Preparation method and application of a Pickering emulsion interface-induced confined polymerization imprinted adsorbent

Abstract

The present invention belongs to the technical field of molecular recognition adsorption separation functional materials, and discloses a preparation method and application of a Pickering emulsion interface-induced confined polymerization imprinted adsorbent. The present invention uses glycidyl methacrylate nanospheres as the matrix material, reacts with iminodiacetic acid and FeCl3 solution to modify carboxyl groups on its surface and chelate ferric ions, and uses this as Pickering particles to stabilize the water-in-oil (O / W) Pickering emulsion with dichloromethane as the oil phase, so that the water-soluble target molecule AMP attracted by ferric ions to the surface of Pickering particles forms a base complementary pairing interaction with the oil-soluble functional monomer 1-(vinylbenzyl) thymine at the oil-water interface of the emulsion; then, ultraviolet light is used to initiate the confined polymerization of MIPs on the surface of Pickering particles, rather than the polymerization in the oil phase or water phase in the prior art. The adsorbent obtained by this invention enables the imprinting process to get rid of the limitation of the different solubilities of the template molecule and the functional monomer in oil or water, and is used for the rapid, large-scale, and precise adsorption and separation of AMP in aqueous solution.

Description

Technical Field

[0001] The present invention belongs to the technical field of preparation of molecular recognition adsorption separation functional materials, and particularly relates to a preparation method and application of a Pickering emulsion interface-induced confined polymerization imprinted adsorbent. Background Art

[0002] Adenosine is a special endogenous purine nucleoside derivative and widely exists in biological systems. Adenosine has good physiological activities, participates in the transmission of genetic information of biological cells, affects protein synthesis and polysaccharide metabolism, and plays a very important regulatory role in the growth, proliferation, differentiation and inhibition of cells in organisms. 5'-Adenosine monophosphate (AMP) is a typical representative of adenosine, a nucleotide found in ribonucleic acid (RNA), soluble in water with its solubility positively correlated with temperature, and poorly soluble in organic solvents. AMP is produced by hydrolysis of adenosine diphosphate (ADP) or adenosine triphosphate (ATP), and consumed by conversion to ADP through adenylate kinase (AMPK). AMP is an important participant in biological life activities and an important intermediate recognized by many new anti-cancer, anti-viral and anti-AIDS drugs, so it is widely demanded in the international market. At present, various methods for synthesizing AMP all face problems such as many by-products in the product and low AMP content. Moreover, AMP has similar structures, polarities and charges to various nucleoside compounds such as ADP, ATP, 2'-deoxyadenosine (dA), 2'-deoxyguanosine (dG) and 2'-deoxycytidine (dC), with some differences in partial functional groups or molecular sizes, which adds difficulty to its precise separation and purification, thus severely restricting the research and application development of high-quality AMP and its derivatives. Therefore, exploring a new method for selective separation and purification of AMP with independent intellectual property rights has important scientific, economic and social significance in the fields of research and development of new anti-cancer and anti-viral drugs, medical detection, etc.

[0003] Molecular recognition is closely related to life activities and has attracted extensive attention in research fields such as drug delivery, catalysis, and immunoassay. Molecular imprinting technology is an effective technique for generating specific binding sites for target molecules in a polymer network. The resulting molecularly imprinted polymers (MIPs) provide artificial receptor-like recognition sites that are selective for the template molecule, enabling highly specific and selective molecular recognition. Compared with natural receptors such as antibodies and enzymes, this artificially synthesized receptor has the advantages of high chemical stability, easy access and storage, low cost, and strong reusability. Although traditional MIPs have significantly improved the adsorption selectivity and affinity of adsorbents, most of their binding sites are buried deep in the highly cross-linked polymer, making it difficult for target molecules to reach the internal binding sites, resulting in problems such as low mass transfer rate, poor accessibility of target molecules, low adsorption capacity, and difficult template elution. The proposed surface molecular imprinting technology effectively solves these problems. Surface molecularly imprinted polymers (SMIPs) refer to grafting MIPs on the surface or near the surface of nanocarrier materials to establish specific binding sites. The ultra-high specific surface area brought by the nanoscale reduces the diffusion distance, making it easier for target molecules to approach the imprinted sites, which is an effective method to improve the mass transfer efficiency and adsorption capacity of imprinted adsorbents.

[0004] Emulsion polymerization is one of the conventional methods for preparing MIPs. Emulsions are formed by emulsifying two immiscible liquids with the help of external forces such as mechanical stirring, ultrasound, hand shaking, and microfluidics. Nanoparticles, functional molecules, and active ingredients are loaded into the emulsified droplet microspheres and form hydrogels after polymerization. In recent years, many studies have focused on the preparation of spherical adsorbents by the emulsion template method, which has the characteristics of controllable size, confined shaping, and batch preparation. General emulsion polymerization requires a large amount of surfactants or block copolymers to maintain the stability of the system before polymerization. The introduction of surfactants or block copolymers will cause new pollution during the preparation process and is likely to remain in the polymerized hydrogel, thus affecting the material properties. Therefore, in subsequent studies, it is inclined to use Pickering emulsions stabilized by solid particles at the oil-water interface to prepare MIPs. This method not only has better emulsion stability but also avoids the problems of pollution caused by surfactants and easy residue. Pickering particles are located at the two-phase interface and are insoluble in any one phase. The surface tension of Pickering particles determines that the prepared Pickering emulsion is a water-in-oil or oil-in-water system with better stability.

[0005] In recent years, researchers have continuously optimized the details of the method for preparing spherical MIPs nanoimprinted adsorbents using the Pickering emulsion template method. For example: (1) Fixing the template molecules on the surface of solid particles so that the recognition sites are located on the polymer surface, further improving the adsorption rate of MIPs. This innovation solves the problem that most recognition sites are located inside highly cross-linked polymer matrices, resulting in reduced mass transfer efficiency. (2) Selecting functional monomers that are soluble in both the aqueous and oil phases, making MIPs have good hydrophilicity and enabling them to exhibit excellent specific molecular recognition performance in aqueous solutions. MIPs hydrogels prepared using Pickering emulsions have been widely applied in various fields such as chemical separation, wastewater treatment, cosmetics, and drug delivery, and have later been extended to scientific research work such as manufacturing advanced materials with complex structures.

