A material for specifically adsorbing bioactive ingredients and a method for preparing the same

Abstract

The application provides a material capable of specifically adsorbing bioactive components and a preparation method of the material. The material can specifically adsorb, has high adsorption efficiency, good release effect, can maintain bioactivity during the release process, and can be used for extracting bioactive components. By changing the physical properties of the surface of the high polymer material, surface recombination and polar groups can be generated, the wettability of the surface of the high polymer material is improved, the activity of the surface of the material is improved, the biological compatibility is improved, and the adsorption of proteins and the biological contact performance are influenced to a certain extent. The increase of the active sites on the surface makes the material have specific adsorption effect on bioactive components, reduces the operation steps and the amount of solvents, optimizes the adsorption and release conditions, improves the activity maintenance degree of the adsorbed bioactive components in the whole process, further improves the adsorption efficiency, and realizes customized extraction effect through layer-by-layer self-assembly preparation.

Description

Technical Field

[0001] This invention relates to the field of materials technology, and mainly to a material that specifically adsorbs bioactive components and its preparation method. Background Technology

[0002] Currently, common techniques for extracting bioactive components include ultrasonic extraction, enzymatic hydrolysis, acid-base extraction, and solvent extraction. Each method has its advantages and disadvantages, but all also have some technical limitations. For example, traditional methods for extracting bioactive components include ultrasonic extraction, enzymatic hydrolysis, acid-base extraction, and solvent extraction. Their respective advantages and disadvantages are as follows: Ultrasonic extraction is a commonly used non-thermodynamic method. Its principle is to promote mass transfer and reaction of substances through the mechanical action and sonochemical effect of ultrasound. Although ultrasonic extraction has advantages such as simple operation and short extraction time, it has the following drawbacks: During ultrasonic extraction, the energy of ultrasound can cause the degradation of some heat-sensitive components, thus affecting the quality of the active ingredients. Furthermore, the effectiveness of ultrasonic extraction is affected by various factors, such as ultrasonic power, extraction time, and solvent type. The selection of operating parameters has a significant impact on the extraction effect, requiring extensive experimentation and optimization.

[0003] Traditional adsorbent materials suffer from poor selectivity and versatility in adsorbing bioactive components. This means they adsorb other biomolecules or substances besides the target component, reducing the specificity of adsorption for bioactive components. Furthermore, the adsorption effect is unstable and easily affected by environmental conditions. Simultaneously, the compatibility between the adsorbent material and the bioactive component is poor, leading to unstable release effects. This results in fluctuations or unevenness in the release of adsorbed bioactive components during the adsorption and release process, and significant loss of activity during adsorption and release, indicating poor retention of bioactivity. Therefore, many adsorbent materials exhibit weaknesses such as limited adsorption capacity for bioactive components, low adsorption efficiency, high extraction costs, and weak impurity removal capabilities.

[0004] Therefore, there is an urgent need to develop a new technology or material to overcome the current difficulty in extracting bioactive components. Summary of the Invention

[0005] To address the above problems, this invention provides a material for specifically adsorbing bioactive components and its preparation method. The material exhibits specific adsorption, high adsorption efficiency, and good release effect, maintaining bioactivity during the release process, and can be used to extract bioactive components. By altering the surface physical properties of the polymer material, surface recombination and polar groups can be generated, improving the wettability of the polymer material surface, thereby enhancing the surface activity and improving biocompatibility. This will have a certain impact on protein adsorption and biocontact performance. Increasing the number of active sites on the surface enables the material to specifically adsorb bioactive components, reducing the number of operation steps and solvent usage, optimizing adsorption and release conditions, and improving the retention of the activity of adsorbed bioactive components throughout the process, further improving adsorption efficiency. Prepared through layer-by-layer self-assembly, the material can achieve customized extraction effects.

[0006] The material that specifically adsorbs bioactive components provided by this invention is called a nanofilter material, and the method for preparing the material that specifically adsorbs bioactive components is called nanofilter technology.

[0007] The material specifically adsorbing bioactive components provided by this invention can be used to extract bioactive substances. For example, in animal or plant tissue fluid, a target compound is first selected, and the material specifically adsorbing bioactive components can be prepared using the method of this invention. After the material is fully contacted with the tissue fluid, the material will specifically adsorb the target compound. After the adsorption is completed, it is taken out and released, and high-purity bioactive substances can be obtained.

[0008] On one hand, the present invention provides a material that specifically adsorbs bioactive substances, wherein the material is: Fe3O4@SiO2@TiO2 nanoparticles crosslinked with bioactive substances, the bioactive substances are eluted, and a material containing three-dimensional holes that match the bioactive molecules is obtained.

[0009] Bioactive components, also known as bioactive substances or physiologically active substances, are compounds with biological activity. They refer to trace or small amounts of substances that have an impact on life phenomena, including polysaccharides, terpenes, sterols, alkaloids, peptides, nucleic acids, proteins, amino acids, glycosides, oils, waxes, resins, plant pigments, mineral elements, enzymes, and vitamins.

[0010] For bioactive components to generate biological activity, their molecules must contain specific chemical structures and spatial conformations. Based on this structure, theoretically, various bioactive components can undergo layer-by-layer self-assembly with the nanoparticles of this invention at the molecular level. The nanofilter materials that specifically adsorb various bioactive substances prepared by the method of this invention are all identical in terms of the specific adsorption mechanism. Therefore, the methods for preparing materials that specifically adsorb various different bioactive substances are all within the protection scope of this invention.

[0011] Furthermore, the Fe3O4@SiO2@TiO2 nanoparticles are a composite formed by Fe3O4@SiO2 nanoparticles and nano-TiO2 through hydrogen bonding, and the Fe3O4@SiO2 nanoparticles are monodisperse silica encapsulated magnetite nanoparticles.

[0012] Due to the small size effect and magnetic dipole attraction, magnetic Fe3O4 nanoparticles rapidly aggregate and deposit when directly exposed to the environment, failing to form a stable dispersion system. Their magnetic surface exhibits strong chemical reactivity, is easily oxidized and demagnetized, is susceptible to acid corrosion, and lacks sufficient surface hydroxyl groups, thus limiting their applications.

[0013] Due to its strong mechanical properties and high chemical stability, SiO2 can shield the interaction of magnetic dipoles when coated on the Fe surface, improving the dispersion of magnetic particles and increasing their biocompatibility. Furthermore, the mesoporous silica surface with its regular pore structure is rich in hydroxyl groups, allowing for the loading of more substances and facilitating further functionalization of the composite particles. Therefore, using SiO2 to modify magnetic Fe3O4 nanoparticles has the following advantages: 1) It possesses excellent biocompatibility and inertness, preventing the aggregation of magnetic nanoparticles and ensuring their stable existence in biological systems; 2) The technology for preparing SiO2-modified Fe3O4 nanoparticles is relatively mature and simple; 3) It is easily further functionalized, can combine with other biomolecules, and facilitates the introduction of other active groups. Simultaneously, the Fe3O4 nanostructure exhibits superparamagnetic properties, which is beneficial for particle recycling and reuse. This novel composite microsphere structure is characterized by uniform size and non-physiological toxicity.

[0014] The Fe3O4@SiO2@TiO2 particles have a size of approximately 600 nm. The SiO2 shell increases Si-OH bonds, which is beneficial for the formation of complexes by nano-TiO2 through hydrogen bonding. Due to its reversible binding with phosphate groups, TiO2 is widely used for the separation and enrichment of phosphate peptides and proteins. The surface functional groups of the core further facilitate TiO2 coating, improving the adsorption performance of the complex and forming a more uniform and stable core-shell structure. This structure also possesses excellent reusability, which pure TiO2 lacks.

[0015] In some embodiments, the bioactive substances include polysaccharides, terpenes, sterols, alkaloids, peptides, nucleic acids, proteins, amino acids, glycosides, oils, waxes, resins, plant pigments, mineral elements, enzymes, and vitamins.

