JP2019082356A - Magnetic particle and detection/separation method - Google Patents

Abstract

To provide a magnetic particle that has a high re-dispersibility and shows a high S/N ratio during a diagnosis or the like using the particle.SOLUTION: The magnetic particle has, on at least its surface, a polymer including a structural unit (A) derived from an aromatic monomer by 20-80 mass% to the whole structural unit constituting the polymer. A ratio S/Sg of a specific surface area S to a spherical equivalent surface area Sg is 6.0 or less.SELECTED DRAWING: None

Description

  The present invention relates to magnetic particles and detection and separation methods.

In recent years, magnetic particles have been able to provide excellent reaction fields in immune reactions between antigens and antibodies, hybridization between DNAs or between DNAs and RNAs, and in particular, their application to diagnostic reagents and pharmaceutical research has become active. There is.
For example, as an example of application to a diagnostic agent, a first test probe such as an antibody or an antigen is immobilized on a carrier. The substance to be tested in the sample is captured on the carrier via the first test probe and then reacts with the second test probe. If the second test probe is labeled with a fluorescent substance or an enzyme, detection can be performed by fluorescence, an enzyme reaction, or the like.

  Immunodiagnostic agents are one of the uses of the magnetic particles for diagnostic agents of the present invention. When the magnetic particles of the present invention are used as an immunodiagnostic agent, it is desirable to have both the property of aggregating in response to the application of an external magnetic field quickly and the property of redispersing particles.

  The property of aggregation upon application of an external magnetic field is necessary for recovering only the components specifically bound to the diagnostic magnetic particles and the diagnostic magnetic particles and removing unnecessary components.

  Also, the property of redispersion of particles is, for example, in order to measure light emission, magnetic particles are uniformly dispersed in a dispersion medium (for example, water), or magnetic particles are aggregated by an external magnetic field to remove unnecessary components. It is necessary to disperse the magnetic particles later.

  So far, as an approach to improve the performance of magnetic particles for diagnostic drugs, noise is reduced by introducing hydrophilic functional groups such as carboxy groups on the surface and reducing nonspecific adsorption of biorelated substances such as proteins and nucleic acids. The detection sensitivity was improved (Patent Document 1).

  In addition, by making the particles porous, it is possible to increase the amount of protein binding, but on the other hand, in order to achieve high sensitivity in immunoassays, a greater amount of protein binding as compared to smooth particles Were required (Patent Document 2).

Patent No. 4985916 JP 2007-95903 A

Conventional magnetic particles such as magnetic particles described in the above patent documents have poor dispersibility (hereinafter also referred to as “redispersion”) after being collected and then dispersed, and diagnostic and the like using such magnetic particles There was a tendency for signal variations to occur and there was room for improvement in terms of signal / noise (S / N) ratio.
The present invention has been made in view of the above problems, and it is an object of the present invention to provide a magnetic particle which is excellent in redispersibility and which exhibits a high S / N ratio at diagnosis etc. using the particle. As an issue.

As a result of earnest studies to solve the above problems, the inventor of the present invention has found that the problems can be solved according to the following configuration example, and has completed the present invention.
The structural example of this invention is as follows.

<1> At least the surface of the polymer comprises 20 to 80% by mass of the structural unit (A) derived from the aromatic monomer with respect to the total structural units constituting the polymer,
The ratio S / Sg of the specific surface area S to the spherical equivalent specific surface area Sg is 6.0 or less
Magnetic particles.

<2> The magnetic particle according to <1>, wherein the aromatic monomer is an aromatic vinyl monomer.
<3> The magnetic particle according to <2>, wherein the aromatic vinyl monomer is divinylbenzene.

<4> The polymer according to <1>, wherein the polymer further contains a structural unit (B) having a functional group capable of chemically bonding to the probe in an amount of 15 to 70% by mass based on all structural units constituting the polymer. Magnetic particle as described in any one of 3>.
<5> The magnetic particle according to <4>, wherein the structural unit (B) is represented by the formula (1).

〔式（１）中、
Ｒ¹は、水素原子またはメチル基を示し、
Ｒ²は、−（Ｃ＝Ｏ）−Ｏ−＊、−（Ｃ＝Ｏ）−ＮＲ⁴−＊（Ｒ⁴は、水素原子またはメチル基を示し、＊は、式（１）中のＲ³と結合する位置を示す）またはフェニレン基を示し、
Ｒ²が−（Ｃ＝Ｏ）−Ｏ−＊である場合、Ｒ³は、水素原子またはプローブと化学結合可能な官能基を有する有機基を示し、Ｒ²が−（Ｃ＝Ｏ）−ＮＲ⁴−＊またはフェニレン基である場合、Ｒ³は、プローブと化学結合可能な官能基を有する有機基を示す。〕

[In the formula (1),
R ¹ represents a hydrogen atom or a methyl group,
R ² represents — (C = O) —O— *, — (C = O) —NR ⁴ — * (R ⁴ represents a hydrogen atom or a methyl group, and * represents R ³ in the formula (1) Indicate a position to bond with) or a phenylene group,
When R ² is — (C = O) —O— *, R ³ is a hydrogen atom or an organic group having a functional group capable of chemically bonding to a probe, and R ² is — (C = O) —NR ^When it is a 4- * or phenylene group, R ³ represents an organic group having a functional group capable of chemically bonding to the probe. ]

  <6> The magnetic particle according to <4> or <5>, wherein the structural unit (B) is represented by Formula (2).

〔式（２）中、
Ｒ¹は、水素原子またはメチル基を示し、
Ｒ⁵は、炭素数２〜６の直鎖または分岐のアルキレン基を示し、
Ｒ⁶は、炭素数２〜６のアルキレン基、シクロヘキシレン基またはフェニレン基を示す。〕

[In the formula (2),
R ¹ represents a hydrogen atom or a methyl group,
R ⁵ represents a linear or branched alkylene group having 2 to 6 carbon atoms,
R ⁶ represents an alkylene group having 2 to 6 carbon atoms, a cyclohexylene group or a phenylene group. ]

  <7> The structural unit (B) is 2- (meth) acryloyloxyethyl succinic acid, 2- (meth) acryloyloxyethyl phthalic acid, 2- (meth) acryloyloxyethyl hexahydrophthalic acid, 2 -Derived from at least one selected from the group consisting of-(meth) acryloyloxypropyl succinic acid, 2- (meth) acryloyloxy propyl phthalic acid, and 2- (meth) acryloyloxy propyl hexahydrophthalic acid The magnetic particle in any one of <4>-<6>.

  <8> The polymer according to <1> to <7>, wherein the polymer further contains a structural unit (C) derived from a weakly hydrophilic monomer in an amount of 10 to 60% by mass based on all structural units constituting the polymer. The magnetic particle as described in any one.

<9> The magnetic particle according to <8>, wherein the structural unit (C) is one or more selected from structural units (C-1) and (C-2).
(C-1) A structural unit derived from a monoacrylate monomer that holds intermediate water when polymerized into a homopolymer and a free water, or a monomethacrylate monomer having the same structure as the side chain of the homopolymer (C- 2) Structural unit derived from the mono (meth) acrylate monomer represented by the formula (3)

〔式（３）中、
Ｒ⁷は、水素原子またはメチル基を示し、
Ｒ⁸は、炭化水素基を示す。〕

[In the formula (3),
R ⁷ represents a hydrogen atom or a methyl group,
R ⁸ represents a hydrocarbon group. ]

<10> The magnetic particle according to <9>, which includes a structural unit (C-1) as the structural unit (C).
The magnetic particle as described in <9> or <10> which is a structural unit to which a <11> structural unit (C-1) originates in Formula (4) or (5).

〔式（４）中、
Ｒ⁹は、水素原子またはメチル基を示し、
Ｒ¹⁰は、炭素数１〜３のアルカンジイル基を示し、
Ｒ¹¹は、炭素数１〜３のアルキル基を示し、
ｎは、１〜５の整数を示す。〕

[In the formula (4),
R ⁹ represents a hydrogen atom or a methyl group,
R ¹⁰ represents an alkanediyl group having 1 to 3 carbon atoms,
R ¹¹ represents an alkyl group having 1 to 3 carbon atoms,
n is an integer of 1 to 5; ]

〔式（５）中、
Ｒ¹²は、水素原子またはメチル基を示し、
ｍは、１または２を示す。〕

[In the formula (5),
R ¹² represents a hydrogen atom or a methyl group,
m represents 1 or 2; ]

<12> The magnetic particle according to any one of <1> to <11>, wherein a number average particle diameter of the magnetic particle is 5 nm to 20 μm.
<13> The magnetic particle according to any one of <1> to <12>, wherein the coefficient of variation (CV value) of the number average particle diameter of the magnetic particle is 20% or less.
<14> The magnetic particle according to any one of <1> to <13>, wherein the average circularity of the magnetic particle is 0.965 to 1.000.

  <15> The magnetic particle according to any one of <1> to <14>, wherein the magnetic particle has a functional group for binding a probe, and the functional group density for binding a probe in the magnetic particle is 1 to 200 μmol / g. Magnetic particles.

