CN118085345A - Preparation method and application of monodisperse magnetic polymer nanoparticles - Google Patents

Preparation method and application of monodisperse magnetic polymer nanoparticles Download PDF

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CN118085345A
CN118085345A CN202311808491.8A CN202311808491A CN118085345A CN 118085345 A CN118085345 A CN 118085345A CN 202311808491 A CN202311808491 A CN 202311808491A CN 118085345 A CN118085345 A CN 118085345A
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magnetic
polymer nanoparticles
solution
magnetic metal
carboxyl
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袁明龙
董瑛
贺正国
岳越
袁明虎
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Sichuan Zuoxin Biomaterial Research Co ltd
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Sichuan Zuoxin Biomaterial Research Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/12—Powdering or granulating
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F212/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by an aromatic carbocyclic ring
    • C08F212/02—Monomers containing only one unsaturated aliphatic radical
    • C08F212/04—Monomers containing only one unsaturated aliphatic radical containing one ring
    • C08F212/06—Hydrocarbons
    • C08F212/08—Styrene
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2325/00—Characterised by the use of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by an aromatic carbocyclic ring; Derivatives of such polymers
    • C08J2325/02—Homopolymers or copolymers of hydrocarbons
    • C08J2325/04—Homopolymers or copolymers of styrene
    • C08J2325/08—Copolymers of styrene
    • C08J2325/14—Copolymers of styrene with unsaturated esters
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
    • C08K3/00—Use of inorganic substances as compounding ingredients
    • C08K3/18—Oxygen-containing compounds, e.g. metal carbonyls
    • C08K3/20—Oxides; Hydroxides
    • C08K3/22—Oxides; Hydroxides of metals
    • C08K2003/2265—Oxides; Hydroxides of metals of iron
    • C08K2003/2275—Ferroso-ferric oxide (Fe3O4)
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
    • C08K2201/00—Specific properties of additives
    • C08K2201/01—Magnetic additives

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Abstract

The present disclosure provides a method for preparing magnetic polymer nanoparticles, which comprises preparing carboxyl microspheres by a dispersion polymerization method, and then preparing monodisperse carboxyl magnetic microspheres by a chemical precipitation method. The preparation method provided by the disclosure is simple and easy to implement, and the prepared magnetic microbeads have uniform particle size and high magnetic responsiveness.

Description

Preparation method and application of monodisperse magnetic polymer nanoparticles
Technical Field
The invention relates to the field of materials, in particular to a preparation method and application of monodisperse magnetic polymer nanoparticles.
Background
The magnetic microsphere is a composite microsphere formed by combining inorganic magnetic particles and organic high molecular polymers. The organic high molecular polymer must allow further modification to attach various ligands and must be stable in defined media. Carboxyl groups are one of the most important surface functional groups of particles for biomedical or biological diagnostic applications. The carboxyl polymer microsphere has the interesting properties of larger specific surface area, stronger adsorption capacity and the like, has very high reactivity with various ligands, particularly biomolecules, and can improve the colloid stability of microsphere dispersion by surface modification with carboxyl to prevent particle aggregation, thus being widely applied. Currently, the main methods for preparing polymeric microspheres are suspension polymerization, emulsion polymerization, dispersion polymerization and seed swelling polymerization. Dispersion polymerization is a relatively new method for preparing monodisperse polymer microspheres with a particle size of 0.5-8.0 μm. And has become the main method for synthesizing carboxyl microsphere. In particular example applications, carrier materials with room temperature superparamagnetism are preferred. This is because the magnetic beads can be easily separated and recovered by an external magnetic device, and the magnetic beads can be easily redispersed by simple shaking after the external magnetic field is removed, the nano Fe 3O4 can be used for magnetic resonance imaging contrast enhancement, catalysis, magnetic separation, magnetic fluid, controllable drug delivery, biosensor, medical diagnosis, and the like due to its low toxicity, inherent biocompatibility, high saturation magnetization, and room temperature superparamagnetism at critical dimensions. The technology for preparing nano Fe 3O4 has a plurality of technologies, such as a microemulsion method, a high-temperature pyrolysis method and a solvothermal method, in particular a chemical coprecipitation method, and is increasingly favored by people due to the advantages of simple operation, low equipment cost, good product magnetism, easy mass production and the like. Magnetic nanoparticles (Fe 3O4 and gamma-Fe 2O3) can be coated into polymers by methods such as emulsion polymerization, soap-free emulsion polymerization, inverse emulsion polymerization, miniemulsion polymerization, suspension polymerization and the like to prepare magnetic polymer composite particles. Suspension polymerization is considered to be one of the most advantageous methods for preparing medium diameter, high saturation magnetization magnetic polymer composite particles. However, the obtained magnetic polymer composite particles have irregular morphology and wide particle size distribution. Preparing microspheres with regular spheres, controlled surface carboxyl content and magnetic content remains challenging.
