JP7312043B2 - Metal complex particles and immunoassay reagents using the same - Google Patents

Description

The present invention relates to metal complex particles and immunoassay reagents using the same.

Conventionally, methods for measuring or purifying samples containing biological substances, such as proteins in biological samples, are known. For example, in order to bind proteins and the like in the biological sample to the surfaces of the particles to which proteins can bind, and remove impurities other than the target protein and the like in the biological samples, the particles are washed and the particles bound with the proteins and the like are collected, Examples include a method of measuring the amount of protein binding and a method of dissociating in a protein dissociation solution for purification.
Alternatively, a method of detecting a target protein (such as an antigen) by reacting an enzyme-labeled antibody against the target protein (antigen, etc.) and then optically measuring the luminescence produced by the reaction between the enzyme and a luminescent reagent (ELISA). law) is widely used.
In the ELISA method, the following Patent Documents 1 and 2 disclose that magnetic particles are used because they can be easily separated and recovered by magnetic force.

Furthermore, particles have been proposed that enable fluorescence labeling by imparting fluorescence to particles having magnetism. For example, Patent Document 3 discloses particles having magnetism and fluorescence by impregnating a polymer layer formed on the surface of ferrite particles with a europium chelate complex.

Moreover, Patent Document 3 discloses a method for obtaining an aqueous suspension in which a europium chelate complex is encapsulated in magnetic particles. , a solution of a low boiling point non-aqueous solvent (preferably acetone) having a high affinity for water in which a europium chelate complex is dissolved is added to swell the polymer layer and incorporate the europium chelate complex at the same time, and then the solvent is evaporated. (2) adding the slightly crosslinked polymer-coated magnetic particles from which water has been removed to an acetone solution in which the europium chelate complex is dissolved to form a polymer layer; It describes a method of obtaining an aqueous suspension of magnetic particles containing a europium chelate complex by swelling and incorporating the europium chelate complex at the same time, evaporating the acetone, and adding water.

Further, in Patent Document 4, (3) slightly crosslinked polymer-coated magnetic particles, from which water has been removed by washing with alcohol or the like, are added to an acetone solution in which a europium chelate complex is dissolved, and the polymer layer is swollen and at the same time the europium chelate complex is is also incorporated, water is added, and acetone is evaporated to obtain an aqueous suspension of europium chelate complex-containing magnetic particles.

Japanese Patent Application Laid-Open No. 2005-062087 International Publication No. 2012-173002 JP 2008-127454 A International Publication No. 2010-029739

However, in the manufacturing method described in Patent Document 3, the maximum diameter of the magnetic particles obtained is 300 nm. Since the separation speed is slow and the fluorescence is absorbed by ferrite, sufficient fluorescence cannot be obtained and the sensitivity is low. Further, in Patent Document 3, there is no specific example of method (1), and when acetone, which is considered preferable, is used, an acetone solution of a europium chelate complex is added to an aqueous suspension of polymer-coated magnetic particles. At that point, the europium complex precipitated and it was difficult to incorporate the required amount into the polymer layer. When a large amount of acetone is used with respect to water so as not to deposit the europium chelate complex, the concentration of the europium chelate complex is lowered, and the europium chelate complex cannot be contained sufficiently. In method (2), the particles coalesced in the step of evaporating acetone, making it difficult to obtain single particles. Also in method (3), when water was added, a chelate complex precipitated and was extremely difficult to remove.

As described above, particles with highly sensitive magnetism and fluorescence that enable rapid separation/recovery with a magnet and enable fluorescent labeling have not been put to practical use as immunoassay reagents. A method for controlling the content of a metal complex having magnetism and fluorescence in the metal complex particles has also not been known.

The present invention provides metal complex particles having both magnetism and fluorescence, which are excellent in magnetism collection, an immunoassay reagent using the same, and a method of manufacturing by controlling the content of the metal complex in the metal complex particles. intended to

The present invention relates to the following contents.
[1] A metal complex particle comprising a magnetic material and a particle size-adjusting composition containing a hydrophobic polymer, wherein the magnetic material is a metal having both magnetism and fluorescence, which is composed of metal ions and ligands. Metal complex particles, wherein the metal complex is metal complex A or metal complex B below, and the content of the particle size adjusting composition in the metal complex particles is less than 60 wt%.
(Metal complex A)
A metal complex A consisting of a metal ion, a β-diketone ligand and a phosphorous hydrophobic neutral ligand and satisfying the following formulas (A1) to (A3): metal ion: β-diketone coordination child = 1: 2 to 3 times equivalent (A1), metal ion: phosphorous hydrophobic neutral ligand = 1: 2 to 3 times equivalent (A2), β-diketone ligand and phosphorus hydrophobic The total double equivalent weight of neutral ligands does not exceed 5 double equivalents (A3)
(Metal complex B)
Metal complex B consisting of a metal ion, a β-diketone ligand and a heterocyclic ligand, and satisfying the following formulas (B1) to (B3): metal ion: β-diketone ligand = 1 : 1 to 3 equivalents (B1), metal ion: heterocyclic ligand = 1: 1 to 3 equivalents (B2), total times of β-diketone ligand and heterocyclic ligand Equivalents never exceed 4-fold equivalents (B3)
[2] The metal complex particle according to [1], wherein the magnetic substance is a magnetic substance that does not contain a metal complex having magnetism and no fluorescence.
[3] The metal complex particles according to [1], wherein the magnetic substance is only the metal complex.
[4] The metal complex particles according to any one of [1] to [3], wherein the metal ion forming the metal complex has a magnetic moment of 7.5 to 10.7.
[5] The metal complex particles according to any one of [1] to [4], wherein the metal ion forming the metal complex is at least one selected from the group consisting of ions of terbium, dysprosium, holmium, and erbium. .
[6] (1) the β-diketone ligand is dibenzoylmethane or thenoyltrifluoroacetone,
(2) the heterocyclic ligand is a ligand selected from 2,2′-bipyridyl, 2,2′:6,2″-terpyridine, or 1,10-phenanthroline;
(3) The metal according to any one of [1], wherein the phosphorus-based hydrophobic neutral ligand is a ligand selected from trioctylphosphine oxide, tri-n-octylphosphine and tributyl phosphate. complex particles.
[7] The metal complex particles according to any one of [1] to [6], wherein the metal complex particles have an average particle size of 1 μm or more and 5 μm or less.
[8] The metal complex particles according to any one of [1] to [7], wherein the content of the metal complex in the metal complex particles is 40w% or more and 98w% or less.
[9] The metal complex particle according to any one of [1] to [8], comprising a supporting substance on the surface of the metal complex particle.
[10] The metal complex particles according to any one of [1] to [9], wherein the supporting substance is an antibody or antigen that specifically reacts with the substance to be detected.
[11] An immunoassay reagent comprising the metal complex particles of [10].
[12] a step of dispersing template particles of the particle size-adjusting composition in water in which a surfactant and/or a dispersion stabilizer are dissolved to obtain a dispersion;
dissolving the metal complex in a hydrophobic organic solvent capable of dissolving both the template particles and the metal complex to obtain a metal complex solution;
adding the metal complex solution to the dispersion to absorb the metal complex together with the hydrophobic organic solvent into the template particles;
and distilling off the hydrophobic organic solvent by heating or under reduced pressure.
[13] The metal complex solution further contains a polymerizable monomer capable of dissolving both the template particles and the metal complex,
The method for producing metal complex particles according to [12], further comprising the step of polymerizing the polymerizable monomer by heating and/or light irradiation after absorbing the metal complex into the template particles.
[14] The method for producing metal complex particles according to [12] or [13], wherein the metal complex is composed of metal ions and ligands and has both magnetism and fluorescence.
[15] The method for producing metal complex particles according to any one of [12] to [14], wherein the hydrophobic organic solvent is ethyl acetate.
[16] The method for producing metal complex particles according to any one of [12] to [15], wherein the template particles are polystyrene or a derivative thereof, or polymethyl methacrylate or a derivative thereof.
[17] The method for producing metal complex particles according to any one of [12] to [16], wherein the polymerizable monomer is polystyrene or a derivative thereof, or polymethyl methacrylate or a derivative thereof.

INDUSTRIAL APPLICABILITY According to the present invention, a metal complex particle having both magnetism and fluorescence, which is excellent in magnetism gathering, an immunoassay reagent using the same, and a method for manufacturing by controlling the content of the metal complex in the metal complex particle is provided.

FIG. 1A is a diagram showing an outline of the metal distribution in the cross section of the particle, and FIG. 1B is a partially enlarged view showing the outline of the metal distribution in the cross section of the particle. 2A and 2B are schematic diagrams of the metal distribution in cross section of a particle, respectively, according to the prior art.

As a result of intensive studies aimed at solving the above problems, the inventors of the present invention have found that among a plurality of rare earth complexes, there are several types of metal complexes having both magnetism and fluorescence. They have also found that by preparing this metal complex as particles having a uniform particle size distribution, it is possible to obtain metal complex particles that are excellent in magnetism collection and have fluorescence emission properties. Furthermore, by using the obtained particles as immunoassay reagents, high magnetism collection and improvement in measurement accuracy (visibility) were achieved, and the above problems were solved. Furthermore, they have found that when a metal complex in which two or more ligands are coordinated to a metal ion is used, the content of the metal complex in the metal complex particles can be controlled. The present invention will be described below with reference to embodiments, but the present invention is not limited to the following embodiments.

[Metal complex particles]
A first aspect of the present invention comprises a magnetic substance and a particle size-adjusting composition containing a hydrophobic polymer, wherein the magnetic substance is a metal complex having both magnetism and fluorescence, which consists of a metal ion and a ligand. wherein the metal complex has two or more ligands, and the content of the particle size adjusting composition in the metal complex particles is less than 60 wt%. The metal complex particles preferably contain only the metal complex as the substance having magnetism.

A second aspect of the present invention is a metal complex particle comprising a magnetic substance and a particle size-adjusting composition containing a hydrophobic polymer, wherein the magnetic substance is composed of metal ions and ligands, magnetic and fluorescent wherein the metal complex is metal complex A or metal complex B below, and the content of the particle size adjusting composition in the metal complex particles is less than 60 wt%.
(Metal complex A)
A metal complex A consisting of a metal ion, a β-diketone-based ligand and a phosphorus-based hydrophobic neutral ligand and satisfying the following formulas (A1) to (A3):
Metal ion: β-diketone ligand = 1: 2 to 3 equivalents (A1),
Metal ion: Phosphorus-based hydrophobic neutral ligand = 1: 2 to 3 equivalents (A2),
The total equivalent amount of the β-diketone ligand and the phosphorus hydrophobic neutral ligand does not exceed 5 equivalents (A3)
(Metal complex B)
A metal complex B consisting of a metal ion, a β-diketone ligand and a heterocyclic ligand and satisfying the following formulas (B1) to (B3):
Metal ion: β-diketone ligand = 1: 1 to 3 equivalents (B1),
Metal ion: heterocyclic ligand = 1: 1 to 3 equivalents (B2),
The total double equivalent weight of the β-diketone ligand and the heterocyclic ligand does not exceed 4 equivalent weights (B3)
In addition, the number of ligands in the above description means an integer within the above numerical range.

