JP2013019889A - Magnetic particle and manufacturing method thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a magnetic particle favorable as a biomaterial binding magnetic carrier excelling in a binding property of a biomaterial, in a recovery property by magnetic force, and in a dispersion property when magnetism is removed, and capable of exhibiting excellent sensitivity at an immunoassay including a short-time washing process.SOLUTION: A magnetic particle (E) includes, in a nonmagnetic metal oxide (D), superparamagnetic metal oxide particles (C) having an average particle diameter of 1-15 nm, with (C) accounting for 60-95 wt% of a total weight of (C) and (D). The average particle diameter of (E) is preferably 1-5 μm. (C) is preferably at least one kind selected from a group including magnetite, γ-hematite, magnetite α-hematite intermediate iron oxide and γ- hematite-α-hematite intermediate iron oxide. (D) is preferably at least one kind selected from a group including silicon oxide, titanium oxide, zirconium oxide, aluminum oxide, calcium oxide, yttrium oxide and tantalum oxide.

Description

  The present invention relates to magnetic particles. More specifically, the present invention relates to magnetic particles used for diagnostic agents, therapeutic agents, protein purification, cell separation and the like.

  Conventionally, as a method for detecting or purifying a protein containing a biological substance, for example, a protein from a biological sample, a protein capable of binding to the protein is used to bind the protein in the biological sample to the surface of the particle. In order to remove impurities other than the target protein, etc., there are known methods of washing the particles, collecting the particles bound with proteins, etc., and detecting the amount of protein bound or dissociating it in a protein dissociation solution and purifying it. Yes.

  In addition, magnetic particles are used because they can be easily separated and collected by magnetic force. For example, in Patent Document 1, a magnetic film in which a silica film is formed on the surface of core particles made of iron oxide is used. Silica particles are known. However, the magnetic particles are ferromagnetic, and even if the magnetic field at the time of recovery is removed, the magnetic material itself shows a temporary magnetic field due to ferromagnetism, and the particles self-associate and the cleaning properties deteriorate. It adversely affects the immune response.

  Furthermore, for the purpose of solving self-association of the magnetic substance itself due to ferromagnetism, for example, Patent Document 2 discloses magnetic silica particles using a superparamagnetic magnetic substance as the magnetic substance. However, when the particle size is small, the magnetic particle content is low, and it takes time to collect the particles by magnetic force. When the particle size is large, the specific surface area is small, so that the magnetic particles are bonded. There is a problem that the amount of protein or the like is small.

JP 2000-256388 A JP 2000-40608 A

  The purpose of the present invention is to bind biological materials, have excellent recoverability by magnetic force, and dispersibility in a state where the magnetic force is removed, and can provide excellent sensitivity in immunoassay including a short washing step. The object is to provide magnetic particles suitable as a carrier.

The inventors of the present invention have arrived at the present invention as a result of intensive studies to achieve the above object. That is, the present invention comprises superparamagnetic metal oxide particles (C) having an average particle diameter of 1 to 15 nm in nonmagnetic metal oxide (D), and is based on the total weight of (C) and (D). Magnetic particles (E) containing 60 to 95% by weight of (C); a magnetic substance-binding magnetic carrier (H) comprising the magnetic particles (E); superparamagnetic metal oxide having an average particle diameter of 1 to 15 nm Dispersion (A) containing 30 to 500% by weight of metal alkoxide (N) and dispersant (K) based on the weight of product particles (C) and (C), water, water-soluble organic solvent (L) And a solution (B) containing a nonionic surfactant (P) and a catalyst (Q) for hydrolysis of a metal alkoxide to form an oil-in-water emulsion. It is a manufacturing method of (E).

  By using the magnetic particle (E) of the present invention, it is excellent in magnetic properties and redispersibility of particles after magnetism collection, and can be detected with high sensitivity in a short time when used as a reagent for detecting biological substances. it can.

<Magnetic particles (E)>
The magnetic particles (E) of the present invention comprise superparamagnetic metal oxide particles (C) having an average particle diameter of 1 to 15 nm and superparamagnetism in a nonmagnetic metal oxide (D) matrix, preferably Particles obtained by dispersing (D) in (C).

  Various known nonmagnetic metal oxides can be used as the nonmagnetic metal oxide (D). Among the nonmagnetic metal oxides (D), silicon oxide, titanium oxide, zirconium oxide, aluminum oxide, calcium oxide, yttrium oxide and tantalum oxide are preferable from the viewpoint of dispersibility of the magnetic particles (E) in water. Silicon oxide More preferred are titanium oxide, zirconium oxide and aluminum oxide, and most preferred is silicon oxide.

The average particle diameter of the superparamagnetic metal oxide particles (C) in the present invention is an average value of particle diameters measured by observing arbitrary 200 (C) with a scanning electron microscope.
The average particle diameter of (C) can be controlled by adjusting the metal ion concentration at the time of preparation of (C) described later. Further, the average particle diameter of the superparamagnetic metal oxide can be controlled also by a usual method such as classification.

  Superparamagnetism refers to a temporary magnetic field that is induced by the individual atomic magnetic moments of a material being aligned in the presence of an external magnetic field, and that when the external magnetic field is removed, partial alignment is impaired and no magnetic field is exhibited. Say.

Examples of the superparamagnetic metal oxide particles (C) having an average particle diameter of 1 to 15 nm and exhibiting superparamagnetism include oxides such as iron, cobalt, nickel, and alloys thereof, but have excellent sensitivity to magnetic fields. Therefore, iron oxide is particularly preferable. A superparamagnetic metal oxide may be used individually by 1 type, or may use 2 or more types together.
When the average particle diameter of (C) is less than 1 nm, synthesis is difficult, and when the average particle diameter exceeds 15 nm, the magnetic properties of the obtained magnetic particles (E) become ferromagnetic, and the magnetic field in the actual application aspect Even if the magnetic material is removed, there is a problem that the magnetic substance itself exhibits a temporary magnetic field and the particles self-associate with each other, resulting in poor cleanability and / or adverse effects on immune reactions and the like.

  As iron oxide, various known iron oxides can be used. Among iron oxides, magnetite, γ-hematite, magnetite-α-hematite intermediate iron oxide and γ-hematite-α-hematite intermediate iron oxide are preferred because of their excellent chemical stability, and have a large saturation magnetization. Magnetite is more preferable because of its excellent sensitivity to an external magnetic field.

