JP7393896B6 - Particles and their manufacturing method - Google Patents

Description

TECHNICAL FIELD The present invention relates to analytical particles for an agglutination method and a method for producing the same.

Among the agglutination methods, the latex immunoagglutination method is used as a method for measuring substances to be measured in biological samples (hereinafter referred to as "specimen") in the field of clinical testing. In this method, a dispersion of particles (hereinafter referred to as "ligand-sensitized particles," "antibody-sensitized particles," or "antigen-sensitized particles") formed by chemically immobilizing antibodies or antigens as a ligand and a target to be measured are used. Mix with a sample that may contain substances (antigens or antibodies). At that time, if the substance to be measured (antibody or antigen) is contained in the sample, the sensitized particles will cause an agglutination reaction, and this agglutination reaction will be expressed as the amount of change in scattered light intensity, transmitted light intensity, absorbance, etc. By optical detection, the presence or absence of the substance to be measured can be identified or quantified.

Improving detection sensitivity and suppressing nonspecific reactions are challenges for particles used in latex immunoagglutination methods. To improve detection sensitivity, it has been proposed to increase the amount of ligand sensitization on particles (Ligand sensitization means immobilization of a ligand on particles. Therefore, ligand sensitized particles have immobilized ligands on particles. (meaning particles). For example, in Patent Document 1, the amount of antibody sensitization is increased by increasing the specific surface area of particles. As the amount of antibody sensitization increases, the amount of antigen that can be detected increases. To suppress non-specific reactions, Patent Document 2 discloses a method of post-coating antibody-sensitized particles with albumin or a hydrophilic polymer in order to avoid adsorption of contaminants in the sample to the particles. The particles with increased antibody sensitizing amount in Patent Document 1 are also post-coated with albumin to suppress non-specific reactions.

Incidentally, in recent years, research has been widely conducted on purifying and quantifying target substances using affinity particles, which are formed by chemically immobilizing particles with a ligand that has an affinity for the target substance. When chemically immobilizing a ligand on the particle surface, a common method is to introduce another reactive functional group such as a carboxy group, amino group, or thiol group, and then chemically react the reactive functional group with the ligand. be. Among these, particles converted to carboxy groups are the most versatile and preferred form for chemically immobilizing a ligand on the particle surface.

JP2006-266970A International Publication No. 2017/138608

However, with the particles described in Patent Documents 1 and 2, it was difficult to achieve both improved detection sensitivity and suppression of nonspecific reactions. In other words, although sensitivity can be improved by increasing the amount of ligand sensitization to the particles, post-coating is necessary to suppress nonspecific reactions. In order to secure an area for post-coating, the amount of ligand sensitization to the particles is limited. In addition, while post-coating is effective in making the particle surface hydrophilic, post-coating is a temporary surface modification based on physical adsorption, and therefore particles that suppress nonspecific reactions and do not require post-coating are desired.

The problem to be solved by the present invention is to provide particles that can be used in an agglutination method and can both improve detection sensitivity and suppress non-specific reactions. Specifically, it is an object of the present invention to provide particles that have low nonspecific adsorption, have a carboxy group for immobilizing a ligand, and have high sensitization efficiency for a ligand, and a method for producing the particles. Another object of the present invention is to provide sensitized particles for agglutination methods in which an antigen or antibody is chemically fixed as a ligand, and a particle dispersion in which the above-mentioned particles or sensitized particles are dispersed in an aqueous solution. . A further object of the present invention is to provide a method for detecting a target substance in a specimen by an agglutination method using the above particles, the above sensitized particles, or the above particle dispersion.

The particles according to the present invention have a core comprising a polymer having a unit derived from a vinyl monomer, or a copolymer having a unit derived from a vinyl monomer and a glycidyl group-containing monomer, The surface of the core is characterized in that it is coated with a polymer of a silane coupling agent having one or more carboxy groups.

Furthermore, the ligand-sensitized particles according to the present invention are characterized in that a ligand is immobilized on the carboxy groups of the particles.

Further, the particle dispersion according to the present invention is characterized in that the above-mentioned particles or the above-mentioned ligand-sensitized particles are dispersed in an aqueous solution.

Furthermore, the method for detecting a target substance in a specimen according to the present invention is characterized by using an agglutination method using the above-mentioned particles, the above-mentioned sensitized particles, or the above-mentioned particle dispersion.

（式（１）においてＸはそれぞれ独立に、Ｏを介して隣り合う式（１）で示される構造単位が有するＳｉとの結合手、前記コアの構造に由来するエポキシ基、アミノ基および水酸基から選択される１つ以上の官能基との結合手、または、ＯＨを表し、式（１）で示される構造単位の少なくとも一つは、前記コアとの結合を有する。
Ｒはそれぞれ独立に、末端に少なくとも１つのカルボキシ基を有する、－ＮＨ－ＣＯ－、－ＮＨ－、－Ｏ－、－Ｓ－、－ＣＯ－、－Ｏ－ＣＯ－を含んでも良い炭素数１以上３０以下の直鎖または分岐アルキル基を表す。） Further, the method for producing particles according to the present invention includes mixing a vinyl monomer, water, and a radical polymerization initiator, or a vinyl monomer, a glycidyl group-containing monomer, water, and a radical polymerization initiator to form a granular polymer. or a step 1 of forming a copolymer to obtain an aqueous dispersion of the granular polymer or copolymer, a surfactant in the aqueous dispersion, and the following formula (1) that the produced particles have: It is characterized by including a step 2 of mixing a silane coupling agent from which the structure represented by is derived and coating the copolymer.

(In formula (1), each X independently represents a bond with Si of a structural unit represented by formula (1) adjacent to each other via O, an epoxy group, an amino group, and a hydroxyl group derived from the structure of the core. At least one of the structural units represented by formula (1) that represents a bond with one or more selected functional groups or OH has a bond with the core.
Each R independently has at least one carboxy group at the end, and has a carbon number of 1 and may include -NH-CO-, -NH-, -O-, -S-, -CO-, -O-CO- Represents a straight chain or branched alkyl group of 30 or more. )

According to the present invention, it is possible to provide particles that have low non-specific adsorption, have carboxyl groups for immobilizing ligands, and have high ligand sensitization efficiency, and a method for producing the same. It is also possible to provide ligand-sensitized particles, particle dispersions, and methods for detecting target substances in samples that use the above particles.

Embodiments of the present invention will be described in detail below, but the technical scope of the present invention is not limited to these embodiments.

The particles according to the present invention will now be described.
The particles according to the present invention are characterized in that the core of the particles is mainly composed of a polymer having a unit derived from a vinyl monomer, or a copolymer having a unit derived from a vinyl monomer and a unit derived from a glycidyl group-containing monomer, and the surface of the core is coated with a polymer of a silane coupling agent having one or more carboxy groups.