[0006] However, to date, there is still an issue worthy of optimization in the research on preparing spherical MIPs nanoimprinted adsorbents using the Pickering emulsion template method, and there has been little discussion on this so far: The Pickering emulsion template method requires that functional components such as template molecules, functional monomers, cross-linking agents, and initiators must be simultaneously and well-dissolved in either the oil phase or the aqueous phase. When the template molecules and functional monomers are only soluble in the oil phase or the aqueous phase respectively, pre-assembly cannot be carried out, resulting in imprinting failure. To solve this problem, highly toxic reagents such as the "universal solvent" dimethyl sulfoxide (DMSO) need to be relied on, and the condition of "immiscible two phases" will be destroyed, preventing the formation of emulsion droplets. Therefore, the present invention proposes a suitable strategy of carrying out polymerization confined at the oil-water interface of a Pickering emulsion stabilized by Pickering particles, enabling the water-soluble target molecule AMP and the oil-soluble functional monomer VBT to assemble and match at the oil-water interface, polymerizing to form a MIPs layer on the surface of the Pickering particles, and using this imprinted adsorbent for the selective separation of water-soluble AMP. While breaking through the above problems, the present invention also significantly improves the adsorption rate, adsorption selectivity, and adsorption capacity for the target molecule AMP, enabling this nanoimprinted adsorbent to achieve efficient separation of AMP in aqueous solutions and actual samples (human urine). Summary of the Invention

[0007] The present invention first synthesizes glycidyl methacrylate (GMA) nanoparticles through a soap-free emulsion polymerization method, and modifies carboxyl groups on its surface and chelates ferric ions as Pickering particles (GMA-IDA-Fe 3 by reacting with iminodiacetic acid (IDA) and an FeCl 3+) By utilizing the electrostatic interaction between it and the phosphate groups carried by AMP, AMP is aggregated onto the surface of Pickering particles. Secondly, the Pickering particles are used to stabilize the water-in-oil (O / W) Pickering emulsion with dichloromethane as the oil phase, enabling the water-soluble target molecule AMP and the oil-soluble functional monomer 1-(vinylbenzyl) thymine (VBT) to assemble and match at the oil-water interface. Then, VBT is polymerized on the surface of the Pickering particles by ultraviolet light irradiation to form an MIPs layer. Finally, the template molecule AMP is eluted to obtain a nano-imprinted adsorbent (GMA-IDA-Fe 3+ @MIPs) grafted with an MIPs layer on the surface of Pickering particles. In the present invention, taking the Pickering emulsion interface as a bridge, the water-soluble target molecule AMP and the oil-soluble functional monomer VBT are assembled and matched at the oil-water interface, constructing a nano-imprinted adsorbent that is not restricted by water / oil solubility for efficient adsorption and separation of AMP molecules. In addition, the surface molecular imprinting technology greatly shortens the diffusion distance, improves the mass transfer rate, and greatly optimizes the processes of specific adsorption and template molecule desorption; the nano-sized lightweight GMA substrate provides a relatively high specific surface area, effectively enhancing the adsorption capacity.

[0008] The present invention provides a preparation method of a confinement polymerization imprinted adsorbent induced by a Pickering emulsion interface, and uses an AMP simulated solution to evaluate the performance of the GMA-IDA-Fe 3+ @MIPs adsorbent for selectively adsorbing and separating AMP molecules. The method comprises the following steps:

[0009] (1) Preparation of GMA nanoparticles:

[0010] Firstly, GMA nanoparticles are prepared by the prior art. The specific steps are as follows: A certain amount of glycidyl methacrylate (GMA), styrene, divinylbenzene (DVB), and a certain volume of deionized water are stirred and mixed evenly in a round-bottom flask, and the mixed system is stirred for a period of time under nitrogen protection at a certain temperature. Subsequently, deionized water dissolved with a certain amount of potassium persulfate (KPS) is added to the flask, and the reaction is carried out for a period of time. After the reaction is completed, the product is collected by centrifugation and washed three times with ethanol and water respectively, and then dried under vacuum to obtain GMA nanoparticles;

[0011] (2) Preparation of Pickering nanoparticles (GMA-IDA-Fe 3+ )

[0012] Take a certain amount of the GMA nanoparticles prepared in step (1), ultrasonically disperse them in deionized water, adjust the pH of the system to 11 with an aqueous NaOH solution, and then add a certain amount of iminodiacetic acid (IDA) thereto. The reaction is carried out under stirring conditions at a certain temperature for a period of time. After the reaction is completed, the product is collected by centrifugation and washed several times with water to obtain GMA-IDA nanoparticles with carboxyl groups grafted on the surface. Transfer the above product to an aqueous solution of FeCl 3 and ultrasonically disperse it evenly. Stir at room temperature for a period of time, collect the product by centrifugation and wash it several times with water. After vacuum drying, GMA-IDA-Fe 3+ nanoparticles are obtained and ground for standby;

[0013] (3) Preparation of Pickering emulsion:

[0014] For the preparation of the aqueous phase: Ultrasonically dissolve a certain amount of the template molecule AMP and a certain amount of the photoinitiator (2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone) completely in a certain volume of Tris-HCl buffer solution, and then add a certain amount of Pickering particles (GMA-IDA-Fe 3+ ) thereto. Ultrasonically disperse them sufficiently to form a suspension, and self-assemble at a certain temperature for a period of time to enable the template molecule AMP to be chelated to the surface of the Pickering particles through electrostatic attraction.