Since the metal complex particles of the present invention have a high content of a magnetic metal complex, they have high magnetism collection and at the same time the amount of magnetism per particle is uniform. response to magnetic force becomes uniform, and dispersion during separation is reduced. Furthermore, since the metal complex has not only magnetism but also fluorescence properties, the use of this metal complex particle as an immunoassay reagent increases the detection sensitivity. In addition, by changing the metal complex species, it is possible to change the fluorescence spectrum of the metal complex particles, which changes the color of the metal complex particles. can be avoided. Further, by using a predetermined combination of metal complexes, a plurality of fluorescence spectra can be observed, thereby making it easier to avoid erroneous diagnosis due to mistaken reagents or the like.

A "metal complex" is a metal ion bound to molecules or ions called ligands.

Compared to particles containing a magnetic substance and a fluorescent substance, the metal complex particles of the present invention have magnetism and fluorescence with the same metal complex. , fluorescence can be effectively expressed. In addition, since the metal complex particles of the present invention can have a high metal complex content, they can exhibit high magnetism collection. Furthermore, since the metal complex particles of the present invention have uniform particle diameters, the responsiveness to magnets is uniform, and uniform magnetism collection is exhibited.

The particle size-adjusting composition contains, as a main component, a polymer (hereinafter also simply referred to as "polymer") obtained by polymerizing (including polycondensation) a polymerizable monomer (hereinafter also simply referred to as "monomer"). Polymers also include copolymers of one or more polymerizable monomers.

Examples of the polymer include (un)saturated hydrocarbons, aromatic hydrocarbons, (unsaturated) fatty acids, aromatic carboxylic acids, (unsaturated) ketones, aromatic ketones, (unsaturated) alcohols, aromatic alcohols, Examples thereof include polymers composed of one or more polymerizable monomers selected from (un)saturated amines, aromatic amines, (un)saturated thiols, aromatic thiols, and organosilicon compounds.
Examples of the above polymers include polyolefins such as polyethylene, polypropylene, polybutylene, polymethylpentene, and polybutadiene; polyethers such as polyethylene glycol, polypropylene glycol, and tetramethylene glycol; polystyrene; poly(meth)acrylic acid; Acrylic acid ester; polyvinyl alcohol; polyvinyl ester; polyvinyl ether; phenolic resin; melamine resin; benzoguanamine resin; allyl resin; furan resin; (un)saturated polyester; epoxy resin; ); acrylonitrile/styrene resin; styrene/butadiene resin; vinyl halide resin; polycarbonate; Examples thereof include polymers obtained by polymerizing one or more of various polymerizable monomers. These polymers may be used alone or in combination of two or more.
The above polymers are produced by radical polymerization, ionic polymerization, coordination polymerization, ring-opening polymerization, isomerization polymerization, cyclization polymerization, elimination polymerization, polyaddition, polycondensation, and addition condensation.

The polymerizable monomer is not particularly limited as long as it is a compound containing one or more polymerizable functional groups in one molecule, even a monofunctional monomer containing one polymerizable functional group in one molecule, polymerizable A polyfunctional monomer containing two or more functional groups in one molecule may be used. Moreover, it may be an oligomer or prepolymer containing one or more polymerizable functional groups in one molecule. In addition, the polymerizable monomers may be used singly or in combination of two or more at any ratio. Further, when two or more polymerizable monomers are used, the combination of two or more polymerizable monomers includes a combination of two or more monofunctional polymerizable monomers, a combination of two or more polyfunctional polymerizable monomers, and one or more or a combination of a monofunctional polymerizable monomer and one or more polyfunctional polymerizable monomers. The molecular weight of these polymers is, for example, 80 or more and 30,000 or less, but is not particularly limited. In the present invention and the specification, the term "molecular weight" refers to the weight-average molecular weight of polymers measured by gel permeation chromatography (GPC) in terms of polystyrene. The weight average molecular weight, which will be described later, is a value measured by the following measurement.
GPC device: APC system (manufactured by Waters)
Column: HSPgel HR MB-M (manufactured by Waters) 6.0 × 150 mm Eluent: tetrahydrofuran (THF), flow rate: 0.5 mL/min, column temperature: 40°C, injection volume: 10 µL, detector: RI

The polymerizable functional group may be a radically polymerizable functional group, an ionically polymerizable functional group, or a coordination polymerizable functional group, and is preferably a radically polymerizable functional group. From the viewpoint of the reactivity of the polymerization reaction, polymerizable groups such as an ethylenically unsaturated bond-containing group, an epoxy group, an oxetane group, and a methylol group can be mentioned, and an ethylenically unsaturated group is more preferable. Examples of ethylenically unsaturated groups include (meth)acryloyloxy, (meth)acryloyl, vinyl, aromatic vinyl and allyl groups, and (meth)acryloyl, vinyl and aromatic vinyl groups. is more preferred. In the present invention and this specification, the term "(meth)acryloyl group" is used to mean at least one of an acryloyl group and a methacryloyl group. The same applies to "(meth)acryloyloxy group", "(meth)acrylate", "(meth)acryl" and the like. As for the polyfunctional monomer, the number of polymerizable groups contained in the compound is two or more per molecule.

Specific examples of the monofunctional monomer having an ethylenically unsaturated group are not particularly limited,
For example, monofunctional monomers having a (meth)acryloyl group include methyl (meth) acrylate, ethyl (meth) acrylate, propyl (meth) acrylate, butyl (meth) acrylate, 2-ethylhexyl (meth) acrylate, lauryl (meth) ) acrylate, cetyl (meth)acrylate, stearyl (meth)acrylate, cyclohexyl (meth)acrylate, isobornyl (meth)acrylate, 2-hydroxyethyl (meth)acrylate, glycerol (meth)acrylate, polyoxyethylene (meth)acrylate, glycidyl (meth) acrylate, trifluoromethyl (meth) acrylate, pentafluoroethyl (meth) acrylate, 3-(meth) acryloxypropylmethyldimethoxysilane, 3-(meth) acryloxypropyltrimethoxysilane, 3-(meth) ) (meth)acrylate monomers such as acryloxypropylmethyldiethoxysilane and 3-(meth)acryloxypropyltriethoxysilane.
Monofunctional monomers having a vinyl group include olefin monomers such as diisobutylene, isobutylene, linearene, ethylene and propylene; conjugated diene monomers such as isoprene and butadiene; vinyl ether monomers such as methyl vinyl ether, ethyl vinyl ether and propyl vinyl ether; vinyl ester monomers such as vinyl butyrate, vinyl laurate and vinyl stearate; halogen-containing vinyl monomers such as vinyl chloride and vinyl fluoride;
Nitrile-containing vinyl monomers such as (meth)acrylonitrile; vinyltrimethoxysilane, vinyltriethoxysilane, dimethoxymethylvinylsilane, dimethoxyethylvinylsilane, diethoxymethylvinylsilane, diethoxyethylvinylsilane, methylvinyldimethoxysilane, ethylvinyldimethoxysilane , methylvinyldiethoxysilane, ethylvinyldiethoxysilane, and other silane alkoxide-containing vinyl monomers.
Monofunctional monomers having an aromatic vinyl group include aromatic vinyl monomers such as styrene, α-methylstyrene, chlorostyrene, vinylnaphthalene, 4-vinylbiphenyl and p-styryltrimethoxysilane.

Specific examples of the polyfunctional monomer having an ethylenically unsaturated group are not particularly limited, and examples of the polyfunctional monomer having a (meth)acryloyl group include tramethylolmethane tetra(meth)acrylate, tetramethylolmethane (meth)acrylate, tetramethylolmethane di(meth)acrylate, trimethylolpropane tri(meth)acrylate, dipentaerythritol hexa(meth)acrylate, dipentaerythritol penta(meth)acrylate, glycerol tri(meth)acrylate, glycerol di(meth)acrylate (Meth)acrylate, (poly)ethylene glycol di(meth)acrylate, (poly)propylene glycol di(meth)acrylate, (poly)tetramethylene glycol di(meth)acrylate, 1,4-butanediol di(meth)acrylate , 9,9-bis[4-(2-acryloyloxyethyloxy)]phenylfluorene and other polyfunctional (meth)acrylate monomers.
Polyfunctional monomers having a vinyl group include 1,4-divinyloxybutane, divinylsulfone, 9,9'-bis(4-allyloxyphenyl)fluorene, ethylmethyldivinylsilane and the like.
Polyfunctional monomers having an aromatic vinyl group include aromatic vinyl monomers such as divinylbenzene, divinylnaphthalene, and divinylbiphenyl.
Polyfunctional monomers having allyl groups include allyl monomers such as triallyl (iso) cyanurate, triallyl trimellitate, diallyl phthalate, diallyl acrylamide, diallyl ether, 9,9′-bis(4-allyloxyphenyl) fluorene. mentioned.

The particle size adjusting composition preferably contains a hydrophobic polymer. In this specification, the term "hydrophobic polymer" refers to a polymer having a solubility in water of 5 w% or less at 25°C, and is a block polymer having a hydrophilic functional group or a hydrophilic block segment. is also included. Also included are those that can be dispersed in water with a predetermined probability.

The magnetic substance is preferably a metal complex composed of metal ions and ligands. It is preferable to use the following metals and ligands that constitute the metal complex.
The metal composing the metal complex is not particularly limited as long as it is bonded with a ligand described later and has magnetic properties and fluorescence properties as a metal complex. is preferably a metal with a magnetic moment of 7.5 or more. Furthermore, it is preferably 9.0 or more. Examples of metals having a magnetic moment of 7.5 or more include terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), and thulium (Tm). Among them, Tb, Dy, Ho, Er, etc. are particularly preferable because their magnetic moments are 9.0 or more. These may be used alone or in combination of two or more.

The ligand that forms a metal complex is not particularly limited as long as it forms a complex with the metal and exhibits magnetism and fluorescence. Hydrophobic interaction with a hydrophobic polymer facilitates the formation of metal complex particles. A rank is preferred. Of these, it is preferable to use two or more of them in combination. In order to obtain stronger magnetism and fluorescence, a β-diketone ligand and a heterocyclic ligand, a β-diketone ligand and a phosphorus hydrophobic It is more preferable to use a combination of neutral neutral ligands, and it is particularly preferable to use a combination of β-diketone-based ligands and phosphorus-based hydrophobic neutral ligands.