The particles according to the present invention preferably have, between the core and the polymer of the silane coupling agent, bonds between one or more functional groups selected from an epoxy group, an amino group, and a hydroxyl group derived from the structure of the core and a functional group possessed by the silane coupling agent.

The bond between one or more functional groups selected from epoxy groups, amino groups, and hydroxyl groups derived from the structure of the core and the functional group possessed by the silane coupling agent may be a non-covalent bond or a covalent bond. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, electrostatic interactions, van der Waals interactions, and hydrophobic interactions. Examples of the covalent bond include a bond formed by a reaction between a hydroxyl group and a silanol group, an amide bond, a bond formed by a reaction between an epoxy group and a carboxy group, and the like.

In this specification, the term "coating" means that it is sufficient to partially cover the core in order to suppress non-specific adsorption of particles, and it is not necessary to completely cover the core. Furthermore, being coated with a polymer may mean any bonding state. That is, it is not necessarily necessary to cover the entire particle, it may be partially covered, and the coated polymer layer does not need to be uniform.

The particles according to the present invention are particles for agglutination, and can be used, for example, as particles for latex immunoagglutination. The particles can immobilize ligands. Since the obtained ligand-sensitized particles bind to the target substance, it becomes possible to measure the target substance by the latex immunoagglutination method.

The particles according to the present invention have carboxy groups on their surfaces that can chemically immobilize antibodies or antigens as ligands.

The core of the particle according to the present invention contains a polymer having a unit derived from a vinyl monomer, or a copolymer having a unit derived from a vinyl monomer and a glycidyl group-containing monomer. In addition, in this specification, a "unit" means the unit structure corresponding to one monomer.

Units derived from vinyl monomers are not limited in their chemical structure as long as the object of the present invention can be achieved, but preferably units derived from styrene monomers, units derived from diene monomers, (meth)acrylic Units derived from monomers can be mentioned. Among these, it is preferable that the unit derived from a vinyl monomer be a unit derived from a styrene monomer. As used herein, "(meth)acrylic" means acrylic and/or methacryl.

The chemical structure of the unit derived from a styrene monomer is not limited as long as the object of the present invention can be achieved, but it is preferably at least one member selected from the group derived from styrenes. Styrenes include styrene, α-methylstyrene, β-methylstyrene, o-methylstyrene, m-methylstyrene, p-methylstyrene, 2,4-dimethylstyrene, pn-butylstyrene, p-tert- Butylstyrene, pn-hexylstyrene, pn-octylstyrene, pn-nonylstyrene, pn-decylstyrene, pn-dodecylstyrene, p-methoxystyrene, p-phenylstyrene, etc. can be mentioned.

The chemical structure of the unit derived from a diene monomer is not limited as long as the object of the present invention can be achieved, but it is preferably at least one type selected from the group derived from dienes. Examples of dienes include isoprene, butadiene, chloroprene, and the like.

The unit derived from a (meth)acrylic monomer is not limited in its chemical structure as long as it can achieve the purpose of the present invention, but is preferably at least one selected from the group derived from (meth)acrylics. It is preferable. Examples of the (meth)acrylics include (meth)acrylic acid alkyl ester monomers and (meth)acrylic acid alkoxyalkyl ester monomers. (Meth)acrylic acid alkyl ester monomers are not particularly limited, but specifically include methyl (meth)acrylate, ethyl (meth)acrylate, n-propyl (meth)acrylate, and isopropyl (meth)acrylate. , n-butyl (meth)acrylate, isobutyl (meth)acrylate, n-hexyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, and cyclohexyl (meth)acrylate. The (meth)acrylic acid alkoxyalkyl ester monomer is not particularly limited, but specifically, methoxymethyl (meth)acrylate, ethoxymethyl (meth)acrylate, 2-methoxyethyl (meth)acrylate, 2-ethoxyethyl (meth)acrylate, 2-propoxyethyl (meth)acrylate, 2-butoxyethyl (meth)acrylate, 3-methoxypropyl (meth)acrylate, and 4-methoxybutyl (meth)acrylate Examples include.

The chemical structure of the unit derived from a glycidyl group-containing monomer is not limited as long as the object of the present invention can be achieved, but it is preferably at least one selected from glycidyl methacrylate and glycidyl acrylate.

Regarding a copolymer having a unit derived from a vinyl monomer and a unit derived from a glycidyl group-containing monomer, the composition ratio of the units is not limited as long as the object of the present invention can be achieved. Preferably, the "unit derived from a styrene monomer"/"unit derived from a glycidyl group-containing monomer" (mol fraction) is 0.1 or more and 10 or less, more preferably 0.2 or more and 5 or less, and even more preferably, It is 0.5 or more and 2 or less. The above preferred range is based on the strength of the particles derived from the "styrenic monomer", the ability to suppress non-specific adsorption of the particles derived from the "glycidyl group-containing monomer", and the reaction efficiency between the carboxy group of the particles and the ligand. This is a value determined by the relationship. When the above relationship is satisfied, there is a good balance between the strength of the particles, the ability to suppress nonspecific adsorption, and the carboxy groups possessed by the particles and the efficiency of the ligand reaction.

（式（１）においてＸはそれぞれ独立に、Ｏを介して隣り合う式（１）で示される構造単位が有するＳｉとの結合手、前記コアの構造に由来するエポキシ基、アミノ基および水酸基から選択される１つ以上の官能基との結合手、または、ＯＨを表し、式（１）で示される構造単位の少なくとも一つは、前記コアとの結合を有する。
Ｒはそれぞれ独立に、末端に少なくとも１つのカルボキシ基を有する、－ＮＨ－ＣＯ－、－ＮＨ－、－Ｏ－、－Ｓ－、－ＣＯ－、－Ｏ－ＣＯ－を含んでも良い炭素数１以上３０以下の直鎖または分岐アルキル基を表す。） (Silane coupling agent)
The polymer of the silane coupling agent having a carboxyl group preferably has a structural unit represented by formula (1).

(In formula (1), each X independently represents a bond with Si of a structural unit represented by formula (1) adjacent to each other via O, an epoxy group, an amino group, and a hydroxyl group derived from the structure of the core. At least one of the structural units represented by formula (1) that represents a bond with one or more selected functional groups or OH has a bond with the core.
Each R independently has at least one carboxy group at the end, and has a carbon number of 1 and may include -NH-CO-, -NH-, -O-, -S-, -CO-, -O-CO- Represents a straight chain or branched alkyl group of 30 or more. )

An example of the silane coupling agent having a polymer structure represented by the above formula (1) is X-12-1135 (manufactured by Shin-Etsu Chemical Co., Ltd.).

The particles according to the invention may be crosslinked. Particles can be crosslinked by using monomers such as divinylbenzene during particle synthesis. Crosslinking of the particles improves the physical strength of the particles, which is advantageous for particle handling (such as centrifugation during manufacturing and ligand immobilization). Using divinylbenzene also improves the solvent resistance of the particles.

