JP5003867B2 - Magnetic particle, method for producing the same, and probe-coupled particle - Google Patents

Description

  The present invention relates to a magnetic particle, a method for producing the same, and a probe binding particle.

  In recent years, magnetic particles can be easily washed by magnetic separation and have excellent reaction fields in studies such as immune reactions between antigens and antibodies, hybridization between DNA or DNA and RNA, and interactions between drug candidates and internal substances. In particular, application to biochemical uses such as diagnostics and pharmaceutical research has become active.

  Among them, as the carrier particles for biochemistry, mainly polystyrene particles are widely used as a physical adsorption sensation effect, and mainly carboxyl group-modified polystyrene particles are used as a chemical bond sensation effect. However, these particles have a large adsorption (hereinafter referred to as “non-specific adsorption” in the present specification) of other undesired physiologically active substances present in test specimens such as cells, proteins and DNA. Nonspecific adsorption impairs the performance of the sensitized particles. That is, non-specific adsorption has been a major obstacle to using these particles.

  As a biochemical carrier with little nonspecific adsorption, gel bodies based on sugar chains such as agarose and sepharose are used, but the activity of the bound probe tends to be low, and a sufficient signal may not be obtained. Many.

  Similarly, magnetic particles are required to reduce non-specific adsorption, and magnetic particles having glycidyl groups introduced on the particle surface have been proposed for that purpose. For example, Japanese Patent Laid-Open No. 2006-131771 proposes a method in which superparamagnetic fine particles are dispersed in styrene and glycidyl methacrylate and then finely dispersed by ultrasonic treatment and then polymerized. However, in this method, the coating of the magnetic material is insufficient and the reduction of nonspecific adsorption is insufficient, and the average particle size is 200 nm or less and the particle size is small, so that the magnetic separation property is also insufficient.

  The present applicant, in Japanese Patent No. 3738847, is capable of easily and efficiently producing diagnostic pharmaceutical particles having a uniform particle size without dropping off of a magnetic material and elution of a substance derived from a magnetic material component such as iron ions. For this purpose, a method for producing magnetic particles including a step of providing a two-layer polymer coat is disclosed, and further, in JP 2005-83904 A and JP 2005-83905 A, magnetic particles with reduced non-specific adsorption are disclosed. However, further reduction of non-specific adsorption is desired.

  Although not necessarily the purpose of reducing non-specific adsorption, as a magnetic particle into which a glycidyl group is introduced, for example, in Japanese Translation of PCT Publication No. 2-501753, a mixture of a magnetic substance and a monomer is polymerized in the presence of a core particle. In addition, a method for further polymer coating has been proposed. However, in this method, the portion of the mixture of the magnetic substance and the monomer that is polymerized on the core particle is very small, and as a result, only a very small amount of the magnetic substance can be taken into the core particle. Inferior.

  In addition, JP 10-505118 A describes a reaction of introducing an amino group into a magnetic particle into which a glycidyl group has been introduced. This magnetic particle is a porous particle for the purpose of ion exchange, Non-specific adsorption is remarkable.

Furthermore, Japanese Patent Application Publication No. 2006-511935 proposes that a magnetic substance is deposited on the surface and inside of a particle and further coated with a polymer having a glycidyl group. However, in this method, when the magnetic particles are deposited to the surface of the particles, the coating with the polymer having a glycidyl group alone is insufficient to coat the magnetic particles, and the nonspecific adsorption property cannot be reduced.
JP 2006-131771 A Japanese Patent No. 3738847 JP 2005-83904 A JP 2005-83905 A Japanese National Publication No. 2-501753 Japanese National Patent Publication No. 10-505118 JP-T 2006-511935

  The present invention relates to a magnetic particle that exhibits low non-specific adsorption of proteins, nucleic acids, and the like, and that expresses high sensitivity and low noise that are particularly outstanding in the fields of biochemistry and pharmaceuticals, a method for producing the same, and probe-binding particles.

  In order to achieve the above object, the present inventors have conducted intensive research. As a result, nonmagnetic child particles having a particle size of d / 2 or less are provided on the surface of magnetic mother particles having a particle size of d, and glycidyl is formed in the outermost layer. Forming a first polymer layer having a group, and introducing a polar group containing at least one atom selected from the group consisting of an oxygen atom, a nitrogen atom and a sulfur atom into the first polymer layer; This produces magnetic particles with high sensitivity and low noise that are outstanding in the biochemical / pharmaceutical field, and with a relatively large surface area per unit weight due to the uneven surface, resulting in a high binding amount of the trapping substance. The present invention has been completed. According to the present invention, it is possible to provide the following magnetic particles, a method for producing the same, and probe-bound particles.

A method for producing magnetic particles according to an aspect of the present invention includes:
A first step of providing non-magnetic child particles having a particle size of d / 2 or less on the surface of magnetic base particles having a particle size of d,
A second step of forming a first polymer layer having a glycidyl group so as to cover the magnetic mother particles and the non-magnetic child particles; and
A third step of introducing a polar group containing at least one atom selected from the group consisting of an oxygen atom, a nitrogen atom and a sulfur atom into the first polymer layer;

  In the present invention, the mother particle refers to a particle that becomes the central body of the magnetic particle of the present invention, and the child particle refers to a particle that covers the surface of the mother particle.

  In the method for producing magnetic particles, the third step may include a reaction for introducing an amino group.

  In the method for producing magnetic particles, the third step may include a reaction for introducing an aldehyde group.

  In the method for producing magnetic particles, the third step may include a reaction for introducing a carboxyl group. In this case, the third step may further include a reaction for converting the carboxyl group into an active ester group.

  In the magnetic particle manufacturing method, the second step is a step of forming a first polymer layer having a plurality of glycidyl groups, and may further include a step of hydrolyzing a part of the glycidyl groups.

In the method for producing magnetic particles, the magnetic mother particles include core particles, a magnetic layer containing superparamagnetic fine particles formed on the surface of the core particles, and a hydrophobic layer provided on the surface of the magnetic layer. A second polymer layer,
The second step may be a step of forming the first polymer layer so as to cover the second polymer layer and the nonmagnetic child particles. in this case,
The magnetic mother particles have a positive or negative surface charge in an aqueous medium, the non-magnetic child particles have a negative or positive surface charge in the aqueous medium, and the first step includes the step of A step of adsorbing the nonmagnetic child particles on the surface of the magnetic mother particles by mixing the magnetic mother particles and the nonmagnetic child particles in the aqueous medium may be included. Furthermore, in this case, the magnetic mother particles can have a positive surface charge in the aqueous medium, and the non-magnetic child particles can have a negative surface charge in the aqueous medium.

The magnetic particles according to one aspect of the present invention are:
Magnetic base particles having a particle size d;
Non-magnetic child particles having a particle diameter of d / 2 or less present on the surface of the magnetic mother particles;
A first polymer layer having an amino group and a 2,3-dihydroxypropyl group provided so as to cover the magnetic mother particle and the non-magnetic child particle;
including.

The magnetic particles according to one aspect of the present invention are:
Magnetic base particles having a particle size d;
Non-magnetic child particles having a particle diameter of d / 2 or less present on the surface of the magnetic mother particles;
A first polymer layer having an aldehyde group and a 2,3-dihydroxypropyl group provided so as to cover the magnetic mother particle and the non-magnetic child particle;
including.

The magnetic particles according to one aspect of the present invention are:
Magnetic base particles having a particle size d;
Non-magnetic child particles having a particle diameter of d / 2 or less present on the surface of the magnetic mother particles;
A first polymer layer having a carboxyl group and a 2,3-dihydroxypropyl group provided so as to cover the magnetic mother particle and the non-magnetic child particle;
including.

The magnetic particles according to one aspect of the present invention are:
Magnetic base particles having a particle size d;
Non-magnetic child particles having a particle diameter of d / 2 or less present on the surface of the magnetic mother particles;
A first polymer layer having an active ester group and a 2,3-dihydroxypropyl group provided so as to cover the magnetic mother particle and the non-magnetic child particle;
including.

  In the magnetic particle, the magnetic mother particle includes a core particle, a magnetic layer including superparamagnetic fine particles formed on the surface of the core particle, and a hydrophobic second polymer provided on the surface of the magnetic layer. And the first polymer layer may be formed to cover the second polymer layer and the non-magnetic child particles.

  The magnetic particles can be used for probe binding.

  A probe-coupled particle according to one embodiment of the present invention includes the magnetic particle and a probe that binds to the magnetic particle.

  Since the first magnetic layer having the polar group is provided so as to cover the magnetic mother particle and the nonmagnetic child particle, the magnetic particle has less nonspecific adsorption of protein, nucleic acid, etc. -High sensitivity and low noise that are outstanding in the pharmaceutical field can be expressed, and a high S / N ratio can be obtained for biochemical tests. Further, since the surface of the magnetic particle is uneven due to the presence of non-magnetic child particles on the surface of the magnetic mother particle, the surface area per unit weight is large, so that the binding amount of the trapping substance is large.

  In addition, according to the above method for producing magnetic particles, it is possible to efficiently produce magnetic particles that have less nonspecific adsorption of proteins, nucleic acids, etc., and that can express high sensitivity and low noise, which are particularly outstanding in the fields of biochemistry and pharmaceuticals. Can do.

  Furthermore, according to the probe binding particles, there is little desorption of the probe, less nonspecific adsorption, and high sensitivity.

  Hereinafter, a magnetic particle, a method for producing the magnetic particle, and a probe binding particle according to an embodiment of the present invention will be described.

1. Magnetic Particle and Method for Producing the Same Magnetic particles according to the present embodiment include a magnetic mother particle having a particle size d, a non-magnetic child particle having a particle size of d / 2 or less present on the surface of the magnetic mother particle, a magnetic mother particle, and And a first polymer layer provided to cover the non-magnetic child particles. In this case, the first polymer layer can include a polar group including at least one atom selected from the group consisting of an oxygen atom, a nitrogen atom, and a sulfur atom.

  The method for producing magnetic particles according to this embodiment includes a first step of providing nonmagnetic child particles having a particle size of d / 2 or less on the surface of magnetic mother particles having a particle size d, the magnetic mother particles, and the nonmagnetic child. A second step of forming a first polymer layer having a glycidyl group so as to coat the particles, and at least one selected from the group consisting of an oxygen atom, a nitrogen atom and a sulfur atom in the first polymer layer A third step of introducing a polar group containing one or more atoms of

1.1. Configuration and Production of Magnetic Particles Magnetic particles according to an embodiment of the present invention include magnetic mother particles and nonmagnetic child particles, and the nonmagnetic child particles are present on the surface of the magnetic mother particles. Here, “the nonmagnetic child particles are present on the surface of the magnetic mother particles” means not only (a) the nonmagnetic child particles are in contact with the surface of the magnetic mother particles, but also, for example, (b ) When the magnetic mother particle includes a second polymer layer to be described later, the non-magnetic child particle is not in contact with the surface of the magnetic mother particle, and the non-magnetic child particle is lifted from the surface of the magnetic mother particle. Including. In the case of (b), a second polymer layer can be present between adjacent non-magnetic particles.

