Resin particle composition, test kit, and manufacturing method for resin particle composition

Abstract

To provide a resin particle composition with superior solvent resistance and high fluorescence intensity.SOLUTION: There is provided with a resin particle composition containing resin particles incorporating a marker substance. The resin particle composition is manufactured by a method comprising the steps of: producing a mixture by mixing the marker substance and a polymerizable monomer using subcritical water; producing a monomer particle dispersion liquid by mixing and cooling the mixture and an emulsifier; and producing the resin particles by causing a polymerization reaction between the monomer particle dispersion liquid and a crosslinkable monomer.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a resin particle composition, a test kit, and a method for producing a resin particle composition. [Background technology]

[0002] Resin particle compositions containing a labeling substance have been found to be useful as fluorescent labels in clinical tests in the fields of biological and medical diagnosis, for example. Depending on the type of labeling substance, the labeling substance is used in a dilute or high-concentration state in which it is uniformly dispersed at the molecular level in a polymer host material or solvent. For example, Patent Document 1 discloses a method of granulating polymer particles in the presence of a fluorescent dye and incorporating the fluorescent dye into the polymer particles during the polymer granulation process. Patent Document 2 discloses an infiltration method in which the polymer particles are swelled in a liquid containing the fluorescent dye to incorporate the fluorescent dye into the polymer particles. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Patent No. 6978081 [Patent Document 2] Patent No. 6769728 Summary of the Invention [Problem to be solved by the invention]

[0004] However, in Patent Document 1, when the monomer oil droplets are micronized using a high-shear dispersing device such as a high-pressure homogenizer, the dispersion liquid becomes hot, and the dissolved fluorescent dye precipitates, so that the amount of fluorescent dye that can be encapsulated in the polymer particles is limited. In Patent Document 2, there is a problem that the fluorescent dye-encapsulated particles made using polymer particles that easily swell in liquid are dissolved in a solvent, resulting in low fluorescence intensity. Thus, Patent Document 1 and Patent Document 2 fail to realize a resin particle composition that has excellent solvent resistance and high fluorescence intensity.

[0005] Therefore, an object of the present invention is to provide a resin particle composition having excellent solvent resistance and high fluorescent intensity. [Means for solving the problem]

[0006] In order to achieve the object of the present invention, the resin particle composition of the present invention is a resin particle composition containing resin particles encapsulating a labeling substance, and is produced by a method including the steps of: mixing the labeling substance and a polymerizable monomer with subcritical water to produce a mixture; mixing and cooling the mixture with an emulsifier to produce a monomer particle dispersion; and performing a polymerization reaction between the monomer particle dispersion and a crosslinkable monomer to produce the resin particles. Effect of the Invention

[0007] According to the present invention, it is possible to provide a resin particle composition having excellent solvent resistance and high fluorescent intensity. [Brief description of the drawings]

[0008] [Figure 1] FIG. 2 is a diagram illustrating a process for producing a monomer particle dispersion using a supercritical water supply device. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The following embodiments are described in detail. Note that the following embodiments do not limit the scope of the invention, and not all of the combinations of features described in the embodiments are essential to the invention. Two or more of the features described in the embodiments may be combined in any combination.

[0010] (Resin particle composition) The resin particle composition according to one embodiment includes resin particles encapsulating a labeling substance. The resin particle composition according to one embodiment is produced by a method including the steps of: mixing the labeling substance and a polymerizable monomer with subcritical water to produce a mixture; mixing and cooling the mixture with an emulsifier to produce a monomer particle dispersion; and carrying out a polymerization reaction between the monomer particle dispersion and a crosslinkable monomer to produce resin particles.

[0011] (Resin particles) The polymerizable monomer according to one embodiment includes at least one of an aromatic monomer, a (meth)acrylic monomer, and a monomer having a functional group. The resin particle according to one embodiment includes, as constituent units, 40 to 95 mass% of an aromatic monomer and / or a (meth)acrylic monomer, 0 to 10 mass% of a monomer having a functional group, and 5 to 60 mass% of a crosslinkable monomer, relative to 100 mass% of the total polymerizable monomers. Here, "100 mass% of the total polymerizable monomers" refers to the mass% of all polymerizable monomers constituting the resin particle, and refers to the total mass% of the polymerizable monomers and the crosslinkable monomers. In addition, "40 to 95 mass% aromatic monomer and / or (meth)acrylic monomer" means that three types of forms (40 to 95 mass% aromatic monomer, 40 to 95 mass% (meth)acrylic monomer, and a mixture of 40 to 95 mass% aromatic monomer and (meth)acrylic monomer) are included.

[0012] The resin particles according to one embodiment have an average particle size of 50 to 500 nm, where the average particle size is measured by a dynamic light scattering method.

[0013] The polymerizable monomer, emulsifier, polymerization initiator, etc. that can be used in the method for producing resin particles of the present invention will be described. In this specification, the term "(meth)acrylic" means both "acrylic" and "methacrylic", and the term "(meth)acrylate" means both "acrylate" and "methacrylate".

[0014] Polymerizable monomers that can be used to produce resin particles are listed below as examples, but are not limited to these monomers. The following monomers can be used alone or in combination of two or more kinds.