[0016] In some embodiments, the bioactive substance includes proteins, which include growth factors, cytokines, and neurotrophic factors.

[0017] In some embodiments, the bioactive substance includes growth factors, including vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), osteosarcoma-derived growth factor (ODGF), transforming growth factor (TGF), insulin-like growth factor (IGF), nerve growth factor (NGF), interleukin-like growth factors, erythropoietin (EPO), and colony-stimulating factor (CSF).

[0018] In some embodiments, the bioactive substance includes epidermal growth factor (EGF).

[0019] In some embodiments, the bioactive substance includes sheep placenta extract.

[0020] On the other hand, the present invention provides a method for preparing a material that specifically adsorbs bioactive substances, wherein Fe3O4@SiO2@TiO2 nanoparticles are cross-linked with bioactive substances, and the bioactive substances are eluted to obtain a material containing three-dimensional holes that match the bioactive molecules.

[0021] Furthermore, the crosslinking includes introducing groups on the surface of the nanoparticles, the groups including at least one of borate groups and amino groups.

[0022] Boric acid groups can form stable ester or ester-amine bonds with nucleophilic sites such as hydroxyl or amino groups in nanomaterials. Amino groups and sulfonic acid groups, which are strongly anionic and hydrophilic functional groups, can bind to colloidal surfaces, exhibiting excellent experimental grafting effects. They are suitable for surface modification of nanoparticles, and their hydrophobicity prevents the aggregation of colloidal particles while their structure allows for better binding of target molecules. Amino groups possess multiple cationic properties, reducing crystallinity, weakening intermolecular hydrogen bonds, and interacting with negative charges, thus reducing molecular crystallinity. Introducing new functional groups gives complexes novel properties. Adding substances with different biocompatibility characteristics alters hydrophobicity and simplifies the preparation process.

[0023] Furthermore, the crosslinking agents used include 2,4-diaminophenylboronic acid and 2-propylamido-2-ethylpropanesulfonic acid.

[0024] In polymer nanomaterials, the introduction of functional groups such as borate and amino groups on the surface can enable nanoparticles to specifically recognize target molecules. 2,4-Diaminophenylboronic acid contains both amino and borate groups, both relatively reactive, and can act as a reaction intermediate. Utilizing the property that 2,4-diaminophenylboronic acid readily forms reversible covalent bonds with cis-neighboring compounds, the relatively weak intermolecular forces can be replaced by relatively strong reversible covalent bonds, thus eluting the bound target molecules and achieving separation and enrichment. 2-Propylamido-2-ethylpropanesulfonic acid has a strong anionic and hydrophilic sulfonic acid functional group, giving it excellent polymerization properties; it can undergo self-polymerization and homopolymerization, as well as copolymerization with various monomers.

[0025] The functional monomer 2,4-diaminophenylboronic acid and the auxiliary functional monomer 2-propylamido-2-ethylpropanesulfonic acid are prepolymerized with the template molecule EGF under certain conditions to enable the monomers to bind well with the template molecule. Then, an initiator and a support are added, and a polymer reaction is carried out under certain conditions to form a copolymer. This copolymer is then eluted, forming cavities on the surface of Fe3O4@SiO2@TiO2 particles that specifically recognize the template molecule. These cavities can specifically recognize EGF in the mixed solution. The synergistic effect of the reversible covalent bonds and hydrogen bonds formed at the same time greatly improves the selectivity and adsorption capacity of Fe3O4@SiO2@TiO2 particles for EGF.

[0026] Furthermore, the molar mass ratio of 2,4-diaminophenylboronic acid and 2-propylamido-2-ethylpropanesulfonic acid is 1:(0.2~1.0).

[0027] The molar ratio of 2,4-diaminophenylboronic acid to 2-propylamido-2-ethylpropanesulfonic acid affects the elution efficiency and loading capacity of nanofilter materials. Elution efficiency and loading capacity are higher in the range of 1:(0.2 to 1.0), which is beneficial to the specific adsorption of bioactive substances by nanofilter materials and the improvement of adsorption rate.

[0028] Furthermore, the elution solution used includes an SDS-HAC solution.

[0029] The SDS-HAC solution is an aqueous solution containing both sodium dodecyl sulfate (SDS) and acetic acid (HAC).

[0030] The composition and concentration of the eluent affect the elution efficiency and carrying capacity of the nanofilter material. Common eluents often influence the three-dimensional cavity structure and number of the nanofilter material of this invention. Specifically, they affect the groups in the three-dimensional cavities that match bioactive molecules, thereby reducing the specificity and adsorption characteristics of the three-dimensional cavities and decreasing the number of three-dimensional cavities with specific and adsorption properties. This invention uses an SDS-HAC solution for elution of the nanofilter material, which is beneficial for the specific adsorption of bioactive substances and improves the adsorption rate.

[0031] Preferably, the elution solution used is a 5% SDS-2% HAC solution.

[0032] After determining the composition of the eluent, the concentration of the eluent affects the elution efficiency and loading capacity of the nanofilter material. The nanofilter material eluted with 5% SDS-2% HAC solution has higher elution efficiency and loading capacity, which is beneficial to the specific adsorption of bioactive substances by the nanofilter material and the improvement of adsorption rate.

[0033] On the other hand, the present invention provides a method for extracting bioactive substances, which involves contacting the material with a mixture containing the target bioactive substance for adsorption, and then transferring it to a buffer solution to release the target bioactive substance.

[0034] The material of this invention specifically adsorbs bioactive components, and its adsorption efficiency for the corresponding bioactive components far exceeds that of any previously reported adsorption material; it has high specificity, enabling specific extraction and enrichment of bioactive substances; it has good release effect, enabling full release; and it can maintain the bioactivity of bioactive components throughout the entire adsorption-release process.

[0035] In some embodiments, the bioactive substance is epidermal growth factor, and the buffer solution has a pH of 7.4.

[0036] In some embodiments, the mixture containing the target bioactive substance includes at least one of a mixed solution and a tissue fluid.

[0037] On the other hand, the present invention provides an application of Fe3O4@SiO2@TiO2 nanoparticles, the application of which includes any one or more of the following: 1) preparing materials that specifically adsorb bioactive components; 2) improving the adsorption efficiency of materials; 3) improving the adsorption specificity of materials for target bioactive components; 4) improving the release effect of adsorbent materials; 5) preparing adsorbent materials that maintain the bioactivity of bioactive components.

[0038] On the other hand, the present invention provides an application of a reagent combination comprising 2,4-diaminophenylboronic acid and 2-propylamido-2-ethylpropanesulfonic acid, wherein the application includes any one or more of the following: 1) preparing materials that specifically adsorb bioactive components; 2) improving the adsorption efficiency of materials; 3) improving the adsorption specificity of materials for target bioactive components; 4) improving the release effect of adsorbent materials; 5) preparing adsorbent materials that maintain the bioactivity of bioactive components; 6) preparing adsorbent materials with high loading capacity.

[0039] On the other hand, the present invention provides an use of an eluent comprising an SDS-HAC solution, the use of which includes any one or more of the following: 1) preparing materials that specifically adsorb bioactive components; 2) improving the adsorption efficiency of the material; 3) improving the adsorption specificity of the material for the target bioactive component; 4) improving the release effect of the adsorbent material; 5) preparing adsorbent materials that maintain the bioactivity of the bioactive component; 6) preparing adsorbent materials with high loading capacity.

[0040] The beneficial effects of this invention include:

[0041] 1. High Selectivity: This specially treated nanoscale polymer material can be directionally designed according to the characteristics of the target analyte, improving the selectivity of extraction. In contrast, traditional extraction methods often require multiple steps or reagents to separate and purify the target analyte, while this specially treated nanoscale polymer material can directly and selectively adsorb the target analyte, reducing the number of steps and reagents used.

[0042] 2. High Efficiency: This specially treated nanoscale polymer material has a large specific surface area and porous structure, providing more adsorption sites and thus improving extraction efficiency. Furthermore, the adsorption rate of this material is faster than traditional methods, significantly shortening the extraction time.