  In the magnetic particles according to the present embodiment, the nonmagnetic child particles can be in at least one of the above states (a) and (b) on the surface of the magnetic mother particles.

  The magnetic particles according to the present embodiment preferably exist so that a plurality of nonmagnetic child particles coat the surface of the magnetic mother particles, and the plurality of nonmagnetic child particles constitute a coating layer having a uniform thickness. More preferably.

  For example, the nonmagnetic child particles may be adsorbed on the surface of the magnetic mother particles, or may be fixed on the surface of the magnetic mother particles or above the surface. When the nonmagnetic child particles are adsorbed on the surface of the magnetic mother particles, the adsorption may be, for example, physical adsorption or chemical adsorption. In addition, as a method for fixing the nonmagnetic child particles on the surface of the magnetic mother particles or above the surface, for example, the magnetic mother particles and the nonmagnetic child particles are coated with another layer (for example, a first polymer layer described later). The method of doing is mentioned.

  In the present invention, “physical adsorption” means adsorption without chemical reaction. The principle of “physical adsorption” includes, for example, hydrophobic / hydrophobic adsorption, melt bonding or adsorption, fusion bonding or adsorption, hydrogen bonding, van der Waals bonding, and the like.

  The particle size of the magnetic particles according to this embodiment is preferably 0.1 to 10 μm, and more preferably 0.2 to 5 μm. When the particle diameter is less than 0.1 μm, sufficient magnetic responsiveness is not exhibited, it takes a considerably long time to separate the particles, and a considerably large magnetic force is necessary to separate them. Therefore, it is not preferable. On the other hand, when the particle diameter exceeds 10 μm, the particles are likely to settle in the dispersion medium, so that an operation of stirring the dispersion medium is necessary when capturing the target substance, and the ratio of the surface area to the weight of the particles Therefore, it may be difficult to capture a sufficient amount of the target substance.

  The magnetic particles according to this embodiment can be used as a dispersion or a dry powder. Examples of the dispersion medium used for the dispersion include an aqueous medium. The aqueous medium is not particularly limited, and examples thereof include water and water containing an aqueous solvent. Examples of the aqueous solvent include alcohols (for example, ethanol, alkylene glycol, monoalkyl ether, etc.). The dispersion medium may contain a dispersant.

  The aqueous dispersion is usually obtained after the third step by washing with an aqueous solvent such as distilled water, adding an aqueous solvent and stirring, homogenizer, ultrasonic treatment, or the like. The solvent-dried powder can be obtained by heat drying, vacuum drying, spray drying, freeze drying or the like of an aqueous dispersion.

  Next, each component constituting the magnetic particle and the method for producing the magnetic particle according to the present embodiment will be described in detail.

1.2. Magnetic mother particle 1.2.1. Configuration and production of magnetic mother particles Magnetic mother particles are known particulate substances that can be collected with a magnet, and the particle size d thereof is preferably 0.1 to 10 μm, more preferably 0.2 to 5 μm, More preferably, it is 0.5-3 micrometers. If the particle size d is less than 0.1 μm, it may take a long time for separation and purification by magnetic force, whereas if the particle size d exceeds 10 μm, the amount of biochemical substance binding may be small.

  The magnetic mother particles may be homogeneous magnetic particles or heterogeneous magnetic particles. However, the homogeneous magnetic particles in the above preferred particle size range are often paramagnetic substances, and re-dispersion in the medium may become difficult if separation and purification by magnetic force is repeated. For this reason, the magnetic mother particles are preferably heterogeneous particles including superparamagnetic fine particles with little residual magnetization.

  Specific examples of such heterogeneous magnetic base particles include (a) particles in which superparamagnetic fine particles are dispersed in a continuous phase of a nonmagnetic material such as an organic polymer, and (b) superparamagnetic. Particles having a secondary aggregate of fine particles as a core and a non-magnetic material such as an organic polymer as a shell, (c) Core particles made of a non-magnetic material such as an organic polymer, and superparamagnet provided on the surface of the core particles And particles including a secondary aggregate layer (magnetic layer) of fine particles. Among these, (c) a magnetic base particle in which a magnetic layer containing superparamagnetic fine particles is formed on the surface of the core particle is preferable in that it has excellent magnetic response and the particle diameter can be uniformly controlled. Hereinafter, the configuration of the magnetic mother particle (c) will be described.

  In the magnetic mother particle (c), for example, the magnetic mother particle can include a core particle and superparamagnetic fine particles (secondary aggregates) present on the surface of the core particle. In this case, the magnetic mother particles can be obtained, for example, by physically adsorbing superparamagnetic fine particles on the surface of the core particles. “Superparamagnetic fine particles are present on the surface of the core particles” means that (ii) when the superparamagnetic fine particles are in contact with the surface of the core particles, (ii) the second polymer layer in which the magnetic mother particles are described later, for example. When the superparamagnetic fine particles are not in contact with the surface of the core particles, the superparamagnetic fine particles are in a state of being lifted from the surface of the core particles. In the magnetic mother particle, the superparamagnetic fine particles can be in at least one of the above states (i) and (ii) on the surface of the core particle.

  In this case, the magnetic mother particle is preferably present so that the magnetic layer covers the surface of the core particle, and more preferably, the plurality of superparamagnetic fine particles constitute a magnetic layer having a uniform thickness. preferable.

1.2.2. Core particle The core particle is basically a non-magnetic substance, and either an organic substance or an inorganic substance can be used, and can be appropriately selected depending on the purpose of use of the diagnostic drug particle. As a typical example of the organic substance, for example, a polymer can be mentioned. Such a polymer is particularly preferably a vinyl polymer, and most preferably crosslinked polystyrene or crosslinked polymethyl methacrylate. These polymers may have a functional group such as a carboxyl group introduced therein.

  The average particle diameter of the core particles is preferably 0.1 to 10 μm, more preferably 0.2 to 5 μm, and most preferably 0.3 to 2 μm. When the average particle diameter of the core particles is less than 0.1 μm, the magnetic separation property may be inferior. On the other hand, when the average particle diameter of the core particles exceeds 10 μm, gravity sedimentation becomes significant, and the reaction field may become non-uniform as particles bound with the probe.

  The material of the core particles is preferably an organic substance such as a polymer from the viewpoints of processability and lightness at the time of compounding. In the present invention, the “average particle size” is obtained by randomly measuring the particle size of 100 particles on an electron micrograph.

  The polymer particles as the core particles having an average particle diameter in the specific range described above can also be obtained, for example, by suspension polymerization of a vinyl monomer or pulverization of a polymer bulk. As a method for producing core particles having a uniform particle size, a swelling polymerization method described in JP-B-57-24369 or a polymerization method previously proposed by the present applicant (Japanese Patent Laid-Open Nos. 61-215602, 61). -215603 and 61-215604).

1.2.3. Magnetic layer The magnetic layer is composed of superparamagnetic fine particles. As the superparamagnetic fine particles, iron oxide-based fine particles having a particle size of 20 nm or less (preferably a particle size of 5 to 20 nm) are typical, and MnFe ₂ O ₄ (Mn = Co, Ni, Mg, Cu, Li _0.5 Fe _0.5 or the like), magnetite expressed by Fe ₃ O ₄ , or γ-Fe ₂ O ₃ , and γ-Fe ₂ O ₃ having strong saturation magnetization and low residual magnetization, and preferably contains one of Fe ₃ O _4.

  The weight ratio of the core particles to the superparamagnetic fine particles (nuclear particles: superparamagnetic fine particles) is preferably 95: 5 to 20:80. If the amount of superparamagnetic particles is less than this range, the magnetic separation property may be inferior. When the amount of superparamagnetic fine particles is larger than this range, the amount of the core particles becomes excessive, and the number of superparamagnetic fine particles that are not complexed may increase.

  The superparamagnetic fine particles preferably have a hydrophobized surface from the viewpoint of the affinity and compatibility between the core particles and the monomer monomer used in the subsequent step. Examples of the method for hydrophobizing the surface of superparamagnetic fine particles include the method described in Japanese Patent No. 3738847. Such superparamagnetic fine particles can also be obtained by precipitating and washing particles from a known magnetic fluid with a suitable poor solvent.

1.2.4. Formation of magnetic layer The method of forming a magnetic layer containing superparamagnetic fine particles on the surface of the core particles is to mix the core particles with the superparamagnetic fine particles and physically adsorb the superparamagnetic fine particles on the surface of the core particles. Therefore, a method of forming a magnetic layer is preferable. As a method of adsorbing superparamagnetic fine particles on the surface of the core particles, for example, by dry blending the core particles and the superparamagnetic fine particles, and applying a physically strong force from the outside to combine both particles The method of producing is mentioned. As a method of applying a physically strong force, for example, a mortar, an automatic mortar, a ball mill, a method using a mechanochemical effect such as a blade pressure type powder compression method, a mechanofusion method, a jet mill, a hybridizer The thing using the impact method in high-speed air currents, such as these, is mentioned. It is desirable that the physical adsorption force is strong in order to efficiently and firmly perform the composite. In order to carry out complexing with a strong physical adsorption force, the peripheral speed of the stirring blade is preferably 15 m / second or more, more preferably 30 m / second or more, and further preferably 40 to 150 m / second in a vessel equipped with a stirring blade. It is possible to carry out compounding. When the peripheral speed of the stirring blade is lower than 15 m / sec, it may not be possible to obtain sufficient energy for adsorbing the superparamagnetic fine particles on the surface of the core particles. In addition, although there is no restriction | limiting in particular about the upper limit of the peripheral speed of a stirring blade, It determines automatically from points, such as an apparatus to be used and energy efficiency.

1.2.5. Hydrophobic Second Polymer Layer Next, the configuration of a hydrophobic second polymer layer (hereinafter also simply referred to as “second polymer layer”) capable of constituting the outermost layer of the magnetic mother particles and a method for forming the same will be described. .

  As described above, the second polymer layer covers the core particle and the magnetic layer in the magnetic mother particle. That is, in the magnetic mother particle, the second polymer layer can be formed so as to further cover the core particle whose surface is covered with superparamagnetic fine particles. By including the second polymer layer in the magnetic mother particles, it is possible to effectively prevent the superparamagnetic fine particles from eluting.

  The monomer for forming the second polymer layer (hereinafter also referred to as “second monomer part”) contains 80% by weight or more of hydrophobic monomer, preferably 95% by weight or more of hydrophobic monomer, and more preferably Contains 98% by weight or more of hydrophobic monomers. If the hydrophobic monomer in the second monomer portion is less than 80% by weight, non-specific adsorption may deteriorate. Here, the hydrophobic monomer is a simple substance or a mixture of polymerizable monomers having a solubility in water at 25 ° C. of 2.5% by weight or less. The hydrophobic monomer may be either a monofunctional (non-crosslinkable) monomer or a crosslinkable monomer, or may be a mixture of a monofunctional monomer and a crosslinkable monomer.

