Amine-functionalized magnetic nanocomposites and their applications in protein separation and purification

Abstract

This invention relates to the field of bioseparation materials technology, and discloses amine-functionalized magnetic nanocomposite materials and their application in protein separation and purification. The material has a core-shell structure, comprising a magnetic iron oxide nanocore, an ordered mesoporous silica intermediate layer, and a covalently grafted polyethyleneimine functional layer. By controlling the matching relationship between the mesopore size and the polymer molecular size, and employing a cross-linking strategy, the material possesses both high magnetic responsiveness, rapid mass transfer performance, and high-density surface amino groups. The material exhibits high adsorption capacity, rapid adsorption kinetics, and good cycling stability during protein separation, making it suitable for the enrichment and purification of proteins in biological samples and for metal chelate affinity separation. This invention solves the technical problems of low protein adsorption capacity, slow kinetics, and poor cycling stability in existing amine-functionalized magnetic materials due to their dense shells, uneven amino group distribution, and limited mass transfer.

Description

Technical Field

[0001] This invention belongs to the field of nanobiomaterials for separation, and more specifically, relates to amine-functionalized magnetic nanocomposites and their application in protein separation and purification. Background Technology

[0002] With the increasing demands for protein separation and purification efficiency in the biomedical field, amine-functionalized magnetic nanocomposites have become a key material platform for the efficient separation of biomacromolecules due to their unique external field response and surface modifiability. However, existing technologies face numerous unresolved technical challenges in material structure design and functional realization.

[0003] Among the main technical routes for constructing amine functionalized layers on the surface of magnetic nanomaterials, the invention patent with publication number CN113967471B adopts a three-step modification route of APTES amination → succinic anhydride carboxylation → PEI amidation, which achieves the adsorption of metal ions by grafting polyethyleneimine (PEI) onto the surface of magnetic mesoporous silica microspheres. However, this technical solution has the following limitations when applied to protein separation: First, the formation of carboxyl-aminoamide bonds causes the PEI molecular chain to be fixed only through the end connection points. Based on the Flory chain segment theory, this single-point anchoring method limits the grafting density to the surface carboxyl density and lacks cross-linking stabilization design, making the PEI layer prone to swelling and detachment under acidic elution conditions; Second, it ignores the matching relationship between pore size and molecular chain size, and the PEI molecular chain (hydrodynamic diameter 8-10 nm) cannot enter the mesoporous channel, resulting in a specific surface area utilization rate of less than 25% for the material with a maximum of 800 m² / g, and the spatial distribution density of functional groups is severely limited.

[0004] The invention patent with publication number CN106693920A constructs a composite functional layer through the layer-by-layer assembly of chitosan-triethylenetetramine-graphene oxide. Although this scheme has shown some effectiveness in the adsorption of heavy metal ions and dyes, its multilayered and dense structural design may face the following challenges when applied to protein separation: Based on Fick's diffusion law analysis, the formation of multilayered and dense shells reduces the diffusion coefficient of protein molecules to 1 / 5-1 / 8 of that in bulk solution, significantly increasing mass transfer resistance; at the same time, the π-π stacking effect of the graphene oxide layers leads to partial shielding of surface amine groups, and based on the Langmuir adsorption model, the effective amine group density is reduced by about 40%; in addition, there is a significant interfacial energy barrier between the hydrophobic graphene layer and the hydrophilic protein, which significantly increases the risk of nonspecific adsorption.

[0005] In addition, the MAAM-EGDMA copolymer coating strategy adopted in the invention patent with announcement number CN115920864B results in the vast majority of amino groups being encapsulated inside the three-dimensional network due to their highly cross-linked and dense shell, making it difficult to effectively expose them to the solution interface. Meanwhile, the amino modification scheme based on carbon nanotubes in the invention patent with announcement number CN112574431B is prone to irreversible aggregation due to the inherent hydrophobicity of the material and strong van der Waals interactions. This not only reduces dispersion stability but may also shield the magnetic core surface, affecting the magnetic field response efficiency.

[0006] Existing technologies either tend to have high cross-linking to ensure stability but sacrifice chain mobility, or low cross-linking to maintain flexibility but lack durability; dense functional layers increase group density but hinder molecular diffusion, while sparse functional layers improve mass transfer but limit adsorption capacity; strong electrostatic interactions ensure adsorption capacity but trigger non-specific binding, while weak interactions improve specificity but reduce separation efficiency.

[0007] Existing technologies have failed to solve the problem of precisely matching the size of functional molecules with the pore size of mesopores: when the PEI molecule size is larger than the pore size, it leads to pore blockage and waste of specific surface area; when the molecule size is much smaller than the pore size, the inner wall of the pore cannot be effectively functionalized, limiting the adsorption capacity. This has prevented existing materials from overcoming the technical bottleneck of adsorption capacity and kinetic rate in protein separation applications. Summary of the Invention

[0008] This invention discloses amine-functionalized magnetic nanocomposites and their application in protein separation and purification, solving the technical problems of low protein adsorption capacity, slow kinetics, and poor cycling stability caused by dense shells, uneven amino group distribution, and limited mass transfer in existing amine-functionalized magnetic materials.

[0009] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0010] This invention mainly addresses the technical problems in the prior art, such as insufficient adsorption capacity, slow kinetics, poor regeneration performance, and severe non-specific adsorption caused by dense shell structure, uneven amino distribution, limited mass transfer, and low accessibility of functional groups.

[0011] The amine-functionalized magnetic nanocomposite material comprises, from the inside out: a magnetic nanocore, a mesoporous silica interlayer, and a covalently grafted polyethyleneimine (PEI) functional layer; wherein the magnetic nanocore is Fe3O4 nanocrystals with a particle size in the range of 8–12 nm and a saturation magnetization of not less than 65 emu / g; the mesoporous silica interlayer has a thickness of 25–35 nm, and its channels are arranged in a two-dimensional hexagonal ordered pattern, with the pore size distribution concentrated in the range of 4.5–6.0 nm, and a specific surface area ratio of [missing information]. The area is not less than 800 m² / g, and the pore volume is not less than 0.9 cm³ / g; the polyethyleneimine functional layer is covalently anchored by a condensation reaction between a silane coupling agent and the silanol groups on the surface of mesoporous silica, and further moderately crosslinked by a crosslinking agent to improve its structural stability in an aqueous environment. The total nitrogen content of free primary amines, secondary amines and tertiary amines in the polyethyleneimine (PEI) functional layer is not less than 2.8 mmol / g, and the amino accessibility rate measured by the Orange II adsorption method is not less than 75%.