Disclosure of Invention
The invention aims to provide a preparation method and application of magnetic polymer nano particles.
In one aspect of the present invention, there is provided a method of preparing magnetic polymer nanoparticles, the method comprising: (1) Combining a solution comprising a magnetic metal source with a solution comprising carboxyl polymer nanoparticles to obtain a mixed solution, (2) adding a precipitant to the mixed solution to form the magnetic polymer nanoparticles.
In some embodiments, the magnetic metal source is selected from the group consisting of a hydrochloride salt of a magnetic metal, a nitrate salt of a magnetic metal, a sulfate salt of a magnetic metal, an oxalate salt of a magnetic metal, or any combination thereof. In some embodiments, the magnetic metal source is an iron salt, such as an iron hydrochloride salt. In some embodiments, the magnetic metal source comprises a trivalent iron salt and a divalent iron salt, preferably the magnetic metal source has a molar ratio of Fe 3+ to Fe 2+ selected from 1.5 to 2.5, e.g., a molar ratio of Fe 3+ to Fe 2+ of 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, or any value therebetween.
In some embodiments, the solution comprising the magnetic metal source is an aqueous solution and/or the solution comprising the carboxyl polymer nanoparticles is an aqueous solution.
In some embodiments, the precipitant is selected from sodium hydroxide, ammonia, sodium carbonate, and sodium acetate. In some embodiments, the precipitating agent is selected from sodium hydroxide. In some embodiments, the precipitant is selected from aqueous ammonia. In some embodiments, the amount of the precipitant is in excess relative to the amount of the magnetic metal source. In the present disclosure, an excess of the amount of the precipitant relative to the amount of the magnetic metal source means that the molar amount of the precipitant is at least 3 times, such as at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, or even at least 20 times the molar amount of the magnetic metal source (e.g., the total molar amount of Fe 3+ and Fe 2+).
In some embodiments, step (2) is performed at a temperature of 40-80 ℃, for example at a temperature selected from 40, 45, 50, 55, 60, 65, 70, 75, 80 ℃ or any value therebetween.
In some embodiments, both step (1) and step (2) are performed under the protection of an inert gas (e.g., nitrogen or helium).
In some embodiments, the carboxypolymer nanoparticle is a carboxypolystyrene-methyl methacrylate copolymer. In some embodiments, the carboxy polymer nanoparticle is prepared by a dispersion polymerization process comprising the steps of:
1-1) dissolving Azobisisobutyronitrile (AIBN) and polyvinylpyrrolidone (PVP, such as PVP-K30) in an aqueous alcohol solution to obtain a first mixed solution;
1-2) placing the first mixed solution in a reaction system, and adding Methyl Methacrylate (MMA) and styrene (St) under the protection of inert gas to obtain a second mixed solution;
1-3) heating the second mixed solution to a prepolymerization temperature for prepolymerization for a certain time, and then heating to the polymerization temperature for polymerization for a certain time to obtain a polystyrene-methyl methacrylate copolymer;
1-4) contacting the polystyrene-methyl methacrylate copolymer with a base selected from sodium hydroxide or potassium hydroxide to perform hydrolysis reaction, so as to prepare the carboxyl polymer nano particles.
In some embodiments, the aqueous alcohol solution is a mixed solution of anhydrous alcohol and water, or a mixed solution of anhydrous alcohol and water. In some embodiments, the mass ratio of alcohol to water in the aqueous alcohol solution is selected from 5:0 to 3:2; preferably 4:1.