<16> The magnetic particle according to any one of <1> to <15>, wherein the magnetic particle has a core-shell structure, and the shell contains the polymer as a main component.
<17> The magnetic particle according to <16>, wherein the thickness of the shell is 0.01 to 0.3 μm.

  <18> The magnetic particle according to any one of <1> to <17>, wherein the magnetic material is not exposed on the surface of the magnetic particle.

  <19> A magnetic particle, wherein a probe is bound to the magnetic particle according to any one of <1> to <18>.

<20> The magnetic particles according to any one of <1> to <19>, which are for in vitro diagnostic agents or for detection of biorelated substances.
The detection / isolation | separation method including the process of detecting or isolate | separating the target substance in a sample using the magnetic particle in any one of <21><1>-<20>.

According to the present invention, it is possible to obtain magnetic particles that are excellent in redispersibility, and exhibit high S / N ratio when the particles are used in a diagnostic method such as CLEIA.
From the above, by using the magnetic particles according to the present invention, the S / N ratio is improved in the case of a diagnostic method such as chemiluminescent enzyme immunoassay (CLEIA), and the sensitivity at the time of detecting the test object is improved. In order to improve, the magnetic particles according to the present invention are effective for a diagnostic method such as CLEIA.

  Hereinafter, preferred embodiments according to the present invention will be described in detail. It is to be understood that the present invention is not limited to only the embodiments described below, but also includes various modifications implemented within the scope of the present invention. In the present specification, the description of “A to B” or the like representing a numerical range is synonymous with “A or more and B or less”, and includes A and B within the numerical range.

«Magnetic particles»
A magnetic particle according to the present invention (hereinafter also referred to as "the present particle") has a weight containing 20 to 80% by mass of a structural unit (A) derived from an aromatic monomer with respect to all structural units constituting the polymer. It is characterized in that the united body is at least on the surface, and the ratio S / Sg of the specific surface area S to the spherical equivalent specific surface area Sg is 6.0 or less.
Since the particles have a predetermined amount of the structural unit (A) and S / Sg is in the above range, they are excellent in dispersibility when dispersed after being magnetically collected once, and by using the particles, CLEIA Etc. can be improved.

The reason why the particles exhibit such an effect is not always clear, but the following reasons can be considered.
That is, by having a predetermined amount of the structural unit (A), the particle surface becomes hard, and when the magnetism is conducted, the particles are difficult to be deformed and densely clogged, and thus redispersion when demagnetized Is considered to be good. Further, that S / Sg is in the above range means that the particle surface is smooth, the first test probe such as antibody or antigen is immobilized on the present particle, and the probe and the antigen Since the first test probe and the second test probe easily come into contact with each other when the second test probe such as antibody or antibody reacts, the S / N ratio in the diagnostic method such as CLEIA is improved. It is thought that it can be done.

<Polymer>
The present particles have at least a polymer containing the structural unit (A). The present particles are preferably particles having a shell containing the polymer on the surface.
The polymer contained in the present particles may be used alone or in combination of two or more.

・ Structural unit (A)
The polymer has a structural unit (A) derived from an aromatic monomer.
When the polymer has a structural unit (A), the surface of the present particle becomes hard, and particles excellent in redispersibility are obtained, and a first inspection probe such as an antibody or an antigen is immobilized on the surface of the present particle When the reaction is carried out, the orientation of the probe is improved, and when the probe reacts with the second test probe such as an antigen or an antibody, the first test probe and the second test probe It is considered that the S / N ratio in the diagnostic method such as CLEIA can be improved because
Further, as the polymer has the structural unit (A), even if the thickness of the shell is thin, the shell can sufficiently cover the surface of the magnetic body, and magnetic particles excellent in surface smoothness close to a true sphere are obtained. It can be easily obtained.
The structural unit (A) contained in the polymer may be used alone or in combination of two or more.

The aromatic monomer is not particularly limited as long as it has an aromatic ring, but styrene, α-methylstyrene, 3-methylstyrene, 4-methylstyrene, t-butylstyrene, N, N-dimethyl-p -Aminoethylstyrene, 2,4-dimethylstyrene, N, N-diethyl-p-aminoethylstyrene, 2,4-diethylstyrene, vinyl naphthalene, vinyl anthracene, halogenated styrene, benzyl (meth) acrylate, naphthyl (meth ) Acrylate, phenoxyethyl (meth) acrylate, phenoxybutyl (meth) acrylate, 2- (1-naphthyloxy) ethyl (meth) acrylate, 2- (2-naphthyloxy) ethyl (meth) acrylate, 6- (1-) Naphthyloxyoxy) hexyl (meth) acrylate, 6- (2) Naphthyloxyoxy) hexyl (meth) acrylate, 8- (1-naphthyloxy) octyl (meth) acrylate, 8- (2-naphthyloxy) octyl (meth) acrylate, 4-vinylbenzoic acid, 4-ethenylphenol, 4 -Monofunctional monomers such as aminostyrene; divinylbenzene, divinylnaphthalene, diallyl phthalate and isomers thereof, trivinylbenzene, divinyltoluene, divinylxylene, divinylbiphenyl, bis (vinylphenyl) methane, bis (vinylphenyl) ethane, bis Examples thereof include crosslinkable (polyfunctional) monomers such as (vinylphenyl) propane and bis (vinylphenyl) butane.
The aromatic monomer preferably contains a crosslinkable monomer from the viewpoint of easily covering the surface of the magnetic substance completely and obtaining particles in which the magnetic substance is not exposed, and the like. Naphthalene is more preferred.

The content of the structural unit (A) is preferably 20 to 80% by mass, based on all structural units constituting the polymer, from the viewpoint of obtaining magnetic particles having more excellent redispersibility and surface smoothness. Is 22 to 65% by mass, more preferably 24 to 50% by mass.
The content of the structural unit (A) can be calculated from the amount of the monomer to be the structural unit (A) with respect to the total amount of monomers used when synthesizing the polymer.

・ Structural unit (B)
The polymer preferably includes a structural unit (B) having a functional group capable of chemically bonding to the probe. The magnetic particles obtained by the polymer having such a structural unit (B) can be easily used for diagnostic agents, drug research and the like.
The structural unit (B) contained in the polymer may be used alone or in combination of two or more.

  As the structural unit (B), the structural unit (A) may have a functional group capable of chemically bonding to the probe, but is preferably a structural unit other than the structural unit (A).

  The functional group capable of chemically bonding to the probe is preferably a group to which a probe such as an antigen or an antibody can be covalently bonded, and may be appropriately selected depending on the desired application. Examples thereof include a carboxylic acid group, a hydroxyl group, an epoxy group, an amino group, a triethyl ammonium group, a dimethylamino group and a sulfonic acid group.

  As the structural unit (B), a structural unit represented by the following formula (1) is preferable in that a target substance can be detected with higher sensitivity in a diagnostic method such as CLEIA using the present particles. .

〔式（１）中、Ｒ¹は、水素原子またはメチル基を示し、Ｒ²は、−（Ｃ＝Ｏ）−Ｏ−＊、−（Ｃ＝Ｏ）−ＮＲ⁴−＊（Ｒ⁴は、水素原子またはメチル基を示し、＊は、式（１）中のＲ³と結合する位置を示す）またはフェニレン基を示し、Ｒ²が−（Ｃ＝Ｏ）−Ｏ−＊である場合、Ｒ³は、水素原子またはプローブと化学結合可能な官能基を有する有機基を示し、Ｒ²が−（Ｃ＝Ｏ）−ＮＲ⁴−＊またはフェニレン基である場合、Ｒ³は、プローブと化学結合可能な官能基を有する有機基を示す。〕

Wherein (1), R ¹ represents a hydrogen atom or a methyl group, R ² is, - (C = O) -O - *, - (C = O) -NR 4 - * (R 4 is R represents a hydrogen atom or a methyl group, * represents a position to be bonded to R ³ in the formula (1)) or a phenylene group, and when R ² is — (C = O) —O— *, R is ³ represents a hydrogen atom or an organic group having a functional group capable of chemically bonding to the probe, and when R ² is — (C = O) —NR ⁴ — * or a phenylene group, R ³ chemically bonds to the probe The organic group which has a possible functional group is shown. ]

Examples of the organic group having a functional group capable of chemically bonding to the probe include a functional group itself capable of chemically bonding to the above-mentioned probe, -R ²⁰ -R ^{21 and the} like. Here, R ²⁰ is a hydrocarbon group having 1 to 12 carbon atoms, preferably a hydrocarbon group having 1 to 8 carbon atoms, or a part of these hydrocarbon groups is an oxygen atom, a nitrogen atom, a sulfur atom, phosphorus R ²¹ is a group substituted with at least one atom selected from the group consisting of atoms and halogen atoms, and R ²¹ is a functional group capable of chemically bonding to the aforementioned probe.

