Particle used for condensation method, inspection particle, reagent, inspection kit, method for manufacturing particle, and method for detecting target

Abstract

To solve the problem in which: an improvement in low concentration sensitivity is demanded in a condensation method, and to provide particles that achieve both an improvement in sensitivity and prevention of nonspecific adsorption.SOLUTION: A particle having a core particle and a shell on a surface of the core particle and used for a condensation method is provided. The core particle has a volume average particle diameter of 200 nm or more and 500 nm or less, and contains a polymer having a structural unit represented by the formula (1). The content ratio of the structural unit represented by the formula (1) to the particle is 40 mass% or more and 85 mass% or less. The shell has a film thickness of 5 nm or more and 50 nm or less, and contains a polymer having a structural unit represented by the formula (2).SELECTED DRAWING: None

Description

Technical Field

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

Background Art

[0002] In recent years, as a simple and rapid immunoassay method, an agglutination method typified by an immunolatex agglutination assay has attracted attention. A method is known in which a dispersion of particles having an antibody or an antigen as a ligand on the surface is mixed with a specimen that may contain a target substance (antigen or antibody). At this time, if the specimen contains the target substance (antibody or antigen), the particles cause an agglutination reaction. Therefore, the presence or absence of a disease can be specified by optically detecting this agglutination reaction as a change amount such as scattered light intensity, transmitted light intensity, or absorbance. The particles used in the agglutination method are desired to have low non-specific adsorption properties, that is, low adsorption properties with substances other than the target substance, for the purpose of reducing noise. As the particles used in the agglutination method, polystyrene particles are widely used, but particles having a structure derived from styrene and glycidyl methacrylate are also known. Patent Document 1 describes particles having a core-shell structure and having a carboxy group or a 2,3-dihydroxypropyl group, which is a reactive functional group, in the shell. These particles are obtained by copolymerizing a monomer having a carboxy group and a monomer having a 2,3-dihydroxypropyl group with seed particles (parent particles, core) such as polystyrene or a styrene-based copolymer by a two-stage swelling polymerization method to add a shell to the seed particles and form a core-shell structure, or a method of forming a core-shell structure by a two-stage swelling polymerization method using a monomer capable of forming a carboxy group or a 2,3-dihydroxypropyl group by hydrolysis has been reported to be preferable.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Non-Patent Literature

[0004]

Non-Patent Literature 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] The inventors synthesized particles having a core - shell structure according to Patent Document 1, obtained a dispersion of particles in which an antibody or an antigen was chemically bonded as a ligand to the particle surface, mixed it with a specimen containing a target substance, and confirmed the 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 using particles synthesized by reducing glycidyl methacrylate for the purpose of improving sensitivity, the desired sensitivity could not be obtained and non - specific adsorption worsened. Also, when using particles prepared by copolymerizing styrene and glycidyl methacrylate without providing a core - shell structure, the desired sensitivity could not be obtained.

[0006] This disclosure has been made in view of these background technologies and problems. Specifically, the object of this disclosure is to provide particles and a manufacturing method capable of achieving both improved sensitivity and suppression of non - specific adsorption in an agglutination method.

Means for Solving the Problems

[0007] This disclosure provides Particles for use in an agglutination method, having core particles and a shell on the surface of the core particles, wherein the core particles have a volume - average particle diameter of 200 nm or more and 500 nm or less, contain a polymer having a structural unit represented by formula (1), and the content ratio of the structural unit represented by formula (1) with respect to the particles is 40% by mass or more and 85% by mass or less, and the shell has a film thickness of 5 nm or more and 50 nm or less and contains a polymer having a structural unit represented by formula (2). [Chemical formula] (R 1 represents a hydrogen atom or a methyl group. R 2 represents a substituted or unsubstituted phenyl group or a naphthyl group. However, in the case of substitution, the substituent is a methyl group or an ethyl group. R 1 and R 2 may be different for each structural unit.) [Chemical formula] (R 3 represents a hydrogen atom or a methyl group, and R 4 represents a group having an epoxy group, a group having a hydroxy group, or a group having a carboxy group. R 3 and R 4 may be different for each structural unit.) [Advantages of the Invention]

[0008] According to the present disclosure, there are provided particles having a core-shell structure, having a high refractive index of the core particles, and having reactive functional groups on the shell, which can improve the sensitivity and have excellent ability to suppress non-specific adsorption in the agglutination method. Furthermore, inspection particles, reagents, inspection kits, methods for detecting target substances, methods for producing the above particles, and methods for detecting target substances can be provided. [Modes for Carrying Out the Invention]

[0009] 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.

[0010] First Embodiment As a first embodiment, the present disclosure provides the following particles. Particles for use in the agglutination method, having core particles and a shell on the surface of the core particles, The core particles have a volume-average particle diameter of 200 nm or more and 500 nm or less, contain a polymer having a structural unit represented by formula (1), and the content ratio of the structural unit represented by formula (1) with respect to the particles is 40% by mass or more and 85% by mass or less. The shell has a film thickness of 5 nm or more and 50 nm or less, contains a polymer having a structural unit represented by formula (2), and the content of the structural unit represented by formula (2) with respect to the particles is 9% by mass or more and 60% by mass or less, and is a particle.

Chemical formula

Chemical formula

[0011] It can also be said that the particles according to this embodiment are particles including a first layer and a second layer in this order. It can also be said that the first layer may be a particle, and the aforementioned core particles have a first layer that is a core particle and the aforementioned shell is a second layer. In addition, in this embodiment, between the first layer and the second layer, and further outside the second layer (on the surface side of the particle), there may be a layer different from the second layer. Hereinafter, regarding the particles according to this embodiment that include a first layer and a second layer in this order, a configuration in which the first layer is a core particle and the second layer is a shell will be described as an example.

[0012] The particles of this embodiment contain a polymer having a structure represented by formula (1), and the content of the structure represented by formula (1) is 40% by mass or more and 85% by mass or less in the particles. Formula (1) is R 2 has a phenyl group or a naphthyl group which may be substituted or unsubstituted. The phenyl group and the naphthyl group are known to increase the refractive index. By having the repeating unit structure represented by formula (1) in the particles in an amount of 40% by mass or more and 85% by mass or less, the refractive index of the particles increases, and the refractive index of the particles themselves also increases. When the reagent containing the particles reacts with the target substance and aggregates, the absorbance increases. However, when the particles with a high refractive index aggregate, the absorbance increases more, so the difference in absorbance before and after aggregation also becomes larger, and the sensitivity is improved. Further, it is preferable that the repeating unit structure represented by formula (1) is contained in the particles in an amount of 50% by mass or more, and more preferably 60% by mass or more. Further, it is preferable that the repeating unit structure represented by formula (1) is contained in the particles in an amount of 65% by mass or less. Further, it is preferable that the content ratio of the structural unit represented by formula (1) to the core particles is 90% by mass or more.

[0013] Note that the core particles may contain a copolymer derived from a polymerization initiator or a vinyl monomer, a coloring material such as a pigment, a dye, a fluorescent agent, or an inorganic filler. The content is preferably 9% by mass or less.

[0014] The core particle size of this embodiment is 200 nm or more and 500 nm or less in volume average particle size. In the inspection by the aggregation method, it is common to use light with a wavelength in the visible light region. In this wavelength region, as the particle size increases up to 1 μm, the absorbance also increases (Non-Patent Document 1). Therefore, when the particles of this embodiment having a core particle size of 200 nm or more in volume average particle size and further having a shell with a film thickness of 5 nm or more aggregate, compared with the case where particles with a core particle size smaller than 200 nm aggregate, the aggregate of the particles after aggregation becomes larger, and the increase in absorbance before and after aggregation is large, so the sensitivity is high. Also, since the core particle size is 500 nm or less, even when the concentration of the target substance is high, the sensitivity increases at a rate of at least a certain level as the concentration increases, and it becomes possible to evaluate the concentration contained in the specimen. Note that the particles according to this embodiment are used in the aggregation method, and the aggregation method can be referred to as a nephelometry method in the sense that it detects the change in turbidity before and after mixing the target substance and the particles. Also, when an antibody, antigen, etc. are bound to the particles to detect an antigen or antibody, the particles according to this embodiment can also be referred to as particles used in the immunonephelometry method.

[0015] The particles of this embodiment have a shell film thickness of 5 nm or more and 50 nm or less and contain a polymer having a structure represented by formula (2). Since the structure represented by formula (2) has any one of an epoxy group, a hydroxy group, and a carboxy group, it has high hydrophilicity. Therefore, by covering the surface of the core particles with a shell layer containing a polymer having a structure represented by formula (2), the hydrophobic portion of the particle surface is covered and the hydrophilicity becomes high. As a result, the hydrophobic interaction with proteins and the like other than the target substance in the specimen is reduced, and non-specific adsorption can be suppressed.

[0016] Also, since the shell film thickness is 5 nm or more and 50 nm or less, it is possible to cover the hydrophobic surface of the core particles without exposing it, and also, the dispersibility of the particles does not become too high, and the aggregation of the particles is not easily suppressed, so the sensitivity is high, and it is possible to achieve both high sensitivity and suppression of non-specific adsorption.

[0017] In this embodiment, the content of the structure represented by the formula (2) with respect to the particles is preferably 9% by mass or more and 60% by mass or less, preferably 9% by mass or more and 53% by mass or less, preferably 9% by mass or more and 39% by mass or less, preferably 16% by mass or more and 39% by mass or less, preferably 20% by mass or more and 39% by mass or less, and preferably 31% by mass or more and 39% by mass or less.