[0015] For the preparation of the oil phase: Ultrasonically dissolve a certain amount of the synthesized functional monomer 1-(vinylbenzyl) thymine (VBT) and a certain amount of ethylene glycol dimethacrylate (EGDMA) completely in a certain volume of dichloromethane;

[0016] (4) Preparation of GMA-IDA-Fe 3+ @MIPs:

[0017] Take the prepared aqueous phase and oil phase in different ratios in a centrifuge tube, shake and ultrasonically emulsify them for a period of time to form a stable oil-in-water (O / W) Pickering emulsion. Gently purge nitrogen into it and then seal the centrifuge tube. Self-assemble at room temperature in the dark for a period of time to enable the template molecule AMP and the functional monomer VBT to form a base complementary pairing interaction at the oil-water interface of the emulsion. Then, transfer the Pickering emulsion into a transparent glass bottle and irradiate it with a UV light source of a fixed wavelength in an incubator at a certain temperature for a period of time. After the isothermal polymerization is completed, collect the product by centrifugation, wash it several times with ethanol and water respectively to remove the residues, then elute the template molecule AMP with a mixed eluent, and wash it with water until it is neutral. After vacuum drying, a nanoimprint adsorbent with an MIPs layer grafted on the surface of the Pickering particles is obtained, denoted as GMA-IDA-Fe 3+ @MIPs.

[0018] In step (1), the dosage ratio of GMA, styrene, DVB, KPS, and deionized water used in the reaction system is 1 mL:(0.157 - 0.235) mL:(0.0218 - 0.0327) mL:(0.024 - 0.036) mg:50 mL; the temperature under nitrogen protection at a certain temperature is 70 °C, the stirring time is 30 min; the reaction time after adding KPS is 5 h.

[0019] In step (2), the dosage of GMA nanoparticles and IDA is: 100 mg:(0.528 - 0.792) mg; the reaction time for a period of time is: 22 - 26 h. The concentration of the NaOH aqueous solution is 2.0 mol / L; the reaction temperature is 70 °C, and the reaction time is 22 - 26 h.

[0020] In step (2), the FeCl 3 concentration of the aqueous solution is: (0.08 - 0.12 mol / L); the stirring time at room temperature for a period of time is: 10 - 14 h.

[0021] In step (3), the concentration of the Tris-HCl buffer solution is 0.05 M, pH = 7.4;

[0022] In the aqueous phase, the dosage ratio of Pickering particles (GMA-IDA-Fe 3+ ), template molecule AMP, photoinitiator 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone, and Tris-HCl buffer solution is 30 mg:16 mg:(2.48 - 3.72) mg:(3 - 5) mL; the self-assembly temperature is 25 °C, and the self-assembly time is 1 - 3 h.

[0023] In the oil phase, the dosage ratio of functional monomer VBT, crosslinking agent EGDMA, and dichloromethane is (8.96 - 13.44) mg:(29.36 - 44.04) μL:(1 - 3) mL;

[0024] In step (4), the volume ratio of different ratios of water to oil (Tris-HCl buffer solution:dichloromethane) is 3:3 to 5:1;

[0025] In step (4), the emulsification time for a period of time is: 4 - 6 min; the self-assembly time of the template molecule AMP and the functional monomer VBT at the emulsion oil-water interface in the dark is 7 - 9 h; the ultraviolet light irradiation for a period of time is: 6 - 10 h;

[0026] In the above method, if the template molecule 5′-adenosine monophosphate (AMP) is not added, then the non-imprinted polymer GMA-IDA-Fe is prepared3+ @NIPs.

[0027] Use of GMA-IDA-Fe prepared by the present invention 3+ @MIPs for the selective adsorption and separation of AMP. Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] The present invention uses a photoinitiation technique, VBT as a functional monomer, and the oil-water interface stabilized by Pickering particles as a bridge to assemble and match the water-soluble target molecule AMP and the oil-soluble functional monomer VBT at the oil-water interface of the Pickering emulsion, and carry out confinement polymerization at the oil-water interface of the emulsion. In addition, the surface molecular imprinting technique greatly shortens the diffusion distance, improves the mass transfer rate, and greatly optimizes the processes of specific adsorption and template molecule desorption; the nano-sized lightweight GMA substrate provides a higher specific surface area and effectively improves the adsorption capacity. In short, this technique enables the imprinting process to get rid of the limitation of different solubilities of the template molecule and the functional monomer in oil or water, and realizes the rapid, large-scale, and precise adsorption of AMP molecules. Description of the Drawings

[0029] Figure 1 1H nuclear magnetic resonance spectrum of the functional monomer 1-(vinylbenzyl) thymine (VBT) prepared in Example 1.

[0030] Figure 2 Scanning electron microscope images of the GMA nanoparticles (a, b), GMA-IDA-Fe 3+ @MIPs (c, d), GMA-IDA-Fe 3+ @MIPs-stabilized Pickering emulsions of different sizes (e, f, g, h) prepared in Example 1 and transmission electron microscope images of ultrasonically dispersed GMA-IDA-Fe 3+ @MIPs (i, j, k, l).

[0031] Figure 3 Bright-field micrographs of the Pickering emulsion prepared in Example 1 at 1 min (a, b) and 48 h (c) after emulsification, and fluorescence micrographs of fluorescein isothiocyanate (FITC)-labeled Pickering particles (d, e) and rhodamine B-labeled oil phase (f, g) under dark field.

[0032] Figure 4 Infrared spectra of GMA, GMA-IDA-Fe 3+ and GMA-IDA-Fe 3+ @MIPs prepared in Example 1.

[0033] Figure 5GMA-IDA-Fe prepared in Example 1 3+ 、GMA-IDA-Fe 3+ @NIPs and GMA-IDA-Fe 3+ @MIPs kinetic data for AMP and their non-linear kinetic model fitting curves.

[0034] Figure 6 GMA-IDA-Fe prepared in Example 1 3+ @NIPs and GMA-IDA-Fe 3+ @MIPs equilibrium data and Langmuir and Freundlich non-linear model fitting curves.

