CN116159545A - Preparation method and application of magnetic molecular imprinting inverse opal photonic crystal microsphere - Google Patents

Abstract

The invention discloses a preparation method and application of a magnetic molecular imprinting inverse opal photonic crystal microsphere, wherein the microsphere is synthesized firstly and can specifically identify AFB ₁ The core-shell type nanoscale magnetic molecular imprinting nanoparticle is self-assembled with polystyrene and silicon dioxide nanoparticle to form a novel molecular imprinting inverse opal microsphere, wherein the magnetic molecular imprinting nanoparticle takes ferroferric oxide as a substrate, and can specifically identify aflatoxin B through synthesis of a bifunctional monomer by a surface molecular imprinting strategy ₁ Is prepared by the molecular imprinting layer of (2). The magnetic molecularly imprinted photonic crystal microsphere prepared by the invention has obvious porosityThe structure and the magnetic performance can rapidly and selectively enrich aflatoxin B in the sample ₁ Compared with the traditional enrichment material immunoaffinity column, the material disclosed by the invention is simple to prepare, low in cost and easy to store and recycle.

Description

A preparation method and application of magnetic molecular imprinted inverse opal photonic crystal microspheres

Technical Field

The invention belongs to the field of separation science, and specifically relates to a preparation method and application of magnetic molecular imprinted inverse opal photonic crystal microspheres.

Background Art

Food safety has always been a focus of people's attention. In the process of food processing, transportation, storage and sales, fungal contamination may occur due to irregular supervision and operation. Aflatoxin is one of the most common mycotoxins. It 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 is the most toxic and is also one of the main pathogenic factors of liver cancer. It is widely present in grain and agricultural products such as peanuts, corn, soybeans, and wheat. Due to its high solubility in fat, after the grain 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 high attention internationally and was identified as a "1A" carcinogen by the International Agency for Research on Cancer in 1987. Due to the low content of aflatoxin in food samples and the presence of matrix interference, efficient and highly selective sample pretreatment technology is required to achieve the extraction and analysis of the target.

At present, the main pretreatment technologies for AFB ₁ in the sample to be tested are 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, while immunoaffinity columns are subject to certain limitations due to their high production cost, sensitivity to environmental factors, easy degradation and denaturation, and inability to be reused. Solid phase extraction is 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 separation and enrichment of AFB ₁ in actual samples.

The prior art discloses a magnetic photonic crystal microsphere for enriching and separating aflatoxin _B1 and its preparation method and application (CN2021109175482). The core-shell type surface molecular imprinted magnetic inverse opal photonic crystal microsphere prepared in the period can selectively extract aflatoxin _B1 in the sample. Compared with the material modified with biological antibodies, it can greatly improve the stability of the material and reduce the material preparation cost. However, the core-shell type surface molecular imprinted magnetic inverse opal photonic crystal microsphere is prepared by using a monofunctional monomer on the surface of the photonic crystal microsphere, so it can only provide a single recognition group, resulting in poor selectivity for the target molecule, and the surface polymer causes certain damage to the ordered porous structure of the inverse opal microsphere, affecting the transfer of the substance, and thus affecting the separation and enrichment time.

Summary of the invention

Purpose of the invention: In view of some problems existing in the prior art and materials, the present invention provides a magnetic molecular imprinted inverse opal photonic crystal microsphere. The magnetic molecular 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, thereby improving specificity and enrichment speed.

The invention also provides the magnetic molecular imprinted inverse opal photonic crystal microsphere and application thereof.

Technical solution: In order to achieve the above-mentioned purpose, the preparation method of a magnetic molecular imprinted inverse opal photonic crystal microsphere described in the present invention comprises the following steps:

(1) Surface modification of magnetic ferroferric oxide: a solution of ethyl orthosilicate is adjusted to be alkaline, and the ferroferric oxide nanoparticles are coated with silica by mechanical stirring in a water bath environment, and then a silane coupling agent is used to graft double-bonded magnetic nanoparticles on the surface;

(2) Preparation of magnetic molecular imprinted nanoparticles: Take the magnetic nanoparticles modified in step (1), add template molecules, bifunctional monomers and cross-linking agents and reaction solvents, and then react; then add initiators under an inert gas atmosphere, and after the reaction is completed, discard the remaining reaction liquid under the action of magnetic force, and after the template molecules are eluted by the product, magnetic molecular imprinted nanoparticles are obtained.

(3) Preparation of magnetic molecular imprinted inverse opal photonic crystal microspheres: The magnetic molecular imprinted nanoparticles prepared in step (2) are mixed with pure water, and then silica nanoparticles and polystyrene nanoparticle emulsion are added to form uniform microspheres through microfluidic self-assembly. After curing and drying, the polystyrene nanoparticles are removed by high-temperature calcination to obtain magnetic molecular imprinted photonic crystal microspheres with an inverse opal structure.

