PH response type magnetic graded porous MOFs composite material and preparation method and application thereof

Abstract

The invention belongs to the technical field of biological materials, and discloses a pH response type magnetic hierarchical porous MOFs composite material and a preparation method thereof, the composite material is formed by emulsifying and cross-linking magnetic MOFs composite nanospheres with Fe3O4 nanoparticles as a core and an MOFs layer as a shell, and the composite material is of a cluster structure. According to the hierarchical porous structure of the composite material, the pore diameter is increased, the specific surface area of MOFs is increased, the yield of protein can be increased, and the application efficiency is improved; the method can be used for protein enrichment and release.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials, and relates to a pH-responsive protein capture and release material, and in particular to a pH-responsive magnetic graded porous MOFs composite material, and a preparation method and application thereof. Background Art

[0002] Metal-Organic Frameworks (MOFs), also known as coordination polymers, are crystalline materials with ultra-porous structures. MOFs are a low-density network composed of multi-dentate bridging ligands (such as carboxylates, sulfonates, or phosphates) and inorganic central nodes (metal ions or metal ion clusters) self-assembled and connected through coordination bonds or intermolecular forces. Due to the almost infinite combination of metals and ligands, MOFs exhibit more unique properties than traditional materials, such as higher porosity, excellent mechanical stability, and easily adjustable surface properties. MOFs have been widely used in different fields as a new type of inorganic-organic hybrid material, such as gas storage, catalysis, nonlinear optics, separation, sensing, imaging, drug release, etc. In recent years, MOFs have also been widely used in the biomedical field, such as drug release, photodynamic therapy, imaging, enzyme catalysis, and protein enrichment. A large number of reports on its protein applications basically focus on improving the capture efficiency of proteins, and there is less research on how to capture and release proteins efficiently. Moreover, the MOFs reported in the literature have a small pore size, and proteins with larger sizes are only adsorbed on the surface of the MOFs composite material, which cannot fully utilize the advantages of MOFs with porous structure and large specific surface area, and the amount of protein captured is small, and there is still a lot of room for improvement; at the same time, the release process of proteins is usually ignored, and the proteins captured by MOFs are difficult to elute from the material. Even if they can be eluted, since the elution process requires the addition of substances that affect protein activity, such as 2-methylimidazole or trifluoroacetic acid, it will seriously affect the further analysis or application of the released proteins (see Liu WL, Lo SH, Singco B., Yang CC, Huang HY, Lin CH, Novel trypsin-FITC@MOF bioreactor efficiently catalyzes protein digestion. J. Mater. Chem. B, 2013, 1, 928-932). Therefore, designing and constructing a MOFs composite material that efficiently captures and releases proteins has become a current research hotspot and urgent need.

[0003] In recent years, hierarchical porous MOFs with different pore sizes have attracted widespread attention in various fields. The specific surface area of ​​MOFs is increased by constructing a unique micro / nano hierarchical pore structure to improve the application efficiency. However, the hierarchical porous structure of MOFs usually needs to be constructed using a template, but the template removal process is cumbersome and will affect the MOF structure. Therefore, developing a mild template-free strategy to construct hierarchical porous MOFs has important scientific significance and application value.

[0004] Magnetic composite materials can quickly collect the desired products under a magnetic field, significantly improve the separation rate, and are easy to operate. They have attracted widespread attention in the field of bioseparation. Because phenylboronic acid affinity chromatography has a unique pH switch property, many boric acid functionalized magnetic materials have been used to enrich or separate glycoproteins and glycopeptides. Generally, small molecular boric acid ligands are modified on the surface of magnetic nanomaterials or polymer boric acid ligands are grafted on them. However, the preparation process of boric acid functionalized magnetic composite materials is relatively cumbersome and time-consuming, and even requires anhydrous and oxygen-free operation, and the reaction conditions are relatively harsh; moreover, in the process of capturing or releasing proteins, the required pH value is strongly acidic or alkaline, which is not conducive to maintaining the activity of proteins, thereby limiting their application in biomedicine. Therefore, how to efficiently capture and release proteins without affecting protein activity has become a bottleneck problem in promoting the progress of magnetic bioseparation and early disease screening. Moreover, there are few reports on the research of magnetic pH MOFs composite materials. Constructing a simple and effective strategy to enable separation carriers to efficiently separate proteins under a mild pH environment has important academic value and broad application development prospects.

[0005] In summary, making MOFs hierarchically porous and multifunctional is a feasible potential method to overcome the above problems. Currently, multifunctional hierarchical porous MOFs for controllable protein separation have not been reported. Summary of the invention

[0006] The purpose of the present invention is to provide a pH-responsive magnetic hierarchical porous MOFs composite material to address the problems existing in the above-mentioned prior art, by constructing a hierarchical porous structure of MOFs and increasing the pore size to improve the capture amount of protein.

[0007] Another object of the present invention is to provide a method for preparing a pH-responsive magnetic hierarchical porous MOFs composite material, and to construct a pH-responsive magnetic hierarchical porous MOFs nanocomposite material for controllable capture and release of proteins by a template-free strategy.

[0008] The third object of the present invention is to provide an application of the pH-responsive magnetic hierarchical porous MOFs composite material in protein enrichment and release.

[0009] This invention intends to combine boric acid affinity chromatography technology with hierarchical porous MOFs to propose for the first time a novel template-free strategy to construct pH-responsive magnetic hierarchical porous MOFs nanocomposites for efficient capture and release of proteins. Figure 1 As shown, firstly, boric acid derivatives are selected as organic ligands to construct pH-responsive magnetic MOFs composite nanospheres, giving the material high magnetic responsiveness and environmental sensing function; then, the colloidal assembly technology is combined with the reverse emulsification cross-linking technology to construct a porous magnetic MOFs composite nanomaterial composed of a large number of assembly unit magnetic MOFs composite nanospheres tightly stacked, giving the material a hierarchical porous structure, and on the basis of the micropores of the magnetic MOFs composite nanospheres, more pore sizes suitable for capturing proteins are constructed, which is beneficial to improving the separation efficiency of proteins; on this basis, a systematic study on the separation of glycoproteins by this multifunctional magnetic hierarchical porous MOFs composite material is carried out.

[0010] Furthermore, the present invention uses two mixed organic ligands: the first organic ligand can give the material pH responsiveness through further hydrolysis; the second organic ligand provides amino cross-linking sites for the subsequent cross-linking step of preparing a hierarchical porous structure, thereby improving the stability of the material. The present invention also explores the effects and laws of material factors such as the particle size, shape, type of boric acid-based ligands, and environmental factors such as incubation medium and time on the recognition, binding and release efficiency of proteins.

[0011] Based on the above analysis, the present invention first provides a pH-responsive magnetic hierarchical porous MOFs composite material, which is assembled by emulsification and cross-linking of magnetic MOFs composite nanospheres with Fe3O4 nanoparticles as the core and MOFs layer as the shell, and has a cluster structure.

