Preparation and application of surface carboxyl functionalized magnetic polymer microspheres

Abstract

The invention discloses a preparation method and application of a magnetic polymer microsphere with a core-shell structure and rich carboxyl groups on the surface. The specific preparation process comprises the following two steps: (1) mixing a dispersing agent and a deep eutectic solvent, dissolving the mixture in a reaction medium, then adding Fe3O4 nanoparticles, and stirring under closed and nitrogen protection conditions; and (2) dissolving an initiator in a polymeric monomer, pouring into the reaction solution, stirring, heating, and continuing the reaction to obtain the carboxyl-rich magnetic polymer microspheres. On the basis of the dispersion polymerization method, a polymer shell can be formed on the nano magnetic particle core in situ, and the surface of the polymer shell is rich in carboxyl functional groups; therefore, obvious interaction with various biomacromolecules can be generated, and the method has good application potential in separation and detection of the biomacromolecules. The prepared microspheres have ideal stability, uniform particle size distribution, good superparamagnetism and excellent dispersity, and show expected performance in adsorption application of macromolecules represented by protein.

Description

Technical Field

[0001] The invention belongs to the technical field of functional polymer materials, and in particular relates to the preparation and application of magnetic polymer microspheres with surface carboxyl functionalization. Background Art

[0002] Magnetic polymer microspheres are magnetic materials composed of inorganic magnetic particles and polymer materials with particle sizes at the micrometer and nanometer levels and spherical morphology. They combine the multiple advantages and functions of the two main components. The inorganic magnetic particles mainly include iron, cobalt, nickel materials and their oxides and alloys, which can easily separate the microspheres with polymer layers from the solid-liquid system under the action of an external magnetic field; while the flexible organic polymer chains give the microspheres surface modifiability. After completing the functionalization of different types of polymers, they have corresponding physical, chemical and functional properties, which are conducive to further application in the fields of biological separation, disease diagnosis and drug controlled release. Therefore, they are currently playing an increasingly important role in chemistry, medicine, life sciences and other fields.

[0003] Fe3O4 nanoparticles have strong saturation magnetization and paramagnetism. In recent years, the research and development of innovative materials with such low-toxic, cheap and easily available raw materials as magnetic centers has become a hot topic. Magnetic microspheres with different microstructures can be prepared from Fe3O4 nanoparticles. Their structures mainly include: (1) core-shell structure, that is, the core is composed of inorganic magnetic particles and the shell is composed of polymer materials; (2) reverse core-shell structure, that is, the core is composed of polymers and the shell is composed of inorganic magnetic particles; (3) sandwich structure, that is, the inner and outer layers are composed of polymers and the middle layer is magnetic particles; (4) dispersed structure, that is, magnetic nanoparticles are randomly distributed in the polymer microsphere system. The synthesis methods of core-shell structure microspheres, which are the main ones, include emulsion and microemulsion polymerization, dispersion polymerization, precipitation polymerization and suspension polymerization. Among them, the dispersion polymerization method is a polymerization method in which monomers soluble in the reaction medium are polymerized to generate polymers insoluble in the reaction medium to form a colloidal stable dispersion system; this method can be used to prepare microspheres with small size, uniform particle size and good monodispersity through simple and fast steps.

[0004] Deep eutectic solvent (DES) is a room temperature liquid product obtained by heating a hydrogen bond acceptor (HBA) and a hydrogen bond donor (HBD) in a certain molar ratio. Compared with traditional organic solvents, it is easy to synthesize, low in cost, high in atom economy, and low in vapor pressure; it also has the advantages of ideal conductivity, stability, solubility, and good biocompatibility. As a new type of green solvent, the research and development and application of DES have attracted much attention from researchers at home and abroad, and has been widely used in many fields such as synthesis, extraction, catalysis, separation, and analysis. For example, in a preparation method and application of a magnetic thermosensitive molecular imprinting polymer based on a low eutectic solvent system (Wan Yiqun, Xiong Huihuang, Wan Hao, Guo Lan, Fan Yong, Xu Mengjia, Guo Xian; Patent No. CN202210133355.2), N-isopropylacrylamide-(3-acrylamidopropyl)trimethylammonium chloride low eutectic solvent (molar ratio 1:1) system is used as a functional monomer, combined with graphite, silica, ferric chloride and other main raw materials, and polymer particles that can be used for template molecule enrichment detection are prepared in 5 steps. For another example, a magnetic low eutectic solvent adsorption material (Chen Yu, Wang Shujun, Wang Xintong, Li Yuting; Patent No. CN202310057705.6) is composed of phytic acid DES, Fe3O4 and lithium cobalt oxide. After reacting at 100°C for 24 hours, it is ground into powder to obtain the corresponding product, which can adsorb dyes such as methylene blue, methyl orange and rhodamine B in wastewater and waste oil. The synthesis conditions of the above materials are relatively complex and time-consuming, and their main targets are small molecules. At present, there is an urgent need to develop more methods with simple synthesis steps and mild reaction conditions to prepare new magnetic materials based on DES, and apply them to the field of biological macromolecules, which are currently less involved.