[0043] 3. Reusable: This specially treated nanoscale polymer material can be reused through a simple regeneration process without losing its adsorption performance, making it more economical and sustainable in large-scale extraction processes. Attached Figure Description

[0044] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0045] Figure 1 Electron micrographs of materials specifically adsorbed with EGF

[0046] Figure 2 EGF release curves of specifically adsorbed EGF materials

[0047] Figure 3 Graph showing the change in EGF elution rate by nanofilter particles with the number of repetitions Detailed Implementation

[0048] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0049] Example 1: Preparation, characterization, and application of materials that specifically adsorb EGF

[0050] I. Preparation of materials specifically adsorbing EGF

[0051] (1) Preparation of Fe3O4@SiO2@TiO2 nanoparticles:

[0052] Fe3O4 (purchased from Suzhou Beaver Company) was dissolved in a mixture of ethanol, deionized water, and ammonia, and sonicated. Tetraethoxysilane (from Shanghai Husheng Technology Co., Ltd.) was then added, and the mixture was continuously stirred at room temperature to obtain Fe3O4@SiO2 (monodisperse silica-encapsulated magnetite nanoparticles). The obtained Fe3O4@SiO2 particles were dissolved in a mixture of ethanol and ammonia, sonicated for 5–15 min, and then heated to 20–60°C. Tetrabutyl titanate (from Shanghai Husheng Technology Co., Ltd.) dissolved in ethanol was then added. The reaction was carried out under a nitrogen atmosphere, with a stirring speed of 100 r / min to 300 r / min and reflux for 6 h to 14 h. The reaction was stopped, and the obtained solid material was washed. The washing process was as follows: the solid material was first washed twice with ultrapure water, then washed five times with ethanol, and then magnetically separated and settled using a neodymium iron boron magnet. After pouring out the supernatant liquid, the solid material was dried at a temperature of 40 to 70 °C for 11 h to 13 h to obtain Fe3O4@SiO2@TiO2 particles.

[0053] (2) Layer-by-layer self-assembly

[0054] Preparation of nanofilter particles: Recombinant human epidermal growth factor (EGF stock solution) (Shenzhen Huashengyuan Gene Engineering Co., Ltd.), 2,4-diaminophenylboronic acid (Sigma-Aldrich), and 2-propylamido-2-ethylpropanesulfonic acid (Sigma-Aldrich) were dissolved in a 0.02 mol / L phosphate buffer solution with a pH of 9.0. The solution was sonicated for 5-10 min, then aged at room temperature for 1-3 h. The Fe3O4@SiO2@TiO2 particles obtained in step two were then added. Under a stirring speed of 100-300 r / min and a nitrogen atmosphere, a phosphate buffer solution of ammonium persulfate (Shanghai Maclean Biochemical Technology Co., Ltd.) was added dropwise at a rate of 1-5 mL / min. The reaction was carried out in an oil bath at 40-80℃ for 24-36 h, stirring until the monomer solution and sol material were completely and uniformly mixed. The mixture was then ultrasonically cleaned for 30 min to remove air bubbles and form a homogeneous system. A 5% [unclear text - likely a specific chemical process] was used. Elute the template nanoparticles with SDS-2% HAC solution until no EGF is detected, then vacuum dry at room temperature until the product reaches constant weight. The resulting material is the nanofilter material, which is stored in a desiccator.

[0055] The molar ratio of EGF to 2,4-diaminophenylboronic acid is 1:(100-1000); the molar ratio of 2,4-diaminophenylboronic acid to 2-propylamido-2-ethylpropanesulfonic acid is 1:(0.2-1.0); the mass ratio of the Fe3O4@SiO2@TiO2 particles to the volume of a 0.02 mol / L phosphate buffer solution with a pH of 9.0 is 1 mg:(0.2-1) mL; the mass ratio of EGF to the obtained Fe3O4@SiO2@TiO2 particles is 1:(35-50); the mass ratio of EGF to ammonium persulfate in the ammonium persulfate phosphate buffer solution is 1:(10-20).

[0056] II. Characterization

[0057] The material that specifically adsorbs EGF was prepared, and its electron micrograph is shown below. Figure 1 As shown in the figure, the black part is the three-dimensional cavity. Its size and fixed arrangement of functional groups are complementary to the template molecule EGF, thus it has the memory function of EGF.

[0058] III. Usage Method

[0059] Specific extraction of EGF from a mixture: Take the obtained material that specifically adsorbs EGF and directly contact it with a mixture containing EGF (mixed solution, tissue fluid, etc.). Shake together in a constant temperature shaker at 37°C for 12 hours or more. After adsorption is complete, remove the material and put the adsorbed nanoparticles into a dialysis bag. Tie the bag tightly and place it in a container containing a buffer solution of pH 7.4. Place the container in a horizontal constant temperature shaker at 37°C and shake for 24 hours or more, or replace the buffer solution periodically. After the release is complete, the buffer solution will contain the extracted EGF.

[0060] The above description uses EGF as an example to illustrate the present invention. EGF can be replaced with other bioactive substances. First, based on similar structures, the method in Example 1 can be applied to growth factor bioactive substances. 2,4-Diaminophenylboronic acid and 2-propylamido-2-ethylpropanesulfonic acid can be prepolymerized with growth factors and then crosslinked to Fe3O4@SiO2@TiO2 particles. Growth factors include vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), osteosarcoma-derived growth factor (ODGF), transforming growth factor (TGF), insulin-like growth factor (IGF), nerve growth factor (NGF), interleukin-like growth factors, erythropoietin (EPO), and colony-stimulating factor (CSF). Specific adsorption materials are prepared for the above-mentioned multiple growth factors. The adsorption effect, specificity, release effect, and bioactivity retention function of the materials are similar.

[0061] Secondly, growth factors belong to the category of proteins. There are a large number of proteins with similar spatial structures and chemical properties to growth factors, such as cytokines and neurotrophic factors. The methods for preparing specific adsorbent materials for growth factors, as well as the preparation and adsorption mechanisms of these materials, are the same. The adsorption effect, specificity, release effect, and biological activity retention of these materials are similar.

[0062] Bioactive substances possess biological activity due to their unique chemical properties and spatial structure, enabling them to undergo biochemical reactions with substances within organisms. Therefore, specific adsorbent materials can be prepared using the unique chemical properties and spatial structure of bioactive substances. These bioactive substances include not only proteins but also polysaccharides, terpenes, sterols, alkaloids, peptides, nucleic acids, amino acids, glycosides, oils, waxes, resins, plant pigments, mineral elements, enzymes, and vitamins.

[0063] Example 2: Comparative Tests of Materials Specifically Adsorbing EGF

[0064] The materials to be tested include:

[0065] The conventional adsorbent material, model G-100DEAE-Cellulose, is manufactured by Whatman and its main components are DEAE cellulose and dextran gel.

[0066] The material was prepared by replacing the Fe3O4@SiO2@TiO2 particles in step three of Example 1 with magnetic Fe3O4 nanoparticles and then performing layer-by-layer self-assembly.

[0067] The material was prepared by replacing the Fe3O4@SiO2@TiO2 particles in step three of Example 1 with Fe3O4@SiO2 and then performing layer-by-layer self-assembly.

[0068] FeCl3·6H2O was dissolved in ethylene glycol and sonicated for about 15 minutes. Then, anhydrous sodium acetate was added, and the mixture was stirred at a speed of 200-400 rpm for half an hour. The mixture was then transferred to a stainless steel reactor and reacted at 180-220℃ for 5-7 hours. After the reaction was stopped, the reactor was removed and allowed to cool naturally to room temperature. The obtained solid was washed three times with anhydrous ethanol and five times with double-distilled water, and then dried for 11-13 hours to obtain Fe3O4 nanoparticles. These nanoparticles were then further processed under a stirring speed of 200-400 rpm. A mixture of isopropanol and distilled water was added to Fe3O4 nanoparticles and mixed until uniformly dispersed. Then, ammonia water was added dropwise. After the addition was complete, tetrabutyl titanate was added dropwise. The mixture was stirred at 200 r / min to 400 r / min at room temperature for 10 h to 14 h. The reaction was then stopped. The obtained solid material was washed five times with double-distilled water and three times with anhydrous ethanol. Then, it was separated and settled by magnetic force using a neodymium iron boron magnet. After the supernatant was poured off, the solid material was dried at 40 to 70 °C for 11 h to 13 h to obtain Fe3O4@TiO2 microparticles.