[0012] In one achievable embodiment, the surface of the Fe3O4 nanoparticles is modified with a hydrophilic polymer compound A for chelating metal ions; the hydrophilic polymer compound A is at least one of polyacrylic acid (PAA), chitosan, carboxymethyl chitosan, polyvinyl pyrrolidone, etc.

[0013] In one achievable embodiment, the MOFs layer comprises metal ions and organic ligands bound to the metal ions; the organic ligands comprise a first organic ligand that imparts pH responsiveness to the material and a second organic ligand for providing cross-linking sites; the first organic ligand is at least one of 4-pyrazole boronic acid pinacol ester, 3,5-dimethylpyrazole-4-boronic acid pinacol ester, 1-methylpyrazole-4-boronic acid pinacol ester, pyrido[2,3-b]pyrazine-7-yl boronic acid pinacol ester, 2-aminopyridine-5-boronic acid pinacol ester, 1-methylimidazole-5-boronic acid pinacol ester, and the like; and the second organic ligand is at least one of aminopyrazole, aminopyrazine, aminopyridine, aminoimidazole, and the like.

[0014] In one achievable embodiment, the oil phase used for emulsification is at least one of liquid paraffin, squalane, caprylic / capric triglyceride, etc.; the non-ionic surfactant used for emulsification is at least one of Span 80, Tween 80, Span 20, Span 60, etc.; the cross-linking agent used for cross-linking is genipin and / or glutaraldehyde.

[0015] The present invention also provides a method for preparing the pH-responsive magnetic graded porous MOFs composite material, which comprises the following steps:

[0016] (1) Preparing pH-responsive magnetic MOFs composite nanospheres; this step includes the following sub-steps:

[0017] (11) Preparation of Fe3O4 nanoparticles;

[0018] (12) coating a transition layer on the surface of Fe3O4 nanoparticles;

[0019] (13) Preparation of pH-responsive magnetic MOFs composite nanospheres;

[0020] A Fe3O4 nanoparticle suspension containing a transition layer is dispersed in a mixed solvent, and a metal salt, a first organic ligand and a second organic ligand are added; the resulting reaction system is reacted at 50-90°C for 2-6 hours under stirring conditions until it becomes reddish brown, and the precipitate is collected and washed to obtain a dispersion containing magnetic MOFs composite nanospheres; the magnetic MOFs composite nanospheres are hydrolyzed to obtain a dispersion containing pH-responsive magnetic MOFs composite nanospheres; the molar ratio of the Fe3O4 nanoparticles containing a transition layer, the metal salt, the first organic ligand and the second organic ligand is (0.1-0.2):1:1.5:0.5;

[0021] (2) Preparing a magnetic graded porous MOF composite material; this step includes the following sub-steps:

[0022] (21) adding a dispersion containing pH-responsive magnetic MOFs composite nanospheres into an oil phase containing a nonionic surfactant to form a uniform emulsion;

[0023] (22) Under stirring conditions at 40–80 °C, an aqueous crosslinking agent solution was added to the emulsion to allow the crosslinking reaction to proceed for 3–6 h. The precipitate was collected and washed to obtain a magnetic graded porous MOF composite material.

[0024] In the above step (1), if Figure 2 As shown, based on the coprecipitation method, hydrophilic polymer polyethyleneimine (PEI) is added during the preparation process, Fe 2+ , Fe 3+Fe3O4 magnetic nanoparticles with a surface rich in positive charge are prepared by co-precipitation with ammonia water; then, they are dispersed in water, interact with the hydrophilic polymer compound A that chelates metal ions, and a transition layer is coated on the surface of the magnetic nanoparticles to obtain Fe3O4 nanoparticles containing a transition layer. Then, the Fe3O4 nanoparticle suspension containing the transition layer is dispersed in a reaction solvent, and metal ions of MOFs and two types of organic ligands are added thereto, and the mixture is heated to a certain temperature and mechanically stirred for several hours. After the reaction is completed, the precipitate is collected with a magnet and repeatedly washed with deionized water for several times. Then, the collected precipitate is hydrolyzed under certain conditions to obtain pH-responsive magnetic MOFs composite nanospheres with a surface rich in boric acid groups.

[0025] In the above step (11), Fe 2+ , Fe 3+ Fe3O4 nanoparticles were synthesized by co-precipitation with ammonia water. Under nitrogen protection, the iron salt was dissolved in water, and then an aqueous solution of a hydrophilic polymer compound B was added. Ammonia water was then dripped into the obtained reaction system, and the reaction was carried out at 60-80°C for 1-3 hours until the solution turned black. The black precipitate was collected and washed to neutrality. Ammonia water was dripped into the obtained reaction system, and the hydroxide ions ionized by the ammonia water combined with the iron ions in the solution to form iron hydroxide precipitates. These iron hydroxide precipitates were dehydrated, oxidized and other steps in the subsequent reaction process, and finally converted into ferroferric oxide. The amount and speed of ammonia water added will affect the formation speed and particle size of the precipitate. Controlling the precipitation speed, slowly adding ammonia water and controlling its dosage can make Fe 2+ and Fe 3+ Uniform precipitation is conducive to the formation of ferroferric oxide particles with uniform particle size and good dispersibility. The iron salt is a divalent iron salt and a trivalent iron salt; preferably, the divalent iron salt is FeCl2 or FeSO4, and the trivalent iron salt is FeCl3 or / and Fe2(SO4)3; the molar volume ratio of the divalent iron salt, the trivalent iron salt, the hydrophilic polymer compound B and the ammonia water is 5mmol:9mmol:0.1mmol:35mL. The washing liquid used for washing is deionized water; the Fe3O4 nanoparticles obtained by washing are dispersed in water to obtain a stable Fe3O4 nanoparticle suspension.

[0026] In the above step (12), the aqueous solution of the hydrophilic polymer compound A is added to the Fe3O4 nanoparticle suspension, and the resulting reaction system is subjected to magnetic stirring at room temperature to 40°C until the zeta potential no longer changes, and the brown product is collected and washed. The magnetic stirring reaction time is 4-36 hours; the mass ratio of the hydrophilic polymer compound A to the Fe3O4 nanoparticles is 2-5:1. The washing liquid used for washing is deionized water. The Fe3O4 nanoparticles containing the transition layer obtained by washing are dispersed in water for later use.