（上記式（Ｘ）中、Ｒ₁及びＲ_２は、同一でも異なっていても良く、チエニル基、トリフルオロメチル基、フルオロメチル基、フリル基、フェニル基、ナフチル基、ベンジル基、ｔｅｒ‐ブチル基、メチル基等を表す。）
このようなβ－ジケトン配位子として、例えばテノイルトリフルオロアセトン、ナフトイルトリフルオロアセトン、ベンゾイルトリフルオロアセトン、フロイルトリフルオロアセトン、ピバロイルトリフルオロアセトン、ヘキサフルオロアセチルアセトン、トリフルオロアセチルアセトン、フルオロアセチルアセトン、ジベンゾイルメタン、ジテノイルメタン、およびフロイルトリフルオロアセトンを挙げることができる。なかでも、テノイルトリフルオロアセトン（ＴＴＡ）、ジベンゾイルメタン（ＤＢＭ）が好ましい。 As the β-diketone ligand, those represented by the following formula (X) can be used.

(In formula (X) above, R ₁ and R ₂ may be the same or different, and are thienyl, trifluoromethyl, fluoromethyl, furyl, phenyl, naphthyl, benzyl, ter-butyl group, methyl group, etc.)
Examples of such β-diketone ligands include thenoyltrifluoroacetone, naphthoyltrifluoroacetone, benzoyltrifluoroacetone, furoyltrifluoroacetone, pivaloyltrifluoroacetone, hexafluoroacetylacetone, trifluoroacetylacetone, fluoroacetylacetone, Mention may be made of dibenzoylmethane, dithenoylmethane, and furoyltrifluoroacetone. Among them, thenoyltrifluoroacetone (TTA) and dibenzoylmethane (DBM) are preferred.

Heterocyclic (heterocyclic anionic) ligands Heterocyclic ligands containing heteroatoms such as nitrogen and oxygen include, for example, 2,2'-bipyridyl, 4,4'-dimethyl -2,2'-bipyridyl, 6,6'-dimethyl-2,2'-bipyridyl, 5,5'-dimethyl-2,2'-bipyridyl, 2,2'-bipyrimidyl, 4,4'-diethyl- 2,2′-bipyridyl, 8-quinolinol, 2,2′:6,2″-terpyridine, 4,4′-dinonyl-2,2′-bipyridyl, di-tert-butylbipyridine, 1,10-phenanthroline , 3,4,7,8-tetramethyl-1,10-phenanthroline, batphenanthroline, 5,6-dimethyl-1,10-phenanthroline, phthalocyanine, 5,15-diphenylporphyrin, tetraphenylporphyrin, 5,10, 15,20-tetra(4-pyridyl)porphyrin, 3-pyrazolone and the like. Among them, 2,2'-bipyridyl, 2,2':6,2''-terpyridine and 1,10-phenanthroline are preferred.

Phosphorus-Based Hydrophobic Neutral Ligands Phosphorus-containing hydrophobic neutral ligands include trioctylphosphine oxide, tri-n-octylphosphine, tributyl phosphate and the like. Among them, trioctylphosphine oxide and tri-n-octylphosphine are particularly preferred.

The content A (w%) of the metal complex in the particles is calculated by formulas (1) to (3) from the residue after decomposition of the polymer and ligand when heated to 1000°C in an inert gas. be done.
After weighing the particles X (g) and firing at 1000° C. in the air, assuming that Y (g) of holmium oxide with a molecular weight of 377.86 (g/mol) remains, the weight of holmium oxide contained in the particles is Z (mol) is calculated by formula (1).
Z (mol) = Y (g)/377.86 (g/mol) Formula (1)
Assuming that the molecular weight of the complex used is M (g/mol), the amount a (g) of the metal complex contained in the particles is calculated by Equation (2).
a(g)=M(g/mol)×Z(mol) Formula (2)
Therefore, the content A (w%) of the metal complex contained in the particles is calculated by the formula (3).
A (w%) = [a (g) / X (g)] × 100 formula (3)
Further, the residue was obtained by heating the particles from 35° C. to 1000° C. at a rate of 5° C./min using a TG-DTA6300 (manufactured by Hitachi High-Tech Co., Ltd.) device and then maintaining the temperature at 1000° C. for 5 minutes. It can be calculated from quantity.

A preferred embodiment of the metal complex particles includes a styrene-based or (meth)acrylate-based hydrophobic polymer as a particle size adjusting composition; a holmium ion, a terbium ion or a dysprosium ion as a metal complex, and a β-diketone-based ligand. and at least one of heterocyclic ligands, or a metal complex with both of the ligands; The content of the metal complex is preferably 40 wt % or more and 98 wt % or less with respect to the total weight of the particles.
The content of the metal complex is preferably 40 wt % or more and 98 wt % or less, although it depends on the type. When it is 40 wt % or more, sufficient magnetism collection is exhibited. When it is 98 w% or less, the shape as particles can be maintained. More preferably, it is 50w% or more, still more preferably 60w% or more.

Another preferred embodiment of the metal complex particles is a combination of two types: a styrene-based or (meth)acrylate-based hydrophobic polymer as the particle size adjusting composition; and a holmium ion, a terbium ion, or a dysprosium ion as the metal complex. a metal complex with a ligand (for example, a combination of a β-diketone ligand and a phosphorus hydrophobic neutral ligand, or a combination of a β-diketone ligand and a heterocyclic ligand) and ; metal complex particles consisting of. The content of the metal complex is preferably 40 wt % or more and 98 wt % or less with respect to the total weight of the particles.
The content of the metal complex is preferably 40 wt % or more and 98 wt % or less, although it depends on the type. When it is 40 wt % or more, sufficient magnetism collection is exhibited. When it is 98 w% or less, the shape as particles can be maintained. More preferably, it is 50w% or more, still more preferably 60w% or more.

(Metal complex A)
A metal complex A consisting of a metal ion, a β-diketone-based ligand and a phosphorus-based hydrophobic neutral ligand and satisfying the following formulas (A1) to (A3):
Metal ion: β-diketone ligand = 1: 2 to 3 equivalents (A1),
Metal ion: Phosphorus-based hydrophobic neutral ligand = 1: 2 to 3 equivalents (A2),
The total equivalent amount of the β-diketone ligand and the phosphorus hydrophobic neutral ligand does not exceed 5 equivalents (A3)
(Metal complex B)
A metal complex B consisting of a metal ion, a β-diketone ligand and a heterocyclic ligand and satisfying the following formulas (B1) to (B3):
Metal ion: β-diketone ligand = 1: 1 to 3 equivalents (B1),
Metal ion: heterocyclic ligand = 1: 1 to 3 equivalents (B2),
The total double equivalent weight of the β-diketone ligand and the heterocyclic ligand does not exceed 4 equivalent weights (B3)

The particle size of the metal complex particles of the present invention is not particularly limited, but the particle size is preferably 500 nm or more and 10 μm or less in order to achieve high magnetism collection. If it is 500 nm or more, the separation speed by the magnet is high, and if it is 10 μm or less, the particles are less likely to settle. More preferably, the particle size is 1 μm or more and 5 μm or less.
The average particle diameter of the particles was determined by observing the particles with a scanning electron microscope and arithmetically averaging the maximum diameters of 50 arbitrarily selected particles in the observed image.
The coefficient of variation (CV value) of the metal complex particles of the present invention is preferably 20% or less in order to improve the reproducibility of measurement results. If it exceeds 20%, the magnetism collection may vary, and the reproducibility of the measurement results may deteriorate. More preferably, it is 10% or less.
The coefficient of variation is obtained by observing the particles with a scanning electron microscope, obtaining the standard deviation of the particle size of 50 arbitrarily selected particles in the observed image, and calculating the CV value of the particle size using the following formula. asked for
CV value (%) = (ρ/Dn) × 100
ρ: standard deviation of particle size Dn: average value of particle size

As the method for producing the metal complex particles of the present invention, the following production methods are exemplified.
(1) A: Step of dissolving both the particle size adjusting composition and the metal complex in a hydrophobic organic solvent capable of dissolving both; B: The solution obtained in Step A is dissolved in a surfactant and/or a dispersion stabilizer A step of forming droplets by adding to an aqueous solution obtained by emulsification, layer-transition emulsification, suspension, etc. C: A step of distilling off the hydrophobic organic solvent by heating or under reduced pressure D: Filtration A method by a process of arranging the particle size by classification or the like.
(2) A: A step of dissolving a polymerizable monomer, a hydrophobic polymerization initiator and a metal complex; B: Droplets after emulsification, suspension, etc. in an aqueous solution in which a surfactant and/or a dispersion stabilizer are dissolved D: polymerizing the particle size-adjusting composition in the presence of a metal complex by heating and/or light irradiation; and E: aligning the particle size by filtration, classification, or the like. In step A, if necessary, a hydrophobic organic solvent capable of dissolving both the polymerizable monomer and the metal complex may be added. In that case, after step D, a step of distilling off the hydrophobic organic solvent by heating or under reduced pressure is added. Moreover, the polymerizable monomer may be the above-mentioned monofunctional monomer, a polyfunctional monomer, or a combination of both.
(3) A: A step of dispersing particles (hereinafter also referred to as "template particles") made of a particle size-adjusted composition that serves as a template in water in which a surfactant and/or a dispersion stabilizer are dissolved; B: the template; A step of preparing a metal complex solution in which the metal complex is dissolved in a hydrophobic organic solvent and/or a polymerizable monomer capable of dissolving both the particles and the metal complex; A step of adding the obtained metal complex solution and allowing the template particles to absorb the metal complex together with the hydrophobic organic solvent and/or the polymerizable monomer, D: polymerizing the polymerizable monomer by heating and/or light irradiation. and distilling off the hydrophobic organic solvent by heating or under reduced pressure. In step B, a hydrophobic polymerization initiator may be added. Moreover, in the case where the polymerizable monomer is not added in the step B, the polymerization step in the step D becomes unnecessary.
etc.
By using particles of a uniform particle size for the particles (template particles) made of the particle size adjusting composition that serves as a template, the particle size of the obtained metal complex particles can be made uniform, and the amount of the metal complex to be included can be made uniform. Therefore, the method (3) is preferably used.