[0015] The aromatic monomer is not particularly limited as long as it dissolves the labeling substance, and examples thereof include one or more selected from the group consisting of styrene, methylstyrene, chlorostyrene, methoxystyrene, α-methylstyrene, p-nitrostyrene, ethylvinylbenzene, vinylnaphthalene, benzyl acrylate, benzyl methacrylate, phenylethyl acrylate, phenylethyl methacrylate, phenylpropyl acrylate, phenylpropyl methacrylate, phenylnonyl acrylate, and phenylnonyl methacrylate.

[0016] The content of the aromatic monomer according to one embodiment is preferably 40% by mass or more, and more preferably 40 to 95% by mass, based on 100% by mass of all polymerizable monomers.

[0017] Examples of the (meth)acrylic monomer include methyl (meth)acrylate, ethyl (meth)acrylate, n-propyl (meth)acrylate, i-propyl (meth)acrylate, n-butyl (meth)acrylate, i-butyl (meth)acrylate, sec-butyl (meth)acrylate, t-butyl (meth)acrylate, n-amyl (meth)acrylate, i-amyl (meth)acrylate, n-hexyl (meth)acrylate, and 2-ethylhexyl. Examples of the methacrylates include alkyl (meth)acrylates having an alkyl group having 1 to 18 carbon atoms, such as (meth)acrylate, n-octyl (meth)acrylate, nonyl (meth)acrylate, decyl (meth)acrylate, dodecyl (meth)acrylate, lauryl (meth)acrylate, and stearyl (meth)acrylate; and cycloalkyl (meth)acrylates, such as cyclohexyl (meth)acrylate and pt-butylcyclohexyl (meth)acrylate.

[0018] The content of the (meth)acrylic monomer according to one embodiment is preferably 40% by mass or more, and more preferably 40 to 95% by mass, based on 100% by mass of all polymerizable monomers.

[0019] As the crosslinkable monomer, one or more selected from the group consisting of allyl methacrylate, ethylene glycol dimethacrylate, ethylene glycol diacrylate, butanediol diacrylate, butanediol dimethacrylate, neopentyl glycol dimethacrylate, hexanediol dimethacrylate, triethylene glycol dimethacrylate, tetraethylene glycol dimethacrylate, trimethylolpropane trimethacrylate, pentaerythritol tetramethacrylate, and divinylbenzene can be used. As the crosslinkable monomer, aromatic divinylbenzene is particularly preferred.

[0020] The content of the crosslinkable monomer according to one embodiment is preferably 5 to 60 mass%, 5 to 50 mass%, or 5 to 40 mass%, more preferably 5 to 30 mass%, based on 100 mass% of the total polymerizable monomers. When the resin particles contain the crosslinkable monomer in the above-mentioned predetermined mass%, the solvent resistance of the resin particles can be further improved, and swelling of the resin particles in a solvent can be suppressed. The solvent resistance refers to the difficulty of eluting the labeling substance to the outside of the resin particles.

[0021] Examples of monomers having functional groups include carboxyl group-containing polymerizable monomers such as (meth)acrylic acid, acrylic acid dimer, 2-hydroxyethyl (meth)acrylate, and 4-hydroxybutyl (meth)acrylate, which are hydroxyalkyl (meth)acrylates reacted with maleic anhydride, succinic anhydride, and phthalic anhydride; hydroxyethyl acrylate, hydroxyethyl methacrylate, 2-hydroxyethyl (meth)acrylate, 2-(3-)hydroxypropyl (meth)acrylate, 4-hydroxybutyl (meth)acrylate, and the like. Examples of the hydroxyalkyl (meth)acrylates include hydroxyalkyl (meth)acrylates such as ethyl (meth)acrylate and glycerol mono(meth)acrylate; aminoalkyl (meth)acrylates such as 2-aminoethyl (meth)acrylate, 2-dimethylaminoethyl (meth)acrylate, 2-aminopropyl (meth)acrylate and 2-butylaminoethyl (meth)acrylate; and amide group-containing (meth)acrylates such as (meth)acrylamide, N-methylolacrylamide and N-butoxymethyl (meth)acrylamide.

[0022] The monomer having a functional group according to an embodiment is preferably 0 to 10% by mass relative to 100% by mass of the total polymerizable monomer. Here, when the resin particles are used as luminescent particles for antigen testing and luminescent particles for antibody testing described below, the monomer having a functional group can be used to bind the resin particles to target substances such as antibodies and antigens. Therefore, when the resin particles of the present invention are not used as luminescent particles for antigen testing and luminescent particles for antibody testing, the monomer having a functional group may not be an essential component for producing the resin particles.

[0023] The functional group according to one embodiment includes at least one of a hydroxy group, a carboxy group, an amino group, and an amide group.

[0024] (labeled substance) The content of the labeling substance according to one embodiment is preferably 0.1 to 15% by mass, and more preferably 0.5 to 10% by mass, based on 100% by mass of the total polymerizable monomers.

[0025] The labeling substance according to one embodiment includes at least one of a fluorescent dye and an aggregation induced luminescence dye (hereinafter also referred to as an AIE dye).

