CN115181215B - 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; the method comprises the steps of taking a high molecular monomer as a reaction monomer, polyethylene glycol, pluronic PE10100, polyvinyl alcohol and tween as dispersing agents, taking dibenzoyl peroxide and ammonium persulfate as initiators, and preparing high molecular microspheres with uniform particle size in green solvent water by using a dispersion polymerization method; and then introducing magnetic ferroferric oxide particles into the high molecular microsphere through chemical coprecipitation of iron ions in an alkaline solution, and introducing functional groups through surface graft polymerization to obtain the immune micrometer magnetic beads for immunodiagnosis, instant detection, gene sequencing and the like. The whole operation process is carried out in aqueous solution without participation of organic solvent, and the preparation method is environment-friendly; the polymer microsphere is obtained by a dispersion polymerization one-step method, and compared with the previous microsphere preparation method by a seed swelling method, the process is simpler and faster, and the process is greatly shortened.
Description
Technical Field
The invention relates to the technical field of biological materials, in particular to a preparation method of immune micron magnetic beads with uniform particle size.
Background
The immune micron magnetic bead is one kind of homogeneous spherical small particle with superparamagnetism and protecting shell, and is formed through combining carrier microsphere and immune ligand. The immune ligand generally comprises antigen, antibody or lectin, etc., the ligand has biospecificity, 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 immune magnetic separation technology is to fix the antibody (or antigen) on the surface of Immune Magnetic Microsphere (IMMS) by utilizing the high specific recognition function of antigen and antibody, form solid phase antibody/antigen complex, and after the action of external magnetic field, the complex is detained, and the magnetic microsphere carries antigen/antibody complex and other components to separate. Because the magnetic microsphere replaces other solid phase carriers to be used for immunoseparation, the method is simple and easy to operate, has high specificity and small loss, and can integrate immunoseparation and enrichment, the method has wide research and application prospects in the fields of medicine, biological separation, food sanitation detection, environment detection and the like.
The immune micron magnetic beads on the current market are mainly dynabeads flown by the United states and magbeads of Japanese JSR, but other enterprises at home and abroad are difficult to prepare immune micron magnetic beads with uniform particle size at present, the main reason is that the process is realized by an oil-water emulsification method, then the immune micron magnetic 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 because of an incompatible two-phase solvent, and the uniform particle size is difficult to obtain.
Patent CN 104492397A discloses a polymerization reaction of a combination of sodium dodecyl benzene sulfonate and PEG4000 as a dispersant, but polystyrene microspheres and magnetic beads with 50-150 microns of non-uniform particle size are obtained, and the definition and requirement of immunomagnetic beads are magnetic beads with uniform particle size, so that the microsphere magnetic beads obtained by the method cannot be used for immunomagnetic beads.
CN 108129614B and US4654267A are emulsified in oil-water two-phase solvent by seed expansion, this process is complicated, it needs to synthesize seed material first, then it can obtain polymer microsphere by oil-in-water emulsification method, and this process needs to use a large amount of oily solvent to participate in emulsification, and the later product needs long time cleaning process to remove oily solvent. In addition, the emulsification process has the participation of two-phase solvents, so that the dispersion of the high polymer monomer in the two phases is not easy to control, and uneven dispersion is often caused, so that the high polymer microspheres with different particle sizes are often obtained.
Disclosure of Invention
The invention aims to provide a preparation method of immune micron magnetic beads with uniform particle size, aiming at the defects and shortcomings of the prior art. The invention discloses a method for preparing polymer microspheres and immune micron magnetic beads with uniform particle size by carrying out dispersion polymerization reaction in water phase and optimizing components and proportion of a dispersing agent.
The aim of the invention is realized by the following technical scheme:
in a first aspect, the present invention relates to an immunomicrometer magnetic bead for immunodiagnosis, instant detection and gene sequencing, said immunomicrometer magnetic bead is a functional polymer layer whose outer shell is covered, and its inner core is a polymer microsphere and Fe dispersed in the interior and surface of the polymer microsphere 3 O 4 Core-shell structure of nanoparticles.
Preferably, the nano magnetic beads are uniform-particle-diameter microspheres with particle diameters of 1-3 microns.
Preferably, the polymer layer of the shell is provided with various functional groups, and polymer microspheres with uniform particle sizes and embedded ferroferric oxide nano particles are arranged inside the polymer layer; the functional group includes carboxyl, amino, epoxy, hydroxyl, and the like.