As a functional group capable of chemically bonding to the probe, a carboxy group and a carboxylate group are preferable.
Examples of the structural unit (B) having a carboxy group or a carboxylic acid group include structural units derived from the following monomers.
Specific examples of the monomer having a carboxy group include unsaturated monocarboxylic acids such as (meth) acrylic acid, crotonic acid, α-chloroacrylic acid and cinnamic acid; maleic acid, maleic anhydride, fumaric acid, itaconic acid, Unsaturated dicarboxylic acids (anhydrides) such as itaconic anhydride, citraconic acid, citraconic anhydride, mesaconic acid; unsaturated polybasic carboxylic acids (anhydrides) having 3 or more valences; 2- (meth) acryloyloxyethyl Succinic acid, 2- (meth) acryloyloxyethyl phthalic acid, 2- (meth) acryloyloxyethyl hexahydrophthalic acid, 2- (meth) acryloyloxypropyl succinic acid, 2- (meth) acryloyloxypropyl Phthalic acid, 2- (meth) acryloyloxypropyl hexahydrophthalic acid, ω-carboxypolycaprolactone mono (meth) acrylic acid There is a rate.
Specific examples of the monomer having a carboxylate group include monomers having a group in which a hydrogen ion of a carboxy group of the monomer having a carboxy group is substituted with a cation such as sodium ion, potassium ion, ammonium ion and the like. .

  As the structural unit (B), in a diagnostic method such as CLEIA using the present particle, it is possible to detect a target substance with higher sensitivity and to easily obtain a particle to which a probe such as a protein is easily bound. The structural unit represented by the following formula (2) is preferable in that

〔式（２）中、Ｒ¹は、水素原子またはメチル基を示し、Ｒ⁵は、炭素数２〜６の直鎖または分岐のアルキレン基を示し、Ｒ⁶は、炭素数２〜６のアルキレン基、シクロヘキシレン基またはフェニレン基を示す。〕

[In formula (2), R ¹ represents a hydrogen atom or a methyl group, R ⁵ represents a linear or branched alkylene group having 2 to 6 carbon atoms, and R ⁶ represents an alkylene having 2 to 6 carbon atoms Group, a cyclohexylene group or a phenylene group is shown. ]

  The structural unit (B) is a 2- (meth) acryloyloxyethyl succinic acid, 2- (meth) acryloyloxyethyl succinic acid, from the viewpoint that the target substance can be detected with higher sensitivity in a diagnostic method such as CLEIA using this particle. (Meth) acryloyloxyethyl phthalic acid, 2- (meth) acryloyloxyethyl hexahydrophthalic acid, 2- (meth) acryloyloxypropyl succinic acid, 2- (meth) acryloyloxy propyl phthalic acid, Particularly preferred is a structural unit derived from at least one selected from the group consisting of 2- (meth) acryloyloxypropyl hexahydrophthalic acid.

With regard to the content of the structural unit (B), in the diagnostic method such as CLEIA using the present particle, the functional group density to which the probe is bound is within the following range from the viewpoint that the target substance can be detected with higher sensitivity. The amount is preferably 10 to 70% by mass, more preferably 15 to 70% by mass, and particularly preferably 22 to 60% by mass, based on all structural units constituting the polymer. Within the above numerical range, the probe binds sufficiently, and the activity of the probe does not easily drop.
The content of the structural unit (B) can be calculated from the amount of the monomer to be the structural unit (B) with respect to the total amount of monomers used when synthesizing the polymer.
In addition, when the said structural unit (A) has a functional group which can be chemically bonded with a probe, although said that the said polymer has a structural unit (B), content of this structural unit Is counted as the amount of structural unit (A).

・ Structural unit (C)
The polymer preferably contains a structural unit (C) derived from a weakly hydrophilic monomer other than the structural units (A) and (B). Nonspecific binding of the probe to the present particle can be suppressed by the polymer having such a structural unit (C), and the sensitivity is higher in a diagnostic method such as CLEIA using the present particle. Target substance can be detected.
The structural unit (C) contained in the polymer may be used alone or in combination of two or more.

  The weakly hydrophilic monomer refers to a monomer having a solubility of 1 to 10 g in 100 g of water at 25 ° C.

  Examples of the structural unit (C) include the following structural unit (C-1) and the following structural unit (C-2). Among these, non-specific binding of the probe to the present particle can be suppressed, and in the diagnostic method such as CLEIA using the present particle, the target substance can be detected with higher sensitivity, etc. From the above, the structural unit (C-1) is preferred. Specifically, when the polymer has a structural unit (C-1), the polymer is prevented from destroying hydration water present on the surface of a protein or the like contained in the sample, and as a result, the protein or the like is not modified. It can prevent. This can reduce nonspecific adsorption.

  The structural unit (C-1) is a monoacrylate monomer that retains intermediate water when it is polymerized to form a homopolymer and coexist with free water, or a monomethacrylate monomer having the same structure as the side chain of the homopolymer (hereinafter “monomer It is a structural unit derived from (C-1). As a monomer (C-1), the said monoacrylate monomer is preferable.

  In the present invention, “to retain intermediate water when polymerized into homopolymer and coexist with free water” means a temperature rising process in a DSC curve obtained by differential scanning calorimetry by differential scanning calorimetry (DSC). It refers to a polymer in which an exothermic peak due to water crystallization is observed below -10 ° C. That is, "intermediate water" refers to water observed as an exothermic peak due to crystallization below -10 ° C in a temperature rising process in a DSC curve obtained by differential scanning calorimetry. Here, the "temperature rising process" refers to a temperature rising process after cooling the magnetic particles to -100 ° C or less in differential scanning calorimetry. Moreover, "free water" refers to water observed as a melting point (endothermic peak) around 0 ° C. in a temperature rising process in a DSC curve obtained by differential scanning calorimetry.

  The monomer (C-1) is represented by the following formula (4) or (5) from the viewpoint that the target substance can be detected with higher sensitivity in a diagnostic method such as CLEIA using the present particles. The compound is preferably a compound, and more preferably a compound represented by the following formula (5).

〔式（４）中、Ｒ⁹は、水素原子またはメチル基を示し、Ｒ¹⁰は独立に、炭素数１〜３のアルカンジイル基を示し、Ｒ¹¹は、炭素数１〜３のアルキル基を示し、ｎは、１〜５の整数を示し、好ましくは１〜３の整数である。〕

[In formula (4), R ⁹ represents a hydrogen atom or a methyl group, R ¹⁰ independently represents an alkanediyl group having 1 to 3 carbon atoms, and R ¹¹ represents an alkyl group having 1 to 3 carbon atoms. N is an integer of 1 to 5 and is preferably an integer of 1 to 3. ]

〔式（５）中、Ｒ¹²は、水素原子またはメチル基を示し、ｍは、１または２を示し、好ましくは１である。〕

[In Formula (5), R ¹² represents a hydrogen atom or a methyl group, m represents 1 or 2, and is preferably 1. ]

  As the monomer (C-1), preferably, methoxyethyl (meth) acrylate, tetrahydrofurfuryl (meth) acrylate, di (ethylene glycol) methyl ether (meth) acrylate, di (ethylene glycol) ethyl ether (meth) acrylate And tri (ethylene glycol) methyl ether (meth) acrylate, tri (ethylene glycol) ethyl ether (meth) acrylate, etc., among them, tetrahydrofurfuryl from the viewpoint of being able to further suppress nonspecific adsorption of protein. (Meth) acrylate is more preferred.

  The structural unit (C-2) is a structural unit derived from a mono (meth) acrylate monomer represented by the following formula (3).

〔式（３）中、Ｒ⁷は、水素原子またはメチル基を示し、Ｒ⁸は、炭化水素基を示す。〕

[In Formula (3), R ⁷ represents a hydrogen atom or a methyl group, and R ⁸ represents a hydrocarbon group. ]

As a hydrocarbon group in R < ⁸ >, Preferably a C1-C30 hydrocarbon group, More preferably, a C1-C24 hydrocarbon group is mentioned.

  As the monomer represented by the formula (3), methyl (meth) acrylate, ethyl (meth) acrylate, propyl (meth) acrylate, butyl (meth) acrylate, 2-ethylhexyl (meth) acrylate, ( Examples include stearyl methacrylate, lauryl (meth) acrylate and cyclohexyl (meth) acrylate.

The content of the structural unit (C) is relative to all structural units constituting the polymer in that the target substance can be detected with higher sensitivity in the diagnostic method such as CLEIA using the present particles. Preferably it is 10-60 mass%, More preferably, it is 20-40 mass%.
The content of the structural unit (C) can be calculated from the amount of the monomer to be the structural unit (C) with respect to the total amount of monomers used in synthesizing the polymer.

-Other structural unit The said polymer may have other structural units other than the said structural unit (A)-(C).
The other structural unit is not particularly limited, and examples include structural units derived from the following monomers.
Examples of the monomer include vinyl esters such as vinyl acetate and vinyl propionate, unsaturated nitriles such as acrylonitrile, and polyfunctional (meth) such as ethylene glycol di (meth) acrylate, trimethylolpropane triacrylate and dipentaerythritol hexaacrylate. An acrylate etc. are mentioned.
Among these, it is preferable to use a polyfunctional (meth) acrylate from the viewpoint that the exposure of the magnetic material on the surface of the present particles can be more easily suppressed.

The content of the other structural unit is preferably 0 to 30% by mass, and more preferably 0 to 20% by mass, based on all structural units constituting the polymer.
The content of the other structural unit can be calculated from the amount of the monomer to be the other structural unit with respect to the total amount of monomers used in synthesizing the polymer.

