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WO2019208669A1 - Particules et leur procédé de production - Google Patents

Particules et leur procédé de production Download PDF

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Publication number
WO2019208669A1
WO2019208669A1 PCT/JP2019/017534 JP2019017534W WO2019208669A1 WO 2019208669 A1 WO2019208669 A1 WO 2019208669A1 JP 2019017534 W JP2019017534 W JP 2019017534W WO 2019208669 A1 WO2019208669 A1 WO 2019208669A1
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Prior art keywords
group
particles
particle
monomer
compound
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English (en)
Japanese (ja)
Inventor
小林　本和
哲士 山本
法重 掛川
文生 山内
健吾 金崎
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Canon Inc
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Canon Inc
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    • 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
    • C08F2/00—Processes of polymerisation
    • C08F2/12—Polymerisation in non-solvents
    • C08F2/16—Aqueous medium
    • C08F2/22—Emulsion polymerisation
    • 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
    • C08F291/00—Macromolecular compounds obtained by polymerising monomers on to macromolecular compounds according to more than one of the groups C08F251/00 - C08F289/00
    • 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
    • C08F8/00—Chemical modification by after-treatment
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/545—Synthetic resin

Definitions

  • the present invention relates to particles and a method for producing the same.
  • Polymer particles are gaining importance in the fields of basic biology and medicine. For example, there is an increasing need for application to in vitro diagnostics using latex agglutination.
  • the desired antigen or antibody can be adsorbed on the particle surface, the sensitivity of the latex agglutination reaction can be increased.
  • many contaminants exist in specimens such as serum and these contaminants are also adsorbed on the particle surface (nonspecific adsorption), thereby inhibiting the adsorption of desired antigens and antibodies to the particle surface.
  • the particles are aggregated through the foreign matter adsorbed on the particles, the measurement accuracy is lowered. Therefore, after adsorbing the desired antibody to the prepared particles, the surface of the particles is coated with BSA (Bovine serum albumin, bovine serum albumin) to suppress the adsorption (nonspecific adsorption) of contaminants on the particle surface.
  • BSA Bovine serum albumin, bovine serum albumin
  • the particle production method according to the present invention is a particle production method including a first step of preparing an emulsion by mixing a radical polymerizable monomer, an organic silane compound, a radical polymerization initiator, a water-soluble polymer, and an aqueous solution.
  • the organosilane compound is a compound having radical polymerizability in which an alkoxy group is bonded to a silicon atom.
  • the particles according to the present invention are particles produced by a step of preparing an emulsion by mixing a radical polymerizable monomer, an organic silane compound, a radical polymerization initiator, a water-soluble polymer, and an aqueous solution, and the organic silane
  • the compound is a compound having radical polymerizability in which an alkoxy group is bonded to a silicon atom.
  • particles and particle production method In the method for producing particles according to the present embodiment, at least a radical polymerizable monomer, an organic silane compound, a radical polymerization initiator, and a water-soluble polymer are mixed with an aqueous solution (aqueous medium) to prepare an emulsion ( First step).
  • the organosilane compound used here is a compound in which an alkoxy group is bonded to a silicon atom and further has radical polymerizability.
  • the particles produced by the production method according to the present embodiment may be referred to as polymer fine particles.
  • Particles produced by the method for producing particles according to the present embodiment have high hydrophilicity on the particle surface due to the presence of silica (silanol group) derived from an organosilane compound, and therefore, nonspecific adsorption can be performed without using BSA. Can be suppressed. Further, since a natural product such as BSA is not used, the difference in the ability to suppress nonspecific adsorption for each particle is small. In other words, when producing particles, the reproducibility of the ability to suppress nonspecific adsorption is high.
  • the polymer fine particle according to the present embodiment is an emulsion obtained by mixing at least a radical polymerizable monomer not containing a silicon atom, an organic silane compound, a radical polymerization initiator, a water-soluble polymer and a molecule having a reactive functional group with an aqueous medium.
  • a liquid is prepared (first step).
  • the molecule having a reactive functional group is a molecule having at least one of a silicon alkoxide group, a carboxyl group, an amino group, a thiol group, and a glycidyl group.
  • the organosilane compound is a compound having an alkoxy group bonded to a silicon atom and further having a radically polymerizable double bond.