  The lower limit of the content of the superparamagnetic metal oxide particles (C) in the magnetic particles (E) is 60% by weight, preferably 65% by weight, and the upper limit is 95% by weight, preferably 80% by weight. When the content of (C) is less than 60% by weight, the obtained magnetism of (E) is not sufficient, so that the separation operation takes time when used as a magnetic substance-binding magnetic carrier, and exceeds 95% by weight. Products are difficult to synthesize and have poor cleanability when used for diagnostics, protein purification, nucleic acid purification, and the like.

  The production method of the superparamagnetic metal oxide particles (C) is not particularly limited, but based on the one reported by Massart, a coprecipitation method using a water-soluble iron salt and ammonia (R. Massart, IEEE Trans. Magn. 1981). , 17, 1247) and a method using an oxidation reaction in an aqueous solution of a water-soluble iron salt.

  The average particle diameter of the magnetic particles (E) is preferably 1 to 5 μm, more preferably 1 to 3 μm. When the average particle size is 1 μm or more, it does not take time for separation and recovery. When it is 5 μm or less, the surface area becomes large, the amount of biological substances bound is high, and the recovery efficiency tends to be high.

The average particle diameter of the magnetic particles (E) of the present invention is an average value of particle diameters measured by observing an arbitrary 200 (E) with a scanning electron microscope.
The average particle size of (E) can be controlled by adjusting the particle size of the oil-in-water emulsion by adjusting the mixing conditions (shearing force, etc.) when preparing the oil-in-water emulsion described later. In addition, (E) The average particle diameter can be set to a desired value also by a method such as a change in conditions of the washing step during production or a normal classification.

<Method for Producing Magnetic Particle (E)>
The method for producing magnetic particles (E) according to the present invention comprises superparamagnetic metal oxide particles (C) having an average particle diameter of 1 to 15 nm, and 30 to 500% by weight of metal alkoxide (N) based on the weight of (C). And a dispersion liquid (A) containing the dispersant (K) and a solution containing water, a water-soluble organic solvent (L), a nonionic surfactant (P) and a catalyst for hydrolysis of metal alkoxide (Q) (B) is mixed and the process of forming an oil-in-water emulsion is included.

After the formation of the oil-in-water emulsion, the metal alkoxide (N) is hydrolyzed and condensed to include the superparamagnetic metal oxide particles (C) in the nonmagnetic metal oxide (D), preferably (D). An aqueous dispersion of magnetic particles (E) in which (C) is uniformly dispersed is obtained.
The obtained aqueous dispersion of magnetic particles (E) is subjected to solid-liquid separation by centrifugation and / or magnetism, washed with water or methanol, and dried to obtain magnetic particles (E).

Dispersion (A)
(A) is a dispersion (A) in which superparamagnetic metal oxide particles (C) are dispersed in a metal alkoxide (N) using a dispersant (K). Examples of the dispersant (K) include organic compounds having one or more carboxyl groups in the molecule, organic compounds having one or more sulfo groups, and organic compounds having one or more carboxyl groups and one or more sulfo groups. Compounds and the like. Specific examples include the organic compounds (K-1) to (K-5) exemplified below, and these may be used alone or in combination of two or more.

(K-1) Organic compound having one carboxyl group:
C1-C30 aliphatic saturated monocarboxylic acid (formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, verargonic acid, lauric acid, myristic acid, stearic acid and behenine Acid etc.), C3-C30 aliphatic unsaturated monocarboxylic acid (acrylic acid, methacrylic acid, oleic acid, stearic acid, etc.), C3-C30 hydroxy aliphatic monocarboxylic acid (glycolic acid, lactic acid and the like) Tartaric acid, etc.), C4-30 alicyclic monocarboxylic acids (cyclopropanecarboxylic acid, cyclopentanecarboxylic acid, cyclohexanecarboxylic acid, etc.), C7-30 aromatic monocarboxylic acids (benzoic acid, cinnamon) Acid, naphthoic acid, etc.), C7-20 hydroxy aromatic monocarboxylic acids (salicylic acid, mandelic acid, etc.) and carbon number To 20 perfluorocarboxylic acids (trifluoroacetic acid, undecafluorohexanoic acid, tridecafluoroheptanoic acid, perfluorooctanoic acid, pentadecafluorooctanoic acid, peptadecafluorononanoic acid, nonadecafluorodecanoic acid, etc.) .

(K-2) Organic compound having one sulfo group:
C1-C30 aliphatic monosulfonic acid (methanesulfonic acid, ethanesulfonic acid, propanesulfonic acid, butanesulfonic acid, hexanesulfonic acid, decanesulfonic acid, undecanesulfonic acid, dodecanesulfonic acid, etc.), C6-C30 Aromatic monosulfonic acid (benzenesulfonic acid, p-toluenesulfonic acid, o-toluenesulfonic acid, m-toluenesulfonic acid, 4-dodecylbenzenesulfonic acid, 4-octylbenzenesulfonic acid, naphthalenesulfonic acid, etc.) and carbon number 1-20 perfluoroalkanesulfonic acid (such as trifluoromethanesulfonic acid) and the like.

(K-3) Organic compounds each having one or more carboxyl groups and sulfo groups:
C2-C30 sulfocarboxylic acid (such as sulfoacetic acid and sulfosuccinic acid) and C7-C30 sulfoaromatic mono- or polycarboxylic acid (o-, m- or p-sulfobenzoic acid, 2,4-disulfo) Benzoic acid, 3-sulfophthalic acid, 3,5-disulfophthalic acid, 4-sulfoisophthalic acid, 2-sulfoterephthalic acid, 2-methyl-4-sulfobenzoic acid, 2-methyl-3,5-disulfobenzoic acid, 4 -Propyl-3-sulfobenzoic acid, 4-isopropyl-3-sulfobenzoic acid, 2,4,6-trimethyl-3-sulfobenzoic acid, 2-methyl-5-sulfoterephthalic acid, 5-methyl-4-sulfo Isophthalic acid, 5-sulfosalicylic acid, 3-oxy-4-sulfobenzoic acid and the like).

(K-4) Organic compound having two or more carboxyl groups:
C2-C30 aliphatic polycarboxylic acid (oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, propane-1,2,3-tricarboxylic acid, Saturated polycarboxylic acids such as citric acid and dodecanedioic acid and unsaturated polycarboxylic acids such as maleic acid, fumaric acid and itaconic acid), aromatic polycarboxylic acids having 8 to 30 carbon atoms (phthalic acid, isophthalic acid, terephthalic acid) Acid, trimellitic acid, pyromellitic acid and the like) and alicyclic polycarboxylic acid having 5 to 30 carbon atoms (cyclobutene-1,2-dicarboxylic acid, cyclopentene-1,2-dicarboxylic acid, furan-2,3-dicarboxylic acid) Acids, bicyclo [2,2,1] hept-2-ene-2,3-dicarboxylic acid and bicyclo [2,2,1] hepta-2,5-diene- , 3-dicarboxylic acid, etc.) and the like.