[0035] Figure 7 GMA-IDA-Fe prepared in Example 1 3+ 、GMA-IDA-Fe 3+ @NIPs and GMA-IDA-Fe 3+ @MIPs single-component adsorption results for dG, dC, dA, AMP, ADP and ATP.

[0036] Figure 8 GMA-IDA-Fe prepared in Example 1 3+ @NIPs and GMA-IDA-Fe 3+ @MIPs regeneration adsorption capacity. Detailed implementation method

[0037] To better enable those skilled in the art to understand the technical solutions of the present invention, the technical solutions of the present invention will be further described below in conjunction with specific embodiments and drawings.

[0038] The identification performance evaluation in the specific implementation method of the present invention is carried out according to the following method:

[0039] Add 2 mL of AMP solution with an initial concentration of 300 μmol L -1 to a 10 mL centrifuge tube, add a certain amount of GMA-IDA-Fe 3+ 、GMA-IDA-Fe 3+ @NIPs and GMA-IDA-Fe 3+ @MIPs adsorbent, place it in a constant temperature water bath oscillator at 25 °C, take it out at a certain time gradient respectively, centrifuge to recover the adsorbent, measure the content of AMP with an ultraviolet-visible spectrophotometer, and calculate the adsorption capacity according to the detection results, for GMA-IDA-Fe 3+ 、GMA-IDA-Fe 3+ @NIPs and GMA-IDA-Fe 3+ Kinetic performance of @MIPs adsorbent. Add 2 mL of AMP solutions with different concentrations into 10 mL centrifuge tubes, and add 2 mg of GMA-IDA-Fe 3+ @NIPs and GMA-IDA-Fe 3+ @MIPs adsorbents. After adsorption, the adsorbents are centrifuged and recovered, the content of AMP is measured by ultraviolet-visible spectrophotometer, and the saturated adsorption capacity is calculated according to the results. Select several nucleoside compounds with similar structures and properties, such as adenosine diphosphate (ADP), adenosine triphosphate (ATP), 2'-deoxyadenosine (dA), 2'-deoxyguanosine (dG) and 2'-deoxycytidine (dC) as selective adsorbates to participate in the study of the specific recognition performance of the adsorbent. The adsorbed GMA-IDA-Fe 3+ @NIPs and GMA-IDA-Fe 3+ @MIPs are eluted with acetic acid / methanol mixed elution solution (9:1, V / V) for AMP, and are used for the second, third and fourth adsorption / desorption cycles to evaluate the regeneration performance of the adsorbent.

[0040] The present invention will be further described below in conjunction with specific implementation examples.

[0041] Example 1:

[0042] (1) Preparation of GMA nanoparticles:

[0043] First, prepare GMA nanoparticles through the prior art. The specific steps are as follows: Stir and mix 1 mL of GMA, 0.184 mL of styrene, 0.0256 mL of DVB and 45 mL of deionized water evenly in a round-bottom flask, and stir the mixed system under nitrogen protection at 70 °C for 0.5 h. Subsequently, add 5 mL of an aqueous solution containing 0.024 g of KPS to the flask and react for 5 h. After the reaction, centrifuge to collect the product, and wash it three times with ethanol and deionized water respectively. After vacuum drying, GMA nanoparticles are obtained;

[0044] (2) Preparation of Pickering nanoparticles (GMA-IDA-Fe 3+ ) :

[0045] Take 0.1 g of the GMA nanoparticles prepared in step (1), ultrasonically disperse them in deionized water, adjust the pH of the system to 11 with an aqueous solution of NaOH (2.0 mol L -1 ), then add 0.528 g of IDA to it, and the reaction is carried out under stirring conditions at 70 °C for 22 h. After the reaction, centrifuge to collect the product and wash it several times with water to obtain GMA-IDA nanoparticles with carboxyl groups on the surface. Transfer the above product to 50 mL of FeCl 3 (0.08 mol L -1In an aqueous solution, it was ultrasonically dispersed uniformly, stirred at room temperature for 10 h, the product was collected by centrifugation and washed several times with water, and GMA-IDA-Fe was obtained after vacuum drying. 3+ The nanoparticles were ground and reserved for use.

[0046] (3) Preparation of Pickering emulsion:

[0047] The preparation of the aqueous phase was as follows: 16 mg of the template molecule AMP and 2.48 mg of the photoinitiator (2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone) were ultrasonically dissolved completely in 3 mL of Tris-HCl buffer solution, and then 30 mg of Pickering particles (GMA-IDA-Fe 3+ ) were added thereto, and it was ultrasonically treated sufficiently to form a suspension, and self-assembled at 25 °C for 1 h, so that the template molecule AMP was chelated to the surface of the Pickering particles by electrostatic attraction.

[0048] The preparation of the oil phase was as follows: 8.96 mg of the synthesized functional monomer VBT and 29.36 μL of EGDMA were ultrasonically dissolved completely in 3 mL of dichloromethane;

[0049] (4) Preparation of GMA-IDA-Fe 3+ @MIPs:

[0050] The Pickering emulsion was prepared with Tris-HCl buffer solution as the aqueous phase, dichloromethane as the oil phase, and GMA-IDA-Fe 3+ as the Pickering particles, and an oil-in-water (O / W) Pickering emulsion was obtained with a water / oil volume ratio of 3:3.

[0051] The prepared aqueous phase and oil phase were taken in a centrifuge tube, shaken manually and ultrasonically for 4 min to form a stable O / W Pickering emulsion. Nitrogen was gently purged into it and then the centrifuge tube was sealed, and self-assembled at room temperature in the dark for 7 h, so that the template molecule AMP and the functional monomer VBT formed a base complementary pairing interaction at the oil / water interface of the emulsion. Then, the Pickering emulsion was transferred into a transparent glass bottle and irradiated with an ultraviolet light source (λ = 254 nm) in an incubator at 25 °C for 6 h. After the constant-temperature polymerization was completed, the product was collected by centrifugation, washed several times with ethanol and water to remove the residues, and then the template molecule AMP was eluted with a methanol / acetic acid mixed elution solution (9:1, V / V), and washed with water until neutral, and a nano-imprinted adsorbent with an MIPs layer grafted on the surface of the Pickering particles was obtained after vacuum drying, denoted as GMA-IDA-Fe 3+ @MIPs.