  • the polymer fine particle according to the present embodiment is a polymer fine particle obtained by copolymerizing a radical polymerizable monomer not containing at least a silicon atom and a compound having a double bond having a radical polymerizable property, wherein an alkoxy group is bonded to the silicon atom. . Furthermore, a molecule having at least one of functional groups of silicon alkoxide group, carboxyl group, amino group, thiol group, and glycidyl group is covalently bonded to the polymer fine particle.
  • radical polymerizable monomer As the radical polymerizable monomer, at least one selected from the group consisting of a styrene monomer, an acrylate monomer, and a methacrylate monomer can be used. Examples thereof include styrene, butadiene, vinyl acetate, vinyl chloride, acrylonitrile, methyl methacrylate, methacrylonitrile, and methyl acrylate. At least one selected from the group of these monomers can be used. That is, from these, it can be used alone or in combination of two or more. Moreover, you may use the monomer which has two or more double bonds in one molecule, for example, divinylbenzene, as a crosslinking agent.
  • organic silane compound examples include vinyltrimethoxysilane, vinyltriethoxysilane, p-styryltrimethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyltrimethoxysilane, and 3-methacryloxypropylmethyldiethoxysilane. , 3-methacryloxypropyltriethoxysilane, 3-acryloxypropyltrimethoxysilane, and the like. At least one selected from the group of these monomers can be used. That is, among these, you may use individually or in combination of multiple types.
  • the radical polymerization initiator can be widely used from azo compounds, organic peroxides and the like. Specifically, 2,2′-azobis (isobutyronitrile), 2,2′-azobis (2,4-dimethylvaleronitrile), 2,2′-azobis (2-methylbutyronitrile), 4 , 4′-azobis (4-cyanovaleric acid), 2,2′-azobis (2-methylpropionamidine) dihydrochloride, 2,2′-azobis (2-methylpropionic acid) dimethyl, tert-butyl hydroperoxide Benzoyl peroxide, ammonium persulfate (APS), sodium persulfate (NPS), potassium persulfate (KPS), and the like.
  • APS ammonium persulfate
  • NPS sodium persulfate
  • KPS potassium persulfate
  • the water-soluble polymer acts as a protective colloid during the synthesis of polymer fine particles, and contributes to control of the particle size of the produced polymer fine particles.
  • a preferable water-soluble polymer one or a combination of polyacrylamide, polyvinyl alcohol, polyethylene oxide, and polyvinylpyrrolidone may be used.
  • the molecular weight is preferably from 10,000 to 1,000,000, more preferably from 30,000 to 50,000. This is because when the molecular weight is less than 10,000, the protective colloid properties are not reduced, and when the molecular weight exceeds 1000000, the viscosity of the aqueous medium increases and it becomes difficult to handle.
  • Some of these water-soluble polymers may be adhered to the surface of the synthesized particles by physical adsorption, chemical adsorption, or the like.
  • a molecule having at least one of a silicon alkoxide group, a carboxyl group, an amino group, a thiol group, and a glycidyl group can be used without particular limitation as long as it contains at least one of these functional groups. .
  • the aqueous solution (aqueous medium) preferably contains 80% or more and 100% or less of water contained in the medium.
  • examples of the aqueous solution include water and water-soluble organic solvents such as methanol, ethanol, isopropyl alcohol, and acetone mixed with water. If the organic solvent other than water is contained in an amount of more than 20%, the polymerizable monomer may be dissolved during the production of the polymer fine particles.
  • the aqueous solution (aqueous medium) preferably has a pH adjusted in advance to 6 or more and 9 or less. If the pH is less than 6 or greater than 9, the alkoxide group in the organosilane compound may react with polycondensation or other functional groups before the formation of polymer fine particles, and the resulting particles may aggregate. In this embodiment, it is not necessary to intentionally polycondensate the alkoxide before forming the polymer fine particles.
  • the pH is preferably adjusted using a pH buffering agent, but may be prepared with an acid or a base.
  • surfactants may be added at a ratio of 10% or less with respect to the aqueous medium.
  • a water-soluble polymer is dissolved in an aqueous medium whose pH is adjusted to 6 to 9.