(K-5) Organic compound having two or more sulfo groups:
C1-C30 aliphatic polysulfonic acid (methionic acid, 1,1-ethanedisulfonic acid, 1,2-ethanedisulfonic acid, 1,1-propanedisulfonic acid, 1,3-propanedisulfonic acid, polyvinylsulfonic acid, etc. ), Aromatic polysulfonic acid having 6 to 30 carbon atoms (m-benzenedisulfonic acid, 1,4-naphthalenesulfonic acid, 1,5-naphthalenedisulfonic acid, 1,6-naphthalenedisulfonic acid, 2,6-naphthalenedisulfonic acid 2,7-naphthalenedisulfonic acid and sulfonated polystyrene, etc.), bis (fluorosulfonyl) imide and bis (perfluoroalkanesulfonyl) imide having 2 to 10 carbon atoms [bis (trifluoromethanesulfonyl) imide, bis (pentafluoroethane) Sulfonyl) imide, bis (nonafluorobutanesulfonate) ) Imide, pentafluoroethanesulfonyl trifluoromethanesulfonyl imide, trifluoromethanesulfonyl heptafluoropropanesulfonyl imide, nonafluorobutanesulfonyl trifluoromethanesulfonyl imide], and the like.

  Among these, an organic compound having 10 to 30 carbon atoms in (K-1) is preferable from the viewpoint of compatibility with the metal alkoxide.

  The amount of the dispersant (K) used is preferably 100 to 2,000% by weight, particularly 250 to 1,000% by weight, based on the weight of the superparamagnetic metal oxide particles (C). When (K) is 100% by weight or more, (C) tends to be easily dispersed in the metal alkoxide solution, and when it is 2,000% by weight or less, an emulsion is formed during dispersion in an aqueous solution in a later step. It tends to be easy to do.

Examples of the metal alkoxide (N) to be used include compounds represented by the following general formula (1).
R ¹ _m M (OR ² ) _n (1)
In General Formula (1), R ¹ and R ² represent a monovalent hydrocarbon group having 1 to 10 carbon atoms that may be substituted with an amino group, a carboxyl group, a hydroxyl group, a mercapto group, or a glycidyloxy group.

  Examples of the hydrocarbon group having 1 to 10 carbon atoms include an aliphatic hydrocarbon group having 1 to 10 carbon atoms (methyl group, ethyl group, n- or iso-propyl group, n- or iso-butyl group, n- or iso group). -Pentyl group, vinyl group and the like), aromatic hydrocarbon groups having 6 to 10 carbon atoms (phenyl group and the like), and aromatic aliphatic groups having 7 to 10 carbon atoms (benzyl group and the like).

M in the general formula (1) represents a metal element. Specific examples of the metal element include Si, Al, Zr, Ti, Ca, Y, and Ta.

In the general formula (1), m and n represent integers, and the sum of m and n is the valence of the metal element. For example, Si is 4, Al is 3, and Ti is 4.
When the valence is 4, m is an integer of 0 to 3, and n is an integer of 1 to 4. When the valence is 3, m is an integer of 0 to 2, and n is an integer of 1 to 3.

In the general formula (1), when M is Si, when n is 1 alkylalkoxysilane, it is necessary to use it together with (alkyl) alkoxysilane where n is 2 to 4. From the viewpoint of the strength of the particles after the reaction and the amount of silanol groups on the particle surface, n is preferably 4.
Specific examples of the metal alkoxide when M is Si include alkoxysilanes such as tetramethoxysilane, tetraethoxysilane, tetraisopropoxysilane and tetrabutoxysilane; alkylalkoxysilanes such as methyltrimethoxysilane and methyltriethoxysilane; Amino groups such as 3-aminopropyltrimethoxysilane, 3-aminopropyl-triethoxysilane, N-2 (aminoethyl) 3-aminopropyltrimethoxysilane and N-2 (aminoethyl) 3-aminopropyltriethoxysilane Alkylalkoxysilanes having an alkyl group substituted with an alkylalkoxysilane having an alkyl group substituted with a carboxyl group such as 7-carboxy-heptyltriethoxysilane and 5-carboxy-pentyltriethoxysilane Silanes; alkyl alkoxysilanes having alkyl groups substituted with hydroxyl groups such as 3-hydroxypropyltrimethoxysilane and 3-hydroxypropylethoxysilane; mercapto groups such as 3-mercaptopropyltrimethoxysilane and 3-mercaptopropyltriethoxysilane And alkylalkoxysilanes having an alkyl group substituted with a glycidyloxy group such as 3-glycidyloxypropyltrimethoxysilane and 3-glycidyloxypropyltriethoxysilane.

When the metal M is Ti in the general formula (1), the metal alkoxide includes trimethoxy titanium, tetramethoxy titanium, triethoxy titanium, tetraethoxy titanium, tetrapropoxy titanium, chlorotrimethoxy titanium, chlorotriethoxy titanium, ethyltri Examples include methoxy titanium, methyl triethoxy titanium, ethyl triethoxy titanium, diethyl diethoxy titanium, phenyl trimethoxy titanium, and phenyl triethoxy titanium.
When the metal M is Al, Zr, Ca, Y and Ta, the metal alkoxide includes trimethoxyaluminum, triethoxyaluminum, tri-n-propoxyaluminum, triisopropoxyaluminum, tri-n-butoxyaluminum, triisobutoxy Aluminum alkoxides such as aluminum, tri-sec-butoxyaluminum and tri-tert-butoxyaluminum; zirconium alkoxides such as zirconium tetraethoxide, zirconium tetrapropoxide and zirconium tetrabutoxide; calcium methoxide, calcium ethoxide, calcium propoxide and Calcium alkoxide such as calcium isopropoxide, yttrium triethoxide, yttrium triisopropoxide side and And yttrium alkoxides such as yttrium trimethoxy ethoxide; tantalum alkoxides such as tantalum pentamethoxide, tantalum pentaethoxide, tantalum pentaisopropoxide, and tantalum pentabutoxide.
A metal alkoxide (N) may be used individually by 1 type, or may use 2 or more types together.

