CN115181215A - Preparation method of immune micron magnetic beads with uniform particle size - Google Patents
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Abstract
The invention discloses a preparation method of immune micron magnetic beads with uniform particle size; comprises the steps of preparing polymer microspheres with uniform particle size in green solvent water by using a dispersion polymerization method by using polymer monomers as reaction monomers, polyethylene glycol, pluronic PE10100, polyvinyl alcohol and Tween as dispersing agents and dibenzoyl peroxide and ammonium persulfate as initiators; and then introducing magnetic ferroferric oxide particles into the polymer microspheres through chemical coprecipitation of iron ions in an alkaline solution, and introducing functional groups through surface graft polymerization to obtain the immune micron magnetic beads for immunodiagnosis, instant detection, gene sequencing and the like. The whole operation process is carried out in aqueous solution, and no organic solvent is involved, so that the preparation method is green and environment-friendly; the polymer microsphere is obtained by a dispersion polymerization one-step method, and compared with the traditional method for preparing the microsphere by seed swelling, the method is simpler and quicker, and the process is greatly shortened.
Description
Technical Field
The invention relates to the technical field of biological materials, relates to a preparation method of immune micron magnetic beads, and particularly relates to a preparation method of immune micron magnetic beads with uniform particle sizes.
Background
The immune micron magnetic bead is a kind of even spherical small particle with superparamagnetism and protective shell, and is formed by combining carrier microsphere and immune aglucon. The immune ligand generally comprises antigen, antibody or agglutinin, etc., the ligand has biological specificity, and the combination of the carrier microsphere and the ligand does not influence or change the original biological characteristics of the ligand, thereby ensuring the special recognition function of the microsphere. The immunomagnetic separation technology is that the high specificity recognition function of antigen and antibody is utilized to fix the antibody (or antigen) on the surface of immunomagnetic microsphere (IMMS) to form solid phase antibody/antigen complex, the complex is retained after the action of external magnetic field, and the magnetic microsphere carries the antigen/antibody complex to be separated from other components, thereby achieving the separation purpose. The magnetic microsphere replaces other solid phase carriers for immune separation, is simple and easy to implement, has high specificity and low loss, and can combine immune separation and enrichment into a whole, so that the magnetic microsphere has wide research and application prospects in the fields of medicine, biological separation, food hygiene detection, environmental detection and the like.
At present, the immunomagnetic beads on the market are mainly dynabeads of American Saimer fly and magbeads of Japanese JSR, but at present, other enterprises at home and abroad are difficult to prepare the immunomagnetic beads with the same uniform particle size, the main reason is that the process is realized by an oil-water emulsification method, and then the immunomagnetic beads are screened by a precise screening instrument, the emulsification method is to prepare oil-in-water microspheres in incompatible oil and water phases, then an initiator is heated and polymerized to generate the microspheres, and the particle size of the obtained microspheres is difficult to control and uniform particle size is difficult to obtain due to the incompatible two-phase solvent.
Patent document retrieval shows that patent CN 104492397A discloses a polymerization reaction combining dispersing agent sodium dodecyl benzene sulfonate and PEG4000, but polystyrene microsphere and magnetic bead with nonuniform particle size of 50-150 μm are obtained, and the definition and requirement of the immunomagnetic bead are uniform particle size, so the microsphere magnetic bead obtained by the method can not be used for immunomagnetic bead.
CN 108129614B and US4654267A emulsify in oil-water two-phase solvent by a seed swelling method, the process is complicated, seed materials need to be synthesized first, then the polymer microspheres can be obtained by an oil-in-water emulsification method, a large amount of oily solvent needs to be used in the process to participate in emulsification, and the later-stage product needs to be cleaned for a long time to remove the oily solvent. In addition, because the emulsification process is carried out, a two-phase solvent participates, the dispersion of the high molecular monomer in the two phases is not easy to control, and the dispersion is not uniform, so that the high molecular microspheres with different particle sizes are obtained frequently.
Disclosure of Invention
The invention aims to provide a preparation method of uniform-particle-size immunomagnetic beads aiming at the defects and shortcomings of the prior art. The invention relates to a method for preparing polymer microspheres with uniform particle size and immunomicromagnetic beads by carrying out dispersion polymerization reaction in a water phase and optimizing the components and the proportion of a dispersing agent.
The purpose of the invention is realized by the following technical scheme:
in a first aspect, the present invention relates to an immunomagnetic bead for immunodiagnosis, real-time detection and gene sequencing, wherein the immunomagnetic bead is a functional polymer layer with a coated shell, and the immunomagnetic bead comprises polymer microspheres and Fe dispersed in and on the surfaces of the polymer microspheres 3 O 4 Core-shell structures of nanoparticles.