The immune micron magnetic bead preparation process is carried out in green solvent water, and the polymer microsphere is prepared by a simple and rapid dispersion polymerization one-step method.
In a second aspect, the invention relates to a preparation method of immune micrometer magnetic beads with uniform particle size, wherein the immune micrometer magnetic beads are provided with a functional polymer layer with a shell as a package, and an inner core is a polymer microsphere and Fe dispersed in the inside and the surface of the polymer microsphere 3 O 4 Core-shell structure of the nanoparticle; the method comprises the following steps:
s1, using water as a solvent, and preparing polymer microspheres with uniform particle size by a one-step dispersion polymerization method of polymer monomers in the presence of a dispersing agent and an initiator;
s2, introducing magnetic ferroferric oxide particles into the high molecular microsphere 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 size.
Preferably, the dispersant is selected from polyethylene glycol, pluronic PE10100, polyvinyl alcohol and tween.
Preferably, the mass ratio of polyethylene glycol, pluronic PE10100, polyvinyl alcohol and tween is (0.05-1): 0.01-1): 0.1-2): 0.01-1. The proportion can fully disperse the monomer and the initiator in the water phase, thereby obtaining the polymer microspheres with uniform particle size 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 a specific embodiment, the above materials may be selected from products sold by Alatine Biotechnology Co., 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 55-90 ℃ and the reaction time is 5-48 hours.
Preferably, each 0.1-2 ml of water corresponds to 0.1-1 g of high molecular 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 initiator; more preferably 0.0001 to 0.1 gram of initiator.
In some embodiments, step S1 is specifically: adding a high molecular monomer, polyethylene glycol PEG, pluronic PE10100, polyvinyl alcohol 1799 type and tween into water, fully dispersing, and heating and polymerizing under the action of an initiator ammonium persulfate APS or dibenzoyl peroxide BPO to obtain the high molecular microsphere.
Preferably, the magnetic ferroferric oxide particles introduced in the step S2 are specifically: the method comprises the steps of taking ferrous chloride or ferrous sulfate and ferric chloride as raw materials, adding the polymer microsphere into a mixed aqueous solution of iron ions, mixing and incubating, then adding ammonia water or sodium hydroxide for heating, and introducing magnetic ferroferric oxide particles into the inside and the outside of the polymer microsphere by a coprecipitation method to obtain micrometer magnetic beads with uniform particle sizes.
Preferably, fe in the mixed aqueous solution of iron ions 3+ The molar concentration of the ions is 0.01 mol/L to 10 mol/L, and the molar ratio of ferric iron to ferrous iron is 1:2 to 2:1.
Preferably, the mixed aqueous solution of iron ions is mixed and incubated for 1 to 48 hours at the temperature of between 0 and 100 ℃ according to the proportion of between 0.1 and 1 gram of the polymer microsphere per 0.1 to 2 milliliters of the mixed aqueous solution of iron ions.
Preferably, ammonia or sodium hydroxide is added to bring the pH of the mixed solution to 11-13.
Preferably, the coprecipitation is carried out at 25 to 90℃for 1 to 24 hours.
Preferably, the functional group introduced in step S2 is specifically: and ultrasonically dispersing the polymer microsphere introduced with the magnetic ferroferric oxide particles in water, then adding a functional monomer, a cross-linking agent and an initiator, and introducing functional groups on the microsphere surface through heating graft polymerization.
Preferably, every 0.1-2 ml of deionized water corresponds to 0.1-1 g of polymer microsphere, 0.1-1 g of functional monomer, 0.001-1 g of cross-linking agent and 0.00001-0.1 g of initiator which are introduced with magnetic ferroferric oxide particles. More preferably 0.05 to 1 gram of cross-linking 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 cross-linking 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 heated graft polymerization is 55-90 ℃ and the reaction time is 5-48 hours.
In some embodiments, step S2 includes:
firstly, ferric chloride, ferrous chloride or ferrous sulfate is dissolved in water, and the molar ratio of ferric iron to ferrous iron is 1:2-2: 1, then mixing and incubating for 1-48 hours at the temperature of 0-100 ℃ according to the proportion of 0.1-1 g of iron ion solution to 0.1-2 ml of polymer microsphere, then adding ammonia water or sodium hydroxide aqueous solution into the incubation solution, leading the pH value of the mixed solution to be 11-13, and reacting for 1-24 hours at the temperature of 25-90 ℃ to obtain the immune micron magnetic beads with uniform particle size.