  The polymer may be synthesized by a conventionally known method, for example, a monomer for inducing the structural unit and, if necessary, auxiliary materials (polymerization initiator, emulsifier, dispersant, surfactant, electrolyte, crosslinking agent , A molecular weight regulator, etc.), and the polymerization can be carried out by polymerization.

<Structure, physical properties, etc. of magnetic particles>
The present particles are not particularly limited in structure or the like as long as the polymer is present on the surface, but (I) particles in which a magnetic substance is dispersed in the continuous phase of the polymer, (II) non-silica such as silica A particle having as a core a particle in which a magnetic substance is dispersed in a continuous phase of a magnetic substance, a particle having a layer containing the polymer as a shell, (III) a magnetic substance or a secondary aggregate thereof as a core (IV) A mother particle having a core particle comprising a nonmagnetic material such as (IV) polymer or silica, and a magnetic material layer containing a magnetic material present on the surface of the core particle or a secondary aggregate thereof The particle etc. which use a particle as a core and make a layer containing the said polymer a shell are mentioned. Among these, particles of (IV) are preferable in that they have excellent magnetic response and can uniformly control the particle size.
The particles of (IV) will be described below.

<Nuclear particle>
The core particle is basically a nonmagnetic substance, and any of an organic substance and an inorganic substance can be used, and can be appropriately selected according to the purpose of use of the present particle, etc. From the viewpoint of ease, particles made of an organic substance such as a polymer are preferred.
The polymer is preferably a vinyl-based polymer, more preferably cross-linked polystyrene, cross-linked acrylate, or cross-linked polymethyl methacrylate. These polymers may have a functional group such as a carboxy group introduced.
Such core particles are described in conventionally known methods, for example, in JP-B-57-24369, JP-A-61-215602, JP-A-61-215603, JP-A-61-215604. It can be manufactured by the method of

The average particle diameter of the core particles is preferably 0.1 to 10 μm, more preferably 0.2 to 5 μm, particularly preferably from the viewpoint of excellent magnetic separation, less occurrence of gravitational sedimentation and uniform reaction field. Is 0.3 to 2 μm.
The average particle size of the core particles is a number average particle size measured using Coulter principle known as an electrical detection zone method.

<Magnetic substance>
The magnetic substance is not particularly limited as long as it is a magnetic material, but an iron oxide-based substance is representative, and MFe ₂ O ₄ (M = Co, Ni, Mg, Cu, Mn, Zn or Li _0.5 Fe _0.5, etc.) Ferrites represented by the formula, magnetite represented by Fe ₃ O ₄ , γ-Fe ₂ O _{3 and the} like. In particular, γ-Fe ₂ O ₃ and Fe ₃ O ₄ are preferable as the magnetic material having high saturation magnetization and low remanent magnetization.

As the magnetic substance, a material having superparamagnetism free from residual magnetism is preferable, from the viewpoint of obtaining magnetic particles that are superior in magnetic collection performance and magnetic separation performance. The magnetic substance having superparamagnetism is not particularly limited. For example, superparamagnetic metal oxides such as various ferrites such as iron trioxide (Fe ₃ O ₄ ) and γ-triiron dioxide (γ-Fe ₂ O ₃ ) are used. The thing is mentioned.

The shape of the magnetic substance is not particularly limited, but is preferably magnetic fine particles, and the particle size of the magnetic fine particles can sufficiently exhibit the characteristics of the magnetic substance, and in particular, the present particles are excellent in magnetic separation performance. It is preferably 5 to 25 nm, more preferably 5 to 20 nm, and still more preferably 8 to 15 nm from the viewpoint of obtaining.
The particle size is an average value of particle sizes of 100 randomly selected particles in an electron micrograph.

It is preferable that the magnetic body is a particle whose surface is subjected to a hydrophobization treatment.
The method for hydrophobizing the surface of the magnetic substance is not particularly limited. For example, there is a method in which a hydrophobic substance having a portion having high affinity for the magnetic substance and a hydrophobic portion in the molecule is brought into contact with the magnetic substance and bound. It can be mentioned. More specifically, examples of the method include a method of obtaining particles from a commercially available oily magnetic fluid and drying the particles.

Examples of the hydrophobic substance include silane compounds represented by silane coupling agents, fatty acids, and surfactants represented by fatty acid soaps. Among these, fatty acids represented by RCOOH (wherein R is a hydrocarbon group) are preferable. The carbon number of the fatty acid is not particularly limited, but is preferably 6 or more and 30 or less, and particularly preferably 10 or more and 24 or less. Specifically, pelargonic acid, capric acid, undecylic acid, lauric acid, tridecylic acid, myristic acid, pentadecylic acid, palmitic acid, heptadecylic acid, stearic acid, nonadecanoic acid, arachic acid, behenic acid, lignoceric acid, cerotic acid, Saturated fatty acids such as heptacosanoic acid, montanic acid and melisic acid, unsaturated fatty acids such as undecylenic acid, oleic acid, oleic acid, elaidic acid, celylic acid, erucic acid, brashidic acid, sorbic acid, linoleic acid, linolenic acid, arachidonic acid Be
As the hydrophobic substance, salts of these fatty acids can also be used.

  The amount of the magnetic material used may be appropriately changed according to the particle size of the base particles and the magnetic material, and is not particularly limited, but it is used in such an amount that a plurality of magnetic materials cover the entire surface of the core particle. It is preferable to use an amount such that the thickness of the magnetic layer present on the surface of the core particle is substantially uniform, and the thickness of the magnetic layer is about the same as the particle size of the magnetic particles. It is more preferable to use in an amount such that the particle diameter of the magnetic fine particles is about twice that of the fine particles.

<Mother particle>
There is no particular limitation on the method of producing the base particles in which the magnetic material layer is formed on the surface of the core particles, but for example, dry blending of core particles such as non-magnetic organic polymer particles and magnetic material is carried out. There is a method of combining both particles by applying an external force from outside.
As a method of applying physical force, for example, a method utilizing mechanochemical effect such as mortar, automatic mortar, ball mill, blade pressure type powder compression method, mechanofusion method, high speed such as jet mill, hybridizer etc. There is a method of utilizing an impact in air flow. In order to carry out complexation efficiently and firmly, it is desirable that the physical adsorption force be strong. The method includes, for example, stirring at a circumferential velocity of the stirring blade of preferably 15 m / s or more, more preferably 30 m / s or more, still more preferably 40 to 150 m / s in a container with a stirring blade. . When the circumferential velocity of the stirring blade is lower than 15 m / sec, the magnetic material may not be sufficiently adsorbed on the surface of the core particle. The upper limit of the peripheral velocity of the stirring blade is not particularly limited, but may be determined in terms of the apparatus used, energy efficiency, and the like.

  It is preferable that the base particles also have the same S / Sg as the following main particles, from the viewpoint that the main particles exhibiting the above effects can be easily obtained.

<shell>
The shell is, for example, a monomer for inducing the structural unit in the presence of the base particles prepared by the above-mentioned method, and, if necessary, auxiliary materials (polymerization initiator, emulsifier, dispersant, surfactant, electrolyte, It can form by performing superposition | polymerization in the liquid in which the crosslinking agent, the molecular weight regulator, etc. were added.

  The shell containing the polymer having the structural unit (B) preferably uses, as the monomer, a monomer having a functional group capable of chemically bonding to the probe, as described in JP-A-2007-262113. Alternatively, it may be formed by a method of generating a functional group capable of chemically bonding to the probe by a reaction in forming a shell, or a method of introducing a functional group capable of chemically bonding to the probe after forming the shell.

  In the shell, even if the thickness of the shell is thin, the shell can completely and completely cover the base particles, and particles having excellent surface smoothness close to a true sphere can be easily obtained. It is preferable that it is a layer which has a polymer as a main component, and content of the said polymer with respect to the whole said shell is preferably 80-100 mass%, More preferably, it is 90-100 mass%.

  The shell covers the base particle so that the magnetic material is not exposed on the surface of the particle, from the viewpoint that the performance of the particle is maintained for a long period of time and magnetic particles excellent in magnetism and redispersibility are obtained. Is preferred.

The thickness of the shell is not particularly limited, but is preferably such a thickness that the magnetic material is not exposed to the surface of the particle, and according to the polymer, even if the thickness of the shell is thin, the shell is completely From the viewpoint that the mother particles can be covered cleanly and the thickness of the shell is thin, the speed of magnetic collection can be increased, etc., it is preferably 0.01 to 0.3 μm, more preferably 0.02 to 0. It is 2 μm. The thickness d (μm) of the shell is calculated by the following equation.
d = (R−r) / 2 (R: particle diameter of magnetic particles (μm), r: particle diameter of base particles (μm))

  It is preferable that the magnetic particles are not exposed on the surface of the present particles. When the magnetic substance is exposed, nonspecific adsorption of biochemical substances and the like tends to increase, and the purity of the separated substances tends to decrease, and the addition of an acid or the like may cause the superparamagnetic fine particles to be dissolved.

  As a method of measuring the degree of exposure of the magnetic substance to the surface of the particles, it is an orthophenylene diamine (OPD) which is one of the substrates used in ordinary enzyme immune reactions and which reacts sensitively with iron. can do. Since OPD is a substrate for enzyme immunoassay, the effect of the magnetic substance exposed on the surface of the present particle can be confirmed in a form closer to an actual assay system.

