Resin particle composition, detection kit, and method for producing resin particle composition

Abstract

The invention relates to a resin particle composition, a detection kit, and a method for producing the resin particle composition. The purpose of the present invention is to provide a resin particle composition having excellent solvent resistance and high fluorescence intensity. The present invention relates to a resin particle composition comprising resin particles in which a marking substance is wrapped, the resin particle composition being produced by a method comprising: a step for producing a mixture by mixing the marking substance and a polymerizable monomer with subcritical water; a step in which the mixture and an emulsifier are mixed and cooled to produce a monomer particle dispersion; and a step for producing the resin particles by performing a polymerization reaction between the monomer particle dispersion liquid and a crosslinkable monomer.

Description

Technical Field

[0001] The invention relates to a resin particle composition, a detection kit and a method for producing the resin particle composition. Background Art

[0002] Resin particle compositions containing marker substances are considered to be useful as fluorescent markers in clinical tests in the fields of biological and medical diagnosis, for example. In addition, depending on the type of marker substance, the marker substance is used in a dilute state or a high concentration state in which the marker substance is uniformly dispersed in a polymer matrix material or a solvent at a molecular level. For example, Patent Document 1 discloses a method of granulating polymer particles in the presence of a fluorescent pigment and introducing a fluorescent pigment into the polymer particles during the granulation process of the polymer. Patent Document 2 discloses an infiltration method of introducing a fluorescent pigment into polymer particles by swelling the polymer particles in a liquid containing a fluorescent pigment.

[0003] Prior art literature

[0004] Patent Literature

[0005] Patent Document 1: Japanese Patent No. 6978081

[0006] Patent Document 2: Japanese Patent No. 6769728 Summary of the invention

[0007] Problems to be solved by the invention

[0008] However, Patent Document 1 has the following problem: when the monomer oil droplets are miniaturized using a high-shear dispersion device such as a high-pressure homogenizer, the dispersion liquid reaches a high temperature, and the dissolved fluorescent pigment precipitates, so the amount of fluorescent pigment that can be encapsulated in the polymer particles is limited. Patent Document 2 has the following problem: the particles encapsulating the fluorescent pigment produced using polymer particles that easily swell in liquids dissolve in the solvent, so the fluorescence intensity is low. As such, Patent Documents 1 and 2 fail to achieve a resin particle composition having excellent solvent resistance and high fluorescence intensity.

[0009] Therefore, an object of the present invention is to provide a resin particle composition having excellent solvent resistance and high fluorescence intensity.

[0010] Solutions for solving problems

[0011] In order to achieve the purpose of the present invention, the resin particle composition of the present invention comprises resin particles encapsulating a marking substance, and the resin particle composition is manufactured by a method comprising the following steps: a step of generating a mixture by mixing the aforementioned marking substance with a polymerizable monomer using subcritical water; a step of generating a monomer particle dispersion by mixing and cooling the aforementioned mixture with an emulsifier; and a step of performing a polymerization reaction between the aforementioned monomer particle dispersion and a crosslinking monomer to generate the aforementioned resin particles, wherein the aforementioned polymerizable monomer comprises at least one of an aromatic monomer, an alkyl (meth)acrylate having an alkyl group with 1 to 18 carbon atoms and a cycloalkyl (meth)acrylate, and at least one of an alkyl (meth)acrylate having a functional group, and the aforementioned resin particles comprise 40 to 95% by mass of the aforementioned aromatic monomer and / or At least any one of the aforementioned (meth)acrylic acid alkyl esters having an alkyl group with 1 to 18 carbon atoms and the aforementioned (meth)acrylic acid cycloalkyl esters, 0 to 10% by mass of the aforementioned (meth)acrylic acid alkyl ester having a functional group, and 5 to 60% by mass of the aforementioned cross-linking monomer as structural units, the aforementioned labeling substance comprises at least any one of a fluorescent dye and an aggregation-induced emission dye, the aromatic monomer is 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, and the functional group comprises at least any one of a hydroxyl group, a carboxyl group, an amino group, and an amide group.

[0012] In order to achieve the purpose of the present invention, the method for producing a resin particle composition of the present invention is a method for producing a resin particle composition containing resin particles wrapped with a marking substance, comprising the following steps: a step of mixing the aforementioned marking substance and a polymerizable monomer with subcritical water to generate a mixture; a step of mixing and cooling the aforementioned mixture and an emulsifier to generate a monomer particle dispersion; and a step of polymerizing the aforementioned monomer particle dispersion with a crosslinking monomer to generate the aforementioned resin particles, wherein the aforementioned polymerizable monomer comprises an aromatic monomer, at least one of (meth)acrylic acid alkyl esters and (meth)acrylic acid cycloalkyl esters having an alkyl group with 1 to 18 carbon atoms, and at least one of (meth)acrylic acid alkyl esters having a functional group, and the aforementioned resin particles contain 40 to 95% by mass of the aforementioned aromatic monomer and / or the crosslinking monomer relative to 100% by mass of the total polymerizable monomers. or at least any one of the aforementioned alkyl (meth)acrylates having an alkyl group with 1 to 18 carbon atoms and the aforementioned cycloalkyl (meth)acrylates, 0 to 10% by mass of the aforementioned alkyl (meth)acrylate having a functional group and 5 to 60% by mass of the aforementioned cross-linking monomer as structural units, the aforementioned labeling substance comprises at least any one of a fluorescent dye and an aggregation-induced emission dye, the aforementioned aromatic monomer is 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, and the aforementioned functional group comprises at least any one of a hydroxyl group, a carboxyl group, an amino group, and an amide group.