[0027] In the above step (13), the mixed solvent is obtained by mixing methanol, acetonitrile, ethanol and deionized water in a volume ratio of 1:1:1:3; the metal salt is ferric nitrate and / or ferric sulfate; the first organic ligand is at least one of 4-pyrazole boric acid pinacol ester, 3,5-dimethylpyrazole-4-boric acid pinacol ester, 1-methylpyrazole-4-boric acid pinacol ester, pyrido[2,3-b]pyrazine-7-yl boric acid pinacol ester, 2-aminopyridine-5-boric acid pinacol ester, 1-methylimidazole-5-boric acid pinacol ester, etc., and the second organic ligand is at least one of aminopyrazole, aminopyrazine, aminopyridine, aminoimidazole, etc.; the precipitate containing magnetic MOFs composite nanospheres is washed several times with deionized water, and the washed magnetic MOFs composite nanospheres are dispersed in deionized water for standby use. The dispersion containing magnetic MOFs composite nanospheres is added to an ethanol solution containing sodium periodate, reacted at 30-50°C and magnetic stirring for 4-12 hours, and then the precipitate is collected and washed; the molar ratio of the first organic ligand to sodium periodate is 1:(5-6); the ethanol solution is an aqueous solution containing ethanol, and the ethanol concentration is 5%-10%. Deionized water is used for washing; the pH-responsive magnetic MOFs composite nanospheres obtained by washing are dispersed in water for use.

[0028] In the above step (2), if Figure 3 As shown, a magnetic hierarchical porous MOFs composite nanomaterial is prepared by a synthetic strategy prepared by a reverse emulsion crosslinking method and a colloidal group. The magnetic MOFs composite nanosphere suspension is added to an emulsifying oil phase containing a non-ionic surfactant and emulsified for several minutes at a certain speed. Then, under mechanical stirring at 40-80°C, an aqueous crosslinking agent solution is added and the crosslinking reaction is carried out for 3-6 hours. The product is collected by an external magnetic field and washed continuously with petroleum ether, ethanol and water several times to obtain a magnetic MOFs composite nanomaterial with a hierarchical porous structure.

[0029] In the above step (21), the nonionic surfactant is at least one of Span 80, Tween 80, Span 20, Span 60, etc. The volume ratio of the dispersion containing pH-responsive magnetic MOFs composite nanospheres to the emulsifier and the nonionic surfactant is 1:50:25. The oil phase used for the emulsification is at least one of liquid paraffin, squalane, caprylic / capric triglyceride, etc.; the emulsification condition is emulsification at a speed of 3000-6000 rpm for 10-20 minutes.

[0030] In the above step (22), the molar ratio of the cross-linking agent to the pH-responsive magnetic MOFs composite nanospheres is (2.5-3.5):1. The cross-linking agent is genipin and / or glutaraldehyde. The washing liquid used for washing is petroleum ether, ethanol and deionized water in sequence. The magnetic graded porous MOF composite material obtained by washing is dispersed in water for standby use.

[0031] The present invention also provides the application of the pH-responsive magnetic graded porous MOFs composite material in protein enrichment and release, achieving protein capture in an environment of pH 4-5 and protein release in an environment of pH 9-11.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1) The pH-responsive magnetic hierarchical porous MOFs composite material provided by the present invention is assembled into a cluster structure by pH-responsive magnetic MOFs composite nanospheres. The hierarchical porous structure increases the pore size and the specific surface area of ​​MOFs, which can increase the capture amount of proteins and improve the application efficiency;

[0034] 2) The preparation method of the pH-responsive magnetic hierarchical porous MOFs composite material provided by the present invention is constructed based on a template-free strategy. First, boric acid derivatives are selected as organic ligands to construct pH-responsive magnetic MOFs composite nanospheres, giving the material high magnetic responsiveness and environmental perception ability; then, by combining colloidal assembly with reverse emulsification cross-linking, a porous magnetic MOFs composite nanomaterial formed by densely stacking a large number of assembly unit magnetic MOFs composite nanospheres is constructed, giving the material a hierarchical porous structure, and on the basis of the micropores of the magnetic MOFs composite nanospheres, more pore sizes suitable for capturing proteins are constructed, which is beneficial to improving the separation efficiency of proteins; this can not only simplify the preparation process and reduce the impact on the MOFs structure, but also reduce the impact on proteins, thereby enhancing the application value of the composite material. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a general idea diagram of the preparation process (A) of the pH-responsive magnetic graded porous MOFs composite material and the protein separation process (B) provided by the present invention.

[0036] Figure 2 The invention provides a preparation route for pH-responsive magnetic MOFs composite nanospheres.

[0037] Figure 3 The invention provides a preparation route for the magnetic graded porous MOF composite material.

[0038] Figure 4The morphology of various particles prepared in the embodiments of the present invention is characterized; wherein A is a transmission electron microscopy image of magnetic Fe3O4 nanoparticles, B is a transmission electron microscopy image of PAA-modified magnetic nanoparticles (Fe3O4 / PAA), C is a transmission electron microscopy image of magnetic MOF nanoparticles (Fe3O4 / PAA / MOFs); D is a transmission electron microscopy image of hydrolyzed magnetic MOF nanoparticles (Fe3O4 / PAA / HMOFs); E is a transmission electron microscopy image of Fe3O4 / PAA / HMOFs clusters; FG are scanning electron microscopy images of Fe3O4 / PAA / HMOFs nanoclusters.

[0039] Figure 5 Dynamic light scattering (DLS) test results (A) and Zeta potential test results (B) of the Fe3O4 / PAA / HMOFs clusters prepared in the examples of the present invention.

[0040] Figure 6 Infrared spectra of various particles prepared in accordance with the embodiments of the present invention; wherein a corresponds to magnetic Fe3O4 nanoparticles, b corresponds to PAA-modified magnetic nanoparticles (Fe3O4 / PAA), c corresponds to magnetic MOF nanoparticles (Fe3O4 / PAA / MOFs), d corresponds to hydrolyzed magnetic MOF nanoparticles (Fe3O4 / PAA / HMOFs), and e corresponds to Fe3O4 / PAA / HMOFs clusters.

[0041] Figure 7 X-ray diffraction patterns of various particles prepared in the embodiments of the present invention and standard XRD patterns of Fe3O4 (JCPDS19-06290) (represented by red solid bars).

[0042] Figure 8 Thermogravimetric analysis curves of various particles prepared in the embodiments of the present invention; wherein a corresponds to magnetic Fe3O4 nanoparticles, b corresponds to PAA-modified magnetic nanoparticles (Fe3O4 / PAA), c corresponds to magnetic MOF nanoparticles (Fe3O4 / PAA / MOFs), d corresponds to hydrolyzed magnetic MOF nanoparticles (Fe3O4 / PAA / HMOFs), and e corresponds to Fe3O4 / PAA / HMOFs clusters.

[0043] Fig. 9 These are the magnetization curves of various particles prepared in the embodiments of the present invention; wherein a corresponds to magnetic Fe3O4 nanoparticles, b corresponds to PAA-modified magnetic nanoparticles (Fe3O4 / PAA), c corresponds to magnetic MOF nanoparticles (Fe3O4 / PAA / MOFs), d corresponds to hydrolyzed magnetic MOF nanoparticles (Fe3O4 / PAA / HMOFs), and e corresponds to Fe3O4 / PAA / HMOFs clusters.

