Particles used in agglutination method, test particles, reagent, test kit, method for producing particles, and method for detecting target substance

Abstract

The present invention relates to a particle used in an aggregation method, a test particle, a reagent, a test kit, a method for producing the particle, and a method for detecting a target substance. In order to achieve both improvement of sensitivity and suppression of non-specific adsorption in an agglutination method, which is required to improve sensitivity at low concentrations, provided is a particle for use in an agglutination method, the particle comprising a core particle having a volume average particle diameter of 200 nm or more and 500 nm or less, and a shell on the surface of the core particle, a polymer having a structural unit represented by formula (1) wherein the content ratio of the structural unit represented by formula (1) is 40 mass% or more and 85 mass% or less with respect to the particles; the shell has a thickness of 5 nm or more and 50 nm or less, and contains a polymer having a structural unit represented by formula (2).

Description

Technical Field

[0001] The present disclosure relates to particles used in an agglutination method, test particles, reagents, test kits, a method for detecting a target substance, and a method for producing the particles. Background Art

[0002] In recent years, agglutination methods such as immunolatex agglutination assays have attracted attention as simple and rapid immunoassays. Known methods include mixing a dispersion of particles having an antibody or antigen as a ligand on the surface with a sample that may contain a target substance (antigen or antibody). At this time, when the sample contains the target substance (antibody or antigen), the particles will cause an agglutination reaction, and thus the presence or absence of a disease can be identified by optically detecting a change in the agglutination reaction as a change in a quantity such as scattered light intensity, transmitted light intensity, absorbance, etc. It is desirable that the particles used in the agglutination method have a small property of adsorbing substances other than the target substance, that is, so-called non-specific adsorption, in order to reduce noise. As particles used in the agglutination method, polystyrene particles are widely used, and particles having a structure derived from styrene and glycidyl methacrylate are also known. Patent Document 1 describes particles having a core-shell structure, in which the shell has a carboxyl group and 2,3-dihydroxypropyl as reactive functional groups. It has been reported that, for these particles, the following methods are preferred: a method of adding a shell to a seed particle (mother particle, core) such as polystyrene or a styrene-based copolymer by copolymerizing a monomer having a carboxyl group and a monomer having 2,3-dihydroxypropyl by a two-stage swelling polymerization method to form a core-shell structure, and a method of forming a core-shell structure by using a monomer capable of forming a carboxyl group or 2,3-dihydroxypropyl by hydrolysis by a two-stage swelling polymerization method. Summary of the Invention

[0003] The present inventors synthesized particles having a core-shell structure according to Patent Document 1 (Japanese Patent Application Laid-Open No. 2007-224213), prepared a dispersion of particles in which an antibody or antigen was chemically bonded as a ligand on the particle surface, mixed the dispersion with a sample containing a target substance, and detected a change in absorbance by an agglutination method. However, depending on the type and concentration of the target substance, a sufficient change in absorbance could not be obtained, and the sensitivity was low. Even when particles having a core-shell structure were synthesized with a reduced amount of glycidyl methacrylate to improve the sensitivity, the desired sensitivity was not obtained, and non-specific adsorption deteriorated.

[0004] In addition, when copolymer particles of styrene and glycidyl methacrylate without a core-shell structure were prepared and used, the desired sensitivity was not obtained either.

[0005] The present disclosure has been made in view of the foregoing background and problems. Specifically, an object of the present disclosure is to provide particles capable of simultaneously improving sensitivity and suppressing non-specific adsorption in an agglutination method and a method for producing the same.

[0006] The present disclosure provides:

[0007] Particles used in an agglutination method, the particles comprising a core particle and a shell on the surface of the core particle, wherein the core particle has a volume average particle diameter of 200 nm or more and 500 nm or less, and the core particle comprises a polymer having a structural unit represented by formula (1), wherein the content ratio of the structural unit represented by formula (1) is 40% by mass or more and 85% by mass or less with respect to the particles; the thickness of the shell is 5 nm or more and 50 nm or less, and comprises a polymer having a structural unit represented by formula (2),

[0008]

[0009] (wherein, R 1 represents a hydrogen atom or a methyl group; R 2 represents a substituted or unsubstituted phenyl group or naphthyl group, provided that when substituted, the substituent is a methyl group or an ethyl group; and for each structural unit, R 1 and R 2 may be different),

[0010]

[0011] (wherein, R 3 represents a hydrogen atom or a methyl group; R 4 represents a group having an epoxy group, a group having a hydroxyl group or a group having a carboxyl group; and for each structural unit, R 3 and R 4 may be different).

[0012] From the description of the following exemplary embodiments, further features of the present invention will become apparent. Detailed Description

[0013] Preferred embodiments of the present invention will now be described in detail.

[0014] Hereinafter, the present disclosure will be described in detail with reference to embodiments, but the technical scope of the present disclosure is not limited to these embodiments.

[0015] First Embodiment

[0016] As a first embodiment, the present disclosure provides the following particles.

[0017] Particles used in an agglutination method, the particles comprising a core particle and a shell on the surface of the core particle, wherein

[0018] The volume-average particle diameter of the core particles is 200 nm or more and 500 nm or less, and the particles contain a polymer having a structural unit represented by the formula (1), wherein the content ratio of the structural unit represented by the formula (1) is 40% by mass or more and 85% by mass or less with respect to the particles; and

[0019] The thickness of the shell is 5 nm or more and 50 nm or less, and the shell contains a polymer having a structural unit represented by the formula (2),

[0020]

[0021] (wherein, R 1 represents a hydrogen atom or a methyl group; R 2 represents a substituted or unsubstituted phenyl group or naphthyl group, provided that when substituted, the substituent is a methyl group or an ethyl group; and for each structural unit, R 1 and R 2 may be different),

[0022]

[0023] (wherein, R 3 represents a hydrogen atom or a methyl group; R 4 represents a group having an epoxy group, a group having a hydroxyl group, or a group having a carboxyl group; and for each structural unit, R 3 and R 4 may be different).

[0024] The particles according to the present embodiment may be referred to as particles sequentially including a first layer and a second layer. The first layer may be a particle, and it can be said that the aforementioned core particle is the first layer and the aforementioned shell is the second layer. It should be noted that in the present embodiment, a layer different from the second layer may be provided between the first layer and the second layer or on the outermost side of the second layer (on the surface side of the particle). Hereinafter, the particles according to the present embodiment will be described by taking the structure of particles sequentially including a first layer and a second layer as an example, wherein the first layer is a core particle and the second layer is a shell.

[0025] The particles according to the present embodiment contain a polymer having a structure represented by the formula (1), wherein the content of the structure represented by the formula (1) is 40% by mass or more and 85% by mass or less of the core particles. Formula (1) is in R 2It has a substituted or unsubstituted phenyl or naphthyl group. It is known that phenyl and naphthyl groups increase the refractive index. By having a repeating unit structure represented by the formula (1) in an amount of 40% by mass or more and 85% by mass or less in the particles, the refractive index of the particles is increased, and thus the refractive index of the particles themselves is also increased. When the reagent containing the particles reacts with the target substance and agglutinates, the absorbance increases. When the particles with a higher refractive index agglutinate, the absorbance becomes larger, so the difference in absorbance before and after agglutination is also larger, and the sensitivity is improved. Preferably, the particles contain 50% by mass or more, more preferably 60% by mass or more of the repeating unit structure represented by the formula (1). It is also preferable that the particles contain 65% by mass or less of the repeating unit structure represented by the formula (1). In addition, preferably, the content ratio of the structural unit represented by the formula (1) is 90% by mass or more relative to the core particles.

[0026] It should be noted that the core particles may contain a polymerization initiator, a copolymer derived from a vinyl monomer, coloring materials such as pigments, dyes or fluorescent agents, and inorganic fillers, etc. The content is preferably 9% by mass or less.

[0027] The volume average particle diameter of the core particles of this embodiment is 200 nm or more and 500 nm or less. In the detection by the agglutination method, light with a wavelength in the visible light region is usually used. In this wavelength region, as the particle diameter increases, the absorbance also increases until the particle diameter reaches 1 μm (Non-Patent Document 1; Medical Electronics and Bioengineering, Vol. 22, No. 4, 39 - 45 (1984)). Therefore, when the particles of this embodiment having a core particle diameter of 200 nm or more and a shell thickness of 5 nm or more agglutinate, compared with the case where the particles with a core particle diameter less than 200 nm agglutinate, the aggregate of the agglutinated particles becomes larger, and the difference in absorbance before and after agglutination becomes larger, so the sensitivity is higher. In addition, when the core particle diameter is 500 nm or less, even when the concentration of the target substance is high, as the concentration increases, the sensitivity increases to a certain extent, so the concentration of the target substance in the sample can be evaluated. Note that the particles according to this embodiment are used in the agglutination method. From the perspective of detecting the change in turbidity before and after mixing the target substance and the particles, the agglutination method can be called a nephelometry method. When the particles bind to an antibody or an antigen to detect the antigen or antibody, this embodiment can also be called the particles used in an immunonephelometry method.

[0028] In the particles of the present embodiment, the thickness of the shell is 5 nm or more and 50 nm or less, and the shell contains a polymer having a structural unit represented by the formula (2). The structure represented by the formula (2) is highly hydrophilic because it has any one of an epoxy group, a hydroxyl group, or a carboxyl group. By covering the surface of the core particle with a shell layer containing a polymer having the structure represented by the formula (2), the hydrophobic part of the particle surface is covered, and the hydrophilicity is improved. As a result, the hydrophobic interaction with proteins other than the target substance in the sample is reduced, and non-specific adsorption can be suppressed.

[0029] In addition, when the thickness of the shell is 5 nm or more and 50 nm or less, the hydrophobic surface of the core particle can be covered without being exposed, the dispersibility of the particles is not too high, and the aggregation of the particles is not easily inhibited, resulting in high sensitivity. Therefore, both high sensitivity and suppression of non-specific adsorption can be achieved simultaneously.

[0030] In the present embodiment, the content ratio of the structural unit represented by the formula (2) relative to the core particle is preferably 9% by mass or more and 60% by mass or less, more preferably 9% by mass or more and 53% by mass or less, more preferably 9% by mass or more and 39% by mass or less, more preferably 16% by mass or more and 39% by mass or less, more preferably 20% by mass or more and 39% by mass or less, and more preferably 31% by mass or more and 39% by mass or less.