  • the water-soluble polymer is 0.01% to 20% by weight, preferably 0.03% to 15% by weight, based on the aqueous medium. If it is 0.01% by weight or less, the amount is not sufficient for controlling the particle diameter, and the effect is not exhibited. On the other hand, if it is 20% by weight or more, the viscosity of the aqueous medium increases, and there is a possibility that stirring cannot be performed sufficiently.
  • a radically polymerizable monomer (A) containing no silicon atom and an organosilane compound (B) having an alkoxy group bonded to the silicon atom and further having a radically polymerizable double bond are added to the aqueous medium.
  • the weight ratio of (A) and (B) is from 4: 6 to 9.7: 0.3.
  • the weight ratio of the aqueous medium and the total amount of (A) and (B) is preferably 5: 5 to 9.5: 0.5.
  • the ratio of the aqueous medium is 5 or less, there is a possibility that aggregation of polymer fine particles to be generated becomes remarkable. Moreover, even if it is 9.5 or more, there is no problem in the production of polymer fine particles, but the production amount may be reduced.
  • a molecule having at least one of a silicon alkoxide group, a carboxyl group, an amino group, a thiol group, and a glycidyl group may be used simultaneously with the above (A) and (B) or the reaction of (A) and (B). It may be added after completion of the process and after the process is completed.
  • Molecules having these functional groups are necessary for immobilizing the antibody.
  • the added amount can be between 0.01 wt% and 60 wt% with respect to the total weight of (A) and (B).
  • the radical polymerization initiator is used after being dissolved in water, a buffering agent or the like.
  • the radical polymerization initiator relative to the total weight of (A) and (B) can be used between 0.5 wt% and 10 wt%.
  • the step of heating the emulsion may be performed as long as the whole emulsion is heated uniformly.
  • the heating temperature can be arbitrarily set between 50 ° C. and 90 ° C., and the heating time can be set between 2 hours and 24 hours.
  • the average particle size of the polymer fine particles is preferably 50 nm or more and 400 nm or less, and more preferably 100 nm or more and 400 nm or less.
  • the coefficient of variation (CV value) of the particle size distribution of the polymer fine particles is preferably 5 or less.
  • the measurement method of the average particle size is mainly a dynamic light scattering method (DLS), laser diffraction method (LD method), observation with an electron microscope, etc., but in this embodiment, the dynamic light scattering method is used. Is preferably used.
  • a dispersion of polymer fine particles can be measured by a dynamic light scattering method, the obtained light intensity can be converted into a number distribution, and the average value can be used as the particle diameter. If the average particle size is less than 50 nm, it may take time for the washing treatment during synthesis. Moreover, when larger than 400 nm, there exists a possibility that the aggregation by sedimentation at the time of a preservation
  • the coefficient of variation (CV value) can be obtained by taking an image of polymer fine particles with an electron microscope, obtaining the diameter of, for example, 100 particles or more, and obtaining the coefficient of variation (CV value) by statistical processing. If the coefficient of variation (CV value) is 5 or more (the distribution range of the particle size is wide), the reaction between the specimen test particles and the antigen may become unstable.
  • the particle size of the polymer fine particles can be controlled by the ratio of the monomer and aqueous medium at the time of synthesis, the amount of water-soluble polymer added, the reaction temperature, and the reaction time.
  • the dispersion of polymer fine particles thus produced is purified by filtration, decantation by centrifugation, ultrafiltration, etc., and after removing unreacted substances and aggregates.
  • the polymer fine particles thus produced are precursor particles for specimen inspection.
  • An antibody can be immobilized on the precursor particles and used as a sample test particle.
  • the immobilization method uses functional groups of polymer fine particles and is immobilized by chemical bonding, physical adsorption, or the like. In the present invention, the method is not limited.
  • the specimen test particles in which the antibody is immobilized on the polymer fine particles aggregate the specimen test particles via the antigen when the antigen is present in the specimen, and the antigen can be detected by observing the aggregation. .
  • aggregation is unlikely to occur.
  • an affinity particle having the particle according to the present embodiment and a ligand bonded to a reactive functional group can be provided.
  • a ligand is a compound that specifically binds to a receptor possessed by a specific target substance.
  • the site where the ligand binds to the target substance is determined and has a high affinity selectively or specifically.