Then, still taking water as a solvent, and reacting the obtained immune micron magnetic beads with a functional monomer, a cross-linking agent and an initiator, namely, 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 glycol dimethacrylate (EGDMA) and 0.001-0.1 g of initiator Ammonium Persulfate (APS) or dibenzoyl peroxide (BPO) are corresponding to each 0.1-2 ml of deionized water; 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, quick and novel method for preparing immune micron magnetic beads, namely, the dispersion polymerization is carried out in a green solvent water phase, the monomer is uniformly dispersed by adding a dispersing agent, and then the initiator is heated for polymerization. Because of homogeneous dispersion polymerization, the preparation of the microspheres is mainly influenced by a dispersing agent, unlike emulsion polymerization which is influenced not only by the dispersing agent but also by a 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 high molecular immune micron magnetic beads by adopting the green solvent water in all the documents and patents at present, and the toxicity of the solvent is avoided. The method is carried out in the water phase, and after all reactions are finished, the products are easy to clean and collect, so that the complicated cleaning process is avoided, the process period is greatly shortened, and the method is very suitable for large-scale industrial production.
3) Solves the problems that the immune micron magnetic beads possibly have nonspecific adsorption and unstable dispersion in complex samples. According to the invention, a functional polymer layer is wrapped on the surface of the immune micrometer magnetic bead by utilizing a grafting polymerization method, and then various functional groups are introduced on the surface of the polymer layer, so that in an aqueous solution, the polymer layer greatly reduces the nonspecific adsorption of the immune micrometer magnetic bead, improves the efficiency and the loading capacity of the immune micrometer magnetic bead for combining target molecules, also increases the dispersion stability of the immune micrometer magnetic bead in the aqueous solution, increases the monodispersity of the immune micrometer magnetic bead in a physiological system such as blood, tissue fluid and the like, and further solves the stability of the immune micrometer magnetic bead in a physiological environment, and lays a foundation for subsequent cell treatment and biological detection.
Drawings
Other features, objects and advantages of the present invention will become more apparent upon reading of the detailed description of non-limiting embodiments, given with reference to the accompanying drawings in which:
FIG. 1 is a microscopic image (eyepiece 10X, objective lens 50X) of 1 micron carboxyl immune micron magnetic beads with uniform particle size prepared by the invention;
FIG. 2 is a microscopic image (eyepiece 10X, objective lens 50X) of 1 micron amino immune micron magnetic beads with uniform particle size prepared according to the invention;
FIG. 3 is a microscopic image (eyepiece 10X, objective lens 50X) of 1 micron epoxy-based immunomicrometer magnetic beads prepared according to the present invention with uniform particle size;
FIG. 4 is a microscopic image (eyepiece 10X, objective lens 50X) of 2.8 μm carboxyl-immune micrometer magnetic beads prepared according to the present invention with uniform particle size;
FIG. 5 is a microscopic image (eyepiece 10X, objective lens 50X) of 2.7 μm amino immune micrometer magnetic beads with uniform particle size prepared according to the present invention;
FIG. 6 is a microscopic image of 2.7 micron epoxy-based immunomicrometer magnetic beads of uniform particle size (eyepiece 10X, objective lens 50X) prepared according to the present invention;
FIG. 7 is a microscopic image of the carboxyl microbeads prepared in comparative example 1;
FIG. 8 is a microscopic image of the carboxyl microbeads prepared in comparative example 2;
FIG. 9 is a microscopic image of the carboxyl microbeads prepared in comparative example 3;
FIG. 10 is a microscopic image of the carboxyl microbeads prepared in comparative example 4;
FIG. 11 is a microscopic image of the carboxyl microbeads prepared in comparative example 5;
FIG. 12 is a microscopic image of the amino microbeads prepared in comparative example 6;
FIG. 13 is a microscopic image of the amino microbeads prepared in comparative example 7.
Detailed Description
The present invention will be described in detail with reference to specific embodiments and drawings. The following examples will assist those skilled in the art in further understanding the present invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications could be made by those skilled in the art without departing from the inventive concept. These are all within the scope of the present invention.