[0031] In the present embodiment, the content of the structure represented by the formula (2) in the particles is 9% by mass or more and 60% by mass or less. Therefore, the hydrophobicity of the particle surface is reduced, and non-specific adsorption can be suppressed.

[0032] In addition, the dispersibility of the particles is not too high, and the aggregation of the particles is not easily inhibited, resulting in high sensitivity. Therefore, both high sensitivity and suppression of non-specific adsorption can be achieved simultaneously.

[0033] As described below, the particles of the present embodiment have a structure including a core particle and a shell covering the core particle, wherein the core has a high refractive index and a large difference in absorbance before and after aggregation, and the shell is highly hydrophilic and can suppress non-specific adsorption. By having such a core-shell structure, the particles of the present embodiment can have both a high refractive index and hydrophilicity on the particle surface, and can balance high-sensitivity detection in the agglutination method and suppression of non-specific adsorption.

[0034] In the present embodiment, preferably, the formula (1) is a structure represented by the formula (1-A). By using the formula (1-A), the core refractive index of the core particle can be improved. In addition, since the structure of the formula (1-A) is highly hydrophobic, it is less likely that the hydrophilic shell component is introduced into the core part:

[0035]

[0036] (wherein, R 10 represents phenyl, tolyl or naphthyl).

[0037] The structure represented by formula (1-A) in the present embodiment is obtained by polymerizing a monomer represented by the following formula (X1). As described below, specific examples of the monomer include styrenes, 1-vinylnaphthalene, and 2-vinylnaphthalene, and styrene, 1-vinylnaphthalene, and 2-vinylnaphthalene are particularly preferred. These monomers can be used alone, or multiple of these monomers can be used simultaneously.

[0038] Preferably, the core particle further has a crosslinked structure. The crosslinked structure is obtained by polymerizing a crosslinkable radical polymerizable monomer which is a monomer having two or more radical polymerizable unsaturated bonds in one molecule. Examples of such crosslinkable monomers include polyfunctional (meth)acrylates such as ethylene glycol diacrylate, ethylene glycol dimethacrylate, trimethylolpropane triacrylate, trimethylolpropane trimethacrylate, pentaerythritol triacrylate, pentaerythritol trimethacrylate, dipentaerythritol hexaacrylate, dipentaerythritol hexamethacrylate; conjugated dienes such as butadiene and isoprene; divinylbenzene; diallyl phthalate; allyl acrylate and allyl methacrylate. Two or more of the crosslinkable radical polymerizable monomers can be used. As the crosslinked structure, the structure represented by formula (3) is more preferred. When the core particle has a crosslinked structure, the particle having a core-shell structure becomes physically stronger, and there is no concern about cracking or chipping even when the centrifugation operation is repeated during purification.

[0039]

[0040] (wherein, Z represents a substituted or unsubstituted phenylene or naphthylene, provided that when substituted, the substituent is methyl or ethyl. For each structural unit, Z can be different.)

[0041] Examples of the crosslinkable radical polymerizable monomer for forming the crosslinked structure of formula (3) include 1,2-divinylbenzene, 1,3-divinylbenzene, 1,4-divinylbenzene, 2,6-diethynylnaphthalene, and 2,7-diethynylnaphthalene. These can be used alone, or multiple of them can be used simultaneously.

[0042] Among the crosslinkable radical polymerizable monomers mentioned as examples, divinylbenzene is preferred.

[0043] Although the reason is not clear, when divinylbenzene is used, the processability during the radical polymerization reaction is excellent, the monomer conversion rate during the formation of the core particles is improved, and the core component is less likely to be introduced into the shell, and thus it is preferred.

[0044] In the present embodiment, it is preferred that the mass ratio of the core to the particle is 50% by mass or more and 95% by mass or less. When the mass ratio of the core to the particle is 50% by mass or more, the content of the core particles having a high refractive index can be increased, the refractive index can be improved, the difference in absorbance before and after aggregation can be increased, and the sensitivity in the region where the concentration of the target substance is low can be improved. When the mass ratio of the core to the particle is 95% by mass or less, the surface of the core particles can be covered with the shell layer, the hydrophilicity of the particle surface can be increased, and the suppression of non-specific adsorption can be improved.

[0045] In the present embodiment, it is preferred that the formula (2) is a structure represented by the formula (2-A). The structure represented by the formula (2-A) has one of a hydroxyl group and a carboxyl group, and the ability to suppress non-specific adsorption is equal to or higher than that of the structure having an epoxy group, and thus it is preferred:

[0046]

[0047] (wherein one of R 31 and R 32 represents a hydroxyl group, and the other represents a hydroxyl group, a group represented by the formula (2-B), or a group represented by the formula (2-C)),

[0048]

[0049] (wherein R 20 represents a single bond or a methylene group, R 22 , R 23 and R 24 each represent a hydrogen atom, a methyl group, a hydroxyl group, or a hydroxymethyl group, and one or more of R 22 , R 23 and R 24 represent a hydroxyl group, Y 1 represents a sulfur atom or an imino group, and *1 represents the bonding position),

[0050]

[0051] (wherein R 25 represents a hydrogen atom, a methyl group, a hydroxyl group, or a carboxyl group; Y 2 represents a sulfur atom or an imino group; Y 3 represents a single bond or a methylene group; and *2 represents the bonding position).

[0052] When the total number of hydroxyl groups (0 or more) and carboxyl groups (0 or more) in formula (2-A) is 2 or more, even under the condition that the thickness of the shell is 5 nm or more and 50 nm or less, non-specific adsorption can be reduced because a structure with high hydrophilicity is included in one structural unit. In addition, in view of even higher hydrophilicity, more preferably, the total number of hydroxyl groups (0 or more) and carboxyl groups (0 or more) in formula (2-A) is 3 or more.

[0053] Specific examples of the structure of formula (2-A) include, but are not limited to, the following formulas (2-A-1) to (2-A-12).

[0054]

[0055] The structure represented by formula (2-A) in the present embodiment is obtained by reacting a polymer obtained by polymerizing a monomer represented by the following formula (X2) with the following formula (X3).

[0056] As shown in formulas (2-A-1) to (2-A-12), the side chain may contain a sulfur atom or a nitrogen atom. From the viewpoint of improving sensitivity, it is more preferable to include a sulfur atom in the side chain. From the viewpoint of suppressing non-specific adsorption, it is more preferable to include a nitrogen atom in the side chain. Since the structure represented by "-S-" has a weak hydrophobic tendency, it is desirable to moderately weaken the water-binding force of the highly hydrophilic side chain and suppress the osmotic aggregation that may occur when the structure represented by "-S-" is mixed with a high-concentration sample, thereby contributing to the improvement of sensitivity. Since the amino group has a hydrophilic tendency, it can contribute to the suppression of non-specific adsorption.

[0057] Specific examples of the monomer represented by (X2) are not particularly limited, but glycidyl (meth)acrylate is preferred.

[0058] Preferably, the content ratio of the structural unit represented by formula (2) with respect to the shell is 90% by mass or more and 100% by mass or less. By setting the content of the structure represented by formula (2) to 90% by mass or more, the hydrophilicity of the shell can be improved, and the suppression of non-specific adsorption can be improved. In order to further suppress non-specific adsorption, more preferably, the content of the structure represented by formula (2) is 95% by mass or more. It should be noted that the shell may contain a polymerization initiator, a copolymer derived from a vinyl monomer, coloring materials such as pigments, dyes or fluorescent agents, and inorganic fillers, etc. The content is preferably 9% by mass or less.

[0059] The particles of the present embodiment may have a structure other than formula (1) or formula (2). Examples of the structure other than formula (1) and formula (2) contained in the particles include, but are not limited to, structures obtained by polymerizing monomers such as styrene-based, acrylic acid-based, and methacrylic acid-based monomers. The particles of the present embodiment may simultaneously have a variety of structures other than formula (1) and formula (2).

[0060] Preferably, when measured by X-ray photoelectron spectroscopy (XPS), the composition ratio of carbon element to oxygen element of the particles is in the range of 2.0 or more and 3.3 or less. In XPS measurement, photoelectrons generated from the outermost surface to about 10 nm are detected. Therefore, XPS is a method that can analyze the surface components of the particles. When the composition ratio is less than 2.0, the oxygen element in the surface layer of the particles is excessive. Although the hydroxyl groups and carboxyl groups are sufficiently connected, the hydrophilicity is very high. Therefore, it is difficult for the particles to aggregate, and the detection sensitivity based on the principle of the aggregation method may be reduced. When the composition ratio is greater than 3.3, the oxygen element in the surface layer of the particles is too little, and the hydrophilicity is insufficient, which is not only unfavorable for the dispersion stability when the particles are stored for a long time, but also may deteriorate the non-specific adsorption property. Therefore, by optimizing the composition ratio of each component in the surface layer of the particles, particles having both excellent sensitivity and excellent non-specific adsorption property can be provided.

[0061] By measuring the test particles of the present embodiment by FT-IR, the ratio of formula (1) to formula (2) can be evaluated. Preferably, the measurement method is the ATR method in which infrared light can penetrate from the outermost surface to about μm size to obtain an FT-IR spectrum. In the infrared absorption spectrum (FT-IR spectrum), when the peak height of the C═C bond of the aromatic ring present at 1500-1650 cm -1 is defined as A and the peak height of the carbonyl group of the ester bond present at 1680-1750 cm -1 is defined as B, in view of suppressing the balance of sensitivity and non-specific adsorption, preferably, the value of A / B of the particles of the present embodiment is 0.85 or more and 5.35 or less.

[0062] When the value of A / B is 0.85 or more, the proportion of formula (1) is sufficiently high relative to the carbonyl group, so the detection sensitivity is improved. When the value of A / B is 5.35 or less, the carbonyl group of the ester bond contained in the particles is sufficiently abundant, that is, the proportion of the hydrophilic formula (2) is large relative to the whole particles. Thereby, the particles obtain hydrophilicity and the non-specific adsorption property is improved.