[0026] A fluorescent dye is a dye that emits fluorescence. When a fluorescent dye absorbs light, the electrons in the dye are excited, and when they return to their ground state, they release excess energy as electromagnetic waves. This electromagnetic wave is fluorescence, and the wavelength of the fluorescence is always longer than the wavelength of the absorbed light (excitation light). The difference between the wavelength of the excitation light and the wavelength of the fluorescence is called the Stokes shift, and the larger this difference is, the easier it is to distinguish the fluorescence from the excitation light, making the dye easier to detect.

[0027] Examples of fluorescent dyes include compounds and derivatives having fluorescent properties such as merocyanine, perylene, acridine, anthracene, perylene, luciferin, pyranine, stilbene, rhodamine, coumarin, 4-(dicyanomethylene)-2-methyl-6-(4-dimethylaminostyryl)-4H-pyran (DCM), pyrromethene, fluorescein, and umbelliferone, and one or more types selected from the above group can be used.

[0028] AIE dyes are aggregation-induced emission dyes (AIE = Aggregation-Induced Emission) that do not emit light in a dilute solution but emit strong light in a solid or aggregated state, and show the opposite behavior to that of general fluorescent dyes. In other words, AIE dyes are dyes that allow for high concentration and high brightness, which are difficult to achieve with normal fluorescent dyes due to concentration quenching, etc.

[0029] Examples of the AIE dye include silole ring-containing compounds and derivatives such as 1,1-dimethyl-2,3,4,5-tetraphenylsilole, 1,1,2,3,4,5-hexaphenylsilole, 1,1-dimethyl-2,5-dianisyl-3,4-diphenylsilole, and 1,1-diallyl-2,3,4,5-tetraphenylsilole, hydrocarbon aromatic compounds and derivatives such as tetraphenylethylene, heteroaromatic compounds and derivatives, and rhodamine compounds and derivatives, and one or more selected from the above group can be used. In particular, the AIE dye is preferably a silole ring-containing compound from the viewpoint of being easily incorporated into resin particles.

[0030] (Polymerization initiator) The polymerization initiator is not particularly limited, and any of those known in the art can be used. For example, the following polymerization initiators generally used in radical polymerization can be appropriately used.

[0031] Examples of the polymerization initiator include persulfates such as potassium persulfate, sodium persulfate, and ammonium persulfate; oils such as 2,2'-azobisisobutyronitrile, 2,2'-azobis(2-methylbutyronitrile), 2,2'-azobis(2,4-dimethylvaleronitrile), 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile), and 2-phenylazo-4-methoxy-2,4-dimethylvaleronitrile. Soluble azo compounds; 2,2'-azobis{2-methyl-N-[1,1-bis(hydroxymethyl)-2-hydroxyethyl]propionamide}, 2,2'-azobis{2-methyl-N-[2-(1-hydroxyethyl)]propionamide}, 2,2'-azobis{2-methyl-N-[2-(1-hydroxybutyl)]propionamide}, 2,2'-azobis[2-(5-methyl-2-imidazolin-2-yl) propane] and its salts, 2,2'-azobis[2-(2-imidazolin-2-yl)propane] and its salts, 2,2'-azobis[2-(3,4,5,6-tetrahydropyrimidin-2-yl)propane] and its salts, 2,2'-azobis{2-[1-(2-hydroxyethyl)-2-imidazolin-2-yl]propane} and its salts, 2,2'-azobis(2-methylpropionamidine) and its salts, Examples of the water-soluble azo compounds include 2,2'-azobis(2-methylpropynamidine) and its salts, and 2,2'-azobis[N-(2-carboxyethyl)-2-methylpropionamidine] and its salts; and organic peroxides such as benzoyl peroxide, cumene hydroperoxide, t-butyl hydroperoxide, t-butyl peroxy-2-ethylhexanoate, and t-butyl peroxyisobutyrate.

[0032] (emulsifier) The emulsifier according to one embodiment includes a nonionic emulsifier having a cloud point.

[0033] The cloud point of a nonionic emulsifier means a temperature at which a dispersion of inorganic or organic particles dispersed in an aqueous medium in the presence of a nonionic emulsifier becomes cloudy during a temperature rise process. The cloud point can be measured under each reaction condition because it is affected by the concentration of the nonionic emulsifier and electrolytes. The cloud point of a nonionic emulsifier is, for example, 1 to 99°C, preferably 20 to 95°C, and more preferably 50 to 90°C. The hydrophilic-lipophilic balance (HLB) of the nonionic emulsifier can be selected from a wide range, and is, for example, about 1 to 20, preferably 5 to 18, and more preferably about 8 to 16.