The number-average particle size of the particles according to the present invention in water is preferably 0.05 μm to 1 μm, more preferably 0.05 μm to 0.5 μm. When the number-average particle size is 0.05 μm to 0.5 μm, the particles have excellent handleability in centrifugation and a large specific surface area, which is a characteristic of the particles. The number-average particle size of the particles is evaluated by dynamic light scattering.

(ligand)
The present invention also relates to ligand-sensitized particles for use in aggregation methods, in which a ligand is immobilized on the particles according to the present invention. Preferably, the ligand is fixed to a carboxy group of the particles according to the present invention.

A ligand is a compound that specifically binds to a receptor possessed by a specific target substance. The site where the ligand binds to the target substance is fixed and has high affinity selectively or specifically. Examples include antigens and antibodies, enzyme proteins and their substrates, signal substances such as hormones and neurotransmitters and their receptors, and nucleic acids, but the ligands are not limited to these. Examples of ligands include antigens, antibodies, antigen-binding fragments (e.g., Fab, F(ab')2, F(ab'), Fv, scFv, etc.), naturally occurring nucleic acids, artificial nucleic acids, aptamers, peptide aptamers, and oligonucleotides. Examples include peptides, enzymes, and coenzymes. The ligand-sensitized particles for aggregation methods in the present invention refer to ligand-sensitized particles for agglutination methods that have selectively or specifically high affinity for a target substance.

In the present invention, conventionally known methods can be applied to the chemical reaction method for chemically immobilizing the carboxy group and the ligand that the particles of the present invention have, within the scope of achieving the object of the present invention. For example, carbodiimide-mediated reactions and NHS ester activation reactions are commonly used chemical reactions. However, the chemical reaction method for chemically fixing the carboxy group and the ligand in the present invention is not limited to these.

In the ligand-sensitized particles according to the present invention, a hydrophilic molecule may be bonded to the remaining carboxy groups (activated esterified) to which no ligand is immobilized. This can also be referred to as active ester inactivation, carboxy group blocking treatment, or masking treatment, and is performed to suppress nonspecific adsorption of proteins to carboxy groups and improve dispersion stability of ligand-sensitized particles. In the ligand-sensitized particles according to the present invention, the hydrophilic molecule bonded to the carboxy group is preferably polyethylene glycol (PEG) or trishydroxymethylaminomethane (Tris). Polyethylene glycol is particularly preferred because it can greatly suppress protein adsorption. As described later in Examples, when inactivating an active ester using polyethylene glycol, the molecular weight of the polyethylene glycol is important, and if the molecular weight is large, the antigen-antibody reaction may be inhibited. The optimum molecular weight of polyethylene glycol is 350 or more and 5000 or less, particularly preferably 1000 or more and 2000 or less. As the polyethylene glycol, polyethylene glycol having a carboxy group or a functional group reactive with active esters, for example, polyethylene glycol having an amino group is preferable, and polyethylene glycol having a primary amine is particularly preferable. Polyethylene glycol may be a linear polymer or a branched polymer. Examples of trishydroxymethylaminomethane (Tris) are shown in the following formula (3), and examples of polyethylene glycol are shown in the following formulas (4) and (5). n in formulas (4) and (5) is an integer of 1 or more indicating the number of oxyethylene units.

The amount of immobilized ligand is also an important factor; if the amount of immobilized ligand is small, the reactivity of the antigen-antibody decreases, which is not preferable. On the other hand, when the amount of immobilized ligand is large, it also causes deterioration in the dispersibility of the ligand-sensitized particles. Although it depends on the particle size, if the average particle size is about 200 nm, the amount of ligand immobilized is preferably 1 μg or more and 500 μg or less per 1 mg of particles. In particular, 10 μg or more and 200 μg or less is preferable.

The ligand-sensitized particles for agglutination according to the present invention can be preferably applied to latex immunoagglutination assays, which use antibodies or antigens as ligands and are widely used in clinical testing, biochemical research, and other fields. When general particles are applied to latex immunoagglutination assays, the target substances, such as antigens (antibodies) and foreign substances in serum, are non-specifically adsorbed to the particle surface, which causes unintended particle agglutination to be detected, hindering accurate measurement. Therefore, in order to reduce noise caused by non-specific adsorption, particles are usually coated with a biological substance such as albumin as a blocking agent to suppress non-specific adsorption to the particle surface. However, since the characteristics of such biological substances vary slightly depending on the lot, particles coated with these substances have different abilities to suppress non-specific adsorption for each coating process. Therefore, there is a problem in stably supplying particles with the same level of ability to suppress non-specific adsorption. In addition, biological substances coated on the particle surface may exhibit hydrophobicity due to denaturation, and are not necessarily excellent in the ability to suppress non-specific adsorption. Biological contamination is also an issue. Patent Document 2 discloses ligand-sensitized particles for latex immunoagglutination that are post-coated to suppress non-specific reactions. However, the post-coating agent is water-soluble and coats the particle surface by physical adsorption, so there is a concern that it may be released by dilution. The ligand-sensitized particles of the present invention are highly hydrophilized particles that have an enhanced ability to suppress the above-mentioned non-specific adsorption. The above problem can be solved without the need for post-coating with albumin or the like.

(particle dispersion)
The particle dispersion liquid for use in the aggregation method according to the present invention is characterized in that the particles according to the present invention or the ligand-sensitized particles according to the present invention are dispersed in an aqueous solution. The particle dispersion according to the present invention preferably contains a surfactant.

(reagent)
The reagent for use in the agglutination method is characterized by containing the ligand-sensitized particles for the agglutination method according to the present invention. The amount of the ligand-sensitized particles for the aggregation method according to the present invention contained in the reagent is preferably 0.001% by mass or more and 20% by mass or less, more preferably 0.01% by mass or more and 10% by mass or less. The reagent may contain a third substance such as a solvent or a blocking agent in addition to the ligand-sensitized particles for the aggregation method according to the present invention, within a range that can achieve the purpose of the present invention. A combination of two or more types of third substances such as a solvent and a blocking agent may be included. Examples of the solvent used include various buffers such as phosphate buffer, glycine buffer, Good's buffer, Tris buffer, and ammonia buffer, but the solvent contained in the reagent is not limited to these.

A kit for use in detecting a target substance in a specimen by the agglutination method is characterized by comprising at least a reagent. The kit preferably further comprises a reaction buffer solution containing albumin (hereinafter, reagent 2) in addition to a reagent (hereinafter, reagent 1). The albumin may be serum albumin or the like, and may be protease-treated. The amount of albumin contained in reagent 2 is approximately 0.001% by mass to 5% by mass, but the kit is not limited to this. A sensitizer for immune agglutination measurement may be contained in both or either of reagent 1 and reagent 2. Examples of sensitizers for immune agglutination measurement include polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, polyalginic acid, etc., but the kit is not limited to these. A surfactant may be contained in both or either of reagent 1 and reagent 2. Since surfactants have the effect of stabilizing particles and proteins, for example, polyoxyethylene sorbitan monolaurate and poly(oxyethylene) octylphenyl ether are preferably used. In addition to Reagent 1 and Reagent 2, the kit may also include a positive control, a negative control, a serum dilution solution, etc. As a medium for the positive control and negative control, serum that does not contain a measurable target substance, physiological saline, or a solvent may be used.