[0013] Effects of the Invention

[0014] According to the present invention, a resin particle composition having excellent solvent resistance and high fluorescence intensity can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a diagram for explaining the production process of a monomer particle dispersion using a supercritical water supply apparatus. DETAILED DESCRIPTION

[0016] The following is a detailed description of the embodiments. It should be noted that the following embodiments do not limit the invention protected by the claims, and the combination of features described in the embodiments is not necessarily required for the invention. Two or more of the multiple features described in the embodiments can be combined arbitrarily.

[0017] (Resin Particle Composition)

[0018] A resin particle composition of one embodiment includes resin particles encapsulating a marker substance. The resin particle composition of one embodiment is manufactured by a method including the following steps: a step of generating a mixture by mixing a marker substance with a polymerizable monomer using subcritical water; a step of generating a monomer particle dispersion by mixing and cooling the mixture with an emulsifier; and a step of performing a polymerization reaction between the monomer particle dispersion and a crosslinkable monomer to generate resin particles.

[0019] (resin particles)

[0020] The polymerizable monomer of one embodiment includes at least any one of an aromatic monomer, a (meth) acrylic monomer, and a monomer having a functional group. The resin particles of one embodiment include 40 to 95% by mass of aromatic monomers and / or (meth) acrylic monomers, 0 to 10% by mass of monomers having functional groups, and 5 to 60% by mass of cross-linking monomers as structural units relative to 100% by mass of all polymerizable monomers. Here, "100% by mass of all polymerizable monomers" represents the mass % of all polymerizable monomers constituting the resin particles, and represents the mass % of the total polymerizable monomers and cross-linking monomers. In addition, "40 to 95% by mass of aromatic monomers and / or (meth) acrylic monomers" means that 3 modes are included (40 to 95% by mass of aromatic monomers, 40 to 95% by mass of (meth) acrylic monomers, and 40 to 95% by mass of a mixture of aromatic monomers and (meth) acrylic monomers).

[0021] The average particle size of the resin particles according to one embodiment is 50 to 500 nm. Here, the average particle size is measured by a dynamic light scattering method.

[0022] The polymerizable monomer (monomer), emulsifier, polymerization initiator, etc. that can be used in the method for producing the resin particles of the present invention are described. It should be noted that the term "(meth)acrylic acid" in this specification means both "acrylic acid" and "methacrylic acid", and the term "(meth)acrylate" means both "acrylate" and "methacrylate".

[0023] The following lists polymerizable monomers that can be used in the production of resin particles as examples, but are not limited to these monomers. In addition, the following monomers can be used alone or in combination of two or more.

[0024] The aromatic monomer is not particularly limited as long as it can dissolve the marker substance, and examples thereof include 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.

[0025] The content of the aromatic monomer in 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.

[0026] Examples of the (meth)acrylic monomer include alkyl (meth)acrylates having an alkyl group having 1 to 18 carbon atoms, such as methyl (meth)acrylate, ethyl (meth)acrylate, n-propyl (meth)acrylate, isopropyl (meth)acrylate, n-butyl (meth)acrylate, isobutyl (meth)acrylate, sec-butyl (meth)acrylate, tert-butyl (meth)acrylate, n-pentyl (meth)acrylate, isopentyl (meth)acrylate, n-hexyl (meth)acrylate, 2-ethylhexyl (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 p-tert-butylcyclohexyl (meth)acrylate.

[0027] The content of the (meth)acrylic acid-based monomer in 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.

[0028] As the crosslinking 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 crosslinking monomer, aromatic divinylbenzene is particularly preferred.

[0029] The content of the crosslinkable monomer in one embodiment is preferably 5 to 60 mass%, 5 to 50 mass%, or 5 to 40 mass%, and more preferably 5 to 30 mass%, relative to 100 mass% of all polymerizable monomers. When the resin particles contain the crosslinkable monomer in the above-specified mass%, the solvent resistance of the resin particles can be further improved, and the swelling of the resin particles in the solvent can be suppressed. Solvent resistance refers to the property that the marking substance is not easily eluted to the outside of the resin particles.

[0030] Examples of the monomer having a functional group include: carboxyl group-containing polymerizable monomers such as monomers obtained by reacting (meth)acrylic acid, acrylic acid dimer, hydroxyalkyl (meth)acrylates such as 2-hydroxyethyl (meth)acrylate and 4-hydroxybutyl (meth)acrylate with maleic anhydride, succinic anhydride or phthalic anhydride; hydroxyalkyl (meth)acrylates such as hydroxyethyl acrylate, hydroxyethyl methacrylate, 2-hydroxyethyl (meth)acrylate, 2-(3-)hydroxypropyl (meth)acrylate, 4-hydroxybutyl (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-hydroxymethylacrylamide and N-butoxymethyl (meth)acrylamide.