As the measurement method, specifically, (a) 1 mg of magnetic particles, (b) 7.8 mg of orthophenylenediamine dihydrochloride, (c) 4.5 μL of 30% hydrogen peroxide water, and (d) 0 It can be evaluated by the absorbance at 450 nm of the magnetic particle dispersion supernatant obtained by mixing 1.5 mL of 1 M aqueous sodium phosphate solution (pH 5) and stirring at 25 ° C. for 30 minutes.
When the magnetic substance is exposed, hydrogen peroxide is reduced by the reduction action of iron, and accordingly, the orthophenylene diamine is dimerized to change to 2,3-diaminophenazine. Since 2,3-diaminophenazine has an absorption maximum at around 492 nm, the degree of magnetic substance exposure can be quantified by measuring the absorbance near this.

The absorbance measurement is carried out, for example, by taking 300 μL of the reaction supernatant and using a microplate reader (type name: Model 450, manufactured by BIO-RAD). The absorbance obtained in this manner is preferably 1.5 or less, more preferably 1.2 or less, and particularly preferably 1.0 or less.
When the absorbance is 1.5 or more, the exposure of the magnetic substance on the particle surface may be too high, which may lead to an increase in nonspecific adsorption.

<S / Sg>
The particles preferably have a ratio S / Sg of the specific surface area S to the spherical equivalent specific surface area Sg of 6.0 or less, preferably 4.0 or less, more preferably 1.5 or less, and the lower limit is preferably small. Although it does not restrict | limit, it is 1.0, for example.
When the S / Sg is in the above range, the smoothness of the particle is increased, so in the diagnostic method such as CLEIA using the particle, the test probe and the luminescent substrate are easily accessible to the surface of the particle and easily bound. The signal goes high. In addition, the cleaning property with respect to the test probe and the luminescent substrate not bound to the particles becomes good, and the noise becomes low. As a result of these, the S / N becomes high, and the target substance can be detected with higher sensitivity.
By using a polymer containing the structural unit (A), and by reducing the thickness of the shell, etc., it is possible to obtain the present particles having S / Sg in the above range.
The S / Sg can be specifically measured by the method described in the following Examples.

<Functional group density to bind the probe>
The present particles preferably have a functional group density for binding a probe of 1 to 200 μmol, more preferably 2 to 90 μmol, particularly preferably 5 to 30 μmol, per 1 g of the particle (A).
When the functional group density for binding the probe is in the above range, the redispersibility is excellent, and by using the particles, it is possible to express high sensitivity which is particularly noticeable in a diagnostic method such as CLEIA.
When the functional group is a carboxy group, the functional group density for binding a probe can be measured by the same method as the measurement of the surface charge amount in the following examples.

<Number average particle size>
The number average particle size of the present particles is preferably 5 nm to 20 μm, more preferably 100 nm to 15 μm, and particularly preferably 200 nm to 10 μm.
The number average particle size can be determined by the Coulter principle known as the electrical detection zone method.
When the number average particle size is in the above range, the present particles having a large amount of probe binding per unit volume can be easily obtained, and by using the obtained present particles, the sensitivity in a diagnostic method such as CLEIA can be excellent. Become. If the particle size is below the lower limit of the above range, separation using centrifugation, magnetic separation, etc. tends to take a long time, and the separation between the washing solvent such as water and the magnetic particles tends to be insufficient. Removal of non-target molecules (for example, biologically relevant substances such as proteins and nucleic acids) may be insufficient, and sufficient purification may not be possible. On the other hand, when the particle size exceeds the upper limit of the above range, the specific surface area decreases, and as a result, the binding amount of the probe decreases, and as a result, the sensitivity may decrease.

CV value
The coefficient of variation (CV value) of the number average particle diameter of the particles is preferably 20% or less in that the target substance can be detected with higher sensitivity in a diagnostic method such as CLEIA using the particles. And more preferably 15% or less.
When the CV value is in the above range, the variation when performing the immunoassay is reduced.

<Average circularity>
The average circularity of the particles is preferably 0.965 to 1.000, more preferably 0.975 to 1.000, and particularly preferably 0.985 to 1.000.
If the average degree of circularity is less than 0.965, the shape of the particles may be heterogeneous, which may result in large measurement variation in immunodiagnosis.
Specifically, the average circularity was measured when the circularity of each of 2000 particles was measured using a flow type particle image analyzer (“FPIA-3000” manufactured by Sysmex Corporation). It is the average value of 2000 roundnesses.
The degree of circularity is a value obtained by dividing the perimeter measured by optically detecting particles by the perimeter of the equivalent circle having the same projected area.

<Magnetic particles bound to a probe>
The particles may be probe-bound particles, depending on the desired application.
The method for binding the probe to the magnetic particle is not particularly limited, and physical adsorption method or chemical bonding method can be mentioned, but the probe can be chemically linked from the point that the probe can be firmly bound to the magnetic particle. Bonding is preferred. Examples of this method include a method of reacting a functional group capable of chemically bonding with a probe on the surface of a magnetic particle and a group in the probe that reacts with the functional group, and a method of using a condensing agent such as carbodiimide.

<probe>
The probe is not particularly limited, but is preferably a substance having specific binding properties, for example, an antigen, an antibody, (strept) avidin, protein A, protein G, proteins such as enzymes, hormones, DNA, RNA, etc. Nucleic acids, lipids, physiologically active sugar chain compounds, various affinity tag capture substances, coenzymes such as biotin, chemical substances having specific physiological activity (or which may have specific physiological activity), etc. Can be mentioned.

In the present particles for diagnostic agents, antigens or antibodies are mainly used as probes. In this case, the antigen or antibody is not particularly limited as long as it is a substance that reacts with the substance to be detected or to be detected, for example, anti-antiplasmin antibody for antiplasmin test, anti-D dimer antibody for D dimer test, FDP test Anti-FDP antibody for anti-tPA antibody for tPA test, anti-TAT complex antibody for TAT test, anti-FPA antibody for FPA test etc. Antigen or antibody for coagulation / fibrinolysis related test; Anti-BFP antibody for BFP test, for CEA test Tumor-associated test antigen or antibody such as anti-CEA antibody, anti-AFP antibody for AFP test, anti-ferritin antibody for ferritin test, anti-CA19-9 antibody for CA19-9 test; anti-apolipoprotein antibody for apolipoprotein test, β2-micro Anti-β2-microglobulin antibody for globulin test, anti-α1- for α1-microglobulin test Antigens or antibodies for serum protein related examinations such as immunoglobulins, anti-immunoglobulin antibodies for immunoglobulin examinations, anti-CRP antibodies for CRP examinations; Antigens or antibodies for endocrine function examinations such as anti-HCG antibodies for HCG examinations; for HBs antigens examinations Anti-HBs antibody, HBs antigen for HBs antibody test, HCV antigen for HCV antibody test, HIV-1 antigen for HIV-1 antibody, HIV-2 antigen for HIV-2 antibody test, HTLV-1 antigen for HTLV-1 test, Mycoplasma Antigens or antibodies for infectious disease related tests such as mycoplasma antigen for toxoplasma test, toxoplasma antigen for toxoplasma test, streptolysin O antigen for ASO test; DNA antigen for anti-DNA antibody test, heat denatured human IgG for RF test etc autonomic related test Antigen or antibody; anti-digoxin antibody for digoxin test, lidocaine test These include antigens or antibodies for drug analysis such as anti- lidocaine antibodies.
As the antibody, either a polyclonal antibody or a monoclonal antibody may be used.

  Further, for the present particles for affinity carrier, mainly, as a probe, protein A, protein G, protein such as enzyme or hormone, nucleic acid such as DNA or RNA, lipid, physiologically active sugar chain compound or the like is used.

  Furthermore, in the present particles for drug discovery, a chemical substance to be analyzed (analyzed chemical substance; corresponds to a probe molecule) as a probe is used to analyze the interaction using a specific interaction with a protein etc. Drug discovery research is possible by analyzing and analyzing chemical substances to be analyzed by selecting and purifying proteins (which correspond to target molecules) that specifically interact with the chemical substance to be analyzed by / or measuring It is.

  As a probe, what was contained in the sample containing a probe may be used. The sample may be used as it is in the present method or may be used in the present method after pretreatment such as separation of the probe.

<Use>
The particles can be suitably used, for example, as a support, carrier or medium in the fields of bioengineering, diagnostics and pharmaceutics, and specifically, for example, separating target substances such as antigens, antibodies, biomolecules, nucleic acids etc. It can be suitably used as means for detecting and / or detecting.
In such an application, magnetic particles to which a probe is not bound may be used as it is, or magnetic particles to which a probe is bound may be used.

