appropriate magnetic amount. According to the method of step (1) above, 15 mg, 30 mg, and 60 mg of magnetic nanoparticles were selected respectively to prepare three different MMIPs, and adsorption experiments were carried out on the template molecule DMC. The results are as Figure 5 shown. The adsorption amounts of MMIPs with magnetic amounts of 15 mg, 30 mg, and 60 mg for DMC were 2.59, 3.16, and 2.12 μg / mg, respectively; the imprinting factors were 1.2, 1.53, and 1.49, indicating that when the magnetic amount was 30 mg, the adsorption amount and selectivity of the prepared polymer were relatively good. Therefore, the magnetic amount was selected as 30 mg.

[0070] 4. Morphology characterization of MMIPs:

[0071] The above-prepared MMIPs (with DMC as the template molecule and ST:MAA = 1:2 as the functional monomer) were characterized by infrared spectroscopy. See Appendix Figure 2 . There was a typical Si-O bending vibration peak at 465 cm -1 . At 805 cm -1 and 1085 cm -1They are the symmetric and asymmetric stretching of Si-O respectively. After modifying Fe3O4-SiO2 with 3-(methacryloyloxy)propyltrimethoxysilane, the C=O group of Fe3O4-SiO2-CH2=CH2 exhibits weak vibration near 1720 cm -1 which indicates that double bond functional groups are successfully grafted onto the surface of Fe3O4-SiO2. Strong characteristic peaks of the C=O groups from MAA (methacrylic acid) and EGDMA (ethylene glycol dimethacrylate) appear near 1730 cm -1 which shows that the organic polymer is successfully attached to the surface of the magnetic nanoparticles.

[0072] Example 3

[0073] The preparation method of Example 3 is the same as that of Example 2, except that: the reaction solvent is dimethyl sulfoxide or N,N-dimethylformamide, and finally it is placed in a shaker at 60 °C and 220 rpm for 24 h.

[0074] Example 4

[0075] 1. Preparation of magnetic molecularly imprinted inverse opal photonic crystal microspheres:

[0076] Using the optimal magnetic molecularly imprinted nanoparticles in Example 2, take a silica nanoparticle solution with a final concentration (mass fraction) of 20%, a PS nanoparticle solution of 10%, and an aqueous solution of magnetic molecularly imprinted nanoparticles of 2.5% and place them in a centrifuge tube. Ultrasonically mix them into a homogeneous emulsion, transfer it to a 5 mL syringe, and another large 50 mL syringe contains methyl silicone oil. Fix the two syringes on a constant flow microfluidic pump respectively. The oil-phase microfluidic flow rate is 10 mL / h, and the emulsion flow rate is 5 mL / h. Using the principle of water-in-oil, the methyl silicone oil truncates the emulsion into micron-sized droplets and collects them in a plastic petri dish containing methyl silicone oil. Then place the petri dish in a 60 °C forced-air drying oven for constant-temperature drying. After drying the water in the droplets, wash them 3 to 4 times with n-hexane and absolute ethanol respectively. After the ethanol evaporates, transfer them to a tubular furnace and slowly raise the temperature to 550 °C for calcination for 3 h to remove the template microspheres (remove PS microspheres), and magnetic molecularly imprinted inverse opal photonic crystal microspheres (MPCM@MIP) can be obtained. Among them, the volume ratio of MMIPs (or MNIPs):SiO2 in the original emulsion (20% silica nanoparticle solution, 10% PS nanoparticle solution, and 2.5% aqueous solution of magnetic molecularly imprinted nanoparticles) is 1:3, and the volume ratio of SiO2:PS is 1:8, and they are characterized by a metallurgical microscope and a scanning electron microscope, as shown in the appendix Figure 6 wherein, Figure 6 a shows that the microspheres prepared by using magnetic nanoparticles modified with molecularly imprinted polymerization as components for synthesizing inverse opal microspheres have good magnetic properties, Figure 6Figure b shows the metallographic microscope image of the magnetic molecularly imprinted inverse opal photonic crystal microspheres, which have obvious bright spots at the center, indicating that the surface structure of the microspheres is regular and has the general characteristics of inverse opal. Figure 6 c and Figure 6 d are the SEM images of the microspheres under 500X and 5000X magnifications respectively, which show that the prepared magnetic molecularly imprinted inverse opal microspheres are overall regular spheres, and their surfaces have obvious ordered porous structures. By comparing with the characterization images of the inverse opal microspheres in CN2021109175482 Figure 2 d, it can be seen that the porous structure on its surface is not damaged. After the existing technology CN2021109175482 modified the surface with molecularly imprinted polymers, the porous structure was blocked by the polymer layer ( Figure 6 f), which caused a certain damage to the ordered porous structure of the inverse opal microspheres, affected the mass transfer, and further affected the separation and enrichment time.