As described above, the template particles made of the particle size-adjusting composition are mainly composed of a polymer obtained by polymerizing a polymerizable monomer, and the polymer also contains a copolymer of one or more polymerizable monomers.
The polymer that mainly constitutes the template particles is not particularly limited as long as it is a hydrophobic polymer, and a polymer obtained by polymerizing the above-mentioned monofunctional polymerizable monomer by radical polymerization, ionic polymerization, coordination polymerization, or the like is used. be done. Among them, polymers/copolymers mainly composed of aromatic vinyl monomers, (meth)acrylate monomers, and vinyl ester monomers are preferably used because of their high solubility in hydrophobic organic solvents, which will be described later. be done. Aromatic vinyl monomers and (meth)acrylates are more preferred.
Examples of methods for producing the template particles include emulsion polymerization, suspension polymerization, soap-free polymerization, dispersion polymerization, miniemulsion polymerization, and microsuspension polymerization; phase inversion emulsification using a polymer solution; and submerged drying. However, since polymer particles having a uniform particle size can be obtained, soap-free polymerization and dispersion polymerization are preferably used.

In the case of the production method (1) or (3), the polymerizable monomer and the initiator may be added and polymerized by heating or the like. Among them, the use of a polyfunctional monomer is preferable because a crosslinked structure can be introduced into the particles.
Note that the polymer obtained by polymerizing the added polymerizable monomer by heating or the like becomes a part of the particle size adjusting composition, and is therefore also referred to as "second particle size adjusting composition".

When particles containing a fluorescent metal complex were prepared by a conventional method (emulsion polymerization method), the fluorescence intensity of the particles decreased due to the quenching effect when the content of the metal complex exceeded a predetermined amount (68% by weight). (See Colloids and Surfaces B: Biointerfaces, 2016, Vol.145, No.1, p.152-159). Therefore, in the conventional method, even if a metal complex having both magnetism and fluorescence is used, due to the quenching effect, it has been difficult to achieve both magnetism and fluorescence as the content of the metal complex increases. On the other hand, in the particles according to one aspect of the present invention, quenching is suppressed by forming the second particle size adjusting composition that forms a crosslinked structure in the particles, and the region has a high metal complex content. Even so, a good balance between fluorescence intensity and magnetism can be maintained.

The upper limit of the content of the particle size adjusting composition in the metal complex particles is preferably less than 60w%, more preferably less than 50w%. The lower limit is preferably 2w% or more, more preferably 5w% or more. When it is 2 wt % or more, the shape of the particles can be easily maintained, and the outflow of the metal complex can be prevented. In addition, the content of the metal complex is preferably 30 wt % or more in order for the metal complex particles of the present invention to exhibit sufficient magnetism collection, so the content of the particle size adjusting composition is inevitably 70 wt % or less. The content of the particle size adjusting composition is calculated by formula (4) using the metal complex content of formulas (1) to (3) above.
Content of particle size adjusting composition (w%) = 100 - metal complex content (w%) formula (4)

In the conventional magnetic particles disclosed in Japanese Patent Application Laid-Open No. 10-55911, as shown in FIG. 2A, the magnetic material (Fe ₃ O ₄ ) was present in the surface layer of the particles in a cross-sectional view. Further, in the conventional magnetic particles disclosed in Japanese Patent Application No. 2010-528640 (Patent _{No. 5574492} ), as shown in FIG. Particles (islands) were present locally, and the fluorescent dye (rare earth element (Eu)) was present on the surface layer of the particles. However, as shown in FIGS. 1A and 1B, in the metal complex particles of the present application, since the particles are formed by the metal complex itself, in a cross-sectional view of the metal complex particles, from the surface layer (surface) to the center (point ), a substantially uniform morphology is formed. That is, as described above, since the content of the metal complex is high, at the same time as having high magnetism collection, the amount of magnetism per particle is uniform. The response is uniform and the variability in separation is small. The particle size-adjusting composition may be locally distributed within the metal complex particles. In this case, the particle size-adjusting composition (islands) is locally or scattered in the metal complex (sea). becomes. Such a form is also one aspect of the present invention.

The hydrophobic organic solvent used in the above production methods (1), (2) and (3) is not particularly limited, but has an SP value of 10 or less and a boiling point for distilling off the hydrophobic organic solvent later. is lower than that of water. For example, ethyl acetate (SP value: 9.1, boiling point: 77.1°C), benzene (SP value: 9.2, boiling point: 80.1°C), diisopropyl ether (SP value: 6.9, boiling point: 69 °C), chloroform (SP value: 9.1, boiling point: 61.2°C), and the like. In addition to these, toluene (SP value: 8.8, boiling point: 110.6°C), butyl acetate (SP value: 8.5, boiling point: 126.1°C), etc. can also be used. These may be used alone or in combination of two or more. Among them, ethyl acetate is preferred.
The SP value is the following formula (A) using the ΔF and Δv values of various atomic groups by Okitsu, described in Toshinao Okitsu, "Adhesion", Kobunshi Publishing Association, Vol. 40, No. 8 (1996), p. 342-350. means the solubility parameter δ calculated by In the case of mixtures and copolymers, it means the solubility parameter δ _mix calculated by the following formula (B).
δ=ΣΔF/ΣΔv (A)
δ _mix = φ ₁ δ ₁ + φ ₂ δ ₂ + ... φ _n δ _n (B)
In the formula, ΔF and Δv represent ΔF and molar volume Δv of various atomic groups according to Okitsu, respectively. φ represents a volume fraction or a molar fraction, and φ ₁ +φ ₂ + . . . φ _n =1.
In this specification, the hydrophobic organic solvent may be referred to as an organic solvent.

Hydrophobic organic solvent: ethyl acetate, metal complex: Ho (holmium)/2,2'-bipyridyl (bpy), particle size control composition: polystyrene;
Hydrophobic organic solvent: ethyl acetate, metal complex: Ho (holmium)/2,2'-bipyridyl (bpy), dibenzoylmethane (DBM), particle size control composition: polystyrene;
Hydrophobic organic solvent: ethyl acetate, metal complex: Ho (holmium)/dibenzoylmethane (DBM), particle size control composition: polystyrene;
Hydrophobic organic solvent: ethyl acetate, metal complex: Ho (holmium)/2,2'-bipyridyl (bpy), thenoyltrifluoroacetone (TTA), particle size control composition: polystyrene;
Hydrophobic organic solvent: ethyl acetate, metal complex: Ho (holmium)/trioctylphosphine oxide (TOPO), thenoyltrifluoroacetone (TTA), particle size control composition: polystyrene;
Hydrophobic organic solvent: ethyl acetate, metal complex: Ho (holmium)/trioctylphosphine oxide (TOPO), dibenzoylmethane (DBM), particle size control composition: polystyrene;
Hydrophobic organic solvent: ethyl acetate, metal complex: Ho (holmium)/2,2'-bipyridyl (bpy), DBM (dibenzoylmethane), particle size control composition: pMMA;
Hydrophobic organic solvent: toluene, metal complex: Ho (holmium)/bpy, dibenzoylmethane (DBM), particle size control composition: polystyrene;
Hydrophobic organic solvent: ethyl acetate, metal complex: Ho (holmium), Dy (dysprosium)/2,2'-bipyridyl (bpy), dibenzoylmethane (DBM), particle size control composition: polystyrene.

The hydrophobic polymerization initiator used in the production methods (1), (2) and (3) is not particularly limited as long as it is soluble in the hydrophobic organic solvent. Specifically, peroxides such as di-tert-butyl peroxide, tert-butyl hydroperoxide, benzoyl peroxide, methyl ethyl ketone peroxide, di (secondary butyl) peroxydicarbonate, azobisisobutyronitrile, 2,2 '-azobis(2-methylbutyronitrile) (V-59), 2,2'-azobis(2,4-dimethylvaleronitrile) (V-65), 2,2'-azobis(4-methoxy-2 ,4-dimethylvaleronitrile) (V-70), dimethyl 2,2'-azobis(isobutyrate) (V-601) and other azo compounds, preferably tert-butyl-2-ethylperoxyhexano Art, di(secondary butyl)peroxydicarbonate, and azobisisobutyronitrile are good.

Dispersants used in the above production methods (1), (2) and (3) include polyalkylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, poly(meth)acrylic acid, hydroxyl groups in side chains, carboxyl groups, and polyalkylene glycol. Examples include water-soluble polymers such as poly(meth)acrylic acid and poly(meth)acrylate having water-soluble groups such as groups, colloidal silica, calcium carbonate, and the like. These may be used alone or in combination of two or more.
The surfactant used in the above production methods (1), (2) and (3) is not particularly limited as long as it is water-soluble, and nonionic surfactants such as polyalkylene glycol alkyl ethers; alkyl sulfates; Anionic surfactants such as alkylbenzenesulfonates, alkylcarboxylates, alkylphosphate salts, alkylsulfuric acid ester salts; cationic surfactants such as alkylamine salts and quaternary alkylammonium salts; betaine type, sulfobetaine and alkylbetaine type amphoteric surfactants. These may be used alone or in combination of two or more.
In the above manufacturing methods (1), (2) and (3), other additives may be used as necessary.
Other additives include electrolytes such as potassium chloride, sodium chloride, sodium acetate, ammonium acetate, sodium citrate, potassium sulfate, sodium sulfate, sodium dihydrogen phosphate, and disodium hydrogen phosphate.
These may be used alone or in combination of two or more.

As one aspect of the method for producing metal complex particles,
A step of preparing an aqueous medium (a) in which a surfactant or a water-soluble polymer is dissolved;
A step of preparing an aqueous medium dispersion (A) in which particles (template particles) made of a particle size adjusting composition are dispersed in the aqueous medium (a);
preparing a hydrophobic organic solvent (b) in which the particle size adjusting composition can be dissolved;
Metal complex solution (B ), and
The step of mixing (A) and (B) to prepare swollen droplets (c) comprising the particle size adjusting composition, the metal complex, and the hydrophobic organic solvent (b);
and removing the hydrophobic organic solvent (b) from the swollen droplets (c).

The metal complex is composed of a holmium ion, a terbium ion or a dysprosium ion and a combination of a β-diketone ligand and a heterocyclic ligand or a combination of a β-diketone ligand and a phosphorus hydrophobic neutral ligand It is preferable to be
The hydrophobic polymer preferably comprises a styrene-based or (meth)acrylate-based hydrophobic polymer.

Using the metal complex particles having both fluorescence and magnetism of the present invention as a carrier, a substance to be measured, a substance analogous to the substance to be measured, or a substance that specifically binds to the substance to be measured (hereinafter collectively referred to as a supporting substance) ) can be used to prepare sensitized metal complex particles. This sensitized metal complex particle is also one aspect of the present invention.