[0012] Furthermore, the magnetic nanonucleus is synthesized via a co-precipitation method, with the specific steps as follows:

[0013] Step 1: Dissolve FeCl2·4H2O and FeCl3·6H2O in deionized water at a molar ratio of 1:2;

[0014] Step 2: After purging with nitrogen to remove oxygen for 30 minutes, add 25wt% ammonia solution dropwise under a 60℃ water bath until the pH value reaches 10.5, and continue stirring for 1 hour.

[0015] Step 3: The obtained black precipitate was magnetically separated, washed three times alternately with ethanol and deionized water, and then vacuum dried at 60°C for 12 hours to obtain Fe3O4 nanocrystals.

[0016] In a preferred embodiment of the present invention, the mesoporous silica intermediate layer is grown in situ on the surface of the magnetic nanonucleus using a sol-gel method, the specific process of which is as follows:

[0017] The Fe3O4 nanocrystals prepared above were ultrasonically dispersed in an ethanol / water mixed solvent (volume ratio 3:1). 0.5M ammonia was added to adjust the pH of the system to 9.0–9.5. Then, tetraethyl orthosilicate (TEOS) was added as a silicon source, with a mass ratio of TEOS to Fe3O4 of 4:1. The reaction was stirred at 30°C for 4 hours. Subsequently, a template agent, cetyltrimethylammonium bromide (CTAB), was introduced at a concentration of 0.1M, and the reaction was continued at 40°C for 12 hours. After the reaction was completed, the product was collected by magnetic separation, washed three times with ethanol, and then refluxed at 80°C for 6 hours to remove the CTAB template, finally obtaining a Fe3O4@SiO2 core-shell structure with an ordered mesoporous structure.

[0018] Furthermore, the construction of the polyethyleneimine functional layer involves two steps:

[0019] Step 1: Introduce the silane coupling agent 3-glycidoxypropyltrimethoxysilane (GPTMS) onto the surface of Fe3O4@SiO2. The dosage is 1.2 mmol of GPTMS per gram of Fe3O4@SiO2. Specifically, Fe3O4@SiO2 is dispersed in anhydrous toluene, GPTMS is added, and the mixture is refluxed at 80°C for 18 hours under nitrogen protection. The product is then magnetically separated, washed alternately with toluene and ethanol, and vacuum dried.

[0020] Step 2: The GPTMS-modified material was dispersed in a 0.5M aqueous solution of polyethyleneimine (PEI molecular weight 25,000 g / mol) and reacted at 60°C for 8 hours to allow the epoxy groups to undergo a ring-opening addition reaction with the primary amine on the PEI molecular chain, forming a stable C–N covalent bond. After the reaction was completed, the material was magnetically separated and washed with deionized water until the pH of the eluent was neutral to obtain the primary amine functionalized material.

[0021] The polyethyleneimine functional layer requires glutaraldehyde cross-linking treatment to enhance its structural integrity in complex biological media. The degree of cross-linking is considered adequate when the PEI functional layer shedding rate is less than 5% after 10 cycles of use under pH 3.0 elution conditions. The shedding rate is determined by nitrogen loss rate: the material is treated 10 times in a glycine-HCl elution solution at pH 3.0. After each elution, the material is collected, vacuum dried, and the nitrogen content is determined by elemental analysis.

[0022] Functional layer shedding rate of material after i-th cycle Calculate using the following formula:

[0023] ;

[0024] in, Indicates the nitrogen content of the initial amine-functionalized material (unit: mmol / g); This indicates the nitrogen content of the material after the i-th adsorption-elution cycle (unit: mmol / g).

[0025] The specific crosslinking conditions are as follows: the primary amine functionalized material is dispersed in a 0.1M glutaraldehyde aqueous solution and stirred at room temperature in the dark for 2 hours. Then, a 0.5M glycine solution is added to quench the unreacted aldehyde groups, and the reaction is continued for 30 minutes. The final product is subjected to magnetic separation, thorough washing with deionized water, and freeze-drying to obtain the amine functionalized magnetic nanocomposite material of the present invention.

[0026] In the amine-functionalized magnetic nanocomposite material of this invention, the introduction of the mesoporous silica intermediate layer not only effectively isolates the magnetic core from the external aqueous environment, preventing Fe3O4 from corroding or decaying under acidic or oxidizing conditions, but also provides a low-resistance diffusion path for macromolecular targets (such as proteins) through its highly ordered mesoporous channels, significantly improving mass transfer efficiency. Meanwhile, the polyethyleneimine functional layer, due to its highly branched structure and abundant tertiary amine groups, can exhibit strong positive charge under physiological pH conditions, thereby efficiently capturing negatively charged protein molecules through electrostatic interactions. In addition, the flexible conformation of the PEI molecular chains can form a hydrophilic microenvironment with a certain buffering capacity on the material surface, effectively inhibiting conformational changes and non-specific denaturation of proteins during adsorption.

[0027] In protein separation and purification applications, the operation procedure of the amine-functionalized magnetic nanocomposite material described in this invention is as follows:

[0028] The amine-functionalized magnetic nanocomposite material was dispersed in the protein solution to be treated at a concentration of 10 mg / mL and adsorbed by shaking at 25 °C for 30 minutes.

[0029] An external magnetic field (magnetic field strength ≥ 0.3T) is then applied to cause the material to settle rapidly, and the supernatant is discarded.

[0030] Next, wash three times with phosphate-buffered saline (PBS) at pH 7.4 to remove weakly adsorbed impurities;

[0031] Finally, the target protein was recovered by elution with glycine-HCl at pH 3.0 for 10 minutes at 25°C.

[0032] After elution, the material is regenerated with 0.1M NaOH solution for 15 minutes, then washed with deionized water until neutral, and can be reused for the next round of separation.

[0033] Furthermore, the functional expandability of the material described in this invention is achieved through the following methods:

[0034] After PEI grafting is completed, some unreacted primary amine groups are retained, which can be further amidated with N-hydroxysuccinimide (NHS) activated carboxylic acid ligands (such as IDA, NTA) or biotin molecules to construct a metal chelate affinity chromatography platform or a biotin-avidin recognition system.

[0035] For example, by dispersing the material in a MES buffer (pH 5.5) of 5 mM NHS and 10 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), activating for 30 minutes, adding 10 mM iminodiacetic acid (IDA), and reacting at 4°C for 12 hours, Ni can be obtained. 2+Chelateable affinity materials for the specific purification of histidine-tagged proteins.

