Composite particles, testing reagents, and methods for producing composite particles

Abstract

To provide composite particles with improved dispersion stability in liquid. [Solution] A composite particle for capturing a target substance, comprising a hollow particle and a polymer particle containing the hollow particle.

Description

[Technical Field]

[0001] The present invention relates to composite particles, reagents for testing, and methods for producing composite particles. [Background technology]

[0002] In the field of biomedicine, composite particles are known for their use in biosensing and drug delivery systems (DDS). One known biosensing method using composite particles involves detecting composite particles that have captured a target substance using a sensor to measure the presence or concentration of that target substance in a sample.

[0003] Patent Document 1 describes polymer particles suitable for surface modification, comprising poly-p-xylene in particulate form having at least one chemically active functional group, wherein the poly-p-xylene has a nanoscale or microscale particle size. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2015-209545 [Overview of the project] [Problems that the invention aims to solve]

[0005] This invention provides composite particles with improved dispersion stability in liquid. [Means for solving the problem]

[0006] The inventors of the present invention have discovered that the dispersion stability in liquid can be improved when the composite particles for capturing target substances include hollow particles and polymer particles containing hollow particles, and have completed the present invention.

[0007] In other words, the present invention provides the following composite particles, a reagent for testing, and a method for producing composite particles.

[0008] [1] A composite particle for capturing a target substance, Hollow particles and, Polymer particles containing the aforementioned hollow particles, A composite particle containing this particle. [2] The composite particle according to [1], wherein the volume-average diameter of the composite particle measured by a wet laser diffraction scattering method is 10.0 μm or more. [3] The composite particle according to [1] or [2], wherein the content of the hollow particles in the composite particle is 0.1% by mass or more and 50% by mass or less. [4] A composite particle according to any one of [1] to [3], wherein the volume-average diameter of the hollow particle measured by a wet laser diffraction scattering method is 1.0 μm or more and 10.0 μm or less. [5] The composite particle according to any one of [1] to [4], wherein the composite particle has at least one selected from the group consisting of an alkynyl group and an azide group. [6] A composite particle according to any one of [1] to [5], further comprising magnetic particles. [7] The composite particle according to [6], wherein the content of the magnetic particles in the composite particle is 0.1% by mass or more and 50.0% by mass or less. [8] N obtained by the following method 75 A composite particle described in any one of [1] to [7], wherein / N3 is 0.55 or greater. (method) Disperse the composite particles in ultrapure water so that the solid content is 2% by mass. Next, dilute with phosphate buffered saline to prepare a measurement sample such that the concentration of the composite particles in the measurement sample is 500 particles / μL or more and 5000 particles / μL or less. Perform a flow cytometry test on the measurement sample using a flow cytometer under the condition of a flow rate of 12 μL / min, and measure the measurement rate N [particles / sec] of the composite particles. Based on the measurement rate N3 3 seconds after the start of measurement and the measurement rate N 75 seconds after the start of measurement 75 , calculate N 75 / N3. [9] The composite particle according to any one of [1] to [8], wherein the target substance comprises a nucleic acid.

[10] The composite particle has a functional group, The composite particle according to any one of [1] to [9], further comprising the target substance bound to the functional group.

[11] The composite particle according to any one of [1] to

[10] , which is used for a test reagent.

[12] A test reagent comprising the composite particle according to any one of [1] to

[11] .

[13] a mixing step of obtaining a mixed liquid comprising a monomer, a polymerization initiator, hollow particles, and a dispersion stabilizer; a step of polymerizing the monomer to obtain composite particles; A method for producing composite particles, comprising:

[14] The mixing step comprises: a mixing step (A) of mixing the monomer, the polymerization initiator, and the hollow particles to obtain a mixed liquid a; a mixing step (B) of mixing the mixed liquid a and the dispersion stabilizer to obtain the mixed liquid; The method for producing composite particles according to

[13] , comprising:

[15] The mixing step (A) comprises: a mixing step (A1) of mixing the monomer and the polymerization initiator to obtain a mixed liquid a1; a mixing step (A2) of mixing a mixed liquid a1 and hollow particles to obtain a mixed liquid a, The method for producing composite particles according to

[14] , comprising

[16] the step of obtaining the composite particles includes a separation step of separating to obtain the composite particles, The method for producing composite particles according to any one of

[13] to

[15] , wherein the separation step includes a centrifugation step of centrifuging the mixed liquid to obtain the composite particles.

[17] the step of obtaining the composite particles includes a separation step of separating to obtain the composite particles, The method for producing composite particles according to any one of

[13] to

[16] , wherein the separation step includes a classification step of obtaining the composite particles from the mixed liquid by classification.