  The usage-amount of a metal alkoxide (N) is 30 to 500 weight% with respect to a superparamagnetic metal oxide particle (C), Preferably it is 40 to 200 weight%. When the metal alkoxide is less than 30% by weight, the surface of (C) becomes difficult to be uniformly coated. When the metal alkoxide exceeds 500% by weight, the content of (C) decreases and the recovery time by magnetic force increases.

The method for producing the dispersion (A) is not particularly limited, and for example, the dispersant (K) can be dissolved in the metal alkoxide (N) and then mixed with the superparamagnetic metal oxide particles (C). Further, after the physical adsorption of the dispersant (K) to the superparamagnetic metal oxide particles (C), excess dispersant can be removed by washing and dispersed in the metal alkoxide (N).

Solution (B)
The solution (B) is obtained by dissolving a nonionic surfactant (P) and a metal alkoxide hydrolysis catalyst (Q) in a mixed solution of water and a water-soluble organic solvent (L).
Examples of the water-soluble organic solvent (L) include alcohols having 1 to 3 carbon atoms (methanol, ethanol, n- or iso-propanol, etc.) having a solubility in water at 25 ° C. of 100 g / 100 g or more of water and 2 carbon atoms. ~ 9 glycols (such as ethylene glycol and diethylene glycol), amides (such as N-methylpyrrolidone), ketones (such as acetone), cyclic ethers (such as tetrahydrofuran), lactones (such as γ-butyrolactone), sulfoxides (such as dimethyl sulfoxide) and nitriles (Acetonitrile and the like).
Among these, C1-C3 alcohol is preferable from a viewpoint of the uniformity of the particle diameter of a magnetic particle (E). A water-soluble organic solvent may be used individually by 1 type, or may use 2 or more types together.
It is preferable that the usage-amount of a water-soluble organic solvent (L) is 100 to 500 weight% with respect to water.

  As nonionic surfactant (P), higher alcohol alkylene oxide (hereinafter, alkylene oxide is abbreviated as AO) adduct; higher alcohol having 8 to 24 carbon atoms (decyl alcohol, dodecyl alcohol, coconut oil alkyl alcohol, octadecyl) 1-20 mol of ethylene oxide (hereinafter abbreviated as EO) and / or 1-20 mol of propylene oxide (hereinafter abbreviated as PO) (including block adducts and / or random adducts) of alcohol and oleyl alcohol) The same shall apply hereinafter), AO adducts of alkylphenols having alkyl of 6 to 24 carbon atoms; EO 1-20 mol and / or PO 1-20 mol adducts of octyl or nonylphenol, polypropylene glycol EO adducts and polyethylene glycol PO adducts; Pluro Fatty acid AO adducts such as sac-type surfactants; EO 1-20 mol of fatty acids having 8 to 24 carbon atoms (decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, coconut oil fatty acid, etc.) And / or PO1-20 mol adducts and polyhydric alcohol type nonionic surfactants; C3-C3 2-8 polyhydric alcohols (glycerin, trimethylolpropane, pentaerythritol, sorbit, sorbitan, etc.) EO and / or PO adducts of the above; fatty acid esters of polyhydric alcohols and EO adducts thereof [TWEEN® 20 and TWEEN® 80 etc.]; alkyl glucosides (N-octyl-β-D-maltosides) N-dodecanoyl sucrose and n-octyl-β-D-glucopyranoside); sugar fatty acid ester Le, fatty acid alkanolamides and their AO adducts (polyoxyethylene fatty acid alkanolamide, etc.); non-ionic surfactants, and the like. These may be used alone or in combination of two or more.

  The amount of the nonionic surfactant (P) used is preferably 100 to 1,000% by weight, particularly 300 to 500% by weight, based on the superparamagnetic metal oxide particles (C). When the content is 100% by weight or more or 1,000% by weight or less, the emulsion is stable, and the particle size distribution of the generated particles is preferably narrowed.

The amount of the solution (B) used is preferably 1,000 to 10,000% by weight, more preferably 1,500 to 4,000% by weight, based on (C). When the content is 1,000% by weight or more or 10,000% by weight or less, the emulsion is stable, and the particle size distribution of the generated particles is preferably narrowed.

As the metal alkoxide hydrolysis catalyst (Q), acids and bases can be used. Specifically, inorganic acids such as hydrochloric acid, organic acids such as acetic acid, inorganic basic compounds such as ammonia, ethanolamine, etc. These amine compounds can be used.
The amount of the hydrolysis catalyst used is preferably 0.01 to 100% by weight, particularly preferably 0.2 to 50% by weight, based on the weight of the metal alkoxide (N).

  The amount of water used is preferably 500 to 3,000% by weight, more preferably 800 to 2,000% by weight, based on (C).

  The dispersion (A) can further contain a water-insoluble organic solvent (M). As the water-insoluble organic solvent (M), an aromatic hydrocarbon solvent having 6 to 16 carbon atoms (toluene, xylene, etc.) having a solubility in water at 25 ° C. of 0.1 g / 100 g or less of water and 5 carbon atoms. -16 aliphatic hydrocarbon solvents (n-heptane, n-hexane, cyclohexane, n-octane, decane, decahydronaphthalene, etc.) and the like. These may be used alone or in combination of two or more.

  The amount of the water-insoluble organic solvent (M) used is preferably 200 to 1,000% by weight, more preferably 250 to 500% by weight, based on the superparamagnetic metal oxide particles (C). When the amount of (M) used is 200% by weight or more, the dispersibility of the superparamagnetic metal oxide is good, and when it is 1,000% by weight or less, the particle diameter of (C) tends to be uniform. .

The method for preparing the solution (B) is not particularly limited. For example, a nonionic surfactant (P), a catalyst for hydrolysis of metal alkoxide (Q), water, and a water-soluble organic solvent (L) are mixed. Can be adjusted.

The mixing method of the dispersion liquid (A) and the solution (B) is not particularly limited, and can be mixed at once using the equipment described below, but from the viewpoint of the uniformity of the particle diameter of the magnetic particles (E). A method of dropping the dispersion (A) into the solution (B) while stirring the solution (B) is preferable.
The amount of (B) used is preferably 2 to 350% by weight relative to (A). Within this range, the emulsion is stable and the particle size distribution of the generated particles is preferably narrowed.

  The conditions for mixing and reacting the dispersion (A) and the solution (B) are preferably 25 to 100 ° C, more preferably 45 to 60 ° C. Moreover, reaction time becomes like this. Preferably it is 0.5 to 5 hours, More preferably, it is 1-2 hours.