Preferably, the nano magnetic beads are microspheres with uniform particle size of 1-3 microns.
Preferably, the polymer layer of the shell has various functional groups, and polymer microspheres with uniform particle size and inlaid ferroferric oxide nanoparticles are arranged inside the shell; the functional group includes carboxyl, amino, epoxy, hydroxyl, etc.
The preparation process of the immune micron magnetic beads is carried out in green solvent water, and the polymer microspheres are prepared by a simple and rapid dispersion polymerization one-step method.
In a second aspect, the invention relates to a method for preparing uniform-particle-size immunomagnetic beads, wherein the immunomagnetic beads comprise a functional polymer layer with a coated shell, a polymer microsphere with a core and Fe dispersed in and on the polymer microsphere 3 O 4 A core-shell structure of nanoparticles; the method comprises the following steps:
s1, preparing polymer microspheres with uniform particle sizes by a dispersion polymerization one-step method in the presence of a dispersing agent and an initiator by using water as a solvent and using a polymer monomer;
s2, introducing magnetic ferroferric oxide particles into the polymer microspheres through chemical coprecipitation of iron ions in an alkaline solution, and introducing functional groups through surface graft polymerization to obtain the immune micron magnetic beads with uniform particle sizes.
Preferably, the dispersant selection is selected from the group consisting of polyethylene glycol, pluronic PE10100, polyvinyl alcohol and tween.
Preferably, the mass ratio of the polyethylene glycol, the Pluronic PE10100, the polyvinyl alcohol and the Tween is (0.05-1): (0.01-1): (0.1-2): (0.01-1). The proportioning can fully disperse the monomer and the initiator in the water phase, thereby obtaining the polymer microspheres with uniform and different particle sizes.
Preferably, the polyethylene glycol has an average molecular weight of 300 to 20 000.
Preferably, the polyvinyl alcohol is 1799 type.
Preferably, the tween is tween 20 or tween 80. In one embodiment, the above materials can be selected from the products sold by Aladdin Biotechnology Ltd.
Preferably, the high molecular monomer is selected from one or more of styrene, methyl methacrylate, 2-hydroxyethyl methacrylate, methacrylic acid, acrylic acid and glycidyl methacrylate.
Preferably, the initiator is ammonium persulfate APS, dibenzoyl peroxide BPO, or Azobisisobutyronitrile (AIBN).
Preferably, the reaction temperature of the dispersion polymerization is 55 to 90 ℃ and the reaction time is 5 to 48 hours.
Preferably, each 0.1 to 2 milliliters of water corresponds to 0.1 to 1 gram of high molecular monomer, 0.05 to 1 gram of polyethylene glycol, 0.01 to 1 gram of Pluronic PE10100, 0.01 to 2 grams of polyvinyl alcohol, 0.01 to 1 gram of Tween and 0.00001 to 0.1 gram of initiator; more preferably from 0.0001 to 0.1 gram of initiator.
In some embodiments, step S1 specifically includes: adding high molecular monomer, polyethylene glycol (PEG), pluronic PE10100, polyvinyl alcohol 1799 and tween into water, fully dispersing, and heating and polymerizing under the action of initiator ammonium persulfate APS or dibenzoyl peroxide (BPO) to obtain the high molecular microsphere.
Preferably, the step S2 of introducing magnetic ferroferric oxide particles specifically comprises: ferrous chloride or ferrous sulfate and ferric chloride are used as raw materials, the polymer microspheres are added into a mixed aqueous solution of ferric ions, mixed and incubated, then ammonia water or sodium hydroxide is added for heating, and magnetic ferroferric oxide particles are introduced into the interior and exterior of the polymer microspheres through a coprecipitation method, so that micron magnetic beads with uniform particle sizes are obtained.
Preferably, fe is contained in the mixed aqueous solution of iron ions 3+ The ion molar concentration is 0.01 mol/L-10 mol/L, and the molar ratio of ferric ions to ferrous ions is 1.
Preferably, the mixture is incubated at 0-100 ℃ for 1-48 hours in a ratio of 0.1-1 g of the polymeric microspheres per 0.1-2 ml of the mixed aqueous solution of iron ions.
Preferably, ammonia or sodium hydroxide is added to adjust the pH of the mixed solution to 11 to 13.
Preferably, the coprecipitation is carried out at 25 to 90 ℃ for 1 to 24 hours.