[18] the step of obtaining the composite particles includes a separation step of separating to obtain the composite particles, The method for producing composite particles according to any one of

[13] to

[17] , wherein the separation step includes a magnetic separation step of obtaining the composite particles from the mixed liquid by magnetic separation. Advantageous Effects of the Invention

[0009] According to the present invention, composite particles having improved dispersion stability in a liquid can be provided. Mode for Carrying Out the Invention

[0010] Hereinafter, embodiments of the present invention will be described. In the present specification, "A to B" indicating a numerical range means from A to B inclusive, unless otherwise specified. In the present specification, the notation "(meth)acrylate" represents a concept encompassing both acrylate and methacrylate. The same applies to similar notations such as "(meth)acrylic".

[0011] Composite Particles The composite particles of the present embodiment are composite particles for capturing a target substance, and include hollow particles and polymer particles containing the hollow particles.

[0012] The inventors have found that the dispersion stability in liquid can be improved for composite particles used to capture target substances by including hollow particles and polymer particles containing hollow particles. The composite particles of this embodiment can improve dispersion stability in liquid.

[0013] (hollow particles) The composite particles of this embodiment include hollow particles. In this embodiment, a hollow particle means a particle having voids inside. The hollow particles may have holes that connect the internal voids to the outside of the hollow particle.

[0014] The hollow particles may include, for example, hollow polymer particles such as hollow crosslinked poly(meth)acrylate particles, hollow crosslinked polystyrene particles, and hollow crosslinked poly(meth)acrylate-polystyrene particles; and hollow inorganic particles such as hollow silica particles and hollow carbon particles, or they may include hollow polymer particles, or they may include at least one selected from the group consisting of hollow crosslinked poly(meth)acrylate particles, hollow crosslinked polystyrene particles, and hollow crosslinked poly(meth)acrylate-polystyrene particles, or they may include at least one selected from the group consisting of hollow crosslinked poly(meth)acrylate particles and hollow crosslinked polystyrene particles.

[0015] The volume-average diameter of hollow particles measured by the wet laser diffraction scattering method may be 1.0 μm or more and 10.0 μm or less, 2.0 μm or more and 9.5 μm or less, 2.5 μm or more and 9.0 μm or less, or 3.0 μm or more and 8.5 μm or less. In this embodiment, the volume-average diameter of hollow particles measured by the wet laser diffraction scattering method is the value measured by the wet laser diffraction scattering method using a laser diffraction particle size distribution analyzer, after preparing a 1% by mass hollow particle dispersion with water as the dispersion medium.

[0016] The content of hollow particles in the composite particles is preferably 0.1% by mass or more and 50% by mass or less, more preferably 1% by mass or more and 40% by mass or less, and even more preferably 5% by mass or more and 30% by mass or less. By setting the content of hollow particles in the composite particles within the above range, the dispersion stability of the composite particles in liquid can be further improved.

[0017] (Polymer particles) The composite particles of this embodiment include polymer particles containing hollow particles.

[0018] It is preferable that the polymer particles contain hollow particles within them. In other words, it is preferable that the polymer particles encapsulate hollow particles. This can further improve the dispersion stability of the composite particles in liquid.

[0019] The polymer particles preferably comprise a polymer containing the following monomer-derived structural units.

[0020] The monomer preferably includes at least one selected from the group consisting of monomers having a (meth)acryloyl group and styrene-based monomers. This can improve the heat resistance of the composite particles. The monomer may also include monomers having a (meth)acryloyl group.

[0021] The monomer having a (meth)acryloyl group may include at least one selected from the group consisting of alkyl (meth)acrylate, hydroxyalkyl (meth)acrylate, and aminoalkyl (meth)acrylate, and may include alkyl (meth)acrylate, and may include at least one selected from the group consisting of methyl (meth)acrylate, ethyl (meth)acrylate, n-butyl (meth)acrylate, t-butyl (meth)acrylate, cyclohexyl (meth)acrylate, and isobornyl (meth)acrylate, and may include methyl (meth)acrylate.

[0022] The monomer having a (meth)acryloyl group may include a difunctional (meth)acrylate, and may include at least one selected from the group consisting of ethylene glycol di(meth)acrylate, diethylene glycol di(meth)acrylate, triethylene glycol di(meth)acrylate, and 1,5-pentanediol di(meth)acrylate, and may also include ethylene glycol di(meth)acrylate.

[0023] The monomers preferably include monomers having a carboxyl group, and more preferably include monomers having both a carboxyl group and a (meth)acryloyl group. The presence of a carboxyl group in the monomer allows for the synthesis of composite particles having a carboxyl group. The presence of both a carboxyl group and a (meth)acryloyl group in the monomer allows for copolymerization with other monomers having a (meth)acryloyl group, enabling the synthesis of composite particles with desired properties (e.g., high heat resistance) depending on the other monomer having a (meth)acryloyl group. The monomers may include, for example, at least one selected from the group consisting of (meth)acrylic acid, mono-2-(meth)acryloyloxyethyl succinic acid, mono-2-(meth)acryloyloxyethyl phthalic acid, and 2-(meth)acryloyloxyethyl hexahydrophthalic acid, and may also include mono-2-(meth)acryloyloxyethyl succinic acid.