In some embodiments, the prepolymerization temperature is selected from 30 to 70 ℃, e.g. 40 to 60 ℃, e.g. 30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70℃ or any value in between. In some embodiments, the time of the prepolymerization may be 20 to 60min, e.g. 30 to 40min, e.g. 20, 25, 30, 35, 40, 45, 50, 55, 60min or any value in between.
In some embodiments, the polymerization temperature is selected from 50 to 100 ℃, such as 60 to 80 ℃, such as 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 ℃, or any value therebetween. In some embodiments, the time of the pre-polymerization may be 18 to 30 hours, such as 20 to 28 hours, such as 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30 hours or any value therebetween.
In some embodiments, the dispersion polymerization process further comprises steps 1-5) of purifying the polystyrene-methyl methacrylate copolymer after steps 1-4), such as washing the polystyrene-methyl methacrylate copolymer with absolute ethanol, absolute methanol, water, or any combination thereof.
In some embodiments, the hydrolysis reaction is carried out at a temperature selected from 50 to 100 ℃, for example at a temperature selected from 60 to 80 ℃, for example 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 ℃, or any value therebetween.
In some embodiments, the carboxyl polymer nanoparticles produced by the dispersion polymerization process are monodisperse carboxyl polymer nanoparticles.
In some embodiments, the monodisperse carboxyl magnetic beads have a particle size of about 0.8 to 10 μm, e.g., about 0.8 to 6 μm, about 1 to 5 μm, or about 1 to 4 μm, e.g., about 0.8, 1, 1.2, 1.5, 1.8, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10 μm, or any value therebetween, preferably, have a particle size of about 2 to 3 μm.
In some embodiments, the dispersion polymerization process is conducted under the protection of an inert gas (e.g., nitrogen or helium).
In some embodiments, the mass ratio of PVP to MMA is selected from 20% to 60%. In some preferred embodiments, the mass ratio of PVP to MMA is selected from 30% to 50%. In still other more preferred embodiments, the mass ratio of PVP to MMA is selected from 35% to 45%.
In another aspect of the present disclosure, there is provided a monodisperse carboxyl magnetic bead prepared using the method described in the present disclosure.
In yet another aspect of the disclosure, there is provided the use of the monodisperse carboxyl magnetic microbeads for the extraction, purification or detection of target molecules in a sample. For example, the monodisperse carboxyl magnetic microbeads are used for cell sorting by covalent coupling with target molecules in a sample, or for concentration determination of target proteins to be detected in a sample, or for affinity chromatography, immunoassay of target molecules such as polypeptides to be detected in a sample, or for extraction and purification of oligonucleotides to be detected in a sample.
Advantageous effects
The monodisperse carboxyl magnetic microbeads prepared by the invention have regular spheres, rough surfaces, uniform particle size and no adhesion agglomeration.
The monodisperse carboxyl magnetic microbeads prepared by the invention have controllable surface carboxyl content, can meet the requirements under different detection conditions, and are convenient to apply.
The monodisperse carboxyl magnetic microbeads prepared by the method have controllable magnetic content, and the particle size of the magnetic microbeads can be adjusted through the mass ratio of alcohol to water, so that different detection requirements are met.
The method is simple and easy to implement, firstly synthesizes hydrolyzed polystyrene-methyl methacrylate polymer microspheres, ensures the uniformity of the magnetic microbeads, uses methyl methacrylate as a functionalizing agent to introduce carboxyl more easily, also facilitates subsequent coating of Fe 3O4,, prepares the magnetic microbeads by using the carboxyl microbeads as templates through a classical thermal precipitation method, and has good effect of coating Fe 3O4, and the prepared magnetic microbeads have uniform particle size and good monodispersity.
The magnetic microbeads prepared by the invention have detection effect similar to JSR.
Drawings
FIG. 1 shows electron microscopy scans of carboxyl microspheres prepared at different solvent alcohol to water ratios (5:0 (a), 4:1 (b), 3:2 (c)) in example 1.
FIG. 2 shows the particle size distribution results of carboxyl microspheres prepared at different solvent alcohol-water ratios (5:0 (a), 4:1 (b), 3:2 (c)) in example 1.
FIG. 3 shows electron microscopy scans of carboxyl microspheres prepared at different PVP-K30 to MMA mass ratios (20.8% (a), 41.6% (b), 62.4% (c)) in example 2.