  • examples include antigens and antibodies, enzyme proteins and their substrates, signal substances such as hormones and neurotransmitters and their receptors, and nucleic acids, but the ligands in this embodiment are not limited thereto.
  • the nucleic acid include deoxyribonucleic acid.
  • the affinity particles in the present embodiment have a high affinity (affinity) selectively or specifically for the target substance.
  • the ligand in the present embodiment is preferably any one of an antibody, an antigen, and a nucleic acid.
  • a conventionally known method can be applied to the chemical reaction method for chemically bonding the reactive functional group of the particle according to the present embodiment and the ligand to the extent that the object of the present invention can be achieved. it can.
  • a catalyst such as 1- [3- (dimethylaminopropyl) -3-ethylcarbodiimide] can be appropriately used.
  • the affinity particle in this embodiment uses an antibody (antigen) as a ligand and an antigen (antibody) as a target substance, it can be preferably applied to an immunolatex agglutination measurement method widely used in areas such as clinical examination and biochemical research. .
  • the test reagent for in-vitro diagnosis in this embodiment that is, the test reagent for use in detecting a target substance in a specimen by in-vitro diagnosis has affinity particles according to this embodiment and a dispersion medium for dispersing the affinity particles.
  • the amount of the affinity particles according to this embodiment contained in the reagent in this embodiment is preferably 0.001% by mass to 20% by mass, and more preferably 0.01% by mass to 10% by mass.
  • the reagent according to the present embodiment may contain a third substance such as a solvent or a blocking agent in addition to the affinity particles according to the present embodiment as long as the object of the present invention can be achieved. Third substances such as solvents and blocking agents may be included in combination of two or more.
  • Examples of the solvent used in the present embodiment include various buffer solutions such as a phosphate buffer solution, a glycine buffer solution, a Good buffer solution, a Tris buffer solution, and an ammonia buffer solution, and are included in the reagent in the present embodiment.
  • the solvent is not limited to these.
  • an antibody or antigen When used for detection of an antigen or antibody in a specimen by latex agglutination, an antibody or antigen can be used as the ligand.
  • a test kit for use in detecting a target substance in a specimen by in-vitro diagnosis includes the reagent and a housing that contains the reagent.
  • the kit according to this embodiment may contain a sensitizer for measuring latex aggregation.
  • the sensitizer for measuring latex agglutination include polyvinyl alcohol, polyvinyl pyrrolidone, polyalginic acid and the like, but the present invention is not limited thereto.
  • the kit according to the present embodiment may include a positive control, a negative control, a serum diluent, and the like.
  • a solvent other than serum and physiological saline containing no measurable target substance may be used as a medium for positive control and negative control.
  • the kit according to the present embodiment can be used in the method for detecting a target substance according to the present embodiment in the same manner as a kit for use in detecting a target substance in a specimen by normal in vitro diagnosis. Further, the concentration of the target substance can be measured by a conventionally known method, and it is particularly suitable for use in detecting the target substance in the specimen by the latex agglutination method.
  • the method for detecting a target substance in a specimen by in-vitro diagnosis according to the present embodiment includes a step of mixing the affinity particles according to the present embodiment and a specimen that may contain the target substance.
  • the mixing of the affinity particles and the specimen according to the present embodiment is preferably performed in the range of pH 3.0 to pH 11.0.
  • the mixing temperature is in the range of 20 ° C. to 50 ° C.
  • the mixing time is in the range of 1 minute to 20 minutes.
  • this detection method uses a solvent.
  • the concentration of the affinity particles according to this embodiment in the detection method according to this embodiment is preferably 0.001% by mass to 5% by mass, more preferably 0.01% by mass to 1% by mass in the reaction system.
  • the detection method according to this embodiment optically detects an agglutination reaction that occurs as a result of mixing the affinity particles according to this embodiment and the specimen, that is, can detect a target substance in the specimen by a latex agglutination method. preferable. Specifically, at least one of a step of mixing a specimen with a test reagent to obtain a mixture, a step of irradiating the mixture with light, and a transmitted light or a scattered light of the light irradiated on the mixture A step of detecting. By optically detecting the agglutination reaction occurring in the mixed solution, the target substance in the specimen can be detected, and the concentration of the target substance can also be measured. As a method for optically detecting the agglutination reaction, the amount of change in these values may be measured using an optical instrument capable of detecting scattered light intensity, transmitted light intensity, absorbance, and the like.