[0052] Figure 11H NMR spectrum of VBT prepared in Example 1. The successful preparation of the functional monomer was demonstrated by analyzing the 1H NMR spectrum of specific functional groups.

[0053] Figure 2 Scanning electron microscope images of GMA nanoparticles (a, b), GMA-IDA-Fe 3+ @MIPs (c, d), and Pickering emulsions with different sizes stabilized by GMA-IDA-Fe 3+ @MIPs (e, f, g, h) prepared in Example 1, and transmission electron microscope images of ultrasonically dispersed GMA-IDA-Fe 3+ @MIPs (i, j, k, l). The structure shows that the GMA nanoparticles are of uniform size, well-dispersed, with a diameter of about 150 nm and a relatively smooth surface; after polymerization, the surface of GMA-IDA-Fe 3+ @MIPs becomes significantly rougher, with a diameter of 190 to 200 nm and an MIPs layer thickness between 20 and 25 nm, proving that the molecularly imprinted polymer was successfully modified on the surface of GMA nanoparticles. After polymerization, the internal oil phase of the Pickering emulsion volatilized, leaving a polymer shell with MIPs-coated and regularly arranged Pickering particles. The different degrees of shrinkage or collapse are attributed to the different original droplet sizes, confirming its hollow structure, that is, MIPs only grew on the surface of Pickering particles at the oil-water interface and did not polymerize inside the oil phase.

[0054] Figure 3 Bright-field micrographs of the Pickering emulsion prepared in Example 1 at 1 min (a, b) and 48 h (c) after emulsification, and fluorescence micrographs of fluorescein isothiocyanate (FITC)-labeled Pickering particles (d, e) and rhodamine B-labeled oil phase (f, g) under dark field. It was observed by bright field that the droplet sizes were relatively uniform, with diameters ranging from 20 to 50 μm; there was no obvious demulsification phenomenon after sealing and standing for 48 h, confirming the excellent stability of the emulsion; it was observed by dark field that the FITC-labeled Pickering particles emitted strong green fluorescence at the droplet edge; the rhodamine B-stained oil phase emitted strong red fluorescence inside the droplet, confirming that the emulsion was an oil-in-water (O / W) Pickering emulsion stabilized by Pickering particles at the interface.

[0055] Figure 4 Infrared spectra of GMA, GMA-IDA-Fe 3+ and GMA-IDA-Fe 3+ @MIPs prepared in Example 1. In GMA, at 3450 cm -1 、1730 cm -1 、1630 cm -1and 908 cm -1 The characteristic peaks at [specific positions] are respectively attributed to O═C-O, C═O, C═C and epoxy bonds in the GMA structure. GMA-IDA-Fe 3+ In [substance], the carboxyl group introduced by IDA makes the peak at 3450 cm -1 obviously enhanced, and the intensity of the characteristic peak of the epoxy bond at 908 cm -1 decreases, indicating that IDA has been successfully grafted onto the surface of GMA nanoparticles. GMA-IDA-Fe 3+ @MIPs, a new peak appears at 3190 cm -1 , which is attributed to the stretching vibration of -NH in the amide bond of VBT, confirming that VBT has been successfully grafted onto the surface of Pickering particles.

[0056] Example 2:

[0057] (1) Preparation of GMA nanoparticles:

[0058] First, GMA nanoparticles are prepared by the prior art. The specific steps are as follows: 1 mL of GMA, 0.23 mL of styrene, 0.032 mL of DVB, and 45 mL of deionized water are stirred and mixed evenly in a round-bottom flask, and the mixed system is stirred under nitrogen protection at 70 °C for 0.5 h. Subsequently, 5 mL of an aqueous solution containing 0.03 g of KPS is added to the flask, and the reaction is carried out for 5 h. After the reaction is completed, the product is collected by centrifugation and washed three times with ethanol and deionized water respectively, and the GMA nanoparticles are obtained after vacuum drying;

[0059] (2) Preparation of Pickering nanoparticles (GMA-IDA-Fe 3+ ):

[0060] Take 0.1 g of the GMA nanoparticles prepared in step (1), ultrasonically disperse them in deionized water, and adjust the pH of the system to 11 with an aqueous solution of NaOH (2.0 mol L -1 ). Subsequently, 0.66 g of IDA is added thereto, and the reaction is carried out under stirring conditions at 70 °C for 24 h. After the reaction is completed, the product is collected by centrifugation and washed several times with water to obtain GMA-IDA nanoparticles with carboxyl groups grafted on the surface. Transfer the above product to 50 mL of FeCl 3 (0.1 mol L -1 ) aqueous solution, ultrasonically disperse it evenly, stir at room temperature for 12 h, collect the product by centrifugation and wash it several times with water, and obtain GMA-IDA-Fe 3+ nanoparticles, which are ground for standby;

[0061] (3) Preparation work of Pickering emulsion:

[0062] The preparation of the aqueous phase is as follows: 16 mg of the template molecule AMP and 3.1 mg of the photoinitiator (2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone) are ultrasonically dissolved completely in 4 mL of Tris-HCl buffer solution. Subsequently, 30 mg of Pickering particles (GMA-IDA-Fe 3+ ) are added thereto, and ultrasonic treatment is carried out sufficiently to form a suspension. Self-assembly is carried out at 25 °C for 2 h, so that the template molecule AMP is chelated to the surface of the Pickering particles through electrostatic attraction.