[0063] The particles of the present embodiment have a particle size of 210 nm or more and 600 nm or less in terms of volume average particle size in the aqueous dispersion, more preferably 230 nm or more and 500 nm or less. When the particles are 230 nm or more, compared with particles smaller than 230 nm, the aggregates of the aggregated particles become larger, and the difference in absorbance before and after aggregation increases, so the sensitivity becomes higher. When the particle size is 600 nm or less, even if the concentration of the target substance is high, as the concentration increases, the sensitivity increases above a certain level, so the concentration of the target substance in the sample can be evaluated.

[0064] The ratio (Dv / Dn) of the volume average particle size (Dv) to the number average particle size (Dn) of the particles of the present embodiment is preferably 1.25 or less. It is known that as the value of Dv / Dn approaches 1, the particle size distribution becomes narrower. When the value of Dv / Dn is 1.25 or less, the variation in particle size is small, and there are no particles with a large particle size, so the size difference from the aggregate after aggregation becomes larger and the sensitivity becomes higher.

[0065] When the particles are dispersed in water at a solid content concentration of 0.1% by mass so that the value of Dv / Dn is 1.25 or less, and the absorbance at an incident light wavelength of 572 nm with a cell length of 10 mm is defined as E, preferably, the value of E / Dv is 8.0×10 -4 or more and 2.0×10 -3 or less. When the value of E / Dv is 8.0×10 -4 or more and 2.0×10 -3 or less, even if the particle size is above a certain size, the absorbance is large, and the difference in absorbance before and after aggregation becomes larger, so the sensitivity becomes higher.

[0066] As a method for producing the core particles, a soap-free emulsion polymerization method is preferred. When the soap-free emulsion polymerization method is used, the particle size distribution becomes uniform, the sensitivity is stable, and the detection limit in the region where the concentration of the target substance is low can be improved.

[0067] The method for forming the core-shell structure is not particularly limited as long as the core-shell structure is formed. However, in view of the improvement in sensitivity and the suppression of non-specific adsorption, a method of adding a monomer for forming a shell to a dispersion of core particles and then adding a water-soluble polymerization initiator to form a shell structure is preferred. Although the details are not clear, the mechanism is considered as follows. When a monomer and a water-soluble polymerization initiator are added to an aqueous dispersion of core particles, the monomer dissolved in water starts to polymerize and form oligomers. As the polymerization progresses, the hydrophobicity of the oligomers increases, and the oligomers become difficult to disperse alone and deposit on the surface of the core particles. On the surface of the particles, the monomer and the oligomers introduced onto the surface of the core particles further react to form a core-shell structure. In the particles thus formed, the shell component is less likely to penetrate into the core region, and thus it is less likely that the refractive index of the core particles decreases. As a result, the difference in absorbance before and after aggregation becomes larger, and the sensitivity is improved. On the other hand, as an effective method for homogenizing the particle size distribution, a two-stage swelling polymerization method of swelling a monomer for forming a shell on core particles and forming a shell structure using a hydrophobic initiator or the like is known. However, in this method, the core particles swell. Therefore, the shell component may penetrate into the core particles, which may lead to a decrease in the refractive index of the core particles. Therefore, when the particles produced by this method are used in an aggregation method, it is difficult to improve the sensitivity.

[0068] Second Embodiment

[0069] As a second embodiment, the present disclosure provides a method for producing particles used in an aggregation method as follows.

[0070] A method for producing particles used in an aggregation method, the method comprising:

[0071] A first step of subjecting a first monomer composition containing a monomer represented by formula (X1) to a polymerization reaction to obtain core particles having a volume average particle diameter of 200 nm or more and 500 nm or less;

[0072] A second step of subjecting a reaction solution including core particles, a second monomer composition containing a monomer represented by formula (X2), and a water-soluble polymerization initiator to a polymerization reaction to obtain particles having a layer of 5 nm or more and 50 nm or less formed on the outside of the core particles,

[0073] The content ratio of the monomer represented by formula (X1) is 90% by mass or more relative to the total amount of the monomers contained in the first monomer composition,

[0074] provided that,

[0075] The content ratio of the unpolymerized monomer represented by formula (X1) is 1000 ppm or less relative to the reaction solution, and

[0076] The content ratio of the monomer represented by the formula (X2) is 9% by mass or more and less than 40% by mass relative to the total amount of the monomers contained in the first monomer composition and the monomers contained in the second monomer composition; and

[0077] In the third step, the particles having the above layer are reacted with the compound represented by the formula (X3),

[0078]

[0079] (wherein, R 11 represents a hydrogen atom or a methyl group, and R 12 represents a substituted or unsubstituted phenyl group or naphthyl group, provided that when substituted, the substituent is a methyl group or an ethyl group),

[0080]

[0081] (wherein, R 13 represents hydrogen or a methyl group, and R 14 represents an ethylene group or a carbonyl group)

[0082]

[0083] (wherein, R 15 represents an amino group or a thiol group, and R 16 and R 17 each represent a hydrogen atom, a methyl group, a group having a hydroxyl group or a group having a carboxyl group, and at least one of R 16 and R 17 represents a group having a hydroxyl group or a group having a carboxyl group).

[0084] Preferably, the content of the monomer represented by the formula (X1) in the reaction solution before the start of the second step is 1000 ppm or less. When the content is 1000 ppm or less, during the formation of the shell in the second step, the amount of the hydrophobic monomer represented by the formula (X1) is small, so that the hydrophobic repeating structure is less likely to be introduced into the shell, and non-specific adsorption can be suppressed.

[0085] The water-soluble polymerization initiator used in this embodiment is not particularly limited, but a water-soluble azo compound or a water-soluble peroxide is preferably used.

[0086] Preferred examples of the water-soluble azo compounds include any one of 4,4'-azobis(4-cyanovaleric acid), 2,2'-azobis[2-methyl-N-(2-hydroxyethyl)propionamide], 2,2'-azobis[N-(2-carboxyethyl)-2-methylpropionamidine] tetrahydrate, 2,2'-azobis(2-methylpropionamidine) dihydrochloride, 2,2'-azobis[2-(2-imidazolin-2-yl)propane], 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride, and 2,2'-azobis[2-(2-imidazolin-2-yl)propane] disulfate dihydrate.

[0087] Preferred examples of the water-soluble peroxides are any one of potassium persulfate, ammonium persulfate, sodium persulfate, tert-butyl hydroperoxide, cumene hydroperoxide, p-menthane hydroperoxide, and diisopropylbenzene hydroperoxide.

[0088] In the present embodiment, for the improvement of sensitivity, the monomer represented by the formula (X1) is preferred because the polymer obtained by polymerizing the monomer has a high refractive index. Examples of the monomer include styrenes, 1-vinylnaphthalene, and 2-vinylnaphthalene, and styrene, 1-vinylnaphthalene, and 2-vinylnaphthalene are particularly preferred. These monomers can be used alone, or multiple of these monomers can be used simultaneously.

[0089] Styrenes: styrene, α-methylstyrene, β-methylstyrene, o-methylstyrene, m-methylstyrene, p-methylstyrene, 2,4-dimethylstyrene, p-n-butylstyrene, p-tert-butylstyrene, p-n-hexylstyrene, p-n-octylstyrene, p-n-nonylstyrene, p-n-decylstyrene, p-n-dodecylstyrene, p-methoxystyrene, p-phenylstyrene, etc.

[0090] In the present embodiment, the monomer represented by the formula (X2) has a glycidyl group in the side chain, so that the glycidyl group in the shell of the polymer containing the formula (X2) can react with the compound represented by the formula (X3), and the carboxyl group or hydroxyl group contained in the formula (X3) can be introduced onto the particle surface. The monomer represented by the formula (X2) is not particularly limited, but (meth)acrylic acid glycidyl ester is preferred.

[0091] In the present embodiment, the monomer represented by the formula (X3) is connected to the shell on the particle surface by reacting the amino group or thiol group in the formula (X3) with the glycidyl group in the shell in the third step to form the formula (2). The monomer represented by the formula (X3) is not particularly limited, and examples include mercaptosuccinic acid, aspartic acid, 3-mercapto-1,2-propanediol, 3-amino-1,2-propanediol, 2-amino-1,3-propanediol, ethanolamine, and tris(hydroxymethyl)aminomethane.

[0092] Third Embodiment

[0093] As a third embodiment, the present disclosure provides the following particles.

[0094] Particles used in an agglutination method, which are produced by:

[0095] A first step of subjecting a first monomer composition containing 40% by mass or more and 85% by mass or less of a monomer represented by the formula (X1) to a polymerization reaction to obtain core particles having a volume average particle diameter of 200 nm or more and 500 nm or less;

[0096] A second step of subjecting a reaction solution including the core particles, a second monomer composition containing a monomer represented by the formula (X2), and a water-soluble polymerization initiator to a polymerization reaction to obtain particles having a layer of 5 nm or more and 50 nm or less formed on the outer side of the core particles,

[0097] The content ratio of the monomer represented by the formula (X1) is 90% by mass or more with respect to the total amount of the monomers contained in the first monomer composition,

[0098] Provided that,

[0099] The content ratio of the unpolymerized monomer represented by the formula (X1) is 1000 ppm or less with respect to the reaction solution in the second step, and

[0100] The content ratio of the monomer represented by the formula (X2) is 9% by mass or more and less than 40% with respect to the total amount of the monomers contained in the first monomer composition and the monomers contained in the second monomer composition; and

[0101] A third step of reacting the particles having the above layer with a compound represented by the formula (X3),

[0102]

[0103] (wherein, R 11 represents a hydrogen atom or a methyl group; R 12 represents a substituted or unsubstituted phenyl group or naphthyl group, provided that when substituted, the substituent is a methyl group or an ethyl group),

[0104]

[0105] (wherein, R 13 represents hydrogen or a methyl group, R 14 represents an ethylene group or a carbonyl group),

[0106]

[0107] (wherein, R 15 represents an amino group or a thiol group, R 16 and R 17each represents a hydrogen atom, a methyl group, a group having a hydroxyl group, or a group having a carboxyl group, and R 16 and R 17 at least one of which represents a group having a hydroxyl group or a group having a carboxyl group).

[0108] Other embodiments

[0109] As a further embodiment, the present disclosure provides a test particle comprising a ligand attached to the surface of a particle of the present disclosure as described above.