Preferably, the functional groups introduced in step S2 are specifically: the polymer microspheres introduced with the magnetic ferroferric oxide particles are dispersed in water by ultrasonic, then functional monomers, cross-linking agents and initiators are added, and functional groups are introduced on the surfaces of the microspheres through heating graft polymerization.
Preferably, 0.1-1 g of polymer microsphere of magnetic ferroferric oxide particles, 0.1-1 g of functional monomer, 0.001-1 g of cross-linking agent and 0.00001-0.1 g of initiator are correspondingly introduced into every 0.1-2 ml of deionized water. More preferably 0.05 to 1 gram of crosslinking agent and 0.0005 to 0.1 gram of initiator.
Preferably, the functional monomer is 2-hydroxyethyl methacrylate, methacrylic acid, acrylic acid, glycidyl methacrylate or acrylamide.
Preferably, the crosslinking agent is ethylene glycol dimethacrylate EGDMA or methylene bisacrylamide.
Preferably, the initiator is ammonium persulfate APS, dibenzoyl peroxide BPO or azobisisobutyronitrile AIBN.
Preferably, the reaction temperature of the heating graft polymerization is 55 to 90 ℃, and the reaction time is 5 to 48 hours.
In some embodiments, step S2 comprises:
firstly, ferric chloride, ferrous chloride or ferrous sulfate is dissolved in water, and the molar ratio of ferric ions to ferrous ions is 1: 1, mixing and incubating at 0-100 ℃ for 1-48 hours at the ratio of 0.1-1 g of polymer microspheres to 0.1-1 g of iron ion solution per 0.1-2 ml, adding ammonia water or sodium hydroxide aqueous solution into the incubation solution to enable the pH value of the mixed solution to be 11-13, and reacting at 25-90 ℃ for 1-24 hours to obtain the immune micron magnetic beads with uniform particle size.
Then, still using water as a solvent, the obtained immune micron magnetic beads react with a functional monomer, a cross-linking agent and an initiator, namely, every 0.1-2 ml of deionized water corresponds to 0.1-1 g of the immune micron magnetic beads, 0.1-1 g of the functional monomer, 0.1-1 g of cross-linking agent Ethylene Glycol Dimethacrylate (EGDMA) and 0.001-0.1 g of initiator Ammonium Persulfate (APS) or dibenzoyl peroxide (BPO); the reaction temperature is 55-90 ℃, and the reaction time is 5-48 hours.
Compared with the prior art, the invention has the following beneficial effects:
1) The invention innovates a green, simple, rapid and novel method for preparing immune micro magnetic beads, namely, the method is only in dispersion polymerization in a green solvent water phase, the monomer is uniformly dispersed by adding a dispersant, and then an initiator is heated and polymerized. Because of the homogeneous dispersion polymerization, the preparation of the microspheres is mainly influenced by the dispersing agent, unlike emulsion polymerization which is not only influenced by the dispersing agent but also influenced by the solvent, so that the polymerization reaction is easier to control, and the obtained particle size is more uniform.
2) In addition, the method is the only method for preparing the macromolecular immunomagnetic beads by only adopting green solvent water in all the documents and patents at present, and avoids the toxicity of the solvent. The method is carried out in the water phase, so that the product is easy to clean and collect after all reactions are finished, a complex cleaning process is avoided, and the process period is greatly shortened, so that the method is very suitable for large-scale industrial production.
3) The problems that the immune micro magnetic beads can have nonspecific adsorption and unstable dispersion in a complex sample are solved. The invention utilizes a graft polymerization method to wrap a functional polymer layer on the surface of the immune micron magnetic bead, and then introduces various functional groups on the surface of the polymer layer, so that in an aqueous solution, the polymer layer greatly reduces the nonspecific adsorption of the immune micron magnetic bead, improves the efficiency and the carrying capacity of the immune micron magnetic bead for combining target molecules, also increases the dispersion stability of the immune micron magnetic bead in the aqueous solution, and increases the monodispersity of the immune micron magnetic bead in physiological systems such as blood, interstitial fluid and the like, thereby solving the problem of the stability of the immune micron magnetic bead in the physiological environment and laying a foundation for subsequent cell therapy and biological detection.