[0018] In this embodiment, the content of the structure represented by the formula (2) with respect to the particles is 9% by mass or more and 60% by mass or less in the particles, so that the hydrophobicity of the particle surface is reduced and non-specific adsorption can be suppressed. In addition, the dispersibility of the particles does not become too high, and the aggregation of the particles is not easily suppressed, so the sensitivity is increased, and it is possible to achieve both high sensitivity and suppression of non-specific adsorption.

[0019] The particles of this embodiment have a structure in which core particles having a high refractive index and a large difference in absorbance before and after aggregation, as described later, are covered with a shell having high hydrophilicity and capable of suppressing non-specific adsorption. By having such a core-shell structure, it is possible to have both a high refractive index and hydrophilicity on the particle surface, and it is possible to achieve both high-sensitivity detection and suppression of non-specific adsorption in the aggregation method.

[0020] In this embodiment, the formula (1) is preferably a structure represented by the formula (1-A). By being the formula (1-A), the core refractive index of the core particles can be increased. In addition, since the structure of the formula (1-A) has high hydrophobicity, it is difficult for the hydrophilic shell component to be incorporated into the core portion.

Chemical formula

[0021] In this embodiment, the structure represented by formula (1-A) can be obtained by polymerizing the monomer represented by formula (X1) described below. Specific examples of the monomer include styrenes, 1-vinylnaphthalene, and 2-vinylnaphthalene as described below, and styrene, 1-vinylnaphthalene, and 2-vinylnaphthalene are particularly preferred. These monomers may be used alone or in combination of two or more.

[0022] The core particles preferably further have a crosslinked structure. The crosslinkable structure is obtained by polymerization using 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 diolefins such as butadiene and isoprene; divinylbenzene, diallyl phthalate, allyl acrylate, allyl methacrylate, etc. Also, two or more crosslinkable radical polymerizable monomers may be used. As the crosslinked structure, the structure represented by formula (3) is more preferred. By the core particles having a crosslinked structure, the particles having a core-shell structure become physically strong, and there is no concern of cracking or chipping even when centrifugation is repeated during purification.

Chemical formula

[0023] The crosslinkable radically polymerizable monomer used to form the crosslinkable structure of formula (3) includes, for example, 1,2-divinylbenzene, 1,3-divinylbenzene, 1,4-divinylbenzene, 2,6-diethynylnaphthalene, 2,7-diethynylnaphthalene. These may be used alone or in combination of two or more. Among the exemplified crosslinkable radically polymerizable monomers, divinylbenzene is preferred. Although the reason is not clear, when using divinylbenzene, it has excellent handling properties during the radical polymerization reaction, the monomer conversion rate during core particle formation is improved, and the core component is less likely to be incorporated into the shell, which is preferable.

[0024] In this embodiment, it is preferable that the mass ratio of the core in the particles is 50% by mass or more and 95% by mass or less. By setting the mass ratio of the core in the particles to 50% by mass or more, the content of core particles with a high refractive index can be increased, the refractive index is improved, the difference in absorbance before and after aggregation becomes large, and the sensitivity in a region where the concentration of the target substance is low can be improved. Further, by setting the mass ratio of the core in the particles to 95% by mass or less, the shell layer can coat the surface of the core particles, improve the hydrophilicity of the particle surface, and improve the suppression of non-specific adsorption.

[0025] In this embodiment, it is preferable that the formula (2) has a structure represented by formula (2-A). The structure represented by formula (2-A) has either a hydroxy group or a carboxy group, and the ability to suppress non-specific adsorption is equal to or higher than that of a structure having an epoxy group, which is preferable.

Chemical formula

Chemical formula

[0026] When the total of the number of hydroxy groups (0 or more) and the number of carboxy groups (0 or more) contained in the structure represented by the above formula (2-A) is 2 or more, a highly hydrophilic structure is contained within one repeating unit. Therefore, even under the condition that the film thickness of the shell is 5 nm or more and 50 nm or less, non-specific adsorption can be reduced. Furthermore, when the total of the number of hydroxy groups (0 or more) and the number of carboxy groups (0 or more) contained in the structure represented by the above formula (2-A) is 3 or more, it is preferable because the hydrophilicity becomes higher.

[0027] Examples of the specific structure of the formula (2-A) are shown in the following (2-A-1) to (2-A-12), but are not limited thereto. [Chemical formula]

[0028] The structure represented by the formula (2-A) of the present embodiment is obtained by reacting a polymer obtained by polymerizing a monomer represented by the formula (X2) described later with the formula (X3) described later. As in the above formulas (2-A―1) to (2-A-12), either a sulfur atom or a nitrogen atom may be included in the side chain. When a sulfur atom is included in the side chain, it is more preferable from the viewpoint of improving sensitivity, and when a nitrogen atom is included in the side chain, it is more preferable from the viewpoint of suppressing non-specific adsorption. Since the sulfide bond has a weak hydrophobic tendency, it can weaken the water confinement force of the highly hydrophilic side chain appropriately and suppress the osmotic pressure aggregation that may occur when mixed with a high-concentration analyte, and thus can contribute to the improvement of sensitivity. On the other hand, since the amino group has a hydrophilic tendency, it can contribute to the reduction of non-specific adsorption. The specific example of the monomer represented by (X2) is not particularly limited, but glycidyl (meth)acrylate is preferable.

[0029] The content ratio of the structural unit represented by formula (2) in the shell is preferably 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 becomes high, and non-specific adsorption suppression can be improved. More preferably, it is 95% by mass or more to further suppress non-specific adsorption. In addition, the shell may contain a copolymer derived from a polymerization initiator or a vinyl monomer, a coloring material such as a pigment, a dye, a fluorescent agent, and an inorganic filler. The content is preferably 9% by mass or less.

[0030] In addition, the particles of the present embodiment may have a structure other than formulas (1) and (2). Examples of the structure other than formulas (1) and (2) included in the particles include structures obtained by polymerizing monomers such as styrenes, acrylates, and methacrylates, but are not particularly limited. Also, as a structure other than formulas (1) and (2), a plurality of structures may be simultaneously possessed.

[0031] When the measurement is performed by X-ray photoelectron spectroscopy (XPS) on the particles, the composition ratio of C element to O element quantified is preferably in the range of 2.0 or more and 3.3 or less. In XPS measurement, since photoelectrons generated from the outermost surface up to about 10 nm are detected, it is a technique capable of analyzing the components of the surface layer of the particles. When the above composition ratio is less than 2.0, the O element in the surface layer of the particles is excessive, and while sufficient hydroxyl groups and carboxyl groups are added, the hydrophilicity is very high, so that particle aggregation hardly occurs, and the detection sensitivity based on the principle of the aggregation method may decrease. Further, when the above composition ratio is greater than 3.3, the O element in the surface layer of the particles is too small and the hydrophilicity is insufficient. Therefore, not only is the dispersion stability disadvantageous when the particles are stored for a long time, but the non-specific adsorption property may deteriorate. Thus, by optimizing the composition ratio of the components of the surface layer of the particles, particles excellent in both sensitivity and non-specific adsorption property can be provided.

[0032] By performing FT-IR measurement on the inspection particles of the present embodiment, the ratio of the formula (1) to the formula (2) can be evaluated. As the measurement method, the ATR method is preferable, and infrared light can penetrate from the outermost surface up to about μm size to obtain an FT-IR spectrum. The particles of the present embodiment have, in the infrared absorption spectrum (FT-IR spectrum), a peak height existing at 1500 to 1650 cm -1 derived from the C═C bond of the aromatic ring as A, and a peak height existing at 1680 to 1750 cm -1 derived from the carbonyl group of the ester bond as B. Then, it is preferable that the value of A / B is 0.85 or more and 5.35 or less in terms of achieving both sensitivity and suppression of non-specific adsorption property.

[0033] When the value of A / B is 0.85 or more, the ratio of the formula (1) to the carbonyl group is sufficiently high, so the detection sensitivity is improved. Further, when the value of A / B is 5.35 or less, the carbonyl groups of the ester bonds contained in the particles are sufficiently many, that is, the ratio of the formula (2) showing hydrophilicity to the whole particles is large. Therefore, the hydrophilicity of the particles is obtained and the non-specific adsorption property is improved.

[0034] The particle size of the particles in this embodiment is the volume average particle size in the aqueous dispersion, which is 210 nm or more and 600 nm or less, and more preferably 230 nm or more and 500 nm or less. Particles with a size of 230 nm or more have larger aggregates of particles after aggregation compared to particles smaller than 230 nm, and the difference in absorbance before and after aggregation increases, resulting in higher sensitivity. Also, when the particle size is 600 nm or less, even when the concentration of the target substance is high, the sensitivity increases at a rate of at least a certain level with the increase in the concentration, enabling the evaluation of the concentration contained in the sample.

[0035] The value of the ratio (Dv / Dn) of the volume average particle size (Dv) to the number average particle size (Dn) of the particles in this embodiment is preferably 1.25 or less. It is known that the closer the value of Dv / Dn is to 1, the narrower the particle size distribution. When the value of Dv / Dn is 1.25 or less, the variation in the particle size is small, and there are no particles with a large particle size. Therefore, the difference in size from the aggregate after aggregation also increases, resulting in higher sensitivity.

[0036] When the particles of this embodiment are dispersed in water at a solid content concentration of 0.1 mass% so that the value of Dv / Dn becomes 1.25 or less, and the absorbance at an incident light wavelength of 572 nm and a cell length of 10 mm is defined as E, the value of E / Dv is 8.0×10 -4 or more and 2.0×10 -3 or less is preferable. 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 larger than a certain size, the absorbance is large, and the difference in absorbance before and after aggregation also increases, resulting in higher sensitivity.