[0077] Example 5

[0078] Using the MPCM@MIP prepared in Example 4 as the material, its adsorption performance was investigated and analyzed.

[0079] Three portions of 4 mg microspheres prepared in Example 4 were respectively taken and placed in 1000 uL of AFB1 solutions with different concentrations of 5 - 50000 ng / mL to determine the enrichment ability of MPCM@MIP and MPCM@NIP (the microspheres prepared with non-imprinted magnetic nanoparticles without adding template molecules in Example 4) for aflatoxin B1. The results are shown in Figure 7 , because MPCM@MIP is prepared from MMIPs that form imprinted cavities on the surface that can specifically recognize AFB1, so MPCM@MIP can separate and enrich more AFB1 than MPCM@NIP. As the concentration of AFB1 increases, the adsorption capacity of MPCM@MIP also gradually increases, and its imprinting factor is up to 8.0 at most.

[0080] Using 1000 ng / mL of AFB1 to determine the adsorption saturation rate of MPCM@MIP and MPCM@NIP, the results are shown in Figure 8 , at this concentration, the highest imprinting factor is 1.74. Because MMIPs are surface molecularly imprinted polymers, their binding sites are exposed on the particle surface, enabling rapid separation and enrichment of the target substance, and the ordered porous structure of MPCM also promotes the transfer of the target molecule. Therefore, MPCM@MIP can rapidly enrich AFB1, and the required saturation enrichment time is 20 min. By comparing the porosity and specific surface area of MIOPCM@MIP prepared under the optimal conditions in Example 3 of CN2021109175482 as shown in Figure 6 h, they are 6.7 nm and 60 m 2 / g, it can be found that the porosity and specific surface area of MPCM@MIP prepared in Example 4 of the present invention Figure 6 g) are 17.3 nm and 159 m 2 / g, which are 2.6 times and 2.7 times that of CN2021109175482 respectively. The larger the specific surface area, the more conducive to the contact between the microspheres and the target molecules, and the larger the porosity, the more conducive to the transfer of substances. Therefore, under the same conditions, the enrichment speed of the present invention is about 7 times that of CN2021109175482, effectively shortening the enrichment time.

[0081] Example 6

[0082] Using the MPCM@MIP prepared in Example 4 as the material, its specificity was investigated and analyzed.