[0005] As a hot topic in the field of functional polymer materials technology in recent years, there are many reports on nano-scale magnetic microspheres, while research on the preparation technology of micron-scale large-particle microspheres is relatively lacking. Micron-scale magnetic microspheres have the advantages of more uniform size, better dispersibility, simple preparation method, controllable and easy process, and convenient modification by functional groups, so they have higher maturity in commercialization; and the magnetic microspheres containing amino, carboxyl, and thiol groups on the surface can be paired with biomacromolecules through physical adsorption or chemical coupling adsorption of activated functional groups, thereby achieving a variety of application purposes on this basis. Based on the above background, the present invention proposes to synthesize micron-scale magnetic microspheres containing carboxyl groups on the surface by dispersion polymerization and use them for the adsorption and separation of protein biomacromolecules. Summary of the invention

[0006] The purpose of the present invention is to provide a method for preparing magnetic polymer microspheres with core-shell structure and surface carboxyl functionalization, and to apply the method to the adsorption of protein biomacromolecules.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] Preparation of magnetic polymer microspheres functionalized with surface carboxyl groups:

[0009] (1) The dispersant and the deep eutectic solvent are mixed and dissolved in an ethanol-water mixture to form a clear system, which is then poured into a three-necked flask equipped with a thermometer and a mechanical stirring device, and then Fe3O4 nanoparticles are added and stirred under closed and nitrogen protection conditions;

[0010] (2) dissolving the initiator in the polymerization monomer and pouring it into the reaction solution in (1), heating it and continuously stirring it under nitrogen protection to react, and cooling the reaction system after completion;

[0011] (3) After the reaction solution in (2) is completely cooled, magnetic separation and screening are performed using a NdFeB magnet to obtain magnetic polymer microspheres (P(DES-co-St)@Fe3O4) with basically uniform particle sizes; after repeated washing three times with anhydrous ethanol and ultrapure water, the microspheres are dispersed in ultrapure water for later use.

[0012] The preparation of the above-mentioned surface carboxyl-functionalized magnetic polymer microspheres is characterized in that the low eutectic solvent in step (1) is choline chloride-acrylic acid (ChCl / AA, molar ratio 1:2) or choline chloride-itaconic acid (ChCl / IA, molar ratio 1:1).

[0013] The preparation of the above-mentioned surface carboxyl functionalized magnetic polymer microspheres is characterized in that the dispersant in step (1) is polyvinyl pyrrolidone (PVP), and the mass ratio of the dispersant to the low eutectic solvent is 1:2 to 2:1.

[0014] The above-mentioned preparation method of magnetic polymer microspheres with surface carboxyl functionalization is characterized in that the stirring time in step (1) is 20 to 40 minutes.

[0015] The preparation of the above-mentioned surface carboxyl functionalized magnetic polymer microspheres is characterized in that the mass ratio of the Fe3O4 nanoparticles to the low eutectic solvent in step (1) is 1:6 to 1:3.

[0016] The preparation of the above-mentioned surface carboxyl functionalized magnetic polymer microspheres is characterized in that the initiator in step (2) is azobisisobutyronitrile (AIBN), and the mass ratio of the initiator to the low eutectic solvent is 1:4 to 1:6.

[0017] The above-mentioned preparation of magnetic polymer microspheres functionalized with surface carboxyl groups is characterized in that the polymerization monomer in step (2) is styrene (St), and the mass ratio of styrene (St) to the low eutectic solvent is 7:0.5-4.

[0018] The above-mentioned preparation method of magnetic polymer microspheres with surface carboxyl functionalization is characterized in that the reaction temperature in step (2) is 60-80° C. and the reaction time is 8-12 hours.