  In the presence of magnetic mother particles, auxiliary materials such as polymerization initiators, emulsifiers, dispersants, electrolytes, cross-linking agents, molecular weight regulators, etc. are added as necessary, and the main raw materials are 80% by weight or more. By polymerizing the second monomer part containing the hydrophobic monomer, a second polymer layer constituting the outermost layer of the magnetic mother particles can be formed. Thus, by forming a 2nd polymer layer by superposition | polymerization, elution of superparamagnetic fine particles can be suppressed and the coupling | bonding with the nonmagnetic child particle mentioned later can be accelerated | stimulated.

  Among the hydrophobic monomers that can be used in the second monomer part, monofunctional monomers include aromatic vinyl monomers such as styrene, α-methylstyrene, and halogenated styrene, methyl acrylate, ethyl acrylate, ethyl methacrylate, and stearyl acrylate. Examples thereof include ethylenically unsaturated carboxylic acid alkyl esters such as stearyl methacrylate, cyclohexyl acrylate, cyclohexyl methacrylate, isobornyl acrylate and isobornyl methacrylate. Among the hydrophobic monomers, crosslinkable monomers include ethylene glycol diacrylate, ethylene glycol dimethacrylate, trimethylolpropane triacrylate, trimethylolpropane trimethacrylate, pentaerythritol triacrylate, pentaerythritol trimethacrylate, dipentaerythritol hexaacrylate. And polyfunctional (meth) acrylates such as dipentaerythritol hexamethacrylate, conjugated diolefins such as butadiene and isoprene, divinylbenzene, diallyl phthalate, allyl acrylate, and allyl methacrylate.

  The second monomer portion may contain 20% by weight or less of a non-hydrophobic monomer (hydrophilic monomer). Among non-hydrophobic monomers, monofunctional monomers include monomers having a carboxyl group such as acrylic acid, methacrylic acid, maleic acid, itaconic acid, 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, glycerol acrylate, glycerol methacrylate , Methoxyethyl acrylate, methoxyethyl methacrylate, polyethylene glycol acrylate, polyethylene glycol methacrylate, 2-dimethylaminoethyl (meth) acrylate, 2-diethylaminoethyl (meth) acrylate, 2-dimethylaminopropyl (meth) acrylate, 3-dimethylamino (Meth) acrylates having hydrophilic functional groups such as propyl (meth) acrylate (for example, hydroxyl group, amino group, alkoxy group, etc.) , Acrylamide, methacrylamide, N-methylol acrylamide, N-methylol methacrylamide, diacetone acrylamide, N- (2-diethylaminoethyl) (meth) acrylamide, N- (2-dimethylaminopropyl) (meth) acrylamide, N- (3-dimethylaminopropyl) (meth) acrylamide, styrenesulfonic acid and its sodium salt, 2-acrylamido-2-methylpropanesulfonic acid and its sodium salt, isoprenesulfonic acid and its sodium salt, N, N-dimethylaminopropyl Examples thereof include copolymers of acrylamide and its copolymerizable monomer such as methyl chloride quaternary salt and allylamine. Among the non-hydrophobic monomers, hydrophilic monomers such as polyethylene glycol diacrylate, polyethylene glycol dimethacrylate, and poly (meth) acrylic ester of polyvinyl alcohol can be exemplified as the crosslinkable monomer. The non-hydrophobic monomer contained in the second monomer part is 20% by weight or less, preferably 5% by weight or less, and more preferably 2% by weight or less.

  The ratio of the crosslinkable monomer (including the hydrophobic monomer and the non-hydrophobic monomer) in the second monomer part is preferably 1 to 40% by weight in 100% by weight of the monomer part constituting the second polymer layer, Preferably, it is 5 to 20% by weight. When the ratio of the crosslinkable monomer in the second monomer part exceeds 40% by weight, the particles may become porous and increase nonspecific adsorption.

  The polymerization initiator is preferably an oil-soluble polymerization initiator from the viewpoint of solubility in water. When a water-soluble polymerization initiator is used, there is a tendency that not only polymerization on the surface of the composite particles but a large amount of new particles in which only a hydrophobic monomer not containing magnetic substance-coated particles is polymerized is generated.

  Examples of oil-soluble polymerization initiators include benzoyl peroxide, lauroyl peroxide, tertiary butyl peroxy 2-ethylhexanate, 3,5,5-trimethylhexanoyl peroxide, peroxide compounds such as azobisisobutyronitrile, azo compounds, etc. Can be mentioned.

  Water-soluble initiators include potassium persulfate, ammonium persulfate, persulfates such as sodium persulfate, hydrogen peroxide, 2-2 azobis (2-aminopropane) mineral acid salt, azobiscyanovaleric acid and its alkali metal salts, and Examples thereof include ammonium salts, and redox initiators in which persulfate, hydrogen peroxide salt and sodium bisulfite, sodium thiosulfate, ferrous chloride, etc. are combined. Among them, persulfate is preferably used. . The ratio of these polymerization initiators to the whole monomer is preferably in the range of 0.01 to 8% by weight.

  As the emulsifier, a commonly used anionic emulsifier, nonionic emulsifier, cationic emulsifier or the like can be used alone or in combination. Examples of anionic emulsifiers include alkali metal salts of higher alcohol sulfates, alkali metal salts of alkylbenzene sulfonic acids, alkali metal salts of dialkyl ester succinic acids, alkali metal salts of alkyl diphenyl ether disulfonic acids, polyoxyethylene alkyl ( In addition to anionic emulsifiers such as sulfate esters of alkylphenyl) ether, phosphate esters of polyoxyethylene alkyl (or alkylphenyl) ether, and formalin condensates of sodium naphthalenesulfonate, Eleminol JS-2, JS- 5 [Product name, manufactured by Sanyo Chemical Co., Ltd.], Latemuru S-120, S-180A, S-180, PD-104 [Product name, manufactured by Kao Corporation], Aqualon HS-10, HS-20, KH -10 [Product name, 1st Work Pharmaceutical Co., Ltd.], ADEKAREASOAP SE-10N, SR-10 [product name, and the like can be mentioned reactive emulsifier such as Asahi Denka Kogyo Co., Ltd.].

  Examples of nonionic emulsifiers include polyoxyethylene alkyl ether and polyoxyethylene alkylphenyl ether, Aqualon RS-20 (manufactured by Daiichi Kogyo Seiyaku Co., Ltd.), Adekari Soap NE-20 (Asahi Denka). And reactive nonionic emulsifiers such as those manufactured by Kogyo Co., Ltd.

  Examples of the cationic emulsifier include alkylamine (salt), polyoxyethylene alkylamine (salt), quaternary alkylammonium (salt), alkylpyridinium (salt), and the like.

  The method for adding the monomer to the polymerization system in the formation of the second polymer layer is not particularly limited, and may be any of a batch method, a division method, or a continuous addition method. The polymerization temperature varies depending on the polymerization initiator, but is usually 10 to 90 ° C, preferably 30 to 85 ° C, and the time required for the polymerization is usually about 1 to 30 hours.

  The thickness of the second polymer layer is preferably 0.005 to 20 μm, more preferably 0.01 to 5 μm. The second polymer layer preferably completely covers the magnetic layer.

  In the magnetic particles according to this embodiment, leakage of superparamagnetic fine particles can be prevented by the second polymer layer constituting the outermost layer of the magnetic mother particles. In particular, in the magnetic particle according to the present embodiment, when the magnetic base particle includes a core particle and a magnetic layer including superparamagnetic fine particles formed on the surface of the core particle, the second polymer layer is the top of the magnetic base particle. By constituting the outer layer, leakage of superparamagnetic fine particles can be effectively prevented.

1.3. Non-magnetic child particles Non-magnetic child particles are basically non-magnetic substances, and both organic substances and inorganic substances can be used, but organic substances are preferred. As a typical example of the organic substance, for example, a polymer can be mentioned. As this polymer, what can be used when manufacturing the above-mentioned core particle can be used. By selecting the monomer component as described above, non-magnetic child particles having a functional group having a positive or negative surface charge on the surface can be obtained. Such a functional group is not particularly limited. For example, amino group, carboxyl group, carbonyl group, aldehyde group, hydroxyl group, mercapto group, sulfone group, isocyanate group, thioisocyanate group, epoxy group, thioepoxy group, aziridine group And an oxazoline group.

  The particle size of the non-magnetic child particles is d / 2 or less, preferably d / 100 to d / 4, more preferably d / 50 to d / 6 with respect to the particle size d of the magnetic mother particles. . If the particle size of the non-magnetic child particles exceeds d / 2, the non-magnetic child particles may not be adsorbed to the magnetic mother particles, and even if adsorbed, the biochemical substance binding amount may be inferior.

  In the magnetic particle according to the present embodiment, the CV (Coefficient of Variation) value of a plurality of non-magnetic child particles present on the surface of one magnetic mother particle is 30% or less, preferably 20% or less, more preferably 10 % Or less.

  As described above, the nonmagnetic child particles are provided so as to cover the surfaces of the magnetic mother particles. Here, as a method of providing the nonmagnetic child particles on the surface of the magnetic mother particles, a method of using a Coulomb attractive force is suitable in addition to a method of physically adsorbing the nonmagnetic child particles on the surface of the magnetic mother particles. As a method using the Coulomb attractive force, for example, a magnetic mother particle having a positive or negative surface charge in an aqueous medium and a non-magnetic child particle having a reverse surface charge (negative or positive) in the aqueous medium are used. The process of mixing is mentioned. Among them, (i) a commonly used anionic functional group such as a carboxyl group can be easily introduced on the surface of the magnetic particle, and (ii) nonspecific adsorption of anionic biological substances such as DNA is reduced. It is preferable that the magnetic mother particle has a positive surface charge. In this case, magnetic mother particles having a positive surface charge and nonmagnetic child particles having a negative surface charge are mixed in an aqueous medium to adsorb the nonmagnetic child particles on the surface of the magnetic mother particles. Here, as the aqueous medium, those exemplified in the column of magnetic particles can be used.

  As a method for adjusting the surface charge of the magnetic mother particle and the non-magnetic child particle to be positive or negative, a known colloid interfacial chemical method may be used. For example, the following methods (a) to (f) are used. Can do.

  (A) As a method for producing magnetic mother particles having a positive surface charge in an aqueous medium, for example, (i) using superparamagnetic fine particles by using iron oxide-based superparamagnetic fine particles without surface treatment; A method of coating the core particles to form an aqueous dispersion having an isoelectric point (usually about pH 9.5) of the superparamagnetic fine particles, (ii) an amine such as N-β (aminoethyl) γ-aminopropyltrimethoxysilane (Iii) a monomer having a cationic group such as N, N-dimethylaminopropylacrylamide is used as a polymerizable monomer when forming the second polymer layer. And / or using an initiator having a cationic group such as 2,2′-azobis (2-methylpropionamidine) dihydrochloride as an auxiliary material, and / or The method of performing polymerization using a cationic emulsifier as an emulsifier can be mentioned.