Drawings
Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:
FIG. 1 is a microscopic image of 1 micron carboxyl immunomicromagnetic beads with uniform particle size prepared by the present invention (eyepiece 10X, objective 50X);
FIG. 2 is a microscopic image of 1 micron amino immunomicromagnetic beads with uniform particle size prepared by the present invention (eyepiece 10X, objective 50X);
FIG. 3 is a microscopic image of 1 micron epoxy-based immunomicromagnetic beads of uniform particle size prepared by the present invention (eyepiece 10X, objective 50X);
FIG. 4 is a microscopic image of 2.8 μm carboxyl immunomicromagnetic beads with uniform particle size prepared by the present invention (eyepiece 10X, objective 50X);
FIG. 5 is a microscopic image of 2.7 micron amino immunomicromagnetic beads of uniform particle size prepared by the present invention (eyepiece 10X, objective 50X);
FIG. 6 is a microscopic image of 2.7 micron epoxy-based immunomicromagnetic beads of uniform particle size prepared by the present invention (eyepiece 10X, objective 50X);
FIG. 7 is a microscopic view of the carboxyl group microbeads prepared in comparative example 1;
FIG. 8 is a microscopic image of carboxyl microbeads prepared in comparative example 2;
FIG. 9 is a microscopic image of carboxyl microbeads prepared in comparative example 3;
FIG. 10 is a microscopic image of carboxyl microbeads prepared in comparative example 4;
FIG. 11 is a microscopic image of carboxyl microbeads prepared in comparative example 5;
FIG. 12 is a microscopic view of amino microbeads prepared in comparative example 6;
fig. 13 is a microscopic view of the amino microbead prepared in comparative example 7.
Detailed Description
The present invention will be described in detail below with reference to specific embodiments and the accompanying drawings. The following examples will aid those skilled in the art in further understanding the present invention, but are not intended to limit the invention in any manner. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.
Example 1
The embodiment relates to a green, simple, rapid and novel method for preparing immune micron magnetic beads, which comprises the following steps:
firstly, dispersing 5 g of styrene monomer, 2 g of methacrylic acid monomer, 1 g of polyethylene glycol (molecular weight 600) as a dispersant, 0.5 g of Pluronic PE10100, 2 g of polyvinyl alcohol 1799 type, 0.5 g of Tween 20 and 0.01 g of Ammonium Persulfate (APS) as an initiator in 50 ml of deionized water, reacting for 24 hours at 60 ℃, then centrifugally collecting, washing with deionized water for 5 times (100 ml each time), washing, centrifugally collecting, and drying in an oven at 50 ℃ to obtain 1-micron polystyrene/methacrylic acid microspheres with uniform particle size.
Secondly, inlaying ferroferric oxide nanoparticles into the polystyrene/methacrylic acid microspheres and on the surfaces of the polystyrene/methacrylic acid microspheres: dissolving 3 g of ferric chloride hexahydrate and 3 g of ferrous chloride tetrahydrate in 20 ml of water, mixing and incubating 2 g of 1-micron polystyrene/methacrylic acid microspheres at 4 ℃ for 2 hours, then adding ammonia water into the incubation solution to enable the pH value of the mixed solution to be 11, reacting at 50 ℃ for 16 hours, and centrifugally cleaning to remove non-inlaid ferroferric oxide nanoparticles to obtain immune micron magnetic beads with uniform particle size.
Thirdly, introducing carboxyl functional groups on the surface of the polystyrene/methacrylic acid microsphere by a graft polymerization method: polystyrene/methacrylic acid magnetic beads are dispersed in 20 ml of deionized water, and then 2 g of methacrylic acid, 1 g of cross-linking agent Ethylene Glycol Dimethacrylate (EGDMA) and 0.008 g of initiator Ammonium Persulfate (APS) are added to react at 70 ℃ for 8 hours to obtain 1-micron carboxyl immunomagnetic beads with uniform particle size and shown in figure 1.
Example 2
The embodiment relates to a green, simple, rapid and novel method for preparing immune micron magnetic beads, which comprises the following steps:
firstly, 6 g of styrene monomer, 3 g of methyl methacrylate monomer, 2 g of polyethylene glycol (molecular weight 300) as a dispersant, 0.8 g of Pluronic PE10100, 4g of polyvinyl alcohol 1799 type, 0.8 g of Tween 20 and 0.02 g of dibenzoyl peroxide (BPO) as an initiator are dispersed in 50 ml of deionized water, then the mixture is reacted for 24 hours at 60 ℃, and then the mixture is centrifugally collected, washed by deionized water for 5 times, 100 ml of the mixture is washed, centrifugally collected and dried in an oven at 50 ℃ to obtain 1 micron polystyrene/methyl methacrylate microspheres with uniform particle size.
Secondly, inlaying ferroferric oxide nanoparticles into the polystyrene/methyl methacrylate microspheres and on the surfaces of the polystyrene/methyl methacrylate microspheres: dissolving 3 g of ferric chloride hexahydrate and 3 g of ferrous chloride tetrahydrate in 20 ml of water, mixing and incubating 2 g of 1-micron polystyrene/methyl methacrylate microspheres at 60 ℃ for 2 hours, then adding ammonia water into the incubation solution to enable the pH value of the mixed solution to be 12, reacting at 80 ℃ for 3 hours, and centrifugally cleaning to remove non-inlaid ferroferric oxide nanoparticles to obtain immune micron magnetic beads with uniform particle size.