Example 1
The embodiment relates to a green, simple, quick and novel method for preparing immune micron magnetic beads, which comprises the following steps:
in the first step, 5 g of styrene monomer, 2 g of methacrylic acid monomer, 1 g of polyethylene glycol (molecular weight 600) as a dispersing agent, 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 are dispersed in 50 ml of deionized water, then reacted for 24 hours at 60 ℃, centrifugally collected, washed 5 times with deionized water, 100 ml of each time, and finally dried in an oven at 50 ℃ after washing and centrifugally collected, so that 1-micrometer polystyrene/methacrylic acid microspheres with uniform particle sizes are obtained.
Secondly, embedding ferroferric oxide nano particles into the polystyrene/methacrylic acid microsphere and on the surface of the polystyrene/methacrylic acid microsphere: 3 g of ferric chloride hexahydrate and 3 g of ferrous chloride tetrahydrate are dissolved in 20 ml of water, 2 g of 1 micrometer polystyrene/methacrylic acid microsphere are mixed and incubated for 2 hours at the temperature of 4 ℃, then ammonia water is added into the incubation solution to enable the pH value of the mixed solution to be 11, the mixed solution reacts for 16 hours at the temperature of 50 ℃, and the non-embedded ferroferric oxide nano particles are removed by centrifugal cleaning, so that the immune micrometer magnetic beads with uniform particle size are obtained.
Thirdly, introducing carboxyl functional groups on the surface of the polystyrene/methacrylic acid microsphere by a graft polymerization method: polystyrene/methacrylic acid magnetic beads were dispersed in 20 ml of deionized water, and then 2 g of methacrylic acid, 1 g of Ethylene Glycol Dimethacrylate (EGDMA), which is a crosslinking agent, and 0.008 g of Ammonium Persulfate (APS), which is an initiator, were added to react at 70℃for 8 hours, to obtain 1 μm carboxyl immunomagnetic beads having a uniform particle size as shown in FIG. 1.
Example 2
The embodiment relates to a green, simple, quick and novel method for preparing immune micron magnetic beads, which comprises the following steps:
in the first step, 6 g of styrene monomer, 3 g of methyl methacrylate monomer, 2 g of polyethylene glycol (molecular weight 300) serving as a dispersing agent, 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) serving as an initiator are dispersed in 50 ml of deionized water, reacted for 24 hours at 60 ℃, centrifugally collected, washed 5 times with deionized water, 100 ml of each time, centrifugally collected, and dried in an oven at 50 ℃ to obtain 1-micrometer polystyrene/methyl methacrylate microspheres with uniform particle sizes.
Secondly, embedding ferroferric oxide nano particles into the polystyrene/methyl methacrylate microsphere and on the surface of the polystyrene/methyl methacrylate microsphere: 3 g of ferric chloride hexahydrate and 3 g of ferrous chloride tetrahydrate are dissolved in 20 ml of water, 2 g of 1 micrometer polystyrene/methyl methacrylate microsphere is mixed and incubated for 2 hours at 60 ℃, then ammonia water is added into the incubation solution to enable the pH value of the mixed solution to be 12, the mixed solution reacts for 3 hours at 80 ℃, and the non-embedded ferroferric oxide nano particles are removed by centrifugal cleaning, so that the immune micrometer magnetic beads with uniform particle size are obtained.
Thirdly, introducing amino functional groups on the surfaces of the polystyrene/methyl methacrylate microspheres by a graft polymerization method: polystyrene/methyl methacrylate magnetic beads were 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) were added to react at 70℃for 24 hours, to obtain 1 μm amino immunomagnetic beads having a uniform particle size as shown in FIG. 2.
Example 3
The embodiment relates to a green, simple, quick and novel method for preparing immune micron magnetic beads, which comprises the following steps:
the immune micrometer magnetic beads in the examples 1 and 2 are introduced with epoxy functional groups on the surfaces of the magnetic beads by a grafting polymerization method: the beads were dispersed in 30 ml of deionized water, and then 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) were added to react at 70℃for 12 hours to obtain 1 μm epoxy immunomagnetic beads having a uniform particle size as shown in FIG. 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:
in the first step, 8 g of styrene monomer, 4g of acrylic acid monomer, 2 g of polyethylene glycol (molecular weight 1000) serving as a dispersing agent, 1 g of Pluronic PE10100, 4g of polyvinyl alcohol 1799 type, 1 g of Tween 20 and 0.5 g of Ammonium Persulfate (APS) serving as an initiator are dispersed in 50 ml of deionized water, then reacted at 70 ℃ for 24 hours, centrifugally collected, washed with deionized water for 5 times, 100 ml of each time, centrifugally collected, and dried in a 50 ℃ oven to obtain 2.7-micrometer polystyrene/acrylic microspheres with uniform particle sizes.