  (B) As a method for producing magnetic mother particles having a negative surface charge in an aqueous medium, for example, (i) using superparamagnetic fine particles by using iron oxide-based superparamagnetic fine particles without surface treatment. (Ii) a method of using superparamagnetic fine particles surface-treated with a long-chain fatty acid or the like, covering the core particles to form an aqueous dispersion exceeding the isoelectric point (usually about pH 9.5) of the superparamagnetic fine particles; iii) using a monomer having an anionic group such as methacrylic acid as a polymerizable monomer and / or using an initiator having an anionic group such as potassium persulfate as an auxiliary material when forming the second polymer layer; and A method of performing polymerization by using an anionic emulsifier as an emulsifier may be used.

  (C) When the nonmagnetic child particles are made of a polymer, a method for producing nonmagnetic child particles having a positive surface charge in an aqueous medium includes N, N-dimethylamino as a polymerizable monomer during polymerization of the polymer particles. Using a monomer having a cationic group such as propylacrylamide and / or using an initiator having a cationic group such as 2,2′-azobis (2-methylpropionamidine) dihydrochloride as an auxiliary material, and / or Or the method of superposing | polymerizing using a cationic emulsifier as an emulsifier is mentioned.

  (D) When the nonmagnetic child particles are made of a metal oxide, a method for producing a nonmagnetic child particle having a positive surface charge in an aqueous medium is a method of forming an aqueous dispersion exceeding the isoelectric point of the metal oxide. Can be mentioned.

  (E) When the non-magnetic child particles are made of a polymer, a method for producing non-magnetic child particles having a negative surface charge in an aqueous medium includes an anionic group such as methacrylic acid as a polymerizable monomer during polymerization of the polymer particles. And / or using an initiator having an anionic group such as potassium persulfate as an auxiliary material and / or using a cationic emulsifier as an emulsifier.

  (F) When the nonmagnetic child particles are made of a metal oxide, a method for producing a nonmagnetic child particle having a negative surface charge in an aqueous medium is a method of forming an aqueous dispersion having a lower isoelectric point of the metal oxide. Can be mentioned.

  In order to adsorb non-magnetic child particles on the surface of magnetic mother particles, magnetic mother particles having a positive surface charge and non-magnetic child particles having a negative surface charge are mixed in an aqueous medium. More preferably, the magnetic mother particles having a positive surface charge in the aqueous medium produced by the methods (a) to (c) above and the negative surface charge produced by the method (e) above. More preferably, the non-magnetic child particles are mixed in an aqueous medium. By adopting such a process, the elution of biochemical reaction interfering substances such as iron ions is further reduced, and the biochemical substance binding amount is further increased.

  In the step of mixing the magnetic mother particles having a positive or negative surface charge in the aqueous medium and the nonmagnetic child particles having the opposite surface charge in the aqueous medium, the nonmagnetic child particles are stirred and / or super A method of gradually adding the magnetic mother particles while dispersing the sound waves is preferable. Further, the mixing ratio of the magnetic mother particles and the non-magnetic child particles varies depending on the ratio of the respective particle sizes, but the non-magnetic child particles that are not adsorbed remain in the state where the adsorption of the non-magnetic child particles is completed. When the ratio is such, the dispersion system is stabilized, and thus the magnetic particles according to this embodiment can be easily produced. The remaining nonmagnetic child particles can be easily separated and purified from the magnetic particles according to the present embodiment by magnetic separation.

  In the step of mixing a magnetic mother particle having a positive or negative surface charge in an aqueous medium and a non-magnetic child particle having a surface charge opposite to this in the aqueous medium, ions that promote binding of the non-magnetic child particles It is preferable to add a functional compound. Such ionic compounds include acidic compounds such as hydrochloric acid, nitric acid and sulfuric acid when the magnetic mother particles have a positive surface charge, and sodium hydroxide and hydroxide when the magnetic mother particles have a negative surface charge. Examples include basic compounds such as potassium and ammonia.

  The mode in which the nonmagnetic child particles are adsorbed on the surface of the magnetic mother particles is preferably one-layer adsorption in which as many nonmagnetic child particles as possible are adsorbed. In such an adsorption mode, the amount of biochemical substance binding is further increased, and the binding of non-specific biochemical substances is reduced, thereby reducing noise.

1.4. First Polymer Layer The magnetic particles according to this embodiment include a first polymer layer that covers the exposed surfaces of the magnetic mother particles and the nonmagnetic child particles. The first polymer layer includes at least one atom selected from the group consisting of oxygen atom, nitrogen atom and sulfur atom. The polar group can be obtained by forming a first polymer layer having a glycidyl group so as to cover the magnetic mother particles and the nonmagnetic child particles, and chemically modifying the glycidyl group of the first polymer layer. .

1.4.1. Configuration and Forming Method of First Polymer Layer First, a configuration of a first polymer layer having a glycidyl group (hereinafter, also simply referred to as “first polymer layer”) and a forming method thereof will be described. As described above, the first polymer layer is provided so as to cover the magnetic mother particles and the nonmagnetic child particles.

  Further, when the surface of the magnetic mother particle is constituted by the second polymer layer, the first polymer layer can be provided so as to cover the second polymer layer and the nonmagnetic child particles covering the second polymer layer.

  The primary purpose of the first polymer layer is to introduce a functional group for forming a suitable low non-specific adsorptive particle surface. The particle surface with low nonspecific adsorption is suitable, for example, as the surface of magnetic particles for probe binding. Further, the first polymer layer has a function of fixing nonmagnetic child particles on the surface of the magnetic mother particles.

  The monomer for forming the first polymer layer (hereinafter also referred to as “first monomer part”) contains 20% by weight or more of glycidyl group-containing monomer, preferably 40% by weight or more of glycidyl group-containing monomer, More preferably, it contains 80% by weight or more of a glycidyl group-containing monomer. As another monomer which comprises a 1st polymer layer, what was illustrated as a monomer used for the said 2nd monomer part can be used. By polymerizing the first monomer portion containing a predetermined amount of the glycidyl group-containing monomer, a first polymer layer having two or more glycidyl groups can be obtained. The number of glycidyl groups contained in the first polymer layer is usually 2 or more.

  The first polymer layer can be formed by basically the same method as the method for forming the second polymer layer. That is, in the presence of the magnetic mother particles having the second polymer layer formed on the surface, auxiliary materials such as a polymerization initiator, an emulsifier, a dispersant, an electrolyte, a crosslinking agent, and a molecular weight modifier were added as necessary. The first polymer layer can be formed by polymerizing the first monomer part as the main raw material in the liquid.

  Here, examples of the copolymerizable monomer containing a glycidyl group include glycidyl acrylate, glycidyl methacrylate, and allyl glycidyl ether.

  The first monomer part preferably further contains a crosslinkable monomer, and the ratio thereof is preferably 1 to 40% by weight, more preferably 5 to 20% by weight in 100% by weight of the first monomer part. is there. When the ratio of the crosslinkable monomer in the first monomer portion exceeds 40% by weight, the particles may become porous and increase nonspecific adsorption.

  The method for adding the monomer to the polymerization system in the formation of the first polymer layer is not particularly limited, and may be any of a batch method, a division method, or a continuous addition method. The polymerization temperature varies depending on the polymerization initiator, but is usually 10 to 90 ° C, preferably 30 to 85 ° C, and the time required for the polymerization is usually about 1 to 30 hours.

  The thickness of the first polymer layer can be made thinner than that of the second polymer layer, and is preferably 0.005 to 5 μm, more preferably 0.005 to 1 μm.

1.4.2. Introduction of polar groups into the first polymer layer The third step, that is, introduction of polar groups into the first polymer layer can be carried out, for example, by chemically modifying the glycidyl groups in the first polymer layer. The third step can include, for example, the following reaction. Two or more of these reactions may be combined.
(A) a reaction for introducing an amino group,
(B) a reaction for introducing an aldehyde group,
(C) -1. Reaction to introduce a carboxyl group,
(C) -2. Reaction that converts carboxyl group into active ester group after introducing carboxyl group

  That is, by chemically modifying the glycidyl group of the first polymer layer, a polar group containing at least one kind of atom selected from the group consisting of an oxygen atom, a nitrogen atom and a sulfur atom can be introduced. Here, the polar group is preferably a functional group capable of reacting with the probe, for example, at least one selected from an amino group, an aldehyde group, a carboxyl group, and an active ester group. For example, when the first polymer layer of the obtained magnetic particles has the polar group and a 2,3-dihydroxypropyl group described later, the binding property with the probe is good and non-specific adsorption is small.

  Hereinafter, the above reactions will be described in order.

1.4.2.1-1. Reaction for Introducing Amino Group (a) More specifically, the reaction for introducing an amino group is performed by causing an aminating agent to act on the particles on which the first polymer layer having a glycidyl group is formed. This is a reaction for introducing an amino group. By this reaction, amino group-introduced magnetic particles are obtained. This amino group-introduced magnetic particle can be suitably used for probe binding.

  Examples of the aminating agent include ammonia, an organic compound having two amino groups in the molecule (diamine), or an organic compound having three or more amino groups in the molecule. An organic compound having two or more amino groups is more preferable.

  Here, examples of the diamine include primary diamines such as ethylenediamine, propylenediamine, and o-phenylenediamine. Examples of organic compounds having three or more amino groups in the molecule include 1,2,3-triaminopropane, tetra (aminomethyl) methane, 1,3,5-triaminobenzene, 1,2,3. , 4-tetraaminobenzene and the like.

  The reaction for introducing an amino group may be carried out by dispersing the dried particles in the aminating agent as they are, or may be carried out in a state where the particles are dispersed in an aqueous solvent. The aqueous solvent is a mixed solvent of a water-soluble organic solvent and water, or water. Here, examples of the water-soluble organic solvent include methanol, ethanol, acetone, dimethylformamide and the like. The preferable temperature and time for the reaction for introducing the amino group vary depending on the concentration of the glycidyl group in the first polymer layer, the presence or absence of the solvent, the type of the solvent, etc., but usually 4 ° C to 100 ° C, preferably 20 ° C to At 80 ° C., it is usually 10 minutes to 48 hours, preferably 1 hour to 24 hours. In the reaction for introducing an amino group, it is not necessary that all glycidyl groups are aminated.

  The amount of amino groups in the magnetic particles according to this embodiment is preferably 0.1 μmol / g to 100 μmol / g, more preferably 0.5 μmol / g to 50 μmol / g. When the amount of amino group is less than 0.1 μmol, the amount of binding of the probe decreases and the signal may be inferior, and when it exceeds 100 μmol / g, nonspecific adsorption may increase.