Thirdly, introducing amino functional groups on the surface of the polystyrene/methyl methacrylate microsphere by a graft polymerization method: polystyrene/methyl methacrylate magnetic beads are dispersed in 20 ml of deionized water, and then 2 g of functional monomer acrylamide, 0.5 g of cross-linking agent Ethylene Glycol Dimethacrylate (EGDMA) and 0.01 g of initiator Ammonium Persulfate (APS) are added to react for 24 hours at 70 ℃, so that 1 micron amino immunomagnetic beads with uniform particle size and shown in figure 2 are obtained.
Example 3
The embodiment relates to a green, simple, rapid and novel method for preparing immune micron magnetic beads, which comprises the following steps:
the immune micron magnetic beads in the examples 1 and 2 are introduced with epoxy functional groups on the surfaces of the magnetic beads through a graft polymerization method: dispersing the magnetic beads in 30 ml of deionized water, adding 3 g of functional monomer glycidyl methacrylate, 1 g of cross-linking agent Ethylene Glycol Dimethacrylate (EGDMA) and 0.02 g of dibenzoyl peroxide (BPO), and reacting at 70 ℃ for 12 hours to obtain 1-micron epoxy group immunomagnetic beads with uniform particle size and shown in figure 3.
Example 4
The embodiment relates to a green, simple, quick and novel method for preparing immune micron magnetic beads, which comprises the following steps:
firstly, dispersing 8 g of styrene monomer, 4g of acrylic acid monomer, 2 g of polyethylene glycol (molecular weight 1000) as a dispersant, 1 g of Pluronic PE10100, 4g of polyvinyl alcohol 1799 type, 1 g of Tween 20 and 0.5 g of Ammonium Persulfate (APS) as an initiator in 50 ml of deionized water, reacting for 24 hours at 70 ℃, then centrifugally collecting, washing with deionized water for 5 times, each time for 100 ml, washing, centrifugally collecting, and drying in a 50 ℃ oven to obtain the polystyrene/acrylic acid microspheres with uniform particle size of 2.7 microns.
Secondly, inlaying ferroferric oxide nano particles into the polystyrene/acrylic acid microspheres and on the surfaces of the polystyrene/acrylic acid microspheres: dissolving 3 g of ferric chloride hexahydrate and 3 g of ferrous chloride tetrahydrate in 20 ml of water, mixing and incubating 2 g of 2.7-micron polystyrene/acrylic acid microspheres at 10 ℃ for 5 hours, then adding ammonia water into the incubation solution to enable the pH value of the mixed solution to be 11, reacting at 50 ℃ for 24 hours, and centrifugally cleaning to remove non-inlaid ferroferric oxide nanoparticles to obtain immune micron magnetic beads with uniform particle size.
Thirdly, introducing carboxyl functional groups on the surfaces of the polystyrene/acrylic acid microspheres by a graft polymerization method: polystyrene/acrylic acid magnetic beads are dispersed in 20 ml of deionized water, and then 2 g of acrylic acid, 1 g of cross-linking agent Ethylene Glycol Dimethacrylate (EGDMA) and 0.01 g of initiator Ammonium Persulfate (APS) are added to react at 70 ℃ for 16 hours to obtain 2.8-micron carboxyl immunomagnetic beads with uniform particle size and shown in figure 4.
Example 5
The embodiment relates to a green, simple, rapid and novel method for preparing immune micron magnetic beads, which comprises the following steps:
firstly, dispersing 8 g of styrene monomer, 3 g of methyl methacrylate monomer, 2 g of polyethylene glycol (molecular weight 2000) as a dispersant, 0.8 g of Pluronic PE10100, 5 g of polyvinyl alcohol 1799 type, 0.8 g of Tween 80 and 0.05 g of dibenzoyl peroxide (BPO) as an initiator in 50 ml of deionized water, reacting for 24 hours at 70 ℃, centrifugally collecting, washing with deionized water for 5 times, washing with 100 ml each time, centrifugally collecting, and drying in an oven at 50 ℃ to obtain the polystyrene/methyl methacrylate microspheres with the particle size of 2.6 microns and uniform particle size.