[0024] The monomers preferably include monomers having a (meth)acryloyl group and monomers having a carboxyl group, and more preferably include alkyl (meth)acrylates and monomers having both a carboxyl group and a (meth)acryloyl group. This makes it possible to synthesize composite particles having a carboxyl group that have high heat resistance. The monomers may include at least one selected from the group consisting of methyl (meth)acrylate, ethyl (meth)acrylate, n-butyl (meth)acrylate, t-butyl (meth)acrylate, cyclohexyl (meth)acrylate, and isobornyl (meth)acrylate, as well as at least one selected from the group consisting of (meth)acrylic acid, mono-2-(meth)acryloyloxyethyl succinic acid, mono-2-(meth)acryloyloxyethyl phthalic acid, and 2-(meth)acryloyloxyethyl hexahydrophthalic acid, and may also include methyl (meth)acrylate and mono-2-(meth)acryloyloxyethyl succinic acid.

[0025] Styrene monomers include styrene and styrene derivatives, etc. Styrene monomers include, for example, at least one selected from the group consisting of styrene, divinylbenzene, 2-methylstyrene, 3-methylstyrene, 4-methylstyrene, 2,4,6-trimethylstyrene, 4-tert-butoxystyrene, 4-methoxystyrene, 3-chlorostyrene, 4-aminostyrene, α-methylstyrene, 2,4-dimethylstyrene, ethylstyrene, isopropylstyrene, butylstyrene, phenylstyrene, cyclohexylstyrene, 4-chloromethylstyrene, clotylbenzene, trivinylbenzene, and vinylnaphthalene.

[0026] (magnetic particles) The composite particles of this embodiment preferably further contain magnetic particles. This is preferable from the viewpoint of applications in medical and bio-fields such as biosensing and drug delivery systems (DDS), as the composite particles exhibit magnetism.

[0027] Generally, the specific gravity of magnetic particles is greater than that of polymer particles; therefore, when composite particles contain magnetic particles, the dispersion stability of the composite particles in liquid may be insufficient. However, with the composite particles of this embodiment, the dispersion stability in liquid can be improved even when the composite particles contain magnetic particles.

[0028] The magnetic particles may include, for example, at least one selected from the group consisting of magnetite (Fe3O4), maghemite (γ-Fe2O3), hematite (α-Fe2O3), manganese ferrite (MnFe2O4), cobalt ferrite (CoFe2O4), nickel ferrite (NiFe2O4), copper ferrite (CuFe2O4), Mn-Zn ferrite, Ni-Zn ferrite, Mn-Mg ferrite, Li ferrite, Cu-Zn ferrite, and goethite (FeO(OH)), or at least one selected from the group consisting of magnetite and maghemite.

[0029] The particle diameter of the magnetic particles may be between 1 nm and 5000 nm, between 3 nm and 1000 nm, or between 10 nm and 300 nm. In this embodiment, the particle diameter of the magnetic particles is the arithmetic mean particle diameter of any 10 magnetic particles when image analysis is performed on a transmission electron microscope (TEM) image. Generally, the specific gravity of magnetic particles is greater than that of polymer particles, so if the particle diameter of the magnetic particles is relatively large, the dispersion stability of the composite particles in liquid may be insufficient. With the composite particles of this embodiment, even if the particle diameter of the magnetic particles is above the lower limit, the dispersion stability in liquid can be improved. Also, if the particle diameter of the magnetic particles is relatively small, it is easier to reduce the variation in the magnitude of magnetism for each composite particle, so from the viewpoint of application in the medical and bio fields, it is preferable that the particle diameter of the magnetic particles is below the upper limit.

[0030] The content of magnetic particles in the composite particles is preferably 0.1% by mass or more and 50.0% by mass or less, more preferably 0.2% by mass or more and 40.0% by mass or less, even more preferably 0.5% by mass or more and 30.0% by mass or less, even more preferably 1.0% by mass or more and 20.0% by mass or less, and even more preferably 1.5% by mass or more and 10.0% by mass or less. By setting the content of magnetic particles in the composite particles to be above the lower limit, the magnetic properties of the composite particles can be improved. By setting the content of magnetic particles in the composite particles to be below the upper limit, the dispersion stability of the composite particles in liquid can be further improved.