[0110] The test particles in the present disclosure have a selective or specific high affinity for a target substance through a ligand attached to the surface of the particle. In particular, preferably, the test particle is a particle in which the ligand is attached to the particle surface by a chemical bond.

[0111] The ligand in the present disclosure is a compound that specifically binds to a receptor possessed by a specific target substance. The site where the ligand binds to the target substance is fixed, and the ligand has a selective or specific high affinity. Examples include antigens and antibodies, enzyme proteins and their substrates, signaling substances such as hormones or neurotransmitters, and their receptors and nucleic acids, but the ligands in the present disclosure are not limited to these. Examples of nucleic acids include deoxyribonucleic acid. The test particles in the present disclosure have a selective or specific high affinity for a target substance. Preferably, the ligand in the present disclosure is any one of an antibody, an antigen, and a nucleic acid.

[0112] As a further embodiment, the present disclosure provides a reagent used in an agglutination assay, characterized in that the particles or test particles of the present disclosure as described above are dispersed in an aqueous solution. The detection target of the reagent is not particularly limited. Examples of the reagent include drugs for in vitro diagnosis, and specifically include an antigen detection reagent for testing an antigen and an antibody detection reagent for testing an antibody. The reagent may be a reagent in which particles without a ligand are dispersed in an aqueous solution, assuming that the user attaches a ligand such as an antibody as required. Alternatively, the reagent may be a reagent in which test particles pre-attached with a ligand are dispersed in an aqueous solution assuming a specific target substance.

[0113] The reagent of the present disclosure contains the particles or test particles of the present disclosure and a dispersion medium for dispersing the particles or test particles. In addition to the particles or test particles of the present disclosure, the reagent of the present disclosure may contain a third substance such as a solvent or a blocking agent within the scope that can achieve the object of the present disclosure. A third substance such as a solvent or a blocking agent may be included in a combination of two or more. Examples of the dispersion medium used in the present disclosure include various buffers such as phosphate buffer, glycine buffer, Good buffer, Tris buffer, and ammonia buffer, but the dispersion medium contained in the reagent of the present disclosure is not limited to these.

[0114] As a further embodiment, the present disclosure provides a test kit. The kit includes the above-mentioned reagent and a container for encapsulating the reagent.

[0115] In addition to the above-mentioned reagent (hereinafter referred to as Reagent 1), the kit in this embodiment may further include a reaction buffer (hereinafter referred to as Reagent 2). One or both of Reagent 1 and Reagent 2 may contain a sensitizer. In addition to Reagent 1 and Reagent 2, the kit of the present disclosure may further include a positive control, a negative control, a serum diluent, a primary antibody or a secondary antibody, etc. As the medium for the positive control and the negative control, serum, saline or a solvent containing no measurable target substance may be used.

[0116] As a further embodiment, the present disclosure provides a method for detecting a target substance in a sample by in vitro diagnosis, the method including mixing a reagent containing the above-mentioned test particles with a sample that may contain the target substance.

[0117] As a further embodiment, the present disclosure provides a method for detecting a target substance in a sample by an agglutination method. The agglutination method according to this embodiment is a method for detecting a target substance in a sample, which includes the steps of mixing a reagent containing the above-mentioned test particles with the sample to obtain a mixed solution; irradiating the mixed solution with light; and detecting at least one of transmitted light or scattered light from the light that has irradiated the mixed solution.

[0118] [Method for measuring volume-average particle diameter and particle size distribution of particles in an aqueous dispersion]

[0119] The method for measuring the volume-average particle diameter (Dv) of the particles in the aqueous dispersion in the present disclosure will be described. The Dv of the particles present in the aqueous dispersion is measured by dynamic light scattering. For example, measurement is performed at 25 °C using a potentiometric analyzer (zetasizer) (ZetasizerUltra: Malvern Panalytical Ltd.). The Dv of the core particles is measured using the aqueous dispersion of the core particles obtained in the above first step. The Dv of the particles having a core-shell structure is measured using the aqueous dispersion of the particles obtained in the above third step.

[0120] The particle size distribution of the particles having a core-shell structure in the present disclosure is obtained by measuring the number-average particle diameter (Dn) using the above-mentioned dynamic light scattering method and calculating the ratio (Dv / Dn) of Dv to Dn.

[0121] [Method for measuring thickness of shell of particles]

[0122] A method for calculating the thickness of the shell of the particles of the present disclosure will be described. The thickness of the shell is calculated by subtracting the volume-average particle diameter of the core particles from the volume-average particle diameter of the particles obtained by measuring the aqueous dispersion of the particles in the above third step, and then dividing by 2.

[0123] [Method for measuring the composition ratio (C / O) of C element and O element of particles]

[0124] The measurement of the composition ratio (C / O) of C element and O element in the present disclosure will be described. The composition ratio of the particles of the present disclosure is measured by X-ray photoelectron spectroscopy (XPS) using freeze-dried particles fixed to indium foil. In the following examples, the following measurement equipment and measurement conditions are used.

[0125] - Measurement equipment: X-ray photoelectron spectrometer: Quantum 2000 (product name, manufactured by ULVAC-PH, Inc.)

[0126] - X-ray source: Monochromatic AlKα

[0127] - X-ray setting: 100μmφ (25W (15KV))

[0128] - Photoelectron takeoff angle: 45 degrees

[0129] - Neutralization condition: Combination of neutralization gun and ionization gun

[0130] - Analysis area: 300×200μm

[0131] - Pass energy: 58.70eV

[0132] - Step size: 0.125eV

[0133] - Analysis software: Maltipak (ULVAC-PHI, Inc.)

[0134] The cumulative number of times during measurement is 15 times for C1S and O1S, and 30 times for N1S. The composition ratio of C element and O element is obtained using the quantitative values of the obtained element amounts (the ratio (atomic %) of the amount of C element and the ratio (atomic %) of the amount of O element with respect to the total amount of the three elements, respectively).

[0135] [Method for measuring the infrared absorption spectrum of particles]

[0136] A method for measuring the ratio of the C═O bond derived from an ester to the C═C bond derived from an aromatic ring in the IR spectrum of the particles according to the present disclosure, that is, the ratio of the ester component to the aromatic ring component in the particles, will be described. The composition ratio of the particles according to the present disclosure obtained by the IR spectrum is measured using freeze-dried particles. In the following examples, the following measurement equipment and measurement conditions are used.

[0137] - Measuring device: Fourier Transform Infrared Spectrometer Spectrum One (PerkinElmer, Inc.)

[0138] - Measuring method: ATR method

[0139] - Crystal: Diamond

[0140] - Measuring range: 4000 cm -1 to 650 cm -1

[0141] - Resolution: 4 cm -1

[0142] - Number of accumulations: 32 times

[0143] - Data analysis: Conducted using absorbance

[0144] Place the freeze-dried powdered particles on the measuring part and press them down using a flat probe. At this time, start the measurement so that the pressure gauge displayed on the measurement software is approximately 100.

[0145] After the measurement, perform quantification and analysis using the absorbance data.

[0146] Define the peak height of the C═C bond derived from the aromatic ring existing at 1500 - 1650 cm -1 as A, and define the peak height of the carbonyl group derived from the ester bond existing at 1680 - 1750 cm -1 as B, and calculate the value of A / B.

[0147] [Measurement method of absorbance of particles]

[0148] The measurement method of the absorbance of the particles described in the present disclosure will be described. The absorbance of the particles can be obtained by measuring the absorbance of the aqueous dispersion of the particles in the present disclosure at a wavelength of 572 nm. In the following examples, an aqueous dispersion of particles at 0.01% by mass is added to the cell, and the absorbance at an incident light of 572 nm and a cell length of 10 mm is measured using a spectrophotometer Biospectrometer manufactured by Eppendorf corporate.

[0149] Examples

[0150] Hereinafter, the present disclosure will be described in detail by way of examples, but the present disclosure is not limited to these examples.

[0151] [Example 1] (Synthesis of Particle 1)

[0152] (Step - 1 / Preparation of Core 1)

[0153] Into a 2 L four-neck separable flask, 71.75 g of styrene (St: KISHIDACHEMICAL CO., LTD.), 1.30 g of divinylbenzene (DVB: KISHIDACHEMICAL CO., LTD.) and 1190.67 g of ion-exchanged water were weighed to prepare a mixed solution. Then, the mixed solution was maintained at 70 °C while stirring at 140 rpm, and nitrogen was flowed at a rate of 200 mL / min to deoxygenate the inside of the four-neck separable flask. Next, a solution separately prepared by dissolving 3.11 g of V-50 (FUJIFILM Wako Pure Chemical Corporation) in 50 g of ion-exchanged water was added to the above mixed solution to initiate soap-free emulsion polymerization. After 48 hours from the start of the polymerization reaction, a dispersion of core particles 1 (hereinafter simply referred to as core) containing a copolymer of St and DVB was obtained. A part of the dispersion was collected and evaluated using dynamic light scattering of core 1 (ZetasizerUltra: Malvern Panalytical Ltd.), showing a volume average particle size of 330 nm.

[0154] (Step - 2 / Preparation of mother particle 1)

[0155] The dispersion was adjusted with ion-exchanged water to prepare 148.29 g of a dispersion of core 1 with a solid content concentration of 2.0%. A part of this dispersion was collected and the contents of St and DVB were evaluated by gas chromatography, showing that the contents of St and DVB were 75 ppm. Next, 1.52 g of glycidyl methacrylate (GMA: KISHIDA CHEMICAL CO., LTD.) was added, and the mixture was maintained at 70 °C while stirring at 100 rpm, and nitrogen was flowed at a rate of 200 ml / min to deoxygenate the four-neck separable flask. Then, a solution separately prepared by dissolving 0.018 g of V-50 in 1 g of ion-exchanged water was added to the above mixed solution to initiate the formation of the shell. By continuing to stir for 17 hours after the start of the reaction, a dispersion of mother particle 1 having a core-shell structure was obtained. After the above dispersion was slowly cooled to room temperature, a part of the dispersion was collected and the polymerization conversion rate was evaluated by gas chromatography, which was confirmed to be approximately 100%.