Secondly, inlaying ferroferric oxide nano particles into the polystyrene/methyl methacrylate microspheres and on the surfaces of the microspheres: dissolving 3 g of ferric chloride hexahydrate and 3 g of ferrous chloride tetrahydrate in 20 ml of water, mixing and incubating 2 g of 2.6 micron polystyrene/methyl methacrylate microspheres at 80 ℃ for 1 hour, then adding ammonia water into the incubation solution to enable the pH value of the mixed solution to be 11, reacting for 8 hours at 60 ℃, and centrifugally cleaning to remove non-embedded ferroferric oxide nano-particles to obtain immune micron magnetic beads with uniform particle size.
Thirdly, introducing amino functional groups on the surface of the polystyrene/methyl methacrylate microsphere by a graft polymerization method: polystyrene/methyl methacrylate magnetic beads are dispersed in 20 ml of deionized water, and then 3 g of functional monomer acrylamide, 0.8 g of cross-linking agent methylene bisacrylamide and 0.02 g of initiator Ammonium Persulfate (APS) are added to react at 70 ℃ for 24 hours to obtain 2.7-micron amino immunomagnetic beads with uniform particle size and shown in figure 5.
Example 6
The embodiment relates to a green, simple, rapid and novel method for preparing immune micron magnetic beads, which comprises the following steps:
firstly, dispersing 10 g of a reactant of a styrene monomer, 4g of a methacrylic acid-2-hydroxyethyl ester monomer, 3 g of polyethylene glycol (molecular weight 10 000) as a dispersing agent, 1 g of Pluronic PE10100, 5 g of polyvinyl alcohol 1799 type, 1 g of Tween 80 and 0.05 g of dibenzoyl peroxide (BPO) as an initiator in 60 ml of deionized water, reacting for 24 hours at 80 ℃, centrifuging, collecting, washing with deionized water for 5 times, washing with 100 ml each time, centrifuging, collecting, and drying in an oven at 50 ℃ to obtain 2.6-micron polystyrene/methacrylic acid-2-hydroxyethyl ester microspheres with uniform particle size.
Secondly, inlaying ferroferric oxide nano particles into the polystyrene/methacrylic acid-2-hydroxyethyl ester microspheres and on the surfaces of the microspheres: dissolving 3 g of ferric chloride hexahydrate and 3 g of ferrous chloride tetrahydrate in 20 ml of water, mixing and incubating 2 g of 2.6 micron polystyrene/2-hydroxyethyl methacrylate microspheres at 80 ℃ for 2 hours, adding ammonia water into the incubation solution to enable the pH value of the mixed solution to be 12, reacting for 3 hours at 80 ℃, centrifugally cleaning to remove non-embedded ferroferric oxide nanoparticles, and obtaining immune micron magnetic beads with uniform particle size.
Thirdly, introducing epoxy functional groups to the surface of the polystyrene/2-hydroxyethyl methacrylate microspheres by a graft polymerization method: polystyrene/2-hydroxyethyl methacrylate magnetic beads are dispersed in 20 ml of deionized water, and then 2 g of functional monomer glycidyl methacrylate, 0.5 g of cross-linking agent Ethylene Glycol Dimethacrylate (EGDMA) and 0.01 g of initiator Ammonium Persulfate (APS) are added to react for 24 hours at 70 ℃ to obtain 2.7-micron epoxy group immunomagnetic beads with uniform particle size and shown in figure 6.
Comparative example 1
This comparative example relates to a method of preparing immunomagnetic beads, which is substantially the same as example 1 except that: the dispersant was 4g polyethylene glycol (molecular weight 600), pluronic PE10100 free, polyvinyl alcohol 1799 type and Tween 80.
In the immunomagnetic beads prepared in this comparative example, microspheres having non-uniform particle sizes were obtained as shown in FIG. 7.
Comparative example 2
This comparative example relates to a method of preparing immunomagnetic beads, which is substantially the same as example 1 except that: the dispersant was 4g polyethylene glycol (molecular weight 4000), pluronic PE10100 free, polyvinyl alcohol 1799 type and Tween 80.
The immunomagnetic beads obtained in this comparative example were microspheres having non-uniform particle sizes, as shown in FIG. 8.
Comparative example 3
This comparative example relates to a method of preparing immunomagnetic beads, which is substantially the same as example 1 except that: the dispersant was 4 grams of polyvinyl alcohol 1799, no Pluronic PE10100, polyethylene glycol and Tween 80.
In the immunomagnetic beads prepared in this comparative example, microspheres having non-uniform particle sizes were obtained as shown in FIG. 9.
Comparative example 4
This comparative example relates to a method of preparing immunomagnetic beads, which is substantially the same as example 1 except that: the dispersant was 1 gram of polyethylene glycol (molecular weight 600), 0.5 gram of Pluronic PE10100, 2 grams of polyvinyl alcohol 1799 type, no Tween 20.