The substance to be measured in the present invention is not particularly limited as long as it is usually measured in this field. Typical examples include proteins, lipoproteins, nucleic acids, immunoglobulins, blood coagulation-related factors, antibodies, enzymes, hormones, cancer markers, heart disease markers, and various drugs contained in the sample. More specifically, proteins such as albumin, hemoglobin, myoglobin, transferrin, and C-reactive protein (CRP); lipoproteins; nucleic acids such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA); such as alkaline phosphatase, amylase, acid phosphatase, γ-glutamyltransferase (γ-GTP), lipase, creatine kinase (CK), lactate dehydrogenase (LDH), glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), renin, protein kinase (PK), tyrosine kinase; immunoglobulins such as IgG, IgM, IgA, IgD, IgE; Fragments thereof, such as Fc portion, Fab portion, F(ab) ₂ portion); Blood coagulation-related factors such as fibrinogen, fibrin degradation products (FDP), prothrombin, thrombin; Anti-streptolysin O antibody, anti-human Antibodies such as hepatitis B virus surface antigen antibody (HBs antigen), anti-human hepatitis C virus antibody, anti-rheumatoid factor; for example, thyroid stimulating hormone (TSH), thyroid hormone (FT3, FT4, T3, T4), parathyroid hormone hormones such as (PTH), human chorionic gonadotropin (hCG) estradiol (E2); cancer markers such as alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), CA19-9, prostate specific antigen (PSA); Heart disease markers such as troponin T (TnT) and human brain natriuretic peptide precursor N-terminal fragment (NT-proBNP); antiepileptic drugs, antibiotics, drugs such as theophylline, and the like.

An analogue (analog) of a substance to be measured in the present invention is a substance that can bind to a binding site for a substance to be measured possessed by a substance that specifically binds to a substance to be measured (a substance that binds to a substance to be measured). Any substance may be used as long as it has a binding site with the target substance-binding substance possessed by the target substance, or in other words, any substance that can compete with the reaction when the target substance and the target substance-binding substance coexist during the reaction. .

Substances that specifically bind to the substance to be measured in the present invention (substance that binds to the substance to be measured) include, for example, reaction between "antigen" - "antibody", reaction between "sugar chain" - "protein", reaction between "sugar chain" - "Lectin" reaction, "enzyme" - "inhibitor" reaction, "protein" - "peptide chain" reaction, "chromosome or nucleotide chain" - "nucleotide chain" reaction, or "nucleotide chain" - "protein , which binds to the substance to be measured or its analogue by mutual reaction such as interreaction. It is a substance that binds to the substance to be measured. For example, when the target substance or its analogue is an "antigen", the target substance-binding substance is an "antibody", and when the target target substance or its analogue is an "antibody", the target substance-binding substance is an "antigen ” (hereinafter the same applies to each of the other above combinations).

Specifically, for example, nucleotide chains (oligonucleotide chains, polynucleotide chains); chromosomes; peptide chains (eg, C-peptide, angiotensin I, etc.); proteins [eg, procalcitonin, immunoglobulin A (IgA), immunoglobulin E ( IgE), immunoglobulin G (IgG), immunoglobulin M (IgM), immunoglobulin D (IgD), β2-microglobulin, albumin, degradation products thereof, serum proteins such as ferritin]; , salivary gland type, X type, etc.), alkaline phosphatase (e.g., hepatic, bone, placental, small intestinal, etc.), acid phosphatase (e.g., PAP, etc.), γ-glutamyltransferase (e.g., renal, pancreatic, hepatic, etc.) ), lipase (eg, pancreatic type, gastric type, etc.), creatine kinase (eg, CK-1, CK-2, mCK, etc.), lactate dehydrogenase (eg, LDH1 to LDH5, etc.), glutamic acid oxaloacetic transaminase (eg, ASTm, ASTs, etc.) ), glutamate pyruvate transaminase (e.g. ALTm, ALTs, etc.), cholinesterase (e.g., ChE1 to ChE5, etc.), leucine aminopeptidase (e.g., C-LAP, AA, CAP, etc.), renin, protein kinase, tyrosine kinase, etc.] and these enzymes inhibitors, hormones (e.g. PTH, TSH, insulin, LH, FSH, prolactin, etc.), receptors (e.g. estrogen, receptors for TSH etc.); ligands (e.g. estrogen, TSH etc.); Diphtheria, meningococcus, gonococcus, staphylococcus, streptococcus, enterobacteria, Escherichia coli, Helicobacter pylori, etc.), viruses (e.g. rubella virus, herpes virus, hepatitis virus, ATL virus, AIDS virus, influenza virus, adenovirus, enterovirus, poliovirus, EB virus, HAV, HBV, HCV, HIV, HTLV, etc.), fungi (e.g., Candida, Cryptococcus, etc.), spirochetes (e.g., Leptospira, Treponema pallidum, etc.), chlamydia, microorganisms such as Mycoplasma; Proteins, peptides, or sugar chain antigens derived from microorganisms; various allergens that cause allergies such as bronchial asthma, allergic rhinitis, atopic dermatitis (e.g., house dust, mites such as Dermatophagoides leopard mite, Dermatophagoides leopard mite, etc., e.g. cedar, cypress) , Pollens such as common barnyard millet, ragweed, giant ragweed, ragweed, and rye, animals such as cats, dogs, and crabs, foods such as rice and egg whites, allergens derived from fungi, insects, wood, drugs, chemical substances, etc.); lipids (eg, lipoproteins, etc.); proteases (eg, trypsin, plasmin, serine proteases, etc.); tumor marker protein antigens (eg, PSA, PGI, PGII, etc.); sugar chain antigens [eg, AFP (eg, L1 to L3, etc.), hCG ( hCG family), transferrin, IgG, thyroglobulin, Decay-accelerating-factor (DAF), carcinoembryonic antigens (such as CEA, NCA, NCA-2, NFA, etc.), CA19-9, PIVKA-II, CA125, prostate-specific antigen , tumor marker sugar chain antigens having special sugar chains produced by cancer cells, ABO sugar chain antigens, etc.]; proteins that bind to (e.g. hyaluronic acid-binding protein, β-glucan-binding protein, etc.); phospholipids (e.g., cardiolipin, etc.); lipopolysaccharides (e.g., endotoxin, etc.); , di-n-butyl phthalate, dicyclohexyl phthalate, benzophenone, octachlorostyrene, di-2-ethylhexyl phthalate, etc.); binding substances; and antibodies against these.

Antibodies used in the present invention include not only immunoglobulin molecules themselves, but also proteolytic enzymes such as papain and pepsin, and decomposition products such as Fab and F(ab') ₂ fragments generated by chemical decomposition. Moreover, the above antibody may be either a polyclonal antibody or a monoclonal antibody. A commonly used method can also be used for obtaining the above antibody.

As the measurement target substance-binding substance as described above, a substance that binds to the measurement target substance or its analogous substance by the reaction between "antigen" and "antibody" or between the reaction between "sugar chain and protein" is preferable. Specifically, an antibody against the substance to be measured or its analogue, an antigen to which the substance to be measured or its analogue binds, or a protein that binds to the substance to be measured or its analogue is preferable, and the substance to be measured or its analogue Antibodies against or antigens that bind to the substance to be measured or similar substances thereof are more preferable. In this specification, when the terms "antigen-antibody reaction", "antigen", and "antibody" are used, in addition to their usual meanings, the above-mentioned compounds capable of agglutinating the sensitized metal complex particles by specific binding reaction are used. It may include both concepts and forms, and should not be interpreted in a limited way.

The method for producing sensitized metal complex particles by immobilizing a supporting substance on the metal complex particles of the present invention is not particularly limited. Conventionally known methods of immobilization by physical and/or chemical bonding can be used. Examples of chemical bonds include at least one organic compound selected from the group consisting of aldehyde, albumin, carbodiimide, streptavidin, biotin, and epoxides, and/or an amino group, a carboxyl group, a hydroxyl group, a mercapto group, and a glycidyloxy group. , a tosyl group, etc. are provided on the surface of the metal complex particles, and the supporting substance is immobilized on the metal complex particles through them. Among these, organic compounds containing a carboxy group or a tosyl group are particularly preferable from the viewpoint of the stability of the metal complex particles themselves and the efficiency of immobilizing the supporting substance.

The bonding amount of the supporting substance in the sensitized metal complex particles of the present invention varies depending on the type of the supporting substance used, and the optimum amount can be appropriately set experimentally. In the present specification, the terms "support", "sensitization" and "immobilization" have their usual meanings and are used synonymously.

The sensitized metal complex particles of the present invention obtained by such a method are coated (blocked) with various polymer compounds or proteins (for example, bovine serum albumin, etc.), if necessary, and then dissolved in an appropriate buffer. It is dispersed and used as a sensitized metal complex particle dispersion. The sensitized metal complex particle dispersion can be used as an immunoassay reagent, and the assay reagent is also one aspect of the present invention. The immunoassay reagent of the present invention can be combined with a diluent (buffer) used for measurement, a standard substance, etc., and used as a measurement reagent kit, and the kit is also one aspect of the present invention.

The diluent is used for diluting the measurement sample and the like. Any buffer solution having a pH of 5.0 to 9.0 can be used as the diluent. Specific examples include phosphate buffer, glycine buffer, Tris buffer, borate buffer, citrate buffer, Good's buffer and the like.

The immunoassay reagents and diluents of the present invention may contain various sensitizers for the purpose of improving the measurement sensitivity and promoting the specific reaction between the substance to be measured and the carrier substance. Examples of the sensitizer include alkylated polysaccharides such as methyl cellulose and ethyl cellulose, pullulan and polyvinylpyrrolidone.

The immunoassay reagents and diluents of the present invention are used for the purpose of suppressing non-specific reactions caused by substances other than the substance to be measured present in the measurement sample, improving the stability of the measurement reagent, etc. , and its degradation products, amino acids, surfactants, and the like. Examples of such additives include proteins such as albumin (bovine serum albumin, ovalbumin), casein and gelatin.

The method for measuring a substance to be measured of the present invention is not particularly limited except that it is performed using the metal complex particles of the present invention. Eiji Ishikawa et al., Igaku Shoin, 1982).

In the method for measuring a substance to be measured of the present invention, the method of contacting the sample, the metal complex particles, the labeled substance-binding substance to be measured, the labeled substance to be measured or a similar substance, etc., includes the usual processes such as stirring and mixing. It suffices that the metal complex particles are dispersed. The reaction time may be appropriately set depending on the substance to be measured, the substance that binds to the substance to be measured, the sandwich method, the competitive method, and the like.

B/F separation (separation of bound labeled antibody and unbound labeled antibody) in the method for measuring a substance to be measured of the present invention is performed, for example, by utilizing the magnetism of the metal complex particles, using a magnet or the like from the outside of the reaction vessel or the like to separate the metal complex particles. are collected, the reaction solution is discharged, a washing solution is added, the magnet is removed, the metal complex particles are mixed and dispersed, and the particles are washed. The above operation may be repeated 1 to 3 times. The cleaning liquid is not particularly limited as long as it is commonly used in this field.