The microspheres of the present invention are synthesized by first synthesizing nanoscale magnetic molecular imprinted nanoparticles that can specifically recognize AFB ₁ , and then using the nanoparticles to self-assemble with polystyrene and silicon dioxide nanoparticles into a novel molecular imprinted inverse opal microsphere. The nanoscale magnetic molecular imprinted nanoparticles are based on ferroferric oxide, and a molecular imprinting layer that can specifically recognize aflatoxin B ₁ is synthesized by using a surface molecular imprinting strategy and a bifunctional monomer.

Wherein, in step (1), ammonia water is used to adjust the pH value 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.

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

In step (2), the magnetic nanoparticles modified in step (1) are added with template molecules, bifunctional monomers, crosslinking agents and reaction solvents, and then placed at 30-40° C. for reaction for 1 to 2 hours.

Preferably, in step (2), the magnetic nanoparticles modified in step (1) are added with template molecules, bifunctional monomers, crosslinking agents and reaction solvents, and then reacted at 37°C for 1 hour. In step (2), an initiator is added under a nitrogen atmosphere, and the reaction is carried out at 60-80°C for 20-24 hours.

Wherein, in step (3), the concentration of the magnetic molecular imprinted nanoparticle aqueous solution is 2-3%, the concentration of the silica nanoparticle emulsion is 15-25%, and the concentration of the polystyrene nanoparticle emulsion is 8-15%.

Preferably, the concentration of the magnetic molecular imprinted nanoparticle aqueous solution is 2.5%, the concentration of the silicon dioxide nanoparticle emulsion is 20%, and the concentration of the polystyrene nanoparticle emulsion is 10%. The above concentrations are all mass fractions.

Wherein, the curing and drying temperature in step (3) is 60-100° C., the time is 16-24 hours, and the high temperature burning temperature is 500-600° C., the time is 2-3 hours.

Preferably, the curing and drying temperature is 60° C., the time is 16 hours, and the high-temperature burning temperature is 550° C., the time is 3 hours.

Preferably, in step (3), the template is eluted by Soxhlet extraction, and during Soxhlet reflux, the eluent is a mixed solution of methanol and acetic acid (volume ratio is 9:1).

Preferably, the particle size of the ferrosoferric oxide is 10-50 nm, the particle size of the silicon dioxide nanoparticles is 5-10 nm, and the particle size of the polystyrene nanoparticles is 250-300 nm.

The magnetic molecular imprinted inverse opal photonic crystal microspheres prepared by the preparation method of the present invention. In the synthesis process of the magnetic molecular imprinted inverse opal photonic crystal microspheres of the present invention, only 2 mg of magnetic molecular imprinted nanoparticles with specific recognition function need to be added to synthesize 60 mg of inverse opal microspheres, the amount of polymer used is small (1/30), the maximum adsorption amount is 5.2 ug/mg, and the selectivity coefficient is between 2.5 and 4.0.

The invention discloses an application of the magnetic molecular imprinted inverse opal photonic crystal microspheres in the selective and efficient separation and enrichment of aflatoxin _B1 .

The microspheres can separate and enrich the target toxin aflatoxin _B1 with high specificity in a short time through an external magnetic field, the time does not exceed 20 minutes, and the maximum imprinting factor is 8.0.

The present invention first coats ferroferric oxide nanoparticles with silicon dioxide, then modifies the surface of the nanoparticles with polymerizable functional groups such as propylene, and finally reacts the nanoparticles with molecular prepolymer solution to obtain magnetic molecular imprinted nanoparticles capable of specifically identifying AFB ₁ , and then mixes the nanoparticles with silicon dioxide nanoparticles and polystyrene nanoparticle emulsions to form magnetic molecular imprinted inverse opal microspheres through a microfluidic system for separation and enrichment of AFB ₁ in actual samples. The core-shell magnetic molecular imprinted nanoparticles are synthesized by using a surface molecular imprinting strategy and a bifunctional monomer to form a molecular imprinting layer capable of specifically identifying AFB _1. Compared with traditional enrichment materials such as antibodies and immunoaffinity columns, the material of the present invention is simple to prepare, low in cost, easy to store and reusable.

Specifically, the present invention mixes ferroferric oxide with a surface coated with silicon dioxide and modified with acryl functional groups with a molecular imprinting prepolymer solution, obtains magnetic molecular imprinting nanoparticles capable of specifically recognizing AFB ₁ through thermal initiation polymerization, and then self-assembles them into magnetic molecular imprinting inverse opal microspheres through a microfluidic system. The microspheres use SiO ₂ nanoparticles with a particle size of 5 to 10 nm, PS nanoparticles with a particle size of 250 to 300 nm, and ferroferric oxide with a particle size of 10 to 50 nm.