[0069] The material was prepared by replacing the Fe3O4@SiO2@TiO2 particles in step three of Example 1 with Fe3O4@TiO2 and then performing layer-by-layer self-assembly.

[0070] The material prepared using Fe3O4@SiO2@TiO2, i.e., the material prepared according to Example 1;

[0071] FeCl3·6H2O was dissolved in ethylene glycol and sonicated for about 15 minutes. Then, anhydrous sodium acetate was added, and the mixture was stirred at a speed of 200-400 rpm for half an hour. The mixture was then transferred to a stainless steel reactor and reacted at 180-220℃ for 5-7 hours. After the reaction was stopped, the reactor was removed and allowed to cool naturally to room temperature. The obtained solid was washed three times with anhydrous ethanol and five times with double-distilled water, and then dried for 11-13 hours to obtain Fe3O4 nanoparticles. These nanoparticles were then further processed under a stirring speed of 200-400 rpm. A mixture of isopropanol and distilled water was added to Fe3O4 nanoparticles and mixed until uniformly dispersed. Then, ammonia water was added dropwise. After the addition was complete, tetraethyl orthosilicate was added dropwise. After the addition was complete, the mixture was stirred at 200 r / min to 400 r / min at room temperature for 10 h to 14 h. The reaction was then stopped, and the obtained solid material was washed. The solid material was first washed 5 times with double-distilled water and then 3 times with anhydrous ethanol. Then, it was separated and settled by magnetic force using a neodymium iron boron magnet. After the supernatant was poured off, the solid material was dried at a temperature of 40 to 70 °C for 11 h to 13 h to obtain Fe3O4@SiO2 microparticles. Fe3O4@SiO2 particles were dissolved in toluene and sonicated for 5–15 min. The mixture was then heated to 20–60 °C, and 3-glycidyloxypropyltrimethoxysilane was added. Under a nitrogen atmosphere, the mixture was stirred at 100–300 r / min and reflux for 6–14 h. The reaction was then stopped, and the resulting solid was washed twice with toluene and five times with ethanol. The solid was then separated and settled using a neodymium iron boron magnet. After discarding the supernatant, the solid was dried at 40–70 °C for 11–13 h to obtain Fe3O4@SiO2@COC particles; where COC is polycarbonate.

[0072] The material was prepared by replacing the Fe3O4@SiO2@TiO2 particles in step three of Example 1 with Fe3O4@SiO2@COC and then performing layer-by-layer self-assembly.

[0073] The material was prepared by replacing the Fe3O4@SiO2@TiO2 particles in step 3 of Example 1 with TiO2 nanoparticles and then performing layer-by-layer self-assembly.

[0074] (1) Adsorption efficiency

[0075] The effectiveness of the technology was first verified by comparing the adsorption efficiency of bioactive components such as EGF on polymer materials before and after the application of nanofiltration technology. A certain concentration of EGF solution was selected and contacted with untreated polymer materials and polymer materials treated with altered surface physical properties, respectively. The adsorption concentration of EGF in both was then measured, and the difference was compared to evaluate the adsorption efficiency of the technology.

[0076] A series of EGF solutions with different concentrations ranging from 0.1 to 5.0 mmol / L were prepared. Seven concentrations were selected: 0.1 mmol / L, 0.5 mmol / L, 0.75 mmol / L, 1.5 mmol / L, 2.5 mmol / L, 3.5 mmol / L, and 4.5 mmol / L. Seven 20.0 mg portions of the nanofilter material obtained in Example 1 were accurately weighed and added to the prepared EGF solutions of different concentrations. The solutions were then shaken in a constant-temperature shaker for 12 hours and filtered. The filtrate was transferred to a 50 mL volumetric flask and diluted to the mark. The absorbance (A) was measured using a UV-Vis spectrophotometer. The concentration of free EGF in the solution could be determined from the absorbance. The adsorption capacity (Q) of this material for EGF could be calculated based on the change in concentration before and after adsorption. Three parallel measurements were performed, and the average value was taken. The formula for calculating the binding capacity is as follows:

[0077] Q = (C0 - C) e V / m

[0078] Ads. (%) = (C0 - C) e ) / C0×100%

[0079] In the formula, Q is the adsorption capacity, C0 (mmol / L) is the concentration of the substrate in the liquid before adsorption, and C... e (mmol / L) is the concentration of the adsorbed substance in the solution after adsorption, V (mL) is the volume of the adsorption solution, m (g) is the mass of the adsorbent, and Ads. (%) is the adsorption rate.

[0080] Table 1: Adsorption rates of different adsorbent materials

[0081] conventional adsorption materials 60 <![CDATA[Prepared using magnetic Fe3O4 nanoparticles]]> 40 <![CDATA[Prepared using Fe3O4@SiO2]]> 70 <![CDATA[Prepared using Fe3O4@TiO2]]> 68 <![CDATA[Prepared using Fe3O4@SiO2@TiO2]]> 93 <![CDATA[Prepared using Fe3O4@SiO2@COC]]> 75 <![CDATA[Prepared using TiO2 nanoparticles]]> 55

[0082] According to the test results, the nanofilter material obtained by the nanofiltering technology of this invention has an EGF adsorption efficiency of over 90% within the equilibrium time, while the existing conventional adsorption materials have an EGF adsorption efficiency of around 60% within the equilibrium time. The product prepared by this invention has a better EGF adsorption effect.

[0083] In the preparation process, the present invention uses magnetic Fe3O4 nanoparticles, Fe3O4@SiO2, and TiO2 nanoparticles in sequence. According to the experimental results, even under the same layer-by-layer self-assembly method, the materials prepared using the above-mentioned substrates are not as good as the nanofilter materials prepared by Fe3O4@SiO2@TiO2 in terms of adsorbing the bioactive substance EGF.

[0084] Using Fe3O4@TiO2 and Fe3O4@SiO2@COC as controls, under the same layer-by-layer self-assembly method, the EGF adsorption rate of the materials prepared by the above two substrates is lower than that of the nano-filter material prepared by Fe3O4@SiO2@TiO2 in this invention. This indicates that the SiO2 shell is indispensable, and when the SiO2 shell is already present, TiO2 modification is indispensable. The experimental results show that only Fe3O4@SiO2@TiO2 nanoparticles can prepare specific adsorption materials with an EGF adsorption rate of over 90%.

[0085] (2) Specific adsorption

[0086] The specific adsorption performance of the technology is verified by comparing the adsorption of different bioactive components on polymer materials before and after using the technology. Various bioactive component solutions can be selected and brought into contact with untreated polymer materials and polymer materials treated with altered surface physical properties, respectively. The adsorption of each component on the materials is then measured, and the specific adsorption performance of the technology is evaluated by comparing the adsorption amounts of each component.

[0087] Nanoparticles: 20 mg; Substrate concentration: 2 mmol·L⁻¹; Substrate volume: 2 mL; Adsorption solvent: NH₄Ac-phosphate buffer (pH = 7.40) (V / V, 7:3); Adsorption time: 24 hours; Adsorption temperature: 25 °C.

[0088] Selective adsorption experiments were conducted using insulin-like growth factor (IGF), a competitive substrate with a structure similar to EGF. The adsorption on polymers was compared, and the adsorption capacity Q of different substrates on conventional adsorption materials and polymers with modified surface physical properties was calculated. The static partition coefficient KD and separation factor α were used to characterize the selective adsorption capacity of the materials for the substrates. The formula for calculating α is as follows:

[0089] KD = cp / cs

[0090] α=KD i / KD j

[0091] Where cp represents the concentration of the substrate bound by the material (umol / g); cs represents the concentration of the substrate in the solution at adsorption equilibrium (umol / mL). i and j represent the substrate and its control, respectively. When i = j, α = 1. A higher KD value indicates a greater binding amount of the material to the substrate; a higher α value indicates better selectivity of the material for the substrate.