[0031] The monomer having a functional group in one embodiment is preferably 0 to 10% by mass relative to 100% by mass of all polymerizable monomers. Here, when the resin particles are used as luminescent particles for antigen detection and luminescent particles for antibody detection described later, 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 detection and luminescent particles for antibody detection, the monomer having a functional group may not be an essential component for manufacturing the resin particles.

[0032] The functional group of one embodiment includes at least any one of a hydroxyl group, a carboxyl group, an amino group, and an amide group.

[0033] (Marking substance)

[0034] The content of the labeling substance in one embodiment is preferably 0.1 to 15% by mass, more preferably 0.5 to 10% by mass, based on 100% by mass of all polymerizable monomers.

[0035] A labeling substance according to one embodiment includes at least one of a fluorescent dye and an aggregation-induced emission dye (hereinafter also referred to as an AIE dye).

[0036] Fluorescent pigments are pigments that emit fluorescence. When fluorescent pigments absorb light, the electrons in the pigment are excited, and when they return to the base state, they release the excess energy as electromagnetic waves. This electromagnetic wave is fluorescence, and the wavelength of 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 fluorescence is called the Stokes shift. The larger the difference, the easier it is to distinguish fluorescence from excitation light and the easier it is to detect the pigment.

[0037] Examples of fluorescent pigments include merocyanine, perylene, acridine, anthracene, luciferin, pyranine, stilbene, rhodamine, coumarin, 4-(dicyanomethylene)-2-methyl-6-(4-dimethylaminophenylvinyl)-4H-pyran (DCM), pyrromethene, fluorescein, umbelliferone and other compounds and derivatives having fluorescent properties, and one or more selected from the above group can be used.

[0038] AIE dyes are aggregation-induced emission dyes (AIE=Aggregation-Induced Emission), which are fluorescent dyes that do not emit light in a dilute solution state, but emit strong light in a solid and aggregated state, showing the opposite behavior of general fluorescent dyes. In other words, AIE dyes are dyes that can achieve high concentration and high brightness, which are difficult to achieve with ordinary fluorescent dyes due to concentration quenching.

[0039] As the AIE dye, there can be listed: 1,1-dimethyl-2,3,4,5-tetraphenylthiole, 1,1,2,3,4,5-hexaphenylthiole, 1,1-dimethyl-2,5-dianisyl-3,4-diphenylthiole, 1,1-diallyl-2,3,4,5-tetraphenylthiole and other silole ring-containing compounds and derivatives, tetraphenylethylene and other hydrocarbon aromatic compounds and derivatives, heteroaromatic compounds and derivatives, rhodamine compounds and derivatives, etc., and one or more selected from the above group can be used. AIE dyes are particularly preferably silole ring-containing compounds from the perspective of being easily encapsulated in resin particles.

[0040] (Polymerization initiator)

[0041] The polymerization initiator is not particularly limited, and any conventionally known initiator can be used. For example, the polymerization initiators generally used in radical polymerization as listed below can be preferably used.

[0042] Examples of the polymerization initiator include persulfates such as potassium persulfate, sodium persulfate, and ammonium persulfate; oil-soluble azo compounds 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; 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-(2-imidazolin-2-yl)propane] and its salts Water-soluble azo compounds such as bis(2-methylpropionamidine) and its salts, 2,2'-azobis(2-methylpropionamidine) and its salts, 2,2'-azobis[N-(2-carboxyethyl)-2-methylpropionamidine] and its salts; organic peroxides such as benzoyl peroxide, cumene hydroperoxide, tert-butyl hydroperoxide, tert-butyl peroxy-2-ethylhexanoate, and tert-butyl peroxyisobutyrate.

[0043] (Emulsifier)

[0044] The emulsifier according to one embodiment includes a nonionic emulsifier having a cloud point.

[0045] The cloud point of a nonionic emulsifier refers to the temperature at which the dispersed system becomes cloudy during the heating process in a system in which inorganic or organic particles are dispersed in an aqueous medium in the presence of a nonionic emulsifier. The cloud point is affected by the concentration of the nonionic emulsifier, electrolytes, etc., and can be measured under various reaction conditions. 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. In addition, the hydrophilic-lipophilic balance (HLB) of the nonionic emulsifier can be selected in a wide range. For example, it is 1 to 20, preferably 5 to 18, and more preferably about 8 to 16.

[0046] The nonionic emulsifier is not particularly limited as long as it satisfies the above conditions. For example, it includes polyoxyethylene alkyl ethers [polyoxyethylene lauryl ether (manufactured by Kao Corporation, EMULGEN 109P, cloud point 83°C, HLB 13.6), polyoxyethylene isodecyl ether (manufactured by Dai-ichi Kogyo Seiyaku Co., Ltd., NOIGEN SD-80, cloud point 80°C, HLB 14.3), polyoxyalkylene branched decyl ether (manufactured by Dai-ichi Kogyo Seiyaku Co., Ltd., NOIGEN XL-100, cloud point 79°C, HLB 14.7), polyoxyethylene tridecyl ether (manufactured by Dai-ichi Kogyo Seiyaku Co., Ltd., NOIGEN TDS-80, cloud point 60°C, HLB 13.3), polyoxyalkylene tridecyl ether (manufactured by Dai-ichi Kogyo Seiyaku Co., Ltd., NOIGEN TDX-50, cloud point 37°C, HLB 9.0), polyoxyethylene oleyl ether (manufactured by Kao Corporation, EMULGEN 409PV, cloud point 55°C, HLB 12.0)], [polyoxyethylene C6-20 alkyl ether (manufactured by Daiichi Kogyo Seiyaku Co., Ltd., NOIGEN EA-137, cloud point 65°C, HLB 13.0)], [polyoxyethylene C6-20 alkylene alkenyl ether having at least one ethylenically unsaturated group (polymerizable unsaturated group) such as an allyl group (manufactured by Kao Corporation, LATEMUL PD-420, cloud point 83°C, HLB 12.6), (manufactured by Kao Corporation, LATEMUL PD-430, cloud point 95°C, HLB 14.4)], etc. In addition, these nonionic emulsifiers can be used alone or in combination of two or more.