Design mechanism: The selective recognition principle of the magnetic molecular imprinted inverse opal photonic crystal microspheres of the present invention: After the surface of magnetic nanoparticles is coated with silica and modified with propylene functional groups by tetraethyl orthosilicate, it is used as a carrier, and the structural analogues of AFB ₁ , DMC, MUAC or MDAC, are used as template molecules, which interact with the bifunctional monomers and form a template-monomer complex. Then, the crosslinker and the bifunctional monomers form a densely crosslinked polymer film on the surface of the magnetic nanoparticles under the catalysis of the initiator, and the template is fixed therein. After the polymerization is completed, the template molecules are eluted by Soxhlet extraction, thereby leaving cavities on the surface of the magnetic nanoparticles that match the spatial configuration and action sites of AFB _1. These cavities have selective recognition ability for the structural analogues of the template molecules, AFB ₁ , and then they are self-assembled with SiO ₂ nanoparticles and PS nanoemulsions into inverse opal structure microspheres. Its unique porous structure is conducive to improving the adsorption amount and the transfer of substances, and can be used for efficient selective separation and enrichment of aflatoxin B ₁ .

Compared with the prior art CN2021109175482, the microspheres are prepared first and then the surface is modified by the imprinted polymer. However, in the pre-polymerization treatment, the surface of the microspheres is not hydroxylated, which affects the grafting of double bonds and further affects the surface coating of the molecular imprinted polymer. In the present invention, the magnetic nanoparticles are first coated with silicon dioxide, and the amount of surface hydroxyl groups is increased and then the double bonds are modified with a silane coupling agent, thereby effectively improving the molecular imprinted polymer grafting. In addition, bifunctional monomers are used for polymerization during the polymer synthesis process, which is beneficial to improve molecular recognition. In the whole synthesis process, the prior art is to prepare microspheres first and then perform molecular imprinting on the surface of the microspheres, while the present invention is to first synthesize magnetic molecular imprinted nanoparticles as components for synthesizing magnetic molecular imprinted photonic crystal microspheres, thereby avoiding molecular imprinting on the surface and destroying the ordered porous structure caused by the coating of the microsphere surface by the polymer layer. On the other hand, the use of magnetic molecular imprinted nanoparticles as components for synthesizing molecular imprinted photonic crystal microspheres preserves the unique ordered porous structure of the inverse opal microspheres intactly, which is beneficial to accelerate material transfer and shorten separation and enrichment time.

The magnetic molecular imprinted inverse opal photonic crystal of the present invention uses magnetic molecular imprinted nanoparticles as adsorption carriers, and the molecular imprinted layer polymerized on its surface can selectively identify and enrich and separate _AFB1 in actual samples, and then self-assemble into inverse opal photonic crystal microspheres through a microfluidic system. Its porous ordered structure can accelerate the transfer of substances and increase the enrichment amount. However, in the prior art CN2021109175482, after the molecular imprinted polymer is modified on the surface, 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 transfer of substances, and further affects the separation and enrichment time. The microspheres of the present invention use a surface imprinting strategy to prepare an _AFB1 molecular imprinting layer on the surface of magnetic nanoparticles, which is conducive to the elution and re-adsorption of template molecules (wherein elution refers to the elution of the template in the process of preparing the material; re-adsorption refers to the adsorption and enrichment of the template molecule or the target object in the process of using the material for testing or application), and overcome the defect of the traditional imprinting method that the imprinting site is embedded too deeply, resulting in reduced adsorption efficiency. In addition, its unique magnetic properties give the microspheres magnetism, and no filtration or centrifugation is required, which facilitates the rapid separation of target molecules in complex matrices. The magnetic molecular imprinting inverse opal photonic crystal microspheres prepared by the present invention can rapidly and selectively enrich and separate _AFB1 in actual samples, and the operation is simple and fast, the cost is low, the stability is good, and it can be reused, and it is a good replacement material for _AFB1 biological antibodies.