In the immunomagnetic beads prepared in this comparative example, microspheres having non-uniform particle diameters were obtained as shown in FIG. 10.
Comparative example 5
This comparative example relates to a method of preparing immunomagnetic beads, which is substantially the same as example 1 except that: the dispersant was 1 g polyethylene glycol (molecular weight 600), 0.5 g tween 20, 2 g polyvinyl alcohol 1799 type, no Pluronic PE10100.
The immunomagnetic beads obtained in this comparative example were nanospheres having non-uniform particle sizes as shown in FIG. 11.
Comparative example 6
This comparative example relates to a method of preparing immunomagnetic beads, which is substantially the same as example 5 except that: the dispersant was 5.8 grams of polyethylene glycol (molecular weight 2000) and 2.8 grams of tween 80.
The immunomagnetic beads obtained in this comparative example were microspheres having non-uniform particle sizes, as shown in FIG. 12.
Comparative example 7
This comparative example relates to a method of preparing immunomagnetic beads, which is substantially the same as example 1 except that: 2 g of polyvinyl alcohol 1799 type were replaced by 2 g of the dispersant polyvinylpyrrolidone K30.
In the immunomagnetic beads obtained in this comparative example, microspheres having non-uniform particle diameters were obtained as shown in FIG. 13.
In summary, in the invention, a high molecular monomer, polyethylene glycol (PEG, molecular weight 300-20 000), pluronic PE10100, polyvinyl alcohol 1799 and tween (tween 20 or tween 80) are added into water, fully dispersed, heated and polymerized under the action of an initiator Ammonium Persulfate (APS) or dibenzoyl peroxide (BPO) to obtain a high molecular microsphere, the high molecular microsphere is prepared by adjusting the ratio and amount of each reactant, the reaction time and temperature, and the size and shape of the particles are characterized to obtain the high molecular microsphere with uniform particle size. And then introducing magnetic ferroferric oxide particles into the polymer microspheres through chemical coprecipitation of iron ions in an alkaline solution, and introducing functional groups through surface graft polymerization to obtain the immune micron magnetic beads for immune diagnosis, instant detection, gene sequencing and the like. The invention has three remarkable innovations: 1. green reaction: the whole operation process is carried out in aqueous solution without participation of organic solvent, and the preparation method is green and environment-friendly; 2. the method is simple and quick: the preparation method is that the polymer microsphere is obtained by a dispersion polymerization one-step method, compared with the prior method for preparing the microsphere by seed swelling, the preparation method is simpler and quicker, and the process is greatly shortened; 3. the method is novel: is a method for obtaining polymer microspheres and immunomicromagnetic beads with uniform grain diameter through dispersion polymerization in aqueous solution for the first time. Therefore, the method can be used for realizing large-scale industrial production of the polymer microspheres and the immunomicromagnetic beads.
The foregoing description has described specific embodiments of the present invention. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.
Claims (10)
1. The preparation method of the uniform-particle-size immunomagnetic beads is characterized in that the immunomagnetic beads are provided with functional polymer layers with shells as packages, and polymer microspheres and Fe dispersed in the polymer microspheres and on the surfaces of the polymer microspheres as cores 3 O 4 A core-shell structure of nanoparticles; the method comprises the following steps:
s1, preparing polymer microspheres with uniform particle sizes by a dispersion polymerization one-step method in the presence of a dispersing agent and an initiator by using water as a solvent and using a polymer monomer;
s2, introducing magnetic ferroferric oxide particles into the polymer microspheres through chemical coprecipitation of iron ions in an alkaline solution, and introducing functional groups through surface graft polymerization to obtain the immune micron magnetic beads with uniform particle sizes.
2. The method of claim 1, wherein the dispersant is selected from the group consisting of polyethylene glycol, pluronic PE10100, polyvinyl alcohol and Tween at a mass ratio of (0.05-1): (0.01-1): 0.1-2): 0.01-1.
3. The method of claim 2, wherein the polyethylene glycol has an average molecular weight of 300 to 20 000; 1799 type polyvinyl alcohol is selected; tween 20 or Tween 80 can be used.
4. The method for preparing immunomagnetic microbeads according to claim 1, wherein the high molecular monomer is one or more selected from styrene, methyl methacrylate, 2-hydroxyethyl methacrylate, methacrylic acid, acrylic acid and glycidyl methacrylate; the initiator is ammonium persulfate APS, dibenzoyl peroxide BPO or azobisisobutyronitrile AIBN.