[0036] In this invention, the molecular weight of polyethyleneimine (PEI) was selected as 25,000 g / mol through systematic optimization. Dynamic light scattering measurements (pH 7.0, 25°C) showed that its branched hydrodynamic diameter was 3.5-5.2 nm (see supplementary data in Example 1), smaller than the mesopore diameter (4.5-6.0 nm). Therefore, some branches can enter the pores to achieve functionalization. When the molecular weight is below 10,000 g / mol, the branching degree is insufficient, resulting in a low amino density per unit mass. When the molecular weight is above 50,000 g / mol, the excessively long molecular chains easily form a blocking effect at the mesopore openings, hindering pore connectivity and reducing the utilization rate of the internal surface area. PEI at 25,000 g / mol, while ensuring a high amino density, has some chain segments or branches with a kinetic diameter smaller than the mesopore diameter (4.5-6.0 nm), allowing PEI to partially enter the pores to achieve pore wall functionalization, thereby maximizing the utilization of the high specific surface area of ​​the mesoporous structure.

[0037] In addition, the control of the degree of crosslinking of glutaraldehyde is crucial: when the concentration of the crosslinking agent is too low (<0.05M), the PEI layer is prone to swelling and shedding during multiple elution processes; when the concentration is too high (>0.2M), it will lead to excessive crosslinking, which will increase the rigidity of the PEI chain and reduce the conformational flexibility and accessibility of the amino group; the present invention uses 0.1M glutaraldehyde, which maintains the high reactivity of the amino group while ensuring structural stability.

[0038] The material of this invention achieves efficient capture primarily through the synergistic action of the following three forces:

[0039] Electrostatic attraction: Under conditions where pH > pI, the net charge on the protein surface is negative, forming Coulomb attraction with the positively charged amino groups on the material surface;

[0040] Hydrogen bonding: –NH– and –OH in PEI (derived from residual epoxide hydrolysis) can form multiple hydrogen bonds with carbonyl groups in the protein backbone or polar groups in the side chains;

[0041] Hydrophobic microregion assistance: Although PEI is generally hydrophilic, its ethylene backbone still has weakly hydrophobic regions that can generate van der Waals forces with hydrophobic residues on the protein surface, thereby enhancing the binding strength.

[0042] The aforementioned multi-mode interaction mechanism enables the material of the present invention to exhibit good adsorption performance in a wide pH range (4.0–9.0) and to be universally applicable to proteins with different isoelectric points.

[0043] To address the problem of non-specific adsorption, this invention suppresses it through the following design:

[0044] The mesoporous SiO2 layer itself has high hydrophilicity and can form a hydration layer to block hydrophobic interactions; after appropriate cross-linking, the PEI functional layer has a uniform surface charge distribution, avoiding strong non-specific binding caused by local high charge density.

[0045] In practice, the PBS washing step can effectively remove impurities adsorbed by weak electrostatic or hydrophobic interactions, while the target protein is retained due to multi-point binding. Experiments show that in serum samples, the material of this invention has a specific enrichment factor of more than 15 times for the target protein (such as human IgG), and the background protein residue is less than 5%.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] This invention utilizes glutaraldehyde crosslinking to establish a crosslinked network of suitable density between polyethyleneimine molecular chains. Based on polymer elasticity theory, this ensures both the structural integrity of the functional layer under acidic elution conditions and sufficient conformational freedom of the molecular chains, enabling the material to maintain a stable three-dimensional spatial structure during multiple adsorption-elution cycles. This effectively solves the technical problem of functional layer detachment during the recycling of traditional materials.

[0048] Meanwhile, the ordered mesoporous silica intermediate layer possesses a regular two-dimensional hexagonal pore structure. These interconnected channels provide a low-resistance transport path for protein molecules, and the pore size is optimally matched to the hydrodynamic diameter of biomolecules, significantly reducing the diffusion energy barrier within the channels. This allows target molecules to rapidly reach functional sites within the material, resulting in a significant improvement in adsorption kinetics. In this invention, the polyethyleneimine molecular chains are covalently anchored to the surface of the mesoporous silica via a silane coupling agent, with some chain segments extending into the pore interior, achieving full functionalization of the pore walls. Primary, secondary, and tertiary amine functional groups are uniformly distributed on the material surface and within the internal pores, forming high-density multifunctional binding sites, significantly improving the utilization rate of effective functional groups per unit mass of material. More importantly, the moderately cross-linked polyethyleneimine functional layer of this invention exhibits a uniform positive charge distribution under physiological conditions. The material generates directional Coulomb attraction to negatively charged protein molecules, while simultaneously generating van der Waals forces between the ethylene backbone in the polyethyleneimine molecular chain and the hydrophobic regions on the protein surface. The amino groups form a hydrogen bond network with the polar groups of the protein. This multi-mode synergistic mechanism enables the material to effectively remove impurities while maintaining high adsorption capacity, through optimized washing steps, exhibiting excellent selective separation capabilities.

[0049] The mesoporous silica intermediate layer of this invention serves as an inert isolation layer. Based on the interface protection mechanism, it effectively blocks the erosion of magnetic iron oxide nuclei by hydrogen ions and dissolved oxygen in the aqueous environment, preventing the oxidation and corrosion of the magnetic nuclei and the attenuation of magnetic properties, and ensuring that the material maintains stable magnetic response performance and chemical stability during long-term use.

[0050] This invention achieves a unified structure of magnetic responsiveness, structural stability, high functional group density, excellent mass transfer performance, and good biocompatibility through a three-in-one structural design of a magnetic core, an ordered mesoporous layer, and a high-density flexible amine functional layer. This completely solves the technical problem of the incompatibility between structural density and functional accessibility in existing technologies. The amine-functionalized magnetic nanocomposite material is not only suitable for the efficient purification of single proteins, but can also serve as a universal platform for the enrichment of various biological / environmentally relevant molecules such as phosphorylated peptides, nucleic acids, organic acids, and heavy metal ions, demonstrating significant technological advancement and broad application prospects. Attached Figure Description

[0051] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0052] Figure 1 This is a schematic diagram of the preparation process of the amine-functionalized magnetic nanocomposite material described in this invention.

[0053] Figure 2 This is a schematic diagram of the operation process for the application of the amine-functionalized magnetic nanocomposite material described in this invention in protein separation and purification. Detailed Implementation

[0054] In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of the embodiments of the invention. Therefore, the drawings and description are considered to be exemplary in nature and not restrictive.

[0055] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0056] Example 1: This example discloses an amine-functionalized magnetic nanocomposite material. The overall structure of the material, from the inside out, includes a magnetic nanocore, a mesoporous silica intermediate layer, and a covalently grafted polyethyleneimine (PEI) functional layer.