The present invention coats the surface of magnetic nanoparticles with silicon dioxide, thereby providing a large number of hydroxyl groups for double bond grafting, thereby facilitating the use of bifunctional monomers to prepare a molecular imprinting layer capable of efficiently extracting and separating aflatoxin _B1 . Unlike a monofunctional monomer that can only provide one molecular recognition group, the bifunctional monomer provides different recognition groups to improve the specificity of the molecular polymer; and then self-assembles into an inverse opal structure photonic crystal microsphere by microfluidics. Different from the defect that the molecular imprinting layer is modified on the surface of the inverse opal photonic crystal microsphere by the surface molecular imprinting technology, resulting in the porous structure on the surface of the microsphere being buried by the polymer layer, the novel magnetic molecular imprinting photonic crystal microsphere prepared by the method uses magnetic molecular imprinting nanoparticles instead of traditional ferroferric oxide as a synthetic component of the molecular imprinting inverse protein microsphere, thereby effectively avoiding the clogging problem caused by the later surface modification, perfectly preserving the ordered porous structure of the inverse protein microsphere, facilitating the transfer of substances, greatly shortening the enrichment time, and increasing the enrichment amount. The magnetic nanoparticles can give magnetic properties to substances, and the molecular imprinting layer thereon has a molecular sieve effect, and can specifically enrich and separate target molecules of complex samples.

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

(1) A novel molecular imprinting polymer that can specifically recognize AFB1 was synthesized using bifunctional monomers, thereby improving the enrichment performance of the target toxin.

(2) The present invention polymerizes the imprinted layer on the surface of magnetic nanoparticles. Its unique magnetic properties make it easy to separate and enrich through an external magnetic field without the need for filtration or centrifugation, saving time and effort.

(3) The overall particle size of the magnetic molecular imprinted nanoparticles prepared by the present invention is nanometer-scale, and they are self-assembled into the interior of photonic crystals to synthesize novel magnetic molecular imprinted inverse opal photonic crystal microspheres.

(4) The magnetic molecular imprinted inverse opal photonic crystal microspheres prepared by the present invention well preserve the ordered porous structure of the inverse protein microspheres, have a high imprinting factor, accelerate the transfer of substances, and shorten the enrichment time.

(5) The magnetic molecular imprinted inverse opal photonic crystal microspheres prepared by 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.

(6) The magnetic molecular imprinted inverse opal photonic crystal microspheres prepared by self-assembly technology can meet the requirements of magnetic properties under different environments by adjusting the amount of magnetization. They are easy to store and can effectively replace traditional solid phase extraction materials.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a technical roadmap for preparing magnetic molecularly imprinted inverse opal photonic crystal microspheres;

FIG2 is an infrared characterization of magnetic molecular imprinted nanoparticles after modification and polymerization;

FIG3 shows the effect of different template molecules on the adsorption performance of magnetic molecular imprinted nanoparticles;

FIG4 shows the effect of different functional monomers on the adsorption performance of magnetic molecular imprinted nanoparticles;

FIG5 shows the effect of different magnetization amounts on the adsorption performance of magnetic molecular imprinted nanoparticles;

FIG6 is a morphological characterization of magnetic molecular imprinted inverse opal photonic crystal microspheres;

FIG7 is an adsorption saturation curve of magnetic molecular imprinted inverse opal photonic crystal microspheres;

FIG8 shows the adsorption kinetics of magnetic molecularly imprinted inverse opal photonic crystal microspheres;

FIG. 9 is a test of adsorption specificity of magnetic molecular imprinted inverse opal photonic crystal microspheres.

DETAILED DESCRIPTION

The experimental methods described in the examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified. The overall technical route for the preparation of magnetic molecular imprinted inverse opal photonic crystal microspheres is shown in FIG1 .

The particle size of the Fe ₃ O ₄ dispersion is 10-50 nm, and the particle size of the SiO ₂ nanoparticles is 5-10 nm. Both were purchased from Sigma-Aldrich.

The particle size of polystyrene nanospheres (PS) nanoparticles was about 300 nm and was purchased from Nanjing Caina Biotechnology Co., Ltd., CAS9006-53-6.

Example 1

1. Surface modification of magnetic nanoparticles:

(1) Silica coating: 1.5 g of Fe ₃ O ₄ dispersion (1.5 g of _{Fe 3} O ₄ aqueous dispersion contains 375 mg of Fe ₃ O ₄ ) was placed in a 1000 mL three-necked flask, 100 mL of 0.1 M HCL was added and ultrasonicated for 15 min, then washed with ultrapure water under an external magnetic field until neutral, 50 mL of ultrapure water and 200 mL of ethanol were added and ultrasonicated for 15 min to make them uniformly mixed, then stirred at 650 r/min in a 50°C water bath for 10 min, then 2.5 mL of 25% ammonia water was added and stirred for 30 min until the pH value of the liquid was between 8.0 and 9.0, and a mixed solvent of TEOS and ethanol (1.2 mL TEOS + 8.8 mL ethanol) was added every 5 min, 1 mL each time, in ten times, and stirred at 50°C for 20 h. After the reaction was completed, the mixture was washed alternately with ethanol and ultrapure water for several times until neutral, and vacuum dried for use.