FIG. 4 shows the particle size distribution results of carboxyl microspheres prepared at different PVP-K30 to MMA mass ratios (20.8% (a), 41.6% (b), 62.4% (c)) in example 2.
FIG. 5 shows electron microscopy scans of carboxyl microspheres prepared at different St to MMA ratios (3:2 (a), 1:1 (b), 1:3 (c)) in example 3.
FIG. 6 shows the particle size distribution results of carboxyl microspheres prepared at different St to MMA ratios (3:2 (a), 1:1 (b), 1:3 (c)) in example 3.
Fig. 7 shows electron microscopy scans of monodisperse carboxyl microspheres (polystyrene-methyl methacrylate microspheres) prepared in example 4 at different viewing angles.
Fig. 8 shows an electron microscope scan of monodisperse carboxyl magnetic microbeads prepared in example 4 at different magnifications.
Fig. 9 shows a graph comparing the effect of detecting protein concentration of monodisperse carboxyl magnetic beads prepared according to example 4 with JSR magnetic beads.
Fig. 10 shows a graph comparing the effect of monodisperse carboxyl magnetic beads prepared according to another embodiment of the present disclosure on detecting protein concentration with JSR magnetic beads.
Fig. 11 shows a graph comparing the effect of monodisperse carboxyl magnetic beads prepared according to another embodiment of the present disclosure on detecting protein concentration with JSR magnetic beads.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. The specific embodiments described herein are for purposes of illustration only and are not to be construed as limiting the invention in any way. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the concepts of the present disclosure. Such structures and techniques are also described in a number of publications.
The invention also provides application of the monodisperse carboxyl magnetic microbeads in detecting the concentration of proteins in blood, in particular to the application of the carboxyl magnetic microbeads as a separation tool in the detection process to combine with antibodies to form a magnetic bead-antibody complex. Homemade magnetic microbeads and Japanese imported magnetic beads (JSR) were used to detect the protein content in serum, and the results were compared, and the serum sample size was 30 cases. Detecting the separation effect of the magnetic microbeads by using a full-automatic chemiluminescence immunoassay analyzer with the model of shine i2900, wherein the concentration of the magnetic microbeads for detection is 100mg/ml, and the pH value is adjusted to 7-8.
The two magnetic microbeads are respectively connected with specific antibodies by a double-antibody sandwich method, and the content of antigen in serum is qualitatively determined under the participation of a detection kit.
The detection steps are all executed strictly according to the operation steps of the full-automatic chemiluminescence immunoassay analyzer with the model of shine i2900, and comprise setting parameters, loading sequence, loading quantity and the like.
Definition of the definition
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly used in the art to which this invention belongs. For the purposes of explaining the present specification, the following definitions will apply, and terms used in the singular will also include the plural and vice versa, as appropriate.
The terms "a" and "an" as used herein include plural referents unless the context clearly dictates otherwise. For example, reference to "a cell" includes a plurality of such cells, equivalents thereof known to those skilled in the art, and so forth.
The term "about" as used herein means a range of + -20% of the numerical values thereafter. In some embodiments, the term "about" means a range of ±10% of the numerical value following that. In some embodiments, the term "about" means a range of ±5% of the numerical value following that.
The term "monodisperse" particles as used herein refers to a dispersion system in which the size, shape, and chemical composition of the particles are substantially completely uniform. "monodispersity" is an important criterion for measuring the quality of nanoparticles. However, the absolute monodisperse particle population is not available because of the complex and harsh processes and conditions for monodisperse particle preparation. Generally monodisperse refers to a population of particles that are uniform in shape and chemical composition and have a very narrow particle size distribution, and may be, for example, monodisperse particles having a size distribution within + -10% of the stated value, for example, 1 μm in diameter, covering a size range from 0.9 μm to 1.1 μm.
In the present disclosure, the term "nanoparticle" has the same meaning as "microbead" or "microsphere" and generally refers to a sample of nano-, sub-micro-, micro-particles having a particle size in the range of 800nm to 10 μm.
Examples and figures are provided below to aid in the understanding of the invention. It is to be understood that these examples and drawings are for illustrative purposes only and are not to be construed as limiting the invention in any way. The actual scope of the invention is set forth in the following claims. It will be understood that any modifications and variations may be made without departing from the spirit of the invention.