  • Example 1 To a 200 ml flask, 90 g of a phosphate buffer pH 7.4 (manufactured by Kishida Chemical Co., Ltd.) was added, and 0.9 g of polyvinylpyrrolidone K-30 (manufactured by Kishida Chemical Co., Ltd., molecular weight 40000) was dissolved. Next, 3.0 g of 3-methacryloxypropyltrimethoxysilane (LS-3380, manufactured by Shin-Etsu Chemical Co., Ltd.) and 9.0 g of styrene (manufactured by Kishida Chemical Co., Ltd.) were added, and stirring was performed for 10 minutes while blowing nitrogen at room temperature. went.
  • LS-3380 3-methacryloxypropyltrimethoxysilane
  • styrene manufactured by Kishida Chemical Co., Ltd.
  • the emulsion in the flask was heated to 70 ° C. in an oil bath.
  • a solution prepared by dissolving 0.3 g of potassium peroxodisulfate (manufactured by Wako Pure Chemical Industries, Ltd.) in 15 ml of phosphate buffer pH 7.4 (manufactured by Kishida Chemical Co., Ltd.) was added to the emulsion heated to 70 ° C. Added. After stirring at 70 ° C. for 7 hours, the mixture was returned to room temperature to obtain a dispersion of polymer fine particles.
  • this dispersion was centrifuged to collect polymer fine particles, and the supernatant was discarded.
  • the recovered polymer fine particles were redispersed in ion-exchanged water and then centrifuged again.
  • the collection of the polymer fine particles with a centrifuge and the redispersion with ion-exchanged water were repeated four times.
  • the dispersion of polymer fine particles thus obtained was adjusted to a concentration of 1.0% by weight.
  • Example 2 To a 200 ml flask, 90 g of phosphate buffer pH 6.4 (Kishida Chemical Co., Ltd.) was added, and 0.9 g of polyvinylpyrrolidone K-30 (Kishida Chemical Co., Ltd., molecular weight 40000) was dissolved. Next, 0.4 g of 3-methacryloxypropyltrimethoxysilane (LS-3380 manufactured by Shin-Etsu Chemical Co., Ltd.) and 11.6 g of styrene (manufactured by Kishida Chemical Co., Ltd.) were added, and stirring was performed for 10 minutes while blowing nitrogen at room temperature. went.
  • LS-3380 manufactured by Shin-Etsu Chemical Co., Ltd.
  • the emulsion in the flask was heated to 70 ° C. in an oil bath.
  • a solution prepared by dissolving 0.3 g of potassium peroxodisulfate (manufactured by Wako Pure Chemical Industries, Ltd.) in 15 ml of water was added to the emulsion heated to 75 ° C. After stirring for 6 hours at 75 ° C., the temperature was returned to room temperature to obtain a dispersion of polymer fine particles.
  • this dispersion was centrifuged to collect polymer fine particles, and the supernatant was discarded.
  • the recovered polymer fine particles were redispersed in ion-exchanged water and then centrifuged again.
  • the collection of the polymer fine particles with a centrifuge and the redispersion with ion-exchanged water were repeated four times.
  • the dispersion of polymer fine particles thus obtained was adjusted to a concentration of 1.0% by weight.
  • Example 3 To a 200 ml flask, 90 g of phosphate buffer pH 9.0 (manufactured by Kishida Chemical Co., Ltd.) was added, and 0.4 g of polyvinylpyrrolidone K-30 (manufactured by Kishida Chemical Co., Ltd., molecular weight 40000) was dissolved. Next, 7.2 g of 3-methacryloxypropyltrimethoxysilane (LS-3380 manufactured by Shin-Etsu Chemical Co., Ltd.) and 4.8 g of styrene (manufactured by Kishida Chemical Co., Ltd.) were added, and stirring was continued for 10 minutes while blowing nitrogen at room temperature. went.
  • phosphate buffer pH 9.0 manufactured by Kishida Chemical Co., Ltd.
  • polyvinylpyrrolidone K-30 manufactured by Kishida Chemical Co., Ltd., molecular weight 40000
  • the emulsion in the flask was heated to 70 ° C. in an oil bath.