[0019] The above-mentioned preparation of surface carboxyl-functionalized magnetic polymer microspheres is characterized in that the magnetic polymer microspheres (P(ChCl / AA-co-St)@Fe3O4, P(ChCl / IA-co-St)@Fe3O4) prepared by two low eutectic solvents can be used in the field of separation and detection of biological macromolecules represented by proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a scanning electron micrograph of the appearance of magnetic polymer microspheres (aP(ChCl / AA-co-St)@Fe3O4, bP(ChCl / IA-co-St)@Fe3O4).

[0021] Figure 2 It is the Fourier transform infrared spectra of magnetic polymer microspheres (P(ChCl / AA-co-St)@Fe3O4, P(ChCl / IA-co-St)@Fe3O4) and Fe3O4 nanoparticles.

[0022] Figure 3 It is the X-ray diffraction pattern of magnetic polymer microspheres (P(ChCl / AA-co-St)@Fe3O4, P(ChCl / IA-co-St)@Fe3O4) and Fe3O4 nanoparticles.

[0023] Figure 4 This is the magnetic response diagram of magnetic polymer microspheres (a. no external magnetic field, b. magnet attraction).

[0024] Figure 5 It is the hysteresis loop diagram of magnetic polymer microspheres.

[0025] Figure 6 This is the adsorption result of magnetic polymer microspheres P(ChCl / AA-co-St)@Fe3O4 for three protein samples. DETAILED DESCRIPTION

[0026] The specific embodiments of the present invention are described in detail below, but the following embodiments are not intended to limit the content of the present invention. The range endpoints and any values ​​described herein are not limited to the precise ranges and values, and these ranges should be understood to include values ​​close to these range values. Some non-essential improvements and adjustments made by technicians in this field according to the invention still fall within the scope of the present invention.

[0027] Example 1

[0028] Synthesis of DES: 0.04 mol of choline chloride (ChCl) and 0.08 mol of acrylic acid (AA) were added into a 50 mL round-bottom flask and placed in an oil bath at 90 °C for continuous heating and stirring reaction until the solid completely disappeared and a clear and transparent liquid ChCl / AA (1:2) was formed.

[0029] Preparation of magnetic microspheres: (1) Weigh 1.0 g polyvinyl pyrrolidone (PVP) and 1.5 g ChCl / AA (1:2) respectively, dissolve them in an ethanol-water mixture to form a clear system, pour them into a three-necked flask equipped with a thermometer and a mechanical stirring device, then add 0.35 g Fe3O4 nanoparticles, and stir and react for 30 min under closed and nitrogen protection conditions;

[0030] (2) Weigh 7.0 g of styrene (St) into a beaker, add 0.3 g of initiator azobisisobutyronitrile (AIBN) while stirring, and after it is completely dissolved, add the mixture of the two into a three-necked flask and mix thoroughly with other components, then increase the reaction temperature to 70°C and continue stirring for 10 hours under nitrogen protection. After the reaction is completed, remove the heat source and cool naturally while stirring;

[0031] (3) After the reaction liquid is completely cooled, a NdFeB magnet is used for magnetic separation and screening to obtain P(ChCl / AA-co-St)@Fe3O4 magnetic polymer microspheres with basically uniform particle sizes. The microspheres are washed alternately with anhydrous ethanol and ultrapure water three times and then dispersed in ultrapure water for later use.

[0032] Example 2

[0033] Synthesis of DES: 0.04 mol of choline chloride (ChCl) and 0.04 mol of itaconic acid (IA) were added into a 50 mL round-bottom flask and placed in an oil bath at 90 °C for continuous heating and stirring reaction until the solid completely disappeared and a clear and transparent liquid ChCl / IA (1:1) was formed.

[0034] Preparation of magnetic microspheres: (1) Weigh 1.0 g polyvinyl pyrrolidone (PVP) and 1.5 g ChCl / IA (1:1) respectively, dissolve them in an ethanol-water mixture to form a clear system, pour them into a three-necked flask equipped with a thermometer and a mechanical stirring device, then add 0.35 g Fe3O4 nanoparticles, and stir and react for 30 min under closed and nitrogen protection conditions;

[0035] (2) Weigh 7.0 g of styrene (St) into a beaker, add 0.3 g of initiator azobisisobutyronitrile (AIBN) while stirring, and after it is completely dissolved, add the mixture of the two into a three-necked flask and mix thoroughly with other components, then increase the reaction temperature to 70°C and continue stirring for 10 hours under nitrogen protection. After the reaction is completed, remove the heat source and cool naturally while stirring;

[0036] (3) After the reaction liquid is completely cooled, a NdFeB magnet is used for magnetic separation and screening to obtain P(ChCl / IA-co-St)@Fe3O4 magnetic polymer microspheres with basically uniform particle sizes. The microspheres are washed alternately with anhydrous ethanol and ultrapure water three times and then dispersed in ultrapure water for later use.