(Detection method)
The method for detecting a target substance in a specimen by the agglutination method according to the present invention is characterized by using the particles according to the present invention, the ligand-sensitized particles according to the present invention, or the particle dispersion according to the present invention. In detecting a target substance, for example, ligand-sensitized particles for an agglutination method and a specimen that may contain the target substance are mixed. The ligand-sensitized particles according to the present invention and the specimen are preferably mixed at a pH of 3.0 or more and pH 11.0 or less. Further, the mixing temperature is 20° C. or more and 50° C. or less, and the mixing time is 10 seconds or more and 30 minutes or less. Further, the concentration of the ligand-sensitized particles in the detection method according to the present invention is preferably 0.001% by mass or more and 5% by mass or less, more preferably 0.01% by mass or more and 1% by mass or less in the reaction system. The detection method according to the present invention, for example, optically detects an agglutination reaction that occurs as a result of mixing ligand-sensitized particles and a specimen, and detects the target substance in the specimen by optically detecting the agglutination reaction. The concentration of the target substance can also be measured. As a method for optically detecting the aggregation reaction, an optical device capable of detecting scattered light intensity, transmitted light intensity, absorbance, etc. may be used to measure the amount of change in these values.

(Production method)
A preferred method for producing particles according to the present invention will be explained.
One aspect of the present invention is a method for producing particles, comprising mixing a vinyl monomer (e.g., styrene), water, and a radical polymerization initiator to form a particulate polymer; Step 1 of mixing a glycidyl group-containing monomer (for example, glycidyl methacrylate), water, and a radical polymerization initiator to form a granular copolymer to obtain the granular polymer or an aqueous dispersion of the granular copolymer. and the aqueous dispersion, a surfactant, and a silane coupling agent, which is the origin of the structure represented by the above formula (1) of the particles after production, are mixed to form the granular polymer or the granular particles. The method is characterized by including a step 2 of coating the copolymer.

In the method for producing particles according to the present invention, in step 1, a vinyl monomer, a glycidyl group-containing monomer, water, and a radical polymerization initiator are mixed to form a granular copolymer, and after step 1, and , before the step 2, it is preferable to include a step 3 in which the epoxy group derived from the glycidyl group-containing monomer in the aqueous dispersion is reacted with ammonia or an amine of a diamine having a primary or secondary amine. . In step 3, amino groups can be introduced into the epoxy groups of the particulate copolymer. Furthermore, the glycidyl group-containing monomer is preferably at least one selected from glycidyl acrylate and glycidyl methacrylate.

The method for forming a granular polymer or granular copolymer is not limited to radical polymerization, as long as the object of the present invention can be achieved. Among radical polymerizations, it is preferable to use emulsion polymerization, soap-free emulsion polymerization, and suspension polymerization, and it is more preferable to use emulsion polymerization or soap-free emulsion polymerization. It is even more preferable to use soap-free emulsion polymerization. Compared with suspension polymerization, emulsion polymerization and soap-free emulsion polymerization can produce granular copolymers with a sharp particle size distribution. In addition, when chemically fixing particles to a ligand, there is a concern that the ligand may be denatured if an anionic surfactant, such as that commonly used in emulsion polymerization, is present. Therefore, soap-free emulsion polymerization is the most preferable method for forming a granular copolymer.

In forming the particulate polymer or particulate copolymer in step 1 of the method for producing particles according to the present invention, it is preferable to further use a crosslinkable radical polymerization monomer. By using a crosslinkable radical polymerization monomer, the resulting granular polymer or granular copolymer becomes physically strong, and there is no fear of cracking or chipping even if centrifugal separation operations are repeated during purification.

Examples of crosslinkable radically polymerizable monomers that can be used are listed below, but the present invention is not limited thereto. Furthermore, two or more types of crosslinkable radically polymerizable monomers may be used. Among the exemplified crosslinkable radical polymerizable monomers, although the reason is not clear, divinylbenzene is preferably used because it has excellent handling properties during the radical polymerization reaction.

Crosslinkable radical polymerizable monomers: diethylene glycol diacrylate, triethylene glycol diacrylate, tetraethylene glycol diacrylate, polyethylene glycol diacrylate, 1,6-hexanediol diacrylate, neopentyl glycol diacrylate, tripropylene glycol diacrylate, polypropylene glycol diacrylate, 2,2'-bis(4-(acryloxydiethoxy)phenyl)propane, trimethylolpropane triacrylate, tetramethylolmethane tetraacrylate, ethylene glycol dimethacrylate, diethylene glycol dimethacrylate, triethylene glycol dimethacrylate, glycol dimethacrylate, tetraethylene glycol dimethacrylate, polyethylene glycol dimethacrylate, 1,3-butylene glycol dimethacrylate, 1,6-hexanediol dimethacrylate, neopentyl glycol dimethacrylate, polypropylene glycol dimethacrylate, 2,2'-bis(4-(methacryloxydiethoxy)phenyl)propane, 2,2'-bis(4-(methacryloxypolyethoxy)phenyl)propane, trimethylolpropane trimethacrylate, tetramethylolmethane tetramethacrylate, divinylbenzene, divinylnaphthalene, and divinyl ether, etc.

It is preferable that the process of forming a particulate polymer or a particulate copolymer in step 1 of the method for producing particles according to the present invention further includes the following step 4. That is, step 4 of further mixing the monomer glycidyl methacrylate and coating the surface of the granular polymer or granular copolymer with polyglycidyl methacrylate.

The radical polymerization initiator is at least 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, 2,2'-azobis[2-(2-imidazolin-2-yl) yl) propane] disulfate dihydrate. The reason for this is that when a glycidyl group-containing monomer is used in Step 1, the epoxy group derived from the glycidyl group-containing monomer is not ring-opened. For example, when potassium persulfate is used as a radical polymerization initiator, the radical polymerization reaction field becomes acidic due to the influence of the initiator residue, and the epoxy group derived from the glycidyl group-containing monomer reacts with water to form glycol. It may be stored away. Moreover, when ammonium persulfate is used as a radical polymerization initiator, the epoxy group derived from the glycidyl group-containing monomer and ammonia may react. In addition, when an anionic radical polymerization initiator having a carboxyl group is used as a radical polymerization initiator, the epoxy group derived from the glycidyl group-containing monomer and the carboxy group derived from the polymerization initiator react, causing the particulate copolymer to aggregate. Put it away.