(2) Surface grafting of double bonds: 400 mg of the dried magnetic particles were placed in a 250 mL three-necked flask, and 100 mL of methanol solution was added and ultrasonicated for 15 min. After the magnetic nanoparticles were mixed, nitrogen was introduced. 7.5 mL of 3-(methacryloyloxy)propyltrimethoxysilane was added dropwise under a nitrogen atmosphere, and ultrasonicated for 10 min. The mixture was placed in a 25°C constant temperature water bath and vigorously mechanically stirred for 24 h. After the reaction was completed, the mixture was washed alternately with ethanol and ultrapure water several times until neutral, and then vacuum dried for use.

Example 2

Using the magnetic nanoparticles prepared in Example 1 as a substrate, magnetic molecular imprinted nanoparticles were synthesized in the following specific steps:

1. Molecular imprinting polymerization:

Using the surface molecular imprinting technique, 30 mg of surface-modified magnetic nanoparticles were placed in a centrifuge tube, template molecules, bifunctional monomers, crosslinkers were added, and the volume was adjusted to 6 mL with a porogen (i.e., reaction solvent acetonitrile), and the reaction was carried out on a shaker at 37°C for 1 hour at a speed of 200 rpm. Then nitrogen was passed for 6 minutes, and then the initiator was added. The centrifuge tube was sealed with a sealing film and placed in a shaker at 60°C and 220 rpm for 24 hours. After the reaction, the reaction liquid was discarded under the action of a magnet to obtain magnetic molecular imprinted nanoparticles (MMIPs) coated with an organic polymer layer.

2. Removal of template molecules:

Using the Soxhlet extraction method, the nanoparticles in step 1 were wrapped with filter paper and placed in a siphon. 120 mL of a methanol-acetic acid (9:1, v/v) mixture was added to the flask. After Soxhlet reflux elution at 80°C for 12 h, 120 mL of pure methanol was replaced to wash away the acetic acid. Magnetic molecularly imprinted nanoparticles were obtained by vacuum drying. The preparation steps of non-imprinted magnetic molecularly imprinted nanoparticles (MNIPs) were the same as steps 1 and 2 above, but no template molecules were added.

3. Optimization of synthesis conditions:

Different template molecules, functional monomers and magnetic addition amounts were investigated according to the methods in steps 1 and 2 above.

(1) Template molecule

Take 30 mg of the magnetic nanoparticles modified with double bonds in Example 1 in a centrifuge tube (the final product of step 2 of Example 1), add 0.03 mmol of template molecules (5,7-dimethoxycoumarin (DMC), 7-acetoxy-4-methylcoumarin (MUAC) or 7-diethylamino-4-methylcoumarin (MDAC)), bifunctional monomer methacrylic acid MAA 0.3 mmol, styrene ST 0.015 mmol and crosslinker ethylene glycol dimethacrylate EGDMA 0.75 mmol and dilute to 6 mL with acetonitrile, shake at 37 ° C for 1 hour, and the speed is 200 rpm. Then, nitrogen is passed for 6 minutes, and the initiator azobisisobutyronitrile AIBN 0.014 mmol is added, and the centrifuge tube is sealed with a sealing film, and finally placed in a 60 ° C shaker at 220 rpm for 24 hours. After the reaction is completed, the template molecules are removed by Soxhlet extraction, and three MMIPs can be obtained.

The adsorption performance of the three prepared magnetic molecular imprinted nanoparticles on the corresponding template molecules is shown in Figure 3. The imprinting factors of the MIPs synthesized with three templates of DMC, MUAC and MDAC on the corresponding templates are respectively: 1.44, 0.97, 1.13 (imprinting factor is the ratio of the adsorption amount of the molecular imprinting polymer on the template molecule to the adsorption amount of the non-molecular imprinting polymer on the template molecule); the adsorption amounts are respectively 1.53, 1.18, 1.28ug/mg, indicating that the imprinting material synthesized with DMC as the template has good specificity for AFB1 and strong adsorption capacity, so DMC is selected as the template molecule in subsequent experiments. The adsorption performance/adsorption amount refers to Example 5, and the adsorption performance of the template molecule is detected as follows: the polymer (three MMIPs) and the template molecule are mixed in the adsorption solvent, and after shaking at room temperature overnight, the supernatant is taken to test the template molecule concentration therein, that is, the template molecule concentration in the solution after adsorption, and the adsorption amount of the polymer is calculated according to the difference between the template molecule concentration before and after adsorption.