[0037] Example 3

[0038] Chemical coupling and adsorption of ovomucoid by magnetic polymer microspheres: (1) Prepare a 2 mg / mL ovomucoid (OVM) standard solution, pipette 0, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75 and 2.00 mL of the ovomucoid standard solution into a 2 mL volumetric flask, dilute with 0.01 M PBS buffer with a pH of 7.2 to 7.4, and use the PBS buffer as the zero solution for determination. Use an ultraviolet spectrometer to measure the absorbance at 280 nm to obtain the linear relationship between the concentration of the ovomucoid solution and the absorbance and establish a standard curve. The same method can be used to obtain the standard curves of bovine serum albumin and bovine hemoglobin at the measurement wavelengths of 280 nm and 408 nm.

[0039] (2) Accurately pipette 0.1 mL of P(ChCl / AA-co-St)@Fe3O4 magnetic microsphere dispersion (10 mg / mL) into a 2 mL centrifuge tube, let it stand on a magnetic separator for 2 min, and discard the supernatant with a pipette; then add a small amount of PBS buffer to the centrifuge tube, place it on a rotating mixer and mix it thoroughly for 5 min, collect the P(ChCl / AA-co-St)@Fe3O4 microspheres by magnetic separation and wash them three times with a small amount of MES solution (0.1 M, pH = 6.0), then add 50 μL each of the newly prepared 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) solution (50 mg / mL, prepared with MES solution) and N-hydroxysuccinimide (NHS) solution (20 mg / mL, prepared with MES solution), mix it thoroughly on a rotating mixer, and activate it at room temperature (25°C) for 30 min. The supernatant was discarded and washed with a small amount of MES solution (0.1M, pH=6.0), and this step was repeated 3 times. After washing, a protein sample solution with an initial concentration of 2 mg / mL was added to the centrifuge tube, and the mixture was gently shaken and placed in a 25°C constant temperature water bath for 2 hours. After the adsorption was completed, a permanent magnet was used for solid-liquid separation, and the absorbance of the supernatant at the above wavelength was measured. The adsorption effect of different proteins was calculated according to formula (1).

[0040] Formula (1) is: Q = (C0-C t )×V / m.

[0041] In the above formula, Q is the adsorption amount, mg / g; C0 is the initial concentration of the adsorbed solution, mg / ml; C t is the concentration of the solution after adsorption, mg / mL; V is the volume of the adsorption solution, mL; m is the mass of the adsorbent, i.e., the magnetic microspheres, g.

[0042] pass Figure 1 The morphology observed by scanning electron microscopy shows that the two samples prepared by the dispersion-polymerization method are in a complete spherical state, with a relatively smooth surface, good roundness and uniformity. In addition, it can be seen from the figure that there is no adhesion between the microspheres and the dispersion is good.

[0043] Through Figure 2 Infrared spectrum analysis shows that 3000cm -1 The characteristic absorption peaks appearing near 1600cm-1 correspond to the symmetrical stretching vibration of the unsaturated carbon-hydrogen bonds on the benzene ring of St. The characteristic absorption peaks produced by the vibration of the benzene ring skeleton in St are -1 Appears near; appears in the fingerprint area 756cm -1 、697cm -1 The two characteristic absorption peaks indicate that the vinyl group in St is monosubstituted, and the peak at 1027 cm -1The in-plane deformation vibration absorption peak of hydrogen on the monosubstituted benzene ring appears at 1700cm -1 The high and sharp absorption peaks appearing around 1400 cm-1 are caused by the C=O stretching vibration of the carboxyl group of acrylic acid / itaconic acid in DES, while the -1 The absorption peaks on the left and right are attributed to the CN bond stretching vibration of choline chloride in DES. In addition, there are characteristic absorption peaks of Fe3O4 in the infrared spectrum of magnetic polymer microspheres. The above results prove that the magnetic polymer microspheres have been successfully synthesized.

[0044] Through Figure 3 X-ray diffraction analysis shows that the pure Fe3O4 sample shows six groups of characteristic diffraction peaks, and the corresponding six groups of characteristic diffraction peaks can also be observed in the X-ray diffraction patterns of P(ChCl / AA-co-St)@Fe3O4 and P(ChCl / IA-co-St)@Fe3O4 magnetic microspheres. This analysis shows that DES and St did not change the crystal form of Fe3O4 particles during the copolymerization process, but only polymerized and wrapped on its surface.