  More specifically, an antigen or antibody such as a protein may be bound to the present particle, and the measurement target may be quantified and characterized by an antigen-antibody reaction with the antibody or antigen to be measured; the antibody is bound to the present particle May be combined with antigens such as viruses, bacteria, cells, hormones, dioxins and other chemical substances, etc. to be recovered and concentrated; and nucleic acid analogues such as DNA are bound to the particles to hybridize nucleic acids. May be used to recover and detect nucleic acids using this method, or to recover and detect chemical substances such as proteins and dyes that bind to nucleic acids; binding avidin or biotin to this particle to recover biotin or a molecule having avidin These particles may be detected by binding an antibody or antigen to the particles, and may be subjected to enzyme immunoassay using colorimetric method or chemiluminescence.

  Moreover, about the diagnostic item which used the conventional 96 well plate etc. as a support | carrier, it can replace with the automatic analyzer which utilized magnetism, and can be used by using this particle | grain. Substances to be diagnosed include proteins of biological origin, hormones such as luteinizing hormone and thyroid stimulating hormone; various cancer cells; proteins serving as cancer markers such as prostate specific marker and bladder cancer marker; hepatitis B virus, Hepatitis C virus, viruses such as herpes simplex virus; bacteria such as gonococci, MRSA; fungi such as candida, cryptocox; protozoa and parasites such as toxoplasma; components such as viruses, bacteria, fungi, protozoa and parasites Proteins and nucleic acids; environmental pollutants such as dioxins; chemicals such as medicines such as antibiotics and antiepileptic agents; and the like.

  The particles can be used as a diagnostic agent. The diagnostic agent contains the present particles, but may contain, in addition to the present particles, conventionally known components. The present particle may be a magnetic particle to which a probe is bound or may be a magnetic particle to which a probe is not bound, but usually, as described above, a magnetic particle to which an antigen or antibody is bound as a probe including.

«Detection and separation method»
The detection and separation method according to the present invention includes the step of detecting or separating a target substance in a sample using the present particles, and diagnosis can be performed using the detection and separation method.
In these methods, since the present particles are used, highly sensitive detection, separation and diagnosis can be performed.

  As an example of the separation method, a method of removing and separating other than the target substance by reacting the functional group on the surface of the particle and the target substance, collecting and washing the target substance, the present particle to which the probe is bound and the target substance And the reaction is followed by current collection and washing to remove and remove non-target substances.

  As an example of the detection method, the present particle to which a probe is bound is reacted with a target substance that specifically binds to the probe, and if necessary, after passing through a magnetism and washing, the target substance is There is a method of detecting.

In these detection / separation methods, it is possible to reduce non-specific reaction, to improve the dispersibility of the present particles, to suppress denaturation of the probe such as antibody, and to further improve the S / N ratio in the diagnostic method such as CLEIA. It is preferable to include the step of binding a blocking agent from the viewpoint of being able to do so.
As a blocking agent, bovine serum albumin (BSA), skimmed milk, gelatin, casein, synthetic polymers and the like are generally used.
The method of binding the blocking agent may be appropriately selected depending on the particles and the blocking agent to be used, and may be carried out by a conventionally known method.

  One example of the diagnostic method is a method of diagnosing based on the presence or absence of detection of a target substance.

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

[Measurement of particle size, CV value and average circularity]
The particle size of each magnetic particle is total counted using a flow type particle image analyzer (model number FPIA-3000) manufactured by Sysmex Corp. for a sample diluted with water so that the solid content concentration is about 0.5%. It asked for setting to 2000 pieces. In addition, the coefficient of variation (CV value) of the number average particle diameter was also calculated based on CV = (standard deviation / number average particle diameter), and 2000 was measured when the circularity of each of 2000 particles was measured. The average circularity was calculated as the average value of the circularities.

[S / Sg]
The specific surface area S (m ² / g) by argon gas adsorption was measured for the heat-dried magnetic particles, using a fully automatic gas vapor adsorption amount measuring apparatus (manufactured by Yuasa Ionics Co., Ltd.) Autosorb-1.

Next, the density d d of the aqueous dispersion containing about 10% by weight of magnetic particles was measured with a 5 [ml] pycnometer. Further, the solid fraction f of the aqueous dispersion is determined from the weight change before and after the aqueous dispersion is dried at 200 ° C. for 5 minutes, and the density of the magnetic particles is calculated from the following formula (A) using d d and f. The ρ (g / m ³ ) was determined. From the determined density ρ of the magnetic particles and the number average particle diameter d (m) of the magnetic particles determined by the measurement of the particle diameter, the surface of the magnetic particles is regarded as a smooth perfect sphere based on the following formula (B) The spherical equivalent specific surface area Sg (m ² / g) of the particles was measured.
"S / Sg" was calculated from S and Sg which were calculated | required. The results are shown in Table 1.
1 / .rho.d = (f / .rho.) + ((1-f) /. Rho.w) (A)
[In equation (A), ρ w is the density of water. ]
Sg = 6 / (d · ρ) (B)

[Measurement of surface charge amount]
0.2 g of magnetic particles were dispersed in 40 g of 0.1 mass% aqueous solution of Tween 20, and 500 μL of 0.1 mol / L aqueous sodium hydroxide solution was added thereto to neutralize the carboxy group present on the particles . To the obtained particle dispersion, 0.01 mol / L sulfuric acid was dropped while measuring the conductivity using a conductivity titration apparatus (794 Basic Titrino, manufactured by Metrohm), and the conductivity titration curve was determined. The surface charge amount (μmol / g) of the magnetic particles was calculated by calculating the amount of sulfuric acid consumed for protonation of the COONa group on the particles from the curve and dividing by the weight of the magnetic particles used. The results are shown in Table 1.

[Measurement of magnetic material exposure (absorbance after addition of OPD)]
An iron detection solution was prepared by mixing 7.8 mg of orthophenylenediamine dihydrochloride, 4.5 μL of 30 mass% hydrogen peroxide water, and 1.5 mL of 0.1 M aqueous sodium phosphate solution (pH 5). 1 mg of magnetic particles was added thereto, and the mixture was stirred at 25 ° C. for 30 minutes to dimerize ortho-phenylenediamine according to the amount of exposure of the magnetic substance (iron) on the surface of the magnetic particles to color the solution. After color development, the solution was subjected to magnetic separation, 300 μL of the supernatant was removed, and the absorbance of the supernatant at 450 nm was measured using a microplate reader (Model 450) to evaluate the degree of exposure of the magnetic particles on the magnetic particle surface. . The higher the absorbance is, the higher the magnetic substance exposure is.

[Redispersion evaluation]
3 mg of the magnetic particles obtained in each example and comparative example were dispersed in 30 μL of water, respectively. The obtained aqueous dispersion was charged into an eppendorf tube, the particles were separated magnetically, and the supernatant was removed. The particles were then dispersed in 270 μL MES buffer (100 mM, pH 5.0), 30 μg anti-PSA antibody was added and incubated for 10 minutes at room temperature. Then, 30 μL of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (5 mg / mL) was added and incubated at room temperature for 1 hour. The cells were washed three times with TBS-T (0.05% by mass Tween 20) buffer to obtain antibody-bound particles. The resulting antibody-bound particles were incubated for 3 hours at room temperature.
0.5 mg of the antibody-bound particles incubated was dispersed in 1.5 mL of TBS-T buffer to obtain a dispersion of antibody-bound particles, and the dispersion was loaded into an eppendorf tube. The tube was attached to a magnetic bead separation stand (Magical Trapper, manufactured by Toyobo Co., Ltd.) and allowed to stand for about 5 minutes to confirm that the particles were collected in the form of pellets. The tube was then removed from the stand to free the particles of magnetism, checked for 1 minute and evaluated according to the following criteria. The results are shown in Table 1.
<Evaluation criteria>
:: Pellets gradually disintegrated and disappeared while staying in place.
○: The pellets were disintegrated little by little while staying in place, but the pellets remained.
Δ: The pellet dropped to the lower part of the tube while gradually becoming smaller.
X: The pellet dropped to the lower part of the tube while being completely retained.

[Chemiluminescence immunoassay (CLEIA)]
A dispersion of antibody-bound particles was prepared in the same manner as in the evaluation of redispersibility, and 30 μL of the dispersion of antibody-bound particles (corresponding to 300 μg of particles) was taken in an eppendorf tube and diluted with 970 μL of TBS-T buffer. 50 μL of this diluted solution was respectively dispensed to wells 1 and 2 of a 96-well white plate (manufactured by Corning).
Subsequently, for noise measurement, 10 μL of BSA / TBS buffer (1% by mass BSA, 137 mmol / L sodium chloride, 2.7 mmol / L potassium chloride, 25 mmol / L Tris-HCl, pH 7.4) is added to well 1 In addition, 0.01 ng of PSA antigen dissolved in 10 μl of BSA / TBS buffer was dispensed to well 2 for signal measurement, and each was reacted at 37 ° C. for 10 minutes.
Thereafter, the particles are separated by magnetic separation, and then washed with TBS-T buffer using a washer for 96-well plate (manufactured by Tecan Japan Ltd., Hydro Flex), and then labeled body fluid (ALP (alkaline phosphatase) 50 μL of the labeled antibody solution was dispensed and allowed to react at 37 ° C. for 10 minutes. After the reaction, the particles are separated by magnetic separation, and then washed with TBS-T buffer using a washer for 96 wells (Hydro Flex), and then a substrate solution of ALP (Lumipulse substrate solution: Fujirebio Co., Ltd.) Made to react at 25 ° C. for 5 minutes, and the luminescence intensity was measured using ARVO X5 (manufactured by PerkinElmer).
The amount of chemiluminescence in well 1 was noise (N), and the amount of chemiluminescence in well 2 was signal (S). Furthermore, the value S / N ratio was calculated by dividing the signal (S) by the noise (N). The results are shown in Table 1.