The radical polymerization initiator is 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride, 2,2'-azobis[2-(2-imidazolin-2-yl)propane]di Must be any of sulfate dihydrate, 2,2'-azobis(2-methylpropionamidine) dihydrochloride, or 2,2'-azobis[2-(2-imidazolin-2-yl)propane] is particularly preferred.

Step 3 is a step of introducing an amino group into the epoxy group derived from the glycidyl group-containing monomer of the particulate copolymer. Generally, an epoxy group can easily react with ammonia or an amine of a diamine having a primary or secondary amine to introduce an amino group. Since the reaction with ammonia is performed under strong basic conditions, the chemical reaction between the epoxy group and water is suppressed, and an amino group can be efficiently introduced into the epoxy group.

Step 3 may further include step 5 of introducing a hydrophilic chain. Step 5 is, for example, a step of introducing trishydroxymethylaminomethane (Tris) to the epoxy group derived from the glycidyl group-containing monomer possessed by the granular copolymer. Since the reaction with trishydroxymethylaminomethane (Tris) is performed under strongly basic conditions, the chemical reaction between the epoxy group and water is suppressed, and trishydroxymethylaminomethane (Tris) can be efficiently introduced to the epoxy group. Trishydroxymethylaminomethane (Tris) may be introduced to the epoxy group before or after the reaction with ammonia, or may be reacted simultaneously with ammonia.

Step 2 is a step of coating the granular polymer or the granular copolymer with a polymer of a silane coupling agent having a carboxy group.

Further, in step 2, it is preferable to add a surfactant. By adding a surfactant, it is possible to improve the dispersibility of particles coated with a polymer of a silane coupling agent having a carboxy group. As the surfactant, for example, nonionic surfactants, anionic surfactants, cationic surfactants, polymeric surfactants, or phospholipids can be used. These surfactants may be used alone or in combination of two or more.

（式（６）において、Ｒ^２１～Ｒ^２４はそれぞれ独立に、－Ｈ、－ＯＣＲ’から選択される。Ｒ’は炭素数１以上１８以下の、飽和または不飽和アルキル基である。また、ｗ１、ｘ１、ｙ１、ｚ１は、ｗ１とｘ１とｙ１とｚ１の総和が１０以上３０以下となる整数である。） Examples of the nonionic surfactants include polyoxyethylene sorbitan fatty acid esters (e.g., compounds represented by formula (6)), Brij (registered trademark) 35, Brij (registered trademark) 58, Brij (registered trademark) 76, Brij (registered trademark) 98, Triton (registered trademark) X-100, Triton (registered trademark) X-114, Triton (registered trademark) X-305, Triton (registered trademark) N-101, Nonidet (registered trademark) P-40, IGEPAL (registered trademark) CO530, IGEPAL (registered trademark) CO630, IGEPAL (registered trademark) CO720, and IGEPAL (registered trademark) CO730.

(In formula (6), R ²¹ to R ²⁴ are each independently selected from -H and -OCR'. R' is a saturated or unsaturated alkyl group having 1 to 18 carbon atoms. w1, x1, y1, and z1 are integers such that the sum of w1, x1, y1, and z1 is 10 or more and 30 or less.)

Examples of the polyoxyethylene sorbitan fatty acid ester represented by formula (6) include Tween (registered trademark) 20, Tween (registered trademark) 40, Tween (registered trademark) 60, Tween (registered trademark) 80, and Tween (registered trademark). 85 etc. can be mentioned.

Examples of the anionic surfactants include sodium dodecyl sulfate, dodecylbenzenesulfonate, decylbenzenesulfonate, undecylbenzenesulfonate, tridecylbenzenesulfonate, nonylbenzenesulfonate, and their sodium, potassium, and ammonium salts. can be mentioned.

Examples of the above-mentioned cationic surfactants include cetyltrimethylammonium bromide, hexadecylpyridinium chloride, dodecyltrimethylammonium chloride, and hexadecyltrimethylammonium chloride.

Examples of the polymer surfactant include polyvinyl alcohol, polyoxyethylene polyoxypropylene glycol, and gelatin. Commercially available polyoxyethylene polyoxypropylene glycol products include Pluronic F68 (manufactured by BASF) and Pluronic F127 (manufactured by BASF).

The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

(Example 1 Synthesis of SG particles)
The following materials were weighed into a 300 mL flask.
・1.2g (1.32mL) styrene (manufactured by Kishida Chemical)
・1.8g (1.68mL) glycidyl methacrylate (manufactured by Kishida Chemical)
・0.04g (0.044mL) divinylbenzene (manufactured by Kishida Chemical Co., Ltd.)
- 115 g (115 mL) of ion-exchanged water These were mixed to obtain a mixed solution.

Thereafter, this mixed solution was maintained at 70° C. while stirring at 200 rpm, and nitrogen bubbling was performed for 30 minutes. Next, nitrogen bubbling was switched to nitrogen flow.

As a polymerization initiator, 0.06 g of 2,2'-azobis(2-methylpropionamidine) dihydrochloride (trade name: V-50, manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in 5 g (5 mL) of pure water, A V-50 solution was obtained.

Radical polymerization (soap-free emulsion polymerization) was started by adding this V-50 solution to the mixed solution (referred to as radical polymerization solution). Two hours after the start of polymerization, 0.3 g (0.28 mL) of glycidyl methacrylate was added to the radical polymerization solution, and the mixture was maintained at 70° C. while stirring at 200 rpm for 7 hours, and then slowly cooled to room temperature. At this point, the contents of the 300 ml flask were sampled and the radical polymerization conversion rate was evaluated using proton NMR, gas chromatography, and gel permeation chromatography, and it was confirmed that it was substantially 100%.

（式（７）中、＊はポリメタクリル酸骨格との結合を示す。）
ＳＧ粒子は、分散媒体をイオン交換水として保存した。ＳＧ粒子の水分散液をＳＧ粒子分散液とする。 Next, the contents of the 300 mL flask (particulate copolymer dispersion solution) were centrifuged (15,000 rpm). The solid content was washed with ion-exchanged water. Centrifugation was performed again and this washing was performed twice. The obtained particles are referred to as SG particles. The surface of the SG particle is coated with polyglycidyl methacrylate having a partial structure represented by formula (7), and as shown in formula (7), an epoxy group is present.

(In formula (7), * indicates a bond with the polymethacrylic acid skeleton.)
The SG particles were preserved using ion-exchanged water as the dispersion medium. An aqueous dispersion of SG particles is referred to as an SG particle dispersion.

Next, 1 g of SG particle dispersion (solid content: 20 mg) was dispersed in 50 mL of ion-exchanged water containing 1% Tween (registered trademark) 20, and stirred at room temperature for 15 minutes. Thereafter, 0.33 mL of a silane coupling agent having a carboxyl group (trade name: X-12-1135, manufactured by Shin-Etsu Chemical Co., Ltd.) was added, and the mixture was stirred overnight at room temperature. This dispersion was centrifuged and the supernatant was discarded. Subsequently, SG particles coated with the polymer of X-12-1135 (hereinafter sometimes referred to as SGX-1 particles) were recovered by repeatedly performing a washing operation with ion-exchanged water.