  Prior to the reaction for introducing an amino group, a part of two or more glycidyl groups may be hydrolyzed. Moreover, you may hydrolyze a part of two or more glycidyl groups simultaneously with reaction which introduce | transduces an amino group. Alternatively, after the reaction for introducing an amino group, all or part of the remaining two or more glycidyl groups may be hydrolyzed.

  By hydrolyzing the glycidyl group, a 2,3-dihydroxypropyl group is generated. When the first polymer layer has 2,3-dihydroxypropyl groups, nonspecific adsorption can be reduced. The hydrolysis of the glycidyl group proceeds, for example, with an appropriate acid catalyst or base catalyst in an aqueous solvent. Preferably, before or after the reaction for introducing the amino group, the glycidyl group is hydrolyzed in an aqueous solvent using an acid catalyst such as sulfuric acid. Thereby, hydrolysis can be performed promptly and reliably. In this case, the preferred temperature for hydrolysis is usually 4 ° C. to 100 ° C., preferably 20 ° C. to 80 ° C., and the preferred time for hydrolysis is usually 5 minutes to 24 hours, preferably 30 minutes to 12 hours. .

  Examples of magnetic particles obtained by the reaction for introducing an amino group include, for example, a magnetic mother particle having a particle size d, a nonmagnetic child particle having a particle size of d / 2 or less present on the surface of the magnetic mother particle, and magnetic properties. Examples thereof include magnetic particles including a first polymer layer having an amino group and a 2,3-dihydroxypropyl group provided so as to cover the mother particle and the nonmagnetic child particle. Here, the magnetic mother particle includes a core particle, a magnetic layer containing superparamagnetic fine particles formed on the surface of the core particle, and a hydrophobic second polymer layer provided on the surface of the magnetic layer, The first polymer layer can be provided to cover the second polymer layer and the nonmagnetic child particles. Since the first polymer layer has an amino group and a 2,3-dihydroxypropyl group, the binding property to the probe is good and nonspecific adsorption is very small.

1.4.2.2. Reaction for Introducing Aldehyde Group (b) More specifically, the reaction for introducing an aldehyde group is performed by converting a glycidyl group contained in the first polymer layer into a diol group by hydrolysis, and using an oxidizing agent to convert the diol group. Is a reaction in which an aldehyde group is generated by oxidative cleavage. By this reaction, aldehyde group-introduced magnetic particles are obtained. The aldehyde group-introduced magnetic particles can be suitably used for probe binding.

  The conditions for hydrolysis of the glycidyl group are as described in the above (a) reaction for introducing an amino group.

  Examples of oxidizing agents suitable for aldehyde aldehydes include known oxidizing agents such as periodic acid, periodate, and lead tetraacetate. Among these, periodates such as sodium periodate and potassium periodate are preferable because the reaction easily proceeds in an aqueous solvent.

  The reaction to introduce an aldehyde group is performed when an aqueous solvent such as periodate is an appropriate oxidizing agent. For example, after hydrolysis of the glycidyl group, the supernatant is removed by washing and magnetic separation, and an aqueous oxidizing agent solution is added. Can be implemented. In the case of an oxidizing agent suitable for an organic solvent such as lead tetraacetate, after hydrolyzing the glycidyl group, the supernatant is removed by washing and magnetic separation, and further dried, and then the dried particles are dispersed in the oxidizing agent solution. It is preferable to carry out. The preferred temperature and time of the reaction for introducing the aldehyde group vary depending on the concentration of glycidyl group in the first polymer layer, the degree of hydrolysis, the type of solvent, etc., but the reaction temperature is usually 4 ° C. to 100 ° C., preferably 20 The reaction time is usually 1 minute to 12 hours, preferably 10 minutes to 6 hours. In the reaction for introducing an aldehyde group, not all glycidyl groups need to be aldehyded.

  The amount of aldehyde groups in the magnetic particles according to this embodiment is preferably 0.1 μmol / g to 100 μmol / g, more preferably 0.5 μmol / g to 50 μmol / g. When the amount of aldehyde groups is less than 0.1 μmol, the amount of binding of the probe decreases and the signal may be inferior. When it exceeds 100 μmol / g, nonspecific adsorption may increase.

  Examples of magnetic particles obtained by a reaction for introducing an aldehyde group include, for example, a magnetic mother particle having a particle size d, a non-magnetic child particle having a particle size of d / 2 or less present on the surface of the magnetic mother particle, and magnetic Examples thereof include magnetic particles including a first polymer layer having an aldehyde group and a 2,3-dihydroxypropyl group provided so as to cover the mother particle and the nonmagnetic child particle. Here, the magnetic mother particle includes a core particle, a magnetic layer containing superparamagnetic fine particles formed on the surface of the core particle, and a hydrophobic second polymer layer provided on the surface of the magnetic layer. The first polymer layer can be provided so as to cover the second polymer layer and the non-magnetic child particles. Since the first polymer layer has an aldehyde group and a 2,3-dihydroxypropyl group, it can be easily combined with the probe by mixing with the probe having an amino group, so that the probe can be easily combined and non-specific. Less adsorption.

1.4.2-3. Reaction for introducing carboxyl group (c) -1. Examples of the reaction for introducing a carboxyl group include (i) a carboxylating agent (for example, dicarboxylic acid, aminocarboxylic acid, or three or more carboxyl groups in the molecule) in the glycidyl group contained in the first polymer layer. (Ii) a carboxylating agent (for example, carboxylic acid anhydride, carboxylic acid chloride) is allowed to act on the hydroxyl group obtained by hydrolyzing the glycidyl group contained in the first polymer layer. Reaction, (iii) in the presence of a suitable dehydration catalyst, an organic compound having two or more carboxyl groups in the molecule to a hydroxyl group obtained by hydrolyzing the glycidyl group contained in the first polymer layer (for example, The reaction etc. which make dicarboxylic acid or the organic compound which has a 3 or more carboxyl group in a molecule | numerator act are mentioned. Through this reaction, carboxyl group-introduced magnetic particles are obtained. The carboxyl group-introduced magnetic particles can be suitably used for probe binding.

  The reaction of (ii) above is preferable because of the ease of control of the amount of carboxyl groups introduced, and (ii) a hydroxyl group obtained by hydrolyzing the glycidyl group contained in the first polymer layer is used as a carboxylating agent. A reaction in which a carboxylic anhydride is allowed to act is particularly preferred. Here, the carboxylic acid anhydride is a polyvalent carboxylic acid anhydride, and specific examples thereof include itaconic anhydride, succinic anhydride, citraconic anhydride, dodecenyl succinic anhydride, tricarbanilic anhydride, glutaric anhydride, maleic anhydride. Aliphatic dicarboxylic anhydrides such as acid, hexahydrophthalic anhydride, methyltetrahydrophthalic anhydride, and hymic anhydride; 1,2,3,4-butanetetracarboxylic dianhydride, cyclopentanetetracarboxylic dianhydride And alicyclic polyvalent carboxylic acid dianhydrides such as phthalic anhydride, pyromellitic anhydride, trimellitic anhydride, and benzophenone tetracarboxylic anhydride. Of these, 1,2-dicarboxylic anhydrides such as succinic anhydride, maleic anhydride, and phthalic anhydride are more preferable.

  As a specific method for allowing a carboxylic acid anhydride to act as a carboxylating agent on the hydroxyl group obtained by hydrolyzing the glycidyl group contained in the first polymer layer, for example, an organic solvent in which the carboxylic acid anhydride is dissolved And a method of dispersing a dried powder of hydrolyzed particles and stirring at room temperature to 80 ° C. for 1 to 24 hours. Examples of the organic solvent used here include, but are not limited to, pyridine, acetone, methyl ethyl ketone, tetrahydrofuran, dimethylformamide, and the like. As the catalyst, sulfuric acid, p-toluenesulfonic acid, zinc chloride, sodium acetate, pyridine, 4-dimethylaminopyridine, 4-pyrrolidinopyridine, and triethylamine may be used. Of these organic solvents and catalysts, pyridine is suitable as an organic solvent and catalyst.

  In addition, it is not necessary for all the hydroxyl groups on the magnetic particles according to the present embodiment to be esterified, and it is preferable that a part of the hydroxyl groups remain as hydroxyl groups without being esterified.

  In the case of the above (i) to (iii), a part of two or more glycidyl groups are hydrolyzed before the reaction for introducing a carboxyl group, or two or more glycidyls are simultaneously formed with the reaction for introducing a carboxyl group. It is preferred to hydrolyze part of the group. In the case of (i) above, all or part of the remaining two or more glycidyl groups may be hydrolyzed after the reaction for introducing a carboxyl group.

  The amount of carboxyl groups in the magnetic particles according to this embodiment is preferably 0.1 μmol / g to 100 μmol / g, more preferably 0.5 μmol / g to 50 μmol / g. If the amount of the carboxyl group is less than 0.1 μmol, the amount of binding of the probe decreases and the signal may be inferior. On the other hand, if it exceeds 100 μmol / g, nonspecific adsorption may increase.

  Examples of magnetic particles obtained by the reaction for introducing a carboxyl group include, for example, magnetic mother particles having a particle size d, non-magnetic child particles having a particle size of d / 2 or less present on the surface of the magnetic mother particles, and magnetic Examples thereof include magnetic particles including a first polymer layer having a carboxyl group and a 2,3-dihydroxypropyl group, which is provided so as to cover the mother particle and the nonmagnetic child particle. Here, the magnetic mother particle includes a core particle, a magnetic layer containing superparamagnetic fine particles formed on the surface of the core particle, and a hydrophobic second polymer layer provided on the surface of the magnetic layer. The first polymer layer can be provided so as to cover the second polymer layer and the non-magnetic child particles. Since the first polymer layer has a carboxyl group and a 2,3-dihydroxypropyl group, non-specific adsorption is small and a carboxyl group widely used in the biochemical field has been introduced. The particles can be utilized.

1.4.2-4. Reaction which converts this carboxyl group into an active ester group after introducing a carboxyl group (c) -2. As a reaction for converting the carboxyl group into an active ester group after introducing the carboxyl group, for example, the carboxyl group-introduced magnetic particles obtained by the reaction for introducing the carboxyl group are further modified with an appropriate activator. And a method of introducing an active ester group. By this reaction, active ester group-introduced magnetic particles are obtained. This active ester group-introduced magnetic particle can be suitably used for probe binding. Suitable activators include N-hydroxysuccinimide and N-hydroxysulfosuccinimide. The active ester group is not particularly limited, and examples thereof include an N-succinimidyloxycarbonyl group and an N-sulfosuccinimidyloxycarbonyl group.

  As a specific method for introducing an active ester group, for example, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide are added to an aqueous dispersion of carboxyl group-introduced magnetic particles. And a method of stirring at room temperature to 80 ° C. for 1 to 24 hours.