[0034] The nonionic emulsifier is not particularly limited as long as it satisfies the above conditions. For example, polyoxyethylene alkyl ether [polyoxyethylene lauryl ether (manufactured by Kao, Emulgen 109P, cloud point 83°C, HLB 13.6), polyoxyethylene isodecyl ether (manufactured by Daiichi Kogyo Seiyaku, Noigen SD-80, cloud point 80°C, HLB 14.3), polyoxyalkylene branched decyl ether (manufactured by Daiichi Kogyo Seiyaku, Noigen XL-100, cloud point 79°C, HLB 14.7), polyoxyethylene tridecyl ether (manufactured by Daiichi Kogyo Seiyaku, Noigen TDS-80, cloud point 60°C, HLB 13.3), polyoxyalkylene tridecyl ether (manufactured by Daiichi Kogyo Seiyaku, Noigen TDX-50, cloud point 37°C, HLB 14.5), polyoxyethylene tridecyl ether (manufactured by Daiichi Kogyo Seiyaku, Noigen TDX-50, cloud point 37°C, HLB 14.6), polyoxyethylene tridecyl ether (manufactured by Daiichi Kogyo Seiyaku, Noigen TDX-50, cloud point 37°C, HLB 14.5 ... 9.0), polyoxyethylene oleyl ether (Kao Corporation, Emulgen 409PV, cloud point 55°C, HLB 12.0), and other polyoxyethylene C6-20 alkyl ethers], [polyoxyethylene styrenated phenyl ether (Dai-ichi Kogyo Seiyaku, Noigen EA-137, cloud point 65°C, HLB 13.0)], [polyoxyethylene C6-20 alkylene alkenyl ethers having at least one ethylenically unsaturated group (polymerizable unsaturated group) such as an allyl group (Kao Corporation, Latemul PD-420, cloud point 83°C, HLB 12.6), (Kao Corporation, Latemul PD-430, cloud point 95°C, HLB 14.4)], and the like. These nonionic emulsifiers can be used alone or in combination of two or more kinds.

[0035] <Application> The test kit according to one embodiment includes a resin particle composition that binds to a target substance in a specimen. Since the resin particle composition of the present invention described above can be used as the resin particle composition of the test kit, detailed description of the resin particle composition is omitted.

[0036] The resin particle composition of the present invention can be used for applications such as luminescent dyes for organic electroluminescence (EL), wavelength conversion dyes for solar cells, and bioimaging. In particular, by modifying the surface of the resin particle composition with an antibody, the resin particle composition is useful as a labeled luminescent particle for antigen testing by the immunochromatography method described below. Specifically, the resin particle composition having an antibody can be suitably used as a luminescent particle for antigen testing as a rapid and simple test kit for novel coronavirus infection. Furthermore, the resin particle composition having an antigen can also be suitably used as a luminescent particle for antibody testing. Currently, gold colloid particles are used as luminescent particles for antigen testing, but due to their low luminescence sensitivity, there is a problem that the detection sensitivity is extremely low at the early stage of infection when the number of viruses is small. In other words, patients suspected of viral infection undergo PCR testing at hospitals. In addition, the mutation cycle of viruses has become extremely fast in recent years, and the explosive spread of viral infections has placed a heavy burden on medical institutions and patients. In light of this background, there is a growing market need for highly sensitive antigen testing kits that can be easily obtained at pharmacies, drugstores, etc., and the present invention can provide a resin particle composition having fluorescence sensitivity that satisfies such needs.

[0037] (Antigen test) Antigen testing is a method in which a mixture of a specimen and a detection reagent is soaked into a flow channel made of fibers such as cellulose, and the presence or absence of a target substance (such as an antigen or antibody) in the specimen is detected by observing the appearance of a colored line indicating the reaction between the detection reagent and the target substance. Antigen test kits are widely used for simple and rapid detection of antigens. Antigen tests are used to detect antigens produced when the body's cells are infected with a virus, and the specimen used is a throat swab. When the throat swab is placed in the test kit, the results are available in about 15 minutes.

[0038] Antigen tests are characterized by being extremely simple and cost-effective compared to PCR tests. However, unlike PCR tests, they cannot amplify the target, so if the test is performed when the amount of virus is low early in the onset of symptoms, it may not be detected correctly. Currently, test results from the second to ninth day after symptoms appear are said to be accurate, and if the test is negative during this period, it is believed that infection with the new coronavirus can be ruled out. There is no doubt that antigen tests are useful, but due to sensitivity issues, antigen tests for the new coronavirus are currently considered to be screening tests. Screening tests are performed with the aim of identifying people who may be infectious, so that infection control measures can be taken.

[0039] (Immunochromatography) The immunochromatography method (1) requires a short time to reach a result, allowing for rapid testing, (2) is easy to operate as the measurement can be performed simply by dropping the sample, and (3) does not require special detection equipment, making it easy to determine the result. Taking advantage of these features, the immunochromatography method is used in a wide range of applications, including COVID-19 testing, influenza testing, pregnancy testing, and medical applications, as well as food and environmental testing, and is being used as a new Point of Care Testing (POCT) method. As a result, the immunochromatography method enables emergency testing and testing during surgery in hospitals, and even testing at home outside of hospitals, making it a highly sought-after testing method, especially for new viruses such as COVID-19.

[0040] The immunochromatography method will be explained in more detail. When a sample containing an antigen is dropped onto the sample dropping section of the reagent device, the sample penetrates into the conjugate pad and flows from the pad to the membrane filter by capillary action while dissolving the labeled antibody. The conjugate pad is made of, for example, glass fiber, polyester, or rayon, and holds the labeled antibody in a dry state. The antigen in the sample binds to the labeled antibody to form an immune complex. The immune complex of "labeled antibody-antigen" that moves through the membrane filter by capillary action further binds to the capture antibody fixed in a line on the membrane to form a "labeled antibody-antigen-capture antibody" complex, which is trapped on the membrane as if it were dammed up. This trapping creates a state in which the luminescent particles derived from the labeled antibody are concentrated on the capture antibody line, making it possible to visually determine the color development.