Synthesis Example 1
Acetone is added to an oil-based magnetic fluid ("EXP series, EMG" manufactured by Farotech Co., Ltd.) to precipitate particles, which are then dried to obtain a ferrite-based magnetic material having a hydrophobized surface. Body fine particles (average primary particle size: 0.01 μm) were obtained.
Next, 61 g of polyacrylic particles (“MX-150”, manufactured by Soken Chemical Co., Ltd., number average particle diameter: 1.5 μm) and 61 g of the hydrophobized magnetic fine particles are well mixed by a mixer, and this mixture is obtained Is treated at a peripheral speed of 100 m / s (16, 200 rpm) of a blade (agitating blade) using a hybridization system NHS-0 type (manufactured by Nara Machinery Co., Ltd.) for 5 minutes, Base particles (number average particle diameter: 1.9 μm) having a body layer on the surface were obtained.

Example 1
200 g of a 0.50% by mass aqueous solution of sodium dodecyl sulfate was charged into a 500 mL separable flask, and then 10 g of the base particles obtained in Synthesis Example 1 were added, dispersed by a homogenizer, and heated at 60 ° C. to maintain the temperature.
Next, in another container, 50 g of a 0.50% by mass aqueous solution of sodium dodecyl sulfate, 5.0 g of divinylbenzene (hereinafter referred to as "DVB"), and 4.0 g of 2-methacryloyloxyethyl succinic acid (hereinafter referred to as "SA"). , 3.0 g of tetrahydrofurfuryl methacrylate (hereinafter referred to as "THFMA") and 0.60 g of tert-butyl 2-ethylperoxyhexanoate (Perbutyl O, manufactured by NOF Corporation) are dispersed, A pre-emulsion was obtained. The entire pre-emulsion was dropped into the 500 mL separable flask maintained at 60 ° C. over 2 hours. After completion of the dropwise addition, the mixture was kept at 60 ° C. and stirred for 30 minutes. Subsequently, the particles in the separable flask were separated magnetically, and then repeatedly washed with distilled water to obtain magnetic particles. The number average particle diameter of the obtained particles was 2.0 μm, the CV value was 11%, and the thickness of the shell was 0.05 μm.

Example 2
The same operation as in Example 1 was performed except that 5.0 g of styrene (hereinafter referred to as "ST") was used instead of 5.0 g of DVB as a raw material for producing a pre-emulsion. The number average particle diameter of the obtained particles was 2.1 μm, the CV value was 12%, and the thickness of the shell was 0.1 μm.

[Example 3]
The same operation as in Example 1 was performed except that 5.0 g of 4-vinylbenzoic acid was used as a raw material for producing a pre-emulsion instead of 5.0 g of DVB. The number average particle diameter of the obtained particles was 2.1 μm, the CV value was 11%, and the thickness of the shell was 0.1 μm.

Example 4
The same procedure as in Example 1 is performed, except that 3.0 g of di (ethylene glycol) methyl ether methacrylate (hereinafter referred to as "DEGMA") is used instead of 3.0 g of THFMA as a raw material for producing a pre-emulsion. The The number average particle diameter of the obtained particles was 2.0 μm, the CV value was 13%, and the thickness of the shell was 0.05 μm.

[Example 5]
The same procedure as in Example 1 was performed, except that 3.0 g of DVB, 2.0 g of SA, and 7.0 g of THFMA were used instead of 5.0 g of DVB, 4.0 g of SA, and 3.0 g of THFMA as raw materials for producing a pre-emulsion. The The number average particle diameter of the obtained particles was 2.1 μm, the CV value was 13%, and the thickness of the shell was 0.1 μm.

[Example 6]
The same procedure as in Example 1 was performed, except that 7.0 g of DVB, 3.0 g of SA, and 2.0 g of THFMA were used instead of 5.0 g of DVB, 4.0 g of SA, and 3.0 g of THFMA as raw materials for producing a pre-emulsion. The The number average particle diameter of the obtained particles was 2.0 μm, the CV value was 11%, and the thickness of the shell was 0.05 μm.

[Example 7]
The same operation as in Example 1 was performed except that as a raw material for producing a pre-emulsion, 9.0 g of DVB and 3.0 g of SA were used instead of 5.0 g of DVB, 4.0 g of SA, and 3.0 g of THFMA. The number average particle size of the obtained particles was 2.0 μm, the CV value was 10%, and the thickness of the shell was 0.05 μm.

Example 8
200 g of a 0.5 wt% aqueous solution of sodium dodecyl sulfate was charged into a 500 mL separable flask, and then 10 g of the base particles obtained in Synthesis Example 1 was charged, dispersed by a homogenizer, and then heated to 60.degree.
Next, 3.0 g of cyclohexyl methacrylate (hereinafter referred to as "CHMA") and 0.015 g of tert-dodecanethiol (hereinafter referred to as "TDT") were added to 15 g of a 0.5 wt% aqueous solution of sodium dodecyl sulfate in another container. And 0.5 g of Perbutyl O were added dropwise to the 500 mL separable flask controlled at 60 ° C. over 2 hours (the first step of shell formation).

  Then, after the obtained liquid is cooled to room temperature, a pre-emulsion in which 0.60 g of perbutyl O is added to and dispersed in 15 g of a 0.5 wt% aqueous solution of sodium dodecyl sulfate is added thereto, and it is kept for 15 hours at room temperature. It stirred. Then, a pre-emulsion prepared by dispersing 5.0 g of DVB, 4.0 g of SA and 3.0 g of THFMA in 15 g of a 0.5 wt% aqueous solution of sodium dodecyl sulfate is added to the liquid stirred for 15 hours at this room temperature. Stir for 2 hours. Then, the temperature was raised to 80 ° C., and the polymerization was further continued for 2 hours to complete the reaction (the second stage of shell formation).

  The obtained aqueous dispersion of magnetic particles was subjected to magnetic purification and gravity sedimentation purification, to obtain an aqueous dispersion of magnetic particles with a solid concentration of 1%.

  In order to remove water from the aqueous dispersion of magnetic particles, the aqueous dispersion was washed twice with 500 mL of acetone. Next, the particles were contacted with acetone (organic solvent) by dispersing the particles in 500 mL of acetone and stirring for 2 hours. Thereby, porous particles were obtained by eluting part of the first-stage and second-stage polymer portions from the particles using acetone. Thereafter, it was washed twice with 500 mL of acetone and further washed with water to remove acetone. The number average particle diameter of the obtained particles was 2.3 μm, the CV value was 15%, and the thickness of the shell was 0.2 μm.

[Example 9]
The same operation as in Example 8 was performed, except that 5.0 g of CHMA and 0.03 g of TDT were used instead of 3.0 g of CHMA and 0.015 g of TDT as raw materials for producing a first shell-forming pre-emulsion. . The number average particle diameter of the obtained particles was 2.3 μm, the CV value was 17%, and the thickness of the shell was 0.25 μm.

Comparative Example 1
The same operation as in Example 1 was performed except that 5.0 g of dipentaerythritol hexaacrylate (hereinafter referred to as "ADPH") was used instead of 5.0 g of DVB as a raw material for producing a pre-emulsion. The number average particle diameter of the obtained particles was 2.4 μm, the CV value was 14%, and the thickness of the shell was 0.25 μm.

Comparative Example 2
2 parts by mass of a 75% di (3,5,5-trimethylhexanoyl) peroxide solution ("Paroyl 355-75 (S)" manufactured by NOF Corporation) is mixed with 20 parts by mass of a 1% aqueous solution of sodium dodecyl sulfate Finely emulsified with an ultrasonic disperser. The resultant was placed in a reactor containing 13 parts by mass of polystyrene particles having a number average particle diameter of 0.77 μm and 41 parts by mass of water, and stirred at 25 ° C. for 12 hours.
In a separate container, 96 parts by mass of styrene and 4 parts by mass of divinylbenzene were emulsified with 400 parts by mass of a 0.1% aqueous solution of sodium dodecyl sulfate. The obtained emulsion was charged into the reactor, stirred at 40 ° C. for 2 hours, heated to 75 ° C., and polymerized for 8 hours. Then, after cooling to room temperature, only particles were removed by centrifugation, and the particles were further washed with water, dried and pulverized to obtain core particles. The number average particle size of the core particles was 1.5 μm.

  Next, acetone is added to an oily magnetic fluid (trade name: "EXP series, EMG", manufactured by Farotech Co., Ltd.) to precipitate particles, which are then subjected to a hydrophobization treatment by drying. Ferrite-based magnetic fine particles (average primary particle diameter: 0.01 μm) having

  Next, 15 g of the core particles and 15 g of the magnetic fine particles are well mixed with a mixer, and this mixture is used to form the circumference of a blade (agitating blade) using a hybridization system NHS-0 (manufactured by Nara Machinery Co., Ltd.). Treatment was carried out at a speed of 100 m / sec (16,200 rpm) for 5 minutes to obtain base particles having a magnetic layer made of magnetic fine particles having a number average particle diameter of 2.0 μm on the surface.