Examples of labeling substances used for labeling measurement target substance-binding substances, measurement target substances, or similar substances include alkaline phosphatase, β-galactosidase, peroxidase, and microperoxidase used in enzyme immunoassay (EIA). , glucose oxidase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, luciferase, tyrosinase, acid phosphatase, etc. ⁹⁹ mTc, ¹³¹ I, used in radioimmunoassay (RIA), for example. Radioactive isotopes such as ¹²⁵ I, ¹⁴ C, ³ H, ³² P, fluorescein, dansyl, fluorescamine, coumarin, naphthylamine or derivatives thereof used in fluorescence immunoassay (FIA), green fluorescent protein ( Fluorescent substances such as GFP), luminous substances such as luciferin, isoluminol, luminol, bis(2,4,6-trifluorophenyl) oxalate, phenol, naphthol, anthracene, or derivatives thereof in the ultraviolet region. Substances with absorption such as 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl, 3-amino-2,2,5,5-tetramethylpyrrolidin-1-oxyl, 2,6- Spin typified by compounds having an oxyl group such as di-t-butyl-α-(3,5-di-t-butyl-4-oxo-2,5-cyclohexadien-1-ylidene)-p-trioxyl Examples thereof include substances having properties as labeling agents. Among these, from the viewpoint of sensitivity and the like, enzymes and fluorescent substances are preferred, alkaline phosphatase, peroxidase and glucose oxidase are more preferred, and peroxidase is particularly preferred.

In order to bind the labeling substance as described above to the substance to be measured, the substance to be measured or a substance analogous thereto, a method commonly used in this field, such as EIA, RIA or FIA, which is known per se, is generally carried out. There is a labeling method known per se [for example, Medical Chemistry Experiment Course, Vol. 8, supervised by Yuichi Yamamura, 1st edition, Nakayama Shoten, 1971; Publishing Co., 1983; Enzyme immunoassay, Eiji Ishikawa, Tadashi Kawai, Kiyoshi Muroi, 2nd edition, Igaku Shoin, 1982, etc., glutaraldehyde method, periodic acid method, maleimide method, pyridyl disulfide method, etc.], etc. You can use it.

The amount of labeling substance to be used may be appropriately set according to the type of labeling substance to be used. For example, it may be contained in a buffer commonly used in this field, such as Tris buffer, phosphate buffer, Veronal buffer, borate buffer, Good's buffer, and the like. Examples of the buffer include those commonly used in this field, such as Tris buffer, phosphate buffer, Veronal buffer, borate buffer, and Good's buffer. It may be within a range that does not inhibit the reaction, and is usually 5 to 9. In addition, such buffers may contain stabilizers such as albumin, globulin, water-soluble gelatin, polyethylene glycol, surfactants, sugars, etc., as long as they do not inhibit the desired antigen-antibody reaction. You can leave it.

As a method for measuring the labeling substance or its activity, a commonly used method can be used without particular limitation. The optical instrument used for the measurement is also not particularly limited, and typically any biochemical automatic analyzer widely used in clinical examination can be used.

EXAMPLES The present invention will be described with reference to examples below, but the present invention should not be construed as being limited to the following examples.

[Examples 1 to 5] Mixing ratio of ligands to be coordinated to metal ions By changing the mixing (addition) ratio of ligand 1 and ligand 2 to be coordinated to metal ions, five types of metal complexes were prepared. prepared. Metal complex particles were prepared using the obtained metal complex, and the content of the metal complex in the obtained metal complex particles was determined.

[Metal Complex Preparation Examples 1 to 5] Synthesis of metal complexes a to e
In an ethanol solution of holmium chloride hexahydrate (HoCl _{3.6H 2} O), 2,2'-bipyridyl ( bpy), dibenzoylmethane (DBM) were added, and after mixing, further diethanolamine was added to adjust the pH to 6-7. After stirring for 24 hours at room temperature, a pale yellow precipitate was obtained. The resulting precipitate was filtered and washed with ethanol to obtain metal complexes a to e having holmium as the metal ion and bpy and/or DBM as the ligand.

[Template particle preparation example 1] Preparation of template particles (II) In a reaction vessel, 500 g of ion-exchanged water, 50 g of styrene (St) (manufactured by NS Styrene Monomer Co., Ltd.) as a monomer constituting the particle size adjusting composition, potassium persulfate Kojunyaku Kogyo Co., Ltd.) was added, and the mixture was stirred at room temperature for 2 hours under a nitrogen atmosphere. After that, the mixture was heated to 70° C. and polymerized for 24 hours to obtain template particles (II) made of polystyrene. The obtained template particles (II) had an average particle size of 1.03 μm and a CV value of 3.8%.

[Metal Complex Particle Preparations 1 to 5] Preparation of metal complex particles of Examples 1 to 5 In 100 g of ion-exchanged water, the content of the template particles (II) was adjusted to 0.1 g, and triethanol lauryl sulfate was added. The above template particles (II) and triethanolamine lauryl sulfate (EMAL TD, manufactured by Kao Corporation) were added so that the amine content was 0.1 g to obtain a template particle (II) dispersion. Next, in Table 1, metal complexes a to e in the amount of g charged in Examples 1 to 5, 0.20 g of styrene (St) (manufactured by NS Styrene Monomer Co., Ltd.) as a monomer for the second particle size adjusting composition, di Metal complexes a to e solutions prepared by dissolving 0.02 g of (secondary butyl) peroxydicarbonate (Luperox 225, manufactured by Arkema Yoshitomi Co., Ltd.) in 4.00 g of ethyl acetate were added to the template particle (II) dispersion. . By stirring at room temperature for 24 hours, composite droplets of the template particles (II) and metal complexes a to e were formed. Thereafter, heating and stirring was performed at 80° C. for 10 hours to polymerize the styrene monomer and simultaneously remove ethyl acetate to obtain Example 1 containing metal complexes a to e and polystyrene (pSt) as the second particle size adjusting composition. ~5 metal complex particles were obtained.
The average particle diameter of the obtained metal complex particles of Examples 1 to 5 is 3.10 μm to 3.23 μm, the CV value is 4.1% to 4.5%, and the metal complex content is 57% to 89%. Met.

Considering that the stable valence of the Ho (holmium) atom is 3 and the coordination number of the Ho (holmium) atom is 8 to 9, ligands are added so that the total valence and coordination number are satisfied. When the metal complex particles of Examples 1 to 5 were prepared using five kinds of metal complexes synthesized with varying ratios, variations in the content of the metal complex were observed in each metal complex particle. By adding DBM in the metal complex synthesis step, an improvement in the content of the metal complex in the metal complex particles was observed. The content of the metal complex synthesized at the addition ratio of 2 equivalents of bpy and 2 equivalents of DBM or 0 equivalents of bpy and 3 equivalents of DBM was the next highest.
From the above, it was confirmed that the content of the metal complex in the metal complex particles can be controlled by changing the addition ratio of bpy and DBM to prepare the metal complex.

[Examples 6 to 8] Combinations of ligands to be coordinated to metal ions Metal complexes were prepared by changing the combinations of ligand 1 and ligand 2 to be coordinated to metal ions to the combinations in Examples 6 to 8. bottom. Metal complex particles were prepared using the obtained metal complex, and the content of the metal complex in the obtained metal complex particles was determined.

[Metal Complex Preparation Examples 6 to 8] Synthesis of metal complexes f to h
In an ethanol solution of holmium chloride hexahydrate (HoCl _{3.6H 2} O), a double equivalent amount of 2,2'-bipyridyl ( bpy), triphenylphosphine oxide (TOPO), 2-thenoyltrifluoroacetone (TTA), and dibenzoylmethane (DBM) are added in combination, and after mixing, further diethanolamine is added to adjust the pH to 6-7. bottom. After stirring for 24 hours at room temperature, a pale yellow precipitate was obtained. After filtering the obtained precipitate and washing with ethanol, the metal ion is holmium, and the ligand is each combination of "bpy and TTA", "TOPO and DBM", and "TOPO and TTA". 6-8 metal complexes were obtained.

[Metal Complex Particle Preparation Examples 6 to 8] Preparation of metal complex particles of Examples 6 to 8 Metal complex particles of Examples 6 to 8 containing metal complexes f to h and polystyrene (pSt) as the second particle size adjusting composition were obtained by the same method as the metal complex particles of Examples 6 to 5.
The average particle diameter of the obtained metal complex particles of Examples 6 to 8 was 3.13 μm to 3.16 μm, the CV value was 4.3% to 4.8%, and the metal complex content was 83% to 94%. Met.

Regarding the types and combinations of ligands to be coordinated to metal ions, three types of "bpy and TTA", "TOPO and DBM", and "TOPO and TTA" were examined, and all metal complexes with high metal complex content were obtained. particles could be obtained.
The content of "bpy and TTA" is equivalent to the content of the metal complex particles of Example 4 obtained by combining "bpy and DBM", and "TOPO and DBM" and "TOPO and TTA" it was high. From the above, it was confirmed that TTA can be used by replacing DBM, and that the content can be improved by replacing bpy with TOPO. Here, TOPO is a phosphorous-based hydrophobic neutral ligand, and the crystallinity of the obtained metal complex is lower than that of "bpy and DBM". It was considered that this difference in crystallinity is related to the fluctuation of the content.

[Examples 9 to 11] Effect of Type and Addition Amount of Second Particle Size Adjusting Composition Metal complex particles having different particle size adjusting compositions were prepared by incorporating the metal complex of Example 4 into template particles (I) made of MMA. The metal complex content of different metal complex particles of the particle size adjusting composition was compared.

In the preparation of the metal complex particles of Example 4, template particles (I) made of MMA and MMA were used as the second particle size adjusting composition, and the metal complex particles of Examples 9 and 11 were prepared as template particles (II) and Metal complex particles of Example 10 were prepared by varying the amount of St in the second particle size adjusting composition.
The average particle diameter of the obtained metal complex particles of Examples 9 to 11 was 3.11 μm to 3.22 μm, the CV value was 4.2% to 4.6%, and the metal complex content was 74% to 8%. Met.

From the results of Example 9, it was confirmed that MMA also has the same effect as St as the second particle size adjusting composition. Moreover, from the results of Examples 10 and 11, it was confirmed that the content of the metal complex can be controlled by the amount of the second particle size adjusting composition added.

[Example 12] Investigation of hydrophobic organic solvent species in metal complex particle preparation process Complex particles were prepared.