[0063] The preparation of the oil phase is as follows: 11.2 mg of the synthesized functional monomer VBT and 36.7 μL of EGDMA are ultrasonically dissolved completely in 2 mL of dichloromethane;

[0064] (4) GMA-IDA-Fe 3+ Preparation of GMA-IDA-Fe

[0065] Preparation of Pickering emulsion: Using Tris-HCl buffer solution as the aqueous phase, dichloromethane as the oil phase, and GMA-IDA-Fe 3+ as Pickering particles, an oil-in-water (O / W) Pickering emulsion is obtained with a water-to-oil volume ratio of 4:2.

[0066] Take the above-prepared aqueous phase and oil phase in a centrifuge tube, shake it manually and ultrasonically for 5 min to form a stable O / W Pickering emulsion. After gently purging nitrogen into it, seal the centrifuge tube and carry out self-assembly in the dark at room temperature for 8 h, so that the template molecule AMP and the functional monomer VBT form a base complementary pairing effect at the oil-water interface of the emulsion. Then, transfer the Pickering emulsion into a transparent glass bottle, and irradiate it with a UV light source (λ = 254 nm) in an incubator at 25 °C for 8 h. After the constant-temperature polymerization is completed, centrifuge to collect the product, wash it several times with ethanol and water to remove the residues, then elute the template molecule AMP with a methanol / acetic acid mixed elution solution (9:1, V / V), and wash it with water until neutral. After vacuum drying, a nanoimprint adsorbent with an MIPs layer grafted on the surface of the Pickering particles is obtained, denoted as GMA-IDA-Fe 3+ @MIPs.

[0067] Example 3:

[0068] (1) Preparation of GMA nanoparticles:

[0069] First, prepare GMA nanoparticles through existing technologies. The specific steps are as follows: Stir and mix 1 mL of glycidyl methacrylate (GMA), 0.276 mL of styrene, 0.0384 mL of divinylbenzene (DVB), and 45 mL of deionized water evenly in a round-bottom flask, and stir the mixed system under nitrogen protection at 70 °C for 0.5 h. Subsequently, add 5 mL of an aqueous solution containing 0.036 g of potassium persulfate (KPS) to the flask and react for 5 h. After the reaction, collect the product by centrifugation and wash it three times with ethanol and deionized water respectively. Obtain GMA nanoparticles after vacuum drying;

[0070] (2) Preparation of Pickering nanoparticles (GMA-IDA-Fe 3+ ):

[0071] Take 0.1 g of the GMA nanoparticles prepared in step (1), ultrasonically disperse them in deionized water, and adjust the pH of the system to 11 with an aqueous solution of NaOH (2.0 mol L -1 ). Subsequently, add 0.792 g of IDA to it, and carry out the reaction under stirring conditions at 70 °C for 26 h. After the reaction, collect the product by centrifugation and wash it several times with water to obtain GMA-IDA nanoparticles with carboxyl groups on the surface. Transfer the above product to 50 mL of FeCl 3 (0.12 mol L -1 ) aqueous solution, ultrasonically disperse it evenly, stir at room temperature for 14 h, collect the product by centrifugation and wash it several times with water, and obtain GMA-IDA-Fe 3+ nanoparticles, grind them for later use;

[0072] (3) Preparation work of Pickering emulsion:

[0073] The preparation work of the aqueous phase is as follows: Ultrasonically dissolve 16 mg of the template molecule AMP and 3.72 mg of the photoinitiator (2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone) completely in 5 mL of Tris-HCl buffer solution. Subsequently, add 30 mg of Pickering particles (GMA-IDA-Fe 3+ ) to it, ultrasonically disperse it sufficiently to form a suspension, and self-assemble at 25 °C for 3 h to enable the template molecule AMP to chelate to the surface of the Pickering particles through electrostatic attraction.

[0074] The preparation work of the oil phase is as follows: Ultrasonically dissolve 13.44 mg of the synthesized functional monomer VBT and 44.04 μL of EGDMA completely in 1 mL of dichloromethane;

[0075] (4) Preparation of GMA-IDA-Fe 3+ @MIPs:

[0076] The Pickering emulsion was prepared with Tris-HCl buffer solution as the aqueous phase, dichloromethane as the oil phase, and GMA-IDA-Fe 3+ as Pickering particles, and an oil-in-water (O / W) Pickering emulsion was obtained with a water-to-oil volume ratio of 5:1.

[0077] Take the prepared aqueous phase and oil phase in a centrifuge tube, shake and ultrasonicate them for 6 min to form a stable O / W Pickering emulsion. After gently purging nitrogen into it, seal the centrifuge tube and perform self-assembly at room temperature in the dark for 9 h to enable the template molecule AMP and the functional monomer VBT to form base complementary pairing at the oil-water interface of the emulsion. Then, transfer the Pickering emulsion into a transparent glass bottle and irradiate it with a UV light source (λ = 254 nm) in an incubator at 25 °C for 10 h. After the constant-temperature polymerization is completed, centrifuge to collect the product, wash it several times with ethanol and water to remove the residues, then elute the template molecule AMP with a methanol / acetic acid mixed elution solution (9:1, V / V), wash it with water until neutral, and obtain a nanoimprint adsorbent with an MIPs layer grafted on the surface of the Pickering particles after vacuum drying, denoted as GMA-IDA-Fe 3+ @MIPs.