[0047] <Purpose>

[0048] The detection kit of one embodiment includes a resin particle composition that binds to a target substance in a test sample. The resin particle composition of the detection kit can be the resin particle composition of the present invention described above, and therefore a detailed description of the resin particle composition is omitted.

[0049] As the use of the resin particle composition of the present invention, luminescent pigments for organic EL, wavelength conversion pigments for solar cells, bioimaging, etc. can be listed. In particular, by modifying antibodies to the surface of the resin particle composition, it is useful as a labeled luminescent particle based on immunochromatography for antigen detection described later. Specifically, the resin particle composition with antibodies can be suitably used as a luminescent particle for antigen detection as a quick and convenient detection kit for new coronavirus infection. Furthermore, the resin particle composition with antigens can also be suitably used as a luminescent particle for antibody detection. The current situation is that colloidal gold particles are used as luminescent particles for antigen detection, but due to the low luminescence sensitivity, there is a problem of extremely low detection sensitivity at the early stage of infection with a small number of viruses. That is, patients suspected of viral infection receive PCR testing in hospitals. In addition, the mutation cycle of the virus has been extremely fast recently, and the burden on medical institutions and patients has increased due to the expansion of explosive viral infections. Against this background, the market demand for highly sensitive antigen detection kits that can be easily purchased in pharmacies, drugstores, etc. is high, and the present invention can provide a resin particle composition with fluorescence sensitivity that meets this demand.

[0050] (Antigen Test)

[0051] Antigen testing refers to a method of detecting the presence of a target substance by allowing a mixture of a test sample and a test reagent to infiltrate a flow path made of fibers such as cellulose, and observing the appearance of a colored line indicating that the target substance (antigen, antibody, etc.) in the test sample has reacted with the test reagent. Antigen test kits are widely used to detect antigens easily and quickly. Antigen testing is used to detect antigens produced when human cells are infected with viruses, and the test sample uses throat swab fluid. When the throat swab fluid is used in the test kit, the result is known in about 15 minutes.

[0052] Antigen testing is much simpler and more cost-effective than PCR testing. However, unlike PCR testing, it is not possible to amplify the object of testing, so if the test is performed at the early stage of the disease when the amount of virus is small, it may not be able to detect it correctly. At present, it is generally believed that the test results from the second to ninth day after the onset of symptoms are accurate, and it is believed that a negative test during this period can deny infection with the new coronavirus. There is no doubt that antigen testing is useful, but due to the problem of sensitivity, the current situation is that antigen testing for the new coronavirus is considered as screening. Screening refers to an examination carried out for the purpose of identifying people who may be infectious, so that infection countermeasures can be taken.

[0053] (Immunochromatography)

[0054] Immunochromatography: (1) The time required for determination is short and rapid detection is possible; (2) The determination can be performed by simply adding a drop of the test sample and the operation is simple; (3) No special detection equipment is required and determination is easy. Taking advantage of these characteristics, immunochromatography can be used for food testing, environmental testing, etc. in addition to COVID-19 testing, influenza testing, pregnancy testing, and medical testing, and is widely used as a new POCT (Point Of Care Testing, instant testing) method. As a result, immunochromatography makes it possible to conduct emergency testing in hospitals, testing during surgery, and even testing at home outside hospitals, and is a testing method that is in high demand, especially for new viruses such as COVID-19.

[0055] The immunochromatography method is described in more detail. When the test sample containing the antigen is dripped into the test sample dripping part of the reagent device, the test sample penetrates into the conjugate pad, and the labeled antibody is dissolved from the pad and flows to the filter membrane due to the capillary phenomenon. The conjugate pad uses articles such as glass fiber, polyester, and rayon to keep the labeled antibody in a dry state. The antigen in the test sample combines with the labeled antibody to form an immune complex. The immune complex of "labeled antibody-antigen" that moves to the filter membrane due to the capillary phenomenon further combines with the capture antibody fixed in a linear shape on the membrane to form a complex of "labeled antibody-antigen-capture antibody", and is captured in a way that is intercepted on the membrane. The state of luminescent particles derived from the labeled antibody formed on the capture antibody line due to this capture is concentrated, and the color development can be visually determined.