[0037] As a method for producing the core particles, soap-free emulsion polymerization is preferable. By using soap-free emulsion polymerization, the particle size distribution becomes uniform, the sensitivity is stabilized, and the detection limit can be improved in the region where the concentration of the target substance is low.

[0038] As a method for forming a core-shell structure, there is no particular limitation as long as a core-shell structure is formed. However, it is preferable in terms of improving sensitivity and suppressing non-specific adsorption to form a shell structure by adding a monomer for forming a shell to a dispersion of core particles and then adding a water-soluble polymerization initiator. Regarding the mechanism, the details are not clear, but it 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 polymerization to form oligomers. As the polymerization progresses, the hydrophobicity of the oligomers increases, and they can no longer be dispersed alone and deposit on the surface of the core particles. On the particle surface, the monomer and oligomer incorporated into the core particle surface further react to form a core-shell structure. The particles formed in this way are less likely to have the shell component penetrate into the core region, so that the refractive index of the core particles is less likely to decrease due to this. As a result, the absorbance difference before and after aggregation also increases, and the sensitivity is improved. On the other hand, the two-stage swelling polymerization method of swelling the monomer for forming a shell on the core particles and forming a shell structure with a hydrophobic initiator or the like is known as an effective method for making the particle size distribution uniform, but in this method, the core particles swell. Therefore, the shell component may penetrate into the core particles and the refractive index of the core particles may decrease. Therefore, when the particles produced by this method are used in the aggregation method, it is difficult to improve the sensitivity.

[0039] Second Embodiment As a second embodiment, the present disclosure provides a method for producing particles used in the following aggregation method. A first step of obtaining core particles having a volume average particle diameter of 200 nm or more and 500 nm or less by polymerizing a first monomer composition containing a monomer represented by formula (X1); A second step of obtaining particles having a layer of 5 nm or more and 50 nm or less formed outside the core particles by polymerizing using a reaction solution containing the core particles, a second monomer composition containing a monomer represented by formula (X2), and a water-soluble polymerization initiator However, the content ratio of the monomer represented by the formula (X1) to the total amount of the monomers contained in the first monomer composition is 90% by mass or more, However, The content ratio of the unpolymerized monomer represented by the formula (X1) to the reaction solution is 1000 ppm or less. The content ratio of the monomer represented by the formula (X2) to the total amount of the monomer contained in the first monomer composition and the monomer contained in the second monomer composition is 9% by mass or more and less than 40% by mass; and A third step of reacting the particles forming the layer with the compound represented by the formula (X3), A method for producing particles used in the aggregation method, comprising:

Chemical formula

Chemical formula

Chemical formula

[0040] In the reaction solution before the start of the second step, the content of the monomer represented by the formula (X1) is preferably 1000 ppm or less. By being 1000 ppm or less, when forming the shell in the second step, the amount of the hydrophobic monomer represented by the formula (X1) is small, and it is difficult for the shell to contain a hydrophobic repeating structure, and non-specific adsorption can be suppressed.

[0041] The water-soluble polymerization initiator used in this embodiment is not particularly limited, but water-soluble azo compounds and water-soluble peroxides are preferably used. As the water-soluble azo compound, 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 is preferably used. As the water-soluble peroxide, any one of potassium persulfate, ammonium persulfate, sodium persulfate, tertiary butyl hydroperoxide, cumyl hydroperoxide, paramethane hydroperoxide, and diisopropylbenzene hydroperoxide is preferably used.

[0042] In this embodiment, the monomer represented by the formula (X1) is preferable for improving the sensitivity because the refractive index of the polymer obtained by polymerizing the monomer is high. For example, styrenes, 1-vinylnaphthalene, and 2-vinylnaphthalene can be mentioned, and styrene, 1-vinylnaphthalene, and 2-vinylnaphthalene are particularly preferable. These monomers may be used alone or in combination of two or more. 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, and p-phenylstyrene, etc.

[0043] In this embodiment, since the monomer represented by the formula (X2) has a glycidyl group in its side chain, the compound represented by the formula (X3) can react with the glycidyl group in the shell having the polymer of the formula (X2), and carboxy groups and hydroxy groups contained in the formula (X3) can be introduced onto the particle surface. The monomer represented by the formula (X2) is not particularly limited, but glycidyl (meth)acrylate is preferred.

[0044] In this embodiment, in the third step, the amino group or thiol group in the formula (X3) reacts and reacts with the glycidyl group in the shell, thereby being added to the shell on the particle surface to form the formula (2). The monomer represented by the formula (X3) is not particularly limited, and examples thereof 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.

[0045] Third Embodiment As a third embodiment, the present disclosure provides the following particles. A first step of performing a polymerization reaction of a reaction system in a first step including 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 obtain core particles having a volume average particle diameter of 200 nm or more and 500 nm or less; A second step of polymerizing a reaction solution in a second step including the core particles, a second monomer composition containing a monomer represented by the 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 outside the core particles However, in the reaction solution of the second step, The content ratio of the unpolymerized monomer represented by the formula (X1) with respect to the reaction solution of the second step is 1000 ppm or less, The content ratio of the monomer represented by the formula (X2) with respect to the total amount of the monomer contained in the first monomer composition and the monomer contained in the second monomer composition is 9% by mass or more and less than 40% by mass; and A third step of reacting the particles on which the layer has been formed with the compound represented by the formula (X3), Particles for use in the aggregation method, produced by .

Chemical formula

Chemical formula

Chemical formula

[0046] Other embodiments The present disclosure provides, as a further embodiment, inspection particles characterized in that a ligand is added to the surface of the particles of the present disclosure described above.

[0047] The inspection particles in the present disclosure have a high affinity for a target substance selectively or specifically by means of a ligand added to the particle surface. In particular, it is preferable that the ligand is added to the particle surface by a chemical bond. In addition, the ligand in the present disclosure refers to a compound that specifically binds to a receptor possessed by a specific target substance. The site where the ligand binds to the target substance is determined and has selective or specifically high affinity. For example, antigen and antibody, enzyme protein and its substrate, signal substances such as hormones and neurotransmitters and their receptors, nucleic acids, etc. are exemplified, but the ligand in the present disclosure is not limited to these. Examples of nucleic acids include deoxyribonucleic acid and the like. The test particle in the present disclosure has selective or specifically high affinity (affinity) for the target substance. It is preferable that the ligand in the present disclosure is any one of an antibody, an antigen, and a nucleic acid.

[0048] As a further embodiment, the present disclosure provides a reagent for use in an agglutination method, characterized in that the particles or test particles of the present disclosure described above are dispersed in an aqueous solution. The test object of the reagent is not particularly limited. Examples of the reagent include, for example, in vitro diagnostic pharmaceuticals, an antigen detection reagent with an antigen as the test object, and an antibody detection reagent with an antibody as the test object. The reagent may be one in which particles not containing a ligand are dispersed in an aqueous solution, assuming that a user adds a ligand such as a desired antibody to the particles. Alternatively, it may be one in which test particles to which a ligand has been added in advance are dispersed in an aqueous solution assuming a specific target substance.

[0049] The reagent in the present disclosure has the particles or test particles in the present disclosure and a dispersion medium for dispersing the test particles. The reagent in the present disclosure may contain a third substance such as a solvent or a blocking agent in addition to the particles or test particles in the present disclosure within a range capable of achieving the object of the present disclosure. Two or more kinds of third substances such as a solvent and a blocking agent may be combined and contained. Examples of the dispersion medium used in the present disclosure include various buffer solutions such as phosphate buffer, glycine buffer, Good buffer, Tris buffer, and ammonia buffer, but the dispersion medium contained in the reagent in the present disclosure is not limited to these.

[0050] As a further embodiment, the present disclosure provides a test kit. The kit has the above reagent and a container containing the above reagent. As a kit in the present embodiment, in addition to the above reagent (hereinafter referred to as reagent 1), a reaction buffer (hereinafter referred to as reagent 2) may be provided. A sensitizer may be contained in both or either one of reagent 1 and reagent 2. Further, the kit in the present disclosure may include, in addition to reagent 1 and reagent 2, a positive control, a negative control, a serum diluent, a primary antibody, a secondary antibody, and the like. As a medium for the positive control and the negative control, in addition to serum and physiological saline that do not contain a measurable target substance, a solvent may be used.

[0051] As a further embodiment, the present disclosure provides a method for detecting a target substance in a sample by in vitro diagnosis, the detection method being characterized by mixing a reagent containing the above inspection particles and a sample that may contain the target substance.

[0052] 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 in the present embodiment is a method for detecting a target substance in a sample, including a step of mixing a reagent containing the above inspection particles and a sample 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 or scattered light of the light irradiated on the mixed solution.

[0053] [Method for Measuring Volume Average Particle Diameter and Particle Size Distribution in Aqueous Dispersion of Particles] A method for measuring the volume average particle diameter (Dv) in an aqueous dispersion of particles in the present disclosure will be described. The Dv of the particles present in the aqueous dispersion is measured by the dynamic light scattering method. For example, it is measured at 25 °C using a zeta sizer (Zeta Sizer Ultra: Malvern Panalytical). As the Dv of the core particles, the aqueous dispersion of the core particles obtained in the first step above is measured. As the Dv of the particles having a core and shell structure, the aqueous dispersion of the particles obtained in the third step above is measured. Further, the particle size distribution of the particles having a core and shell structure in the present disclosure is calculated by measuring the number average particle diameter (Dn) by the above dynamic light scattering method and taking the ratio (Dv / Dn) of Dv to Dn.