[0092] Table 2: Specificity of different adsorbent materials

[0093]

[0094]

[0095] Compared with existing adsorption materials, the nanofilter material of the present invention has a relative separation factor α of 6.87, which indicates that it has a specific adsorption effect on EGF, while the relative separation factor α of existing conventional adsorption materials is 1, which does not have any specific adsorption effect.

[0096] In the preparation process, the present invention uses magnetic Fe3O4 nanoparticles, Fe3O4@SiO2, and TiO2 nanoparticles in sequence. According to the experimental results, even under the same layer-by-layer self-assembly method, the material prepared using the above-mentioned substrate has poor specificity for EGF, which is far inferior to the nano-filter material of the present invention.

[0097] Using Fe3O4@TiO2 and Fe3O4@SiO2@COC as controls, under the same layer-by-layer self-assembly method, the EGF adsorption specificity of the materials prepared by the above two substrates is not as good as that of the nano-filter material prepared by Fe3O4@SiO2@TiO2 in this invention. This indicates that the SiO2 shell is indispensable, and when the SiO2 shell is already present, TiO2 modification is indispensable. The experimental results show that only Fe3O4@SiO2@TiO2 nanoparticles can be used to prepare adsorption materials with high specificity.

[0098] According to the experimental results, the partition coefficient K of the nanofilter particles for EGF is much larger than that for other substrates such as IGF, indicating that the nanofilter particles can effectively separate EGF from other substrates. The relative separation factor α shows that the nanofilter particles have a stronger ability to separate EGF from competing substrates than untreated polymer materials and conventional adsorbents. This is because the nanofilter particles differ significantly in spatial structure from the polymers with the same composition as the other two materials. The three-dimensional cavities contained in the nanofilter particles, with their size and fixedly arranged functional groups, are complementary to the template molecule EGF, thus exhibiting a memory function for EGF.

[0099] (3) Release effect

[0100] The effectiveness of the technology is verified by comparing the release effect of the adsorbed bioactive components by the polymer material before and after using the technology. The adsorbed polymer material is contacted with a suitable solution, and the concentration change of the released bioactive components is observed and measured. The effectiveness of the technology is evaluated by comparing the release effects.

[0101] For each of the above materials, 5 mg of nanoparticles were weighed, placed in a dialysis bag, sealed, and then placed in a container containing 5 ml of pH 7.4 buffer solution. The container was placed in a horizontal constant temperature shaker at 37°C (frequency 72 r / min) and the timer was started. Samples were taken at intervals, and the EGF content was determined using an EGF ELISA kit (abcam). The ELISA test method was performed according to the kit instructions, and the corresponding amount of fresh buffer solution was added. The EGF release concentration was measured once every 24 hours, as shown in Table 3.

[0102] Table 3: 24h EGF release concentration of different materials

[0103] conventional adsorption materials 60 <![CDATA[Prepared using magnetic Fe3O4 nanoparticles]]> 65 <![CDATA[Prepared using Fe3O4@SiO2]]> 75 <![CDATA[Prepared using Fe3O4@TiO2]]> 70 <![CDATA[Prepared using Fe3O4@SiO2@TiO2]]> 91 <![CDATA[Prepared using Fe3O4@SiO2@COC]]> 75 <![CDATA[Prepared using TiO2 nanoparticles]]> 66

[0104] Compared with existing adsorption materials, the nanofilter material of the present invention releases more than 90% of EGF concentration in 24 hours, showing a good release effect. In contrast, existing conventional adsorption materials release only 60% of EGF concentration in 24 hours after adsorption, showing a poor release effect.

[0105] In the preparation process, the present invention uses magnetic Fe3O4 nanoparticles, Fe3O4@SiO2, and TiO2 nanoparticles in sequence. According to the experimental results, even under the same layer-by-layer self-assembly method, the material prepared using the above-mentioned substrate has a lower EGF release concentration after 24 hours, which is far less than that of the nano-filter material of the present invention.

[0106] Using Fe3O4@TiO2 and Fe3O4@SiO2@COC as controls, under the same layer-by-layer self-assembly method, the EGF release effect of the materials prepared by the above two substrates is not as good as that of the nano-filter material prepared by Fe3O4@SiO2@TiO2 in this invention. This indicates that the SiO2 shell is indispensable, and when the SiO2 shell is already present, TiO2 modification is indispensable. The experimental results show that only Fe3O4@SiO2@TiO2 nanoparticles can be used to prepare adsorbent materials with good release effect.

[0107] Determination of in vitro release curves of nanoparticles: Weigh 5 mg of nanoparticles, place them in a dialysis bag, seal it tightly, and then place it in a container containing 5 ml of pH 7.4 buffer solution. Place the container in a horizontal constant-temperature shaker at 37°C (frequency 72 r / min) and start timing. Take 100 μL samples at regular intervals, determine the EGF content using ELISA, and add the corresponding amount of fresh buffer solution to plot the nanoparticle release curves.

[0108] The EGF release curve of the nanofilter material of the present invention is as follows: Figure 2 As shown.

[0109] (4) Maintenance of bioactivity

[0110] The effectiveness of the technology is verified by comparing the retention of adsorbed bioactive components by polymer materials before and after its application. The adsorbed bioactive components are then appropriately treated, and the differences in their activity before and after treatment are compared. The effectiveness of the technology is evaluated by comparing the retention rates.

[0111] EGF, a bioactive component extracted from conventional adsorbent materials, untreated polymeric materials, and polymeric materials with modified surface physical properties, was thoroughly mixed by ultrasonication and then added to cell culture plates at 100 μL per well. Eight replicates were established for each concentration, and a blank control (100 μL of culture medium per well) was also included to ensure consistent EGF concentration after adsorption and release from different materials. After incubation at 37°C with 5% CO2 for 24 h, 20 μL of MTT working solution was added to each well, and the plate was incubated at 37°C with 5% CO2 for 4 h. All operations were performed under aseptic conditions. The liquid in the culture plate was discarded, and 150 μL of DMSO was added to each well. The plate was mixed on a shaker for 10 min, and the absorbance (OD value) at 490 nm was measured using a microplate reader. The experiment was repeated three times.

[0112] Succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-purple formazan crystals, which are then deposited in the cells. Dead cells lack this function. Dimethyl sulfoxide (DMSO) can dissolve the formazan in the cells. The OD value, measured at 570 nm using a microplate reader, can indirectly reflect the number of living cells. Within a certain cell number range, the amount of MTT crystals formed is directly proportional to the cell number. The number of living cells is determined based on the measured OD value; the higher the OD value, the greater the cell number or the stronger the activity. The OD value is used as the cell proliferation rate, and the results are shown in Table 4.

[0113] Table 4: Bioactivity of EGF Released by Different Materials

[0114] conventional adsorption materials 0.2553±0.022 <![CDATA[Prepared using magnetic Fe3O4 nanoparticles]]> 0.1853±0.023 <![CDATA[Prepared using Fe3O4@SiO2]]> 0.2555±0.040 <![CDATA[Prepared using Fe3O4@TiO2]]> 0.2355±0.040 <![CDATA[Prepared using Fe3O4@SiO2@TiO2]]> 0.3155±0.040 <![CDATA[Prepared using Fe3O4@SiO2@COC]]> 0.2655±0.040 <![CDATA[Prepared using TiO2 nanoparticles]]> 0.251±0.040

[0115] Compared with existing adsorption materials, the nanofilter material of the present invention exhibits the highest cell proliferation rate at the same EGF concentration. This is because the released EGF retains high biological activity, which is beneficial for cell proliferation. In contrast, existing conventional adsorption materials release EGF with poor cell growth stimulation after adsorption.