[0083] Take three portions of 4 mg microspheres prepared in Example 4 and add them to 2 mL centrifuge tubes. Respectively add 1000 uL of 1000 ng / mL AFB1, AFB2, AFG2, OTA, ZEN, and shake them on a shaker at 200 rpm at room temperature for 4 h for the adsorption experiment. After the adsorption is completed, the adsorbed supernatant can be taken out without centrifugation using a magnet, and the supernatant is detected using an HPLC system. The results are as Figure 9 shown. MPCM@MIP has good recognition ability for AFB1 and its structural analogs AFB2 and AFG2. Compared with OTA and ZEN, MPCM@MIP has higher binding selectivity for AFB1, with an imprinting factor of 1.73 and selectivity coefficients of 2.62 and 3.69. Compared with CN2021109175482 (Example 5), this imprinting factor increases by 0.26. This is mainly because during the synthesis process, the addition amount of magnetic molecularly imprinted polymer nanoparticles is relatively low (only about 2 mg of magnetic molecularly imprinted nanoparticles are contained in every 60 mg of photonic crystal microspheres). The overall addition amount can be converted by mass conversion to only 0.13 mg of magnetic molecularly imprinted polymer nanoparticles that can specifically recognize AFB1 in every 4 mg of microspheres, and a small amount of nanoparticles shows good selectivity. Compared with using 2 mg of molecularly imprinted microspheres in each mL of the sample to be tested in CN2021109175482 (Patent CN2021109175482 prepares molecularly imprinted polymers on the surface of the microspheres, so the whole has recognition sites and is a pure molecularly imprinted photonic crystal microsphere, while in the present invention, magnetic molecularly imprinted nanoparticles are first synthesized and then assembled into the microspheres, and only the magnetic nanoparticles assembled into the interior of the microspheres have the overall recognition sites), only 0.13 mg of the molecularly imprinted polymer with recognition in the present invention is used, which is significantly lower than the above patent. The polymer with specific recognition of AFB1 per unit mass in the present invention has more obvious selectivity.

[0084] Example 7

[0085] Separate and enrich aflatoxin B1 in actual samples, and perform quantitative analysis in combination with HPLC.

[0086] (1) Preparation of spiked samples

[0087] Select corn, wheat, and rice as real samples. Weigh 5 g of each sample in four portions into 50 mL centrifuge tubes, dilute the original AFB1 solution (2 mg / mL) with methanol to 1000 ng / mL. Take 0 mL, 0.125 mL, 0.25 mL, and 1.25 mL respectively and add them to the weighed sample test tubes, then make up the volume to 10 mL, shake well to mix the sample and the toxin evenly, place it in a fume hood and let it stand overnight for the methanol to evaporate. Add 0.5 g of sodium chloride and 25 mL of a mixed solution of methanol and water (V / V = 8:2) to the sample after the methanol has evaporated, homogenize in a homogenizer for 5 min, and then extract the toxin by ultrasonic treatment for 30 min. After complete extraction, filter it multiple times with qualitative filter paper and a 0.22 μm filter head and store it in a -20 °C refrigerator.

[0088] Take an appropriate amount of the filtered sample solution, dry it, and add an equal amount of a mixed solution of methanol and water (V / V = 1:1) to prepare actual sample solutions with concentrations of 0 ng / mL, 25 ng / kg, 50 ng / kg, and 250 ng / kg.

[0089] (7) Toxin extraction and enrichment

[0090] Weigh 4 mg of MPCM@MIP and add it to 1 mL of the above actual sample solution, shake at 200 rpm at room temperature for 4 h, and then discard the supernatant under the action of a magnet. Finally, elute the adsorbed AFB1 with a mixed solution of methanol and acetic acid (V / V = 9:1), and collect the eluate.

[0091] (3) HPLC quantitative analysis of aflatoxin B1

[0092] First, place the eluate in a 60 °C oven to dry. After the liquid has evaporated completely, add 300 μL of trifluoroacetic acid and derivatize for 15 minutes; then dry the sample with nitrogen and redissolve it with 200 μL of mobile phase (methanol / water = 1 / 1, v / v). Before the sample is subjected to liquid chromatography detection, filter it through a filter with a pore size of 0.2 μm. HPLC analysis conditions: C18 Agilent XDB (4.6 × 250 mm, 5 μm); methanol / water (45 / 55, v / v) as the mobile phase, flow rate 1.0 mL / min; fluorescence detector, excitation / emission wavelengths of 365 / 435 nm; injection volume 20 μL; column temperature 30 °C.

[0093] (4) Results of spiked recovery rate

[0094] By measuring the spiked recovery rate, the method for the quantitative analysis of aflatoxin B1 in actual samples by solid-phase extraction combined with HPLC established above was further evaluated. As shown in Table 1, in corn, wheat, and rice samples, the spiked recovery rates of AFB1 were 78.57%-91.94%, 92.85%-105.07%, and 85.72%-98.39%, respectively, indicating that MPCM@MIP could effectively capture AFB1 from actual samples and had high accuracy and specificity.