  The equipment for mixing the dispersion (A) and the solution (B) is not particularly limited as long as it is generally commercially available as an emulsifier or a disperser. For example, a homogenizer (manufactured by IKA), Polytron (Registered trademark, manufactured by Kinematica) and batch type emulsifiers such as TK auto homomixer (manufactured by Primics), Ebara Milder (manufactured by Ebara Seisakusho), TK Philmix, TK pipeline homomixer (manufactured by Primics), Colloid mill (manufactured by Shinko Pantech Co., Ltd.), clear mix (manufactured by M Technique Co., Ltd.), slasher, trigonal wet pulverizer (manufactured by Mitsui Miike Chemical Co., Ltd.), Captron (manufactured by Eurotech Co., Ltd.) and fine flow mill (Pacific Kiko Co., Ltd.) Manufactured), etc., microfluidizer (manufactured by Mizuho Kogyo Co., Ltd.), nanomizer (registered trademark, nano High pressure emulsifiers such as Iser) and APV Gaurin (made by Gaurin), membrane emulsifiers such as membrane emulsifier (made by Chilling Industries Co., Ltd.), vibration type such as Vibro mixer (registered trademark, manufactured by Chilling Industries Co., Ltd.) Examples include an emulsifier and an ultrasonic emulsifier such as an ultrasonic homogenizer (manufactured by Branson). From the viewpoint of uniform particle size, APV Gaurin, homogenizer, TK auto homomixer, Ebara Milder, TK Philmix, TK A pipeline homomixer and a clear mix (manufactured by M Technique) are preferred.

  The magnetic particle (E) of the present invention is used as a magnetic carrier for binding biological substances because the superparamagnetic metal oxide particles (C) are included in the nonmagnetic metal oxide (D) and are not present on the particle surface. In many cases, many biological substances can be bound.

  When the magnetic particle (E) of the present invention is used as a biological carrier for binding a biological material, the biological material can be physically adsorbed to (E), but from the viewpoint of binding the biological material more efficiently, glutaraldehyde, albumin It is preferable that at least one organic compound selected from the group consisting of carbodiimide, streptavidin, biotin, and metal alkoxide (N) having a functional group is bonded to the surface of (E). Of these organic compounds, a metal alkoxide (N) having a functional group is more preferable from the viewpoint of binding a specific biological substance.

  Examples of the functional group possessed by the metal alkoxide (N) include an amino group, a carboxyl group, a hydroxyl group, a mercapto group, and a glycidyloxy group. (N) Even if each molecule has different types of functional groups. Good.

  As a method for bonding (N) having a functional group to the surface of (E), the alkoxide (N1) having an alkyl group substituted with the aforementioned amino group, carboxyl group, hydroxyl group, mercapto group or glycidyloxy group is used. And a method of treating (E) with (N1) after preparing (E) using an alkoxide (N2) having no substituent.

  As a specific example of the latter method, (E) is dispersed in a solvent so that its concentration is 1 to 50% by weight, and the solution of (N1) is added to this dispersion, followed by adding water for 1 hour or more at room temperature. The method of performing a decomposition reaction and a condensation reaction is mentioned.

  The solvent in this method is appropriately selected according to the solubility of the metal alkoxide to be used. When (N1) soluble in water is used, it is preferable to use water or a mixed solvent of water-alcohol, etc., and dissolve in water. When (N1) having an alkyl group substituted with a glycidyloxy group which is difficult to form is used, it is preferable to use butyl acetate or the like.

  It is preferable that the usage-amount of (N1) is 0.1 to 5 weight% with respect to the magnetic particle (E) before a process. If it is 0.1% by weight or more, the number of functional groups to be introduced is sufficient, and if it is 5% by weight or less, the amount of functional groups on the particle surface is further increased, which is preferable.

The method for binding glutaraldehyde, albumin, carbodiimide, streptavidin, or biotin to the surface of the magnetic particle (E) is not particularly limited. For example, the binding can be performed as follows.
Glutaraldehyde having an aldehyde group and biotin having a carboxyl group can be bound to the surface of (E) by reacting with a metal alkoxide having an amino group bound to the surface. In addition, albumin and streptavidin having an amino group and carbodiimide having a carbodiimide group can be bonded to the surface of (E) by reacting with a metal alkoxide having a carboxyl group bonded to the surface.

  The biological substance in the present invention means a substance derived from an organism, but is not limited to the biological substance itself, and includes substances having an interaction with the biological substance. For example, carbohydrates, proteins, peptides, nucleic acids, cells, Examples include substances that can be used for fixing microorganisms, drugs or drug candidate substances, harmful substances such as environmental hormones, or other biological substances such as biotin.

  The biological material protein or peptide immobilized on the magnetic particles (E) is preferably either an antibody or an antigen, or a biological receptor or a ligand having specific binding properties to each other. In the case of an antibody / antigen, when the protein to be purified is an antibody, the protein or peptide that becomes the antigen is immobilized on the particle. On the other hand, when the protein to be purified becomes an antigen, the protein or peptide that becomes an antibody is immobilized on the particle. The same applies to biological receptors / ligands as in the case of antibodies / antigens.

  The method for binding the biological substance to the magnetic particles (E) is not particularly limited. For example, when the biological substance is an antibody or an antigen, it has an amino group, and thus reacts with (E) having glutaraldehyde bound to the surface. The biological material can be bound to the surface of (E).

  EXAMPLES Hereinafter, although an Example demonstrates this invention further, this invention is not limited to these. Hereinafter, unless otherwise specified, “%” represents “% by weight” and “parts” represents “parts by weight”.

Example 1
In a reaction vessel, 2.7 parts of iron (III) chloride hexahydrate, 1.0 part of iron (II) chloride tetrahydrate and 375 parts of water were dissolved and heated to 50 ° C. Was added dropwise over a period of 1 hour, and the mixture was stirred for 1 hour, and magnetite particles (C-1) were submerged in water. Got. 10.5 parts of oleic acid which is a dispersing agent (K-1) was added to the obtained magnetite particles (C-1), and stirring was continued for 2 hours. After cooling to room temperature, the operation of washing the magnetite particles adsorbed with oleic acid obtained by solid-liquid separation by decantation with 50 parts of water was performed three times. The obtained magnetite particles adsorbed with oleic acid are charged into a container, 5.7 parts of decane (M-1) and 2.2 parts of tetraethoxysilane (N-1) are added and mixed, and the dispersion (A-1 ) Was prepared.