[0116] In the preparation process, the present invention uses magnetic Fe3O4 nanoparticles, Fe3O4@SiO2, and TiO2 nanoparticles in sequence. According to the experimental results, even under the same layer-by-layer self-assembly method, the EGF released by the material prepared using the above-mentioned substrate has a far less effective effect on stimulating cell growth than the nanofilter material of the present invention.

[0117] Using Fe3O4@TiO2 and Fe3O4@SiO2@COC as controls, under the same layer-by-layer self-assembly method, the EGF-stimulating cell growth effect released by the materials prepared from the above two substrates is not as good as that of the nano-filter material prepared by Fe3O4@SiO2@TiO2 in this invention. This indicates that the SiO2 shell is indispensable, and when the SiO2 shell is already present, TiO2 modification is indispensable. The experimental results show that only Fe3O4@SiO2@TiO2 nanoparticles can be used to prepare an adsorbent material with the highest effect of releasing EGF to stimulate cell growth.

[0118] Example 3: Screening of functional groups on material surface

[0119] Introducing polar groups, such as hydroxyl and carboxyl groups, into the surface of polymer nanomaterials improves the wettability of the polymer surface and has a certain impact on the adsorption and biocontact properties of peptides, thereby improving the activity of the material surface and enhancing biocompatibility.

[0120] By comparing the grafting efficiency and grafting rate of materials with different functional groups introduced.

[0121] Weigh the surface-modified nanoparticles W1(G) (repeatedly weigh several times until the mass difference is less than 0.01g), then place them in a vacuum drying oven and vacuum dry at room temperature until the product reaches constant weight. Take them out and weigh W2 (repeatedly weigh several times until the mass difference is less than 0.01g).

[0122] Grafting rate % = [(W1-W2) / W1] * 100%

[0123] Grafting efficiency % = [(W1-W2) / W2] * 100%

[0124] (1) Method for introducing hydroxyl groups: Recombinant human epidermal growth factor (EGF stock solution) (Shenzhen Huashengyuan Gene Engineering Co., Ltd.), 3-glycidyl etheroxypropyltriethoxysilane (Aladdin) were dissolved in a 0.02 mol / L phosphate buffer solution with a pH of 9.0. The solution was ultrasonically treated for 5 min to 10 min, and then aged at room temperature for 1 h to 3 h. Then, Fe3O4@SiO2@TiO2 particles obtained in step two were added. Under the conditions of stirring speed of 100 r / min to 300 r / min and nitrogen atmosphere protection, ammonium persulfate (Shanghai Maclean Biochemical Technology Co., Ltd.) phosphate buffer solution was added at a dropping rate of 1 mL / min to 5 mL / min. The reaction was carried out in an oil bath at a temperature of 40 to 80℃ for 24 h to 36 h. The mixture was stirred until the monomer solution and sol material were completely and uniformly mixed. The mixture was then ultrasonically cleaned in an ultrasonic cleaner for 30 min to remove air bubbles in the sol gel and form a homogeneous system. 5% SDS-2% was used. Elute the template nanoparticles with HAC solution until no EGF is detected, and then vacuum dry at room temperature until the product reaches constant weight.

[0125] (2) Method for introducing carboxyl groups: Recombinant human epidermal growth factor (EGF stock solution) (Shenzhen Huashengyuan Gene Engineering Co., Ltd.) and 3-aminopropyltrimethoxysilane (Aladdin) were dissolved in a 0.02 mol / L phosphate buffer solution with a pH of 9.0. The solution was ultrasonically treated for 5 min to 10 min, and then aged at room temperature for 1 h to 3 h. Then, Fe3O4@SiO2@TiO2 particles obtained in step two were added. Under the conditions of stirring speed of 100 r / min to 300 r / min and nitrogen atmosphere protection, ammonium persulfate (Shanghai Maclean Biochemical Technology Co., Ltd.) phosphate buffer solution was added at a dropping rate of 1 mL / min to 5 mL / min. The reaction was carried out in an oil bath at a temperature of 40 to 80℃ for 24 h to 36 h. The mixture was stirred until the monomer solution and sol material were completely and uniformly mixed. The mixture was then ultrasonically cleaned in an ultrasonic cleaner for 30 min to remove air bubbles in the sol gel and form a homogeneous system. 5% SDS-2% SDS was used. Elute the template nanoparticles with HAC solution until no EGF is detected, and then vacuum dry at room temperature until the product reaches constant weight.

[0126] (3) Method for introducing amino groups: Recombinant human epidermal growth factor (EGF stock solution) (Shenzhen Huashengyuan Gene Engineering Co., Ltd.), 3-aminopropyltriethoxysilane (Aladdin), and 2,4-diaminophenylboronic acid (Sigma-Aldrich) were dissolved in a 0.02 mol / L phosphate buffer solution with a pH of 9.0. The solution was ultrasonically treated for 5 min to 10 min, and then aged at room temperature for 1 h to 3 h. Then, Fe3O4@SiO2@TiO2 particles obtained in step two were added. Under the conditions of stirring speed of 100 r / min to 300 r / min and nitrogen atmosphere protection, ammonium persulfate (Shanghai Maclean Biochemical Technology Co., Ltd.) phosphate buffer solution was added at a dropping rate of 1 mL / min to 5 mL / min. The reaction was carried out in an oil bath at a temperature of 40 to 80 °C for 24 h to 36 h. The mixture was stirred until the monomer solution and sol material were completely and uniformly mixed. The mixture was then ultrasonically cleaned in an ultrasonic cleaner for 30 min to remove air bubbles in the sol gel and form a homogeneous system. 5% SDS-2% was used. Elute the template nanoparticles with HAC solution until no EGF is detected, and then vacuum dry at room temperature until the product reaches constant weight.

[0127] (4) Method for introducing sulfonic acid groups: Recombinant human epidermal growth factor (EGF stock solution) (Shenzhen Huashengyuan Gene Engineering Co., Ltd.) and 2-propylamido-2-ethylpropanesulfonic acid (Sigma-Aldrich) were dissolved in a 0.02 mol / L phosphate buffer solution with a pH of 9.0. The solution was ultrasonically treated for 5 min to 10 min, and then aged at room temperature for 1 h to 3 h. Then, Fe3O4@SiO2@TiO2 particles obtained in step two were added. Under the conditions of stirring speed of 100 r / min to 300 r / min and nitrogen atmosphere protection, ammonium persulfate (Shanghai Maclean Biochemical Technology Co., Ltd.) phosphate buffer solution was added at a dropping rate of 1 mL / min to 5 mL / min. The reaction was carried out in an oil bath at a temperature of 40 to 80 °C for 24 h to 36 h. The mixture was stirred until the monomer solution and sol material were completely and uniformly mixed. The mixture was then ultrasonically cleaned in an ultrasonic cleaner for 30 min to remove air bubbles in the sol gel and form a homogeneous system. 5% SDS-2% was used. Elute the template nanoparticles with HAC solution until no EGF is detected, and then vacuum dry at room temperature until the product reaches constant weight.

[0128] (5) Method for introducing boric acid groups: Recombinant human epidermal growth factor (EGF stock solution) (Shenzhen Huashengyuan Gene Engineering Co., Ltd.) and 2,4-diaminophenylboronic acid (Sigma-Aldrich) were dissolved in a 0.02 mol / L phosphate buffer solution with a pH of 9.0. The solution was ultrasonically treated for 5 min to 10 min, and then aged at room temperature for 1 h to 3 h. Then, Fe3O4@SiO2@TiO2 particles obtained in step two were added. Under the conditions of stirring speed of 100 r / min to 300 r / min and nitrogen atmosphere protection, ammonium persulfate (Shanghai Maclean Biochemical Technology Co., Ltd.) phosphate buffer solution was added at a dropping rate of 1 mL / min to 5 mL / min. The reaction was carried out in an oil bath at a temperature of 40 to 80 °C for 24 h to 36 h. The mixture was stirred until the monomer solution and sol material were completely and uniformly mixed. The mixture was then ultrasonically cleaned in an ultrasonic cleaner for 30 min to remove air bubbles in the sol gel and form a homogeneous system. 5% SDS-2% SDS was used. Elute the template nanoparticles with HAC solution until no EGF is detected, and then vacuum dry at room temperature until the product reaches constant weight.