Example 1:
Monodisperse carboxyl P (St-MMA) microspheres were prepared in this example at different solvent alcohol to water ratios to select the optimum alcohol to water ratio.
1) Preparation of P (St-MMA) microspheres:
6g PVP-K30 and 0.75g AIBN are weighed and respectively dissolved in mixed solutions (total mass is 300 g) with the mass ratio of alcohol to water of 5:0, 4:1 and 3:2, 14.42g methyl methacrylate is added, 15g styrene (St) is added under the protection of nitrogen after uniform stirring, polymerization is pre-initiated for 30min at 50 ℃, and polymerization is initiated for 24h at 70 ℃. After the reaction is finished, cooling to room temperature, centrifuging at 5000r/min for 3min, pouring out supernatant, washing the precipitate with absolute ethyl alcohol and water for 3 times respectively, obtaining a preliminary product at 5000r/min for 3min, and carrying out the whole reaction process under the protection of nitrogen.
2) Preparation of carboxyl P (St-MMA) microspheres:
Dissolving the obtained product in 250g of pure water, adding 5.12g of sodium hydroxide particles, heating to 70 ℃, reacting overnight, purifying the microspheres with pure water under the centrifugal conditions of 5000r/min and 3min, washing to neutrality, and drying in vacuum at 35 ℃ for standby.
The scanning result of an electron microscope of the prepared monodisperse carboxyl microsphere is shown in figure 1, and the particle size distribution result of the microsphere is shown in figure 2. The result shows that the microsphere prepared under the alcohol-water ratio of 4:1 has regular sphere, uniform particle size of about 2-3 mu m and no adhesion aggregation.
Example 2:
Monodisperse carboxyl P (St-MMA) microspheres were prepared at different PVP-K30/MMA ratios in this example to select the most appropriate PVP-K30/MMA ratio.
3G, 6g, 9g PVP-K30 and 0.75g AIBN are respectively weighed and dissolved in a mixed solution (total mass is 300 g) with an alcohol-water ratio of 4:1, 14.42g MMA is added, after stirring uniformly, 15g styrene is added under the protection of nitrogen, polymerization is pre-initiated for 30min at 50 ℃, and then polymerization is initiated for 24h at 70 ℃. The remaining steps were the same as in example 1.
The scanning result of an electron microscope of the prepared monodisperse carboxyl microsphere is shown in figure 3, and the particle size distribution result of the microsphere is shown in figure 4. The results showed that microspheres prepared from 6g PVP-K30 and 14.42g MMA (i.e. PVP-K30/MMA (wt.%) =41.6%) had regular spheres with a uniform particle size of about 2-3 μm and were free from blocking agglomerates.
Example 3:
Monodisperse carboxyl P (St-MMA) microspheres were prepared in this example at different molar ratios of St to MMA to select the most appropriate molar ratio of St to MMA.
9G PVP-K30 and 0.75g AIBN are weighed and dissolved in a mixed solution (total mass is 300 g) with an alcohol-water ratio of 4:1, nitrogen is replaced for 3 times, a reaction system is filled with nitrogen, st and MMA (St: MMA=3:2, 1:1 and 1:3) with different molar ratios are added, a uniform solution is formed at room temperature, the solution is placed in a water bath kettle with 50 ℃ and mechanically stirred for 30min at a speed of 200rpm, and then the temperature is raised to 70 ℃ for reaction for 24h. The remaining steps were the same as in example 1.
The scanning result of an electron microscope of the prepared monodisperse carboxyl microsphere is shown in figure 5, and the particle size distribution result of the microsphere is shown in figure 6. The result shows that the prepared microsphere has regular spherical shape and uniform particle size of about 2-3 mu m, and is not adhered to the aggregation.
Example 4:
In this example, the magnetic microbeads were prepared using the optimal ratios of the raw materials in examples 1 to 3, respectively.
1) Monodisperse carboxyl P (St-MMA) microspheres were prepared.