Zeta potential (Zetasizer: Malvern) measurements showed that the zeta potential of the SG particles was 42 mV. On the other hand, the zeta potential of the SGX-1 particles was -58 mV. The carboxyl groups were ionized in ion-exchanged water and became negatively charged, and it is believed that the zeta potential of the particles changed from positive to negative when they were coated with a polymer of the silane coupling agent X-12-1135, which has a carboxyl group. Energy dispersive X-ray analysis (EDX) (product name: FE-SEM S-4800, manufactured by Hitachi High-Technologies) measurements showed that Si was detected in the SGX-1 particles. This also confirmed that the SGX-1 particles were coated with a silane coupling agent.

（式（８）中、＊はポリメタクリル酸骨格に結合する部分を示す。）
ＳＧＮ粒子は、分散媒体をイオン交換水として保存した。ＳＧＮ粒子の水分散液をＳＧＮ粒子分散液とする。 (Example 2 Synthesis of SGN particles)
19 g (solid content: 1.98 g) of the SG particle dispersion prepared in Example 1 was transferred to a 200 mL flask. In ice water containing salt, 28% ammonia water (55.4 g) was added dropwise to the SG particle dispersion using a dropping funnel over about 15 minutes while being irradiated with ultrasonic waves. Thereafter, the SG particle dispersion liquid was transferred to an autoclave vessel equipped with a rotor, the vessel was tightly stoppered, and then stirred in an oil bath at 70°C for 24 hours. As a result, 50 times the molar amount of ammonia was reacted with the molar amount of glycidyl methacrylate in the SG particles. Subsequently, it was slowly cooled to room temperature, and centrifugation (15,000 rpm) and washing with ion-exchanged water were repeated three times. The obtained particles are particles in which amino groups have been introduced onto the surface of SG particles, and are called SGN particles. The surface of the SGN particle is coated with polyglycidyl methacrylate having a partial structure represented by formula (8), and as shown in formula (8), amino groups are present.

(In formula (8), * indicates a portion bonded to the polymethacrylic acid skeleton.)
The SGN particles were preserved with ion-exchanged water as the dispersion medium. The aqueous dispersion of SGN particles is referred to as an SGN particle dispersion.

SGN particles were coated with the polymer of X-12-1135 by the same operation as described in Example 1, and the SGN particles coated with the obtained polymer of X-12-1135 (hereinafter referred to as SGX- 2 particles) were collected.

From the measurement of zeta potential, the zeta potential of the SGN particles was 56 mV. On the other hand, the zeta potential of the SGX-2 particles was -45 mV. Similar to the SGX-1 particles, it is thought that the zeta potential of the particles changed from positive to negative due to being coated with the polymer of silane coupling agent X-12-1135 having a carboxy group. Si was detected from the SGX-2 particles by EDX measurement. This also confirmed that the SGX-2 particles were coated with the silane coupling agent.

(Comparative Example 1 Polystyrene particles)
As Comparative Example 1, polystyrene particles having a carboxyl group (trade name: Imtex P0113, manufactured by JSR, particle size 188 nm) (hereinafter also simply referred to as Imtex) were diluted with ion-exchanged water to a concentration of 0.1% by mass. The dispersion liquid obtained was used.

(Example 3: Evaluation of non-specific reaction (non-specific agglutination) of particles)
The non-specific reaction (non-specific agglutination) of the particles was evaluated by immunoturbidimetry. The immunoturbidimetry is a method in which human serum is brought into contact with particles, and non-specific particle agglutination is measured with an absorbance meter using turbidity as an index. If non-specific agglutination occurs, the absorbance increases. To measure the absorbance, an ultraviolet-visible spectrophotometer (product name: GeneQuant 1300, manufactured by GE Healthcare) was used, and the sample was injected into a plastic cell (minimum sample volume 70 μL) and measured with an optical path length of 10 mm. The measurement method is specifically shown below.

4 μL of human serum and 60 μL of R1 buffer solution (phosphate buffer solution) were mixed in a plastic cell and heated at 37°C for 5 minutes. The SGX-1 particles according to Example 1 and the SGX-2 particles according to Example 2 were each dispersed in ion-exchanged water to prepare a particle dispersion solution (particle concentration 0.1% by mass). 30 μL of the obtained SGX-1 particle dispersion solution, SGX-2 particle dispersion solution, and Imtex dispersion solution prepared in Comparative Example 1 (particle concentration 0.1% by mass) were each added to R1 buffer solution (64 μL) containing human serum. Next, the mixture was quickly pipetted while being careful not to introduce air bubbles, and a sample was obtained. The absorbance of the sample at a wavelength of 572 nm was measured and determined as Abs1. The sample was also heated at 37°C for 5 minutes, and then the absorbance of the sample at a wavelength of 572 nm was measured and determined as Abs2. For each particle dispersion of Examples 1 and 2 and Comparative Example 1, the value obtained by subtracting Abs1 from Abs2 was multiplied by 10,000 to obtain the ΔOD×10,000 value. If the ΔOD×10,000 value was 2,000 or more, it was determined that a non-specific reaction had occurred.

The results are shown in Table 1.

The SGX-1 and SGX-2 particles of this example had a ΔOD x 10,000 value of 2,000 or less, and no non-specific reaction was observed. On the other hand, Imtex, used as a comparative example, had a ΔOD x 10,000 value of 10,000 or more. It can be considered that the increase in absorbance in the dispersion is due to particle aggregation caused by non-specific adsorption of the particles in the dispersion, and it was found that Imtex caused non-specific aggregation due to serum. It was confirmed that the SGX-1 and SGX-2 particles of this example have a superior ability to suppress non-specific adsorption compared to Imtex.

Example 4 Preparation of antibody-sensitized particles
0.1 mL (1 mg of SGX-1 particles) of a dispersion of SGX-1 particles (concentration 1.0% by mass) prepared using ion-exchanged water was transferred to a microtube (volume 1.5 mL). Furthermore, 0.12 mL of activation buffer (25 mM 2-morpholinoethanesulfonic acid (MES), pH 6.0) was added to the microtube, and the mixture was centrifuged at 4° C. and 15,000 rpm (20,400 g) for 5 minutes. After centrifugation, the supernatant was discarded (decanted) using a pipetter.

Subsequently, 0.12 mL of activation buffer (25 mM MES, pH 6.0) was added, and the frequency was adjusted to 28 kHz using an ultrasonic cleaner (trade name: 3-frequency ultrasonic cleaner MODEL VS-100III, manufactured by As One). and dispersed. Next, centrifugation was performed at 4° C. and 15,000 rpm (20,400 g) for 5 minutes. The supernatant was discarded using a pipette, 0.12 mL of activation buffer (25 mM MES, pH 6.0) was added, and the mixture was dispersed using ultrasound in the same manner as above. Thereafter, it was centrifuged at 4° C. and 15,000 rpm (20,400 g) for 5 minutes.