[0056] <Method for producing resin particle composition>

[0057] A method for producing a resin particle composition according to one embodiment includes the following steps: (i) a step of generating a mixture by mixing a labeling substance and a polymerizable monomer with subcritical water; (ii) a step of generating a monomer particle dispersion by mixing and cooling the mixture and an emulsifier; and (iii) a step of performing a polymerization reaction between the monomer particle dispersion and a cross-linking monomer to generate resin particles.

[0058] Hereinafter, the monomer particle dispersion is also referred to as “oil droplets” or “fine particles” or the like.

[0059] Here, generally speaking, in order to disperse the oil droplets (monomer particle dispersion) in which the marker substance is dissolved into microparticles, it is known that there are microparticle technologies using high-pressure homogenizers, ultrasonic homogenizers, etc., and these existing microparticle technologies require large physical forces (such as shear force and cavitation destructive force) during stirring and dispersion. During this stirring, it is confirmed that the marker substance is precipitated to the outside of the oil droplets. The inventors have discovered the use of subcritical water emulsification technology for the purpose of suppressing the precipitation of the marker substance to the outside of the oil droplets. The following is an overview of the existing microparticle technology.

[0060] (Existing micro-granulation technology)

[0061] (1) Rotary stirring emulsification: As an emulsification device that uses a common mechanical stirring technology combined with a stirring fixture (stirring blades, turbines, etc.), there are propeller mixers, homogenizer mixers, dispersers, etc. It has a rotary stirring structure and is used for conventional emulsion production. It is intermittent and can produce micron-sized oil droplets depending on the stirring time.

[0062] (2) Ultrasonic homogenizer emulsification: This method uses cavitation generated by ultrasound to promote emulsification. It is intermittent and can produce submicron-sized oil droplets depending on the processing time.

[0063] (3) High-pressure homogenizer emulsification: This is a method of applying shear force to a liquid by using a high-pressure pump to deliver the liquid to a narrow flow path or by causing the liquids to collide with each other. This method has a high emulsifying power and is therefore suitable for the miniaturization of droplets. This is a continuous flow method and can produce submicron-sized oil droplets by repeating the number of passes, but the repeated operation is complicated.

[0064] Below, refer to Figure 1 A method for producing a resin particle composition using a subcritical water emulsification technique will be described.

[0065] Figure 1 This is a diagram for explaining the production process of a monomer particle dispersion using a supercritical water supply device. Figure 1 The supercritical water supply device "SFW-E40S" of the supercritical water emulsification device (manufactured by AKICO Co., Ltd.) that can supply subcritical water to supercritical water is used for subcritical water emulsification treatment. The device has: a "mixing section" formed by an inflow passage for two raw materials and a mixer for the inflow liquid to merge; a "supercritical-subcritical process section" configured immediately after the mixing section for making water into hot water in a supercritical state or a subcritical state; a "mixing section" for mixing the liquid coming out of the "supercritical-subcritical process section" with an emulsifier; and a "cooling section" for rapidly cooling the subcritical liquid mixed with the emulsifier at a rate of about 150°C / second to obtain an emulsion with a particle size of nanometers.

[0066] Here, the process of the method for producing the resin particle composition (i) corresponds to the process from the mixing section located at the most upstream to the subcritical process section. The process of the above-mentioned method for producing (ii) corresponds to the process from the mixing section located downstream of the subcritical process section to the cooling section. It should be noted that the process of the above-mentioned method for producing (iii) is based on a conventional polymerization method, so detailed description is omitted.

[0067] The present invention adds a nonionic emulsifier having a cloud point to the liquid coming out of the supercritical-subcritical process section. The purpose of the present invention is to obtain a dispersion of polymerizable monomers finely emulsified in water from a liquid raw material containing a polymerizable monomer in which a marker substance dissolves under milder conditions. Therefore, the conditions of the supercritical-subcritical process section are set to a water temperature of 250°C to 370°C close to the critical temperature and a high temperature and high pressure of 25MPa to form subcritical water. Therefore, as follows Figure 1 The supercritical-subcritical process section is referred to as the "subcritical process section" in the figure. Here, the subcritical water emulsification is supplementally explained.

[0068] (Description of subcritical water emulsification)

[0069] It is known that the relative dielectric constant of water will be greatly reduced near the critical point. For example, it is known that the relative dielectric constant of water will be reduced to the same level as methanol near 200°C, to the same level as acetone near 300°C, and to the same level as organic solvents such as ethyl acetate near the critical point, and can be compatible and dissolved with many oils. In particular, since many organic oils are used as the object, the following three steps (dissolution-precipitation-emulsification stabilization process) are carried out in a continuous flow manner in the subcritical region below the critical point of water to prepare a micro-emulsion in a short time, which is the micro-emulsification technology. The technology of emulsifying oil into nano-sized particles in a short time (specifically in seconds) using a continuous flow method (single pass) without stirring is called micro-emulsification technology. In this way, nano-sized particles can be generated in a short time, so the precipitation and shedding of the marker substance to the outside of the oil droplets will not occur. The present invention can produce uniform and tiny monomer oil droplets that cannot be achieved by the prior art.

[0070] (1) Dissolution process Use subcritical water to temporarily dissolve the oil component and evenly disperse it

[0071] (2) Precipitation process Use rapid cooling to separate the temporarily dissolved oil components

[0072] (3) Emulsification process Stabilization of precipitated particles using emulsifiers (coagulation inhibition)

[0073] Through the above steps (1) to (3), nano-sized fine oil droplets can be obtained in a bottom-up manner.