[0031] The magnetic particles may be modified with a coupling agent. The coupling agent includes, for example, a silane coupling agent. The silane coupling agent may include at least one selected from the group consisting of silane coupling agents having a (meth)acryloyl group such as 3-(meth)acryloxypropyltrimethoxysilane, 3-(meth)acryloxypropylmethyldimethoxysilane, 3-(meth)acryloxypropylmethyldiethoxysilane, and 3-(meth)acryloxypropyltriethoxysilane; silane coupling agents having a vinyl group such as vinyltrimethoxysilane, vinyltriethoxysilane, and dimethoxymethylvinylsilane; and silane coupling agents having a styryl group such as p-styryltrimethoxysilane, and may also include 3-(meth)acryloxypropyltrimethoxysilane.

[0032] The magnetic particles may be modified with dispersants such as fatty acid-based dispersants, polymer-based dispersants, cationic dispersants, and anionic dispersants.

[0033] (Other components of the composite particle) The volume-average diameter of the composite particles measured by wet laser diffraction scattering is preferably 10.0 μm to 100.0 μm, more preferably 15.0 μm to 75.0 μm, even more preferably 20.0 μm to 50.0 μm, and even more preferably 25.0 μm to 40.0 μm. In this embodiment, the volume-average diameter of the composite particles measured by wet laser diffraction scattering is the value measured by wet laser diffraction scattering using a laser diffraction particle size distribution analyzer after preparing a composite particle dispersion with a concentration of 1% by mass using water as the dispersion medium. By setting the volume-average diameter of the composite particles to be below the above upper limit, the dispersion stability of the composite particles in liquid can be further improved. Furthermore, from the viewpoint of application in the medical and bio-fields, it is preferable to set the volume-average diameter of the composite particles to be above the above lower limit. For example, by setting the volume-average diameter of the composite particles to be above the above lower limit, it is possible to make it easier to analyze target substances.

[0034] In some cases, composite particles are dispersed in a liquid with a specific gravity lower than that of the composite particles themselves. In this case, if the volume-average diameter of the composite particles is relatively large, the dispersion stability of the composite particles in the liquid may be insufficient. With the composite particles of this embodiment, by setting the volume-average diameter of the composite particles within the above range, it is possible to further improve the dispersion stability of the composite particles in the liquid while enabling applications in the medical and biotechnology fields.

[0035] N obtained by the following method 75 / N3 may be 0.55 or higher, 0.60 to 1.10, 0.70 to 1.05, or 0.75 to 1.00.

[0036] (method) Disperse the composite particles in ultrapure water such that the solid content is 2% by mass. Next, dilute with phosphate-buffered saline to prepare a measurement sample such that the concentration of composite particles in the measurement sample is 500 particles / μL or more and 5000 particles / μL or less. Using a flow cytometer, perform a flow cytometry test on the measurement sample under the condition of a flow rate of 12 μL / min, and measure the counting rate N [particles / sec] of the composite particles. The counting rate N3 3 seconds after the start of measurement, and the counting rate N 75 seconds after the start of measurement 75 based on which, N 75 / N3 is calculated.

[0037] N 75 / N3 is an index representing the dispersion stability of the composite particles in a liquid. When N 75 / N3 is within the range of 0 or more and 1 or less and close to 1, this means that the change in the counting rate from 3 seconds after the start of measurement to 75 seconds after the start of measurement is small, and the dispersion stability of the composite particles in the liquid is high. Note that N 75 / N3 may exceed 1 due to factors such as the specific gravity of the composite particles being smaller than that of the dispersion medium or measurement errors.

[0038] The composite particles of the present embodiment are composite particles for capturing a target substance. The target substance may include, for example, biological-related substances such as nucleic acids (DNA, RNA, etc.), proteins (enzymes, antibodies, aptamers, receptors, etc.), peptides (glutathione, etc.), carbohydrates, lipids, and blood-derived substances (platelets, erythrocytes, leukocytes, etc.), and may also include nucleic acids.

[0039] The composite particles preferably have functional groups. When the composite particles have functional groups, the substance can be immobilized on the composite particles by reacting them with a substance capable of bonding to the functional group, which is preferable from the viewpoint of applications in the medical and bio-fields. Specifically, the composite particles may have at least one selected from the group consisting of alkynyl groups, azide groups, carboxyl groups, hydroxyl groups, amino groups, and epoxy groups, or at least one selected from the group consisting of alkynyl groups and azide groups. The alkynyl group may be a substituted or unsubstituted linear alkynyl group having 2 to 30 carbon atoms, at least one selected from the group consisting of ethynyl groups, propargyl groups, and trimethylsilylethynyl groups, or simply an ethynyl group. The alkynyl group may be a substituted or unsubstituted cyclic alkynyl group, at least one selected from the group consisting of cyclooctinyl groups, azacyclooctinyl groups, and dibenzocyclooctinyl groups. The composite particle may have at least one selected from the group consisting of an ethynyl group, a propargyl group, and an azide group, or it may have at least one selected from the group consisting of an ethynyl group and an azide group.