(2) Functional monomer

The interaction between the monomer and the template determines the strength of the imprinting material's ability to select and recognize the target molecule. Therefore, it is crucial to select a suitable functional monomer. According to the method in step (1) above, methyl methacrylate (MAA), 4-vinyl pyridine (4-VP), styrene (ST) and acrylamide (AAm) were selected as monomers to react with the template molecule DMC to prepare different magnetic molecular imprinted nanoparticles, and adsorption experiments were carried out on the template molecules. The results are shown in Figure 4a). The adsorption amounts of DMC by MMIPs prepared with MAA, 4-VP, ST and AAm as functional monomers are 1.53, 0.51, 3.34, and 0.63, respectively. 0.16ug/mg; the imprinting factors are 1.44, 1.02, 1.08 and 1 respectively. In order to improve the adsorption amount and selectivity, ST and MAA are used as bifunctional monomers, and their ratio is optimized. The result is shown in Figure 4b. However, when ST:MAA＝1:2 (molar ratio), the adsorption amount of DMC is 2.06ug/mg, and the imprinting factor is 1.62. The other imprinting factors are 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 is selected as the bifunctional monomer.

In addition, the method of Example 2 was used to examine the magnetic molecular imprinting polymers prepared by using the template molecule acetoxy-4-methylcoumarin (MUAC) or 7-diethylamino-4-methylcoumarin (MDAC) and the monomers 4-vinylpyridine (4-VP) or styrene (ST). The effects were also significantly inferior to the polymers prepared by using the template molecule as DMC and the bifunctional monomer as ST:MAA=1:2.

(3) Adding magnetism

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

4. MMIPs morphology characterization:

The MMIPs prepared above (template molecule is DMC, functional monomer is ST:MAA＝1:2) were characterized by infrared spectroscopy, see Figure 2. There is a typical Si-O bending vibration peak at 465cm ^-1 , 805cm- ¹ and 1085cm ^-1 are symmetric and asymmetric stretching of Si-O respectively. After Fe ₃ O ₄ -SiO ₂ was modified with 3-(methacryloyloxy)propyltrimethoxysilane, the C＝O group of Fe ₃ O ₄ -SiO ₂ -CH ₂ ＝CH ₂ showed weak vibration near 1720cm ^-1 , indicating that the double bond functional group was successfully grafted on the surface of Fe ₃ O ₄ -SiO _2. Strong characteristic peaks of C＝O groups from MAA (methacrylic acid) and EGDMA (ethylene glycol dimethacrylate) appeared near 1730cm ^-1 , indicating that the organic polymer was successfully attached to the surface of magnetic nanoparticles.

Example 3

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 the mixture is finally shaken at 60° C. and 220 rpm for 24 hours.

Example 4

1. Preparation of magnetic molecular imprinted inverse opal photonic crystal microspheres:

The optimal magnetic molecular imprinted nanoparticles in Example 2 were used, and a final concentration (mass fraction) of 20% of a silicon dioxide nano solution, 10% of a PS nano solution, and 2.5% of a magnetic molecular imprinted nanoparticle aqueous solution were placed in a centrifuge tube, ultrasonically mixed to form a uniform emulsion, and transferred to a 5 mL syringe. Another large 50 mL syringe was filled with methyl silicone oil. The two syringes were fixed on a constant current microfluidic pump, respectively, with an oil phase microfluidic flow rate of 10 mL/h and an emulsion phase flow rate of 5 mL/h. Using the principle of water-in-oil, the methyl silicone oil cut the emulsion into micron-sized droplets and collected them in a plastic culture dish containing methyl silicone oil. Then, the culture dish was placed in a 60°C forced air drying oven to dry the water in the droplets, and then washed with n-hexane and anhydrous ethanol for 3 to 4 times, and after the ethanol evaporated, it was transferred to a tube furnace and slowly heated to 550°C for calcination for 3 hours to remove the template microspheres (PS microspheres), and magnetic molecular imprinted inverse opal photonic crystal microspheres (MPCM@MIP) were obtained. Among them, the volume ratio of MMIPs (or MNIPs): SiO ₂ in the original emulsion (20% silica nano solution, 10% PS nano solution and 2.5% magnetic molecular imprinted nanoparticle aqueous solution) was 1:3, and the SiO ₂ :PS volume ratio is 1:8, and it is characterized by metallographic microscope and scanning electron microscope, see Figure 6, wherein Figure 6a shows that the microspheres prepared by using modified molecular imprinting polymerized magnetic nanoparticles as components for synthesizing inverse opal microspheres have good magnetic properties, Figure 6b is a metallographic microscope image of magnetic molecular imprinting inverse opal photonic crystal microspheres, with obvious bright spots in the center, indicating that the surface structure of the microspheres is regular and has the general characteristics of inverse opal, and Figures 6c and 6d are SEM images of the microspheres under 500X and 5000X magnifications, respectively, which show The prepared magnetic molecularly imprinted inverse opal microspheres are regular spherical as a whole, and their surface has an obvious ordered porous structure. By comparing the characterization diagram of the inverse opal microspheres in Figure 2d of CN2021109175482, it can be seen that the porous structure on its surface is not destroyed. In the prior art CN2021109175482, after the surface is modified with molecular imprinting polymer, the porous structure is blocked by the polymer layer (Figure 6f), which causes certain damage to the ordered porous structure of the inverse opal microspheres, affects the transfer of substances, and thus affects the separation and enrichment time.