[0074] <Production of Monomer Particle Dispersion>

[0075] Table 1 shows the raw material formulations 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 Co., Ltd. Evaluation of precipitation of fluorescent pigments, etc. (including AIE pigments) The monomer particle dispersion was irradiated with UV light to confirm the precipitation of fluorescent pigments, etc. "None", "Little" and "Slightly More" in the column of precipitation of fluorescent pigments, etc. in Table 1 respectively indicate an increase in the amount of precipitation of fluorescent pigments, etc. Since the monomer particle dispersion of Production Example 11 does not contain fluorescent pigments, etc., it is excluded from the evaluation of precipitation of fluorescent pigments (indicated by "-"). It should be noted that the polymerizable monomer includes the main component monomers of Table 1 and monomers having functional groups. The amount of fluorescent pigment (relative to the monomer) represents the compounding amount (wt%) of the fluorescent pigment, etc. relative to 100% by mass of the polymerizable monomer used in the production of the monomer particle dispersion. Table 2 shows the raw material formulations and test results of the resin particle compositions of Examples 1 to 5 and Comparative Examples 1 to 8.

[0076] [Table 1]

[0077]

[0078] (Explanation of abbreviations, etc.)

[0079] HPS: Hexaphenylsilole

[0080] ST: Styrene

[0081] MMA: Methyl Methacrylate

[0082] mAAc: methacrylic acid

[0083] AM: Acrylamide

[0084] 2HEMA: 2-Hydroxyethyl Methacrylate

[0085] Part by mass of the main component monomer: Part by mass of the monomer having a functional group = 90:10 (the same for Production Examples 1 to 11)

[0086] Homodisperse (manufactured by PRIMIX Corporation: MARKII Model 2.5, stirring condition: rotation speed 5000 rpm)

[0087] Ultrasonic homogenizer (Mitsui Electric Seiki Co., Ltd.: UX-600, stirring condition: frequency 20KHz)

[0088] High-pressure homogenizer (manufactured by SMT Co., Ltd.: LAB2000, stirring conditions: 5 cycles at a pressure of 200 MPa)

[0089] [Table 2]

[0090]

[0091] (Explanation of abbreviation)

[0092] DVB: divinylbenzene

[0093] TMP: Trimethylolpropane trimethacrylate

[0094] AIBN: 2,2'-azobis(isobutyronitrile)

[0095] KPS: Potassium Persulfate

[0096] HPS: Hexaphenylsilole

[0097] The amount of the crosslinking monomer (wt%) is converted into weight parts by the following calculation formula: weight parts = 100X / (1-X), where X represents weight %

[0098] <Production Example 1: Preparation of Monomer Particle Dispersion>

[0099] Based on Table 1, as liquid raw materials, ion exchange water, a polymerizable monomer liquid in which a marker substance is dissolved (anthracene: styrene: methacrylic acid (= 10 parts: 90 parts: 10 parts)), and an emulsifier dilution liquid (a 22.5% diluted aqueous solution of NOIGEN XL-100 (trade name, manufactured by Dai-ichi Kogyo Seiyaku Co., Ltd., polyoxyalkylene branched decyl ether, turbidity point = 79°C, HLB 14.7)) were prepared.

[0100] since Figure 1 In the inflow passage of the supercritical water emulsification device, ion exchange water as a liquid raw material is flowed in at a rate of 29.5 ml / min, and a polymerizable monomer liquid (a mixed solution of anthracene, styrene and methacrylic acid) in which a marker substance is dissolved is flowed in at a rate of 4.3 ml / min. In the subcritical process section, under the condition of high temperature and high pressure of water forming subcritical water at 300°C, the polymerizable monomer in which a marker substance is dissolved is dissolved in the ion exchange water. Next, an emulsifier diluent (a nonionic emulsifier solution having a turbidity point) is added to the liquid coming out of the subcritical process section at a rate of 5.2 ml / min at the initial stage of forming monomer particles, and the liquid is cooled in the cooling section, thereby obtaining a monomer particle dispersion liquid formed of monomer microparticles having an average particle size of 150 nm and a polymerizable monomer concentration of 10% in which a marker substance is dissolved.

[0101] The particle size distribution of the obtained monomer particles showed a sharp normal distribution, and it was successfully confirmed that it was a dispersion formed by uniform microparticles. The average particle size and particle size distribution of the obtained monomer particles were obtained by dynamic light scattering. The average particle size (diameter, D50) based on the dynamic light scattering method was determined by diluting the monomer particle dispersion with water to an appropriate concentration using FPAR-1000 (trade name, manufactured by Otsuka Electronics Co., Ltd.) at 25°C. In addition, the obtained monomer particle dispersion was irradiated with a UV lamp to observe the dissolved state of the marker substance, and no precipitation of the marker substance was found. Thus, it was confirmed that the marker substance was encapsulated in the monomer particles.