[0044] Fig.10 Isotherm curve (A) obtained by N2 adsorption / desorption test of Fe3O4 / PAA / HMOFs clusters prepared in an embodiment of the present invention and the calculated pore size distribution of Fe3O4 / PAA / HMOFs clusters.

[0045] Fig.11 Schematic diagram of the capture and release process of proteins by the pH-responsive magnetic hierarchical porous MOFs composite material in the experimental example of the present invention.

[0046] Fig.12 SDS-PAGE images of the supernatant (S) and protein-material complex (C) after Fe3O4 / PAA / HMOFs clusters were co-incubated with TRF under different pH conditions (C Protein =0.5 mg / mL, 300 μL protein solution; C Material =15 mg / mL, 20 μL material; incubation time is 1 h); wherein A corresponds to the supernatant, B corresponds to the protein-material complex, Marker is the molecular weight standard, and TRF is the protein solution before the protein and material are co-incubated.

[0047] Fig.13 The SDS-PAGE images of the eluate (E) and the eluted material (C) after Fe3O4 / PAA / HMOFs clusters were co-incubated with TRF in pH=5 buffer (C Protein =0.5mg / ml, 100μL protein solution, C Material =15 mg / mL, 35 μL material, incubation solution with pH=5, 100 μL elution solution, incubation time 5 min, elution time 60 min); lane 1 is the molecular weight standard (Marker); lane 2 is the TRF before co-incubation; lanes 3-12, the eluent (E) and the material after elution (C).

[0048] Fig.14 SDS-PAGE images of the supernatant (S) and protein-material complex (C) after co-incubation of Fe3O4 / PAA / HMOFs clusters with TRF at different incubation times (C Protein =0.5mg / ml, 100μL protein solution, C Material =15 mg / ml, 30 μL material, pH=5).

[0049] Fig.15 The SDS-PAGE images of the eluate and the eluted material after Fe3O4 / PAA / HMOFs clusters were co-incubated with TRF in pH=5 buffer and eluted with pH=9 (A), pH=10 (B), and pH=11 (C) buffers for different times (C Protein=0.5 mg / mL, 100 μL protein solution; C Material =15 mg / mL, 35 μL material, incubation solution with pH=5, 200 μL elution solution, incubation time is 5 min); lane 1 is the molecular weight standard (Marker); lane 2 is the TRF before co-incubation; lanes 3-12, the eluate (E) and the material after elution (C).

[0050] Fig.16 The supernatant (S) and protein-material complex (C) of Fe3O4 / PAA / HMOFs clusters after co-incubation with TRF in pH = 5 buffer, and the SDS-PAGE image of the eluate (E) and the eluted material (C) after elution with pH = 10 buffer (C Protein =0.5 mg / mL, 100 μL protein solution; C Material =15 mg / mL, 35 μL material, incubation solution at pH=5, 200 μL elution solution at pH=10, incubation time is 5 min, elution time is 60 min); lane 1 is the molecular weight standard (Marker); lane 2 is the TRF before co-incubation; lanes 3-4, the supernatant (S) and protein-material complex (C) after co-incubation; lanes 5-6, after co-incubation at pH5, the eluate (E) and the eluted material (C) after elution with pH=10 buffer.

[0051] Fig.17 The adsorption amount and adsorption rate of Fe3O4 / PAA / HMOFs clusters after incubation with TRF in different pH buffers.

[0052] Fig.18 This is the elution rate of captured proteins after Fe3O4 / PAA / HMOFs clusters were incubated with TRF in pH=5 buffer and treated with different elution solutions.

[0053] Fig.19 The elution rate of proteins at different times using pH = 10 eluent after Fe3O4 / PAA / HMOFs clusters were incubated with TRF in pH = 5 buffer.

[0054] Fig. 20 The adsorption amount and adsorption rate of Fe3O4 / PAA / HMOFs clusters incubated with TRF in pH = 5 buffer for different time periods.

[0055] Fig.21 The adsorption amount and adsorption rate of Fe3O4 / PAA / HMOFs clusters after incubation with four proteins (TRF / BHB / BSA / LYZ) in pH = 5 buffer and elution with pH = 10 eluent for 4 cycles.

[0056] Fig. 22Fluorescence spectra of Fe3O4 / PAA / HMOFs clusters, four proteins (TRF / BHB / BSA / LYZ), and Fe3O4 / PAA / HMOFs clusters incubated with four proteins (TRF / BHB / BSA / LYZ). DETAILED DESCRIPTION

[0057] The following will clearly and completely describe the technical solutions of various embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0058] Example 1 Preparation of pH-responsive magnetic hierarchical porous MOFs composite material

[0059] This example prepares a pH-responsive magnetic hierarchical porous MOFs composite material according to the following steps:

[0060] (1) Preparing pH-responsive magnetic MOFs composite nanospheres; this step includes the following sub-steps:

[0061] (11) Preparation of Fe3O4 nanoparticles.

[0062] 100 mL of deionized water was added to a 250 mL flask and heated to 50 ° C. Nitrogen was bubbled in to remove oxygen for half an hour. Then, under nitrogen protection, 1000 mg of FeCl2·4H2O and 2448 mg of FeCl3·6H2O were added. When they were completely dissolved to form a yellow solution, 2 mL of 100 mg / mL polyethyleneimine (PEI, Mw 2000) solution was added. Then, under stirring, 35 mL of ammonia water was added dropwise to the reaction system and reacted at 80 ° C for 1 h. When the obtained black mixture was cooled to room temperature, deionized water was added to the suspension and precipitated under an external magnetic field. The black sediment was collected and washed with deionized water several times until pH = 7. Finally, Fe3O4 nanoparticles were stored in 100 mL of deionized water to obtain a stable Fe3O4 nanoparticle suspension.

[0063] (12) A transition layer is coated on the surface of Fe3O4 nanoparticles.

[0064] 40 mL of PAA aqueous solution (25 mg / mL) was added to 40 mL of the above Fe3O4 nanoparticle suspension and reacted at room temperature under magnetic stirring for 24 hours. The brown product was collected under an external magnetic field and washed three times with deionized water to obtain Fe3O4 / PAA nanospheres. Finally, the Fe3O4 / PAA nanospheres were stored in 10 mL of deionized water for later use.

[0065] (13) Preparation of pH-responsive magnetic MOFs composite nanospheres.

[0066] 1 mL of the above Fe3O4 / PAA nanosphere suspension was dispersed into 18 mL of a mixed solvent containing methanol (3 ml), acetonitrile (3 ml), ethanol (3 ml) and deionized water (9 ml), and 1 mmol Fe(NO3)3·9H2O, 1.5 mmol 4-pyrazole boric acid pinacol ester (PAPE) and 0.5 mmol 3-aminopyrazole (AP) were added thereto. Then, the mixture was heated to 80°C and mechanically stirred for 4 hours. The color of the product changed from black to reddish brown. The precipitate was collected with a magnet and washed repeatedly with deionized water for 3 times. Finally, the obtained Fe3O4 / PAA / MOFs nanospheres were dispersed in deionized water (1 mL) for later use.