[0078] Experimental Example 1:

[0079] Take 2 mL of an AMP solution with an initial concentration of 300 μmol L -1 and add it to 10-mL centrifuge tubes respectively, and add 2 mg of GMA-IDA-Fe in Example 1 3+ , GMA-IDA-Fe 3+ @MIPs and GMA-IDA-Fe 3+ @NIPs adsorbents respectively, place them in a water bath oscillator at 25 °C, and take them out at 1, 3, 5, 10, 15, 20, 30, 40, 60, 80 min; separate the adsorbent and the solution by centrifugation. The concentration of AMP in the filtrate was measured by calculation using a UV spectrophotometer at a wavelength of 259 nm, and the time to reach adsorption equilibrium was obtained according to the results. The results show that the adsorption capacities of the three adsorbents follow the order of GMA-IDA-Fe Figure 5 and the time to reach adsorption equilibrium was calculated. The results show that the adsorption capacities of the three adsorbents follow the order of GMA-IDA-Fe 3+ @MIPs > GMA-IDA-Fe 3+ @NIPs > GMA-IDA-Fe 3+ , attributed to the fact that the presence of MIPs enables more specific recognition sites for capturing AMP to be established on the surface of the adsorbent. The adsorption capacity of GMA-IDA-Fe 3+ @MIPs is stronger than that of GMA-IDA-Fe 3+@NIPs, attributed to the 3D imprinted cavities, enhance the recognition ability for AMP; in addition, their surface adsorption of AMP increases rapidly within 0 to 10 min, reaching about 94.53% of the total adsorption capacity of AMP, and then slowly reaches equilibrium at 30 min as the available active binding sites decrease, indicating that the adsorbent has fast adsorption kinetics.

[0080] Test Example 2:

[0081] Take 2 mL of AMP solutions with initial concentrations of 30, 60, 100, 200, 300, 500, 700, 900, 1000 μmol L -1 and add them to 10 mL centrifuge tubes. Then add 2 mg of GMA-IDA-Fe 3+ @MIPs and GMA-IDA-Fe 3+ @NIPs from Example 1. Place the test solutions in a water bath at 25 °C and shake for 1.0 h. Then, centrifuge to separate the adsorbent and the solution. The concentrations of the unadsorbed AMP molecules are measured using a UV-visible spectrophotometer at a wavelength of 259 nm, and the adsorption capacity is obtained based on the results. Figure 6 The results show that at 25 °C, when the adsorption equilibrium is reached, the maximum adsorption capacity of GMA-IDA-Fe 3+ @MIPs for AMP is 98.236 μmol g -1 , and the maximum adsorption capacity of GMA-IDA-Fe 3+ @NIPs for AMP is 67.967 μmol g -1 . At the same temperature, the maximum adsorption amount of GMA-IDA-Fe 3+ @MIPs is higher than that of GMA-IDA-Fe 3+ @NIPs, indicating that GMA-IDA-Fe 3+ @MIPs is an adsorbent that can effectively recognize AMP.

[0082] Test Example 3:

[0083] Select dG, dC, dA, AMP, ADP, and ATP as competitive nucleoside compounds. Prepare solutions of the above 6 compounds with a concentration of 300 μmol L -1 . Take 2 mL of each solution and add them to centrifuge tubes. Then add 2 mg of GMA-IDA-Fe 3+ , GMA-IDA-Fe 3+ @MIPs and GMA-IDA-Fe 3+For the @NIPs adsorbent, after placing the test solution in a water bath shaker at 25 °C for 1.0 h, the adsorbent and the solution were centrifuged and separated. The molecular concentrations of several nucleoside compounds after adsorption were measured using a UV-visible spectrophotometer at the corresponding wavelengths, and the following was obtained based on the results Figure 7 . The results showed that GMA-IDA-Fe 3+ @MIPs' adsorption capacities for the 5 compounds followed the order of AMP﹥ADP﹥dA﹥dG﹥dC﹥ATP; GMA-IDA-Fe 3+ @MIPs' imprinting factors (IF) for dG, dC, dA, AMP, ADP, and ATP were 1.23, 1.20, 1.32, 1.74, 1.42, and 0.85 respectively, confirming that GMA-IDA-Fe 3+ @MIPs had good selectivity for AMP. Therefore, it can be inferred that there were imprinting sites on the surface of GMA-IDA-Fe 3+ @MIPs that were consistent with the shape and size of AMP, making GMA-IDA-Fe 3+ @MIPs have good adsorption specificity for AMP.

[0084] Test Example 4:

[0085] Take 2 mL of AMP solution with an initial concentration of 300 μmol L -1 and add it to 10 mL centrifuge tubes respectively. Add 2 mg of GMA-IDA-Fe 3+ @MIPs and GMA-IDA-Fe 3+ @NIPs adsorbents from Example 1 respectively, place them in a water bath shaker at 25 °C, take them out after 1.0 h, separate the adsorbent and the solution by centrifugation, calculate and measure the AMP concentration in the filtrate at a wavelength of 259 nm using a UV spectrophotometer, and record the results. Then, the adsorbents were eluted with an acetic acid / methanol mixed elution solution (9:1, V / V) to elute AMP, washed with water until the regenerated adsorbents were neutral, and used for the next adsorption / desorption cycle after being fully dried. The adsorption capacity data after four cycles of adsorption-desorption processes are as Figure 8 shown. The results showed that after four cycles, the adsorption capacity of GMA-IDA-Fe 3+ @MIPs for AMP decreased from 27.897 μmol g -1 to 23.766 μmol g -1 , retaining more than 85.19% of the original adsorption capacity. The decrease in adsorption capacity may be due to the loss of imprinting sites on the material surface after multiple cycles of contact between the solution and the material, or it may be caused by the incomplete release of imprinting sites during desorption. This result indicates that GMA-IDA-Fe 3+@MIPs have good adsorption and regeneration capabilities and have good application prospects.

[0086] Explanation: The above embodiments are only used to illustrate the present invention and do not limit the technical solutions described in the present invention; therefore, although this specification has described the present invention in detail with reference to the above various embodiments, those of ordinary skill in the art should understand that the present invention can still be modified or equivalently replaced; and all technical solutions and their improvements that do not depart from the spirit and scope of the present invention shall be covered within the scope of the claims of the present invention.