1-1) Preparation of P (St-MMA) microspheres: 6g PVP-K30 was weighed out, and dissolved in a mixed solution of 0.75g AIBN, 240g absolute ethanol and 60g pure water, and 14.42g MMA and 15g St were added under nitrogen protection, and the rotational speed was adjusted to 200r/min, and polymerization was pre-initiated at 50℃for 30min, followed by initiation of polymerization at 70℃for 24 hours. After the reaction is finished, cooling to room temperature, centrifuging at 5000r/min for 3min, pouring out supernatant, washing the precipitate with absolute ethyl alcohol and water for 3 times respectively, and centrifuging at 5000r/min for 3min again to obtain a primary product. The whole reaction process is carried out under the protection of nitrogen.
1-2) Preparation of carboxyl P (St-MMA) microspheres. Dissolving the obtained product in 250g of pure water, adding 5.12g of sodium hydroxide particles, heating to 70 ℃, reacting overnight, purifying the microspheres with pure water under the centrifugal conditions of 5000r/min and 3min, washing to neutrality, and drying in vacuum at 35 ℃ for standby.
2) Preparing monodisperse carboxyl magnetic microbeads.
2-1) 0.086G of Fe 2+ salt and 0.235g of Fe 3+ salt are weighed in a molar ratio of 1:2 and dissolved in 10ml of pure water, and then 1ml of hydrochloric acid is added and the solution is completely dissolved by ultrasound.
2-2) 1G of the carboxyl microsphere prepared in 1-2) above was weighed and dispersed in 200ml of pure water, and the temperature was raised to 80 ℃.
2-3) Rapidly adding the aqueous solution of 2-1) into 2-2), stirring uniformly, and immediately adding excessive ammonia water solution to perform classical heat precipitation reaction to form magnetic microbeads.
2-4) The above processes are all carried out under nitrogen protection.
2-5) After the reaction is finished, cooling to room temperature, separating the magnetic microbeads by magnetic precipitation, washing the precipitate with 200ml of pure water, and repeating for 3-5 times to obtain the pure monodisperse carboxyl magnetic microbeads. The magnetic beads obtained were regular in shape and uniform in size as observed under a scanning electron microscope (fig. 7 and 8).
Example 5:
In this example, the effect of the monodisperse carboxyl magnetic beads obtained in example 4 on protein separation was measured.
The magnetic beads prepared in example 4 were combined with IL-6 antibody, and the effect of protein isolation was detected by using a detection kit.
The JSR magnetic microbeads are combined with IL-6 antibody, and the effect of separating protein is detected by a detection kit.
And comparing the detection results, wherein the correlation coefficient R 2 of the two detection results is 0.9755. As shown in fig. 9.
Example 6:
In this example, the protein separation effect of magnetic microbeads having different magnetic contents was measured.
Magnetic microbeads were prepared under otherwise identical conditions by substituting 0.086g of Fe 2+ salt and 0.235g of Fe 3+ salt in step 2-1) of example 4 with 0.172g of Fe 2+ salt and 0.468g of Fe 3+ salt.
And combining the prepared magnetic microbeads with IL-6 antibodies, and detecting the effect of separating proteins by using a detection kit.
The JSR magnetic microbeads are combined with IL-6 antibody, and the effect of separating protein is detected by a detection kit.
Compared with the magnetic microbeads prepared in example 5, the magnetic microbeads prepared in this example have a protein separation effect closer to that of JSR magnetic beads and a better separation effect, as shown in fig. 10, with a correlation coefficient R 2 of 0.9925.
Example 7:
In this example, the protein separation effect of magnetic microbeads having different magnetic contents was measured.
Magnetic beads were prepared under the same conditions except that 1g of the carboxyl microsphere used in step 2-2) of example 5 was changed to 3 g.
And combining the prepared magnetic microbeads with IL-6 antibodies, and detecting the effect of separating proteins by using a detection kit.
The JSR magnetic microbeads are combined with IL-6 antibody, and the effect of separating protein is detected by a detection kit.
In contrast to the above detection results, the correlation coefficient R 2 of the two detection results is 0.9933, as shown in fig. 11. Compared with the magnetic beads prepared in examples 5 and 6, the magnetic beads prepared in the example have a separation effect on proteins, which is closer to that of JSR magnetic beads and better.
The technical scheme of the invention is not limited to the specific embodiment, and all technical modifications made according to the technical scheme of the invention fall within the protection scope of the invention.