  The amount of the activated ester group in the magnetic particles according to the present embodiment is preferably 0.1 μmol / g to 100 μmol / g, more preferably 0.5 μmol / g to 50 μmol / g. When the amount of the activated ester group is less than 0.1 μmol, the amount of binding of the probe is small and the signal may be inferior. On the other hand, when it exceeds 100 μmol / g, nonspecific adsorption may increase.

  As an example of magnetic particles obtained by a reaction for introducing an active ester group, for example, a magnetic mother particle having a particle size d, a non-magnetic child particle having a particle size of d / 2 or less present on the surface of the magnetic mother particle, Examples thereof include magnetic particles including a first polymer layer having an active ester group and a 2,3-dihydroxypropyl group, which is provided so as to cover the magnetic mother particle and the nonmagnetic child particle. Here, the magnetic mother particle is a core particle, a magnetic layer containing superparamagnetic fine particles formed on the surface of the core particle, a hydrophobic second polymer layer provided on the surface of the magnetic layer, And the first polymer layer can be provided to cover the second polymer layer and the non-magnetic child particles. When the first polymer layer has an active ester group and a 2,3-dihydroxypropyl group, it can be easily combined with the probe by mixing with the probe having an amino group, and thus the binding with the probe is easy, and There is little peeling of the probe and non-specific adsorption.

1.5. Applications The magnetic particles according to the present embodiment can be suitably used as probe-binding particles. More specifically, the affinity for compound carrier particles in biochemical fields and chemical binding carrier particles for diagnostic agents, etc. It can be used as a carrier, and can exhibit particularly high sensitivity and low noise as magnetic particles for probe binding for immunoassay and proteome to which a primary probe such as an antigen or antibody is bound.

  In the probe-binding magnetic particles according to the present embodiment, the substances to be tested are biological substances, chemical substances, and living organisms contained in the immunological test reagent and the sample to be tested. In the present invention, “biologically related substance” refers to all substances related to a living body. Examples of the biological substance include substances contained in the living body, substances derived from the substance contained in the living body, and substances that can be used in the living body.

  The biological substance is not particularly limited. For example, proteins (for example, enzymes, antibodies, aptamers, receptors, etc.), peptides (for example, glutathione), nucleic acids (for example, DNA and RNA), carbohydrates, lipids, hormones ( For example, luteinizing hormone, human chorionic gonadotropin, thyroid stimulating hormone, insulin, glucagon, growth hormone, etc.) and other cells or substances (eg, various blood cells including various blood cells such as platelets, red blood cells, white blood cells, etc.) Substances, various floating cells, and proteins and nucleic acids that are constituents of viruses, bacteria, fungi, protozoa, parasites, etc.). More specifically, examples of proteins include biological proteins, proteins that serve as markers for various cancers such as prostate-specific markers, bladder cancer markers, and the like.

  The chemical substance to be inspected is not particularly limited, and examples thereof include environmental pollutants such as dioxins and pharmaceuticals (for example, antibiotics, anticancer agents, antiepileptic agents, etc.).

  The organism to be examined is not particularly limited. For example, various cancer cells, various floating cells, viruses (for example, hepatitis B virus, hepatitis C virus, herpes simplex virus, HIV virus, rubella virus, influenza virus, etc.), Examples include bacteria (for example, Neisseria gonorrhoeae, MRSA, E. coli, etc.), fungi (for example, Candida, ringworm, Cryptocox, alpergillus, etc.), protozoa and parasites (for example, Toxoplasma, malaria, etc.).

  Among the magnetic particles for binding probes according to the present embodiment, according to the aldehyde group-introduced particles and active ester group-introduced particles, the probe and the particles are mixed on the surface of the particles when actually used. Can be chemically coupled.

  Among the probe-bonding magnetic particles according to the present embodiment, according to the amino group-introduced particles and carboxyl group-introduced particles, the amino group or carboxyl group is introduced on the surface of the particle. By activating the carboxyl group of the probe or the particle with a known activator such as carbodiimide and mixing the probe and the particle, the probe can be chemically bonded to the surface of the particle.

  After binding the probe to the surface of the particle, excess probe is washed and unreacted active groups are inactivated as necessary. As the inactivating agent, it is preferable to use an inactivating agent containing a hydroxyl group such as ethanolamine or tris (hydroxymethylamino) methane. Further, after the probe is bonded to the surface of the particle, a blocking operation that is usually performed is not necessary, but a blocking agent such as albumin, skim milk, or casein may be used in combination in the inactivation step described above. Thereafter, the process may be shifted to a normal process using particles.

  The probe that can be carried on the probe-binding magnetic particles according to the present embodiment is a protein (antigen or antibody), nucleic acid, or compound, and of these, the antigen or antibody is preferable. In this case, the antigen or antibody is not particularly limited as long as it reacts with a component generally contained in a subject. For example, anti-antiplasmin antibody for antiplasmin test, anti-D dimer antibody for D dimer test Anti-FDP antibody for FDP test, Anti-tPA antibody for tPA test, Anti-thrombin = antithrombin complex antibody for TAT test, Anticoagulation-related test antigen or antibody such as anti-FPA antibody for FPA test; Anti-BFP for BFP test Anti-Apolipoprotein for testing apolipoprotein such as antibodies, anti-CEA antibodies for CEA testing, anti-AFP antibodies for AFP testing, anti-ferritin antibodies for testing ferritin, anti-CA19-9 testing for CA19-9 Antibody, anti-β2-microblob antibody for β2-microblob test, α1-microglob Anti-α1-microglobulin antibody for serum test, anti-immunoglobulin antibody for immunoglobulin test, serum protein-related test antigen or antibody such as anti-CRP antibody for CRP test; endocrine function test antigen such as anti-HCG antibody for HCG test or Antibody: Anti-HBs antibody for HBs antigen test, HBs antigen for HBs antibody test, HCV antigen for HCV antibody test, HIV-1 antigen for HIV-1 antibody, HIV-2 antigen for HIV-2 antibody test, HTLV-1 test HTLV-1 antigen, Mycoplasma antigen for Mycoplasma test, Toxoplasma antigen for Toxoplasma test, Streptridine O antigen for ASO test, etc. Antigen-related test antigen or antibody; Anti-DNA antibody test DNA antigen, RF test heat-modified human Antigens or antibodies for autoimmune related tests such as IgG; antidigoxin anti And antigens for drug analysis such as anti-lidocaine antibodies for lidocaine testing, and the like, but are not limited thereto. As the antibody, either a polyclonal antibody or a monoclonal antibody may be used.

  In addition, the magnetic particles for probe binding according to the present embodiment are used as affinity carriers for sensitizing proteins such as enzymes and hormones, nucleic acids such as DNA and RNA, lipids, or bioactive sugar chain compounds to the particle surface by a chemical binding method. Can also be used. Furthermore, a chemical substance to be analyzed (chemical substance to be analyzed; corresponding to a ligand molecule) is immobilized on the probe-binding magnetic particles according to the present embodiment by chemical bonding, and a specific interaction with a protein substance or the like is used. By analyzing and / or measuring the interaction, a protein or the like (corresponding to the target molecule) having a specific interaction with the chemical substance to be analyzed can be selected and purified.

  Specifically, the ligand molecule to be bound to the particle is not particularly limited as long as it is a substance having a functional group capable of reacting with at least one of the functional groups of the probe-binding magnetic particle according to the present embodiment. Nucleic acids, peptide nucleic acids, hormones, proteins with a molecular weight of 500 to 1,000,000, sugar chains, polysaccharides, cells, aptamers, viruses, enzymes, various affinity tag capture substances, coenzymes such as biotin, and specific physiological activity A chemical substance or the like (or possibly having a specific physiological activity) can be used.

2. Examples Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto. In each example and comparative example, the evaluation was performed by the following method.

2.1. Evaluation method 2.1.1. Antibody binding amount The particles obtained in each of the examples described below and the particles obtained in the comparative example were chemically bonded to an anti-AFP (α-fetoprotein) antibody, and then the antibody was bound to the particles by the BCA (Bicinchoninic Acid) method. The amount was evaluated.

2.1.2. Signal measurement by CLEIA (chemiluminescent enzyme immunoassay) 10 μl of a dispersion of particles (corresponding to 50 μg of particles) obtained by sensitizing anti-AFP antibody and obtained in each of Examples and Comparative Examples to be described later is placed in a test tube and calf fetus The mixture was mixed with 50 μl of a standard sample of AFP antigen (manufactured by Nippon Biotest) diluted to 100 ng / mL with serum (FCS), and reacted at 37 ° C. for 10 minutes. After separating the particles by magnetic separation and removing the supernatant, an anti-AFP antibody labeled with alkaline phosphatase (hereinafter referred to as “ALP”) as a secondary antibody (manufactured by Fujirebio Inc., attached to Lumipulse AFP-N) Using reagent) 40 μl was added and allowed to react at 37 ° C. for 10 minutes. Next, after magnetic separation and removal of the supernatant, the particles obtained by repeating washing three times with PBS were dispersed in 50 μl of 0.01% Tween 20, and transferred to a new tube. After adding 100 μl of ALP substrate solution (Lumipulse substrate solution: manufactured by Fujirebio Inc.) and reacting at 37 ° C. for 10 minutes, the amount of chemiluminescence was measured. For the measurement of chemiluminescence, a chemiluminescence measuring device (trade name: Lumat LB9507) manufactured by Bertol Japan KK was used.

2.1.3. Noise measurement The amount of chemiluminescence as noise was measured by the same method except that the signal was measured by CLEIA (chemiluminescence enzyme immunoassay) and not mixed with a standard sample.

2.2. Synthesis example (production of magnetic particles)
2.2.1. Synthesis Example 1 (Preparation of core particles)
1 g of 75% di (3,5,5-trimethylhexanoyl) peroxide solution (“Perroyl 355-75 (S)” manufactured by NOF Corporation, hereinafter referred to as “parroyl”) is mixed with 10 g of 1% sodium dodecyl sulfate aqueous solution. And finely emulsified with an ultrasonic disperser. This was put into a reactor containing 6.5 g of polystyrene particles having a particle size of 0.77 μm and 20.5 g of water, and stirred at 25 ° C. for 12 hours. In a separate container, a solution obtained by emulsifying 48 g of styrene and 2 g of divinylbenzene with 200 g of a 0.1% sodium dodecyl sulfate aqueous solution was placed in the reactor, stirred at 40 ° C. for 2 hours, then heated to 75 ° C. and heated to 8 ° C. Polymerized for hours. After cooling to room temperature, the particles taken out by centrifugation were further washed with water, dried and pulverized to obtain core particles (1). The number average particle diameter of the core particles (1) was 1.5 μm.