[0156] (Step - 3 / Preparation of particle 1 having a core-shell structure)

[0157] To an aqueous dispersion containing mother particles 1, an aqueous solution prepared in advance and dissolved with mercaptosuccinic acid (MSA, FUJIFILM Wako Pure Chemical Corporation) and 3-mercapto-1,2-propanediol (MPD: FUJIFILM Wako Pure Chemical Corporation) (the molar ratio of 3-mercapto-1,2-propanediol and mercaptosuccinic acid is 6:4 (mole fraction), and the total molar amount of MSA and MPD is equal to the molar amount of the above glycidyl methacrylate) was added, and then triethylamine (KISHIDA CHEMICAL CO., LTD.) was added to adjust the pH to 10. Then, the mixture was heated to 70 °C while stirring at 200 rpm and maintained in this state for another 18 hours to obtain a dispersion of particles 1 having a core-shell structure. The operation of separating particles 1 from the dispersion by a centrifuge and redispersing particles 1 in ion-exchanged water was repeated 8 times to purify particles 1, and finally particles 1 were stored in an aqueous dispersion adjusted to 1.0 mass% with respect to particles 1.

[0158] The evaluation results of the particle physical properties of particles 1 are shown in Table 1. The amount of formula (1) in core 1, the content of formula (2) in particles 1, the content of formula (2) in the shell, and the mass ratio of core 1 to particles 1 are shown in Table 2.

[0159] IR measurement was also performed on particles 1. The peak height of the C═C bond derived from the aromatic ring present at 1500 - 1650 cm -1 was defined as A, and the peak height of the carbonyl group derived from the ester bond present at 1680 - 1750 cm -1 was defined as B, and the value of A / B was calculated, and the result was 4.05.

[0160] [Table 1]

[0161] Table 1

[0162]

[0163] Ratio E / D of absorbance (E) of particles to Dv V : When an aqueous solution of 0.1 wt% particles was added to a cell, and the absorbance at an incident light of 572 nm with a cell length of 10 mm was set as E and the volume average particle diameter of the particles was set as D V the value of E / D V at that time.

[0164] [Table 2] Table 2

[0165]

[0166] Table 2 (continued)

[0167]

[0168] [Example 2] (Synthesis of Particle 2)

[0169] (Step - 1 / Preparation of Core 2)

[0170] A dispersion of Core 2 was obtained by the same experimental operation as in Example 1, except that the rotational speed of stirring was changed from 140 rpm to 200 rpm. A part of the dispersion was collected and evaluated using dynamic light scattering of Core 2 (Zetasizer Ultra: Malvern Panalytical Ltd.), showing a volume - average particle size of 395 nm.

[0171] (Step - 2 / Preparation of Mother Particle 2)

[0172] A dispersion of Mother Particle 2 was obtained by the same experimental operation as in Example 1, except that the dispersion of Core 1 was changed to the dispersion of Core 2 in Step - 2 of Example 1. Before adding GMA, the contents of St and DVB in the dispersion of Core 2 were 83 ppm. The polymerization conversion rate was evaluated using gas chromatography and confirmed to be approximately 100%.

[0173] (Step - 3 / Preparation of Particle 3 with Core - Shell Structure)

[0174] A dispersion of Particle 2 with a core - shell structure was obtained by the same experimental operation as in Step - 3 of Example 1.

[0175] The evaluation results of the particle physical properties of Particle 2 are shown in Table 1. The amount of Formula (1) of Core 2, the content of Formula (2) in Particle 2, the content of Formula (2) in the shell, and the mass ratio of Core 2 to Particle 2 are shown in Table 2.

[0176] [Example 3] (Synthesis of Particle 3)

[0177] (Step - 1 / Preparation of Core 3)

[0178] A dispersion of Core 3 was obtained by the same experimental operation as in Example 1, except that in Step - 1 of Example 1, the amount of St used was 99.40 g, the amount of DVB was 1.80 g, the amount of ion - exchanged water was 1150.19 g, the amount of V - 50 was 4.31 g, and the rotational speed of stirring was changed from 140 rpm to 200 rpm. A part of the dispersion was collected and evaluated using dynamic light scattering of Core 3 (Zetasizer Ultra: Malvern Panalytical Ltd.), showing a volume - average particle size of 498 nm.

[0179] (Step - 2 / Preparation of Mother Particle 3)

[0180] The dispersion was adjusted with ion - exchanged water to prepare 148.27 g of a dispersion 148 of core 3 with a solid component concentration of 2.5%. A part of this dispersion was collected, and the contents of St and DVB were evaluated by gas chromatography, showing that the contents of St and DVB were 82 ppm. Next, 2.37 g of GMA was added, and the mixture was maintained at 70 °C while stirring at 100 rpm, and nitrogen was flowed at a rate of 200 mL / min to deoxygenate the four - neck separable flask. Then, a solution separately prepared by dissolving 0.018 g of V - 50 (FUJIFILM Wako Pure Chemical Corporation) in 1 g of ion - exchanged water was added to the above - mentioned mixed solution to initiate the formation of the shell. By continuing to stir for 17 hours after the start of the reaction, a dispersion containing mother particle 3 having a core - shell structure was obtained. After slowly cooling the above - mentioned dispersion to room temperature, a part of the dispersion was collected, and the polymerization conversion rate was evaluated by gas chromatography, which was confirmed to be approximately 100%.

[0181] (Step - 3 / Preparation of Particle 3 Having a Core - Shell Structure)

[0182] A dispersion of particle 3 having a core - shell structure was obtained by the same experimental operation as in Step - 3 of Example 1.

[0183] The evaluation results of the particle physical properties of particle 3 are shown in Table 1. The amount of formula (1) of core 3, the content of formula (2) in particle 3, the content of formula (2) in the shell, and the mass ratio of core 3 to particle 3 are shown in Table 2.

[0184] [Example 4](Synthesis of Particle 4)

[0185] (Step - 1 / Preparation of Core 4)

[0186] Into a 2 L four-neck separable flask, 25.35 g of styrene (St: KISHIDA CHEMICAL CO., LTD.), 0.46 g of divinylbenzene (DVB: KISHIDA CHEMICAL CO., LTD.), and 1501.02 g of ion-exchanged water were weighed to prepare a mixed solution. Then, the mixed solution was maintained at 70 °C while stirring at 140 rpm, and nitrogen was flowed at a flow rate of 200 mL / min to deoxygenate the inside of the four-neck separable flask. Next, a solution separately prepared by dissolving 1.10 g of V-50 (FUJIFILM Wako Pure Chemical Corporation) in 30 g of ion-exchanged water was added to the above mixed solution to initiate soap-free emulsion polymerization. By reacting and stirring from the start of polymerization for 48 hours, a dispersion of core 4 containing a copolymer of St and DVB was obtained. A part of the dispersion was collected and evaluated using dynamic light scattering of core 4 (Zetasizer Ultra: MalvernPanalytical Ltd.), showing a volume average particle size of 203 nm.

[0187] (Step - 2 / Preparation of mother particle 4)

[0188] The dispersion was adjusted with ion-exchanged water to prepare 133.0 g of a dispersion of core 4 with a solid content concentration of 2.0%. A part of the dispersion was collected and the contents of St and DVB were evaluated by gas chromatography, showing that the contents of St and DVB were 20 ppm. Next, 1.43 g of GMA was added, and the mixture was maintained at 70 °C while stirring at 100 rpm, and nitrogen was flowed at a flow rate of 200 mL / min to deoxygenate the four-neck separable flask. Then, a solution separately prepared by dissolving 0.0323 g of V-50 (FUJIFILM Wako Pure Chemical Corporation) in 1 g of ion-exchanged water was added to the above mixed solution to initiate the formation of the shell. By continuing to stir for 17 hours after the start of the reaction, a dispersion containing mother particle 4 with a core-shell structure was obtained. After slowly cooling the above dispersion to room temperature, a part of the dispersion was collected and the polymerization conversion rate was evaluated by gas chromatography, which was confirmed to be approximately 100%.

[0189] (Step - 3 / Preparation of particle 4 with a core-shell structure)

[0190] A dispersion of particle 4 with a core-shell structure was obtained through the same experimental operation as in Step - 3 of Example 1.

[0191] The evaluation results of the particle physical properties of particle 4 are shown in Table 1. The amount of formula (1) of core 4, the content of formula (2) in particle 4, the content of formula (2) in the shell, and the mass ratio of core 4 to particle 4 are shown in Table 2.

[0192] [Example 5] (Synthesis of Particle 5)

[0193] (Step - 1 / Preparation of Core 5)

[0194] Except that in Step - 1 of Example 1, the usage amount of St was changed from 71.75 g to 67.58 g, and the usage amount of DVB was changed from 1.30 g to 5.02 g, a dispersion of core 5 was obtained through the same experimental operation. A part of the dispersion was collected and evaluated using dynamic light scattering of core 5 (Zetasizer Ultra: Malvern Panalytical Ltd.), showing a volume - average particle size of 332 nm.

[0195] (Step - 2 / Preparation of Mother Particle 5)

[0196] Except that in Step - 2 of Example 1, the usage amount of GMA was changed from 1.52 g to 0.99 g, and the usage amount of V - 50 was changed from 0.018 g to 0.016 g, a dispersion of mother particle 5 was obtained through the same experimental operation. Before adding GMA, the contents of St and DVB in the dispersion of core 5 were 83 ppm. The polymerization conversion rate was evaluated using gas chromatography and confirmed to be approximately 100%.

[0197] (Step - 3 / Preparation of Particle 5 with Core - Shell Structure)

[0198] Through the same experimental operation as in Step - 3 of Example 1, a dispersion of particle 5 with a core - shell structure was obtained.

[0199] The evaluation results of the particle physical properties of particle 5 are shown in Table 1. The amount of formula (1) of core 5, the content of formula (2) in particle 5, the content of formula (2) in the shell, and the mass ratio of core 5 to particle 5 are shown in Table 2.

[0200] [Example 6] (Synthesis of Particle 6)

[0201] (Step - 1 / Preparation of Core 6)

[0202] Except that in Step - 1 of Example 1, 35.88 g of St and 35.88 g of 1 - vinylnaphthalene (1VN: FUJIFILM Wako Pure Chemical Corporation) were used instead of 71.75 g of St, a dispersion of Core 6 was obtained by the same experimental operation. A part of the dispersion was collected and evaluated using dynamic light scattering of Core 6 (Zetasizer Ultra: Malvern Panalytical Ltd.), showing a volume - average particle size of 329 nm.