Secondly, embedding ferroferric oxide nano particles into the polystyrene/acrylic acid microsphere and on the surface of the polystyrene/acrylic acid microsphere: 3 g of ferric chloride hexahydrate and 3 g of ferrous chloride tetrahydrate are dissolved in 20 ml of water, 2 g of 2.7-micrometer polystyrene/acrylic acid microsphere are mixed and incubated for 5 hours at the temperature of 10 ℃, then ammonia water is added into the incubation solution to enable the pH value of the mixed solution to be 11, the mixed solution reacts for 24 hours at the temperature of 50 ℃, and the non-embedded ferroferric oxide nano particles are removed by centrifugal cleaning, so that the immune micrometer magnetic beads with uniform particle size are obtained.
Thirdly, introducing carboxyl functional groups on the surface of the polystyrene/acrylic acid microsphere by a graft polymerization method: polystyrene/acrylic beads were dispersed in 20 ml deionized water, and then 2 g acrylic acid, 1 g cross-linking agent Ethylene Glycol Dimethacrylate (EGDMA) and 0.01 g initiator Ammonium Persulfate (APS) were added to react at 70℃for 16 hours, to obtain 2.8 μm carboxyl immunomagnetic beads having a uniform particle size as shown in FIG. 4.
Example 5
The embodiment relates to a green, simple, quick and novel method for preparing immune micron magnetic beads, which comprises the following steps:
in the first step, 8 g of a styrene monomer, 3 g of a methyl methacrylate monomer, 2 g of polyethylene glycol (molecular weight 2000) as a dispersing agent, 0.8 g of Pluronic PE10100, 5 g of polyvinyl alcohol 1799 type, 0.8 g of Tween 80 and 0.05 g of a dibenzoyl peroxide (BPO) as an initiator are dispersed in 50 ml of deionized water, then reacted for 24 hours at 70 ℃, and then centrifugally collected, washed 5 times with deionized water, 100 ml of each time, and finally dried in an oven at 50 ℃ after washing and centrifugally collected, so that 2.6-micrometer polystyrene/methyl methacrylate microspheres with uniform particle sizes are obtained.
Secondly, embedding ferroferric oxide nano particles into the polystyrene/methyl methacrylate microsphere and on the surface of the polystyrene/methyl methacrylate microsphere: 3 g of ferric chloride hexahydrate and 3 g of ferrous chloride tetrahydrate are dissolved in 20 ml of water, 2 g of 2.6 micrometer polystyrene/methyl methacrylate microspheres are mixed and incubated for 1 hour at 80 ℃, then ammonia water is added into the incubation solution to enable the pH value of the mixed solution to be 11, the mixed solution reacts for 8 hours at 60 ℃, and the non-embedded ferroferric oxide nano particles are removed by centrifugal cleaning, so that the immune micrometer magnetic beads with uniform particle size are obtained.
Thirdly, introducing amino functional groups on the surfaces of the polystyrene/methyl methacrylate microspheres by a graft polymerization method: polystyrene/methyl methacrylate magnetic beads were 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) were added to react at 70 ℃ for 24 hours, to obtain 2.7 μm amino immunomagnetic beads having a uniform particle size as shown in fig. 5.
Example 6
The embodiment relates to a green, simple, quick and novel method for preparing immune micron magnetic beads, which comprises the following steps:
in the first step, 10 g of styrene monomer, 4g of 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, 1 g of Tween 80 and 0.05 g of dibenzoyl peroxide (BPO) as an initiator are dispersed in 60 ml of deionized water, then reacted for 24 hours at 80 ℃, and then centrifugally collected, washed 5 times with deionized water, 100 ml of each time, and finally dried in an oven at 50 ℃ after washing and centrifugally collected, so that 2.6-micrometer polystyrene/methacrylic acid-2-hydroxyethyl ester microspheres with uniform particle sizes are obtained.