[0102] <Manufacturing Example 2 to Manufacturing Example 11: Preparation of Monomer Particle Dispersion>

[0103] The monomer particle dispersions of Production Examples 2 to 5 were prepared based on Table 1 and the same production method as in Production Example 1, and thus detailed descriptions are omitted. On the other hand, the monomer particle dispersions of Production Examples 6 to 11 were prepared based on the raw material formulations in Table 1 using an oil droplet production apparatus (homogeneous disperser, high pressure homogenizer, ultrasonic homogenizer) different from that of Production Examples 1 to 5.

[0104] <Example 1: Preparation of resin particle composition>

[0105] Into a 300 ml four-necked round-bottom flask equipped with a stirrer, a thermometer, and a reflux condenser, 100 g of the anthracene-containing monomer particle dispersion having an average particle size of 150 nm obtained in Production Example 1, 10 wt % (1.1 g in terms of weight) of DVB as a crosslinking 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 170 rpm for 60 minutes while nitrogen substitution was performed at 25° C. and atmospheric pressure. 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 the polymerization treatment, the mixture was cooled to obtain an anthracene-containing resin particle composition having a concentration of 5% and an average particle size of 160 nm.

[0106] The average particle size (D50) and particle size distribution of the resin particle composition obtained by polymerization were determined by the dynamic light scattering method in the same manner as in the manufacturing example. In addition, the obtained resin particle composition was irradiated with a UV lamp to observe the dissolution state of the marker substance, and no precipitation of the marker substance was found. The obtained fine resin particle composition was further observed using an electron microscope, and no aggregates were observed other than the resin particle composition, confirming that the marker substance was encapsulated in the resin particles.

[0107] <Examples 2 to 5, Comparative Examples 1 to 8: Preparation of Resin Particle Compositions>

[0108] The resin pellet compositions of Examples 2 to 5 and Comparative Examples 1 to 8 were produced based on the raw material formulations in Table 2 and the same production method as in Example 1, and therefore detailed descriptions thereof are omitted.

[0109] <Test method>

[0110] The performance was evaluated by performing the following tests 1 to 3 using the resin pellet compositions of Examples 1 to 5 and Comparative Examples 1 to 8. Note that the resin pellet compositions of Comparative Examples 7 and 8 were tested in Test Examples 1 to 3 after the following step of absorbing a marker substance.

[0111] (Description of the absorption process of the marker substance)

[0112] The purpose of this step is to confirm whether the fluorescence intensity is improved when the resin particle composition of Comparative Examples 7 to 8 absorbs the labeling substance. The 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 with an amount of HPS to reach 1 wt% relative to the resin particle content of the prepared resin particle dispersion and stirred for 5 minutes to allow the resin particle composition to absorb HPS. Then, the obtained dispersion is subjected to ultracentrifugal separation to precipitate the resin particles, and after removing the supernatant, pure water is added to redisperse the particles to obtain a resin particle dispersion.

[0113] (Test Example 1: Confirmation of the Presence of Fluorescent Dyes and the Like Outside Resin Particles)

[0114] The resin particle dispersion was collected and dried at 60° C. for 1 hour, and then a scanning electron microscope (SEM) manufactured by JEOL Ltd. was used to confirm whether or not fluorescent dyes and the like (including AIE dyes) were present outside the resin particles.

[0115] (Evaluation Criteria)

[0116] None: No fluorescent dye or the like was confirmed outside the resin particles.

[0117] Almost none: Almost no fluorescent dye or the like is confirmed outside the resin particles.

[0118] Little: A small amount of fluorescent pigment or the like is observed outside the resin particles.

[0119] Slightly more: A small amount of fluorescent pigment etc. is confirmed outside the resin particles.

[0120] Many: A large amount of fluorescent dye and the like were confirmed outside the resin particles.

[0121] (Test Example 2 Confirmation of Fluorescence Intensity (After Water Washing))

[0122] The obtained resin particle dispersion was subjected to ultracentrifugal separation to precipitate the resin particles, and after removing the supernatant, pure water was added to redisperse the resin particles. This operation was repeated three times, and the precipitated resin particles were diluted with pure water for the fourth time to prepare a 1 mg / mL dispersion, which was photographed using a fluorescence microscope BZ-X800 manufactured by KEYENCE CORPORATION to confirm the fluorescence intensity.

[0123] (Evaluation Criteria)

[0124] ◎: Very strong ○: Strong △: Slightly strong ×: Weak

[0125] (Test Example 3 Confirmation of Fluorescence Intensity (THF Dispersion Liquid))

[0126] The obtained resin particle dispersion was subjected to ultracentrifugal separation to precipitate the resin particles, and after removing the supernatant, pure water was added to redisperse the resin particles. This operation was repeated 3 times, and the precipitated resin particles were diluted with THF in the fourth time to prepare a 10 mg / mL dispersion, and the dispersion was photographed using a fluorescence microscope BZ-X800 manufactured by KEYENCE CORPORATION to confirm the fluorescence intensity.