  • a solution prepared by dissolving 0.3 g of potassium peroxodisulfate (manufactured by Wako Pure Chemical Industries, Ltd.) in 15 ml of water was added to the emulsion heated to 80 ° C. After stirring for 4 hours at 80 ° C., the temperature was returned to room temperature to obtain a dispersion of polymer fine particles.
  • this dispersion was centrifuged to collect polymer fine particles, and the supernatant was discarded.
  • the recovered polymer fine particles were redispersed in ion-exchanged water and then centrifuged again.
  • the collection of the polymer fine particles with a centrifuge and the redispersion with ion-exchanged water were repeated four times.
  • the dispersion of polymer fine particles thus obtained was adjusted to a concentration of 1.0% by weight.
  • Example 4 The 1.0% by weight polymer fine particle dispersion prepared in Example 1 was diluted to 0.1% by weight, and aminopropyltrimethoxysilane was added at a rate of 0.05% by weight with respect to the solution. Stirring was performed to modify the surface of the polymer fine particles with amino groups.
  • Example 5 The 1.0 wt% polymer fine particle dispersion prepared in Example 1 was diluted to 0.1 wt%, 2 wt% of 28 wt% ammonia water, and 0.05 wt% of mercaptopropylpropyltrimethoxysilane with respect to the solution. It was input at a rate. And it stirred at room temperature for 14 hours, and modified the surface of the polymer fine particle with the thiol group.
  • Example 6 The 1.0% by weight polymer fine particle dispersion prepared in Example 1 was diluted to 0.1% by weight, and aminopropyltrimethoxysilane was added at a rate of 0.05% by weight with respect to the solution. Stirring was performed to modify the surface of the polymer fine particles with amino groups. Succinic anhydride dissolved in dimethyl sulfoxide was added at a rate of 0.1 wt% to the polymer fine particle dispersion whose surface was modified with amino groups adjusted to 0.1% and stirred at room temperature for 14 hours. The surface of the polymer fine particle was modified with a carboxyl group.
  • Example 7 The 1.0% by weight polymer fine particle dispersion prepared in Example 1 was diluted to 0.1% by weight and bound to Si-tag fusion protein A (antibody binding protein (protein A) manufactured by Silicon Bio Co., Ltd.) and silanol groups. Fusion protein with the peptide to be added). Then, Si-tag fusion protein A was immobilized on the surface of the polymer fine particles.
  • Si-tag fusion protein A antibody binding protein (protein A) manufactured by Silicon Bio Co., Ltd.
  • Example 8 In the 1.0 wt% polymer fine particle dispersion prepared in Example 1, 0.5 wt% of 3-glycidyloxypropyltrimethoxysilane, 0.5 wt% of glycine, and 2 wt% of 28 wt% ammonia water were added. , And stirred at room temperature for 14 hours. The surface of the polymer fine particles was modified with glycidyl groups.
  • Example 9 A dimethyl sulfoxide solution of trimethoxypropyl succinic anhydride was added at a ratio of 0.4 wt% to the 1.0 wt% polymer fine particle dispersion prepared in Example 1 and stirred at room temperature for 14 hours. The surface of the polymer fine particles was modified with a dicarboxyl group.
  • CRP Anti-C-reactive protein
  • each polymer fine particle dispersion was centrifuged at 15000 rpm (20400 g) for 15 minutes to precipitate the fine particles. After removing the supernatant, the pellet of fine particles was redispersed with a buffer solution, and water-soluble carbodiimide WSC was added thereto. Further N-hydroxysuccinimide was added. The fine particle dispersion was stirred at room temperature for 30 minutes, and then the particles were collected by centrifugation. The microparticles were washed with buffer. The microparticles were redispersed in a buffer and anti-CRP antibody was added to a final antibody concentration of 100 ⁇ g / mL. After stirring for 180 minutes at room temperature, the particles were collected by centrifugation.
  • the particles were sufficiently washed with a buffer to obtain microparticles bound with an anti-CRP antibody.
  • the binding of the antibody was confirmed by measuring the amount of decrease in the antibody concentration in the buffer containing the antibody by BCA assay. It was found that about 100 to 500 antibodies were bound per particle.