2.2.2. Synthesis Example 2 (Preparation of magnetic mother particles (1))
After adding acetone to oily magnetic fluid (trade name: “EXP series”, manufactured by Ferrotec Co., Ltd.) to precipitate and precipitate the particles, this is dried to obtain a ferrite-based surface having a hydrophobized surface. Superparamagnetic fine particles (1) (average primary particle size: 0.01 μm) were obtained.

  15 g of the above core particles (1) and 15 g of the above superparamagnetic fine particles (1) are mixed well with a mixer, and this mixture is mixed with a blade (stirring) using a hybridization system NHS-0 type (manufactured by Nara Machinery Co., Ltd.). The magnetic base particles (1) (average number particle diameter: 2.0 μm) having a magnetic layer composed of superparamagnetic fine particles (1) on the surface are obtained by processing at a peripheral speed of 100 m / sec (16200 rpm) of the wings). It was.

2.2.3. Synthesis Example 3 (Preparation of magnetic mother particle (A-1) having second polymer layer)
30 g of the magnetic mother particles (1) obtained in Synthesis Example 2 above, 0.25% of a nonionic emulsifier “Emulgen 150” (manufactured by Kao Corporation) and a cationic emulsifier “Cotamine 24P” (Kao Corporation) )) 750 g of an aqueous solution containing 0.25% was put into a 1 L separable flask and sufficiently dispersed. In a separate container, 150 g of an aqueous solution containing 0.25% of a nonionic emulsifier “Emulgen 150” and 0.25% of a cationic emulsifier “Cotamine 24P” was placed, and 30 g of cyclohexyl methacrylate and N, N-dimethylaminopropylacrylamide were used as monomers. 7.5 g of a tertiary butyl peroxy 2-ethylhexanate (manufactured by NOF Corporation; Perbutyl O) as a polymerization initiator was added and mixed to prepare a monomer emulsion. The temperature of the separable flask was raised to 60 ° C. while purging with N ₂ gas under stirring at 200 rpm using a squid stirring blade, and then the monomer emulsion was continuously added to the separable flask over 2 hours. After completion of the continuous addition, stirring was continued at 80 ° C. for 2 hours to complete the reaction, and magnetic mother particles having a mother particle coat layer (second polymer layer) formed on the surface were obtained. The magnetic mother particle aqueous dispersion was magnetically purified and centrifuged. The magnetic mother particle produced as described above is referred to as magnetic mother particle (A-1). It was 2.8 micrometers as a result of measuring the particle size d of a magnetic mother particle (A-1) by measurement of 100 particle diameters of a transmission electron micrograph.

2.2.4. Synthesis Example 4 (Preparation of hetero-aggregated magnetic particles)
1000 g of an aqueous dispersion containing 5 g of non-magnetic child particles (B-1) made of styrene / methacrylic acid = 95/5 copolymer and having an average particle diameter of 0.1 μm is placed in a beaker and subjected to indirect ultrasonic waves in a water bath. Then, 500 g of an aqueous solution containing 6 g of magnetic mother particles (A-1) prepared in another container, 50 g of 0.1M HCl, and 0.5% of a nonionic emulsifier “Emulgen 150” was dropped into the beaker, and the magnetic mother Nonmagnetic child particles (B-1) were adsorbed on the particles (A-1). The obtained particle dispersion was magnetically purified to obtain magnetic particles (1) in which the nonmagnetic child particles (B-1) were adsorbed to the magnetic mother particles (A-1).

  When this magnetic particle (1) was observed by SEM, it was observed that the nonmagnetic small particles (B-1) were adsorbed on the surfaces of the magnetic mother particles (A-1).

2.2.5. Synthesis Example 5 (Formation of first polymer layer)
6 g of magnetic particles (1) and 150 g of an aqueous solution containing 0.5% sodium dodecylbenzenesulfonate were charged into a 500 mL separable flask and sufficiently dispersed. In a separate container, 0.75 g of an aqueous solution containing 0.5% sodium dodecylbenzenesulfonate was added, and 0.15 g of glycidyl methacrylate and 0.0214 g of trimethylolpropane triacrylate as monomers, and 0.0075 g of parroyl as a polymerization initiator were added thereto. The monomer emulsion was prepared by adding and mixing. To the separable flask, the whole amount of the monomer emulsion was added while purging with N ₂ gas while stirring at 200 rpm using a squid type stirring blade. The temperature was raised to 80 ° C. and stirring was continued for 3 hours. Magnetic particles (2) in which a first polymer layer was formed on the surfaces of A-1) and nonmagnetic particles (1) were obtained.

  The aqueous dispersion of magnetic particles (2) was magnetically purified and centrifuged. When the magnetic particles (2) were observed with an SEM, the nonmagnetic child particles (B-1) were adsorbed on the surface of the magnetic mother particles (A-1), and the magnetic mother particles (A-1) (second polymer layer) and It was observed that a new polymer layer (first polymer layer) was formed so as to cover the nonmagnetic particle (B-1). In addition, a transmission electron micrograph of the cross section of the magnetic particle (2) was taken, and the average thickness of the first polymer layer measured at 30 locations having different cross sections was 0.01 μm.

2.3. Example (Introduction of polar group into first polymer layer)
2.3.1. Example 1 (Carboxyl group-introduced probe binding particles)
10 g of 1% sulfuric acid aqueous solution is added to 1.0 g of particles isolated by magnetic separation from the aqueous dispersion of magnetic particles (2) obtained from the above synthesis example, and the particles are dispersed by irradiation with indirect ultrasonic waves for 20 minutes. Subsequently, the dispersion liquid of the particles was stirred at 60 ° C. for 5 hours (hydrolysis of glycidyl group).

  Subsequently, the operation of isolating the particles by magnetic separation, dispersing in pure water, magnetic separation and washing was repeated 5 times. 1.0 g of the obtained dried particles were washed with 10 ml of pyridine and then dispersed in 5 ml of pyridine. A succinic anhydride 3 g / 25 ml pyridine solution was added and reacted at 60 ° C. for 2 hours (introduction of carboxyl group).

  After the reaction, the particles are separated using magnetism, washed 3 times with acetone, then 3 times with 0.1 M aqueous sodium hydroxide solution and 4 times with distilled water, and then dispersed in distilled water. A 1% dispersion containing 0 g of carboxyl group-introduced magnetic particles (Ca-2) was obtained.

  500 μl of a 1 wt% aqueous dispersion of carboxyl group-introduced magnetic particles (Ca-2) was taken in a tube, magnetically separated by a magnetic stand, and the supernatant was removed. After washing 3 times with 100 mM MES (pH 5), 500 μl of the same buffer was dispersed, and 0.1 mg of anti-AFP antibody and 0.05 mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) were added thereto. The mixture was added and stirred at room temperature for 5 hours. After completion of the reaction, the supernatant was removed by magnetic separation. Next, after washing 5 times with PBS (−) buffer, the particles were dispersed with 500 μl of PBS (−) buffer to obtain a dispersion of probe (antibody) -bound magnetic particles (antibody-sensitized particles). .

  Table 1 shows the antibody binding amount of the obtained antibody-sensitized particles, signal measurement by CLEIA, and noise measurement results.

2.3.2. Example 2 (Active ester group-introduced probe binding particles)
100 mL of a 1% dispersion containing the carboxyl group-introduced magnetic particles (Ca-2) obtained in Example 1 was placed in a beaker, and the supernatant was removed by magnetic separation. The particles were washed 3 times with 100 mM MES (pH 5), dispersed in 100 mL of the same buffer, 0.16 g of N-hydroxysuccinimide and 0.18 g of EDC were added, and the dispersion of the particles was allowed to stand at room temperature for 2 hours. Stirring was performed (introduction of an active ester group).

  After completion of the reaction, the magnetic separation and dispersion were repeated and washed five times, and the particles were further dispersed in distilled water to obtain a 1% dispersion containing 1.0 g of active ester group-introduced magnetic particles (Ac-2). It was.

  500 μl of a 1 wt% aqueous dispersion of active ester group-introduced magnetic particles (Ac-2) was placed in a tube and magnetically separated using a magnetic stand, and the supernatant was removed. The particles were washed three times with 100 mM MES (pH 5), dispersed in 500 μl of the same buffer, 0.1 mg of anti-AFP antibody was added thereto, and the mixture was stirred at room temperature for 2 hours. After completion of the reaction, the supernatant was removed by magnetic separation. Next, after washing 5 times with PBS (−) buffer, the particles are dispersed with 500 μl of PBS (−) buffer to obtain a dispersion of probe (antibody) -bound magnetic particles (antibody-sensitized particles). It was.

  Table 1 shows the antibody binding amount of the obtained antibody-sensitized particles, signal measurement by CLEIA, and noise measurement results.

2.3.3. Example 3 (Amino group-introduced probe binding particles)
After repeating the operation of dispersing the particles isolated by magnetic separation from the aqueous dispersion of the magnetic particles (2) having the first polymer layer having a glycidyl group dispersed in acetone, magnetically separating and washing, five times, The particles were dispersed again in acetone, the supernatant was removed by magnetic separation, and the particles were dried. Next, 0.50 g of the particles were placed in a 100 ml flask, 25 g of ethylenediamine was added, and then dispersed by irradiation with indirect ultrasonic waves for 20 minutes, and then heated and stirred at 50 ° C. for 3 hours in a nitrogen atmosphere (amino) Group introduction).

  After cooling, the particles are isolated by magnetic separation, dispersed in distilled water, magnetically separated and washed, and then the supernatant is removed by magnetic separation. Then, 1% sulfuric acid is removed. The particles were added to 5 g of an aqueous solution, dispersed by irradiation with indirect ultrasonic waves for 20 minutes, and then stirred at 60 ° C. for 5 hours (hydrolysis of residual glycidyl groups).

  Subsequently, the operation of isolating the particles by magnetic separation, dispersing in pure water, magnetic separation and washing 5 times was repeated, followed by drying to give 0.49 g of amino group-introduced magnetic particles (Am-2) Got.

  The amino group-introduced magnetic particles (Am-2) were diluted and dispersed in pure water to a concentration of 1 wt% to prepare an aqueous dispersion. Next, 500 μl of this aqueous dispersion was taken in a tube and magnetically separated using a magnetic stand, and the supernatant was removed. After washing 3 times with 100 mM MES (pH 5), dispersed in 500 μl of the same buffer, 0.1 mg of anti-AFP antibody was added thereto, 0.05 mg of EDC was further added, and the mixture was stirred for 3 hours at room temperature. It was. After completion of the reaction, the supernatant was removed by magnetic separation. Next, after washing 5 times with PBS (−) buffer, the particles were dispersed with 500 μl of PBS (−) buffer to obtain a dispersion of probe (antibody) -bound magnetic particles (antibody-sensitized particles). .

  Table 1 shows the antibody binding amount of the obtained antibody-sensitized particles, signal measurement by CLEIA, and noise measurement results.