The supernatant was discarded using a pipette, and 60 μL of each of the WSC solution and Sulfo NHS solution were added and dispersed using ultrasound. Here, the WSC solution was obtained by dissolving 50 mg of water-soluble carbodiimide (WSC) in 1 mL of activation buffer. Further, Sulfo NHS solution is obtained by dissolving 50 mg of sodium N-hydroxysulfosuccinimide (Sulfo NHS) in 1 mL of activation buffer.

Thereafter, the carboxy groups of the particles were converted into active esters by stirring at room temperature for 30 minutes. Centrifugation was performed at 4° C. and 15,000 rpm (20,400 g) for 5 minutes, and the supernatant was discarded with a pipette. 0.2 mL of immobilization buffer (25 mM MES, pH 5.0) was added and dispersed using ultrasound in the same manner as above. Centrifugation was performed at 4° C. and 15,000 rpm (20,400 g) for 5 minutes, and the supernatant was discarded with a pipette. 50 μL of immobilization buffer was added per 1 mg of SGX-1 particles, and the carboxy group-activated particles were dispersed using ultrasound in the same manner as above.

An anti-human C-reactive protein (CRP) antibody (polyclonal antibody) was diluted with an immobilization buffer to 100 μg/50 μL (hereinafter also referred to as antibody solution). 50 μL of the antibody solution was added to 50 μL of a solution of carboxy group-activated SGX-1 particles (containing 1 mg of SGX-1 particles), and the particles were dispersed using ultrasound in the same manner as above. The amount of antibody to be charged is 100 μg per 1 mg of SGX-1 particles (100 μg/mg). The tube was agitated for 60 minutes at room temperature to allow the antibody to immobilize on the carboxy groups of the particles. Then, it was centrifuged at 15,000 rpm (20,400 g) for 5 minutes at 4°C, and the supernatant was discarded with a pipette.

Add 0.24 mL of an active ester inactivation buffer containing Tris (1M Tris, pH 8.0 containing 0.1% Tween (registered trademark) 20) and disperse using ultrasound in the same manner as above. Ta. After stirring at room temperature for 1 hour to bind Tris to the remaining activated ester, the mixture was allowed to stand at 4° C. overnight.

Next, it was centrifuged at 4° C. and 15,000 rpm (20,400 g) for 5 minutes, and the supernatant was discarded with a pipette. 0.2 mL of washing/storage buffer (10 mM hydroxyethylpiperazine ethanesulfonic acid (HEPES), pH 7.9) was added and dispersed using ultrasound in the same manner as above. After repeating the washing operation with 0.2 mL of the washing/storage buffer twice, 1.0 mL of the washing/storage buffer was added and dispersed using ultrasound in the same manner as above.

Since almost no loss of particles was observed during the sensitization step, the final concentration of antibody-sensitized particles was 0.1% by mass. The obtained dispersion of antibody-sensitized particles was stored in a refrigerator and redispersed using ultrasound when used. The obtained antibody-sensitized particles are referred to as antibody-sensitized SGX-1 particles.

(Comparative Example 2 Antibody sensitization to polystyrene particles)
As Comparative Example 2, Imtex used in Comparative Example 1 was sensitized with an antibody. Antibody-sensitized polystyrene particles according to Comparative Example 2 were obtained in the same manner as in Example 4, except that the SGX-1 particles used in Example 4 were changed to Imtex. The obtained particles are used as antibody-sensitized polystyrene particles.

(Example 5) Measurement of antibody sensitization efficiency
It was confirmed by protein quantification that the antibody was sensitized (fixed) to the particles for each of the antibody-sensitized SGX-1 particles according to Example 4 and the antibody-sensitized polystyrene particles according to Comparative Example 2. Specifically, this is a method in which the antibody-sensitized particles are reacted with a BCA reagent.

First, 25 μL (particle amount: 25 μg) of a dispersion of antibody-sensitized particles (0.1% solution) was collected. 7 mL of solution A and 140 μL of solution B of the protein assay BCA kit (manufactured by Wako Pure Chemical Industries, Ltd.) were mixed to prepare AB solution. 200 μL of AB solution was added to 25 μL of the particle solution, and the mixture was incubated at 60° C. for 30 minutes. The solution was centrifuged at 4° C. and 15,000 rpm (20,400 g) for 5 minutes, and 200 μL of the supernatant was collected with a pipettor.

The absorbance of the collected solution at a wavelength of 562 nm was measured along with a standard sample using a multimode microplate reader (trade name: SynergyMX, manufactured by BioTek). Here, the standard samples used were antibodies diluted with 10 mM HEPES to several concentrations ranging from 0 to 200 μg/mL.

Using the standard curve obtained from the measurement results for the standard sample, the amount of antibody in the collected solution was calculated. The amount of antibody sensitization to the particles (the amount of antibody immobilized per particle weight (μg/mg)) was determined by dividing the calculated antibody amount by the particle weight (here, 0.025 mg). Sensitization efficiency was determined from the amount of antibody charged.

The results are shown in Table 2.

It was found that the antibody-sensitized SGX-1 particles according to Example 4, which are the antibody-sensitized particles according to this example, had higher sensitization efficiency than the antibody-sensitized polystyrene particles according to Comparative Example 2.

The high sensitization efficiency of the particles according to this example will be explained. In the particles according to this example, the carboxy group is located near the surface, and the particle surface is coated with a silane coupling agent. In other words, the surface charge is negative and uniform, making it extremely hydrophilic. Hydrophilic surfaces exhibit a high ability to suppress nonspecific adsorption. During sensitization, the antibody is in a cationic state and electrostatically attracts the carboxy group region of the particle, which is the minus surface. As a result, the antibody is concentrated on the particle surface, and the reaction between the antibody and the carboxy group on the particle surface is greatly promoted. As a result, it is thought that the rate of antibody sensitization to particles is improved. Since the NHS-activated ester with a carboxyl group is rapidly hydrolyzed in water, the concentration effect of the antibody on the particle surface is an important process. On the other hand, since the particle surface of commercially available polystyrene is hydrophobic, physical adsorption of antibodies occurs to hydrophobic regions other than the carboxy group region. As a result of physical adsorption involving repeated desorption and adsorption, commercially available polystyrene particles are thought to have low sensitization efficiency.

(Example 6 Sensitivity evaluation of antibody-sensitized particles to human CRP antigen)
The sensitivity of antibody-sensitized particles was evaluated by latex immunoagglutination method. Specifically, antibody-sensitized particles are reacted with the antigen to form aggregates of immune complexes, the aggregates are irradiated with light, and the attenuation (absorbance) of the irradiated light due to scattering is measured using an absorbance meter. It's a method. Depending on the amount of antigen contained in the sample, the proportion of aggregates increases and the absorbance increases. In evaluating sensitivity, it is desirable that the increase in absorbance (expressed as ΔOD×10,000 value) at a predetermined prostate-specific antigen (PSA) concentration is large. The absorbance was measured using an ultraviolet-visible spectrophotometer (trade name: GeneQuant 1300, manufactured by GE Healthcare), and the sample was injected into a plastic cell and measured at an optical path length of 10 mm. The measurement method is specifically shown below.