[0129] Simultaneously, amino and borate groups are introduced: as described in Example 1.

[0130] Table 5: Introduction of functional groups and grafting efficiency, grafting rate

[0131] hydroxyl 72.5 23.6 carboxyl 90.8 28.1 amino 88.6 30.6 sulfonic acid group 78.3 27.8 Boronic acid group 76.9 26.9 Boric acid group + amino 95.2 36.5

[0132] According to the experimental results, among the above methods of introducing groups, the grafting efficiency and grafting rate of simultaneously introducing amino and boric acid groups are the highest. This indicates that simultaneously introducing amino and boric acid groups is most beneficial for surface modification of the nano-filter material of the present invention. On the other hand, the high grafting efficiency and grafting rate affect the performance of the adsorption material, indicating that simultaneously introducing amino and boric acid groups is most beneficial for improving the performance of the adsorption material.

[0133] To specifically obtain the performance improvement of the nanofilter material of the present invention by different functional groups, the adsorption efficiency, specificity, release effect and bioactivity retention of the adsorbent materials prepared by introducing different functional groups were tested according to the method of Example 2. The results are shown in Table 6.

[0134] Table 6: Performance Tests of Adsorbent Materials with Different Groups

[0135]

[0136] Taking all factors into consideration, this patent introduces boric acid groups and amino groups onto the surface of polymer nanomaterials. Compared to introducing a single group, the simultaneous introduction of boric acid groups and amino groups significantly increases adsorption efficiency, specificity, release effect, and retention of bioactivity. The boric acid groups and amino groups work synergistically to improve the performance of the nanofilter material of this invention because: boric acid groups can form stable ester or ester-amine bonds with amino groups in the nanomaterials; amino groups and the strongly anionic and hydrophilic functional groups of sulfonic acid groups can combine with the colloidal surface, exhibiting excellent experimental grafting effects for surface modification of nanoparticles; their hydrophobicity prevents the aggregation of colloidal particles, and their structure allows for better binding of target molecules; amino groups possess multiple cationic properties, reducing crystallinity, weakening intermolecular hydrogen bonds, and interacting with negative charges, thus reducing molecular crystallinity. Introducing new functional groups gives the complex new properties, allowing for the addition of substances with different biocompatibility, altering hydrophobicity, and simplifying the preparation process.

[0137] Example 4: Experiment on layer-by-layer self-assembly crosslinking agents

[0138] The layer-by-layer self-assembly method of this invention first requires the introduction of a crosslinking agent. This agent enables the alternating adsorption of negatively charged template nanoparticles and positively charged bioactive component particles such as EGF. Through electrostatic interactions, the template particles and bioactive components such as EGF are alternately self-assembled onto the surface of the crosslinked EGF and other bioactive component particle substrate. The crosslinking agent plays a crucial role in this process. According to Example 3, boric acid groups and amino groups should be introduced to allow for the alternating adsorption of bioactive components and nanoparticles. Further testing is needed on crosslinking agents incorporating boric acid groups and amino groups.

[0139] Determination of adsorption capacity: Accurately weigh 0.10 g of nanofilter particles prepared using different crosslinking agents, add them to 10 mL of 0.5 mg / L EGF solution, and adjust the pH to 9.0 with 10 mL of 0.02 mol / L phosphate buffer solution. Shake in a constant temperature shaker at 25℃ and 150 r / min for 10 min. After the reaction is complete, separate the reaction solution using a magnetic field, and use liquid chromatography to determine the residual amount of EGF in the supernatant. Calculate the adsorption capacity of the nanofilter particles for EGF. The adsorption capacity Q of this material for EGF can be obtained based on the change in concentration before and after adsorption. Perform three parallel determinations and take the average value. The formula for calculating the binding capacity is as follows:

[0140] Q = (C0 - C) e V / m

[0141] In the formula, Q is the adsorption capacity, C0 (mmol / L) is the concentration of the substrate in the liquid before adsorption, and C... e (mmol / L) is the concentration of the adsorbed substance in the solution after adsorption, V (mL) is the volume of the adsorbent solution, and m (g) is the mass of the adsorbent.

[0142] Elution efficiency determination: Accurately weigh 0.10 g of nanofilter particles prepared using different crosslinking agents, add them to 10 mL of 0.5 mg / L EGF solution, and adjust the pH to 9.0 with 10 mL of 0.02 mol / L phosphate buffer solution. Shake in a constant temperature shaker at 25℃ and 150 r / min for 10 min. After the reaction is complete, separate the reaction solution using a magnetic field, and use liquid chromatography to determine the residual amount of EGF in the supernatant. Calculate the adsorption capacity of the nanofilter particles for EGF. Then, elute the used adsorbent with 5% SDS-2% HAC solution for 30 min, and measure the EGF content in the eluent using liquid chromatography to obtain the elution efficiency of the nanofilter particles.

[0143] In the early stages of this invention, various reagents were used as crosslinking agents in the layer-by-layer self-assembly step. Fe3O4@SiO2@TiO2 nanoparticles were prepared using the method described in Example 1. Subsequently, layer-by-layer self-assembly was carried out under the action of different crosslinking agents. Except for the different crosslinking agents, all other steps were performed in accordance with Example 1. The loading capacity and elution efficiency of the prepared materials were measured. The loading capacity represents the highest adsorption mass per unit mass of material, and the elution efficiency represents the proportion of adsorbed bioactive molecules that can be eluted. The results are shown in Table 7.

[0144] Table 7: Loading capacity and elution efficiency of materials prepared under different crosslinking agents

[0145]

[0146] According to the experimental results, the loading capacity and elution efficiency of the materials prepared under different crosslinking agents are different. This is because the crosslinking agent affects the binding of EGF and nanoparticles during the layer-by-layer self-assembly process and has a huge impact on the formation of three-dimensional cavities. 2,4-Diaminophenylboronic acid and 2-propylamido-2-ethylpropanesulfonic acid are the preferred crosslinking agents. At this time, the loading capacity and elution efficiency are the highest and significantly exceed those of other crosslinking agents. Moreover, the combined use of 2,4-diaminophenylboronic acid and 2-propylamido-2-ethylpropanesulfonic acid is significantly better than the effect of using any one crosslinking agent alone.

[0147] The present invention suggests the following possible mechanisms of action:

[0148] In polymer nanomaterials, the introduction of functional groups such as borate and amino groups on the surface can enable nanoparticles to specifically recognize target molecules. 2,4-Diaminophenylboronic acid contains both amino and borate groups, both relatively reactive, and can act as a reaction intermediate. Utilizing the property that 2,4-diaminophenylboronic acid readily forms reversible covalent bonds with cis-neighboring compounds, the relatively weak intermolecular forces can be replaced by relatively strong reversible covalent bonds, thus eluting the bound target molecules and achieving separation and enrichment. 2-Propylamido-2-ethylpropanesulfonic acid has a strong anionic and hydrophilic sulfonic acid functional group, giving it excellent polymerization properties; it can undergo self-polymerization and homopolymerization, as well as copolymerization with various monomers.

[0149] The functional monomer 2,4-diaminophenylboronic acid and the auxiliary functional monomer 2-propylamido-2-ethylpropanesulfonic acid are prepolymerized with the template molecule EGF under certain conditions to enable the monomers to bind well with the template molecule. Then, an initiator and a support are added, and a polymer reaction is carried out under certain conditions to form a copolymer. This copolymer is then eluted, forming cavities on the surface of Fe3O4@SiO2@TiO2 particles that specifically recognize the template molecule. These cavities can specifically recognize EGF in the mixed solution. The synergistic effect of the reversible covalent bonds and hydrogen bonds formed at the same time greatly improves the selectivity and adsorption capacity of Fe3O4@SiO2@TiO2 particles for EGF.