[0054] [Method for Measuring Film Thickness of Particle Shell] A method for calculating the film thickness of the shell of the particles in the present disclosure will be described. The film 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 obtained in the above third step and dividing the result by 2.

[0055] [Method for Measuring Composition Ratio (C / O) of C Element to O Element of Particles] The measurement of the composition ratio (C / O) of the C element to the O element in the present disclosure will be described. The composition ratio of the particles in the present disclosure by X-ray photoelectron spectroscopy (XPS) is measured using particles fixed to an indium foil in a freeze-dried state. In the following examples, the measurement apparatus and measurement conditions were as follows. ·Measurement apparatus: X-ray photoelectron spectrometer: Quantum2000 (trade name, manufactured by ULVAC-PHI, Inc.) ·X-ray source: Monochromatic AlKα ·Xray Setting: 100μmφ (25W (15KV)) ·Photoelectron extraction angle: 45 degrees ·Neutralization condition: Combined use of a neutralization gun and an ion gun ·Analysis area: 300×200μm ·Pass Energy: 58.70eV ·Step size: 0.125eV ·Analysis software: Maltipak (PHI) The number of integrations during measurement is 15 for C1S and O1S, and 30 for N1S. Using the quantitative values of the obtained elemental amounts (the ratio (atomic%) of the amount of the C element to the total amount of the three elements and the ratio (atomic%) of the amount of the O element), the composition ratio of the C element to the O element is determined.

[0056] [Method for Measuring Infrared Absorption Spectrum of Particles] The method for measuring the ratio of the C=O bond derived from the ester and the C=C bond derived from the aromatic ring in the IR spectrum of the particles in the present disclosure, that is, the ratio of the ester component and the aromatic ring component in the particles, will be described. The component ratio based on the IR spectrum of the particles in the present disclosure is measured using freeze-dried particles. In the following examples, the measuring apparatus and measuring conditions were as follows. · Measuring apparatus: Fourier transform infrared spectrometer Spectrum One (manufactured by PerkinElmer, Inc.) · Measuring method: ATR method · Crystal: Diamond · Measuring range: 4000 cm -1 ~650 cm -1 · Resolution: 4 cm -1 · Number of accumulations: 32 times · Data analysis: Performed with absorbance The freeze-dried particles in powder form are placed on the measuring part, and a flat probe is used to apply pressure and hold them down. At this time, the measurement is started after the pressure gauge displayed on the measurement software reaches about 100. After the measurement, quantification and analysis are performed using the absorbance data. Let the height of the peak maximum at 1500 - 1650 cm -1 existing due to the C=C bond of the aromatic ring be A, and the height of the peak maximum at 1680 - 1750 cm -1 existing due to the carbonyl group of the ester bond be B, then the value of A / B is calculated.

[0057] [Method for Measuring the Absorbance of Particles] The method for measuring the absorbance of the particles in the present disclosure will be described. The absorbance of the particles can be measured for the aqueous dispersion of the particles in the present disclosure at a wavelength of 572 nm. In the following examples, a 0.01 mass% aqueous dispersion of the particles was added to a cell, and the absorbance at a cell length of 10 mm for incident light at 572 nm was measured using a spectrophotometer Biospectrometer manufactured by Eppendorf.

Example

[0058] Hereinafter, the present disclosure will be described in detail with reference to examples, but the present disclosure is not limited to these examples.

[0059] [Example 1] (Synthesis of Particle 1) (Step - 1 / Preparation of Core 1) 71.75 g of styrene (St: Kishida Chemical Co., Ltd.), 1.30 g of divinylbenzene (DVB: Kishida Chemical Co., Ltd.), and 1190.67 g of ion - exchanged water were weighed into a 2 - L four - neck separable flask and mixed to form a mixed solution. Then, while stirring this mixed solution at 140 rpm, it was maintained at 70 °C, and the inside of the four - neck separable flask was deoxygenated by performing a nitrogen flow at a flow rate of 200 ml / min. Next, a solution prepared separately by dissolving 3.11 g of V - 50 (Fuji Film Wako Pure Chemical Corporation) in 50 g of ion - exchanged water was added to the above - mentioned mixed solution to initiate soap - free emulsion polymerization. By reacting for 48 hours from the start of polymerization, a dispersion of core particles 1 composed of a copolymer of St and DVB was obtained. A part of it was sampled and evaluated using dynamic light scattering of Core 1 (Zetasizer ultra: Malvern Panalytical). As a result, the volume - average particle diameter was 330 nm.

[0060] (Step - 2 / Preparation of Mother Particle 1) It was adjusted with ion - exchanged water to 148.29 g of a dispersion of Core Particle 1 with a solid - content concentration of 2.0%. A part of this dispersion was sampled and evaluated for the contents of St and DVB using gas chromatography. As a result, the contents of St and DVB were 75 ppm. Next, 1.52 g of glycidyl methacrylate (GMA: Kishida Chemical Co., Ltd.) was added, and while stirring at 100 rpm, it was maintained at 70 °C, and the inside of the four - neck separable flask was deoxygenated by performing a nitrogen flow at a flow rate of 200 ml / min. Then, a solution prepared separately by dissolving 0.018 g of V - 50 in 1 g of ion - exchanged water was added to the above - mentioned mixed solution to initiate the formation of the shell. By continuously stirring for 17 hours after the start of the reaction, a dispersion containing mother particle 1 having a core - shell structure was obtained. After slowly cooling the above - mentioned dispersion to room temperature, a part of it was sampled and evaluated for the polymerization conversion rate using gas chromatography. As a result, it was confirmed that it was substantially 100%.

[0061] (Production of Particles 1 with Core-Shell Structure in Project-3) An aqueous solution prepared in advance by dissolving mercaptosuccinic acid (MSA, Wako Pure Chemical Industries, Ltd.) and 3-mercapto-1,2-propanediol (MPD: Wako Pure Chemical Industries, Ltd.) in the aqueous dispersion containing mother particles 1 (the molar ratio of 3-mercapto-1,2-propanediol to mercaptosuccinic acid is 6:4 (mole fraction), and the total number of moles of MSA and MPD is equal to the number of moles of the above glycidyl methacrylate) was added, and triethylamine (Kishida Chemical Co., Ltd.) was added to adjust the pH to 10. Next, while stirring the above at 200 rpm, the temperature was raised to 70 °C, and this state was maintained for another 18 hours to obtain a dispersion of particles 1 having a core-shell structure. Particles 1 were separated from the above dispersion by a centrifuge, and the operation of redispersing particles 1 in ion-exchanged water was repeated 8 times to purify particles 1, and finally, they were stored in the state of an aqueous dispersion adjusted so that particles 1 became 1.0% by mass. The results of evaluating the particle physical properties of particles 1 are shown in Table 1. In addition, the amount of formula (1) of 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 in particles 1 are shown in Table 2. In addition, IR measurement was performed on particles 1. The peak height at 1500 - 1650 cm -1 derived from the C=C bond of the aromatic ring was designated as A, and the peak height at 1680 - 1750 cm -1 present in the carbonyl of the ester bond was designated as B. When the value of A / B was calculated, it was 4.05.

[0062] [Table 1]

[0063] [Table 2-1] [Table 2-2]

[0064] [Example 2] (Synthesis of Particle 2) (Step - 1 / Preparation of Core 2) A dispersion of Core 2 was obtained by the same experimental procedure as in Example 1, except that the rotation speed of stirring in Example 1 was changed from 140 rpm to 200 rpm. A part of it was sampled and evaluated using dynamic light scattering of Core 2 (Zetasizer ultra: Malvern Panalytical). As a result, the volume - average particle diameter was 395 nm.

[0065] (Step - 2 / Preparation of Mother Particle 2) In Example 1 and Step - 2, a dispersion of Mother Particle 2 was obtained by the same experimental procedure, except that the dispersion of Core 1 was changed to that of Core 2. The St and DVB contents of the dispersion of Core 2 before adding GMA were 83 ppm. When the polymerization conversion rate was evaluated using gas chromatography, it was confirmed that it was substantially 100%.

[0066] (Step - 3 / Preparation of Particle 3 with Core - Shell Structure) A dispersion of Particle 2 with a core - shell structure was obtained by the same experimental procedure as in Step - 3 of Example 1. The results of evaluating the particle physical properties of Particle 2 are shown in Table 1. Also, 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 in Particle 2 are shown in Table 2.

[0067] [Example 3] (Synthesis of Particle 3) (Step - 1 / Preparation of Core 3) 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, and the amount of V - 50 was 4.31 g. A dispersion of Core 3 was obtained by the same experimental procedure as in Example 1, except that the rotation speed of stirring was changed from 140 rpm to 200 rpm. A part of it was sampled and evaluated using dynamic light scattering of Core 2 (Zetasizer ultra: Malvern Panalytical). As a result, the volume - average particle diameter was 498 nm.