Secondly, embedding ferroferric oxide nano particles into the polystyrene/2-hydroxyethyl methacrylate microsphere and on the surface of the microsphere: 3 g of ferric chloride hexahydrate and 3 g of ferrous chloride tetrahydrate are dissolved in 20 ml of water, 2 g of 2.6 micrometer polystyrene/2-hydroxyethyl methacrylate microspheres are mixed and incubated for 2 hours at 80 ℃, then ammonia water is added into the incubation solution to enable the pH value of the mixed solution to be 12, the mixed solution reacts for 3 hours at 80 ℃, and the non-embedded ferroferric oxide nano particles are removed by centrifugal cleaning, so that the immune micrometer magnetic beads with uniform particle size are obtained.
Thirdly, introducing epoxy functional groups on the surfaces of the polystyrene/methacrylic acid-2-hydroxyethyl microspheres by a graft polymerization method: polystyrene/2-hydroxyethyl methacrylate magnetic beads were 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) were added to react at 70℃for 24 hours, to obtain 2.7 μm epoxy-based immunomagnetic beads having a uniform particle size as shown in FIG. 6.
Comparative example 1
This comparative example relates to a method for preparing immunomagnetic beads, which is basically the same as example 1, except that: the dispersant was 4g polyethylene glycol (molecular weight 600), no Pluronic PE10100, polyvinyl alcohol 1799 type and tween 80.
The immunomagnetic beads prepared in this comparative example gave microspheres having non-uniform particle size as shown in FIG. 7.
Comparative example 2
This comparative example relates to a method for preparing immunomagnetic beads, which is basically the same as example 1, except that: the dispersant was 4g polyethylene glycol (molecular weight 4000), no Pluronic PE10100, polyvinyl alcohol 1799 type and tween 80.
The immunomagnetic beads prepared in this comparative example gave microspheres having non-uniform particle size as shown in FIG. 8.
Comparative example 3
This comparative example relates to a method for preparing immunomagnetic beads, which is basically the same as example 1, except that: the dispersant was 4 grams of polyvinyl alcohol 1799 type, free of Pluronic PE10100, polyethylene glycol and tween 80.
The immunomagnetic beads prepared in this comparative example gave microspheres having non-uniform particle size as shown in FIG. 9.
Comparative example 4
This comparative example relates to a method for preparing immunomagnetic beads, which is basically the same as example 1, except that: the dispersing agent is 1 g of polyethylene glycol (molecular weight 600), 0.5 g of Pluronic PE10100, 2 g of polyvinyl alcohol 1799 type and no Tween 20.
The immunomagnetic beads prepared in this comparative example gave microspheres having non-uniform particle size as shown in FIG. 10.
Comparative example 5
This comparative example relates to a method for preparing immunomagnetic beads, which is basically the same as example 1, except that: the dispersing agent is 1 g of polyethylene glycol (molecular weight 600), 0.5 g of Tween 20, 2 g of polyvinyl alcohol 1799 type and no Pluronic PE10100.
The immunomagnetic beads prepared in this comparative example gave nanospheres of non-uniform particle size as shown in FIG. 11.
Comparative example 6
This comparative example relates to a method for preparing immunomagnetic beads, which is basically 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 prepared in this comparative example gave microspheres having non-uniform particle diameters as shown in FIG. 12.
Comparative example 7
This comparative example relates to a method for preparing immunomagnetic beads, which is basically the same as example 1, except that: 2 g of polyvinyl alcohol 1799 were replaced by 2 g of the dispersant polyvinylpyrrolidone K30.
The immunomagnetic beads prepared in this comparative example gave microspheres having non-uniform particle diameters as shown in FIG. 13.
In summary, the invention adds macromolecule monomer, polyethylene glycol (PEG, molecular weight 300-20 000), pluronic PE10100, polyvinyl alcohol 1799 type and tween (tween 20 or tween 80) into water, fully disperses, obtains macromolecule microsphere by heating and polymerizing under the action of initiator Ammonium Persulfate (APS) or dibenzoyl peroxide (BPO), prepares macromolecule microsphere by adjusting the proportion and amount of each reactant, reaction time and temperature, and characterizes the size and shape of the particle, etc. to obtain macromolecule microsphere with uniform particle diameter. And then introducing magnetic ferroferric oxide particles into the high molecular microsphere through chemical coprecipitation of iron ions in an alkaline solution, and introducing functional groups through surface graft polymerization to obtain the immune micrometer magnetic beads for immunodiagnosis, 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, thus being a green and environment-friendly preparation method; 2. simple and rapid: the preparation method is to obtain the polymer microsphere by a dispersion polymerization one-step method, and compared with the previous method for preparing the microsphere by a seed swelling method, the preparation method is simpler and faster, and the process is greatly shortened; 3. novel: the method is a method for obtaining polymer microspheres and immune micron magnetic beads with uniform particle size through dispersion polymerization in aqueous solution for the first time. Therefore, the method of the invention can be used for realizing the large-scale industrial production of the polymer microsphere and the immune micron magnetic bead.