  In a reaction vessel, 39.0 parts of 25% ammonia (Q-1) aqueous solution, 55.4 parts of isopropanol (L-1), 2.9 parts of sorbitan monooleate (“Ionet S-80” manufactured by Sanyo Chemical Industries, Ltd.) And polyoxyethylene (addition mole number 20 moles) alkyl ether (Sanyo Chemical Industry Co., Ltd. “Emalmin 200”) (P-1) 2.0 parts and mixed using a clear mix (Mtechnic Co., Ltd.), After the temperature was raised to 50 ° C., the dispersion (A-1) was added dropwise over 1 hour while stirring at a clear mix rotation speed of 6,000 rpm, and then reacted at 50 ° C. for 1 hour. After the reaction, the mixture was centrifuged at 2,000 rpm for 5 minutes to remove the supernatant containing fine particles. 50 parts of water was added to the obtained solid phase to disperse the particles, and after centrifugation at 1,000 rpm for 10 minutes, the operation of removing the supernatant containing fine particles was performed 10 times. Subsequently, 50 parts of water is added to the obtained solid phase to disperse the particles and centrifuged at 500 rpm for 5 minutes to precipitate particles having a large particle size and contain particles having a target particle size. The supernatant (1) was collected. After adding 50 parts of water to the remaining solid phase and centrifuging at 500 rpm for 5 minutes, the operation of recovering the supernatant (2) was performed twice, and the particles having the target particle size present in the solid phase were recovered. . Next, with respect to the supernatants (1) and (2), particles are collected using a magnet, and the collected particles are dried at 80 ° C. for 8 hours to obtain the magnetic particles (E-1) of the present invention. It was.

Example 2
Magnetic particles (E-2) were obtained in the same manner as in Example 1 except that the rotation speed of the clear mix during emulsification was changed from 6,000 rpm to 7,500 rpm.

Example 3
Magnetic particles (E-3) were obtained in the same manner as in Example 1 except that the amount of tetraethoxysilane charged was changed from 2.2 parts to 0.5 parts.

Example 4
Magnetic particles (E-4) were obtained in the same manner as in Example 3, except that the rotation speed of the clear mix during emulsification was changed from 6,000 rpm to 7,500 rpm.

Example 5
Magnetic particles (E-5) were obtained in the same manner as in Example 1 except that the amount of water mixed with 3.8 parts of 25% ammonia water at the time of magnetite particle preparation was changed from 100 parts to 33.8 parts.

Example 6
Magnetic particles (E-6) were obtained in the same manner as in Example 5 except that the rotational speed of the clear mix during emulsification was changed from 6,000 rpm to 7,500 rpm.

Example 7
Magnetic particles (E-7) were obtained in the same manner as in Example 5 except that the amount of tetraethoxysilane charged was changed from 2.2 parts to 0.5 parts.

Example 8
Magnetic particles (E-8) were obtained in the same manner as in Example 7 except that the rotation speed of the clear mix during emulsification was changed from 6,000 rpm to 7,500 rpm.

Example 9
The amount of water mixed with 3.8 parts of 25% ammonia water at the time of magnetite particle production was changed from 100 parts to 55 parts, and the charge amount of tetraethoxysilane was changed from 2.2 parts to 1.2 parts, In the same manner as in Example 1, magnetic particles (E-9) were obtained.

Example 10
Magnetic particles (E-10) were obtained in the same manner as in Example 2 except that the amount of tetraethoxysilane charged was changed from 2.2 parts to 2.2 parts of tetraethoxytitanium [Wako Pure Chemical Industries, Ltd.]. It was.

Example 11
Magnetic particles (E-11) were obtained in the same manner as in Example 2 except that the amount of tetraethoxysilane charged was changed from 2.2 parts to 1.8 parts of tetraethoxysilane and 0.4 parts of tetraethoxytitanium.

Example 12
2.2 parts of γ-hematite particles (manufactured by Sigma Aldrich Japan Co., Ltd.) having an average particle size of 15 nm were mixed with 5.7 parts of decane and 2.2 parts of tetraethoxysilane to obtain a dispersion (A-2). . Magnetic particles (E-12) were obtained in the same manner as in Production Example 1 except that the dispersion (A-1) was replaced with the dispersion (A-2).

Example 13
Magnetic particles (E-13) were obtained in the same manner as in Example 6 except that the amount of tetraethoxysilane charged was changed from 2.2 parts to 2.2 parts of triethoxyaluminum [Wako Pure Chemical Industries, Ltd.]. It was.

Example 14
0.8 parts of γ-hematite particles were mixed with 5.7 parts of decane and 3.5 parts of tetraethoxysilane to obtain a dispersion (A-3). Magnetic particles (E-14) were obtained in the same manner as in Example 12 except that the dispersion (A-2) was replaced with the dispersion (A-3).

Comparative Example 1
Comparative magnetic particles (E-1 ′) were obtained in the same manner as in Example 4, except that the amount of tetraethysilane was changed from 0.5 part to 8.0 parts.

Comparative Example 2
Comparative magnetic particles (E-2 ′) were obtained in the same manner as in Example 9 except that the amount of tetraethoxysilane charged was changed from 1.2 parts to 12.5 parts.

Comparative Example 3
100 parts of ferrous sulfate was dissolved in 1,000 parts of water, and with stirring, an aqueous solution in which 28.8 parts of sodium hydroxide was dissolved in 500 parts of water was added dropwise over 1 hour. The mixture was heated and heated to air, and air was blown into the suspension to oxidize for 8 hours. Then, 2.2 parts of magnetite particles obtained by centrifugation were mixed with 5.7 parts of decane and 2.2 parts of tetraethoxysilane and dispersed. A liquid (R-1) was obtained. Comparative magnetic particles (E-3 ′) were obtained in the same manner as in Example 2 except that the dispersion (A-1) was replaced with the dispersion (R-1).

Comparative Example 4
Comparative magnetic particles (E-4 ′) were obtained in the same manner as in Comparative Example 34 except that the amount of tetraethoxysilane charged was changed from 2.2 parts to 12.5 parts.

Comparative Example 5
A dispersion (R-2) was obtained by mixing 2.2 parts of magnetite particles (manufactured by Sigma Aldrich Japan Co., Ltd.) having an average particle diameter of 22 nm with 5.7 parts of decane and 2.2 parts of tetraethoxysilane. Comparative magnetic particles (E-5 ′) were obtained in the same manner as in Production Example 1 except that the dispersion (A-1) was replaced with the dispersion (R-2).