  • the antibody-bound microparticles obtained in this example were stably dispersed in a buffer solution and did not need to be post-coated with BSA.
  • Anti-CRP antibody was bound to the polymer microparticles prepared in Example 5.
  • EMCS N- (6-Maleimidocaproyloxy) succinimide
  • the polymer fine particle dispersion was centrifuged at 15000 rpm (20400 g) for 15 minutes to precipitate the fine particles. After removing the supernatant, the pellet of fine particles was redispersed with a buffer solution, and a premaleidized anti-CRP antibody was added thereto. After stirring for 180 minutes at room temperature, the particles were collected by centrifugation.
  • the particles were sufficiently washed with a buffer to obtain microparticles bound with an anti-CRP antibody.
  • the binding of the antibody was confirmed by measuring the amount of decrease in the antibody concentration in the buffer containing the antibody by BCA assay. It was found that about 100 to 500 antibodies were bound per particle.
  • the antibody-bound microparticles obtained in this example were stably dispersed in a buffer solution and did not need to be post-coated with BSA.
  • Anti-CRP antibody was bound to the polymer microparticles prepared in Example 7.
  • the polymer fine particle dispersion of Example 7 was centrifuged at 15000 rpm (20400 g) for 20 minutes to precipitate the fine particles. After removing the supernatant, the pellet of microparticles was redispersed with a buffer solution, and anti-CRP antibody was added thereto so that the final antibody concentration was 200 ⁇ g / mL. After stirring at 4 ° C. for 16 hours, the particles were collected by centrifugation. The particles were sufficiently washed with a buffer to obtain microparticles bound with an anti-CRP antibody.
  • the binding of the antibody was confirmed by measuring the amount of decrease in the antibody concentration in the buffer containing the antibody by BCA assay. It was found that about 500 antibodies were bound per particle.
  • the antibody-bound microparticles obtained in this example were stably dispersed in a buffer solution and did not need to be post-coated with BSA.
  • Anti-CRP antibody was bound to the polymer fine particles of Comparative Example 1 and Comparative Example 2.
  • the polymer fine particle dispersions of Comparative Example 1 and Comparative Example 2 were centrifuged at 15000 rpm (20400 g) for 20 minutes to precipitate the fine particles. After removing the supernatant, the pellet of fine particles was redispersed with a buffer solution, and water-soluble carbodiimide WSC was added thereto. Further, an anti-CRP antibody was added so that the final antibody concentration was 100 ⁇ g / mL. After stirring for 180 minutes at room temperature, the BSA solution was added. Thereafter, particles coated with BSA were recovered by centrifugation.
  • the particles were washed with a buffer to obtain particles bound with BSA-coated anti-CRP antibody.
  • the binding of the antibody was confirmed by measuring the amount of decrease in the antibody concentration in the buffer containing the antibody by BCA assay. It was found that about 500 antibodies were bound per particle. However, immediately after the antibody was bound to the particles, it was confirmed by visual observation that the fine particles aggregated and precipitated, and thus could not be used for the subsequent evaluation of the latex agglutination reaction.
  • ⁇ Non-specific aggregation inhibition evaluation 60 ⁇ l of human serum solution diluted 15 times with 30 ⁇ l of each of the polymer fine particle dispersions prepared in Examples 1 to 9 and the polymer particle dispersion for immunodiagnostic used in Comparative Examples 1 and 2 was added at 37 ° C. Incubated for 5 minutes. Absorbance at 527 nm was measured before and after incubation, and the amount of change in absorbance before and after was measured three times.
  • FIG. 1 shows an average of three times. When the amount of change in absorbance was less than 0.1, non-specific aggregation was regarded as being suppressed, and when it was 0.1 or more, non-specific aggregation was evaluated. The results are shown in FIG.
  • FIG. 1 shows the average value of the absorbance change rate of three times. From FIG. 1, it can be seen that an agglutination reaction occurs due to the antigen-antibody reaction when CRP is added, and that the difference can be clearly recognized as compared with the increase rate when CRP is not added.
  • the fine particles according to this example can bind an antibody without using BSA, and the bound antibody has an antigen recognition ability.
  • the fine particles of this example can be suitably used as test particles that utilize an immune reaction, such as antibody-sensitized latex used in latex agglutination.