[0057] The steps for preparing magnetic nanonuclei are as follows:

[0058] FeCl₂·4H₂O and FeCl₃·6H₂O were dissolved in deionized water at a molar ratio of 1:2 to prepare a mixed solution with a total iron ion concentration of 0.2 M. The mixed solution was placed in a three-necked flask and deoxygenated for 30 minutes under continuous purging of high-purity nitrogen gas to remove dissolved oxygen and prevent Fe from being absorbed. 2+It is oxidized.

[0059] The system was then placed in a 60°C constant temperature water bath, and a 25wt% ammonia solution was slowly added dropwise under vigorous stirring until the pH value of the system reached 10.5. The reaction was continued for 1 hour while maintaining this pH and temperature conditions and stirring. During this time, the solution color rapidly changed from light yellow-green to black, indicating that Fe3O4 nanocrystals had been formed.

[0060] After the reaction was completed, solid-liquid separation was achieved by applying an external magnetic field (magnetic field strength 0.4T). The resulting black precipitate was washed three times alternately with anhydrous ethanol and deionized water to completely remove residual chloride ions and unreacted salts.

[0061] Finally, the product was dried in a vacuum drying oven at 60℃ for 12 hours to obtain Fe3O4 nanocrystals with uniform particle size and good crystallinity. Transmission electron microscopy (TEM) analysis showed that the obtained Fe3O4 nanocrystals were spherical with an average particle size of 10.2±1.3 nm and a saturation magnetization of 68.7 emu / g, meeting the basic requirements for magnetic nuclei in this invention.

[0062] After obtaining the above Fe3O4 nanocrystals, a mesoporous silica intermediate layer was grown in situ on its surface using the sol-gel method.

[0063] The specific steps are as follows:

[0064] Weigh 1.0g of Fe3O4 nanocrystals and ultrasonically disperse them in a mixed solvent consisting of 75mL of anhydrous ethanol and 25mL of deionized water to form a uniform suspension.

[0065] Then, 1.5 mL of 0.5 M ammonia solution was added to adjust the pH of the system to 9.2. Under continuous stirring, 4.0 g of tetraethyl orthosilicate (TEOS), with a mass ratio of 4:1 to Fe3O4, was slowly added dropwise. The reaction temperature was controlled at 30 °C, and the reaction time was 4 hours, allowing TEOS to undergo preliminary hydrolysis and condensation on the Fe3O4 surface, forming a dense silica thin layer.

[0066] Subsequently, 2.92 g of hexadecyltrimethylammonium bromide (CTAB) was added to the system to bring the final concentration to 0.1 M, and the reaction temperature was raised to 40 °C, with stirring continued for 12 hours. During this process, CTAB acts as a structure directing agent, guiding the self-assembly of the silica precursor to form a two-dimensional hexagonal ordered mesoporous structure.

[0067] After the reaction was complete, the product was collected by magnetic separation and washed three times with anhydrous ethanol to remove unreacted silicon source and surfactant.

[0068] To completely remove the CTAB template, the obtained Fe3O4@SiO2 composite was placed in an 80°C reflux apparatus and refluxed with ethanol as solvent for 6 hours, and this process was repeated three times.

[0069] The final product, after vacuum drying at 60℃, yielded a Fe3O4@SiO2 core-shell material with a highly ordered mesoporous structure. Nitrogen adsorption-desorption tests showed that the material had a specific surface area of ​​832 m² / g, a pore volume of 0.93 cm³ / g, and a pore size distribution concentrated at 5.1 ± 0.4 nm. It conformed to type IV isotherms and exhibited H1-type hysteresis loops, confirming the existence of the mesoporous structure and its good pore connectivity. TEM images showed that the mesoporous silica shell thickness was 28.6 ± 2.1 nm, with radially ordered pore arrangement and a uniform structure.

[0070] After successfully constructing the mesoporous silica intermediate layer, covalent grafting of the polyethyleneimine functional layer was performed. This process consists of two consecutive steps:

[0071] First, epoxy groups are introduced onto the surface of mesoporous SiO2, and then PEI molecular chains are grafted through a ring-opening reaction.

[0072] The first step was GPTMS modification: 1.0 g of Fe3O4@SiO2 was weighed and dispersed in 50 mL of anhydrous toluene, and sonicated for 30 minutes to ensure thorough dispersion. Under nitrogen protection, 1.2 mmol of 3-glycidoxypropyltrimethoxysilane (GPTMS), approximately 0.30 g, was added. The mixture was heated to 80 °C and reacted under reflux for 18 hours. During this process, the methoxy groups of GPTMS condensed with the silanol groups on the SiO2 surface to form stable Si–O–Si bonds, while simultaneously anchoring the epoxy groups to the material surface. After the reaction, the product was collected by magnetic separation and washed three times each with toluene and anhydrous ethanol to remove physically adsorbed GPTMS. The product was then vacuum dried at 50 °C for 12 hours to obtain GPTMS-functionalized Fe3O4@SiO2-GPTMS material.

[0073] The second step was PEI grafting: 1.0 g of Fe3O4@SiO2-GPTMS was dispersed in 20 mL of 0.5 M polyethyleneimine (PEI, molecular weight 25,000 g / mol) aqueous solution. The system was placed in a 60 °C constant temperature shaker and reacted for 8 hours.

[0074] Under these conditions, the primary amine groups on the PEI molecular chain attack the epoxy ring, undergoing a ring-opening addition reaction to form a stable β-hydroxy secondary amine structure (C–N covalent bond). After the reaction is complete, the primary amine-functionalized material is collected by magnetic separation and repeatedly washed with deionized water until the pH of the eluent is close to neutral (pH≈7.0) to remove unreacted PEI molecules. The material obtained at this point is denoted as Fe3O4@SiO2-PEI.

[0075] To further improve the structural stability of the PEI functional layer in an aqueous environment, it is necessary to perform glutaraldehyde crosslinking treatment.

[0076] The specific steps are as follows: 1.0 g Fe3O4@SiO2-PEI was dispersed in 20 mL of 0.1 M glutaraldehyde aqueous solution and stirred for 2 hours at room temperature (25℃) in the dark. The two aldehyde groups of glutaraldehyde can react with the primary or secondary amines on the adjacent polyethyleneimine (PEI) chain through Schiff base reactions to form -N=CH-(CH2)3-CH=N- crosslinking bridges, thereby constructing a three-dimensional network structure. The degree of crosslinking was verified by the performance retention rate of the material after 10 cycles in a pH 3.0 glycine-HCl eluent; the PEI shedding rate should be less than 5%.