Example 5

The MPCM@MIP prepared in Example 4 was used as the material, and its adsorption performance was investigated and analyzed.

Three 4 mg microspheres prepared in Example 4 were taken and placed in 1000 uL of 5 to 50000 ng/mL _AFB1 solutions to determine the enrichment capacity of MPCM@MIP and MPCM@NIP (microspheres prepared by 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 by MMIPs with imprinted cavities on the surface that can specifically recognize AFB1, MPCM@MIP can separate and enrich more AFB1 than MPCM@NIP. As the concentration of AFB1 increases, the adsorption amount of MPCM@MIP also gradually increases, and its maximum imprinting factor is 8.0.

The adsorption saturation rate of MPCM@MIP and MPCM@NIP was measured with 1000 ng/mL AFB1. The results are shown in Figure 8. At this concentration, the highest imprinting factor was 1.74. Because MMIPs are surface molecular imprinting polymers, their binding sites are exposed on the particle surface, which can achieve rapid separation and enrichment of target substances. The ordered porous structure of MPCM also promotes the transfer of target molecules. Therefore, MPCM@MIP can quickly 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 of Example 3 in CN2021109175482 as shown in Figure 6h, which are 6.7 nm and 60 m ² /g, respectively, it can be found that the porosity and specific surface area of MPCM@MIP prepared in Example 4 of the present invention (Figure 6g) 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 it is to the contact between the microspheres and the target molecules, and the larger the porosity, the more conducive it is to the transfer of substances. Therefore, under the same conditions, the enrichment rate of the present invention is about 7 times that of CN2021109175482, which effectively shortens the enrichment time.

Example 6

The MPCM@MIP prepared in Example 4 was used as the material to investigate and analyze its specificity.

Take three portions of 4 mg microspheres prepared in Example 4 and add them to a 2 mL centrifuge tube, add 1000 uL 1000 ng/mL AFB1, AFB2, AFG2, OTA, and ZEN respectively, and shake at 200 rpm for 4 hours at room temperature for adsorption experiments. After the adsorption is completed, the adsorption supernatant can be taken out using a magnet without centrifugation, and the supernatant is detected using an HPLC system. As shown in Figure 9, 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), the imprinting factor increased by 0.26. This is mainly because during the synthesis process, the amount of magnetic molecular imprinted polymer nanoparticles added is low (each 60 mg of photonic crystal microspheres contains only about 2 mg of magnetic molecular imprinted nanoparticles). The overall added amount can be converted into only 0.13 mg of magnetic molecular imprinted polymer nanoparticles that can specifically recognize AFB1 in each 4 mg of microspheres through mass conversion, and a small amount of nanoparticles shows better selectivity. Compared with CN2021109175482, which uses 2 mg of molecularly imprinted microspheres per mL of test sample (patent CN2021109175482 prepares molecularly imprinted polymers on the surface of microspheres, so the whole has recognition sites and is a pure molecularly imprinted photonic crystal microsphere, while the present invention first synthesizes magnetic molecularly imprinted nanoparticles and then assembles them into microspheres. The overall recognition sites are only available to the magnetic nanoparticles assembled into the microspheres), the molecularly imprinted polymer with recognition in the present invention only uses 0.13 mg, which is significantly lower than the above-mentioned patent. The polymer with specific recognition of AFB1 per unit mass of the present invention has more obvious selectivity.

Example 7

Aflatoxin B1 in actual samples was separated and enriched, and quantitative analysis was performed in combination with HPLC.

(1) Preparation of spiked samples

Corn, wheat and rice were selected as real samples. Weigh 5g of sample in four portions in 50mL centrifuge tubes, dilute AFB ₁ stock solution (2mg/mL) with methanol to 1000ng/mL. Take 0mL, 0.125mL, 0.25mL and 1.25mL respectively and add them to the weighed sample tubes, and dilute to 10mL. Shake to mix the sample and toxin evenly, place in a fume hood and let stand overnight until the methanol evaporates. Add 0.5g of sodium chloride and 25mL of a mixed solution of methanol and water (V/V=8:2) to the sample after methanol evaporates, homogenize in a homogenizer for 5min, and then extract the toxin by ultrasound for 30min. After complete extraction, filter several times with qualitative filter paper and a 0.22um filter head and store in a -20℃ refrigerator.