[0068] (Step - 2 / Preparation of Mother Particle 3) The dispersion liquid of core particles 1 with a solid content concentration of 2.5% was adjusted with ion-exchanged water to 148.27 g. When a part of this dispersion liquid was sampled and the contents of St and DVB were evaluated using gas chromatography, the contents of St and DVB were 82 ppm. Next, 2.37 g of GMA was added, and while stirring at 100 rpm and maintaining at 70 °C, nitrogen flow was carried out at a flow rate of 200 ml / min to deoxygenate the inside of the above four-neck separable flask. Then, a solution prepared separately by dissolving 0.018 g of V-50 (Fuji Film Wako Pure Chemical Corporation) in 1 g of ion-exchanged water was added to the above mixed solution to start the formation of the shell. By continuously stirring for 17 hours after the start of the reaction, a dispersion liquid containing mother particles 1 having a core-shell structure was obtained. After the above dispersion liquid was gradually cooled to room temperature, a part of it was sampled, and when the polymerization conversion rate was evaluated using gas chromatography, it was confirmed that it was substantially 100%.

[0069] (Step - 3 / Preparation of Particles 3 Having a Core-Shell Structure) A dispersion liquid of particles 3 having a core-shell structure was obtained by the same experimental operation as in Example 1 and Step - 3. The results of evaluating the particle physical properties of particles 3 are shown in Table 1. Also, the amount of formula (1) of core 3, the content of formula (2) in particles 3, the content of formula (2) in the shell, and the mass ratio of core 3 in particles 3 are shown in Table 2.

[0070] [Example 4](Synthesis of Particles 4) (Step - 1 / Preparation of Core 4) In 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 and mixed to form a mixed solution. Then, while stirring this mixed solution at 140 rpm, it was maintained at 70 °C, and nitrogen flow was carried out at a flow rate of 200 ml / min to deoxygenate the inside of the four-neck separable flask. Next, a solution prepared separately by dissolving 1.10 g of V-50 (Fuji Film Wako Pure Chemical Industries, Ltd.) in 30 g of ion-exchanged water was added to the above mixed solution to initiate soap-free emulsion polymerization. By reacting and continuously stirring for 48 hours from the start of polymerization, a dispersion of core particles 1 composed of a copolymer of St and DVB was obtained. A part of it was sampled and evaluated using dynamic light scattering of core 4 (Zetasizer ultra: Malvern Panalytical), and the volume average particle diameter was 203 nm.

[0071] (Step - 2 / Preparation of mother particles 4) It was adjusted with ion-exchanged water to obtain 133.0 g of a dispersion of core particles 4 with a solid content concentration of 2.0%. A part of this dispersion was sampled and evaluated for the contents of St and DVB using gas chromatography. As a result, the contents of St and DVB were 20 ppm. Next, 1.43 g of GMA was added, and while stirring at 100 rpm, it was maintained at 70 °C, and nitrogen flow was carried out at a flow rate of 200 ml / min to deoxygenate the inside of the four-neck separable flask. Then, a solution prepared separately by dissolving 0.0323 g of V-50 (Fuji Film Wako Pure Chemical Industries, Ltd.) in 1 g of ion-exchanged water was added to the above mixed solution to initiate shell formation. By continuously stirring for 17 hours after the start of the reaction, a dispersion containing mother particles 1 having a core-shell structure was obtained. After slowly cooling the above dispersion to room temperature, a part of it was sampled and evaluated for the polymerization conversion rate using gas chromatography. As a result, it was confirmed that it was substantially 100%.

[0072] (Step - 3 / Preparation of particles 4 having a core-shell structure) A dispersion of particles 4 having a core-shell structure was obtained by the same experimental operation as in Example 1 and Step - 3. The results of evaluating the particle physical properties of Particle 4 are shown in Table 1. Also, the amount of Formula (1) in 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 in Particle 4 are shown in Table 2.

[0073] [Example 5] (Synthesis of Particle 5) (Step - 1 / Preparation of Core 5) In Step - 1 of Example 1, except that the amount of St used was changed from 71.75 g to 67.58 g and the amount of DVB used was changed from 1.30 g to 5.02 g, a dispersion of Core 5 was obtained by the same experimental operation. A part of it was sampled and evaluated using dynamic light scattering (Zetasizer ultra: Malvern Panalytical) of Core 5, and the volume - average particle diameter was 332 nm.

[0074] (Step - 2 / Preparation of Mother Particle 5) In Step - 2 of Example 1, except that the amount of GMA used was changed from 1.52 g to 0.99 g and the amount of V - 50 used was changed from 0.018 g to 0.016 g, a dispersion of Mother Particle 5 was obtained by the same experimental operation. The contents of St and DVB in the dispersion of Core 5 before adding GMA were 83 ppm. Also, when the polymerization conversion rate was evaluated using gas chromatography, it was confirmed that it was substantially 100%.

[0075] (Step - 3 / Preparation of Particle 5 with Core - Shell Structure) A dispersion of Particle 5 with a core - shell structure was obtained by the same experimental operation as in Step - 3 of Example 1. The results of evaluating the particle physical properties of Particle 5 are shown in Table 1. Also, the amount of Formula (1) in 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 in Particle 5 are shown in Table 2.

[0076] [Example 6] (Synthesis of Particle 6) (Step - 1 / Preparation of Core 6) In Step-1 of Example 1, instead of 71.75 g of St, 35.88 g of St and 35.88 g of 1-vinylnaphthalene (1VN: FUJIFILM Wako Pure Chemical Corporation) were used, and a dispersion of Core 6 was obtained by the same experimental operation otherwise. A part of it was sampled and evaluated using the dynamic light scattering of Core 6 (Zetasizer ultra: Malvern Panalytical), and the volume average particle diameter was 329 nm.

[0077] (Step-2 / Preparation of Mother Particle 6) In Example 1 and Step-2, a dispersion of Mother Particle 6 was obtained by the same experimental operation except that the dispersion of Core 1 to Core 6 was changed. The contents of St and DVB in the dispersion of Core 6 before adding GMA were 75 ppm. When the polymerization conversion rate was evaluated using gas chromatography, it was confirmed that it was substantially 100%.

[0078] (Step-3 / Preparation of Particle 6 Having Core-Shell Structure) A dispersion of Particle 6 having a core-shell structure was obtained by the same experimental operation as in Step-3 of Example 1. The results of evaluating the particle physical properties of Particle 6 are shown in Table 1. Also, 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 in Particle 6 are shown in Table 2.

[0079] [Example 7](Synthesis of Particle 7) (Step-1 / Preparation of Core 7) In Step-1 of Example 1, instead of 71.75 g of St, 35.88 g of St and 35.88 g of 4-methylstyrene (MSt: FUJIFILM Wako Pure Chemical Corporation) were used, and a dispersion of Core 7 was obtained by the same experimental operation otherwise. A part of it was sampled and evaluated using the dynamic light scattering of Core 7 (Zetasizer ultra: Malvern Panalytical), and the volume average particle diameter was 331 nm.

[0080] (Step-2 / Preparation of Mother Particle 7) In Example 1 and Step - 2, except that the dispersion of Core 1 to Core 7 was changed, the dispersion of mother particle 7 was obtained by the same experimental operation. The St and DVB contents of the dispersion of Core 7 before adding GMA were 88 ppm. When the polymerization conversion rate was evaluated using gas chromatography, it was confirmed that it was substantially 100%.

[0081] (Production of Particles 7 with Core - Shell Structure in Step - 3) By the same experimental operation as in Step - 3 of Example 1, a dispersion of particles 7 with a core - shell structure was obtained. The results of evaluating the particle physical properties of particles 7 are shown in Table 1. Also, the amount of Formula (1) of Core 7, the content of Formula (2) in particle 7, the content of Formula (2) in the shell, and the mass ratio of Core 7 in particle 7 are shown in Table 2.

[0082] [Example 8] (Synthesis of Particles 8) Using the mother particle 1 prepared in Example 1, a dispersion of particles 8 with a core - shell structure was obtained by the same experimental operation as in Example 1, except that 3 - amino - 1,2 - propanediol (3APD: Tokyo Chemical Industry Co., Ltd.) was used instead of MPD in Step - 3 of Example 1. The results of evaluating the particle physical properties of particles 8 are shown in Table 1. Also, the amount of Formula (1) of Core 1, the content of Formula (2) in particle 8, the content of Formula (2) in the shell, and the mass ratio of Core 1 in particle 8 are shown in Table 2.

[0083] [Example 9] (Synthesis of Particles 9) Using the mother particle 1 prepared in Example 1, a dispersion of particles 9 with a core - shell structure was obtained by the same experimental operation as in Example 1, except that 2 - amino - 1,3 - propanediol (2APD: Tokyo Chemical Industry Co., Ltd.) was used instead of MPD in Step - 3 of Example 1. The results of evaluating the particle physical properties of particles 9 are shown in Table 1. Also, the amount of Formula (1) of Core 1, the content of Formula (2) in particle 9, the content of Formula (2) in the shell, and the mass ratio of Core 1 in particle 9 are shown in Table 2.

[0084] [Example 10] (Synthesis of Particles 10) Using the mother particles 1 prepared in Example 1, a dispersion of particles 10 having a core-shell structure was obtained by the same experimental operation as in Example 1, except that 2-amino-2-hydroxymethyl-1,3-propanediol (Tris: Kishida Chemical Co., Ltd.) was used instead of MPD in Step 3 of Example 1. The results of evaluating the particle properties of particles 10 are shown in Table 1. Also, 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 in particles 10 are shown in Table 2.

[0085] [Example 11] (Synthesis of particles 11) A dispersion of particles 11 having a core-shell structure was obtained by the same experimental operation as in Example 1, except that 1.42 g of GMA was used instead of DVB in Step 3 of Example 1. The results of evaluating the particle properties of particles 11 are shown in Table 1. Also, 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 in particles 11 are shown in Table 2.