The foregoing describes specific embodiments of the present invention. It is to be understood that the invention is not limited to the particular embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the claims without affecting the spirit of the invention.
Claims (9)
1. A preparation method of immune micrometer magnetic beads with uniform particle size is characterized in that the immune micrometer magnetic beads are provided with a functional polymer layer with a shell as a package, and an inner core is a polymer microsphere and Fe dispersed in the inside and the surface of the polymer microsphere 3 O 4 Core-shell structure of the nanoparticle; the method comprises the following steps:
s1, using water as a solvent, and preparing polymer microspheres with uniform particle size by a one-step dispersion polymerization method of polymer monomers in the presence of a dispersing agent and an initiator; the dispersant is selected from polyethylene glycol, pluronic PE10100, polyvinyl alcohol and tween with the mass ratio of (0.05-1), (0.01-1), (0.1-2) and (0.01-1);
s2, introducing magnetic ferroferric oxide particles into the high molecular microsphere 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 size.
2. The method for preparing an immunomicrometer magnetic bead according to claim 1, wherein the polyethylene glycol has an average molecular weight of 300-20 000; the polyvinyl alcohol is 1799 type; tween is tween 20 or tween 80.
3. The method for preparing the immune micro-magnetic beads according to claim 1, wherein the high molecular monomer is one or more selected from the group consisting of 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.
4. The method for preparing immunomicrometer magnetic beads according to claim 1, wherein the reaction temperature of the dispersion polymerization is 55-90 ℃ and the reaction time is 5-48 hours.
5. The method for preparing immune micro-magnetic beads according to claim 1, wherein each 0.1-2 ml of water corresponds to 0.1-1 g of high molecular 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 initiator.
6. The method for preparing immune micro-magnetic beads according to claim 1, wherein the introduction of magnetic ferroferric oxide particles in step S2 is specifically: the method comprises the steps of taking ferrous chloride or ferrous sulfate and ferric chloride as raw materials, adding the polymer microsphere into a mixed aqueous solution of iron ions, mixing and incubating, then adding ammonia water or sodium hydroxide for heating, and introducing magnetic ferroferric oxide particles into the inside and the outside of the polymer microsphere by a coprecipitation method to obtain micrometer magnetic beads with uniform particle sizes.
7. The method for preparing immunomicrometer magnetic beads according to claim 6, wherein Fe in the mixed aqueous solution of iron ions 3+ The molar concentration of the ions is 0.01 mol/L-10 mol/L, and the molar ratio of ferric iron to ferrous iron is 1:2-2:1; mixing and incubating at 0-100 ℃ for 1-48 hours according to the proportion of 0.1-1 g of the polymer microsphere corresponding to 0.1-2 ml of the mixed aqueous solution of the 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 the temperature of between 25 and 90 ℃.
8. The method for preparing the immune micrometer magnetic bead according to claim 1, wherein the functional group introduced in the step S2 is specifically: and ultrasonically dispersing the polymer microsphere introduced with the magnetic ferroferric oxide particles in water, then adding a functional monomer, a cross-linking agent and an initiator, and introducing functional groups on the microsphere surface through heating graft polymerization.
9. The method for preparing immune micro-magnetic beads according to claim 8, wherein each 0.1-2 ml of deionized water corresponds to 0.1-1 g of polymer microsphere, 0.1-1 g of functional monomer, 0.001-1 g of cross-linking agent and 0.00001-0.1 g of initiator which are introduced with magnetic ferroferric oxide particles; the functional monomer is methacrylic acid-2-hydroxyethyl ester, 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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| 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 |
| 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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| US20110293690A1 (en) * | 2010-05-27 | 2011-12-01 | Tyco Healthcare Group Lp | Biodegradable Polymer Encapsulated Microsphere Particulate Film and Method of Making Thereof |
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Patent Citations (4)
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|---|---|---|---|---|
| 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 |
| 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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