[0067] Add 1 mL of the above Fe3O4 / PAA / MOFs nanospheres to a 7% ethanol solution containing 7.8 mmol NaIO4 (mixed with ethanol and deionized water, ethanol concentration is 7%). React for 6 h at 40°C with magnetic stirring, then collect the precipitate with a magnet and wash the precipitate repeatedly with deionized water for 3 times. Finally, disperse the hydrolyzed Fe3O4 / PAA / HMOFs nanospheres in deionized water (1 mL) for later use.

[0068] (2) Preparing a magnetic graded porous MOF composite material; this step includes the following sub-steps:

[0069] (21) 200 μL of the above Fe3O4 / PAA / HMOFs nanosphere suspension was added to 10 mL of liquid paraffin containing 5 mL of Span 80 and emulsified at 4000 rpm for 10 min.

[0070] (22) Under mechanical stirring at 60 °C, 200 μL of genipin aqueous solution (125 mg mL -1 ), and the cross-linking reaction was carried out for 4 hours. The product was collected by applying an external magnetic field and washed three times with petroleum ether, ethanol and water. Finally, the prepared Fe3O4 / PAA / HMOFs clusters were dispersed in deionized water (concentration of 15 mg / mL).

[0071] The structures and properties of the prepared magnetic Fe3O4 nanoparticles, PAA-modified magnetic nanoparticles (Fe3O4 / PAA), magnetic MOF nanoparticles (Fe3O4 / PAA / MOFs), and hydrolyzed magnetic MOF nanoparticles (Fe3O4 / PAA / HMOFs) were characterized below.

[0072] (1) The morphology of magnetic Fe3O4 nanoparticles, PAA-modified magnetic nanoparticles (Fe3O4 / PAA), magnetic MOF nanoparticles (Fe3O4 / PAA / MOFs), and hydrolyzed magnetic MOF nanoparticles (Fe3O4 / PAA / HMOFs) were tested. The test results are as follows: Figure 4 As shown in the figure, these nanospheres are spherical particles of about 10nm, indicating that the PAA coating, MOFs coating and hydrolysis reaction have no obvious effect on the size and morphology of the nanoparticles. After the reverse emulsification cross-linking reaction, a spherical Fe3O4 / PAA / HMOFs cluster with uniform size of about 1μm was successfully formed. It can be seen from the transmission electron microscopy and scanning electron microscopy that the surface of the cluster is relatively rough and is composed of densely packed nanoparticles.

[0073] (2) Dynamic light scattering (DLS) and Zeta potential tests were performed on the Fe3O4 / PAA / HMOFs clusters. The test results are as follows: Figure 5 As shown in A and B.

[0074] from Figure 5 As can be seen, the dynamic light scattering (DLS) test shows that the prepared Fe3O4 / PAA / HMOFs clusters have a narrow size distribution, with a size of about 1 micron, which is consistent with the SEM and TEM data.

[0075] from Figure 5 B shows that the Zeta potentials of magnetic Fe3O4 nanoparticles, PAA-modified magnetic nanoparticles (Fe3O4 / PAA), magnetic MOF nanoparticles (Fe3O4 / PAA / MOFs), hydrolyzed magnetic MOF nanoparticles (Fe3O4 / PAA / HMOFs) and Fe3O4 / PAA / HMOFs clusters in deionized water are +42 mV (because the polyethyleneimine on the surface of Fe3O4 nanoparticles contains amino groups, which shows positive charge), -27 mV (because after encapsulating polyacrylic acid PAA, PAA contains a large number of carboxyl groups, which shows negative charge), +32 mV (because the MOFs shell contains a large amount of Fe 3+ , which shows positive charge), -37mV (because a large number of boric acid groups are exposed after hydrolysis of the pinacol ester in the MOFs shell, which shows negative charge), -34mV (because the negative charge is slightly reduced after cross-linking with genipin). This charge reversal proves that a series of composite materials have been successfully synthesized. Moreover, the Zeta potential of the prepared clusters in aqueous solution is as high as -34mV, which will be beneficial for further application in protein adsorption.

[0076] (3) The microstructure, magnetic properties and porous structure of the samples were analyzed by FTIR, XRD, TGA, VSM and BET.

[0077] like Figure 6 As shown, all samples show the 575 cm- -1 The magnetic Fe3O4 nanoparticles have a strong characteristic absorption band at 2849 cm -1 The characteristic peak of aliphatic NH stretching vibration of PEI is shown at 813 cm. The PAA-modified magnetic nanoparticles (Fe3O4 / PAA) show the characteristic absorption peak of PAA: 813 cm -1 is the stretching frequency of C-COOH, 1694 cm -1 is the C=O stretching frequency, 1120cm -1 is the C-OH stretching frequency, 1540 cm -1 is the asymmetric stretching frequency of COO-, indicating that PAA has been successfully coated on the surface of the nanoparticles. The magnetic MOF nanoparticles (Fe3O4 / PAA / MOFs) show the characteristic absorption peaks of 3-aminopyrazole and 4-pyridinium pinacol ester: 1562-1455 cm -1 The fingerprint peak of 3-aminopyrazole and 4-pyridine pinacol ester is 702 cm -1 is the in-plane bending of the pyrazole ring, 632 cm -1 is the torsional vibration of the pyrazole ring; 1369cm -1 and 1357cm -1 It is the characteristic peak of BO. After hydrolysis, the characteristic peak of pyrazole ring vibration moves to 716cm -1 and 641cm -1 The Fe3O4 / PAA / HMOFs clusters formed by colloidal assembly and inverse emulsification crosslinking of nanoparticles restrict the planar bending of the pyrazole ring, so the 716cm -1 The in-plane bending of the pyrazole ring is weakened.

[0078] like Figure 7 As shown, the XRD patterns show the crystal structures of magnetic Fe3O4 nanoparticles, PAA-modified magnetic nanoparticles (Fe3O4 / PAA), magnetic MOF nanoparticles (Fe3O4 / PAA / MOFs), hydrolyzed magnetic MOF nanoparticles (Fe3O4 / PAA / HMOFs) and Fe3O4 / PAA / HMOFs clusters. All samples have six characteristic peaks, 30.50°(220), 35.84°(311), 45.46°(400), 55.90°(422), 57.38°(511) and 62.90°(440), which are consistent with the standard XRD data card of Fe3O4 (JCPDS No.19-06290). The results show that the synthesized Fe3O4 / PAA / HMOFs clusters retain the crystal structure of magnetite.