[0150] Next, keeping other reaction conditions unchanged, the molar ratio of 2,4-diaminophenylboronic acid and 2-propylamido-2-ethylpropanesulfonic acid was adjusted to obtain a suitable molar ratio. The adsorbent material was prepared according to the method in Example 1, with the only difference being the molar ratio between the crosslinking agents. The elution efficiency and loading capacity were tested, and the results are shown in Table 8.

[0151] Table 8: Molar mass ratio of crosslinking agent, elution efficiency, and loading capacity

[0152] 1：0.2 98 24.67 1：1 95 25.89 1：1.2 92 19.16

[0153] According to the experimental results, the molar mass ratio of the crosslinking agent affects the elution efficiency and carrying capacity. The preferred molar mass ratio of 2,4-diaminophenylboronic acid and 2-propylamido-2-ethylpropanesulfonic acid is 1:(0.2-1).

[0154] Example 5: Experiment on layer-by-layer self-assembled eluent

[0155] In Example 4, we have demonstrated through experiments and theoretical studies that 2,4-diaminophenylboronic acid and 2-propylamido-2-ethylpropanesulfonic acid are the best layer-by-layer self-assembly crosslinking agents for preparing specific adsorption of bioactive components (especially EGF). After the crosslinking of EGF with nanoparticles is completed, it is necessary to elute the EGF to obtain the three-dimensional vacancies that specifically adsorb EGF. Therefore, it is necessary to further study how to elute the adsorbed nanofilter material to maximize the protection of the three-dimensional vacancies structure and obtain the most three-dimensional vacancies.

[0156] In the early research of this invention, various eluents were tested. The nanofilter material was prepared according to the method of Example 1, except that the eluent was changed. The elution efficiency and carrying capacity were measured according to the method of Example 3. The results are shown in Table 9.

[0157] Table 9: Elution Buffer Composition and Elution Efficiency

[0158] dichloromethane 90 6.48 2% HAC 82 13.65 methanol 34 2.45 5% SDS-2% HAC 98 24.67

[0159] According to the experimental results, different eluents have different elution efficiencies, and the composition of the eluent has a significant impact on the loading capacity of the nanofilter material. This is because the choice of eluent affects the elution efficiency, which in turn affects the number of three-dimensional vacuoles. Furthermore, the eluent also affects the stability of the three-dimensional vacuoles. Some eluents can destroy the groups in the three-dimensional vacuoles that are complementary to EGF, leading to a decrease in adsorption efficiency and specificity. The eluent with only SDS-HAC component can produce high elution efficiency and high loading capacity, making it the optimal choice.

[0160] Keeping other reaction conditions constant and optimizing the eluent solution ratio, 0.10 g of nanoparticles were accurately weighed and added to 10 mL of a 0.5 mg / L EGF solution. 10 mL of 0.02 mol / L phosphate buffer solution was added to adjust the pH to 9.0. The mixture was shaken in a constant-temperature shaker at 25 °C and 150 rpm for 0.5, 1.5, 3.0, 6.0, 9.5, 12.5, and 15.0 minutes, respectively. After the reaction was complete, the reaction solution was separated using a magnetic field. The concentration of remaining EGF in the supernatant was then determined by liquid chromatography, yielding the change in EGF adsorption capacity of the nanofilter particles with contact time. The adsorption capacity continuously increased with treatment time. It can also be seen that in the early stage of the system, the adsorption capacity of nanoparticles for EGF is continuously increasing. However, after a certain contact time, the adsorption capacity of the entire system reaches its maximum value, and the adsorption capacity of nanoparticles for EGF in the system does not change significantly. This indicates that the adsorption capacity of nanoparticles for EGF has reached saturation at this point. The adsorption capacity of nanoparticles at this point is calculated. The results are shown in Table 10.

[0161] Table 10: Eluent Ratio, Elution Efficiency, and Load Capacity

[0162] 5% SDS-2% HAC 98 24.67 8% SDS-3% HAC 88 17.89 10% SDS-4% HAC 80 9.29

[0163] Based on the test results, the preferred eluent solution ratio is: 5% SDS - 2% HAC.

[0164] Example 6: Stability test of the substrate

[0165] The reusability of the adsorption capacity of nanofilter particles for EGF was investigated. Specifically, 0.10 g of nanofilter particles were accurately weighed and added to 10 mL of a 0.5 mg / L EGF solution. The pH was adjusted to 9.0 by adding 10 mL of 0.02 mol / L phosphate buffer solution, and the mixture was shaken for 10 min at 25℃ and 150 rpm. After the reaction was complete, the reaction solution was separated using a magnetic field. The supernatant was then analyzed by liquid chromatography to determine the residual EGF content and calculate the adsorption capacity of the nanofilter particles for EGF. The used adsorbent was then eluted with 5% SDS-2% HAC solution for 30 min, and the EGF content in the eluent was measured by liquid chromatography to obtain the elution rate of the nanofilter particles. The nanofilter particles were then reused, and the elution rate of the adsorbent after multiple uses was measured. A curve showing the change in the elution rate of the nanofilter particles for EGF with the number of reuses was obtained, as shown in the figure. Figure 3 As shown.

[0166] The elution rate of the nanofilter particles still reached over 95% after repeated elution, which indicates that the nanofilter particles have good chemical stability. The molecular imprinted layer they are coated with was not damaged by frequent elution with acidic eluent, and the specific recognition and adsorption functional groups on it remained stable. This is beneficial to the nanofilter particle material having a long service life, which is conducive to long-term storage and reduces the replacement rate of nanofilter particle material, thereby reducing the cost of use.

[0167] The embodiments described above provide a detailed explanation of the technical solution of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, or similar substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a material that specifically adsorbs bioactive substances, characterized in that, Crosslinking Fe3O4@SiO2@TiO2 nanoparticles with bioactive substances, followed by elution of the bioactive substances, yields a material containing three-dimensional holes matching the bioactive molecules. The Fe3O4@SiO2@TiO2 nanoparticles are a composite formed by Fe3O4@SiO2 nanoparticles and nano-TiO2 through hydrogen bonding. The Fe3O4@SiO2 nanoparticles are monodisperse silica-encapsulated magnetite nanoparticles. The bioactive substance is epidermal growth factor. The eluent used for elution is an aqueous solution containing both sodium dodecyl sulfate and acetic acid.

2. The method as described in claim 1, characterized in that, The elution process uses an aqueous solution containing both sodium dodecyl sulfate and acetic acid, specifically a 5% sodium dodecyl sulfate-2% acetic acid solution.

3. The method as described in claim 2, characterized in that, The crosslinking includes introducing groups on the surface of the nanoparticles, the groups including at least one borate group and an amino group.

4. The method as described in claim 3, characterized in that, The crosslinking agents used include 2,4-diaminophenylboronic acid and 2-propylamido-2-ethylpropanesulfonic acid.

5. The use of Fe3O4@SiO2@TiO2 nanoparticles in the preparation of materials that improve the adsorption rate and 24h release rate of specific adsorption of epidermal growth factor, characterized in that, The material is: Fe3O4@SiO2@TiO2 nanoparticles cross-linked with epidermal growth factor, and the epidermal growth factor is eluted to obtain a material containing three-dimensional cavities matching the epidermal growth factor; the Fe3O4@SiO2@TiO2 nanoparticles are a composite formed by Fe3O4@SiO2 nanoparticles and nano-TiO2 through hydrogen bonding, and the Fe3O4@SiO2 nanoparticles are monodisperse silica-encapsulated magnetite nanoparticles.

6. A method for extracting bioactive substances, characterized in that, The material prepared by the method described in any one of claims 1-4 is contacted with a mixture containing the target bioactive substance for adsorption, and then transferred to a buffer solution to release the target bioactive substance; the bioactive substance is epidermal growth factor.

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