1 μL of CRP solution (CRP concentration 5 μg/mL) was used as a sample, and this sample and 50 μL of R1 buffer were mixed in a plastic cell and heated at 37° C. for 5 minutes. Add 50 μL of a dispersion solution of antibody-sensitized SGX-1 particles (particle concentration 0.1% by mass, 10 mM HEPES, pH 7.9, 0.01% by mass Tween (registered trademark) 20) to 51 μL of R1 buffer containing CRP. did. Thereafter, the sample was quickly pipetted while being careful not to introduce air bubbles. The absorbance of the sample at a wavelength of 572 nm was read and designated as Abs1'. Further, after heating the sample at 37°C for 5 minutes, the absorbance of the sample at a wavelength of 572 nm was read and designated as Abs2'. The value obtained by subtracting Abs1' from Abs2' was calculated and multiplied by 10,000 to obtain the ΔOD×10,000 value.

The results are shown in Table 3.

In the antibody-sensitized SGX-1 particles according to this example, an increase in ΔOD×10,000 was observed in the presence of CRP. This is the result of the antibody-sensitized particles binding to the antigen CRP to form particle aggregates, which were found to function as particles for use in the latex immunoagglutination method.

Claims (14)

a particle having a core including a copolymer having a unit derived from a styrene-based monomer and a unit derived from a glycidyl group-containing monomer, the surface of the core being coated with a polymer of a silane coupling agent having one or more carboxy groups ;
a bond between the core and the polymer of the silane coupling agent includes a structure derived from a glycidyl group of the glycidyl group-containing monomer,
The particle, characterized in that a composition ratio (mol fraction) of the unit derived from the styrene-based monomer to the unit derived from the glycidyl group-containing monomer in the copolymer (the unit derived from the styrene-based monomer/the unit derived from the glycidyl group-containing monomer) is 0.1 or more and 10 or less . In the copolymer, the composition ratio (mol fraction) of units derived from the styrene monomer and units derived from the glycidyl group-containing monomer (units derived from the styrene monomer/units derived from the glycidyl group-containing monomer) The particle according to claim 1 , wherein the number of units derived from the particle is 0.5 or more and 2 or less . The particles according to claim 1 , wherein the polymer of the silane coupling agent has a structural unit represented by formula (1).

(In formula (1), each X independently represents a bond with Si of a structural unit represented by formula (1) adjacent to each other via O, an epoxy group, an amino group, and a hydroxyl group derived from the structure of the core. At least one of the structural units represented by formula (1) that represents a bond with one or more selected functional groups or OH has a bond with the core.
Each R independently has at least one carboxy group at the end, and has a carbon number of 1 and may include -NH-CO-, -NH-, -O-, -S-, -CO-, -O-CO- Represents a straight chain or branched alkyl group of 30 or more. ) The particles according to any one of claims 1 to 3 , having a number average particle size of 0.05 µm or more and 0.5 µm or less. The core is a particle containing a polymer having a unit derived from a vinyl monomer, or a copolymer having a unit derived from a vinyl monomer and a unit derived from a glycidyl group-containing monomer, and the surface of the core is 1 coated with a polymer of a silane coupling agent having three or more carboxy groups,
The particles, wherein the silane coupling agent is X-12-1135. Ligand-sensitized particles in which a ligand is immobilized on the particles according to any one of claims 1 to 5 . The ligand-sensitized particle according to claim 6 , wherein the ligand is fixed to a carboxy group of the particle. The ligand-sensitized particles according to claim 6 or 7 , wherein the amount of ligand immobilized is 1 μg or more and 500 μg or less per 1 mg of particles. A particle dispersion in which the particles according to any one of claims 1 to 5 or the ligand-sensitized particles according to any one of claims 6 to 8 are dispersed in an aqueous solution. The particle dispersion according to claim 9 , comprising a surfactant. An aggregation method using the particles according to any one of claims 1 to 5 , the ligand-sensitized particles according to any one of claims 6 to 8 , or the particle dispersion according to claims 9 or 10 . A method for detecting a target substance in a sample. A method for producing particles, the method comprising:
Step 1 of mixing a vinyl monomer, a glycidyl group-containing monomer, water, and a radical polymerization initiator to form a granular copolymer as a core to obtain an aqueous dispersion of the core ;
Step 2 of mixing into the aqueous dispersion a silane coupling agent, which is the origin of the structure represented by the following formula (1) of the particles after production, and coating the core with a polymer of the silane coupling agent; ,

(In formula (1), each X independently represents a bond with Si of a structural unit represented by formula (1) adjacent to each other via O, an epoxy group, an amino group, and a hydroxyl group derived from the structure of the core. At least one of the structural units represented by formula (1) that represents a bond with one or more selected functional groups or OH has a bond with the core.
Each R independently has at least one carboxy group at the end, and has a carbon number of 1 and may include -NH-CO-, -NH-, -O-, -S-, -CO-, -O-CO- Represents a straight chain or branched alkyl group of 30 or more. )
After the step 1 and before the step 2, the epoxy group derived from the glycidyl group-containing monomer in the aqueous dispersion is reacted with ammonia or an amine of a diamine having a primary or secondary amine. Step 3 and
A method for producing particles, comprising: The method for producing particles according to claim 12 , wherein the glycidyl group-containing monomer is at least one selected from glycidyl acrylate and glycidyl methacrylate. A method for producing particles, the method comprising:
A granular copolymer is formed by mixing a vinyl monomer, water, and a radical polymerization initiator, or a granular copolymer is formed by mixing a vinyl monomer, a glycidyl group-containing monomer, water, and a radical polymerization initiator. Step 1 of forming a core and obtaining an aqueous dispersion of the core;
Step 2 of mixing into the aqueous dispersion a silane coupling agent, which is the origin of the structure represented by the following formula (1) of the particles after production, and coating the core with a polymer of the silane coupling agent; ,
including;

(In formula (1), each X is independently selected from a bond with Si that the structural unit shown in formula (1) adjacent to each other via O, an epoxy group derived from the core, an amino group, and a hydroxyl group) At least one of the structural units represented by formula (1), which represents a bond with one or more functional groups or OH, has a bond with the core.
Each R independently has at least one carboxy group at the end, and has a carbon number of 1 and may include -NH-CO-, -NH-, -O-, -S-, -CO-, -O-CO- Represents a straight chain or branched alkyl group of 30 or more. )
A method for producing particles, characterized in that the silane coupling agent is X-12-1135.