2.3.4. Example 4 (aldehyde group-introduced probe binding particles)
5 g of 1% sulfuric acid aqueous solution is added to 0.5 g of particles isolated by magnetic separation from an aqueous dispersion of magnetic particles (2) having a first polymer layer having a glycidyl group and irradiated with indirect ultrasonic waves for 20 minutes. And then stirred at 60 ° C. for 5 hours (hydrolysis of glycidyl group).

  Subsequently, the operation of isolating the particles by magnetic separation, dispersing in pure water, magnetically separating and washing was repeated five times. Furthermore, 20 mL of a 5.6 mg / mL sodium periodate aqueous solution was added to the particles isolated by magnetic separation, and the mixture was reacted for 1 hour at room temperature with stirring (introduction of aldehyde group).

  Next, the particles were isolated by magnetic separation, and the amount of formaldehyde in the supernatant was quantified using a Glycoprotein Carbohydrate Estimation Kit manufactured by PIERCE. As a result, it was confirmed that 11 μmol of aldehyde groups were introduced per gram of particles. Then, the operation of dispersing the particles in distilled water, magnetically separating and washing was repeated 5 times, and then dispersed in distilled water to contain 1% containing 0.49 g of aldehyde group-introduced magnetic particles (AL-2). A dispersion was obtained.

  500 μl of a 1 wt% aqueous dispersion of aldehyde group-introduced magnetic particles (AL-2) was taken in a tube, magnetically separated by a magnetic stand, and the supernatant was removed. After washing 3 times with citrate-carbinate buffer pH 10, it was dispersed in 500 μl of the same buffer, 0.1 mg of anti-AFP antibody was added thereto, and the mixture was stirred at room temperature for 5 hours. After completion of the reaction, the supernatant was removed by magnetic separation. Subsequently, 500 μl of tris-HCl buffer (pH 7.4) was added, and the mixture was stirred at room temperature for 2 hours. Furthermore, after washing 5 times with PBS (−) buffer, the particles are dispersed with 500 μl of PBS (−) buffer to obtain a dispersion of probe (antibody) -bound magnetic particles (antibody-sensitized particles). It was.

  Table 1 shows the antibody binding amount of the obtained antibody-sensitized particles, signal measurement by CLEIA, and noise measurement results.

2.3.5. Comparative Example 1 (example in which a polar group is not introduced into the first polymer layer)
Magnetic base particles (A-1) having a magnetic layer made of fine particles on the surface were obtained according to the same process procedure as in “Synthesis Examples 1 to 4”. Next, in the same procedure as in Synthesis Example 5, except that 0.075 g of styrene and 0.075 g of methacrylic acid were used instead of 0.15 g of glycidyl methacrylate and 0.0214 g of trimethylolpropane triacrylate, magnetic particles (2) were formed. Corresponding magnetic particles (2-1) were obtained. The first polymer layer of the magnetic particle (2-1) does not contain a glycidyl group.

  500 μl of a 1 wt% aqueous dispersion of magnetic particles (2-1) was taken in a tube, magnetically separated by a magnetic stand, and the supernatant was removed. The particles were washed three times with 100 mM MES (pH 5), and then dispersed in 500 μl of the same buffer, to which 0.1 mg of anti-AFP antibody, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) 0 .05 mg was added and stirred at room temperature for 5 hours. After completion of the reaction, the supernatant was removed by magnetic separation. Next, after washing 5 times with PBS (−) buffer, the particles were dispersed with 500 μl of PBS (−) buffer to obtain a dispersion of probe (antibody) -bound magnetic particles (antibody-sensitized particles). .

  Table 1 shows the antibody binding amount of the obtained antibody-sensitized particles, signal measurement by CLEIA, and noise measurement results.

2.3.6. Comparative Example 2 (example not containing non-magnetic child particles)
Magnetic base particles (1) having a magnetic layer made of superparamagnetic fine particles on the surface were obtained according to the same procedure as in “Synthesis Examples 1 and 2”. Next, 375 g of a 0.5% by weight aqueous solution of sodium dodecylbenzenesulfonate was charged into a 1 L separable flask, then 15 g of magnetic mother particles (1) were charged, dispersed with a homogenizer, and heated to 60 ° C. Methyl methacrylate (MMA) 18 g, trimethylolpropane trimethacrylate (TMP) 2 g, and paroyl 0.4 g were dispersed in 100 g of a 0.5 wt% aqueous solution of sodium dodecylbenzenesulfonate in a separate container. The pre-emulsion was dropped into the 1 L separable flask controlled at 60 ° C. over 1 hour 30 minutes (formation of the second polymer layer).

  After completion of the dropwise addition, the mixture was kept at 60 ° C. and stirred for 1 hour, and then 10.5 g of glycidyl methacrylate and 1.5 g of TMP as monomers, and paroyl were added to 75 g of a 0.5 wt% aqueous solution of sodium dodecylbenzenesulfonate in a separate container. A pre-emulsion in which 0.3 g was added and dispersed was dropped into the 1 L separable flask controlled at 60 ° C. over 1 hour and 30 minutes. Thereafter, the temperature was raised to 75 ° C., and polymerization was further continued for 2 hours to complete the reaction (formation of the first polymer layer).

  Next, the particles in the separable flask were separated using magnetism and washed with distilled water. The magnetic particle (2-2) in which the 1st polymer layer which has a glycidyl group was formed by the above process was obtained.

  By using the magnetic particles (2-2) instead of the magnetic particles (2), the carboxyl group-introduced magnetic particles (Ca-2-2) and the active ester groups are obtained by the same procedure as in the above “Examples 1-4”. Introduced magnetic particles (Ac-2-2), amino group-introduced magnetic particles (Am-2-2), and aldehyde group-introduced magnetic particles (AL-2-2) were obtained.

  Further, using these magnetic particles (Ca-2-2), (Ac-2-2), (Am-2-2), and (AL-2-2), the same as in "Examples 1-4" The anti-AFP antibody was sensitized by a technique to obtain a dispersion of probe (antibody) -bound magnetic particles (antibody-sensitized particles).

  Table 1 shows the antibody binding amount of the obtained antibody-sensitized particles, signal measurement by CLEIA, and noise measurement results.

2.3.7. Comparative Example 3 (example in which the first polymer layer having a glycidyl group is not formed)
In the formation of the first polymer layer, in the same procedure as in Comparative Example 1 except that 10.5 g of cyclohexyl methacrylate and 1.5 g of methacrylic acid were used instead of 10.5 g of glycidyl methacrylate and 1.5 g of TMP, magnetic particles (2 -3) was obtained.

  500 μl of a 1 wt% aqueous dispersion of magnetic particles (2-3) was placed in a tube and magnetically separated using a magnetic stand, and the supernatant was removed. After washing 3 times with 100 mM MES (pH 5), 500 μl of the same buffer was dispersed, and 0.1 mg of anti-AFP antibody and 0.05 mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) were added thereto. The mixture was added and stirred at room temperature for 5 hours. After completion of the reaction, the supernatant was removed by magnetic separation. Next, after washing 5 times with PBS (−) buffer, the particles were dispersed with 500 μl of PBS (−) buffer to obtain a dispersion of probe (antibody) -bound magnetic particles (antibody-sensitized particles). .

  Table 1 shows the antibody binding amount of the obtained antibody-sensitized particles, signal measurement by CLEIA, and noise measurement results.

  According to Table 1, the magnetic particles obtained in Examples 1 to 3 have polar groups (amino groups, active ester groups, carboxyl groups, aldehyde groups) so as to cover the magnetic mother particles and the nonmagnetic child particles. By providing the first polymer layer, there is little non-specific adsorption of proteins, nucleic acids, etc., so that high sensitivity and low noise that are outstanding in the biochemistry / pharmaceutical field are expressed, and high S for biochemical testing. It can be seen that the / N ratio can be obtained. In addition, since the magnetic particles obtained in Examples 1 to 3 have uneven surfaces due to the presence of non-magnetic child particles so as to cover the surfaces of the magnetic mother particles, Because of the large surface area, the amount of antibody binding is large.

  On the other hand, since the magnetic particles obtained in Comparative Example 1 did not introduce polar groups into the first polymer layer, the noise was larger than the magnetic particles obtained in Examples 1 to 3. Guessed. Moreover, since the magnetic particles obtained in Comparative Example 2 do not contain non-magnetic child particles, it is presumed that the amount of antibody binding was less than that of the magnetic particles obtained in Examples 1 to 3. Furthermore, since the 1st polymer layer does not contain a glycidyl group, it is estimated that the magnetic particle obtained by the comparative example 3 was noisy compared with the magnetic particle obtained by Examples 1-3.

Claims (9)

A first step of providing non-magnetic child particles having a particle size of d / 2 or less on the surface of magnetic base particles having a particle size of d,
A second step of forming a first polymer layer having a glycidyl group so as to cover the magnetic mother particles and the non-magnetic child particles; and
A method for producing magnetic particles, comprising a third step of introducing an amino group, an aldehyde group, or a carboxyl group into the first polymer layer. A first step of providing non-magnetic child particles having a particle size of d / 2 or less on the surface of magnetic base particles having a particle size of d,
A second step of forming a first polymer layer having a glycidyl group so as to cover the magnetic mother particles and the non-magnetic child particles; and
A third step of introducing a carboxyl group into the first polymer layer and further converting the carboxyl group into an active ester group
A method for producing magnetic particles , comprising: The second step is a step of forming a first polymer layer having a plurality of glycidyl groups,
The method for producing magnetic particles according to claim 1, further comprising a step of hydrolyzing a part of the glycidyl group. Magnetic base particles having a particle size d;
Non-magnetic child particles having a particle diameter of d / 2 or less present on the surface of the magnetic mother particles;
A first polymer layer having an amino group and a 2,3-dihydroxypropyl group provided so as to cover the magnetic mother particle and the non-magnetic child particle;
Magnetic particles containing Magnetic base particles having a particle size d;
Non-magnetic child particles having a particle diameter of d / 2 or less present on the surface of the magnetic mother particles;
A first polymer layer having an aldehyde group and a 2,3-dihydroxypropyl group provided so as to cover the magnetic mother particle and the non-magnetic child particle;
Magnetic particles containing Magnetic base particles having a particle size d;
Non-magnetic child particles having a particle diameter of d / 2 or less present on the surface of the magnetic mother particles;
A first polymer layer having a carboxyl group and a 2,3-dihydroxypropyl group provided so as to cover the magnetic mother particle and the non-magnetic child particle;
Magnetic particles containing Magnetic base particles having a particle size d;
Non-magnetic child particles having a particle diameter of d / 2 or less present on the surface of the magnetic mother particles;
A first polymer layer having an active ester group and a 2,3-dihydroxypropyl group provided so as to cover the magnetic mother particle and the non-magnetic child particle;
Magnetic particles containing The magnetic particle according to claim 4, which is used for probe binding. A probe-binding particle comprising the magnetic particle according to claim 4 and a probe that binds to the magnetic particle.