[0040] The composite particles preferably have functional groups on their surface. This makes them more receptive to substances that can bond with the functional groups.

[0041] If the composite particles have a carboxyl group, composite particles having an alkynyl group can be synthesized by a condensation reaction with a primary amine having an alkynyl group (e.g., propargylamine).

[0042] If the composite particles contain an alkynyl group, the target substance (e.g., azide-modified DNA) can be immobilized on the composite particles by a click reaction with the azide-modified target substance. Similarly, if the composite particles contain an azide group, the target substance (e.g., alkyne-modified DNA) can be immobilized on the composite particles by a click reaction with the alkyne-modified target substance. By immobilizing the target substance on the composite particles in this way, the target substance can be made easier to analyze.

[0043] If the composite particles have functional groups, the composite particles may further contain a target substance bonded to the functional groups.

[0044] The composite particles of this embodiment are preferably used in a testing reagent. That is, the testing reagent of this embodiment contains the composite particles of this embodiment.

[0045] (Method for manufacturing composite particles) The method for producing composite particles according to this embodiment comprises a mixing step of obtaining a mixture containing a monomer, a polymerization initiator, hollow particles, and a dispersion stabilizer, and a step of polymerizing the monomer to obtain composite particles. By polymerizing the monomer after the mixing step, it is possible to easily adjust the physical properties of the composite particles (for example, the degree of hollowness of the composite particles).

[0046] The monomer includes, for example, at least one selected from the group consisting of the (meth)acrylic monomers and styrene monomers described above.

[0047] The polymerization initiator may include a thermal polymerization initiator, an azo polymerization initiator and a thermal radical polymerization initiator such as a peroxide, or an azo polymerization initiator. The azo polymerization initiator may include, for example, at least one selected from the group consisting of 2,2'-azobis(2,4-dimethylvaleronitrile), 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile), 2,2'-azobis(isobutyronitrile), 2,2'-azobis(2-methylbutyronitrile), 1,1'-azobis(cyclohexane-1-carbonnitrile), and azobis(isobutyrate)dimethyl.

[0048] The dispersion stabilizer may include a polyvinyl alcohol-based dispersion stabilizer, or a partially saponified polyvinyl alcohol-based dispersion stabilizer.

[0049] The mixing step preferably includes a mixing step (A) in which a monomer, a polymerization initiator, and hollow particles are mixed to obtain a mixture a, and a mixing step (B) in which mixture a and a dispersion stabilizer are mixed to obtain a mixture. By adding the dispersion stabilizer after mixing step (A), it is possible to easily adjust the diameter of the emulsion containing each component, and thus it is possible to easily adjust the particle size of the composite particles.

[0050] The mixing step (A) preferably includes a mixing step (A1) in which a monomer and a polymerization initiator are mixed to obtain a mixture a1, and a mixing step (A2) in which mixture a1 and hollow particles are mixed to obtain a mixture a. Adding hollow particles after mixing step (A1) makes it easier to disperse the hollow particles in the mixture, and thus makes it easier to produce the desired composite particles.

[0051] If the mixture contains a polymerization inhibitor, mixing step (B) is preferably a step of mixing mixture a, a dispersion stabilizer, and a polymerization inhibitor to obtain a mixture. By adding the polymerization inhibitor in mixing step (B) after mixing step (A), the generation of fine particles can be suppressed, and the composition and particle size of the composite particles can be easily adjusted.

[0052] The process of obtaining composite particles preferably includes a separation step to obtain composite particles. This allows for the removal of impurities other than the composite particles, thereby improving the purity of the composite particles.

[0053] Specifically, the separation step may include a centrifugation step in which the mixture is centrifuged to obtain composite particles. The separation step may include a classification step in which composite particles are obtained from the mixture by classification. The separation step may include a magnetic separation step in which composite particles are obtained from the mixture by magnetic separation.

[0054] Although embodiments of the present invention have been described above, these are merely examples, and various other configurations can be adopted. Furthermore, the present invention is not limited to the embodiments described above, and modifications, improvements, etc., within the scope that can achieve the objectives of the present invention are included in the present invention. [Examples]

[0055] The present invention will be described in detail below with reference to the examples. However, the present invention is not limited in any way to the descriptions in these examples.