When toluene was used as the hydrophobic organic solvent in Example 4, the average particle size and CV value were comparable to those when ethyl acetate was used, but the metal complex content decreased.

From the results of Example 12, it was confirmed that the metal complex content can be controlled by using solvents with different SP values in the step of preparing metal complex particles.

[Examples 13 and 14] Examination of types of metal ions When using metal complex i using Dy (dysprosium) as the metal ion, when using an equal mixture of metal complex d and metal complex i as the metal complex , examined the content of the metal complex.

The metal complex content of the metal complex particles was high both when metal complex i was used and when an equivalent mixture of metal complex d and metal complex i was used.
Dy (dysprosium) is a metal having both magnetism and fluorescence. It was confirmed that according to the production method of the present invention, metal complexes having different fluorescence intensities and magnetisms can be contained in metal complex particles at a high content rate. In particular, particles containing two kinds of metal complexes are novel, and have an unprecedented effect that one metal complex particle emits a plurality of fluorescences. It is thought that the application will be expanded.

[Comparative Examples 1 and 2] Examination of the amount of the second particle size adjusting composition to be added Metal complex particles were prepared by setting the amount of the second particle size adjusting composition charged in Example 4 or Example 9 to 22.4 g, respectively. compared.

When the order of the number of grams charged of the second particle size adjusting composition was changed by two orders of magnitude, the content of the metal complex was remarkably lowered in both St and MMA. Although the second particle size adjusting composition can control the content of the metal complex, it was found to be ineffective when the amount is excessive.

[Comparative Example 3] Investigation of Hydrophobic Organic Solvent Species in the Metal Complex Particle Preparation Process Using the metal complex of Example 4, acetone, which has an SP value of 9.4 but is a hydrophilic organic solvent that dissolves in water, was added to the metal complex particles. The case of application to the preparation process was confirmed.

When acetone, which is a hydrophilic organic solvent with an SP value of 9.4 but is soluble in water, is applied to the metal complex particle preparation step, the content of the metal complex in the metal complex particles is reduced to that of the hydrophobic organic solvent with an SP value of 9.1. was significantly lower than when using It is presumed that one of the reasons for this decrease in the content rate is that the metal complex is likely to exist in the medium outside the template particles when using a hydrophilic organic solvent (acetone) that dissolves in water. The production method of the present invention is more advantageous by selecting two or more types of ligands used for metal complex formation, adjusting the mixing ratio thereof, and selecting a hydrophobic organic solvent with an SP value of 9.1. It was confirmed that it can be used for

[Evaluation method]
*Magnetic collection evaluation A spectrophotometer (U-3900H, manufactured by Hitachi, Ltd.) was set in the spectrophotometer with a magnet (3000 G, W10 mm x D10 mm x H5 mm) applied via a spacer (W10 mm x D10 mm x H10 mm) in advance. A sample (1.2 mL) was placed in a quartz cell, the absorbance was measured after 5 seconds to 125 seconds, and the magnetic collection rate was calculated using Equation (6).
Magnetic collection rate (%) = [{(absorbance 5 seconds after sample addition) - (absorbance 125 seconds after sample addition)}/(absorbance 5 seconds after sample addition)] x 100 Equation (6)
[criterion]
4+: The magnetic collection rate is 80% or more 3+: The magnetic collection rate is 61% or more and less than 70% 2+: The magnetic collection rate is 41% or more and less than 60% 1+: The magnetic collection rate is 21% or more and less than 40% 0.5+: The magnetic collection rate is 11% Not less than 20% 0: Magnetic collection rate is less than 10% The obtained results are summarized in Tables 1, 2 and 3.

[Preparation Example 1]
Synthesis of metal complex (aa) In an ethanol solution of holmium chloride hexahydrate (HoCl3 6H2O), 1-fold equivalent amount of 2,2'-bipyridyl (bpy), 3-fold, etc. is added to holmium chloride hexahydrate. Amount of dibenzoylmethane (DBM) was added and, after mixing, the pH was adjusted to 6-7 by adding more diethanolamine. After stirring for 24 hours at room temperature, a pale yellow precipitate was obtained. The resulting precipitate was filtered and washed with ethanol to obtain a metal complex (aa) having holmium as the metal ion and bpy and DBM as the ligands.

[Preparation Example 2]
Synthesis of Metal Complex (bb) As a ligand, holmium as a metal ion and bpy and TTA as a ligand were prepared in the same manner as in Preparation Example 1, except that thenoyltrifluoroacetone (TTA) was used instead of DBM. A metal complex (bb) was obtained.

[Preparation Example 3]
Synthesis of Metal Complex (cc) The metal ion is dysprosium and the ligands are bpy and DBM in the same manner as in Preparation Example 1, except that dysprosium chloride (DyCl3) is used instead of holmium chloride hexahydrate. , to obtain the metal complex (cc).

[Preparation Example 4]
Synthesis of Metal Complex (dd) Terbium chloride hexahydrate (TbCl3.6H ₂ O) was used instead of holmium chloride hexahydrate, and the metal ion was terbium. A metal complex (dd) with the ligands bpy and DBM was obtained.

[Preparation Example 5]
Synthesis of Metal Complex (ee) As a ligand, 2,2′:6,2″-terpyridine (terpy) was used instead of bpy, and the metal ion was holmium in the same manner as in Preparation Example 1. , metal complexes (ee) were obtained in which the ligands were terpy and DBM.

[Preparation Example 6]
Synthesis of Metal Complex (ff) As the ligand, 1,10-phenanthroline monohydrate (phen) was used instead of bpy, and the metal ion was holmium and coordinated in the same manner as in Preparation Example 1. A metal complex (ff) whose children are phen and DBM was obtained.

[Preparation Example 7]
Synthesis of metal complex (gg) Preparation example except that europium chloride hexahydrate (EuCl3.6H ₂ O) was used instead of holmium chloride hexahydrate, and thenoyltrifluoroacetone (TTA) was used as the ligand. A metal complex (gg) having europium as the metal ion and TTA as the ligand was obtained by the same operation as in 1.

[Preparation Example 8]
Preparation of Template Particles (I) In a reaction vessel, 500 g of ion-exchanged water, 50 g of methyl methacrylate (MMA) (manufactured by Wako Pure Chemical Industries, Ltd.) and potassium persulfate (manufactured by Wako Pure Chemical Industries, Ltd.) as monomers constituting the particle size-adjusting composition. ) was added and stirred for 2 hours at room temperature under a nitrogen atmosphere. After that, the mixture was heated to 70° C. and polymerized for 24 hours to obtain template particles (I) made of polymethyl methacrylate.
The obtained template particles (I) had an average particle diameter of 1.06 μm and a CV value of 3.3%.

[Preparation Example 9]
Preparation of Template Particles (II) Template particles (II) made of polystyrene were prepared in the same manner, except that the monomer constituting the particle size adjustment composition was changed from MMA to styrene (St) (manufactured by NS Styrene Monomer Co., Ltd.). got
The obtained template particles (II) had an average particle size of 1.03 μm and a CV value of 3.8%.

[Example 15]
Preparation of metal complex particles (1) Template particles (I) and triethanolamine lauryl sulfate (EMAL TD, manufactured by Kao Corporation) were added to deionized water to obtain a dispersion of template particles (I). Next, a metal complex (aa) solution prepared by dissolving 2.70 g of metal complex (aa) in 4.00 g of ethyl acetate was added to the template particle (I) dispersion. By stirring at room temperature for 24 hours, composite droplets of the template particles (I) and the metal complex (aa) were formed. After that, the mixture was heated and stirred at 80° C. for 10 hours to remove ethyl acetate, thereby obtaining metal complex particles (1) of the present invention.
The obtained metal complex particles (1) had an average particle diameter of 3.21 µm, a CV value of 3.8%, and a metal complex content of 96.4 w%.

[Example 16]
The template particles (I) and lauryl sulfate were mixed so that the content of the template particles (I) was 0.1 g in 100 g of ion-exchanged water and the content of triethanolamine lauryl sulfate was 0.1 g. Triethanolamine (Emal TD, manufactured by Kao Corporation) was added to deionized water to obtain a template particle (I) dispersion. Next, 2.50 g of the metal complex (aa), 0.20 g of methyl methacrylate (manufactured by Wako Pure Chemical Industries, Ltd.) as a monomer for the second particle size adjusting composition, di (secondary butyl) peroxydicarbonate (Luperox 225, Arkema Yoshitomi) A solution of metal complex (a) prepared by dissolving 0.02 g of (manufactured by Co., Ltd.) in 4.00 g of ethyl acetate was added to the template particle (I) dispersion. By stirring at room temperature for 24 hours, composite droplets of the template particles (I) and the metal complex (aa) were formed. Thereafter, the metal complex particles (2) containing polymethyl methacrylate (pMMA) as the second particle size adjusting composition are obtained by heating and stirring at 80° C. for 10 hours to polymerize methyl methacrylate and simultaneously remove ethyl acetate. Obtained.
The obtained metal complex particles (2) had an average particle size of 3.20 μm, a CV value of 4.2%, and a metal complex content of 89.1% by weight.

[Example 17]
The procedure of Example 2 was repeated except that divinylbenzene (DVB) (DVB960, manufactured by NS Styrene Monomer Co., Ltd.) was used as the monomer for the second particle size adjusting composition. Metal complex particles (3) containing divinylbenzene (pDVB) were obtained.
The obtained metal complex particles (3) had an average particle size of 3.18 μm, a CV value of 4.1%, and a metal complex content of 88.1% by weight.

[Example 18]
The second particle size adjustment was performed in the same manner as in Example 2, except that the template particles (II) were used and styrene (St) (manufactured by NS Styrene Monomer Co., Ltd.) was used as the monomer for the second particle size adjustment composition. Metal complex particles (4) containing polystyrene (pSt) as a composition were obtained.
The obtained metal complex particles (4) had an average particle diameter of 3.10 μm, a CV value of 4.3%, and a metal complex content of 89.0 wt %.

[Example 19]
Metal complex particles (5) were obtained in the same manner as in Example 17, except that metal complex (bb) was used as the metal complex.
The obtained metal complex particles (5) had an average particle diameter of 3.12 μm, a CV value of 4.2%, and a metal complex content of 88.2 wt %.

[Example 20]
The procedure of Example 17 was repeated except that 2.00 g of the metal complex (aa), 0.70 g of divinylbenzene, and 0.08 g of di(secondary butyl)peroxydicarbonate were used to obtain metal complex particles (6) got
The obtained metal complex particles (6) had an average particle diameter of 3.20 μm, a CV value of 3.8%, and a metal complex content of 71.4 w %.