[0041] <Method of producing resin particle composition> A method for producing a resin particle composition according to one embodiment includes the steps of: (i) mixing a labeling substance and a polymerizable monomer with subcritical water to produce a mixture; (ii) mixing the mixture with an emulsifier and cooling the mixture to produce a monomer particle dispersion; and (iii) polymerizing the monomer particle dispersion with a crosslinkable monomer to produce resin particles.

[0042] Hereinafter, the monomer particle dispersion liquid will also be referred to as "oil droplets" or "microparticles".

[0043] Generally, in order to disperse oil droplets (monomer particle dispersion liquid) in which a marker substance is dissolved into fine particles, microparticle-forming techniques using a high-pressure homogenizer, ultrasonic homogenizer, etc. are known. However, these existing microparticle-forming techniques require a large physical force (e.g., shear force, destructive force due to cavitation) during stirring and dispersion. It has been confirmed that the marker substance is more likely to precipitate outside the oil droplets during this stirring. The inventors have found that subcritical water emulsification technology can be used to suppress the precipitation of the marker substance outside the oil droplets. The following is an overview of the existing microparticle-forming techniques.

[0044] (Existing microparticle technology) (1) Rotary mixing type emulsification: Emulsification equipment that applies general-purpose mechanical mixing technology by combining mixing tools (mixing blades, turbines, etc.) includes propeller mixers, homomixers, and dispersers. These have a rotary mixing structure and are used for general emulsion production. They are batch-type and can adjust the oil droplet size by changing the mixing time. (2) Ultrasonic homogenizer emulsification: This method uses cavitation generated by ultrasonic waves to promote emulsification. It is a batch type method that can adjust the oil droplet size to submicron size by changing the processing time. (3) High-pressure homogenizer emulsification: This method applies shear force to liquids by passing them through a narrow channel using a high-pressure pump or by colliding liquids against each other. This method has a high emulsifying power and is therefore suitable for making droplets finer. Submicron-sized oil droplets can be prepared by repeating the number of passes using a continuous flow system, but the repeated operations are cumbersome.

[0045] Hereinafter, a method for producing a resin particle composition using subcritical water emulsification technology will be described with reference to FIG.

[0046] Figure 1 is a diagram explaining the manufacturing process of a monomer particle dispersion liquid using a supercritical water supplying device. The supercritical water supplying device "SFW-E40S" capable of supplying subcritical water to supercritical water, which is one of the supercritical water emulsifiers (manufactured by AKICO) shown in Figure 1, is used for the subcritical water emulsification process. This device has a "mixing section" consisting of inflow paths for two raw materials and a mixer where the inflowing liquids join, a "supercritical / subcritical process section" for turning the water placed after the mixing section into hot water in a supercritical state or subcritical state, a "mixing section" for mixing the liquid discharged from the "supercritical / subcritical process section" with an emulsifier, and a "cooling section" for rapidly cooling the subcritical liquid mixed with the emulsifier at a speed of about 150°C / sec to obtain an emulsion with nano-sized particles.

[0047] Here, the steps of the resin particle composition production method (i) correspond to the steps from the mixing section located at the most upstream to the subcritical process section. The steps of the above production method (ii) correspond to the steps from the mixing section located downstream of the subcritical process section to the cooling section. Note that the steps of the above production method (iii) are based on a general polymerization method, so a detailed description is omitted.

[0048] In the present invention, a nonionic emulsifier having a cloud point is added to the liquid coming out of the supercritical / subcritical process section. The object of the present invention is to obtain a dispersion of polymerizable monomer finely emulsified in water from a liquid raw material containing a polymerizable monomer in which a labeling substance dissolves under milder conditions, so the conditions in the supercritical / subcritical process section are set to high temperature and high pressure, where the water temperature is 250°C to 370°C, which is just before the critical temperature, and the water becomes subcritical at 25 MPa. For this reason, the supercritical / subcritical process section is hereinafter referred to as the "subcritical process section" as shown in Figure 1. Here, a supplementary explanation of subcritical water emulsification is given.

[0049] (Explanation of subcritical water emulsification) It is known that the relative dielectric constant of water drops significantly near the critical point. For example, it is known that the relative dielectric constant of water drops to the same level as that of organic solvents such as methanol at around 200°C, acetone at around 300°C, and ethyl acetate near the critical point, making it compatible with and soluble in many oils. In particular, in order to target many organic oils, the following three processes (dissolution, precipitation, and emulsion stabilization processes) are carried out in a continuous flow system in the subcritical region below the critical point of water to prepare a microemulsion in a short time, which is the microemulsion technology. The microemulsion technology is a technology that emulsifies oil into nano-sized particles in a short time (specifically, in seconds) using a continuous flow system (one pass) without stirring. In this way, nano-sized particles can be generated in a short time, so there is no precipitation or falling off of the labeled substance outside the oil droplets. The present invention can produce uniform and small monomer oil droplets that could not be achieved by conventional technologies. (1) Dissolution process: The oil components are dissolved once in subcritical water and then uniformly dispersed. (2) Precipitation process: The oil components that were once dissolved are precipitated by rapid cooling. (3) Emulsification process ⇒ Emulsifiers are used to stabilize the precipitated particles (preventing coalescence) By the above steps (1) to (3), nano-sized oil droplet particles can be obtained by a bottom-up method.