[0056] (1) Preparation of magnetic particles modified with a silane coupling agent 60 g of FeCl2·4H2O (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and 135 g of FeCl3·6H2O (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were added to a separable flask. Then, 1500 mL of pure water, which had been pre-purged with nitrogen by nitrogen bubbling, was added to dissolve the FeCl2·4H2O and FeCl3·6H2O. Next, 400 mL of 28% by weight aqueous ammonia was poured in, and the mixture was stirred at 200 rpm for 30 minutes in a 30°C oil bath, and magnetic particles were obtained by magnetic recovery. The obtained magnetic particles were washed by repeating the process of adding water and magnetic recovery five times. Water was added to the washed magnetic particles so that the amount of magnetic particles was 20 parts by mass per 100 parts by mass of slurry to prepare a slurry. 320 g of diethylamine and 64 g of slurry were added to a reaction vessel and treated in an ultrasonic bath for 15 minutes. 9.1 g of distilled water and 31.4 g of 3-methacryloxypropyltrimethoxysilane (manufactured by Tokyo Chemical Industry Co., Ltd.) as a silane coupling agent were added, and the mixture was treated in an ultrasonic bath for 30 minutes. The reaction vessel was then placed in a shaker and stirred at 100 rpm for 24 hours, after which the particles were recovered by centrifugation. The recovered particles were washed by adding acetone and centrifugation three times, and then air-dried to obtain magnetic particles (median diameter: 146 nm) modified with the silane coupling agent.

[0057] (2) Synthesis of composite particles (Example 1) 2.99 parts by mass of methyl methacrylate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), 0.43 parts by mass of ethylene glycol dimethacrylate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and 0.86 parts by mass of mono-2-methacryloyloxyethyl succinic acid (manufactured by Kyoeisha Chemical Co., Ltd.) were mixed, and then 0.26 parts by mass of 2,2'-azobis(2,4-dimethylvaleronitrile) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added as a polymerization initiator. The mixture was treated in an ultrasonic bath using a tabletop ultrasonic cleaner (manufactured by Yamato Scientific Co., Ltd., product name: CPX5800H-J) to completely dissolve the polymerization initiator. 0.13 parts by mass of magnetic particles modified with the silane coupling agent prepared in (1) above were added, and the mixture was treated in an ultrasonic bath for 15 minutes to completely disperse the magnetic particles modified with the silane coupling agent. 0.46 parts by mass of hollow particles (manufactured by Sekisui Chemical Co., Ltd., product name: TP-HS series, volume average diameter: 3.7 μm, coefficient of variation: 34.26%, resin composition: cross-linked polystyrene) were added. Using a rotor / stator homogenizer (manufactured by IKA Japan Co., Ltd., product name: T18 digital ULTRA-TURRAX, shaft generator: S18N-10G), the mixture was stirred at 8000 rpm for 5 minutes in an ice bath to completely disperse the hollow particles and obtain a monomer mixture.

[0058] 0.03 parts by mass of sodium nitrite (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to a four-neck separable flask. Then, ion-exchanged water and an aqueous solution of partially saponified polyvinyl alcohol (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., degree of saponification: 88 mol%, degree of polymerization: 1500) were added as a dispersion stabilizer, in a ratio of 93.0 parts by mass of ion-exchanged water and 2.0 parts by mass of polyvinyl alcohol to obtain an aqueous medium. The monomer mixture was added to the aqueous medium and stirred at 3000 rpm for 10 minutes in an ice bath using a rotor / stator homogenizer (manufactured by IKA Japan Co., Ltd., product name: T18 digital ULTRA-TURRAX, shaft generator: S18N-19G) to obtain a suspension in which the monomer mixture was dispersed as droplets in the aqueous medium.

[0059] A four-necked separable flask was fitted with a stirring blade, a reflux condenser, a nitrogen purge tube, and a thermometer. Nitrogen purging was performed by blowing nitrogen gas through a 23°C water bath at 150 rpm for 30 minutes while stirring. Subsequently, the water bath was heated to 70°C and suspension polymerization was carried out for 24 hours.

[0060] After polymerization, the suspension was passed through a filter (Yotoriyama Co., Ltd., 200 mesh) to remove aggregates. Next, the filtrate was washed by centrifugation followed by the addition of water three times, followed by centrifugation followed by the addition of ethanol once, and followed by centrifugation followed by the addition of water once. The washed liquid was subjected to wet classification using wire mesh sieves with mesh openings of 32 μm and 20 μm, and the particles remaining on the 20 μm wire mesh sieve were collected. Further magnetic separation was performed using a magnet. The material to be dried was transferred to a 50 mL glass bottle, and the glass bottle was placed in a vacuum dryer (AS ONE Corporation, product name: AVO-250NS-D). Using a vacuum pump (Sato Vacuum Co., Ltd., product name: P135D, 0.67 Pa or less), the material was vacuum dried overnight at 50°C to obtain a dried powder. 0.5 parts by mass of the obtained dried powder was mixed with 10 parts by mass of 1 N hydrochloric acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and the particles were washed by exposing the mixture to the acid for 60 minutes. The particles were then purified by magnetic separation, and a purified suspension was obtained by adding water and irradiating with ultrasound.