[0079] like Figure 8As shown in the figure, the quantitative information of magnetic Fe3O4 nanoparticles, PAA-modified magnetic nanoparticles (Fe3O4 / PAA), magnetic MOF nanoparticles (Fe3O4 / PAA / MOFs), hydrolyzed magnetic MOF nanoparticles (Fe3O4 / PAA / HMOFs) and Fe3O4 / PAA / HMOFs clusters were analyzed by TGA test. When the temperature is below 200℃, the sample loses water; when the temperature is above 200℃, the organic molecules PAA, PEI, PAPE and PA in a series of composite materials decompose. The mass of magnetic Fe3O4 nanoparticles decreased by 12% (a) due to the small amount of polyethyleneimine on the surface; the mass of PAA-modified magnetic nanoparticles (Fe3O4 / PAA) decreased by 36% (b); the mass of Fe3O4 / PAA / MOFs nanoparticles decreased by 38% (c); the mass of hydrolyzed magnetic MOF nanoparticles (Fe3O4 / PAA / HMOFs) and Fe3O4 / PAA / HMOFs clusters decreased by 40% and 42%, respectively. Therefore, the magnetic content of Fe3O4 / PAA / HMOFs clusters is about 58%.

[0080] like Fig. 9 As shown, the magnetic properties of magnetic Fe3O4 nanoparticles, PAA-modified magnetic nanoparticles (Fe3O4 / PAA), magnetic MOF nanoparticles (Fe3O4 / PAA / MOFs), hydrolyzed magnetic MOF nanoparticles (Fe3O4 / PAA / HMOFs) and Fe3O4 / PAA / HMOFs clusters at room temperature (300 K) were detected by VSM. All samples are magnetic, and the average saturation magnetization values ​​are 66emu / g, 50emu / g, 46emu / g, 33emu / g, and 30emu / g, respectively.

[0081] like Fig.10 As shown in Figure 2, the N2 adsorption / desorption isotherms show that the surface area of ​​the Fe3O4 / PAA / HMOFs cluster is estimated to be 153 m 2 / g, and the pore volume of the Fe3O4 / PAA / HMOFs cluster was estimated to be 0.42 cm according to the Barrett–Joyner–Halenda (BJH) method. 3 / g. The pore sizes of the Fe3O4 / PAA / HMOFs clusters were estimated to be approximately 3.7nm, 4.1nm, 7.1nm, and 59.9nm according to the Barrett–Joyner–Halenda (BJH) method, proving that the prepared clusters do contain a hierarchical porous structure.

[0082] Experimental example

[0083] In this experimental example, single protein systems transferrin (TRF), bovine serum albumin (BSA), lysozyme (LYZ), and bovine hemoglobin (BHB) were used as model proteins to investigate the protein adsorption capacity and reusability of the prepared magnetic hierarchical porous MOFs composite nanomaterials.

[0084] According to the relevant evaluation standards of biomaterials, the effect of magnetic hierarchical porous MOFs composite materials on protein activity was evaluated. The activity of glycoproteins released from magnetic hierarchical porous MOFs composite nanomaterials was studied using advanced methods and characterization techniques such as fluorescence spectroscopy (FLU).

[0085] (1) Protein capture and release

[0086] In this part, transferrin (TRF) is used as a model protein to investigate the capture and release of proteins by the prepared Fe3O4 / PAA / HMOFs clusters. TRF is a single-chain glycoprotein with a sugar content of about 6%. It is the main iron-containing protein in plasma and is responsible for transporting iron absorbed by the digestive tract and iron released by red blood cell degradation. Abnormal transferrin content in the blood is related to many diseases, such as heart failure, iron deficiency anemia, malnutrition, transferrin deficiency, etc. Therefore, in this application example, transferrin is used as a model protein.

[0087] like Fig.11 As shown, the capture and release steps of the protein are as follows:

[0088] A) Protein capture: Fig.11 As shown, TRF was dissolved in BR buffer solutions of different pH values ​​to prepare 0.5 mg / mL TRF solutions; the TRF protein solution was co-incubated with the above Fe3O4 / PAA / HMOFs clusters; magnetic separation was performed to collect the supernatant and precipitate, and the precipitate was washed three times with protein-free incubation solution and dispersed into 20 μL incubation solution. 12 μL of the protein mixture before incubation, the above supernatant, and the precipitate suspension were taken for SDS-PAGE electrophoresis analysis;

[0089] B) Release of protein: Fig.11 As shown, the above-mentioned protein-bound material (ie, precipitate) was dispersed into 200 μL BR buffer solution of different pH values, and the protein was eluted; the eluate and the eluted material were subjected to SDS-PAGE electrophoresis analysis.

[0090] First, the effects of different pH on the capture and release of glycoproteins were investigated according to the above steps to determine the optimal capture and release pH values.

[0091] Transferrin and materials were co-incubated under different pH conditions (C Protein =0.5 mg / mL, 300 μL protein solution; CMaterial =15 mg / mL, 20 μL material; incubation time 1 h), followed by magnetic separation, collecting the supernatant and precipitate, and washing the precipitate (i.e., protein-nanosphere complex) twice with BR buffer and redispersing it in BR buffer. Then, the supernatant and protein-nanosphere complex were analyzed by SDS-PAGE. Fig.12 As shown in Figure 3, under acidic conditions, TRF can be successfully captured by Fe3O4 / PAA / HMOFs clusters (lanes 3-5 in B). In contrast, in neutral or alkaline media, TRF is rarely captured or even not captured (lanes 6-10 in B). Since the initial amount of TRF is high (150 μg, 300 μL) and the material content is reduced (300 μg, 20 μL), TRF cannot be completely captured by the nanomaterials, and some TRF remains in the supernatant (lanes 2-9 in A). By comparing the colors of the bands of the supernatant (S) or the protein-nanosphere complex (C), the residual amount of TRF in the supernatant is the lightest at pH = 4 and pH = 5 (lanes 2-3 in A), and the amount of adsorbed protein is the largest at pH = 4-6 in the bands of the protein-nanosphere complex (lanes 3-5 in B), indicating that the optimal incubation conditions are pH = 4 and 5. In subsequent experiments, pH = 5 was selected as the incubation condition. Among the bands of the protein-nanosphere complex, the TRF capture amount was minimal at pH = 7-11 (lanes B 6-10), indicating that pH = 7-11 could be used as a potential elution condition.

[0092] Fe3O4 / PAA / HMOFs clusters were incubated in protein buffer at pH 5 (C Protein =0.5mg / ml, 100μL protein solution, C Material =15 mg / mL, 35 μL material, incubation solution with pH=5, incubation time 5 min), the protein-nanosphere complex was eluted with buffers of different pH (100 μL elution solution, elution time 60 min), and the optimal elution conditions were investigated. The test results are shown in Fig.13 As shown in Figure 2, as the pH value of the eluent increases from 7 to 11, the content of TRF in the eluent (E) increases, and the residual amount of TRF on the nanospheres (C) decreases. At pH = 9-11, all captured TRF can be completely released from the nanospheres. The difference in capture or release behavior under different pH media demonstrates the pH responsiveness of Fe3O4 / PAA / HMOFs clusters.