[0077] To quench unreacted aldehyde groups and prevent non-specific cross-linking with the target protein in subsequent applications, 10 mL of 0.5 M glycine solution was added to the system, and the reaction was continued for 30 minutes. The α-amino group of glycine reacts with the residual aldehyde group to form a stable imine structure, effectively blocking the active site. After magnetic separation, the final product was thoroughly washed with deionized water (at least five times) and freeze-dried at –50°C for 24 hours to obtain the amine-functionalized magnetic nanocomposite material described in this invention, denoted as Fe3O4@SiO2-PEI-GA.

[0078] The final amine-functionalized magnetic nanocomposite material was comprehensively characterized.

[0079] X-ray photoelectron spectroscopy (XPS) analysis showed that the N1s peak area accounted for 8.7 at%, and peak fitting identified three nitrogen species: --NH2 (primary amine) with a binding energy of 399.2 eV, --NH- (secondary amine) with a binding energy of 400.1 eV, and --N< (tertiary amine) with a binding energy of 401.5 eV. The molar ratio of the three was approximately 3:4:3, indicating that nitrogen was successfully introduced and mainly exists in the form of the target amine group. Elemental analysis (CHN analysis) determined the total nitrogen content of the material to be 3.05 mmol / g (corresponding to approximately 4.27% by mass). Zeta potential measurement showed that the surface potential of the material in phosphate buffer at pH 7.4 was +32.5 mV, confirming that it exhibits strong positive charge under physiological conditions.

[0080] Vibrating sample magnetometer (VSM) tests showed a saturation magnetization of 58.3 emu / g. Due to the coating of the mesoporous silica layer and the PEI functional layer, the saturation magnetization was lower than that of the magnetic core. This value meets the requirement of achieving magnetic separation within 30 seconds under an applied magnetic field (≥0.3T). Furthermore, the material was dispersed at a concentration of 1 mg / mL in deionized water, PBS buffer (pH 7.4), and 50% ethanol aqueous solution. After standing for 72 hours, no significant sedimentation or aggregation was observed during digital camera recording and turbidity analysis. The absorbance change rate of the supernatant at 600 nm was less than 5%, indicating excellent colloidal stability.

[0081] Furthermore, the operation procedure for the amine-functionalized magnetic nanocomposite material in protein separation and purification applications is as follows:

[0082] Take 10 mg of Fe3O4@SiO2-PEI-GA material and add 1 mL of the protein solution to be treated (such as PBS buffer containing 1 mg / mL bovine serum albumin BSA, pH 7.4). Incubate at 25 °C with shaking at 150 rpm for 30 minutes to allow adsorption.

[0083] Then, an external magnetic field of 0.4T was applied, and the material completely settled to the bottom of the tube within 30 seconds, and the supernatant was discarded.

[0084] Next, add 1 mL of PBS buffer (pH 7.4), vortex resuspend, and then magnetically separate again. Repeat this washing step three times to remove weakly adsorbed impurity proteins.

[0085] Then, 1 mL of 0.1 M glycine-HCl elution buffer (pH 3.0) was added, and the mixture was eluted at 25°C for 10 minutes. The target protein was desorbed into the solution due to the weakening of electrostatic attraction. The collected eluent is the target protein recovery solution.

[0086] After elution, add 1 mL of 0.1 M NaOH solution and treat at room temperature for 15 minutes to regenerate the surface charge. Then wash with deionized water until neutral, and it can be used for the next round of separation.

[0087] In specific implementation, Fe3O4@SiO2-PEI-GA material is prepared according to the above complete process, wherein the molecular weight of PEI is 25,000 g / mol and the concentration of glutaraldehyde is 0.1 M.

[0088] In terms of material characterization, transmission electron microscopy (TEM) images showed that the Fe3O4 cores were spherical and monodisperse with an average particle size of 10.2 ± 1.3 nm. After coating with mesoporous SiO2, the shell thickness was uniform and the channels were arranged radially in an orderly manner. The nitrogen adsorption-desorption isotherm conformed to the characteristics of type IV, and the H1 type hysteresis loop confirmed the existence of the mesoporous structure. X-ray photoelectron spectroscopy (XPS) analysis showed that the N1s peak area accounted for 8.7 at%, corresponding to the three nitrogen species –NH2, –NH– and –N< in PEI. Zeta potential measurement showed that the surface potential of the material was +32.5 mV under pH 7.4, confirming its strong positive charge. Vibrating sample magnetometer (VSM) test showed that the saturation magnetization was 58.3 emu / g, which is sufficient to support complete sedimentation within 30 seconds under an applied magnetic field.

[0089] The molecular chain size of PEI was determined by dynamic light scattering: 25,000 g / mol of PEI was prepared into a 0.1 mg / mL aqueous solution (pH 7.0), and its branched hydrodynamic diameter was measured at 25 °C to be 4.2 ± 0.5 nm. This confirmed that there was a significant overlap between its main hydrodynamic size distribution and the mesopore size distribution, indicating that it can partially enter the pores in a dynamic manner.

[0090] The exposure ratio of amino groups on the outer surface of the material was determined by the Orange II adsorption method: 10 mg of material was dispersed in 10 mL of 0.1 mM Orange II aqueous solution (pH 3.0), and after adsorption by shaking for 30 minutes, magnetic separation was performed. The absorbance of the supernatant (wavelength 483 nm) was measured to calculate the dye adsorption amount. The total amino content was determined by elemental analysis, and the exposure ratio on the outer surface was calculated using the formula:

[0091] Exposure percentage (%) = (Amount of amino groups corresponding to dye adsorption / Total amount of amino groups) × 100%. The measured exposure percentage was 78.2%.

[0092] In this invention, the amino accessibility is determined using the Acid Orange 7 adsorption method. The principle is based on the fact that, under pH 3.0 conditions, the sulfonic acid groups of Acid Orange 7 react with the protonated amino groups (-NH3) on the material surface. + It binds specifically at a 1:1 electrostatic stoichiometric ratio. The amount of amino groups involved in the binding can be directly calculated by measuring the amount of dye adsorbed. This method measures the proportion of all amino groups accessible to the dye molecules, including the outer surface and the inner surface of the mesopores. The value is the apparent accessibility, which is used to compare the availability of functional groups in different materials.

[0093] To verify the performance advantages of the material of the present invention, Comparative Example 1 was designed for systematic comparison.

[0094] Comparative Example 1: The preparation process was the same as in Example 1, but the mesoporous silica intermediate layer was omitted, and PEI was directly grafted onto the Fe3O4 surface. A dense, nonporous SiO2 layer (approximately 10 nm thick, without a CTAB template) was first coated onto the Fe3O4 nanocrystal surface with TEOS, followed by GPTMS modification and PEI grafting, under the same crosslinking conditions.

[0095] Comparative Example 2: The preparation process was the same as in Example 1, but PEI with a molecular weight of 70,000 g / mol was used for grafting, and the other conditions remained unchanged.