Next, 375 g of an aqueous solution (hereinafter referred to as a "dispersant aqueous solution") containing 0.25% by weight of sodium dodecyl sulfate and 0.25% by weight of a nonionic emulsifier (trade name: "Emulgen 150", manufactured by Kao Corporation) The mixture was charged into a 1 L separable flask, and then 15 g of the base particles having the magnetic layer was charged, dispersed by a homogenizer, and then heated to 60 ° C.
In a separate container, 150 g of an aqueous dispersant solution, 27 g of methyl methacrylate (hereinafter referred to as "MMA"), 3 g of trimethylolpropane trimethacrylate (hereinafter referred to as "TMP") and di (3,5,5-trimethylhexanoyl) per A pre-emulsion in which 0.6 g of oxide (Nippon Oil Co., Ltd .; Peroyl 355) was added and dispersed was added dropwise to the 1 L separable flask controlled to 60 ° C. over 1 hour and 30 minutes. After completion of the dropwise addition, the mixture was kept at 60 ° C. and stirred for 1 hour.
Pre-emulsion in which 75 g of aqueous dispersant solution, 13.5 g of glycidyl methacrylate (hereinafter referred to as "GMA"), 1.5 g of TMP and 0.35 g of peroyl were added and dispersed in the obtained 1 L separable flask after 1 hour of stirring Was dripped over 1 hour and 30 minutes. Thereafter, the temperature was raised to 75 ° C., and polymerization was continued for another 2 hours to complete the reaction. Subsequently, 60 ml of 1 mol / L sulfuric acid was added to a 1 L separable flask in which the reaction was completed, and the mixture was stirred at 60 ° C. for 6 hours. Subsequently, the particles in the separable flask obtained using magnetism were separated and repeatedly washed with distilled water. From the above, magnetic particles having a 2,3-dihydroxypropyl group were obtained (hereinafter referred to as “A-1 particles”).

  1.0 g of dry particles obtained by vacuum drying A-1 particles was washed with 10 ml of pyridine and then dispersed in 5 ml of pyridine. Thereto, a solution of 3 g of succinic anhydride dissolved in 25 ml of pyridine was added and stirred at 60 ° C. for 2 hours. After the reaction, the particles were separated magnetically and washed three times with acetone, then three times with 0.1 M aqueous sodium hydroxide solution, and four times with distilled water to obtain carboxy group-containing particles. The number average particle size of this carboxy group-containing particle is 2.9 μm, the CV value is 14%, and the thickness of the shell is 0.45 μm.

Comparative Example 3
The same operations as in Example 1 were carried out except that 1.0 g DVB, 3.0 g SA, and 8.0 g THFMA were used as raw materials for preparing the pre-emulsion instead of 5.0 g DVB, 4.0 g SA, and 3.0 g THFMA. went. The number average particle diameter of the obtained particles was 2.3 μm, the CV value was 15%, and the thickness of the shell was 0.2 μm.

Comparative Example 4
The same operations as in Example 1 were carried out except that 2.0 g of DVB, 3.0 g of SA, and 7.0 g of THFMA were used as raw materials for producing a pre-emulsion instead of 5.0 g of DVB, 4.0 g of SA, and 3.0 g of THFMA. went. The number average particle diameter of the obtained particles was 2.2 μm, the CV value was 13%, and the thickness of the shell was 0.15 μm.

Comparative Example 5
The same operation as in Example 1 was performed except that 10.0 g of DVB and 2.0 g of SA were used instead of 5.0 g of DVB, 4.0 g of SA, and 3.0 g of THFMA as raw materials for producing a pre-emulsion. The number average particle size of the obtained particles was 2.0 μm, the CV value was 10%, and the thickness of the shell was 0.05 μm.

Comparative Example 6
The same operation as in Example 8 was performed, except that 6.7 g of CHMA and 0.07 g of TDT were used instead of 3.0 g of CHMA and 0.015 g of TDT as raw materials for producing a first shell-forming pre-emulsion. . The number average particle diameter of the obtained particles was 2.5 μm, the CV value was 18%, and the thickness of the shell was 0.3 μm.

Claims (21)

At least the surface of the polymer has 20 to 80% by mass of a structural unit (A) derived from an aromatic monomer relative to all structural units constituting the polymer,
The ratio S / Sg of the specific surface area S to the spherical equivalent specific surface area Sg is 6.0 or less
Magnetic particles.   The magnetic particle according to claim 1, wherein the aromatic monomer is an aromatic vinyl monomer.   The magnetic particles according to claim 2, wherein the aromatic vinyl monomer is divinylbenzene.   The polymer according to any one of claims 1 to 3, wherein the polymer further contains a structural unit (B) having a functional group capable of chemically bonding to the probe in an amount of 15 to 70% by mass based on all structural units constituting the polymer. Magnetic particles according to one. The magnetic particle according to claim 4, wherein the structural unit (B) is represented by formula (1).

[In the formula (1),
R ¹ represents a hydrogen atom or a methyl group,
R ² represents — (C = O) —O— *, — (C = O) —NR ⁴ — * (R ⁴ represents a hydrogen atom or a methyl group, and * represents R ³ in the formula (1) Indicate a position to bond with) or a phenylene group,
When R ² is — (C = O) —O— *, R ³ is a hydrogen atom or an organic group having a functional group capable of chemically bonding to a probe, and R ² is — (C = O) —NR ^When it is a 4- * or phenylene group, R ³ represents an organic group having a functional group capable of chemically bonding to the probe. ] The magnetic particle according to claim 4 or 5, wherein the structural unit (B) is represented by the formula (2).

[In the formula (2),
R ¹ represents a hydrogen atom or a methyl group,
R ⁵ represents a linear or branched alkylene group having 2 to 6 carbon atoms,
R ⁶ represents an alkylene group having 2 to 6 carbon atoms, a cyclohexylene group or a phenylene group. ]   The structural unit (B) is 2- (meth) acryloyloxyethyl succinic acid, 2- (meth) acryloyloxyethyl phthalic acid, 2- (meth) acryloyloxyethyl hexahydrophthalic acid, 2- (meth) The present invention is derived from at least one selected from the group consisting of acryloyloxypropylsuccinic acid, 2- (meth) acryloyloxypropylphthalic acid, and 2- (meth) acryloyloxypropylhexahydrophthalic acid. The magnetic particle as described in any one of 4-6.   The polymer according to any one of claims 1 to 7, wherein the polymer further contains a structural unit (C) derived from a weakly hydrophilic monomer in an amount of 10 to 60% by mass based on all structural units constituting the polymer. Magnetic particles as described. The magnetic particle according to claim 8, wherein the structural unit (C) is one or more selected from structural units (C-1) and (C-2).
(C-1) A structural unit derived from a monoacrylate monomer that holds intermediate water when polymerized into a homopolymer and a free water, or a monomethacrylate monomer having the same structure as the side chain of the homopolymer (C- 2) Structural unit derived from the mono (meth) acrylate monomer represented by the formula (3)

[In the formula (3),
R ⁷ represents a hydrogen atom or a methyl group,
R ⁸ represents a hydrocarbon group. ]   The magnetic particle according to claim 9, comprising a structural unit (C-1) as the structural unit (C). The magnetic particle according to claim 9 or 10, wherein the structural unit (C-1) is a structural unit derived from the formula (4) or (5).

[In the formula (4),
R ⁹ represents a hydrogen atom or a methyl group,
R ¹⁰ represents an alkanediyl group having 1 to 3 carbon atoms,
R ¹¹ represents an alkyl group having 1 to 3 carbon atoms,
n is an integer of 1 to 5; ]

[In the formula (5),
R ¹² represents a hydrogen atom or a methyl group,
m represents 1 or 2; ]   The magnetic particles according to any one of claims 1 to 11, wherein a number average particle diameter of the magnetic particles is 5 nm to 20 m.   The magnetic particle according to any one of claims 1 to 12, wherein the coefficient of variation (CV value) of the number average particle diameter of the magnetic particle is 20% or less.   The magnetic particle according to any one of claims 1 to 13, wherein an average circularity of the magnetic particle is 0.965 to 1.000.   The magnetic particle according to any one of claims 1 to 14, wherein the magnetic particle has a functional group for binding a probe, and the functional group density for binding a probe in the magnetic particle is 1 to 200 μmol / g. .   The magnetic particles according to any one of claims 1 to 15, wherein the magnetic particles have a core-shell structure, and the shell contains the polymer as a main component.   The magnetic particles according to claim 16, wherein the thickness of the shell is 0.01 to 0.3 m.   The magnetic particle according to any one of claims 1 to 17, wherein the magnetic substance is not exposed on the surface of the magnetic particle.   The magnetic particle which the probe couple | bonded with the magnetic particle as described in any one of Claims 1-18.   The magnetic particles according to any one of claims 1 to 19, which are for in vitro diagnostic agents or for detection of biological substances.   A detection / separation method comprising the step of detecting or separating a target substance in a sample using the magnetic particles according to any one of claims 1 to 20.