Take an appropriate amount of filtered sample solution and dry it, then add an equal amount of a mixture 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.

(7) Toxin extraction and enrichment

Weigh 4 mg of MPCM@MIP and add it to 1 mL of the actual sample solution. After shaking at 200 rpm for 4 h at room temperature, 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.

(3) HPLC quantitative analysis of aflatoxin _B1

First, the eluent was placed in a 60°C oven to dry, and after the liquid evaporated, 300 μL of trifluoroacetic acid was added and derivatized for 15 minutes; then the sample was blown dry with nitrogen and reconstituted with 200 μL of mobile phase (methanol/water = 1/1, v/v). Before the sample was tested by liquid chromatography, it was filtered with 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 mobile phase, flow rate 1.0 mL/min; fluorescence detector, excitation/emission wavelength 365/435 nm; injection volume 20 μL; column temperature 30°C.

(4) Spike recovery results

The above-mentioned solid phase extraction combined with HPLC quantitative analysis of aflatoxin B1 in real samples was further evaluated by measuring the spike recovery rate. As shown in Table 1, the spike recovery rates of AFB1 in corn, wheat and rice samples were 78.57%-91.94%, 92.85%-105.07% and 85.72%-98.39%, respectively, indicating that MPCM@MIP can effectively capture AFB1 from real samples with high accuracy and specificity.

Table 1. Spiked recovery of aflatoxin B ₁

Claims (10)

1. A preparation method for magnetic molecularly imprinted inverse opal photonic crystal microspheres, characterized in that, comprising the steps: (1) Surface modification of magnetic ferroferric oxide: adjust the solution of ethyl orthosilicate to make it alkaline, and mechanically stir ferric ferric oxide nanoparticles in a water bath environment for silica coating, and then use silyl The coupling agent makes the magnetic nanoparticles with double bonds grafted on the surface; (2) Preparation of magnetic molecularly imprinted nanoparticles: take the modified magnetic nanoparticles in step (1), add template molecules, bifunctional monomers, cross-linking agents and reaction solvents, and react; then in an inert gas atmosphere , adding an initiator, discarding the remaining reaction liquid under the action of magnetic force after the reaction, and obtaining the magnetic molecularly imprinted nanoparticles after the template molecule is eluted from the product. (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 emulsions , through microfluidic self-assembly to form uniform microspheres, after curing and drying, high-temperature burning to remove polystyrene nanoparticles, and obtain inverse opal structure magnetic molecularly imprinted photonic crystal microspheres. 2. the preparation method according to claim 1 is characterized in that, adopting ammoniacal liquor to regulate the solution pH of tetraethyl orthosilicate in step (1) is 8.0～9.0, and mechanical stirring speed is 400-750r/min, and constant temperature water bath is 50°C, the silane-based coupling agent is 3-(methacryloyloxy)propyltrimethoxysilane. 3. preparation method according to claim 1, is characterized in that, template molecule described in step (2) is preferably 5,7-dimethoxycoumarin, and bifunctional monomer is methacrylic acid and styrene , 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. preparation method according to claim 1 is characterized in that, in step (2), get the magnetic nanoparticle that is modified in step (1), after adding template molecule, bifunctional monomer and linking agent and reaction solvent , placed at 30-40 ° C for 1 to 2 hours. 5 . The preparation method according to claim 1 , characterized in that, in step (2), under a nitrogen atmosphere, an initiator is added, and the mixture is placed at 60-80° C. for 20-24 hours. 6. The preparation method according to claim 1, characterized in that, the concentration of the magnetic molecularly imprinted nanoparticle aqueous solution in step (3) is 2 to 3%, and the concentration of the silica nanoparticle emulsion is 15 to 25%. %, the concentration of the polystyrene nano particle emulsion is 8-15%. 7. The preparation method according to claim 1, characterized in that, the curing and drying temperature in step (3) is 60-100°C, the time is 16-24 hours, the high-temperature burning temperature is 500-600°C, and the time is 2 to 3 hours. 8. A magnetic molecularly imprinted inverse opal photonic crystal microsphere prepared by the preparation method according to claim 1. 9. The application of the magnetic molecularly imprinted inverse opal photonic crystal microsphere according to claim 8 in the selective and efficient separation and enrichment of aflatoxin _B1 . 10. The application according to claim 9, characterized in that, the microspheres can achieve the separation and enrichment of the target toxin Aspergillus toxin _B1 with high specificity in a relatively short period of time by means of an external magnetic field, and the time does not exceed 20 minutes. Imprinting factor up to 8.0.

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