[0095] Table 1. Spiked recovery rates of aflatoxin B1

[0096]

[0097]

Claims

1. A preparation method of magnetic molecularly imprinted inverse opal photonic crystal microspheres, characterized in that, It includes the following steps: (1) Surface modification of magnetic iron oxide: Adjust the solution of tetraethyl orthosilicate to be alkaline, and perform silica coating on the magnetic iron oxide nanoparticles through mechanical stirring in a water bath environment. Then, use a silane coupling agent to form magnetic nanoparticles with grafted double bonds on the surface; (2) Preparation of magnetic molecularly imprinted nanoparticles: Take the modified magnetic nanoparticles in step (1), add a template molecule, a bifunctional monomer, a crosslinking agent, and a reaction solvent, and then carry out the reaction. Subsequently, in an inert gas atmosphere, add an initiator. After the reaction is completed, discard the remaining reaction liquid under the action of magnetic force. After the product is eluted with the template molecule, magnetic molecularly imprinted nanoparticles are obtained; (3) Preparation of magnetic molecularly imprinted inverse opal photonic microspheres: Take the magnetic molecularly imprinted nanoparticles prepared in step (2), mix them with pure water, and then add silica nanoparticles and polystyrene nanoparticle emulsion. Then, form uniform microspheres through microfluidic self-assembly. After curing and drying, remove the polystyrene nanoparticles by high-temperature calcination to obtain inverse opal-structured magnetic molecularly imprinted photonic crystal microspheres; The template molecule described in step (2) is 5,7-dimethoxycoumarin, and the bifunctional monomers are methacrylic acid and styrene; the molar ratio of methacrylic acid to styrene is 2:

1.

2. The preparation method according to claim 1, wherein In step (1), ammonia water is used to adjust the pH of the tetraethyl orthosilicate solution to 8.0 - 9.0, the mechanical stirring speed is 400 - 750 r / min, the constant temperature water bath is 50 °C, and the silane coupling agent is 3-(methacryloyloxy)propyltrimethoxysilane.

3. The preparation method according to claim 1, wherein In step (2), the crosslinking agent is ethylene glycol dimethacrylate, the initiator is azobisisobutyronitrile, and the reaction solvent is acetonitrile or dimethyl sulfoxide or N,N-dimethylformamide.

4. The preparation method according to claim 1, characterized in that, In step (2), take the modified magnetic nanoparticles in step (1), add a template molecule, a bifunctional monomer, a crosslinking agent, and a reaction solvent, and place them at 30 - 40 °C for reaction for 1 - 2 hours.

5. The preparation method according to claim 1, wherein, In step (2), in a nitrogen atmosphere, add an initiator and place it at 60 - 80 °C for reaction for 20 - 24 h.

6. The preparation method according to claim 1, wherein In step (3), the concentration of the aqueous solution of the magnetic molecularly imprinted nanoparticles is 2 - 3%, the concentration of the silica nanoparticle emulsion is 15 - 25%, and the concentration of the polystyrene nanoparticle emulsion is 8 - 15%.

7. The preparation method according to claim 1, characterized in that, In step (3), the curing and drying temperature is 60 - 100 °C, the time is 16 - 24 hours, the high-temperature calcination temperature is 500 - 600 °C, and the time is 2 - 3 hours.

8. A magnetic molecularly imprinted inverse opal photonic crystal microsphere prepared by the preparation method described in claim 1.

9. An application of the magnetic molecularly imprinted inverse opal photonic crystal microsphere described in claim 8 in the selective and efficient separation and enrichment of aflatoxin B1.

10. The application according to claim 9, wherein The microspheres can complete the separation and enrichment of the target toxin aflatoxin B1 with high specificity in a short time through an external magnetic field, and the time does not exceed 20 minutes, and the imprinting factor is up to 8.0.

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