Claims

1. Preparation method of Pickering emulsion interface-induced confined polymerization imprinted adsorbent, Characterized in that, Comprising the following steps: (1) Prepare GMA nanoparticles for later use: (2) Preparation of Pickering nanoparticles GMA-IDA-Fe 3+ : Take a certain amount of GMA nanoparticles prepared in step (1), ultrasonically disperse them in deionized water, adjust the pH of the system with an aqueous NaOH solution, then add a certain amount of iminodiacetic acid IDA thereto, and carry out the reaction under stirring conditions at a certain temperature for a period of time. After the reaction is completed, centrifuge to collect the product and wash it several times with water to obtain GMA-IDA nanoparticles with carboxyl groups grafted on the surface. Transfer the above product to an aqueous solution of FeCl 3 Ultrasonically disperse it evenly, stir at room temperature for a period of time, centrifuge to collect the product and wash it several times with water, and obtain GMA-IDA-Fe 3+ nanoparticles after vacuum drying, grind them for standby; (3) Preparation of Pickering emulsion: The preparation of the aqueous phase is as follows: A certain amount of template molecule 5'-monophosphate adenosine (AMP) and a certain amount of photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone are ultrasonically dissolved in a certain volume of Tris-HCl buffer solution. Subsequently, a certain amount of Pickering particles GMA-IDA-Fe is added thereto. 3+ , and it is ultrasonically treated sufficiently to form a suspension. It is self-assembled for a period of time at a certain temperature, so that the template molecule AMP is chelated to the surface of the Pickering particles through electrostatic attraction. Preparation of the oil phase: Dissolve a certain amount of synthesized functional monomer 1-(vinylbenzyl) thymine VBT and a certain amount of ethylene glycol dimethacrylate EGDMA in a certain volume of dichloromethane by ultrasonic treatment until completely dissolved; (4)GMA-IDA-Fe 3+ Preparation of @MIPs: Take the prepared aqueous phase and oil phase in different proportions in a centrifuge tube, shake it by hand and sonicate it for a period of time to form a stable oil-in-water (O / W) Pickering emulsion. After gently purging nitrogen into it, seal the centrifuge tube and let it self-assemble at room temperature in the dark for a period of time, so that the template molecule AMP and the functional monomer VBT form base complementary pairing at the oil-water interface of the emulsion; then, transfer the Pickering emulsion into a transparent glass bottle, irradiate it with a UV light source of a fixed wavelength in an incubator at a certain temperature for a period of time, after the constant temperature polymerization is completed, centrifuge to collect the product, wash it several times with ethanol and water to remove the residues, then elute the template molecule AMP with a mixed eluent, and wash it with water until neutral, after vacuum drying, a nano-imprinted adsorbent with a MIPs layer grafted on the surface of Pickering particles is obtained, denoted as GMA-IDA-Fe 3+ @MIPs.

2. The preparation method according to claim 1, Characterized in that, In step (1), the preparation steps of GMA nanoparticles are as follows: Stir and mix evenly a certain amount of glycidyl methacrylate GMA, styrene, divinylbenzene DVB and a certain volume of deionized water in a round-bottom flask, and stir the mixed system under nitrogen protection at a certain temperature for a period of time. Subsequently, add an aqueous solution containing a certain amount of potassium persulfate KPS to the flask, react for a period of time, centrifuge to collect the product after the reaction, and wash it three times with ethanol and deionized water respectively, and obtain GMA nanoparticles after vacuum drying.

3. The preparation method according to claim 2, Characterized in that, In step (1), the dosage ratio of GMA, styrene, DVB, KPS, and deionized water used in the reaction system is 1 mL: (0.157 - 0.235) mL: (0.0218 - 0.0327) mL: (0.024 - 0.036) g: 45 mL; The temperature under nitrogen protection at a certain temperature is 70 °C, and the stirring time is 30 min; The reaction time after adding KPS is 5 h.

4. The preparation method according to claim 1, Characterized in that, In step (2), the dosage of GMA nanoparticles and IDA is 100 mg: (0.528 - 0.792) mg; The concentration of the NaOH aqueous solution is 2.0 mol / L, and the pH of the system is adjusted to 11 with the NaOH aqueous solution; The reaction temperature is 70 °C, and the reaction time is 22 - 26 h.

5. The preparation method according to claim 1, Characterized in that, In step (2), the concentration of the FeCl 3 aqueous solution is 0.08 - 0.12 mol / L; the stirring time at room temperature is 10 - 14 h.

6. The preparation method according to claim 1, Characterized in that, In step (3), the concentration of the Tris-HCl buffer solution is 0.05 M, pH = 7.4; In the aqueous phase described, the Pickering particles GMA-IDA-Fe 3+ , the template molecule AMP, the photoinitiator 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone, and the Tris-HCl buffer solution are used in a ratio of 30 mg: 16 mg: (2.48 - 3.72) mg: (3 - 5) mL; The self-assembly temperature is 25 °C, and the self-assembly time is 1 - 3 h.

7. The preparation method according to claim 1, Characterized in that, In step (3), in the oil phase, the dosage ratio of the functional monomer VBT, the cross-linking agent EGDMA, and dichloromethane is (8.96 - 13.44) mg: (29.36 - 44.04) μL: (1 - 3) mL.

8. The preparation method according to claim 1, Characterized in that, In step (4), The volume ratio of the water phase to the oil phase is 3:3 to 5:1; The emulsification time is 4 - 6 min; The self-assembly time of the template molecule AMP and the functional monomer VBT at the emulsion oil-water interface in the dark is 7 - 9 h; The temperature of the incubator is 25 °C, the wavelength of the ultraviolet light source is λ = 254 nm, and the irradiation time of the ultraviolet light is 6 - 10 h.

9. The preparation method according to claim 1, characterized in that in step (4), the mixed eluent is a mixed solution of methanol / acetic acid, wherein the volume ratio of methanol to acetic acid is 9:

1.

10. The Pickering emulsion interface-induced confined polymerization imprinted adsorbent prepared by the preparation method according to any one of claims 1 to 9 is used for the selective adsorption and separation of AMP.

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