[0127] (Evaluation Criteria)

[0128] ◎: Very strong ○: Strong △: Slightly strong ×: Weak

[0129] <Test Results>

[0130] Table 2 shows the test results of Examples 1 to 5 and Comparative Examples 1 to 8. Examples 1 to 5 show excellent 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 have excellent fluorescence intensity in the solvent (THF). On the other hand, Comparative Examples 1 to 5 (when no cross-linking monomer is included) show that the fluorescent pigment, etc., fall off outside the resin particles and the fluorescence intensity decreases. In addition, Comparative Example 6 (when a cross-linking monomer is included) shows that the solvent resistance of the resin particles is slightly improved, but does not show a fluorescence intensity sufficient for practical use. Comparative Examples 7 to 8 (when the labeling substance is absorbed in the subsequent process) show that the fluorescent pigment, etc. attached to the surface of the resin particles is easy to fall off and the fluorescence intensity decreases. Based on the above test results, it is speculated that the use of the monomer particle dispersion prepared by the subcritical water emulsification technology of the present invention and the cross-linking monomer to prepare the resin particles helps to improve solvent resistance. It is speculated that this can prevent the labeling substance wrapped in the resin particles from leaking out and achieve high fluorescence intensity.

[0131] As described above, the resin particle composition of the present invention has excellent solvent resistance and has remarkable effects of high fluorescence intensity.

[0132] It should be noted that it is difficult for a person skilled in the art to directly determine the specific structure and properties of the monomer particle dispersion and the resin particles of the present invention, and it should be noted that detailed information about them is still unclear. In addition, it should be noted that determining the specific structure and properties of the monomer particle dispersion and the resin particles of the present invention would require a person skilled in the art to pay excessive experiments and prepare special measuring equipment, etc., which would cause huge economic expenses, which is not realistic from this point of view.

[0133] The present invention is not limited to the above-described embodiments, and various modifications and changes can be made within the scope of the gist of the invention.

Claims

1. A resin particle composition, The resin particle composition comprises resin particles encapsulating a marking substance, and the resin particle composition is produced by a method comprising the following steps: A step of generating a mixture by mixing the labeling substance and a polymerizable monomer with subcritical water; A step of generating a monomer particle dispersion by mixing and cooling the mixture and an emulsifier; and a step of performing a polymerization reaction between the monomer particle dispersion and a crosslinking monomer to produce the resin particles; The polymerizable monomer includes an aromatic monomer, at least one of alkyl (meth)acrylates and cycloalkyl (meth)acrylates having an alkyl group with 1 to 18 carbon atoms, and at least one of alkyl (meth)acrylates having a functional group. The resin particles contain, as structural units, 40 to 95% by mass of the aromatic monomer and / or at least any one of the (meth)acrylic acid alkyl esters having an alkyl group with 1 to 18 carbon atoms and the (meth)acrylic acid cycloalkyl esters, 0 to 10% by mass of the (meth)acrylic acid alkyl ester having a functional group, and 5 to 60% by mass of the crosslinking monomer, relative to 100% by mass of all polymerizable monomers. The labeling substance comprises at least one of a fluorescent pigment and an aggregation-induced emission pigment, The aromatic monomer is 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, The functional group includes at least any one of a hydroxyl group, a carboxyl group, an amino group, and an amide group.

2. The resin particle composition according to claim 1, wherein The resin particles contain 0.1 to 15% by mass of the marker substance relative to 100% by mass of all polymerizable monomers.

3. The resin particle composition according to claim 1, wherein 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.

4. The resin particle composition according to claim 1, wherein The crosslinking monomer is 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.

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

6. The resin particle composition according to any one of claims 1 to 5, wherein The fluorescent pigment comprises at least one selected from the group consisting of merocyanine, perylene, acridine, anthracene, luciferin, solvent green 7, stilbene, rhodamine, coumarin, 4-(dicyanomethylene)-2-methyl-6-(4-dimethylaminophenylvinyl)-4H-pyran (DCM), pyrrole, fluorescent yellow, umbelliferone, and derivatives thereof, The aggregation-induced emission pigment includes one or more selected from the group consisting of silole ring-containing compounds and derivatives, hydrocarbon aromatic compounds and derivatives, heteroaromatic compounds and derivatives, and rhodamine compounds and derivatives.

7. A detection kit, The invention comprises the resin particle composition according to claim 1 which is bound to a target substance in a test sample.

8. A method for producing a resin particle composition, The resin particle composition comprises resin particles encapsulating a marking substance, and the manufacturing method comprises the following steps: A step of generating a mixture by mixing the labeling substance and the polymerizable monomer with subcritical water; A step of generating a monomer particle dispersion by mixing and cooling the mixture and an emulsifier; and a step of performing a polymerization reaction between the monomer particle dispersion and a crosslinking monomer to generate the resin particles; The polymerizable monomer includes an aromatic monomer, at least one of alkyl (meth)acrylates and cycloalkyl (meth)acrylates having an alkyl group with 1 to 18 carbon atoms, and at least one of alkyl (meth)acrylates having a functional group. The resin particles contain, as structural units, 40 to 95% by mass of the aromatic monomer and / or at least any one of (meth)acrylic acid alkyl esters and (meth)acrylic acid cycloalkyl esters having an alkyl group with 1 to 18 carbon atoms, 0 to 10% by mass of the (meth)acrylic acid alkyl ester having a functional group, and 5 to 60% by mass of the crosslinking monomer, relative to 100% by mass of all polymerizable monomers. The labeling substance comprises at least one of a fluorescent pigment and an aggregation-induced emission pigment, The aromatic monomer is 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, The functional group includes at least any one of a hydroxyl group, a carboxyl group, an amino group, and an amide group.

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