  The obtained magnetic particles (E-1) to (E-14) and (E-1 ′) to (E-5 ′) are superparamagnetic metal oxide particles on the surface of the magnetic particles (E) by the following method. The degree of exposure of (C) was confirmed. In addition, the average particle size of the superparamagnetic metal oxide particles (C), the average particle size of the magnetic particles (E), and the content of the superparamagnetic metal oxide particles (C) are measured, and the magnetic collection, redispersibility, Aggregability, detergency and sensitivity in immunoassay in a short time were evaluated. The results are shown in Table 1.

<Exposure of superparamagnetic metal oxide particles (C) on the surface of magnetic particles (E)>
After dispersing 5.0 mg of magnetic particles (E) in 5 mL of 0.1 M hydrochloric acid aqueous solution and mixing by inversion mixing at room temperature for 1 hour, 10 mg of salicylic acid was added to determine the presence or absence of iron (III) ions in the solution. It was confirmed by the color test of III). Since none of the magnetic particles (E) exhibited coloration, it was found that the magnetite particles in each magnetic particle (E) were included in silica or the like and were not exposed on the surface of the magnetic particles (E). .

<Measuring method of average particle diameter of superparamagnetic metal oxide particles (C)>
Arbitrary 200 superparamagnetic metal oxides [in the examples, obtained by solid-liquid separation of the magnetite particles (C) in water by decantation, washing with water, and drying. ] Was observed with a scanning electron microscope (model number JSM-7000F, manufacturer name JEOL Ltd.) to measure the particle diameter, and the average value was taken as the average particle diameter.

<Measuring method of average particle diameter of magnetic particles (E)>
About 200 arbitrary magnetic particles (E), it observed with the said scanning electron microscope, the particle diameter was measured, and the average value was made into the average particle diameter.

<Method for Measuring Content of Superparamagnetic Metal Oxide Particles in Magnetic Particle (E)>
Arbitrary 20 magnetic particles (E) were observed with the above-mentioned scanning electron microscope, and the content of superparamagnetic metal oxide was measured using an energy dispersive X-ray spectrometer (model number INCA Wave / Energy, manufacturer: Oxford). The average value was measured and set as the content X. Further, the silicon content was measured by the same measurement, and the average value was defined as the content Y. The content of superparamagnetic metal oxide particles was determined by the following calculation formula.
Content of superparamagnetic metal oxide particles (%) = 100 × (X) / (X + Y)

<Evaluation Method of Magnetic Collection of Magnetic Particle (E)>
1.0 mg of magnetic particles (E) are dispersed in 2 mL of ion-exchanged water and placed in a glass container having a mouth inner diameter × body diameter × total height = φ10.3 mm × φ12.0 mm × 35 mm, 1 cm × 1 cm × 1 cm neodymium magnet Was attached to the side surface, and an absorbance of 600 nm was measured using an ultraviolet-visible spectrophotometer (apparatus model number UV-1800, manufacturer name: Shimadzu Corporation), and the time until the initial absorbance reached 20% was measured. The shorter the time, the better the magnetic collection.

<Method for evaluating redispersibility of magnetic particles (E)>
1.0 mg of magnetic particles (E) are dispersed in 2 mL of ion-exchanged water and placed in a glass container having a mouth inner diameter × body diameter × total height = φ10.3 mm × φ12.0 mm × 35 mm, 1 cm × 1 cm × 1 cm neodymium magnet On the side to collect the magnetic particles (E) completely, remove the supernatant, sufficiently separate the neodymium magnet from the side, add 2 mL of ion-exchanged water to the particles, and pipette 3 times. The mixture was observed with a microscope within 10 seconds, and the ratio of the number of aggregated particles to the total number of particles in the field of view was calculated. The smaller this ratio, the better the redispersibility.

<Method for evaluating reaggregation of magnetic particles (E)>
The number of aggregated particles relative to the total number of particles in the field of view is the same as the above redispersibility evaluation method except that the time until observation with a microscope is changed from within 10 seconds to 5 minutes after mixing by pipetting three times. The percentage of was calculated. The smaller this ratio, the better the reaggregation property.

<Evaluation method of detergency and sensitivity in immunoassay in a short time>
An α-fetoprotein (hereinafter abbreviated as AFP) antibody was immobilized on the surface of the magnetic particle (E), the following immunoassay was performed, and detergency and sensitivity were determined according to the following criteria.
For detergency, the average amount of luminescence was measured when immunoassay was performed using a standard solution having an AFP concentration of 0 ng / mL. The smaller the amount of emitted light, the better the detergency.
Regarding the sensitivity, the difference in the amount of luminescence when the immunoassay was performed using the standard solution having an AFP concentration of 0 ng / mL and 2 ng / mL was determined. The greater the difference in the amount of luminescence, the higher the sensitivity.

Preparation of anti-AFP antibody-bound magnetic particles:
40 mg of magnetic particles (E) are added to a polyethylene bottle with a lid containing 40 mL of an acetone solution containing 1 wt% γ-aminopropyltriethoxysilane, reacted at 25 ° C. for 1 hour, and after collecting the particles with a magnet, the liquid is aspirated. Removed with suction. Next, 40 mL of deionized water was added, the cap was closed, and the polystyrene bottle was slowly inverted and stirred twice. After collecting the particles with a magnet, the liquid was sucked and removed with an aspirator to wash the magnetic particles (E). This washing operation was performed three times. Next, the magnetic particles (E) after washing were added to a polyethylene bottle with a lid containing 40 mL of an aqueous solution containing 2% by weight glutaraldehyde, and reacted at 25 ° C. for 1 hour. Then, 40 mL of deionized water was added, the cap was closed, the polystyrene bottle was slowly inverted and stirred twice, and after collecting the particles with a magnet, the liquid was sucked and removed with an aspirator to wash the magnetic particles (E). This washing operation was performed three times. Further, this washed magnetic particle (E) is a polyethylene bottle with a lid containing 40 mL of 0.02 M phosphate buffer (pH 8.7) containing anti-AFP monoclonal antibody (purchased from Dako Japan) at a concentration of 20 μg / mL. And reacted at 25 ° C. for 1 hour. After the reaction, the anti-AFP antibody-containing phosphate buffer was removed to prepare anti-AFP antibody-bound magnetic particles (E). This was immersed in 40 mL of 0.02 M phosphate buffer (pH 7.2) containing 0.1% bovine serum albumin and stored at 5 ° C.