Claims (13)

1. A method of preparing magnetic polymer nanoparticles, the method comprising:
1) Combining a solution containing a magnetic metal source with a solution containing carboxyl polymer nanoparticles to obtain a mixed solution;
2) Adding a precipitant to the mixed solution to form the magnetic polymer nanoparticles.
2. The method of claim 1, wherein the magnetic metal source is selected from the group consisting of a hydrochloride salt of a magnetic metal, a nitrate salt of a magnetic metal, a sulfate salt of a magnetic metal, an oxalate salt of a magnetic metal, or any combination thereof.
3. The method of claim 1, wherein the magnetic metal source is an iron salt, more preferably a mixture of a trivalent iron salt and a divalent iron salt, further preferably wherein the molar ratio of Fe 3+ to Fe 2+ in the magnetic metal source is selected from 1.5 to 2.5, preferably wherein the molar ratio of Fe 3+ to Fe 2+ is 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5 or any value therebetween.
4. The method of preparation according to claim 1, characterized in that the solution comprising the magnetic metal source is an aqueous solution and/or the solution comprising the carboxylic polymer nanoparticles is an aqueous solution.
5. The method of claim 1, wherein the precipitant is selected from the group consisting of sodium hydroxide, ammonia, sodium carbonate, and sodium acetate.
6. The method according to claim 1, wherein the step (2) is performed at a temperature of 40 to 80 ℃.
7. The method of claim 1, wherein both the step (1) and the step (2) are performed under the protection of an inert gas.
8. The method of claim 1, wherein the carboxypolymer nanoparticle is a carboxypolystyrene-methyl methacrylate copolymer.
9. The preparation method according to claim 1, wherein the carboxyl polymer nanoparticles are prepared by a dispersion polymerization method comprising the steps of:
1-1) dissolving Azobisisobutyronitrile (AIBN) and polyvinylpyrrolidone (PVP) in an aqueous alcohol solution to obtain a first mixed solution;
1-2) placing the first mixed solution in a reaction system, and adding Methyl Methacrylate (MMA) and styrene (St) under the protection of inert gas to obtain a second mixed solution;
1-3) heating the second mixed solution to a prepolymerization temperature for prepolymerization for a certain time, and then heating to the polymerization temperature for polymerization for a certain time to obtain a polystyrene-methyl methacrylate copolymer;
1-4) contacting the polystyrene-methyl methacrylate copolymer with a base selected from sodium hydroxide or potassium hydroxide to perform a hydrolysis reaction, thereby preparing the carboxyl polymer nanoparticles;
Specifically, the dispersion polymerization method is carried out under the protection of inert gas;
Specifically, the PVP/MMA mass ratio is selected from 20% -60%, preferably from 30% -50%;
Specifically, the aqueous alcohol solution is absolute ethyl alcohol or a mixed solution of absolute methyl alcohol and water, and preferably, the mass ratio of alcohol to water in the aqueous alcohol solution is selected from 5:0-3:2;
Specifically, the prepolymerization temperature is selected from 30 to 70 ℃, preferably 40 to 60 ℃;
Specifically, the pre-polymerization time is 20 to 60min, preferably 30 to 40min;
Specifically, the polymerization temperature is selected from 50 to 100 ℃, preferably 60 to 80 ℃;
specifically, the time for the prepolymerization is 18 to 30 hours, preferably 20 to 28 hours.
10. The method of claim 9, wherein the dispersion polymerization method further comprises steps 1-5) of purifying the polystyrene-methyl methacrylate copolymer after steps 1-4).
11. The preparation method according to claim 9, characterized in that the hydrolysis reaction is carried out at a temperature selected from 50-100 ℃, preferably at a temperature selected from 60-80 ℃, more preferably at a temperature of 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 ℃ or any value in between.
12. Monodisperse magnetic polymer nanoparticles obtainable by the preparation process according to any of claims 1 to 11.
13. Use of monodisperse magnetic polymer nanoparticles according to claim 12 for the extraction, purification or detection of target molecules in a sample, preferably the target molecules are polypeptides or oligonucleotides which specifically bind to the monodisperse magnetic polymer nanoparticles.
CN202311808491.8A 2023-12-25 2023-12-25 Preparation method and application of monodisperse magnetic polymer nanoparticles Pending CN118085345A (en)

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