[0203] (Step - 2 / Preparation of Mother Particle 6)

[0204] Except that in Step - 2 of Example 1, the dispersion of Core 1 was changed to the dispersion of Core 6, a dispersion of Mother Particle 6 was obtained by the same experimental operation. Before adding GMA, the contents of St and DVB in the dispersion of Core 6 were 75 ppm. The polymerization conversion was evaluated using gas chromatography and confirmed to be approximately 100%.

[0205] (Step 3 / Preparation of Particle 6 with Core - Shell Structure)

[0206] A dispersion of Particle 6 with a core - shell structure was obtained by the same experimental operation as in Step - 3 of Example 1.

[0207] The evaluation results of the particle physical properties of Particle 6 are shown in Table 1. The amount of Formula (1) of Core 6, the content of Formula (2) in Particle 6, the content of Formula (2) in the shell, and the mass ratio of Core 6 to Particle 6 are shown in Table 2.

[0208] [Example 7](Synthesis of Particle 7)

[0209] (Step - 1 / Preparation of Core 7)

[0210] Except that in Step - 1 of Example 1, 35.88 g of St and 35.88 g of 4 - methylstyrene (MSt: FUJIFILM Wako Pure Chemical Corporation) were used instead of 71.75 g of St, a dispersion of Core 7 was obtained by the same experimental operation. A part of the dispersion was collected and evaluated using dynamic light scattering of Core 7 (Zetasizer Ultra: Malvern Panalytical Ltd.), showing a volume - average particle size of 331 nm.

[0211] (Step - 2 / Preparation of Mother Particle 7)

[0212] A dispersion of mother particle 7 was obtained by the same experimental procedure as in Example 1, except that in Step - 2 of Example 1, the dispersion of core 1 was changed to a dispersion of core 7. Before adding GMA, the contents of St and DVB in the dispersion of core 7 were 88 ppm. The polymerization conversion was evaluated by gas chromatography and confirmed to be approximately 100%.

[0213] (Step - 3 / Preparation of Particles 7 with Core - Shell Structure)

[0214] A dispersion of particles 7 with a core - shell structure was obtained by the same experimental procedure as in Step - 3 of Example 1.

[0215] The evaluation results of the particle physical properties of particles 7 are shown in Table 1. The amount of formula (1) of core 7, the content of formula (2) in particles 7, the content of formula (2) in the shell, and the mass ratio of core 7 to particles 7 are shown in Table 2.

[0216] [Example 8](Synthesis of Particles 8)

[0217] A dispersion of particles 8 with a core - shell structure was obtained by the same experimental procedure as in Example 1, except that mother particle 1 prepared in Example 1 was used and 3 - amino - 1,2 - propanediol (3APD: Tokyo Chemical Industry Co., Ltd.) was used instead of MPD in Step - 3 of Example 1.

[0218] The evaluation results of the particle physical properties of particles 8 are shown in Table 1. The amount of formula (1) of core 1, the content of formula (2) in particles 8, the content of formula (2) in the shell, and the mass ratio of core 1 to particles 8 are shown in Table 2.

[0219] [Example 9](Synthesis of Particles 9)

[0220] A dispersion of particles 9 with a core - shell structure was obtained by the same experimental procedure as in Example 1, except that mother particle 1 prepared in Example 1 was used and 2 - amino - 1,3 - propanediol (2APD: Tokyo Chemical Industry Co., Ltd.) was used instead of MPD in Step - 3 of Example 1.

[0221] The evaluation results of the particle physical properties of particles 9 are shown in Table 1. The amount of formula (1) of core 1, the content of formula (2) in particles 9, the content of formula (2) in the shell, and the mass ratio of core 1 to particles 9 are shown in Table 2.

[0222] [Example 10](Synthesis of Particles 10)

[0223] A dispersion of particles 10 having a core-shell structure was obtained by the same experimental operations as in Example 1, except that the mother particles 1 prepared in Example 1 were used, and 2-amino-2-hydroxymethyl-1,3-propanediol (Tris: KISHIDA CHEMICAL CO., LTD.) was used instead of MPD in Step-3 of Example 1.

[0224] The evaluation results of the particle physical properties of particles 10 are shown in Table 1. The amount of formula (1) in core 1, the content of formula (2) in particles 10, the content of formula (2) in the shell, and the mass ratio of core 1 to particles 10 are shown in Table 2.

[0225] [Example 11] (Synthesis of particles 11)

[0226] A dispersion of particles 11 having a core-shell structure was obtained by the same experimental operations as in Example 1, except that 1.42 g of GMA was used instead of DVB in Step-3 of Example 1.

[0227] The evaluation results of the particle physical properties of particles 11 are shown in Table 1. The amount of formula (1) in core 1, the content of formula (2) in particles 11, the content of formula (2) in the shell, and the mass ratio of core 1 to particles 11 are shown in Table 2.

[0228] [Example 12] (Synthesis of particles 12)

[0229] A dispersion of particles 12 having a core-shell structure was obtained by the same experimental operations as in Example 1, except that 0.67 g of GMA and 0.05 g of DVB were used instead of 0.72 g of GMA used in Step-2 of Example 1.

[0230] The evaluation results of the particle physical properties of particles 12 are shown in Table 1. The amount of formula (1) in core 12, the content of formula (2) in particles 12, the content of formula (2) in the shell, and the mass ratio of core 12 to particles 12 are shown in Table 2.

[0231] [Example 13] (Synthesis of particles 13)

[0232] A dispersion of particles 13 having a core-shell structure was obtained by the same experimental operations as in Example 1, except that 0.63 g of GMA and 0.09 g of DVB were used instead of 0.72 g of GMA used in Step-2 of Example 1.

[0233] The evaluation results of the particle physical properties of particles 13 are shown in Table 1. The amount of formula (1) in core 13, the content of formula (2) in particles 13, the content of formula (2) in the shell, and the mass ratio of core 13 to particles 13 are shown in Table 2.

[0234] [Example 14] (Synthesis of Particle 14)

[0235] A dispersion of particles 14 having a core - shell structure was obtained by the same experimental procedure as in Example 1, except that the total molar amount of MSA and MPD used in Step - 3 of Example 1 was changed to 0.5 equivalents instead of the molar amount and equivalents of GMA in Step - 2.

[0236] The evaluation results of the particle physical properties of particles 14 are shown in Table 1. The amount of formula (1) in core 14, the content of formula (2) in particles 14, the content of formula (2) in the shell, and the mass ratio of core 14 to particles 14 are shown in Table 2.

[0237] [Example 15] (Particle 15)

[0238] In Step - 1 of Example 1, the amount of St used was set to 85.61 g, the amount of DVB used was set to 1.55 g, the amount of ion - exchanged water used was set to 1120.12 g, the amount of V - 50 used was set to 3.72 g, and the rotational speed of stirring was further changed from 140 rpm to 200 rpm. In addition, in Step - 2 of Example 1, the amount of GMA used was changed to 0.45 g. Otherwise, a dispersion of core 15 was obtained by the same experimental procedure as in Example 1. A part of the dispersion was collected and evaluated using dynamic light scattering of core 15 (Zetasizer Ultra: Malvern Panalytical Ltd.), showing a volume - average particle size of 498 nm.

[0239] [Comparative Example 1] (Synthesis of Comparative Particle 1)

[0240] The core 1 produced in Step - 1 of Example 1 was adjusted to a solid content of 5.0 g with 500 g of water. An organic solvent (ShellSol TK 0.1 g) and 9.71 g of GMA were successively added thereto and stirred, then 2 g of AIBN (azobis(isobutyronitrile)) was added, and the mixture was slowly stirred at 75 °C for 24 hours to form a polymer part. Then, the reaction solution was cooled and then filtered through a 500 - mesh wire screen. The particles were washed with distilled water for centrifugation to obtain a dispersion of comparative particles 1 having a core - shell structure.

[0241] The evaluation results of the particle physical properties of comparative particles 1 are shown in Table 1.

[0242] [Comparative Example 2] (Synthesis of Comparative Particle 2)

[0243] In the following formulation St / GMA / DVB / V - 50 / H 2O = 1.2 / 1.8 + 0.3 / 0.04 / 0.06 / 110 (g), after nitrogen replacement of St, GMA, DVB, V-50 and ion-exchanged water, a polymerization reaction was carried out at 70 °C for 24 hours. The polymerization was a soap-free emulsion polymerization described in Japanese Patent Application Laid-Open No. 2000-351814. Two hours after the start of the polymerization, 0.3 g of GMA was added so that the surface of the obtained comparative mother particle 2 was completely covered with GMA. The obtained comparative mother particle 2 was precipitated by centrifugation (15,000 rpm, 15 min, 4 °C), the supernatant was decanted, and then redispersed in 200 ml of water. The above operation was repeated 3 times to wash the comparative mother particle 2, and finally the obtained comparative mother particle 2 was dispersed in water.

[0244] To introduce an amino group into the washed comparative mother particle 2 (0.25 g), NH 4 OH (55.3 mmol; 50 times the amount of GMA units) was added and adjusted to pH = 11 with 1N HCl. The mixture was reacted at 70 °C for 24 hours while stirring with a stirrer to open the epoxy group of GMA. Next, an example of the immobilization of ethylene glycol diglycidyl ether (EGDE; FUJIFILM Wako Pure Chemical Corporation) in the above-obtained comparative mother particle 2 is shown. An excess of EGDE was added so that the amount (mol) was 100 times the amount of amino groups in about 62.5 mg of the comparative mother particle 2, and it was stirred at 30 °C for 24 hours at pH 11 (adjusted with 1N NaOH) to covalently bond the epoxy group of EGDE to the amino group on the comparative mother particle 2. An excess of EGDE was added to prevent the epoxy groups at both ends of one EGDE molecule from being simultaneously immobilized to the SG particles. This produced EGDE particles in which EGDE was bound to the mother particle 2. After the reaction, the EGDE particles were washed three times with water by centrifugation.