Production of peroxidase-labeled anti-AFP antibody:
Anti-AFP polyclonal antibody (purchased from Dako Japan), horseradish-derived peroxidase (Toyobo, hereinafter abbreviated as POD), literature (S Yoshitake, M Imagawa, E Ishikawa, Etol; J. Biochem, Vol. 92, 1982, 1413-1424), a POD-labeled anti-AFP antibody was prepared and stored frozen at -30 ° C.

AFP immunoassay:
After collecting the particles in the bovine serum albumin-containing phosphate buffer containing 25 μg of anti-AFP antibody-bound magnetic particles (E) with a magnet, the solution is removed with an aspirator, and 0.5 mL of physiological saline is added to remove the particles. After performing the washing operation of removing the liquid after the magnetic collection with an aspirator three times, the AFP concentration prepared with the phosphate buffer containing the magnetic particles (E) and 1% by weight of bovine serum albumin is 2 ng / 0.025 mL of standard AFP solution of mL and buffer for immune reaction [0.1% bovine serum albumin, 0.85% sodium chloride and 0.5% polyoxyethylene sorbitan monolaurate (surfactant) 0.02 mL of the contained 0.02 M phosphate buffer (pH 7.2)] was placed in a test tube and reacted at 37 ° C. for 3 minutes in the test tube to form a particle / AFP complex. After the reaction, collect the particles from the outside of the test tube with a magnet for 10 seconds, remove the liquid in the test tube with an aspirator, add 0.5 mL of physiological saline to disperse the particles, collect the magnetic flux, and then remove the liquid with an aspirator The operation was performed 3 times.

Subsequently, 0.025 mL of an immune reaction buffer containing 200 nM of POD-labeled anti-AFP antibody was injected into the test tube, reacted at 37 ° C. for 3 minutes in the test tube, and the particle / AFP / POD-labeled anti-AFP antibody complex was Formed. After the reaction, collect the particles from the outside of the test tube with a magnet for 10 seconds, remove the liquid in the test tube with an aspirator, add 0.5 mL of physiological saline to disperse the particles, collect the magnetic flux, and then remove the liquid with an aspirator The operation was performed twice.
Finally, 0.07 mL of luminescent reagent A [0.1 M Tris / hydrochloric acid buffer (pH 9.0) containing 0.18 g / L luminol and 0.1 g / L 4- (cyanomethylthio) phenol] Add 0.01% hydrogen peroxide solution 0.07mL at the same time, and let it react for luminescence at 37 ° C for 43 seconds. After adding the luminescence reagent, the average luminescence amount for 43 to 45 seconds was measured with the luminescence detector [BLR-201 (manufactured by Aroka). ): Use photomultiplier tube]. In addition, instead of the standard AFP solution having an AFP concentration of 2 ng / mL, a standard AFP solution having an AFP concentration of 0 ng / mL was used, and the same operation was performed as a background.

表１から、本発明の磁性粒子（Ｅ）は、集磁性、再分散性及び再凝集性に優れており、免疫測定の感度が高く、洗浄性にも優れることが判った。

From Table 1, it was found that the magnetic particles (E) of the present invention are excellent in magnetism collection, redispersibility and reaggregation, have high sensitivity for immunoassay and are excellent in detergency.

  As described above, the magnetic particles (E) of the present invention are excellent in magnetism collection, redispersibility, and reaggregation, and can be highly sensitive in a short time, so that they are particularly useful as a carrier for diagnostic agents. is there.

Claims (16)

Superparamagnetic metal oxide particles (C) having an average particle diameter of 1 to 15 nm are contained in the nonmagnetic metal oxide (D), and (C) is added to the total weight of (C) and (D). Magnetic particles (E) containing 60 to 95% by weight.   The nonmagnetic metal oxide (D) is at least one nonmagnetic metal oxide selected from the group consisting of silicon oxide, titanium oxide, zirconium oxide, aluminum oxide, calcium oxide, yttrium oxide, and tantalum oxide. The magnetic particle (E) according to 1. The magnetic particle (E) according to claim 1 or 2, wherein the nonmagnetic metal oxide (D) is silicon oxide.   The magnetic particle (E) according to any one of claims 1 to 3, wherein the average particle diameter is 1 to 5 µm.   The magnetic particle (E) according to any one of claims 1 to 4, wherein the superparamagnetic metal oxide particle (C) comprises iron oxide (F).   6. The iron oxide (F) is at least one iron oxide selected from the group consisting of magnetite, γ-hematite, magnetite-α-hematite intermediate iron oxide and γ-hematite-α-hematite intermediate iron oxide. Magnetic particles (E) described in 1.   The surface is bonded with at least one organic compound selected from the group consisting of glutaraldehyde, albumin, carbodiimide, streptavidin, biotin and a metal alkoxide (G) having a functional group. Magnetic particles (E) described in 1.   The functional group of the metal alkoxide (G) having the functional group is at least one functional group selected from the group consisting of an amino group, a carboxyl group, a hydroxyl group, a mercapto group, and a glycidyloxy group. Magnetic particles (E).   A magnetic carrier (H) for binding biological materials comprising the magnetic particles (E) according to any one of claims 1 to 8.   The magnetic carrier (H) according to claim 9, wherein the biological substance (J) is immobilized.   The magnetic carrier (H) according to claim 10, wherein the biological substance (J) is one of an antibody and an antigen having specific binding to each other, or one of a biological receptor and a ligand.   Superparamagnetic metal oxide particles (C) having an average particle diameter of 1 to 15 nm, a dispersion (A) containing 30 to 500% by weight of metal alkoxide (N) and dispersant (K) based on the weight of (C) ) And a solution (B) containing water, a water-soluble organic solvent (L), a nonionic surfactant (P), and a catalyst (Q) for hydrolysis of metal alkoxide to produce an oil-in-water emulsion. The manufacturing method of the magnetic particle (E) characterized by including the process to form.   The production method according to claim 12, wherein the dispersion (A) further contains a water-insoluble organic solvent (M).   The manufacturing method according to claim 12 or 13, wherein the dispersant (K) is an organic compound having 10 to 30 carbon atoms and having one carboxyl group in the molecule.   The said water-insoluble organic solvent (M) is a C6-C16 aromatic hydrocarbon solvent and / or a C5-C16 aliphatic hydrocarbon solvent, The any one of Claims 12-14. Manufacturing method.   The said water-soluble organic solvent (L) is a C1-C3 alcohol, The manufacturing method of any one of Claims 12-15.