[0086] [Example 12] (Synthesis of particles 12) A dispersion of particles 12 having a core-shell structure was obtained by the same experimental operation 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. The results of evaluating the particle properties of particles 12 are shown in Table 1. Also, 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 in particles 12 are shown in Table 2.

[0087] [Example 13] (Synthesis of particles 13) A dispersion of particles 13 having a core-shell structure was obtained by the same experimental operation 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. The results of evaluating the particle physical properties of particle 13 are shown in Table 1. Also, the amount of formula (1) of core 13, the content of formula (2) in particle 13, the content of formula (2) in the shell, and the mass ratio of core 13 in particle 13 are shown in Table 2.

[0088] [Example 14] (Synthesis of Particle 14) A dispersion of particle 14 having a core-shell structure was obtained by the same experimental operation as in Example 1, except that the total number of moles of MSA and MPD used in Step - 3 of Example 1 was changed to 0.5 equivalents instead of being equal to the number of moles of GMA in Step - 2. The results of evaluating the particle physical properties of particle 14 are shown in Table 1. Also, the amount of formula (1) of core 14, the content of formula (2) in particle 14, the content of formula (2) in the shell, and the mass ratio of core 14 in particle 14 are shown in Table 2.

[0089] [Example 15] (Particle 15) In Step - 1 of Example 1, the amount of St used was 85.61 g, the amount of DVB was 1.55 g, the amount of ion-exchanged water was 1120.12 g, and the amount of V - 50 was 3.72 g. Furthermore, the rotation speed of stirring was 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 3 was obtained by the same experimental operation as in Example 1. A part of it was sampled and evaluated using dynamic light scattering (Zetasizer ultra: Malvern Panalytical) of core 2, and the volume average particle diameter was 498 nm.

[0090] [Comparative Example 1] (Synthesis of Comparative Particle 1) The core 1 produced in Step - 1 of Example 1 was adjusted to 500 g of water so that the solid content was 5.0 g. An organic solvent (0.1 g of Shellsol TK) and 9.71 g of GMA were added thereto in this order and stirred. Then, 2 g of AIBN (azobisisobutyronitrile) was added and slowly stirred at 75 °C for 24 hours to form a polymer part. Next, after cooling this reaction solution, it was filtered through a 500 - mesh wire net. A dispersion of comparative particle 1 having a core-shell structure was obtained by washing these particles with distilled water using centrifugation. The results of evaluating the particle physical properties of Comparative Particle 1 are shown in Table 1.

[0091] [Comparative Example 2] (Synthesis of Comparative Particle 2) St, GMA, DVB, V-50 and ion-exchanged water were used in the following formulation: St / GMA / DVB / V-50 / H20 = 1.2 / 1.8 + 0.3 / 0.04 / 0.06 / 110 (g). After nitrogen substitution, a polymerization reaction was carried out at 70 °C for 24 hours. The polymerization was carried out by soap-free emulsion polymerization described in JP 2000-351814. After 2 hours from the start of polymerization, 0.3 g of GMA was added to completely cover the surface of the obtained Comparative Mother Particle 2 with GMA. The obtained Comparative Mother Particle 2 was precipitated by centrifugation (15,000 rpm, 15 min, 4 °C). After decantation of the supernatant, it was redispersed in 200 ml of water. The above operation was repeated 3 times to wash Comparative Mother Particle 2, and finally it was dispersed in water. To introduce an amino group into 0.25 g of this washed Comparative Mother Particle 2, NH 4 OH (55.3 mmol; equivalent to 50 times the amount of GMA unit) was added, and the pH was adjusted to 11 with 1N HCl. The reaction was carried out at 70 °C for 24 hours with stirring by a stirrer to open the epoxy group of GMA. Next, an example of immobilization on Comparative Mother Particle 2 obtained above using ethylene glycol diglycidyl ether (EGDE; Wako Pure Chemical Industries) is shown. An excess of EGDE was charged so as to be 100 times the amount (mol) of the amino group of about 62.5 mg of Comparative Mother Particle 2, and the mixture was stirred at 30 °C for 24 hours at pH 11 (adjusted with 1N NaOH) to form a covalent bond between the epoxy group of EGDE and the amino group on Comparative Mother Particle 2. An excess amount of EGDE was added to prevent the epoxy groups at both ends of one molecule of EGDE from being simultaneously immobilized on the SG particles. As a result, EGDE particles in which EGDE was bound to Mother Particle 2 were obtained. After the reaction, it was washed 3 times with water by centrifugation. To introduce an amino group into 0.25 g of this washed EGDE particle, NH 4OH (55.3 mmol; equivalent to 50 times the amount of GMA units) was added and the pH was adjusted to 1 with 1N HCl. The reaction was carried out at 70 °C for 24 hours while stirring with a stirrer. Then, it was centrifugally purified using ion-exchanged water under the conditions of 4 °C, 27000G, and 20 minutes for 3 times, and then redispersed in methanol so that the solid content fraction became 1 mass%. Next, 0.88 g of succinic anhydride (Tokyo Chemical Industry Co., Ltd.) was added to the dispersion 2' weighed so that the particles were 0.20 g, and 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 EGDE terminal with succinic anhydride, thereby obtaining Comparative Particle 2. The results of evaluating the particle physical properties of Comparative Particle 2 are shown in Table 1.

[0092] [Comparative Example 3] (Synthesis of Comparative Particle 3) 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 form a mixed solution, and Comparative Core 3 was prepared by the same experimental operation as in Step-1 of Example 4 except for this. Then, instead of 1.43 g of GMA in Step-2 of Example 4, 1.31 g of GMA was used, and Comparative Particle 3 was obtained by the same experimental operations as in Step-2 and Step-3 of Example 4 except for this. The results of evaluating the particle physical properties of Comparative Particle 3 are shown in Table 1.

[0093] [Comparative Example 4] (Synthesis of Comparative Particle 4) 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 form 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. Except for this, a dispersion of Comparative Core 4 was obtained by the same experimental operation as in Example 1. Then, Comparative Particle 4 was obtained by the same experimental operations as in Step-2 and Step-3 of Example 1. The results of evaluating the particle physical properties of Comparative Particle 4 are shown in Table 1.

[0094] [Comparative Example 5] (Synthesis of Comparative Particle 5) In Step-2 of Example 1, 0.092 g of GMA was used instead of 1.52 g of GMA, and Comparative Particle 5 was obtained by the same experimental procedure as in Example 1 except for this change. Table 1 shows the results of evaluating the particle physical properties of Comparative Particle 5.

[0095] [Comparative Example 6] (Synthesis of Comparative Particle 6) In Step-2 of Example 4, 10.01 g of GMA was used instead of 1.43 g of GMA, and Comparative Particle 6 was obtained by the same experimental procedure as in Example 1 except for this change. Table 1 shows the results of evaluating the particle physical properties of Comparative Particle 6. In addition, for Comparative Examples 1 to 6, the amount of the core of Formula (1), the content of Formula (2) in the comparative particles, the content of Formula (2) in the shell, and the mass ratio of the core in the comparative particles are shown in Table 2.

[0096] [Evaluation 1] Preparation of test particles by sensitizing particles with an antibody and evaluation of the agglutination sensitivity of the test particles (Preparation of test particles by sensitizing particles with an antibody) For Particles 1 to 15 and Comparative Particles 1 to 6 prepared in Examples 1 to 15 and Comparative Examples 1 to 6, respectively, 180 μL of a 1.7 mass% water-suspension was taken into a 1.5 mL microtube, and 90 μL of a 5.0% aqueous solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 90 μL of a 5.0% aqueous solution of N-hydroxysulfosuccinimide sodium were added thereto, followed by stirring at room temperature for 30 minutes to activate the carboxyl groups, thereby obtaining a particle dispersion (activated particle dispersion). After centrifugal washing, 270 μL of a phosphate buffer-physiological saline (hereinafter referred to as PBS) at pH 7.2 was added, and the above particles were dispersed by ultrasonic waves. To this, 5 μL of a 15.0 mg / mL dispersion of monoclonal mouse anti-human C-reactive protein (anti-CRP antibody), Clone C5 (Funakoshi Co., Ltd.) was added, and the mixture was stirred at room temperature for 3 hours to sensitize the particles with the antibody, thereby obtaining test particles. After centrifugal washing of these test particles, 1 mL of PBS was added and they were stored in a dispersed state.

[0097] (Evaluation of Aggregation Sensitivity of Detection Particles) The standard serum for CRP was diluted with PBS to a concentration of 0.75 mg / dL to obtain a CRP sample solution. A mixed solution (hereinafter referred to as R1+) was prepared by mixing 1 μL of the CRP sample solution and 50 μL of a buffer solution (PBS containing 0.01% Tween 20), and incubated at 37°C. Next, 50 μL of a dispersion of each of the above detection particles that had been sufficiently dispersed again by ultrasonic waves before use (particle concentration 0.1% by mass, referred to as R2) was mixed with R1+. The absorbance at a wavelength of 572 nm was measured for the mixed solution (volume 101 μL) immediately after stirring. The absorbance measurement was performed using a spectrophotometer Biospectrometer manufactured by Eppendorf. Then, after allowing this mixed solution to stand at 37°C for 5 minutes, the absorbance at a wavelength of 572 nm was measured again, and the value (R+) of the change amount of absorbance ΔABS × 10000 was calculated. The results were summarized in Table 3. It is expected that the larger the value of R+ for the detection particles, the higher the sensitivity of detecting the target substance when used for sample testing as particles for the agglutination method.