[0050] <Production of Monomer Particle Dispersion> Table 1 shows the raw material blends and evaluation results of the monomer particle dispersions of Production Examples 1 to 11. The average oil droplet diameter (nm) and polydispersity index (pd) in Table 1 are values ​​measured using a particle size measuring device FPAR-1000 manufactured by Otsuka Electronics. In the evaluation of precipitation of fluorescent dyes, etc. (including AIE dyes), UV light was applied to the monomer particle dispersions to confirm the precipitation of fluorescent dyes, etc. Table 1 shows that the amount of precipitation of fluorescent dyes, etc. increases in the order of "none", "low", and "slightly high" in the column of precipitation of fluorescent dyes, etc. The monomer particle dispersion of Production Example 11 does not contain fluorescent dyes, etc., so it was not subject to the evaluation of precipitation of fluorescent dyes (shown as "-"). The polymerizable monomer includes the main component monomer in Table 1 and a monomer having a functional group. The amount of fluorescent dye (relative to monomer) represents the blend amount (wt%) of fluorescent dyes, etc. relative to 100% by mass of polymerizable monomer used in the production of the monomer particle dispersion. Table 2 shows the raw material blends and test results of the resin particle compositions of Examples 1 to 5 and Comparative Examples 1 to 8.

[0051] [Table 1] (Explanation of Abbreviations, etc.) HPS: Hexaphenylsilole ST: styrene MMA: Methyl methacrylate mAAc: methacrylic acid AM: Acrylamide 2HEMA: 2-hydroxyethyl methacrylate Parts by weight of main monomer: Parts by weight of monomer having functional group = 90:10 (common to Production Examples 1 to 11) Homo Disper (Primix: MARKII Model 2.5, mixing conditions: rotation speed 5000 rpm) Ultrasonic homogenizer (Mitsui Electric Seiki Co., Ltd.: UX-600 type, mixing conditions: frequency 20KHz) High-pressure homogenizer (SMT: LAB2000, mixing conditions: 5 cycles at 200MPa pressure)

[0052] [Table 2] (Explanation of Abbreviations) DVB: Divinylbenzene TMP: Trimethylolpropane trimethacrylate AIBN: 2,2'-azobis(isobutyronitrile) KPS: Potassium persulfate HPS: Hexaphenylsilole Formula for converting the amount of crosslinkable monomer (wt%) to parts by weight: parts by weight = 100X / (1-X), where X represents wt%

[0053] <Production Example 1: Preparation of Monomer Particle Dispersion> Based on Table 1, ion-exchanged water, a labeled substance-dissolving polymerizable monomer liquid (anthracene:styrene:methacrylic acid (=10 parts:90 parts:10 parts)), and a diluted emulsifier solution (a 22.5% diluted aqueous solution of Noigen XL-100 (trade name, Daiichi Kogyo Seiyaku Co., Ltd., polyoxyalkylene branched decyl ether, cloud point = 79°C, HLB 14.7)) were prepared as liquid raw materials.

[0054] As liquid raw materials, ion-exchanged water and a labeled substance-dissolved polymerizable monomer solution (a mixture of anthracene, styrene, and methacrylic acid) were introduced at a rate of 29.5 ml / min and 4.3 ml / min, respectively, from the inflow passage of the supercritical water emulsification device in Fig. 1, and in the subcritical process section, the labeled substance-dissolved polymerizable monomer was dissolved in the ion-exchanged water under high temperature and high pressure conditions where the water became subcritical water at 300°C. Next, an emulsifier dilution solution (a nonionic emulsifier solution with a cloud point) was added to the liquid coming out of the subcritical process section at a rate of 5.2 ml / min at the initial stage when the monomer particles started to form, and the solution was cooled in the cooling section, to obtain a monomer particle dispersion solution with a labeled substance-dissolved polymerizable monomer concentration of 10% and consisting of monomer microparticles with an average particle size of 150 nm.

[0055] The particle size distribution of the obtained monomer particles showed a sharp normal distribution, and it was confirmed that the dispersion was composed of uniform fine particles. The average particle size and particle size distribution of the obtained monomer particles were determined by dynamic light scattering. The average particle size (diameter, D50) was measured by dynamic light scattering at 25°C using FPAR-1000 (trade name, manufactured by Otsuka Electronics Co., Ltd.) after diluting the monomer particle dispersion to an appropriate concentration with water. In addition, when the obtained monomer particle dispersion was irradiated with a UV lamp and the dissolved state of the labeling substance was observed, no precipitation of the labeling substance was observed. This confirmed that the labeling substance was encapsulated in the monomer particles.