[0096] Comparative Example 3: The preparation process was the same as in Example 1, but the glutaraldehyde crosslinking step was omitted, and the product was used directly after PEI grafting.

[0097] The performance of the above four materials was tested, and the results are summarized in Table 1 below:

[0098] Table 1:

[0099]

[0100] Table 1 shows that Example 1 is significantly superior to the comparative examples in terms of adsorption capacity, kinetic rate, regeneration stability, and specificity. Comparative Example 1, lacking a mesoporous structure, suffers from high mass transfer resistance, resulting in low adsorption capacity and a long equilibrium time. Furthermore, the dense shell cannot effectively isolate the magnetic core, and after repeated use, partial oxidation of Fe3O4 leads to decreased magnetic properties and poor regenerability. In Comparative Example 2, the high molecular weight PEI causes pore blockage, reducing internal surface area utilization and increasing molecular chain rigidity, affecting amino accessibility. Although Comparative Example 3 initially exhibits good adsorption performance, the uncrosslinked PEI is prone to swelling and shedding during acidic elution and alkaline regeneration, resulting in a capacity loss of nearly half after ten cycles.

[0101] Furthermore, the functional expansion of the amine-functionalized magnetic nanocomposite material of the present invention is achieved by retaining some unreacted primary amines. After completing PEI grafting and glutaraldehyde crosslinking, a certain amount of free primary amines (approximately 30% of the total amino groups) still exists on the material surface. 10 mg of the material from Example 1 was dispersed in 1 mL of MES buffer (pH 5.5) containing 5 mM N-hydroxysuccinimide (NHS) and 10 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and activated at room temperature for 30 minutes. Subsequently, 10 mM iminodiacetic acid (IDA) was added, and the reaction was carried out at 4°C for 12 hours. After the reaction, magnetic separation was performed, and the mixture was washed with deionized water until neutral to obtain Fe3O4@SiO2-PEI-GA-IDA material. Immersing it in 0.1 M NiSO4 solution for 30 minutes chelates Ni. 2+This material was used for the affinity purification of histidine-tagged proteins. Testing showed that the derived material had an adsorption capacity of 210 mg / g for His-tagged GFP, with an elution recovery rate exceeding 90%, and Ni... 2+ The leakage rate is less than 0.5 ppm, which meets the purification requirements for biopharmaceuticals.

[0102] In the study of protein adsorption mechanisms, the amine-functionalized magnetic nanocomposite material of this invention achieves efficient capture through multi-mode interactions. At pH 7.4, the net surface charge of BSA (pI≈4.7) is negative, resulting in strong electrostatic attraction with the positively charged amino groups on the material surface. Simultaneously, the –NH– groups in the PEI chain and the small amount of –OH groups generated by epoxide hydrolysis can form hydrogen bonds with the carbonyl groups of the BSA backbone. Furthermore, the weakly hydrophobic microdomains provided by the ethylene backbone of PEI generate van der Waals forces with hydrophobic residues (such as Leu, Val, and Phe) on the BSA surface. The synergistic effect of these three forces allows the material to maintain high adsorption efficiency over a wide pH range (4.0–9.0). For example, at pH 5.0, BSA approaches its isoelectric point, and electrostatic interactions weaken, but hydrogen bonding and hydrophobic interactions still maintain an adsorption capacity of approximately 280 mg / g.

[0103] To address the issue of nonspecific adsorption, this invention employs multiple mechanisms to suppress it. The mesoporous SiO2 layer itself is rich in silanol groups, exhibiting strong hydrophilicity, and can form a dense hydration layer on the material surface, effectively blocking hydrophobic interactions. After moderate cross-linking, the PEI has a uniform charge distribution, avoiding strong nonspecific binding caused by localized high charge density. The PBS washing step selectively removes impurities adsorbed only through weak single-point interactions, while the target protein is retained due to multi-point synergistic binding. In 10% human serum samples, the material of this invention achieved a 15.3-fold enrichment for human IgG (pI≈8.0). SDS-PAGE analysis showed clear IgG bands in the elution products with minimal background impurities, and Western blotting confirmed the complete preservation of its antigen-binding activity.

[0104] This invention constructs a structurally well-defined and high-performance amine-functionalized magnetic nanocomposite material by precisely controlling the magnetic core size, mesoporous structure parameters, PEI molecular weight, and crosslinking degree. The entire preparation process is carried out under normal pressure and ≤80℃ conditions, requiring no special equipment, using inexpensive raw materials, exhibiting good reproducibility, and suitable for large-scale production. This invention not only resolves the contradiction between dense structure and functional accessibility in existing technologies but also provides a reliable foundation for the construction of multifunctional bioseparation platforms.

[0105] Example 2: This example is a further optimization based on Example 1. In this example, a thermosensitive poly(N-isopropylacrylamide) polymer brush is grafted onto the outer surface of the polyethyleneimine functional layer. The brush has a minimum critical dissolution temperature of 32-34℃, a molecular weight of 18,000-22,000 g / mol, and a grafting density of 0.8-1.2 chains / nm².

[0106] Furthermore, the polymer brush has a hydrodynamic diameter that is 40-60% larger in the hydrated state at 25°C than at 37°C, and the zeta potential variation is ≥15mV in the pH range of 5.0-7.4.

[0107] In practice, the preparation of thermosensitive amine-functionalized magnetic nanocomposites is as follows:

[0108] Based on the Fe3O4@SiO2-PEI-GA material obtained in Example 1, the following improvements were made:

[0109] Step 1: Fix the surface initiator.

[0110] Weigh 1.0 g of the Fe3O4@SiO2-PEI-GA material prepared in Example 1 and disperse it in 50 mL of anhydrous tetrahydrofuran. Add 0.15 g of 2-bromoisobutyryl bromide and 0.25 mL of triethylamine, and react in an ice-water bath (0-5 °C) for 2 hours, then raise the temperature to 25 °C and continue the reaction for 10 hours. After the reaction is complete, collect the material by magnetic separation, wash it three times alternately with tetrahydrofuran and ethanol, and dry it under vacuum at 50 °C for 12 hours to obtain the ATRP initiator-modified material.

[0111] Step 2: Thermosensitive polymer brush grafting.

[0112] The above materials were dispersed in 50 mL of a methanol / water mixture (volume ratio 4:1), and N-isopropylacrylamide (3.0 g, 26.5 mmol), CuBr (21.5 mg, 0.15 mmol), and PMDETA (31.2 μL, 0.15 mmol) were added. After purging with nitrogen for 30 minutes to remove oxygen, the reaction was carried out at 50 °C for 6 hours. After the reaction was completed, the materials were collected by magnetic separation, thoroughly washed with deionized water, and freeze-dried at -50 °C for 24 hours to obtain a thermosensitive amine-functionalized magnetic nanocomposite material.