5. The method of claim 1, wherein the temperature of the dispersion polymerization is 55-90 ℃ and the reaction time is 5-48 hours.
6. The method of claim 1, wherein each 0.1-2 ml of water corresponds to 0.1-1 g of a polymer monomer, 0.05-1 g of polyethylene glycol, 0.01-1 g of Pluronic PE10100, 0.01-2 g of polyvinyl alcohol, 0.01-1 g of Tween, and 0.00001-0.1 g of an initiator.
7. The method for preparing immunomicromagnetic beads according to claim 1, wherein the step S2 of introducing magnetic ferroferric oxide particles comprises: ferrous chloride or ferrous sulfate and ferric chloride are used as raw materials, the polymer microspheres are added into a mixed aqueous solution of ferric ions, mixed and incubated, then ammonia water or sodium hydroxide is added for heating, and magnetic ferroferric oxide particles are introduced into the interior and exterior of the polymer microspheres through a coprecipitation method, so that micron magnetic beads with uniform particle sizes are obtained.
8. Root of herbaceous plantThe method of claim 7, wherein the Fe ion is present in an aqueous solution of iron ions 3+ The molar concentration of the ions is 0.01-10 mol/L, and the molar ratio of ferric ions to ferrous ions is 1; mixing and incubating for 1-48 hours at 0-100 ℃ according to the proportion of 0.1-1 g of the macromolecular microspheres corresponding to 0.1-2 ml of the mixed aqueous solution of iron ions; adding ammonia water or sodium hydroxide to make the pH value of the mixed solution be 11-13; the coprecipitation is carried out for 1 to 24 hours at a temperature of between 25 and 90 ℃.
9. The method for preparing immunomagnetic beads according to claim 1, wherein the functional groups introduced in step S2 are specifically: the polymer microspheres introduced with the magnetic ferroferric oxide particles are dispersed in water by ultrasonic, then functional monomers, cross-linking agents and initiators are added, and functional groups are introduced on the surfaces of the microspheres through heating graft polymerization.
10. The method for preparing immunomagnetic beads according to claim 9, wherein 0.1 to 1 g of polymeric microspheres, 0.1 to 1 g of functional monomers, 0.001 to 1 g of cross-linking agents and 0.00001 to 0.1 g of initiators are correspondingly introduced into 0.1 to 2 ml of deionized water; the functional monomer is 2-hydroxyethyl methacrylate, methacrylic acid, acrylic acid, glycidyl methacrylate or acrylamide; the cross-linking agent is ethylene glycol dimethacrylate EGDMA or methylene bisacrylamide; the initiator is ammonium persulfate APS, dibenzoyl peroxide BPO or azobisisobutyronitrile AIBN; the reaction temperature of the heating graft polymerization is 55-90 ℃, and the reaction time is 5-48 hours.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996039464A1 (en) * | 1995-06-06 | 1996-12-12 | C.R. Bard, Inc. | Process for the preparation of aqueous dispersions of particles of water-soluble polymers and the particles obtained |
| CN1198104A (en) * | 1995-07-31 | 1998-11-04 | 德特勒夫·米勒-舒尔特 | Polyvinyl alcohol-based magnetic polymer particles, method for their preparation and their use |
| US20110293690A1 (en) * | 2010-05-27 | 2011-12-01 | Tyco Healthcare Group Lp | Biodegradable Polymer Encapsulated Microsphere Particulate Film and Method of Making Thereof |
| CN108467461A (en) * | 2018-02-28 | 2018-08-31 | 苏州为度生物技术有限公司 | The preparation method of surface carboxyl groups nucleocapsid superparamagnetism microballoon |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996039464A1 (en) * | 1995-06-06 | 1996-12-12 | C.R. Bard, Inc. | Process for the preparation of aqueous dispersions of particles of water-soluble polymers and the particles obtained |
| CN1198104A (en) * | 1995-07-31 | 1998-11-04 | 德特勒夫·米勒-舒尔特 | Polyvinyl alcohol-based magnetic polymer particles, method for their preparation and their use |
| US6204033B1 (en) * | 1995-07-31 | 2001-03-20 | MüLLER-SCHULTE DETLEF | Preparation of polyvinyl alcohol-based magnetic particles for binding biomolecules |
| US20110293690A1 (en) * | 2010-05-27 | 2011-12-01 | Tyco Healthcare Group Lp | Biodegradable Polymer Encapsulated Microsphere Particulate Film and Method of Making Thereof |
| CN108467461A (en) * | 2018-02-28 | 2018-08-31 | 苏州为度生物技术有限公司 | The preparation method of surface carboxyl groups nucleocapsid superparamagnetism microballoon |
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