[0045] By comparison Figure 4 It can be seen from the state with or without the external magnetic field that in the absence of an external magnetic field, the aqueous dispersion of P(DES-co-St)@Fe3O4 magnetic microspheres appears as a dark gray suspension, and there will be no agglomeration or sedimentation. If a nepheline iron boron magnet is placed on one side of the sample bottle, all the P(DES-co-St)@Fe3O4 magnetic microspheres can be quickly attracted to that side by the magnet, and the original aqueous dispersion will appear transparent. On the other hand, after removing the external magnetic field, shaking the sample bottle can restore it to a suspended state. This shows that P(DES-co-St)@Fe3O4 has good magnetic responsiveness and dispersibility, and it can be attracted and separated from the system very quickly when there is an external magnetic field.

[0046] By observation Figure 5 From the hysteresis loop, it can be seen that the hysteresis curves of the two P(DES-co-St)@Fe3O4 magnetic microspheres are both closed curves without obvious hysteresis phenomenon, indicating that they have superparamagnetism like pure Fe3O4. In addition, the magnetic saturation intensity of P(ChCl / AA-co-St)@Fe3O4 is higher than that of P(ChCl / IA-co-St)@Fe3O4.

[0047] By observation Figure 6 From the separation results, it can be seen that the magnetic aggregate microspheres can produce differentiated adsorption on different types of protein targets, which proves the feasibility of using such materials for the separation and detection of such macromolecules.

Claims

1. Preparation and application of magnetic polymer microspheres functionalized with surface carboxyl groups, characterized in that: The preparation process comprises: (1) The dispersant and the deep eutectic solvent are mixed and dissolved in an ethanol-water mixture to form a clear system, which is then poured into a three-necked flask equipped with a thermometer and a mechanical stirring device, and then Fe3O4 nanoparticles are added and stirred under closed and nitrogen protection conditions; (2) dissolving the initiator in the polymerization monomer and pouring it into the reaction solution in (1), heating it and continuously stirring it under nitrogen protection to react, and cooling the reaction system after completion; (3) After the reaction liquid in (2) is completely cooled, magnetic separation and screening are performed using a nepheline iron boron magnet to obtain magnetic polymer microspheres with basically uniform particle sizes; after washing with anhydrous ethanol and ultrapure water alternately for three times, the microspheres are dispersed with ultrapure water for later use.

2. The preparation and application of a surface carboxyl functionalized magnetic polymer microsphere according to claim 1, characterized in that: The low eutectic solvent in step (1) is choline chloride-acrylic acid (molar ratio 1:2) or choline chloride-itaconic acid (molar ratio 1:1).

3. The preparation and application of a surface carboxyl functionalized magnetic polymer microsphere according to claim 1, characterized in that: The dispersant in step (1) is polyvinyl pyrrolidone, and the mass ratio of the dispersant to the low eutectic solvent is 1:2 to 2:

1.

4. The preparation and application of a surface carboxyl functionalized magnetic polymer microsphere according to claim 1, characterized in that: The stirring time of step (1) is 20 to 40 minutes.

5. The preparation and application of a surface carboxyl functionalized magnetic polymer microsphere according to claim 1, characterized in that: In step (1), the mass ratio of the Fe3O4 nanoparticles to the low eutectic solvent is 1:6 to 1:

3.

6. The preparation and application of a surface carboxyl functionalized magnetic polymer microsphere according to claim 1, characterized in that: The initiator in step (2) is azobisisobutyronitrile, and the mass ratio of azobisisobutyronitrile to the low eutectic solvent is 1:

5.

7. The preparation and application of a surface carboxyl functionalized magnetic polymer microsphere according to claim 1, characterized in that: The polymerization monomer in step (2) is styrene, and the mass ratio of styrene to the low eutectic solvent is 7:0.5-4.

8. The preparation and application of a surface carboxyl functionalized magnetic polymer microsphere according to claim 1, characterized in that: The reaction temperature of step (2) is 60-80° C. and the reaction time is 8-12 h.

9. The preparation and application of a surface carboxyl functionalized magnetic polymer microsphere according to claim 1, characterized in that: The prepared magnetic polymer microspheres can be used in the field of separation and detection of biological macromolecules represented by proteins.

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