[0056] <Production Examples 2 to 11: Preparation of Monomer Particle Dispersion> The monomer particle dispersions of Production Examples 2 to 5 were prepared based on the same production method as in Table 1 and Production Example 1, and therefore detailed explanations will be omitted. On the other hand, the monomer particle dispersions of Production Examples 6 to 11 were prepared based on the raw material composition in Table 1 using oil droplet producing devices (Homodisper, high-pressure homogenizer, ultrasonic homogenizer) different from those of Production Examples 1 to 5.

[0057] Example 1: Preparation of resin particle composition Into a 300 ml four-neck round-bottom flask equipped with a stirrer, a thermometer, and a reflux condenser, 100 g of the anthracene-containing monomer particle dispersion liquid having an average particle diameter of 150 nm obtained in Preparation Example 1, 10 wt% (1.1 g by weight) of DVB as a crosslinkable monomer relative to 100 mass% of all polymerizable monomers, and 0.5 g of AIBN as a polymerization initiator were added, and the mixture was stirred at 25°C and atmospheric pressure at a rotation speed of 170 rpm for 60 minutes while replacing with nitrogen. Then, 120.9 g of ion-exchanged water was added, and the mixture was stirred at an internal temperature of 75°C for 6 hours. After polymerization treatment, the mixture was cooled to obtain an anthracene-containing resin particle composition having a concentration of 5% and an average particle diameter of 160 nm.

[0058] The average particle size (D50) and particle size distribution of the resin particle composition obtained by polymerization were measured by dynamic light scattering in the same manner as in the manufacturing example. The resin particle composition obtained was irradiated with a UV lamp to observe the dissolved state of the marker substance, and no precipitation of the marker substance was observed. Furthermore, when the fine resin particle composition obtained was observed using an electron microscope, no aggregates or the like were observed other than the resin particle composition, and it was confirmed that the marker substance was encapsulated in the resin particles.

[0059] <Examples 2 to 5 and Comparative Examples 1 to 8: Preparation of resin particle compositions> The resin particle compositions of Examples 2 to 5 and Comparative Examples 1 to 8 were produced based on the raw material compositions shown in Table 2 and the same production method as in Example 1, and therefore detailed explanations will be omitted.

[0060] <Test Method> The resin particle compositions of Examples 1 to 5 and Comparative Examples 1 to 8 were used to carry out the performance evaluations in the following Test Examples 1 to 3. For the resin particle compositions of Comparative Examples 7 and 8, the following labeling substance absorption step was carried out, and then the tests in Test Examples 1 to 3 were carried out.

[0061] (Explanation of the absorption process of the labeling substance) The purpose of this step is to confirm whether the fluorescence intensity is improved when a labeling substance is absorbed into the resin particle compositions of Comparative Examples 7 and 8. A labeling substance (hereinafter, HPS: hexaphenylsilole) is dissolved in tetrahydrofuran (hereinafter, also referred to as THF) to prepare a THF dispersion in which HPS is dissolved at a concentration of 1 g / L. The THF dispersion is mixed in an amount such that HPS is 1 wt% relative to the resin particle content of the prepared resin particle dispersion, and stirred for 5 minutes to absorb HPS into the resin particle composition. The obtained dispersion is then subjected to ultracentrifugation to settle the resin particles, and after removing the supernatant, pure water is added to redisperse the particles to obtain a resin particle dispersion.

[0062] (Test Example 1: Confirmation of the presence of fluorescent dyes outside resin particles) The resin particle dispersion liquid was collected and dried at 60° C. for 1 hour, and then a JEOL scanning electron microscope (SEM) was used to confirm whether fluorescent dyes (including AIE dyes) were present outside the resin particles. (Evaluation Criteria) None: No fluorescent dye or the like was found outside the resin particles. Almost none: Almost no fluorescent dyes, etc. were found outside the resin particles. Slight: A small amount of fluorescent dye, etc. was confirmed outside the resin particles. Somewhat large: A small amount of fluorescent dye, etc. was confirmed outside the resin particles. Large amount: A large amount of fluorescent dyes, etc. were confirmed outside the resin particles.

[0063] (Test Example 2: Confirmation of Fluorescence Intensity (After Washing with Water)) The resin particles were precipitated by ultracentrifugation, the supernatant was removed, and pure water was added to redisperse the resin particles. This procedure was repeated three times, and the precipitated resin particles in the fourth run were diluted with pure water to prepare a 1 mg / mL dispersion. This dispersion was photographed using a Keyence BZ-X800 fluorescence microscope to confirm its fluorescence intensity. (Evaluation Criteria) ◎: Very strong ○: Strong △: Slightly strong ×: Weak

[0064] (Test Example 3: Confirmation of Fluorescence Intensity (THF Dispersion)) The resin particles were precipitated by ultracentrifugation, the supernatant was removed, and pure water was added to redisperse the resin particles. This procedure was repeated three times, and the precipitated resin particles from the fourth run were diluted with THF to prepare a 10 mg / mL dispersion. This dispersion was photographed using a Keyence BZ-X800 fluorescence microscope to confirm its fluorescence intensity. (Evaluation Criteria) ◎: Very strong ○: Strong △: Slightly strong ×: Weak