[0245] To introduce an amino group into 0.25 g of the washed EGDE particles, NH 4 OH (55.3 mmol; 50 times the amount of GMA units) was added and adjusted to pH = 1 with 1N HCl. The mixture was reacted at 70 °C for 24 hours while stirring with a stirrer. Then, it was centrifuged at 4 °C for 20 minutes at 3 times, 27,000 g with ion-exchanged water, and then redispersed in methanol so that the solid content was 1% by mass. Next, 0.88 g of anhydrous succinic acid (Tokyo Chemical Industry Co., Ltd.) was added to the dispersion 2' weighed to contain 0.20 g of particles. The mixture was shaken at 30 °C for 5 hours to react the primary amine of the particles with an amino group introduced at the end of EGDE with anhydrous succinic acid, thereby obtaining comparative particle 2.

[0246] The evaluation results of the particle physical properties of Comparative Particle 2 are shown in Table 1.

[0247] [Comparative Example 3] (Synthesis of Comparative Particle 3)

[0248] Except that in Step - 1 of Example 1, 12.68 g of St, 0.23 g of DVB, and 1501.02 g of ion - exchanged water were weighed into a 2 - L four - neck separable flask to prepare a mixed solution, Comparative Core 3 was obtained through the same experimental operation as in Step - 1 of Example 4. Then, except that 1.31 g of GMA was used instead of 1.43 g of GMA in Step - 2 of Example 4, Comparative Particle 3 was obtained through the same experimental operations as in Step - 2 and Step - 3 of Example 4.

[0249] The evaluation results of the particle physical properties of Comparative Particle 3 are shown in Table 1.

[0250] [Comparative Example 4] (Synthesis of Comparative Particle 4)

[0251] Except that in Step - 1 of Example 1, 99.40 g of St, 1.80 g of DVB, and 1150.15 g of ion - exchanged water were weighed into a 2 - L four - neck separable flask to prepare a mixed solution, the rotation speed of stirring was changed from 140 rpm to 200 rpm, and the addition amount of V - 50 was changed to 4.31 g, a dispersion of Comparative Core 4 was obtained through the same experimental operation as in Example 1. Then, Comparative Particle 4 was obtained through the same experimental operations as in Step - 2 and Step - 3 of Example 1.

[0252] The evaluation results of the particle physical properties of Comparative Particle 4 are shown in Table 1.

[0253] [Comparative Example 5] (Synthesis of Comparative Particle 5)

[0254] Except that in Step - 2 of Example 1, 0.092 g of GMA was used instead of 1.52 g of GMA, Comparative Particle 5 was obtained through the same experimental operation as in Example 1.

[0255] The evaluation results of the particle physical properties of Comparative Particle 5 are shown in Table 1.

[0256] [Comparative Example 6] (Synthesis of Comparative Particle 6)

[0257] Except that in Step - 2 of Example 4, 10.01 g of GMA was used instead of 1.43 g of GMA, Comparative Particle 6 was obtained through the same experimental operation as in Example 1.

[0258] The evaluation results of the particle physical properties of Comparative Particle 6 are shown in Table 1.

[0259] For Comparative Examples 1 to 6, the amount of the formula (1) in the core, the content of the formula (2) in the comparative particles, the content of the formula (2) in the shell, and the mass ratio of the core to the comparative particles are shown in Table 2.

[0260] [Evaluation 1] Evaluation of the preparation of test particles by antibody sensitization of particles and the agglutination sensitivity of the test particles

[0261] (Preparation of test particles by antibody sensitization of particles)

[0262] For each of Particles 1 to 15 and Comparative Particles 1 to 6 prepared in Examples 1 to 15 and Comparative Examples 1 to 6, 180 μL of a 1.7 mass% aqueous suspension was placed in a 1.5 mL microtube, and then 90 μL of an aqueous solution of 5.0% 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 90 μL of an aqueous solution of 5.0% sodium N-hydroxysulfosuccinimide hydrochloride were added thereto. The mixture was stirred at room temperature for 30 minutes to activate the carboxyl groups, thereby obtaining a particle dispersion (activated particle dispersion).

[0263] After centrifugal washing, 270 μL of a phosphate buffer-saline solution (hereinafter referred to as PBS) at pH 7.2 was added, and the above particles were dispersed by ultrasonic waves.

[0264] 5 μL of a 15.0 mg / mL dispersion of monoclonal mouse anti-human C-reactive protein antibody (anti-CRP antibody) (Funakoshi Co., Ltd.), clone C5, was added thereto, and the mixture was stirred at room temperature for 3 hours to sensitize the particles with the antibody, thereby obtaining test particles. Each of these test particles was centrifugally washed and then 1 mL of PBS was added, and the test particles were stored in a dispersed state.

[0265] (Evaluation of the agglutination sensitivity of the test particles)

[0266] The standard serum of CRP was diluted with PBS to a concentration of 0.75 mg / dL to obtain a CRP sample solution. 1 μL of the CRP sample solution was mixed with 50 μL of a buffer solution (PBS containing 0.01% Tween 20) to prepare a mixed solution (hereinafter referred to as R1+), and the mixture was incubated at 37°C.

[0267] Next, R1+ was mixed with 50 μL of a dispersion (particle concentration: 0.1% by mass, referred to as R2) prepared by ultrasonically dispersing each test particle dispersion again immediately before use. The absorbance of the mixed solution (volume: 101 μL) at a wavelength of 572 nm was measured immediately after stirring. The absorbance measurement was performed using a spectrophotometer Biospectrometer manufactured by Eppendorf corporate. Then, the mixed solution was allowed to stand at 37 °C for 5 minutes, and then the absorbance at a wavelength of 572 nm was measured again, and the value of the change in absorbance ΔABS×10000 (R+) was calculated. The results are summarized in Table 3.

[0268] It is expected that the higher the value of R+ of the test particle, the more sensitive the target substance can be detected when the test particle is used as a particle for the agglutination method in sample testing.

[0269] Table 3 shows the evaluation results of the test particles of Particles 1 to 15 and Comparative Particles 1 to 6. For any of Particles 1 to 15, the value of the change in absorbance ΔABS×10000 was 10000 or more.

[0270] On the other hand, for any of Comparative Particles 1 to 3, the value of ΔABS×10000 was less than 10000. For Comparative Particles 1 and 2, as shown in Table 2, the contents of the structure represented by the formula (1) in the core particles were 50% by mass and 63% by mass, respectively. Therefore, the proportion of the low refractive index substance was higher than that of Particles 1 to 15, and thus the refractive index of the core particle was low.

[0271] As a result, the difference in absorbance before and after agglutination became small, and the sensitivity was low. For Comparative Particle 3, as shown in Table 1, the particle size of the core was 146 nm in terms of volume average particle size, which was smaller than that of Particles 1 to 15. Therefore, the aggregate of the particles after agglutination was small, and the difference in absorbance before and after agglutination was small. In addition, the E / Dv values of Comparative Particles 1 to 3 were less than 8.0×10 -4 , and even when the particle size was above a certain size, the absorbance was small and the difference in absorbance before and after agglutination was also small. For Comparative Particle 4, the particle size of the core particle was 521 nm, which was larger than that of Particles 1 to 15, and the value of ΔABS×10000 was 7,000, so the sensitivity was low. For Comparative Particle 6, the value of ΔABS×10000 was as low as 2000. This is because the thickness of the shell was thick, 62 nm, and the dispersibility of the particles was high, so it was difficult to cause agglutination.

[0272] [Evaluation 2] Evaluation of non-specific adsorption of particles

[0273] (Emulsion evaluation)

[0274] For Particles 1 to 15 and Comparative Particles 1 to 6, dispersions were prepared by dispersing each in phosphate buffer to a concentration of 0.1% by mass. Next, for 30 μL of each dispersion, 60 μL of an emulsion solution containing triolein, lecithin, free fatty acids, bovine albumin, and tris buffer was added, and the absorbance of the resulting dispersion at a wavelength of 572 nm was measured immediately after stirring. The absorbance measurement was performed using a spectrophotometer Biospectrometer manufactured by Eppendorf corporate. After allowing these dispersions to stand at 37 °C for 5 minutes, the absorbance at a wavelength of 572 nm was measured again, and the change in absorbance ΔABS×10000 was calculated. Based on the value of ΔABS×10000, they were evaluated as follows.

[0275] A: ΔABS×10000 is 30 or less

[0276] B: ΔABS×10000 is greater than 30 and 50 or less

[0277] C: ΔABS×10000 is greater than 50 and 100 or less

[0278] D: ΔABS×10000 is greater than 100 and 500 or less

[0279] E: ΔABS×10000 is greater than 500

[0280] Table 3 shows the evaluation results of Particles 1 to 15 and Comparative Particles 1 to 6. The evaluation results of Particles 1 to 15 were all B or higher. Since the change in the absorbance of the dispersion can be considered to be due to the occurrence of particle aggregation as a result of non-specific adsorption of the particles in the dispersion, it was confirmed that the particles of the present disclosure are excellent in the ability to suppress non-specific adsorption. On the other hand, the evaluation result of Comparative Particle 5 was E, so non-specific adsorption could not be sufficiently suppressed. It is thought that this is because the thickness of the shell of Comparative Particle 5 is 3 nm, which is thinner than that of Particles 1 to 15, and thus a part of the hydrophobic core particle is exposed on the surface of Comparative Particle 5.

[0281] (Human Sample Evaluation)

[0282] For particles 1 to 15 and comparative particles 1 to 6, a dispersion (P solution) was prepared by dispersing each particle in a phosphate buffer solution (containing 0.01% Tween 20) to a concentration of 0.1% by mass. Next, for 50 μL of each dispersion, 55 μL of a sample diluent (Q solution) containing a normal human sample (serum sample, 5 μL) and a phosphate buffer solution (50 μL) was added, and the absorbance of the resulting mixed solution at a wavelength of 572 nm was measured immediately after stirring. The absorbance measurement was performed using a spectrophotometer Biospectrometer manufactured by Eppendorf corporate. After leaving these dispersions to stand at 37 °C for 5 minutes, the absorbance at a wavelength of 572 nm was measured again, and the change in absorbance ΔABS×10000 was calculated. Based on the value of ΔABS×10000, they were evaluated as follows.

[0283] A: ΔABS×10000 is 30 or less

[0284] B: ΔABS×10000 is greater than 30 and 80 or less

[0285] C: ΔABS×10000 is greater than 80 and 100 or less

[0286] D: ΔABS×10000 is greater than 100 and 500 or less

[0287] E: ΔABS×10000 is greater than 500

[0288] The results of each particle are shown in Table 3.