[0098] Table 3 shows the evaluation results of the detection particles of Particles 1 to 15 and Comparative Particles 1 to 6. For all of Particles 1 to 15, the value of the change amount of absorbance ΔABS × 10000 was 10000 or more. On the other hand, for all 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 content of the structure represented by formula (1) in the core particles was 50% by mass and 63% by mass, respectively, which was higher than that of Particles 1 to 15. Since the proportion of substances with a lower refractive index increased, the refractive index of the core particles decreased. As a result, the difference in absorbance before and after aggregation was also small, resulting in low sensitivity. Also, as shown in Table 1, for Comparative Particle 3, the core particle size was 146 nm in volume average particle size, which was smaller than that of Particles 1 to 15, and the aggregate of particles after aggregation was also smaller, so the difference in absorbance before and after aggregation became smaller. In addition, the E / Dv values of Comparative Particles 1 to 3 were 8.0×10 -4It is less than that, and even if the particle size is a certain size or more, the absorbance becomes small, and the difference in absorbance before and after aggregation becomes small. Also, 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 7000, indicating low sensitivity. Also, for Comparative Particle 6, the value of ΔABS×10000 was as low as 2000. This is because the shell film thickness was 62 nm, which was thick, and the dispersibility of the particles was high, making it difficult for aggregation to occur.

[0099] [Evaluation 2] Evaluation of non-specific adsorption to particles (Milk fat globule evaluation) For each of Particles 1 to 15 and Comparative Particles 1 to 6, a dispersion was prepared by dispersing them in a phosphate buffer so as to be 0.1% by mass. Next, 60 μL of a milk fat globule liquid composed of triolein, lecithin, free fatty acid, bovine albumin, and Tris buffer was added to 30 μL of each dispersion, and the absorbance at a wavelength of 572 nm was measured for the dispersion immediately after stirring. The absorbance measurement was performed using a spectrophotometer Biospectrometer manufactured by Eppendorf. Then, 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 amount of absorbance ΔABS×10000 was calculated. The evaluation was made as follows according to the value of ΔABS×10000. A: ΔABS×10000 is 30 or less B: ΔABS×10000 is greater than 30 and 50 or less C: ΔABS×10000 is greater than 50 and 100 or less D: ΔABS×10000 is greater than 100 and 500 or less E: ΔABS×10000 is greater than 500

[0100] The evaluation results of Particles 1 to 15 and Comparative Particles 1 to 6 are shown in Table 3. The evaluation results of Particles 1 to 15 were all B or above. Since the change in absorbance of the dispersion can be considered to be due to particle aggregation caused by non-specific adsorption occurring on 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, indicating that non-specific adsorption could not be sufficiently suppressed. This is presumably because the film thickness of the shell of Comparative Particle 5 was 3 nm, which was thinner than that of Particles 1 to 15, and a part of the hydrophobic core particles was exposed on the particle surface in Comparative Particle 5.

[0101] (Human Specimen Evaluation) Dispersion liquids (P liquids) were prepared by dispersing Particles 1 to 15 and Comparative Particles 1 to 6 in a phosphate buffer solution (containing 0.01% Tween 20) to a concentration of 0.1% by mass, respectively. Next, 55 μL of a specimen dilution liquid (Q liquid) composed of a human normal specimen (serum specimen, 5 μL) and a phosphate buffer solution (50 μL) was added to 50 μL of each dispersion liquid, and the absorbance at a wavelength of 572 nm was measured for the mixed liquid immediately after stirring. The absorbance measurement was performed using a spectrophotometer Biospectrometer manufactured by Eppendorf. Then, after leaving these dispersion liquids to stand at 37 °C for 5 minutes, the absorbance at a wavelength of 572 nm was measured again, and the change amount of absorbance ΔABS×10000 was calculated. The evaluation was based on the value of ΔABS×10000 as follows. A: ΔABS×10000 is 30 or less B: ΔABS×10000 is greater than 30 and 80 or less C: ΔABS×10000 is greater than 80 and 100 or less D: ΔABS×10000 is greater than 100 and 500 or less E: ΔABS×10000 is greater than 500 The results for each particle are shown in Table 3.

[0102] The evaluation results of Particles 1 to 15 and Comparative Particles 1 to 6 are shown in Table 3. For Particles 1 to 15, the evaluation results were all from A to C, and the value of the change amount ΔABS×10000 was less than 100. Since the change in the absorbance of the dispersion is considered to be due to inter-particle aggregation resulting from non-specific adsorption occurring on 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, for Comparative Particle 5, the evaluation result was E, and the value of the change amount ΔABS×10000 was 1000, indicating that non-specific adsorption could not be sufficiently suppressed. This is presumably because the shell film thickness of Comparative Particle 5 is 3 nm, which is thinner than that of Particles 1 to 15, and in Comparative Particle 5, a part of the hydrophobic core particles is exposed on the particle surface.

[0103] As described above, for Particles 1 to 15 of the present disclosure, the aggregation sensitivity is higher than that in Evaluation 1, and non-specific adsorption is suppressed more than in Evaluation 2, enabling both improvement in sensitivity and suppression of non-specific adsorption. On the other hand, in Comparative Examples 1 to 4, non-specific adsorption was suppressed, but the aggregation sensitivity was low. In Comparative Example 6, the sensitivity was low. Also, in Comparative Example 5, the aggregation sensitivity was high, but non-specific adsorption could not be suppressed. That is, in Comparative Examples 1 to 6, it was not possible to achieve both improvement in aggregation sensitivity and suppression of non-specific adsorption.

[0104] [Table 3]

[0105] The disclosure of the present embodiment includes the following configurations and methods. (Configuration 1) Particles for use in an aggregation method, having 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, contains a polymer having a structural unit represented by formula (1), and the content ratio of the structural unit represented by formula (1) with respect to the particle is 40% by mass or more and 85% by mass or less, and the shell has a film thickness of 5 nm or more and 50 nm or less and contains a polymer having a structural unit represented by formula (2). [Chemical formula] (R 1 represents a hydrogen atom or a methyl group. R 2 represents a substituted or unsubstituted phenyl group or a naphthyl group. However, in the case of substitution, the substituent is a methyl group or an ethyl group. R 1 and R 2 may be different for each structural unit.) [Chemical formula] (R 3 represents a hydrogen atom or a methyl group, and R 4 represents a group having an epoxy group, a group having a hydroxy group, or a group having a carboxy group. R 3 and R 4 may be different for each structural unit.) (Constitution 2) The particles according to Constitution 1, wherein the content of the structural unit represented by the formula (2) with respect to the particles is 9% by mass or more and 60% by mass or less. (Constitution 3) The particles according to Constitution 2, wherein the content of the structural unit represented by the formula (2) with respect to the particles is 9% by mass or more and 39% by mass or less. (Constitution 4) The particles according to Constitution 3, wherein the content of the structural unit represented by the formula (2) with respect to the particles is 20% by mass or more and 39% by mass or less. (Constitution 5) The particles according to any one of Constitutions 1 to 4, wherein the content ratio of the structural unit represented by the formula (1) with respect to the core particles is 90% by mass or more. (Constitution 6) The particles according to any one of Constitutions 1 to 5, wherein the composition ratio of the C element to the O element quantified when measured by X-ray photoelectron spectroscopy (XPS) of the particles is 2.0 or more and 3.3 or less. (Constitution 7) The particles according to any one of Constitutions 1 to 6, wherein the content ratio of the structural unit represented by the formula (2) in the shell is 90% by mass or more and 100% by mass or less. (Configuration 8) In the core particles, the polymer having the structural unit represented by the formula (1) contains the structural unit represented by the formula (3). The particles according to any one of Configurations 1 to 7, wherein: [Chemical formula] (Z represents a substituted or unsubstituted phenylene group or a naphthalene group. However, in the case of substitution, the substituent is a methyl group or an ethyl group. Z may be different for each structural unit.) (Configuration 9) The content ratio of the core particles with respect to the particles is 50% by mass or more and 95% by mass or less. The particles according to any one of Configurations 1 to 8, wherein: (Configuration 10) In the FT-IR spectrum of the particles, the peak height existing at 1500 to 1650 cm -1 derived from the C=C bond of the aromatic ring is A, and the peak height existing at 1680 to 1750 cm -1 derived from the carbonyl group of the ester bond is B. The value of A / B is 0.85 or more and 5.35 or less. The particles according to any one of Configurations 1 to 9, wherein: (Configuration 11) The ratio (Dv / Dn) of the volume average particle diameter (Dv) and the number average particle diameter (Dn) of the particles is 1.25 or less. The particles according to any one of Configurations 1 to 10, wherein: (Configuration 12) When the particles are dispersed in water at a solid content concentration of 0.1% by mass such that the ratio (Dv / Dn) of the volume average particle diameter (Dv) and the number average particle diameter (Dn) becomes 1.25 or less, when the absorbance at a cell length of 10 mm for incident light with a wavelength of 572 nm is E, the value of E / Dv is 8.0×10 -4 or more and 2.0×10 -3 or less. The particles according to any one of Configurations 1 to 11, wherein: (Configuration 13) The formula (1) is represented by the formula (1-A). The particles according to any one of Configurations 1 to 12, wherein: [Chemical formula] (R 10 represents a phenyl group, a tolyl group, or a naphthyl group.) (Constitution 14) The particle according to any one of Constitutions 1 to 13, wherein the formula (2) is represented by the formula (2-A). [Chemical formula] (R 31 and R 32 One of them is a hydroxy group, and the other represents a hydroxy group, a group represented by the formula (2-B), or a group represented by the formula (2-C).) [Chemical formula] (R 20 represents a single bond or a methylene group. R 22 , R 23 , R 24 represent a hydrogen atom, a methyl group, a hydroxy group, or a hydroxymethyl group, and one or more of R 22 , R 23 , and R 24 represent a hydroxy group. Y 1 represents a sulfur atom or an imino group. * 1 indicates the bonding position.) [Chemical formula] (R 25 represents a hydrogen atom, a methyl group, a hydroxy group, or a carboxy group. Y 2 represents a sulfur atom or an imino group. Y 3 represents a single bond or a methylene group. * 2 indicates the bonding position.) (Constitution 15) A first step of obtaining core particles having a volume average particle diameter of 200 nm or more and 500 nm or less by polymerizing a first monomer composition containing a monomer represented by the formula (X1); A second step of obtaining particles having a layer of 5 nm or more and 50 nm or less formed on the outer side of the core particles by polymerizing using a reaction solution containing the core particles, a second monomer composition containing a monomer represented by the formula (X2), and a water-soluble polymerization initiator However, the content ratio of the monomer represented by the formula (X1) to the total amount of the monomers contained in the first monomer composition is 90% by mass or more, However, the content ratio of the unpolymerized monomer represented by the formula (X1) to the reaction solution is 1000 ppm or less, the content ratio of the monomer represented by the formula (X2) to the total amount of the monomers contained in the first monomer composition and the monomers contained in the second monomer composition is 9% by mass or more and less than 40% by mass; and a third step of reacting the particles having the layer formed thereon with a compound represented by the formula (X3), Particles for use in the aggregation method, produced thereby.