[0065] <Test Results> Table 2 shows the test results of Examples 1 to 5 and Comparative Examples 1 to 8. Examples 1 to 5 showed superior properties (solvent resistance and high fluorescence intensity) in Test Examples 1 to 3 compared to Comparative Examples 1 to 8. In particular, Examples 1 to 5 had superior fluorescence intensity in a solvent (THF). On the other hand, Comparative Examples 1 to 5 (when no crosslinkable monomer was contained) showed that the fluorescent dye, etc., fell out of the resin particles and that the fluorescence intensity decreased. Comparative Example 6 (when a crosslinkable monomer was contained) showed that the solvent resistance of the resin particles was slightly improved, but did not show sufficient fluorescence intensity for practical use. Comparative Examples 7 to 8 (when a labeling substance was absorbed in a post-process) showed that the fluorescent dye, etc., attached to the resin particle surface easily fell out and that the fluorescence intensity decreased. From the above test results, it is presumed that the production of resin particles using a monomer particle dispersion produced by the subcritical water emulsification technology of the present invention and a crosslinkable monomer contributes to the improvement of solvent resistance. This is believed to prevent leakage of the labeling substance encapsulated in the resin particles, making it possible to achieve high fluorescence intensity.

[0066] As described above, the resin particle composition of the present invention has the remarkable effects of having excellent solvent resistance and high fluorescent intensity.

[0067] It should be understood that it is difficult for a person skilled in the art to directly specify the specific structures and properties of the monomer particle dispersion liquid and resin particles according to the present invention, and it should be noted that detailed information thereof has not yet been made clear. In addition, it should be noted that specifying the specific structures and properties of the monomer particle dispersion liquid and resin particles according to the present invention is not realistic in the sense that it would require a person skilled in the art to make extremely excessive economic expenditures due to excessive experiments and preparation of special measuring devices, etc.

[0068] The invention is not limited to the above-described embodiment, and various modifications and variations are possible within the scope of the gist of the invention.

Claims

1. A resin particle composition comprising resin particles encapsulating a labeling substance, mixing the labeling substance and the polymerizable monomer in subcritical water to produce a mixture; mixing and cooling the mixture with an emulsifier to form a monomer particle dispersion; carrying out a polymerization reaction between the monomer particle dispersion and a crosslinkable monomer to produce the resin particles. Resin particle composition.

2. the polymerizable monomer includes at least one of an aromatic monomer, a (meth)acrylic monomer, and a monomer having a functional group, the resin particles contain, as constituent units, 40 to 95% by mass of the aromatic monomer and / or the (meth)acrylic monomer, 0 to 10% by mass of the monomer having a functional group, and 5 to 60% by mass of the crosslinkable monomer, relative to 100% by mass of the total polymerizable monomers; The resin particle composition according to claim 1 .

3. The resin particles contain 0.1 to 15% by mass of the labeling substance relative to 100% by mass of the total polymerizable monomers. The resin particle composition according to claim 1 .

4. The average particle size of the resin particles is 50 to 500 nm. Here, the average particle size is measured by a dynamic light scattering method. The resin particle composition according to claim 1 .

5. The aromatic monomer is at least one selected from the group consisting of styrene, methylstyrene, chlorostyrene, methoxystyrene, α-methylstyrene, p-nitrostyrene, ethylvinylbenzene, vinylnaphthalene, benzyl acrylate, benzyl methacrylate, phenylethyl acrylate, phenylethyl methacrylate, phenylpropyl acrylate, phenylpropyl methacrylate, phenylnonyl acrylate, and phenylnonyl methacrylate. The resin particle composition according to claim 2 .

6. The crosslinkable monomer is at least one selected from the group consisting of allyl methacrylate, ethylene glycol dimethacrylate, ethylene glycol diacrylate, butanediol diacrylate, butanediol dimethacrylate, neopentyl glycol dimethacrylate, hexanediol dimethacrylate, triethylene glycol dimethacrylate, tetraethylene glycol dimethacrylate, trimethylolpropane trimethacrylate, pentaerythritol tetramethacrylate, and divinylbenzene. The resin particle composition according to claim 2 .

7. The functional group includes at least one of a hydroxy group, a carboxy group, an amino group, and an amide group. The resin particle composition according to claim 2 .

8. The (meth)acrylic monomer includes at least one of an alkyl (meth)acrylate having an alkyl group having 1 to 18 carbon atoms and a cycloalkyl (meth)acrylate. The resin particle composition according to claim 2 .

9. The emulsifier is a nonionic emulsifier having a cloud point. The resin particle composition according to claim 1 .

10. The labeling substance includes at least one of a fluorescent dye and an aggregation-induced luminescent dye. The resin particle composition according to claim 1 .

11. The resin particle composition according to claim 1, which binds to a target substance in a specimen. Test kit.

12. A method for producing a resin particle composition containing resin particles encapsulating a labeling substance, comprising: mixing the labeling substance and the polymerizable monomer with subcritical water to produce a mixture; mixing and cooling the mixture with an emulsifier to form a monomer particle dispersion; carrying out a polymerization reaction between the monomer particle dispersion and a crosslinkable monomer to produce the resin particles. A method for producing a resin particle composition.

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