[Example 21]
The procedure of Example 3 was repeated except that 1.60 g of metal complex (aa), 1.10 g of divinylbenzene, and 0.11 g of di(secondary butyl)peroxydicarbonate were used to obtain metal complex particles (7). got
The obtained metal complex particles (7) had an average particle diameter of 3.18 µm, a CV value of 3.9%, and a metal complex content of 55.6 w%.

[Example 22]
The procedure of Example 3 was repeated except that 1.20 g of the metal complex (aa), 1.50 g of divinylbenzene, and 0.15 g of di(secondary butyl)peroxydicarbonate were used to obtain metal complex particles (8). got
The obtained metal complex particles (8) had an average particle diameter of 3.17 μm, a CV value of 4.1%, and a metal complex content of 40.2% by weight.

[Reference example 1]
2.50 g of metal complex (aa), 0.20 g of styrene, 13 g of chloroform, 0.002 g of di(secondary butyl) peroxydicarbonate, polyvinyl alcohol (Gosenol GH20, manufactured by Nippon Gosei Co., Ltd.) 5% by weight aqueous solution 13 g, 1.5 g of triethanolamine lauryl sulfate and 150 g of ion-exchanged water were mixed and stirred at 2500 rpm using a homogenizer. Thereafter, the mixture was transferred to a reaction tank, stirred at 200 rpm for 2 hours under a nitrogen stream, and then heated and stirred at 80° C. for 10 hours to polymerize styrene and simultaneously remove chloroform. After completion of the reaction, centrifugation was repeated to obtain metal complex particles (9) containing polystyrene (pSt) as a particle size adjusting composition.
The obtained metal complex particles (9) had an average particle diameter of 3.31 μm, a CV value of 12.8%, and a metal complex content of 90.2% by weight.

[Reference example 2]
Metal complex particles (10) of the present invention were obtained in the same manner as in Reference Example 1, except that the centrifugation conditions for the centrifugation operation after completion of the reaction in Reference Example 1 were changed and the centrifugation operation was repeated.
The obtained metal complex particles (10) had an average particle diameter of 3.42 µm, a CV value of 17.8%, and a metal complex content of 91.3 w%.

[Example 23]
Metal complex particles (11) were obtained in the same manner as in Example 17, except that metal complex (cc) was used as the metal complex.
The obtained metal complex particles (11) had an average particle size of 3.22 μm, a CV value of 4.8%, and a metal complex content of 89.3 wt %.

[Example 24]
Metal complex particles (12) were obtained in the same manner as in Example 17, except that metal complex (dd) was used as the metal complex.
The obtained metal complex particles (12) had an average particle size of 3.16 μm, a CV value of 4.4%, and a metal complex content of 88.7% by weight.

[Example 25]
Metal complex particles (13) were obtained in the same manner as in Example 17, except that metal complex (ee) was used as the metal complex.
The obtained metal complex particles (13) had an average particle size of 3.22 μm, a CV value of 5.2%, and a metal complex content of 89.2% by weight.

[Example 26]
Metal complex particles (14) were obtained in the same manner as in Example 17, except that metal complex (ff) was used as the metal complex.
The obtained metal complex particles (14) had an average particle size of 3.06 μm, a CV value of 4.9%, and a metal complex content of 87.5% by weight.

[Reference example 3]
The same operation as in Example 17 was performed except that the charged amount of the metal complex (aa) was 0.50 g, and the charged amount of divinylbenzene as the second particle size adjusting composition was 2.20 g. Metal complex particles (15 ).
The obtained metal complex particles (15) had an average particle diameter of 3.18 µm, a CV value of 5.1%, and a metal complex content of 14.8 w%.

[Reference Example 4]
Metal complex particles (16) of the present invention were obtained in the same manner as in Reference Example 1, except that the centrifugation conditions for the centrifugation operation after completion of the reaction in Reference Example 1 were changed and the centrifugation operation was repeated. The obtained metal complex particles (16) had an average particle diameter of 3.33 μm, a CV value of 25.6%, and a metal complex content of 90.4 wt %.

[Reference Example 5]
Metal complex particles (17) were obtained in the same manner as in Example 17, except that metal complex (g) was used as the metal complex. The obtained metal complex particles (17) had an average particle diameter of 3.15 µm, a CV value of 4.8%, and a metal complex content of 88.6 w%.

[Evaluation method]
*Magnetic collection evaluation A spectrophotometer (U-3900H, manufactured by Hitachi, Ltd.) was set in the spectrophotometer with a magnet (3680G, W10mm x D10mm x H5mm) attached in advance via a spacer (W10mm x D10mm x H10mm). A sample (1.2 mL) was placed in a quartz cell, the absorbance was measured after 5 seconds to 125 seconds, and the magnetic collection rate was calculated using Equation (6).
Magnetic collection rate (%) = [{(absorbance 5 seconds after sample addition) - (absorbance 125 seconds after sample addition)}/(absorbance 5 seconds after sample addition)] x 100 Equation (6)
[criterion]
○○○: magnetic collection rate of 61% or more ○○: magnetic collection rate of 41% or more and less than 60% ○: magnetic collection rate of 21% or more and less than 40% ×: magnetic collection rate of less than 10% Summarize the results obtained. 4, Tables 5 and 6.

[Reference Example 6]
In the manufacturing method of Patent Document 3 (Japanese Patent Application Laid-Open No. 2008-127454), as shown in FIG. exist. Therefore, the color of the particles is also brown to black. Also, the particle size is about 0.2 μm, and the content of ferrite particles is about 5 wt %. The 120s magnetization was 8% and the relative fluorescence intensity was 36.

[Reference Example 7]
In the production method described in JP-A-10-55911, the magnetic material (Fe3O4) exists in the surface layer of the particles as shown in FIG. 2A. Moreover, since it does not contain a fluorescent dye, it has no fluorescence. The ferrite content was 18.3w% and the 120s magnetism collection was 83%.

[Reference Example 8]
In the manufacturing method described in the reference patent (Japanese Patent Publication No. 59-500691), the magnetic material (Fe3O4) exists inside the particles. Moreover, since it does not contain a fluorescent dye, it has no fluorescence. The ferrite content was 31.2w% and the 120s magnetism collection was 95%.

Claims (17)

A metal complex particle comprising a magnetic material and a particle size adjusting composition containing a hydrophobic polymer,
The magnetic substance includes a metal complex having both magnetism and fluorescence, consisting of metal ions and ligands,
The metal complex is the following metal complex A or metal complex B,
(Metal complex A)
A metal complex A consisting of a metal ion, a β-diketone-based ligand and a phosphorus-based hydrophobic neutral ligand and satisfying the following formulas (A1) to (A3):
Metal ion: β-diketone ligand = 1: 2 to 3 equivalents (A1),
Metal ion: Phosphorus-based hydrophobic neutral ligand = 1: 2 to 3 equivalents (A2),
The total equivalent amount of the β-diketone ligand and the phosphorus hydrophobic neutral ligand does not exceed 5 equivalents (A3)
(Metal complex B)
A metal complex B consisting of a metal ion, a β-diketone ligand and a heterocyclic ligand and satisfying the following formulas (B1) to (B3):
Metal ion: β-diketone ligand = 1: 1 to 3 equivalents (B1),
Metal ion: heterocyclic ligand = 1: 1 to 3 equivalents (B2),
The total double equivalent weight of the β-diketone ligand and the heterocyclic ligand does not exceed 4 equivalent weights (B3)
The content of the particle size adjusting composition in the metal complex particles is less than 60 w%,
The metal complex particles have a particle size of 500 nm or more and 10 μm or less and a coefficient of variation (CV value) of the particle size of the metal complex particles of 20% or less. 2. The metal complex particle according to claim 1, wherein the magnetic substance is a magnetic substance that does not contain a metal complex having magnetism and no fluorescence. 2. The metal complex particle according to claim 1, wherein the magnetic substance is only the metal complex. 4. The metal complex particles according to any one of claims 1 to 3, wherein the metal ion forming the metal complex has a magnetic moment of 7.5 to 10.7. 5. The metal complex particle according to claim 1, wherein the metal ion forming said metal complex is at least one selected from the group consisting of ions of terbium, dysprosium, holmium and erbium. (1) the β-diketone ligand is dibenzoylmethane or thenoyltrifluoroacetone,
(2) the heterocyclic ligand is a ligand selected from 2,2′-bipyridyl, 2,2′:6,2″-terpyridine, or 1,10-phenanthroline;
(3) The phosphorus-based hydrophobic neutral ligand is a ligand selected from trioctylphosphine oxide, tri-n-octylphosphine and tributyl phosphate, according to any one of claims 1 to 5. Metal complex particles as described. 7. The metal complex particles according to claim 1, wherein the metal complex particles have a particle size of 1 μm or more and 5 μm or less. 8. The metal complex particle according to any one of claims 1 to 7, wherein the content of said metal complex in said metal complex particle is 40w% or more and 98w% or less. 9. The metal complex particle according to any one of claims 1 to 8, comprising a supporting substance on the surface of said metal complex particle. The metal complex particle according to any one of claims 1 to 9, wherein the supporting substance is an antibody or antigen that specifically reacts with the substance to be detected. An immunoassay reagent comprising the metal complex particles according to claim 10 . a step of dispersing template particles made of a particle size-adjusting composition in water in which a surfactant and/or a dispersion stabilizer are dissolved to obtain a dispersion;
dissolving the metal complex in a hydrophobic organic solvent capable of dissolving both the template particles and the metal complex to obtain a metal complex solution;
adding the metal complex solution to the dispersion to absorb the metal complex together with the hydrophobic organic solvent into the template particles;
and a step of distilling off the hydrophobic organic solvent by heating or reducing pressure ,
A method for producing metal complex particles, wherein the metal complex particles have a particle size of 500 nm or more and 10 μm or less and a coefficient of variation (CV value) of the particle size of the metal complex particles of 20% or less. The metal complex solution further contains a polymerizable monomer capable of dissolving both the template particles and the metal complex,
13. The method for producing metal complex particles according to claim 12, further comprising a step of polymerizing the polymerizable monomer by heating and/or light irradiation after absorbing the metal complex into the template particles. 14. The method for producing metal complex particles according to claim 12, wherein the metal complex is composed of metal ions and ligands and has both magnetism and fluorescence. The method for producing metal complex particles according to any one of claims 12 to 14, wherein the hydrophobic organic solvent is ethyl acetate. The method for producing metal complex particles according to any one of claims 12 to 15, wherein the template particles are polystyrene or a derivative thereof, or polymethyl methacrylate or a derivative thereof. The method for producing metal complex particles according to any one of claims 12 to 16, wherein the polymerizable monomer is polystyrene or a derivative thereof, or polymethyl methacrylate or a derivative thereof.

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