[0093] At the same time, the effects of incubation time and elution time on TRF capture and release were investigated to determine the optimal incubation time and elution time. Fig.14As shown in the figure, with the increase of incubation time, the amount of captured TRF remains basically unchanged, indicating that the captured TRF can reach adsorption saturation within 5 minutes. Further extending the incubation time cannot significantly increase the amount of captured TRF, because the surface of the nanospheres has been coated with a large amount of TRF, and the repulsion between TRF molecules hinders further capture. Fig.15 As shown, different pH buffers were used for different elution times. At pH = 10 and 11, all captured TRFs could be completely released after 60 min (see Figure 2). Fig.15 B and C), there is almost no TRF residue on the Fe3O4 / PAA / HMOFs clusters. Therefore, the milder conditions of pH = 10 and elution time of 60 min were selected as the optimal elution conditions. These results indicate that Fe3O4 / PAA / HMOFs clusters can be captured or released in a short time (about 60 minutes), which will be beneficial to the rapid separation and recovery of proteins in Fe3O4 / PAA / HMOFs clusters.

[0094] In order to improve the protein separation efficiency, the optimized scheme was used to carry out protein adsorption and elution experiments. The results are as follows Fig.16 As shown. After the Fe3O4 / PAA / HMOFs clusters were co-incubated with TRF in an incubation solution at pH = 5 for 5 minutes, there was no TRF residue in the supernatant obtained by magnetic separation; the material bonded with protein showed obvious TRF bands, indicating that all TRF in the solution had been adsorbed to the surface of the material. Therefore, it can be estimated that the adsorption amount is as high as 111 mg / g and the adsorption rate is as high as 100%. When the pH is adjusted to 10, the protein is easily and completely eluted from the surface of the material, indicating that the material can capture and release proteins in a pH-responsive manner.

[0095] (2) Protein adsorption

[0096] In this part, transferrin (TRF), bovine serum albumin (BSA), lysozyme (LYZ), and bovine hemoglobin (BHB) were used as model proteins to investigate the protein adsorption capacity and reuse rate of the prepared Fe3O4 / PAA / HMOFs clusters.

[0097] The four proteins were dissolved in BR buffer solution (pH = 5) to prepare a 0.5 mg / mL protein solution. 525 μg of Fe3O4 / PAA / HMOFs clusters were added to 100 μL of protein solution and shaken at 35 ° C for 5 minutes. Under the action of an external magnetic field, the material was removed from the protein solution. The concentration of unadsorbed protein was measured by the BCA kit. The adsorption amount of protein can be estimated from the following equation:

[0098] q=(C o -C)V o / W;

[0099] q represents the equilibrium adsorption capacity of the material (mg·g -1 );C o and C represent the initial concentration and equilibrium concentration of the protein, respectively (mg·mL -1 );V o represents the volume of the aqueous phase (mL); W represents the weight of the magnetic composite material (g).

[0100] Fig.17 The adsorption amount and adsorption rate of Fe3O4 / PAA / HMOFs clusters after co-incubation with TRF in different pH buffers are given. It can be seen from the figure that the adsorption amount of Fe3O4 / PAA / HMOFs clusters with TRF at different pH values ​​decreases with the increase of pH value, and the adsorption amount and adsorption rate are the highest at pH 4-5, up to 113 mg·g -1 , 96%. Therefore, the subsequent incubation condition was selected to be a relatively mild pH = 5, and the result was consistent with the SDS-PAGE result.

[0101] Fig.18 The elution rates of captured proteins after incubation of Fe3O4 / PAA / HMOFs clusters with TRF at pH=5 buffer and different eluents are given. After incubation of Fe3O4 / PAA / HMOFs clusters with TRF at pH=5, the proteins could not be eluted by water or incubation solution (pH=5). As the pH increased, the elution rate gradually increased. When the pH reached 9-11, the elution rate was high, reaching about 77%, which was consistent with the results of SDS-PAGE analysis.

[0102] Fig.19 The elution rates of proteins at different times of incubation of Fe3O4 / PAA / HMOFs clusters with TRF in pH=5 buffer and elution with pH=10 buffer are given. After incubation of Fe3O4 / PAA / HMOFs clusters with TRF in pH=5, the elution rate increases gradually with the increase of elution time. When the pH reaches 60 minutes, the elution rate is high, reaching about 74%. As the time continues to increase to 120 minutes, the elution rate does not increase significantly, which is consistent with the results of SDS-PAGE analysis.

[0103] Fig. 20 The adsorption amount and adsorption rate of Fe3O4 / PAA / HMOFs clusters incubated with TRF at pH = 5 buffer for different time periods are given. When Fe3O4 / PAA / HMOFs clusters are incubated with TRF at pH = 5, the adsorption amount and adsorption rate gradually increase with the increase of incubation time, and the adsorption can be quickly saturated, with the adsorption amount reaching 117 mg·g in just 3 minutes. -1 , and the adsorption amount did not increase significantly with time.

[0104] Fig.21 The adsorption amount and adsorption rate of Fe3O4 / PAA / HMOFs clusters for four proteins were given to evaluate their reusability. The results show that the Fe3O4 / PAA / HMOFs cluster material provided by the present invention can be reused, and after being reused for 4 times, the separation efficiency is still as high as more than 70%, indicating that the material has great potential in pH-responsive protein separation.

[0105] (3) Biological activity of protein

[0106] The structure of a protein is closely related to its activity. This section characterizes the effects of adsorption and desorption processes on the secondary structure of a protein through fluorescence testing. Any change in the local environment of the fluorophore results in a change in the fluorescence intensity of the protein.

[0107] like Fig. 22 As shown in the figure, when the excitation wavelength is 280nm, after the material is co-incubated with four proteins, the fluorescence of the protein is quenched due to the interaction with the Fe3O4 / PAA / HMOFs clusters. The fluorescence emission wavelength of LYZ shifts from 337nm to 335nm (LYZ-M); the fluorescence emission wavelength of BSA shifts from 339nm to 337nm (BSA-M); the fluorescence emission wavelength of BHB shifts from 326nm to 327nm (BHB-M); the fluorescence emission wavelength of TRF shifts from 321nm to 324nm (TRF-M); the fluorescence emission wavelength of the material is 307nm and 323nm, and the intensity is very weak, with no obvious effect. In summary, after co-incubation, the fluorescence emission wavelengths of all proteins did not move significantly, indicating that the material has no obvious effect on the conformation of the protein.

[0108] Those skilled in the art will appreciate that the embodiments herein are intended to help readers understand the principles of the present invention, and should be understood that the protection scope of the present invention is not limited to such specific statements and embodiments. Those skilled in the art can make various other specific variations and combinations that do not deviate from the essence of the present invention based on the technical revelations disclosed by the present invention, and these variations and combinations are still within the protection scope of the present invention.

Claims

1. A pH-responsive magnetic hierarchical porous MOFs composite material, characterized in that: The magnetic MOFs composite nanospheres with Fe3O4 nanoparticles as core and MOFs layer as shell are assembled by emulsification and cross-linking, presenting a cluster structure.