[0061] Magnetic separation was performed on the purified suspension using a magnet. The material to be dried was transferred to a 50 mL glass bottle, and the glass bottle was placed in a vacuum dryer. Using a vacuum pump, it was vacuum dried overnight at 50°C to obtain an acid-washed dried powder. 0.12 g of the acid-washed dried powder was mixed with 0.11 g of 1-hydroxybenzotriazole monohydrate (manufactured by Tokyo Chemical Industry Co., Ltd.) and 2.4 g of deionized water, and the particles were dispersed by ultrasonic irradiation. 0.72 mL of 0.1 N hydrochloric acid, 0.27 mL of a 0.27 mol / L aqueous solution of propargylamine (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and 0.014 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were added, and the mixture was shaken overnight at room temperature using a shaker (manufactured by AS ONE Corporation). Next, the particles were washed by adding 0.1 N hydrochloric acid once after centrifugation, and by adding deionized water three times after centrifugation, thereby obtaining composite particles with alkynyl groups introduced onto their surface.

[0062] Furthermore, a portion of the purified suspension before magnetic separation was pipetted, vacuum-dried, and dried to obtain a dried powder. Differential thermal-thermogravimetric analysis (TG-DTA) was performed using a differential thermal-thermogravimetric analyzer (Shimadzu Corporation "DTG-60A") in accordance with JIS K 0129:2005. 5 mg of the dried powder was placed in a platinum cell and set in the measurement unit. Under nitrogen flow, the measurement unit was heated from room temperature to 800°C at a heating rate of 10°C / min, held at 800°C for 2 minutes, and then the mass of the residue was measured. The mass of the residue was taken as the mass of magnetic particles in the composite particles, and the content of magnetic particles in the composite particles was determined.

[0063] (Example 2) In the wet classification operation, composite particles with alkynyl groups introduced on their surface were obtained in the same manner as in Example 1, except that the particles remaining on the 32 μm mesh sieve were collected using wire mesh sieves with mesh openings of 45 μm and 32 μm.

[0064] (Example 3) Composite particles with alkynyl groups introduced on their surface were obtained in the same manner as in Example 1, except that hollow particles manufactured by Sekisui Chemical Co., Ltd., product name: TP-HS series, volume average diameter: 8.0 μm, coefficient of variation: 32.97%, resin composition: cross-linked polymethyl methacrylate were used.

[0065] (Example 4) In the wet classification operation, composite particles with alkynyl groups introduced on their surface were obtained in the same manner as in Example 3, except that the particles remaining on the 32 μm mesh sieve were collected using wire mesh sieves with mesh openings of 45 μm and 32 μm.

[0066] (Comparative Example 1) 1.86 parts by mass of methyl methacrylate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), 1.85 parts by mass of ethylene glycol dimethacrylate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and 0.93 parts by mass of mono-2-methacryloyloxyethyl succinic acid (manufactured by Kyoeisha Chemical Co., Ltd.) were mixed, and then 0.28 parts by mass of 2,2'-azobis(2,4-dimethylvaleronitrile) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added as a polymerization initiator. The mixture was treated in an ultrasonic bath using a tabletop ultrasonic cleaner (manufactured by Yamato Scientific Co., Ltd., product name: CPX5800H-J) to completely dissolve the polymerization initiator. 0.14 parts by mass of magnetic particles modified with the silane coupling agent prepared in (1) above were added, and the mixture was treated in an ultrasonic bath for 15 minutes to completely disperse the magnetic particles modified with the silane coupling agent and obtain a monomer mixture. Composite particles with alkynyl groups introduced on their surface were obtained in the same manner as in Example 1.

[0067] (Comparative Example 2) In the wet classification operation, composite particles with alkynyl groups introduced on their surface were obtained in the same manner as in Comparative Example 1, except that the particles remaining on the 32 μm mesh sieve were collected using wire mesh sieves with mesh openings of 45 μm and 32 μm.

[0068] (3) Measurement of the average volume diameter A 1% by mass dispersion of composite particles was prepared using water as the dispersion medium. The volume-average diameter and coefficient of variation of the composite particles were measured using a wet laser diffraction scattering method with a laser diffraction particle size distribution analyzer (Beckman Coulter, product name: LS 13 320).