Chemical formula

Chemical formula

Chemical formula

Chem.

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Claims

1. A particle for use in an aggregation method, comprising a core particle and a shell on the surface of the core particle, The core particles have a volume average particle size of 200 nm or more and 500 nm or less, The particle contains a polymer having a structural unit represented by formula (1), and the content ratio of the structural unit represented by formula (1) relative to the particle is 40% by mass or more and 85% by mass or less, 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). 【Chemistry 1】 (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. R １ and R ２ may be different for each structural unit.) 【Chemistry 2】 (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. R ３ and R ４ may be different for each structural unit.)

2. 2. The particle according to claim 1, wherein the content of the structural unit represented by the formula (2) relative to the particle is 9% by mass or more and 60% by mass or less.

3. 3. The particle according to claim 2, wherein the content of the structural unit represented by the formula (2) relative to the particle is 9% by mass or more and 39% by mass or less.

4. 4. The particle according to claim 3, wherein the content of the structural unit represented by the formula (2) relative to the particle is 20% by mass or more and 39% by mass or less.

5. 2. The particle according to claim 1, wherein the content of the structural unit represented by formula (1) relative to the core particle is 90 mass % or more.

6. 2. The particle according to claim 1, characterized in that the composition ratio of C element to O element quantified when the particle is measured by X-ray photoelectron spectroscopy (XPS) is 2.0 or more and 3.3 or less.

7. 2. The particle according to claim 1, wherein the content of the structural unit represented by the formula (2) in the shell is 90% by mass or more and 100% by mass or less.

8. 2. The particle according to claim 1, wherein in the core particle, the polymer having the structural unit represented by formula (1) contains a structural unit represented by formula (3). 【Chemistry 3】 (Z represents a substituted or unsubstituted phenylene group or naphthalene group, provided that, in the case of substitution, The substituent is a methyl group or an ethyl group. Z may be different for each structural unit.

9. 2. The particle according to claim 1, wherein the content of the core particle in the particle is 50% by mass or more and 95% by mass or less.

10. In the FT-IR spectrum of the particles, a peak at 1500 to 1650 cm originating from a C═C bond in an aromatic ring is observed. －１ The peak height present in the carbonyl group of the ester bond is A, and the peak height present in the carbonyl group of the ester bond is 1680 to 1750 cm －１ 2. The particle according to claim 1, wherein the value of A / B is 0.85 or more and 5.35 or less, where B is the height of the peak present at the peak.

11. 2. The particle according to claim 1, wherein the ratio (Dv / Dn) of the volume average particle diameter (Dv) to the number average particle diameter (Dn) of the particle is 1.25 or less.

12. When the particles are dispersed in water at a solid content concentration of 0.1% by 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, the value of E / Dv is 8.0×10 －４ That's it, 2.0 x 10 －３ 2. The particle according to claim 1, characterized in that:

13. The particle according to claim 1, characterized in that the formula (1) is represented by formula (1-A). 【Chemistry 4】 (R １０ represents a phenyl group, a tolyl group, or a naphthyl group.

14. The particle according to claim 1, characterized in that the formula (2) is represented by formula (2-A). 【Chemistry 5】 (R ３１ and R ３２ one of the groups is a hydroxy group, and the other is a hydroxy group, a group represented by formula (2-B), or a group represented by formula (2-C). 【Chemistry 6】 (R ２０ represents a single bond or a methylene group. R ２２ , R ２３ , R ２４ represents a hydrogen atom, a methyl group, a hydroxy group, or a hydroxymethyl group; R ２２ , R ２３ , and R ２４ One or more of represents a hydroxy group. Y １ represents a sulfur atom or an imino group. ＊ 1 indicates the bond position.) 【Chemistry 7】 (R ２５ represents a hydrogen atom, a methyl group, a hydroxy group, or a carboxy group. Y ２ represents a sulfur atom or an imino group. Y ３ represents a single bond or a methylene group. ＊ 2 indicates the bond position.)

15. 15. The particle according to claim 14, wherein the content of the structural unit represented by the formula (2) relative to the particle is 9% by mass or more and 60% by mass or less.

16. a first step of polymerizing a first monomer composition containing a monomer represented by formula (X1) to obtain core particles having a volume average particle size of 200 nm or more and 500 nm or less; A second step of obtaining particles having a layer of 5 nm or more and 50 nm or less formed on the outside of the core particle by polymerization using a reaction liquid containing the core particle, a second monomer composition containing a monomer represented by formula (X2), and a water-soluble polymerization initiator. provided that the content ratio of the monomer represented by formula (X1) relative to the total amount of monomers contained in the first monomer composition is 90 mass% or more, however, The content of the unpolymerized monomer represented by the formula (X1) in the reaction liquid is 1000 ppm or less, The content ratio of the monomer represented by the formula (X2) with respect to the total amount of the monomers contained in the first monomer composition and the monomers contained in the second monomer composition is 9% by mass or more and less than 40% by mass; and a third step of reacting the layered particles with a compound represented by formula (X3); Particles for use in the agglomeration process, produced by 【Chemistry 8】 (R １１ R represents a hydrogen atom or a methyl group. １２ represents a substituted or unsubstituted phenyl group or naphthyl group, and in the case of a substitution, the substituent is a methyl group or an ethyl group. 【Chemistry 9】 (R １３ represents hydrogen or a methyl group, R １４ represents an ethylene group or a carbonyl group.) 【Chemistry 10】 (R １５ R represents an amino group or a thiol group. １６ , R １７ R represents a hydrogen atom, a methyl group, a group having a hydroxyl group, or a group having a carboxyl group. １６ , R １７ At least one of represents a group having a hydroxy group or a group having a carboxy group.

17. 2. A test particle comprising the particle according to claim 1, having a ligand attached to its surface.

18. A test particle comprising the particle according to claim 11 having a ligand attached to its surface.

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

20. 20. A test kit comprising the reagent according to claim 19 and a container containing the reagent.

21. 20. A method for detecting a target substance in a specimen by in vitro diagnosis, comprising mixing the reagent according to claim 19 with a specimen which may contain the target substance.

22. 20. A method for detecting a target substance in a specimen by an agglutination method, comprising the steps of: mixing a specimen which may contain the target substance with the reagent according to claim 18 to obtain a mixed solution; irradiating the mixed solution with light; and detecting at least one of transmitted light and scattered light from the light irradiated to the mixed solution.

23. a first step of polymerizing a first monomer composition containing a monomer represented by formula (X1) to obtain core particles having a volume average particle size of 200 nm or more and 500 nm or less; A second step of obtaining particles having a layer of 5 nm or more and 50 nm or less formed on the outside of the core particle by polymerization using a reaction liquid containing the core particle, a second monomer composition containing a monomer represented by formula (X2), and a water-soluble polymerization initiator. provided that the content ratio of the monomer represented by formula (X1) relative to the total amount of monomers contained in the first monomer composition is 90 mass% or more, however, The content of the unpolymerized monomer represented by the formula (X1) in the reaction liquid is 1000 ppm or less, The content ratio of the monomer represented by the formula (X2) with respect to the total amount of the monomers contained in the first monomer composition and the monomers contained in the second monomer composition is 9% by mass or more and less than 40% by mass; and a third step of reacting the layered particles with a compound represented by formula (X3); A method for producing particles for use in an aggregation method, comprising: 【Chemistry 11】 (R １１ R represents a hydrogen atom or a methyl group. １２ represents a substituted or unsubstituted phenyl group or naphthyl group, and in the case of a substitution, the substituent is a methyl group or an ethyl group. 【Chemistry 12】 (R １３ represents hydrogen or a methyl group, R １４ represents an ethylene group or a carbonyl group.) 【Chemistry 13】 (R １５ R represents an amino group or a thiol group. １６ , R １７ R represents a hydrogen atom, a methyl group, a group having a hydroxyl group, or a group having a carboxyl group. １６ , R １７ At least one of represents a group having a hydroxy group or a group having a carboxy group.

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