  • microparticles having non-specific adsorption of serum proteins could be obtained without using BSA while having a scaffold capable of binding a ligand such as an antibody.
  • grains using an immune reaction for example, the antibody-sensitized latex used by latex agglutination method.

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Abstract

L'invention concerne un procédé de production de particules comportant une première étape de préparation d'une émulsion en mélangeant un monomère polymérisable par voie radicalaire, un composé organosilane, un initiateur de polymérisation par voie radicalaire, un polymère hydrosoluble et une solution aqueuse, le composé organosilane étant un composé qui présente une aptitude à la polymérisation par voie radicalaire et qui possède un groupe alcoxy lié à un atome de silicium.
PCT/JP2019/017534 2018-04-27 2019-04-25 Particules et leur procédé de production Ceased WO2019208669A1 (fr)

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
WO2021075426A1 (fr) * 2019-10-15 2021-04-22 キヤノン株式会社 Particules et procédé de production de particules
WO2022209952A1 (fr) * 2021-03-31 2022-10-06 キヤノン株式会社 Particules et leur procédé de production

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4435048A4 (fr) * 2021-11-26 2025-10-22 Canon Kk Particules pour examens d'échantillon

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JPH01174506A (ja) * 1987-12-28 1989-07-11 Soken Kagaku Kk 架橋重合体粒子の製造方法
JPH05330887A (ja) * 1992-05-29 1993-12-14 Kanebo Nsc Ltd 変性ポリビニルアルコ−ルを用いたポリマ−エマル ジョン混入セメントの水中養生硬化方法
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WO2005097844A1 (fr) * 2004-03-31 2005-10-20 Sumitomo Bakelite Co., Ltd. Particule polymere
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JP2007204333A (ja) * 2006-02-03 2007-08-16 Showa Highpolymer Co Ltd セメントモルタル用エマルジョンおよびそれを配合したセメントモルタル組成物
JP2008527093A (ja) * 2005-01-05 2008-07-24 ワッカー ケミー アクチエンゲゼルシャフト 架橋性のシラン変性混合ポリマー

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JPS585302A (ja) * 1981-04-29 1983-01-12 レ−ム・ゲゼルシヤフト・ミツト・ベシユレンクテル・ハフツング 生物学的に作用を有する物質を固定するための核一外皮構造を有する再分散性ポリマ−ラテツクス、該ラテツクスの水性分散液、診断試薬の製法、抗原抗体反応検出法、抗体、微生物、ヴイ−ルス及び酵素の固定法、バイオ
JPH01174506A (ja) * 1987-12-28 1989-07-11 Soken Kagaku Kk 架橋重合体粒子の製造方法
JPH05330887A (ja) * 1992-05-29 1993-12-14 Kanebo Nsc Ltd 変性ポリビニルアルコ−ルを用いたポリマ−エマル ジョン混入セメントの水中養生硬化方法
JP2003277455A (ja) * 2002-03-25 2003-10-02 Jsr Corp ポリマー粒子の製造方法、ポリマー粒子および生理活性物質担体
WO2005097844A1 (fr) * 2004-03-31 2005-10-20 Sumitomo Bakelite Co., Ltd. Particule polymere
JP2005330394A (ja) * 2004-05-20 2005-12-02 Nippon Synthetic Chem Ind Co Ltd:The 再分散性アクリル系合成樹脂エマルジョン粉末組成物およびその製造方法
JP2008527093A (ja) * 2005-01-05 2008-07-24 ワッカー ケミー アクチエンゲゼルシャフト 架橋性のシラン変性混合ポリマー
JP2007204333A (ja) * 2006-02-03 2007-08-16 Showa Highpolymer Co Ltd セメントモルタル用エマルジョンおよびそれを配合したセメントモルタル組成物

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021075426A1 (fr) * 2019-10-15 2021-04-22 キヤノン株式会社 Particules et procédé de production de particules
JP2021063225A (ja) * 2019-10-15 2021-04-22 キヤノン株式会社 粒子、及び粒子の製造方法
US12479991B2 (en) 2019-10-15 2025-11-25 Canon Kabushiki Kaisha Particle and method for producing particle
WO2022209952A1 (fr) * 2021-03-31 2022-10-06 キヤノン株式会社 Particules et leur procédé de production

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