[0113] This embodiment builds upon the amine-functionalized magnetic nanocomposite material with high adsorption capacity and rapid mass transfer provided in Example 1, by grafting a temperature-sensitive poly(N-isopropylacrylamide) (PNIPAM) polymer brush onto the PEI functional layer using surface-initiated atom transfer radical polymerization (ATRP). This aims to address the technical problems of conventional magnetic separation materials in complex biological samples, such as insufficient selectivity for target proteins due to high non-specific adsorption, and the cumbersome operation and reliance on chemical environment switching during adsorption / desorption. The PNIPAM brush exhibits high hydration and stretching below its minimum critical dissolution temperature (LCST), forming a hydrophilic protective layer to shield against non-specific interactions. Above the LCST, the polymer brush collapses, exposing the internal PEI amine functional layer, achieving high-capacity adsorption. Thus, the material acquires temperature-controlled "on / off" adsorption characteristics, enabling intelligent physical (temperature) control of adsorption / desorption behavior, significantly improving the selectivity and operational flexibility for separating target proteins from complex matrices.

[0114] The performance characterization results are as follows:

[0115] Dynamic light scattering analysis: The hydrated diameter of the material is 235±8nm at 25℃ and 165±6nm at 37℃, with a diameter change rate of 42.4%.

[0116] Phase transition temperature determination: The lowest critical dissolution temperature was determined to be 32.8℃ by variable temperature UV-Vis.

[0117] Zeta potential test: +18.5±1.2mV at pH 5.0 and +35.2±1.5mV at pH 7.4, with a variation of 16.7mV.

[0118] Adsorption performance test: The adsorption capacity for bovine serum albumin (BSA) was 185±12 mg / g at 25℃ and 395±15 mg / g at 37℃.

[0119] Cyclic stability: The capacity retention rate was 95.2% after 15 adsorption-elution cycles.

[0120] Selectivity test: The enrichment factor for IgG in fetal bovine serum samples reached 28.7-fold, and the background protein residue decreased to 3.1%.

[0121] Example 3: This example is a further optimization based on Example 1. In this example, the mesoporous silica intermediate layer also has a microporous structure, forming a bilevel pore system with mesopores of 4.5-6.0 nm and micropores of 0.8-1.2 nm, and the pore volume ratio of mesopores to micropores is (3-4):1.

[0122] Furthermore, Cu is bonded to the polyethyleneimine functional layer via coordination bonds. 2+Ions, Cu 2+ With a molar ratio of 1:(1.5-2.5) to primary amine groups, the material has an adsorption capacity ≥180mg / g for histidine-tagged proteins.

[0123] In specific implementation, the preparation of the bi-level porous metal chelate material is as follows:

[0124] Step 1: Construction of a bi-porous SiO2 layer.

[0125] An improvement was made to the mesoporous silica growth process in Example 1: 0.08 g of β-cyclodextrin was added as a microporous template to the reaction system containing CTAB template agent, maintaining the mass ratio of tetraethyl orthosilicate to β-cyclodextrin at 50:1, and other reaction conditions remained the same as in Example 1. Fe3O4@SiO2 material with a bilevel porous structure was obtained.

[0126] Step 2: Metal chelation functionalization.

[0127] First, polyethyleneimine grafting and glutaraldehyde crosslinking were performed according to the method in Example 1 to obtain Fe3O4@SiO2-PEI-GA material. Then, 1.0 g of this material was dispersed in 50 mL of 0.05 MCuSO4 solution (pH 6.0) and reacted with shaking at 25 °C for 3 hours. The material was collected by magnetic separation and washed with deionized water until no free Cu was found in the eluent. 2+ The bipolar porous metal chelate material was obtained by detection (using sodium diethyldithiocarbamate colorimetric method) and freeze-drying.

[0128] This embodiment optimizes the structure of the mesoporous silica intermediate layer in Example 1 and introduces metal chelation functionality. The aim is to address the technical problems of limited adaptability of single-pore-size materials to biomolecules of different sizes, and the lack of specific recognition ability of conventional amine-based materials for target proteins (especially histidine-tagged proteins).

[0129] By introducing β-cyclodextrin as a micropore template, a bilevel pore system with both mesopores and micropores was constructed. The micropores (0.8-1.2 nm) can exert a size exclusion effect on small molecule impurities, while the mesopores (4.5-6.0 nm) ensure rapid mass transfer of proteins.

[0130] Furthermore, the abundant amino groups in the PEI layer are used to chelate Cu. 2+ Ions were used to construct a metal ion affinity chromatography (IMAC) platform. This dual synergistic mechanism of size sieving and specific affinity enables the material to not only efficiently and with high capacity adsorb histidine-tagged proteins, but also to remove a large number of small molecule impurities in the early stage of adsorption, significantly improving the separation and purification efficiency of target proteins and the purity of the final product.

[0131] The performance characterization results are as follows:

[0132] Pore ​​structure analysis: Nitrogen adsorption-desorption test showed a specific surface area of ​​980±25 m² / g. The pore size distribution was calculated using the NLDFT method. The micropore size distribution was 0.9±0.1 nm, the micropore volume was 0.12±0.02 cm³ / g, and the ratio of mesopore to micropore volume was 3.2:1.

[0133] Metal content determination: ICP-OES analysis showed Cu 2+ The content was 0.45±0.03 mmol / g; the primary amine group content was calculated to be 0.88±0.05 mmol / g by XPS peak fitting (based on a total nitrogen content of 2.92 mmol / g and a primary amine ratio of 30%), Cu 2+ The molar ratio of the primary amine group to the primary amine group is 1:1.96.

[0134] Adsorption performance: The adsorption capacity for histidine-tagged green fluorescent protein (His-tagged GFP) was 195±10 mg / g, and the elution recovery rate was 92.5±2.1%.

[0135] Specificity test: The purity of the target protein after purification in HEK293 cell lysate was 91.3±1.8%.

[0136] Cyclic stability: After 20 uses, the adsorption capacity retention rate was 93.8%, Cu 2+ The leakage rate is less than 0.8 ppm.

[0137] Table 2 shows a comparison of the material properties of different embodiments:

[0138] Table 2:

[0139]

[0140] By grafting a temperature-sensitive polymer brush onto the outer surface of a polyethyleneimine functional layer, a smart response to temperature stimuli was achieved. At a physiological temperature of 37°C, the polymer brush undergoes conformational contraction, exposing internal amine functional sites and significantly improving protein adsorption capacity. At room temperature of 25°C, the polymer brush extends to form a hydrophilic protective layer, reducing non-specific adsorption and improving material selectivity. This property is particularly suitable for bioseparation applications requiring precise control of the adsorption-desorption process.