[0069] (4) Flow cytometry The composite particles were dispersed in ultrapure water to a solid content of 2% by mass. Next, the sample was diluted with phosphate-buffered saline (PBS, NaCl: 137 mM, KCl: 2.7 mM, Na2HPO4: 10 mM, KH2PO4: 1.8 mM) to prepare a sample for measurement, with a composite particle concentration between 500 and 5000 particles / μL. Flow cytometry was performed on the sample using a flow cytometer (Becton Dickinson, product name: BD LSRFortessa X-20) at a flow rate of 12 μL / min (measurement speed: LOW) to measure the composite particle velocity N [particles / second]. The instrument parameters were Voltage: 130 and Threshold: 200. Voltage is a parameter related to forward scattering intensity, and Threshold is a parameter related to noise reduction. Measurement speed N3 3 seconds after the start of measurement, and measurement speed N 75 seconds after the start of measurement 75 Based on N 75 / N3 was calculated.

[0070] (5) Assessment of sedimentation A dispersion was prepared by dispersing the composite particles in ultrapure water in a 2 mL microcentrifuge tube so that the solid content was 2% by mass. After shaking the microcentrifuge tube and letting it stand for 1 minute, the dispersion was visually observed and the sedimentation properties of the composite particles were evaluated according to the following criteria. A: No composite particles were observed settling in the dispersion. B: Composite particles that settled in the dispersion were observed.

[0071] Table 1 shows the evaluation results for each example and comparative example.

[0072] [Table 1]

Claims

1. A composite particle for capturing a target substance, Hollow particles and, Polymer particles containing the aforementioned hollow particles, A composite particle containing this particle.

2. The composite particle according to claim 1, wherein the volume-average diameter of the composite particle, as measured by a wet laser diffraction scattering method, is 10.0 μm or more.

3. The composite particle according to claim 1 or 2, wherein the content of the hollow particles in the composite particle is 0.1% by mass or more and 50% by mass or less.

4. The composite particle according to claim 1 or 2, wherein the volume-average diameter of the hollow particle, as measured by a wet laser diffraction scattering method, is 1.0 μm or more and 10.0 μm or less.

5. The composite particle according to claim 1 or 2, wherein the composite particle has at least one selected from the group consisting of an alkynyl group and an azide group.

6. The composite particle according to claim 1 or 2, further comprising magnetic particles.

7. The composite particle according to claim 6, wherein the content of the magnetic particles in the composite particle is 0.1% by mass or more and 50.0% by mass or less.

8. N obtained by the following method ７５ / N ３ The composite particle according to claim 1 or 2, wherein the ratio is 0.55 or higher. (method) The composite particles are dispersed in ultrapure water so that the solid content is 2% by mass. Then, the composite particles in the sample for measurement are diluted with phosphate-buffered saline so that the concentration of composite particles in the sample is between 500 particles / μL and 5000 particles / μL, and the sample for measurement is prepared. Using a flow cytometer, a flow cytometry test is performed on the sample for measurement under the condition of a flow rate of 12 μL / min, and the measured velocity N [particles / second] of the composite particles is measured. The measured velocity N 3 seconds after the start of measurement is... ３ , and the measured speed N 75 seconds after the start of measurement ７５ Based on N ７５ / N ３ Calculate.

9. The composite particle according to claim 1 or 2, wherein the target substance comprises nucleic acid.

10. The composite particles have functional groups, The composite particle according to claim 1 or 2, further comprising the target substance bonded to the functional group.

11. A composite particle according to claim 1 or 2, used in a diagnostic reagent.

12. A diagnostic reagent comprising the composite particles described in claim 1 or 2.

13. A mixing step to obtain a mixture containing monomers, polymerization initiators, hollow particles, and dispersion stabilizers, A step of polymerizing the monomer to obtain composite particles, A method for producing composite particles, comprising:

14. The aforementioned mixing step is A mixing step (A) involves mixing the monomer, the polymerization initiator, and the hollow particles to obtain a mixed solution a. A mixing step (B) to obtain the mixture by mixing the aforementioned mixture a with the dispersion stabilizer, A method for producing composite particles according to claim 13, including the method described in claim 13.

15. The mixing step (A) is as follows: A mixing step (A1) to obtain a mixed solution a1 by mixing the monomer and the polymerization initiator, A mixing step (A2) involves mixing mixture a1 with hollow particles to obtain mixture a, A method for producing composite particles according to claim 14, including the method described in claim 14.

16. The step of obtaining the composite particles includes a separation step of separating the particles to obtain the composite particles, The method for producing composite particles according to any one of claims 13 to 15, wherein the separation step includes a centrifugation step of centrifuging the mixed liquid to obtain the composite particles.

17. The step of obtaining the composite particles includes a separation step of separating the particles to obtain the composite particles, The method for producing composite particles according to any one of claims 13 to 15, wherein the separation step includes a classification step of obtaining the composite particles from the mixed liquid by classification.

18. The step of obtaining the composite particles includes a separation step of separating the particles to obtain the composite particles, The method for producing composite particles according to any one of claims 13 to 15, wherein the separation step includes a magnetic separation step of obtaining the composite particles from the mixed liquid by magnetic separation.

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