[0289] Table 3 shows the evaluation results of particles 1 to 15 and comparative particles 1 to 6. The evaluation results of particles 1 to 15 are all A to C, and the value of the change amount ΔABS×10000 is less than 100. Since the change in the absorbance of the dispersion can be considered to be due to the occurrence of particle aggregation as a result of non-specific adsorption of the particles in the dispersion, it was confirmed that the particles of the present disclosure are excellent in the ability to suppress non-specific adsorption. On the other hand, the evaluation result of comparative particle 5 is E, and the value of the change amount ΔABS×10000 is 1000, so non-specific adsorption cannot be sufficiently suppressed. It is considered that this is because the thickness of the shell of comparative particle 5 is 3 nm, which is thinner than that of particles 1 to 15, and therefore, a part of the hydrophobic core particle is exposed on the particle surface of comparative particle 5.

[0290] As described above, from the results of Evaluation 1, Particles 1 to 15 of the present disclosure have high agglutination sensitivity, and from the results of Evaluation 2, non-specific adsorption is suppressed, indicating that both improvement in sensitivity and suppression of non-specific adsorption can be achieved simultaneously. On the other hand, in Comparative Examples 1 to 4, non-specific adsorption is suppressed, but the agglutination sensitivity is low. The sensitivity of Comparative Example 6 is low. In Comparative Example 5, although the agglutination sensitivity is high, non-specific adsorption cannot be suppressed. In other words, in Comparative Examples 1 to 6, the improvement in agglutination sensitivity and the suppression of non-specific adsorption cannot be balanced.

[0291] [Table 3]

[0292] Table 3

[0293]

[0294] According to the present disclosure, by having a core-shell structure, a high refractive index of the core particles, and reactive functional groups in the shell, particles having improved sensitivity and the ability to suppress non-specific adsorption in the agglutination method can be provided. In addition, a test particle, a reagent, a test kit, a method for detecting a target substance, and a method for producing the particle can be provided.

[0295] Although the present invention has been described with reference to exemplary embodiments, it should be understood that the present invention is not limited to the disclosed exemplary embodiments. The scope of the appended claims should be given the broadest interpretation so as to cover all such modifications as well as equivalent structures and functions.

Claims

1. A particle for use in an agglutination method, the particle comprising a core particle and a shell on the surface of the core particle, wherein The core particles have a volume average particle diameter of 200 nm or more and 500 nm or less, and contain a polymer having a structural unit represented by formula (1), wherein the content ratio of the structural unit represented by formula (1) is 40% by mass or more and 85% by mass or less relative to the particles; and The shell has a thickness of 5 nm or more and 50 nm or less and comprises a polymer having a structural unit represented by formula (2), in, R 1 represents a hydrogen atom or a methyl group; R 2 represents substituted or unsubstituted phenyl or naphthyl, provided that, when substituted, the substituent is methyl or ethyl; and For each structural unit, R 1 and R 2 Can be different, Among them, R 3 represents a hydrogen atom or a methyl group; R 4 represents a group having an epoxy group, a group having a hydroxyl group, or a group having a carboxyl group; and For each structural unit, R 3 and R 4 Can be different. 2 . The particle according to claim 1 , wherein a content of the structural unit represented by formula (2) is 9% by mass or more and 60% by mass or less relative to the particle. 3 . The particle according to claim 2 , wherein a content of the structural unit represented by formula (2) is 9% by mass or more and 39% by mass or less relative to the particle. 4 . The particle according to claim 3 , wherein a content of the structural unit represented by formula (2) is 20% by mass or more and 39% by mass or less relative to the particle. 5 . The particle according to claim 1 , wherein a content ratio of the structural unit represented by formula (1) is 90% by mass or more relative to the core particle. The particles according to claim 1 , wherein a composition ratio of C element to O element of the particles, which is quantitatively measured by X-ray photoelectron spectroscopy (XPS), is 2.0 or more and 3.3 or less. 7 . The particle according to claim 1 , wherein a content ratio of the structural unit represented by formula (2) relative to the shell is 90% by mass or more and 100% by mass or less.

8. The particle according to claim 1, wherein the polymer having the structural unit represented by formula (1) in the core particle has a structural unit represented by formula (3): in, Z represents a substituted or unsubstituted phenylene group or naphthylene group, provided that, when substituted, the substituent is a methyl group or an ethyl group; and Z may be different for each structural unit. 9 . The particle according to claim 1 , wherein a content ratio of the core particle is 50% by mass or more and 95% by mass or less relative to the particle.

10. The particle according to claim 1, wherein In the FT-IR spectrum of the particles, the -1 The peak height of the C=C bond originating from the aromatic ring is defined as A and the peaks existing at 1680-1750 cm -1 When the peak height of the carbonyl group derived from the ester bond at is defined as B, the value of A / B is 0.85 or more and 5.35 or less. The particles according to claim 1 , wherein a value of a ratio Dv / Dn of a volume average particle diameter Dv to a number average particle diameter Dn of the particles is 1.25 or less.

12. The particle according to claim 1, wherein When the particles are dispersed in water at a solid content concentration of 0.1 mass % so that the ratio Dv / Dn of the volume average particle diameter Dv to the number average particle diameter Dn is 1.25 or less, and the absorbance of incident light with a wavelength of 572 nm at a cell length of 10 mm is defined as E, the value of E / Dv is 8.0×10 -4 Above and 2.0×10 -3 the following.

13. The particle according to claim 1, wherein the formula (1) is represented by formula (1-A): in, R 10 represents phenyl, tolyl or naphthyl.

14. The particle according to claim 1, wherein the formula (2) is represented by formula (2-A): in, R 31 and R 32 One of them represents a hydroxyl group, and the other represents a hydroxyl group, a group represented by the formula (2-B) or a group represented by the formula (2-C), Among them, R 20 represents a single bond or a methylene group; R 22 , R 23 and R 24 Each represents a hydrogen atom, a methyl group, a hydroxyl group or a hydroxymethyl group, and R 22 , R 23 and R 24 One or more of represents a hydroxyl group; Y 1 represents a sulfur atom or an imino group; and *1 indicates the bonding position, Among them, R 25 represents a hydrogen atom, a methyl group, a hydroxyl group or a carboxyl group; Y 2 represents a sulfur atom or an imino group; Y 3 represents a single bond or a methylene group; and *2 indicates the bonding position.

15. A particle for use in an agglutination method, produced by: In a first step, a first monomer composition containing a monomer represented by formula (X1) is subjected to a polymerization reaction to obtain core particles having a volume average particle diameter of 200 nm or more and 500 nm or less; In the second step, a polymerization reaction is carried out using a reaction solution including the core particle, a second monomer composition including a monomer represented by formula (X2), and a water-soluble polymerization initiator to obtain particles having a layer of 5 nm or more and 50 nm or less formed on the outside of the core particle, The content ratio of the monomer represented by formula (X1) is 90 mass % or more relative to the total amount of monomers contained in the first monomer composition, The condition is, The content ratio of the unpolymerized monomer represented by the formula (X1) is 1000 ppm or less relative to the reaction solution, and The content ratio of the monomer represented by formula (X2) is 9% by mass or more and less than 40% by mass relative to the total amount of the monomers contained in the first monomer composition and the monomers contained in the second monomer composition; and In the third step, the particles having the layer are reacted with a compound represented by formula (X3), in, R 11 represents a hydrogen atom or a methyl group; and R 12 represents a substituted or unsubstituted phenyl or naphthyl group, provided that, when substituted, the substituent is a methyl group or an ethyl group, Among them, R 13 represents hydrogen or methyl, and R 14 represents an ethylene group or a carbonyl group, Among them, R 15 represents an amino group or a thiol group; R 16 and R 17 Each represents a hydrogen atom, a methyl group, a group having a hydroxyl group, or a group having a carboxyl group; and R 16 and R 17 At least one of represents a group having a hydroxyl group or a group having a carboxyl group.

16. A test particle comprising a ligand attached to the surface of the particle according to any one of claims 1 to 15.

17. A reagent for use in an agglutination method, wherein the particles according to any one of claims 1 to 15 are dispersed in an aqueous solution.

18. A test kit comprising: the reagent according to claim 17; and a container encapsulating the reagent.

19. A method for detecting a target substance in a sample by in vitro diagnosis, the method comprising mixing the reagent according to claim 17 with a sample that may contain the target substance.

20. A method for detecting a target substance in a sample by agglutination method, the method comprising: A step of mixing the reagent according to claim 17 with a sample that may contain a target substance to obtain a mixed solution; a step of irradiating the mixed solution with light; and a step of detecting at least one of transmitted light and scattered light from the light that has irradiated the mixed solution.

21. A method for producing particles used in an agglutination method, the method comprising: In a first step, a first monomer composition containing a monomer represented by formula (X1) is subjected to a polymerization reaction to obtain core particles having a volume average particle diameter of 200 nm or more and 500 nm or less; In the second step, a polymerization reaction is carried out using a reaction solution including the core particle, a second monomer composition including a monomer represented by formula (X2), and a water-soluble polymerization initiator to obtain particles having a layer of 5 nm or more and 50 nm or less formed on the outside of the core particle, The content ratio of the monomer represented by formula (X1) is 90 mass % or more relative to the total amount of monomers contained in the first monomer composition, The condition is, The content ratio of the unpolymerized monomer represented by the formula (X1) is 1000 ppm or less relative to the reaction solution, and The content ratio of the monomer represented by formula (X2) is 9% by mass or more and less than 40% by mass relative to the total amount of the monomers contained in the first monomer composition and the monomers contained in the second monomer composition; and In the third step, the particles having the layer are reacted with a compound represented by formula (X3), Among them, R 11 represents a hydrogen atom or a methyl group; and R 12 represents a substituted or unsubstituted phenyl or naphthyl group, provided that, when substituted, the substituent is a methyl group or an ethyl group, Among them, R 13 represents hydrogen or methyl, and R 14 represents an ethylene group or a carbonyl group, Among them, R 15 represents an amino group or a thiol group; R 16 and R 17 Each represents a hydrogen atom, a methyl group, a group having a hydroxyl group, or a group having a carboxyl group; and R 16 and R 17 At least one of represents a group having a hydroxyl group or a group having a carboxyl group.

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