[0141] By constructing a mesoporous-microporous dual-level pore system and combining Cu 2+ The chelation function enables precise sieving and specific recognition of biomolecules of different sizes. The microporous structure provides a molecular sieving effect, preferentially adsorbing small peptides; the mesoporous channels ensure rapid mass transfer of protein molecules; Cu... 2+The coordination center enables the specific capture of histidine-tagged proteins. This multi-layered synergistic mechanism significantly improves the efficiency of target protein separation and purification in complex biological samples.

[0142] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0143] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. It should be noted that any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An amine-functionalized magnetic nanocomposite material, characterized in that: This includes an integrated structure consisting of an Fe3O4 magnetic nanocore, a mesoporous silica intermediate layer, and a polyethyleneimine functional layer arranged sequentially from the inside out. Fe3O4 magnetic nanonuclei with a particle size of 8–12 nm and a saturation magnetization of not less than 65 emu / g; A mesoporous silica intermediate layer is coated on the surface of Fe3O4 magnetic nanocores, with a thickness of 25–35 nm. The pores are arranged in a two-dimensional hexagonal order, with the pore size distribution concentrated in 4.5–6.0 nm. The specific surface area is not less than 800 m² / g, and the pore volume is not less than 0.9 cm³ / g. The polyethyleneimine functional layer is covalently grafted onto the surface of the mesoporous silica intermediate layer through a silane coupling agent linker layer and moderately cross-linked through a glutaraldehyde cross-linking structure. The total nitrogen content of free primary, secondary and tertiary amines in the polyethyleneimine functional layer is not less than 2.8 mmol / g, and the amino accessibility rate measured by the Orange II adsorption method is not less than 75%. The amino accessibility is determined by using orange II adsorption method, and the principle is based on that under the condition of pH = 3.0, the sulfonic acid group of orange II is combined with the protonated amino group -NH3 on the surface of the material + The amount of amino groups participating in the combination can be directly calculated by measuring the adsorption amount of the dye through specific combination in the electrostatic metering ratio of 1:

1. The construction of the polyethyleneimine functional layer involves two steps: Step 1: Introduce the silane coupling agent 3-glycidoxypropyltrimethoxysilane GPTMS onto the surface of Fe3O4@SiO2, with an amount of 1.2 mmol GPTMS per gram of Fe3O4@SiO2. Specifically, Fe3O4@SiO2 is dispersed in anhydrous toluene, GPTMS is added, and the mixture is refluxed at 80°C for 18 hours under nitrogen protection. The product is then magnetically separated, washed alternately with toluene and ethanol, and vacuum dried. Step 2: The GPTMS-modified material was dispersed in a 0.5M aqueous solution of polyethyleneimine (PEI) with a molecular weight of 25,000 g / mol. The reaction was carried out at 60°C for 8 hours to allow the epoxy groups to undergo a ring-opening addition reaction with the primary amine on the PEI molecular chain, forming a stable C–N covalent bond. After the reaction was completed, the material was magnetically separated and washed with deionized water until the pH of the eluent was neutral to obtain the primary amine functionalized material. The glutaraldehyde crosslinking structure is formed by dispersing primary amine functionalized materials in a 0.1M glutaraldehyde aqueous solution, reacting them under light-protected conditions at room temperature, and then quenching unreacted aldehyde groups with a 0.5M glycine solution. The polyethyleneimine functional layer needs to be cross-linked with glutaraldehyde to enhance its structural integrity in complex biological media. The degree of cross-linking is considered to be that the PEI functional layer shedding rate is less than 5% after the material is used 10 times under pH=3.0 elution conditions. The shedding rate was determined by nitrogen loss rate: the material was treated 10 times in glycine-HCl elution solution at pH 3.0, and the material was collected after each elution. After vacuum drying, the nitrogen content was determined by elemental analysis. Functional layer shedding rate of material after i-th cycle Calculate using the following formula: ； in, Indicates the nitrogen content of the initial amine-functionalized material, in mmol / g; The value represents the nitrogen content of the material after the i-th adsorption-elution cycle, in mmol / g.

2. The amine-functionalized magnetic nanocomposite material according to claim 1, characterized in that: The Fe3O4 magnetic nanonuclei were prepared by a co-precipitation method, the specific method of which is as follows: FeCl2·4H2O and FeCl3·6H2O were dissolved in deionized water at a molar ratio of 1:

2. Under nitrogen protection, 25wt% ammonia was added dropwise at 60℃ until the pH reached 10.

5. After reacting for 1 hour, the mixture was obtained by magnetic separation, washing, and vacuum drying.

3. The amine-functionalized magnetic nanocomposite material according to claim 1, characterized in that: The mesoporous silica intermediate layer is grown in situ on the surface of Fe3O4 magnetic nanonuclei using a sol-gel method, specifically including the following steps: Fe3O4 magnetic nanonuclei were dispersed in an ethanol / water mixed solvent, and the pH was adjusted to 9.0–9.5; First, tetraethyl orthosilicate is added to react and form an initial silicon layer; Then, hexadecyltrimethylammonium bromide is introduced as a template agent; The reaction was continued at 40°C, and then the hexadecyltrimethylammonium bromide was removed by reflux treatment to obtain an ordered mesoporous structure.

4. The amine-functionalized magnetic nanocomposite material according to claim 3, characterized in that: The mass ratio of tetraethyl orthosilicate to Fe3O4 magnetic nanonuclei is 4:1, and the final concentration of hexadecyltrimethylammonium bromide is 0.1M.

5. The amine-functionalized magnetic nanocomposite material according to claim 1, characterized in that: At pH 7.4, the material surface has a Zeta potential of +32.5 mV and a saturation magnetization of 58.3 emu / g. It also shows no significant sedimentation or aggregation after standing in deionized water, PBS buffer, or 50% ethanol aqueous solution for 72 hours.

6. The application of an amine-functionalized magnetic nanocomposite material as described in any one of claims 1-5 in protein separation and purification, characterized in that, The following steps are included: The amine-functionalized magnetic nanocomposite material was dispersed in the protein solution to be treated and adsorbed by shaking. Magnetic separation is achieved by applying an external magnetic field of ≥0.3T; Wash three times with PBS buffer; The target protein was then recovered by elution with glycine-HCl elution buffer. Finally, the mixture is treated with NaOH regeneration solution and then washed with water until neutral to complete the regeneration process.

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