2. The pH-responsive magnetic graded porous MOFs composite material according to claim 1, characterized in that: The surface of the Fe3O4 nanoparticles is modified with a hydrophilic molecular compound A for chelating metal ions as a transition layer; the hydrophilic molecular compound A is at least one of polyacrylic acid PAA, chitosan, carboxymethyl chitosan, and polyvinyl pyrrolidone.

3. The pH-responsive magnetic graded porous MOFs composite material according to claim 1, characterized in that: The MOFs layer contains metal ions and organic ligands bound to the metal ions; the organic ligands include a first organic ligand that imparts pH responsiveness to the material and a second organic ligand for providing cross-linking sites; the first organic ligand is at least one of 4-pyrazole boronic acid pinacol ester, 3,5-dimethylpyrazole-4-boronic acid pinacol ester, 1-methylpyrazole-4-boronic acid pinacol ester, pyrido[2,3-b]pyrazine-7-yl boronic acid pinacol ester, 2-aminopyridine-5-boronic acid pinacol ester, and 1-methylimidazole-5-boronic acid pinacol ester, and the second organic ligand is at least one of aminopyrazole, aminopyrazine, aminopyridine, and aminoimidazole.

4. The pH-responsive magnetic graded porous MOFs composite material according to claim 1, characterized in that: The oil phase used for emulsification is at least one of liquid paraffin, squalane, and caprylic / capric triglyceride; the nonionic surfactant used for emulsification is at least one of Span 80, Tween 80, Span 20, and Span 60; and the cross-linking agent used for cross-linking is genipin and / or glutaraldehyde.

5. A method for preparing a pH-responsive magnetic hierarchical porous MOFs composite material, characterized in that: The following steps are involved: (1) Preparing pH-responsive magnetic MOFs composite nanospheres; this step includes the following sub-steps: (11) Preparation of Fe3O4 nanoparticles; (12) coating a transition layer on the surface of Fe3O4 nanoparticles; (13) Preparation of pH-responsive magnetic MOFs composite nanospheres; A Fe3O4 nanoparticle suspension containing a transition layer is dispersed in a mixed solvent, and a metal salt, a first organic ligand and a second organic ligand are added; the resulting reaction system is reacted at 50-90°C for 2-6 hours under stirring conditions until it becomes reddish brown, and the precipitate is collected and washed to obtain a dispersion containing magnetic MOFs composite nanospheres; the magnetic MOFs composite nanospheres are hydrolyzed to obtain a dispersion containing pH-responsive magnetic MOFs composite nanospheres; the molar ratio of the Fe3O4 nanoparticles containing a transition layer, the metal salt, the first organic ligand and the second organic ligand is (0.1-0.2):1:1.5:0.5; (2) Preparing a magnetic graded porous MOF composite material; this step includes the following sub-steps: (21) adding a dispersion containing pH-responsive magnetic MOFs composite nanospheres into an oil phase containing a nonionic surfactant to form a uniform emulsion; (22) Under stirring conditions at 40–80 °C, an aqueous crosslinking agent solution was added to the emulsion to allow the crosslinking reaction to proceed for 3–6 h. The precipitate was collected and washed to obtain a magnetic graded porous MOF composite material.

6. The method for preparing the pH-responsive magnetic graded porous MOFs composite material according to claim 5, characterized in that: In step (11), under nitrogen protection, the iron salt is dissolved in water, and then the hydrophilic polymer compound B aqueous solution is added, and then ammonia water is dropped into the obtained reaction system, and the reaction is carried out at 60-80° C. for 1-3 hours until the solution turns black, and the black precipitate is collected and washed to neutrality; the hydrophilic polymer compound B is polyethyleneimine, ethylenediamine or glucosamine; the iron salt is a divalent iron salt and a trivalent iron salt; the molar volume ratio of the divalent iron salt, the trivalent iron salt, the hydrophilic polymer compound B and the ammonia water is 5mmol:9mmol:0.1mmol:35mL.

7. The method for preparing the pH-responsive magnetic graded porous MOFs composite material according to claim 6, characterized in that: The divalent iron salt is FeCl2 or / and FeSO4, and the trivalent iron salt is FeCl3 or / and Fe2(SO4)3.

8. The method for preparing the pH-responsive magnetic graded porous MOFs composite material according to claim 5, characterized in that: In step (12), an aqueous solution of a hydrophilic polymer compound A is added to a suspension of Fe3O4 nanoparticles, and the resulting reaction system is subjected to magnetic stirring at room temperature to 40°C until the zeta potential of the material no longer changes, and the brown product is collected and washed; the mass ratio of the hydrophilic polymer compound A to the Fe3O4 nanoparticles is 2-5:

1.

9. The method for preparing the pH-responsive magnetic graded porous MOFs composite material according to claim 5, characterized in that: In step (13), the mixed solvent is obtained by mixing methanol, acetonitrile, ethanol and deionized water in a volume ratio of 1:1:1:3; the metal salt is ferric nitrate and / or ferric sulfate; the first organic ligand is at least one of 4-pyrazole boronic acid pinacol ester, 3,5-dimethylpyrazole-4-boronic acid pinacol ester, 1-methylpyrazole-4-boronic acid pinacol ester, pyrido[2,3-b]pyrazine-7-yl boronic acid pinacol ester, 2-aminopyridine-5-boronic acid pinacol ester, and 1-methylimidazole-5-boronic acid pinacol ester; and the second organic ligand is at least one of aminopyrazole, aminopyrazine, aminopyridine and aminoimidazole.

10. The method for preparing the pH-responsive magnetic graded porous MOFs composite material according to claim 5, characterized in that: In step (13), the dispersion containing magnetic MOFs composite nanospheres is added to an ethanol solution containing sodium periodate, and the mixture is reacted at 30-50° C. and magnetically stirred for 4-12 hours, and then the precipitate is collected and washed; the molar ratio of the first organic ligand to sodium periodate is 1:(5-6).

11. The method for preparing the pH-responsive magnetic graded porous MOFs composite material according to claim 5, characterized in that: In step (21), the volume ratio of the dispersion containing pH-responsive magnetic MOFs composite nanospheres to the oil phase and the non-ionic surfactant is 1:(50-75):(15-30); the emulsification condition is emulsification at a rotation speed of 3000-6000 rpm for 10-20 min; In step (22), the molar ratio of the cross-linking agent to the pH-responsive magnetic MOFs composite nanospheres is (2.5-3.5):

1.

12. Use of the pH-responsive magnetic hierarchical porous MOFs composite material according to any one of claims 1 to 4 in protein enrichment and release.

13. The use according to claim 12, characterized in that: The pH-responsive magnetic graded porous MOFs composite material can capture proteins in an environment with a pH of 4-5, and can release proteins in an environment with a pH of 9-11.

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