JP7363327B2 - Magnetic particles for antibody binding and their manufacturing method - Google Patents

Description

The present invention relates to antibody-binding magnetic particles used in immunodiagnosis and a method for producing the same.

Magnetic particles are used as carriers for supporting physiologically active substances for immunodiagnosis by utilizing their magnetic properties. However, when binding physiologically active substances such as antibodies to the surface of magnetic particles, the reaction is carried out in an acidic aqueous solution, which causes problems such as detachment of the magnetic substance and elution of iron ions, resulting in the outflow of substances derived from the magnetic substance components. there were. Since iron acts as an interfering substance in immune reactions, there is a problem that the sensitivity of immunodiagnosis decreases and the number of physiologically active substances that can be sensitized is limited.Therefore, magnetic particles with less leakage of magnetic components are desired. There is. In addition, magnetic particles used as carriers for immunodiagnosis have features such as low noise that suppresses nonspecific adsorption of components unrelated to immune reactions, and high signal that can support many physiologically active substances. There is a need for magnetic particles that can achieve high sensitivity. Furthermore, since magnetic particles are used in immunodiagnosis by being dispersed in an aqueous solution, there is a need for magnetic particles that have good dispersibility in water.

In order to solve these problems, we have developed immunodiagnostic particles in which the surface of magnetic particles having a ferrite coating layer on the core particle surface is coated with an organic silane coupling agent having a functional group such as an amino group, and further a carboxyl group is introduced. It has been proposed (for example, see Patent Document 1). In addition, after adsorbing nanomagnetic material coated with a hydrophobic organosilane coupling agent onto core particles, the nanomagnetic material was coated with a hydrophobic polymer so that it was buried, and functional groups were further introduced onto the surface. Particles for diagnostic reagents have been proposed (see, for example, Patent Document 2).

Patent No. 2979414 Patent No. 3738847

In Patent Document 1, the elution of iron can be suppressed by treating the surface with an organic silane coupling agent, but there is a problem that the suppression is insufficient.

In Patent Document 2, the surface of the core particle is coated with a magnetic material treated with a hydrophobic organosilane coupling agent, and then a hydrophobic polymer layer is formed to suppress the elution of iron. The polymer layer has the problem of worsening the dispersibility of magnetic particles in water. Furthermore, since the nanomagnetic material is buried in the polymer by being coated with a hydrophobic polymer layer, the specific surface area of the magnetic particles is small, and a large amount of magnetic particles is required to support the desired amount of physiologically active substances such as antibodies. The problem was that it was necessary.

The present invention was made in view of the above problems, and has excellent acid resistance, little elution of iron even when treated with an acidic aqueous solution, good dispersibility in water, and low nonspecific adsorption. Another object of the present invention is to provide magnetic particles that have a large specific surface area, support a large amount of physiologically active substances, and are capable of increasing the sensitivity of immunodiagnosis.

In view of the above points, the present inventors have conducted extensive research, and as a result have found that the above-mentioned problems can be solved by using magnetic particles, and have completed the present invention.

Each aspect of the present invention is [1] to [4] shown below.
[1] A magnetic particle having a core particle, a first coating layer formed on the surface of the core particle, and a second coating layer formed on the surface of the first coating layer,
The first coating layer is a crosslinked inorganic layer containing nanomagnetic material,
The second coating layer is a polymer layer having at least one hydrophilic group-containing structural unit selected from a hydroxyl group and a polyethylene glycol group, and forms a covalent bond with the first coating layer, and forms a covalent bond with the first coating layer. Magnetic particles for antibody binding characterized by having a carboxyl group at the end of the polymer chain on the opposite side from the binding.
[2] The magnetic particle for antibody binding according to [1], wherein the crosslinked inorganic layer of the first coating layer contains silica.
[3] The antibody-binding magnetic particle according to [1] or [2], wherein the polymer layer that is the second coating layer has a crosslinked structure.
[4] The method for producing antibody-binding magnetic particles according to any one of [1] to [3], which comprises the following steps (i) to (v).
(i) A step of physically adsorbing the nanomagnetic material on the surface of the core particle (ii) A step of forming a crosslinked inorganic layer on the surface of the particle to which the nanomagnetic material is adsorbed (iii) A step of physically adsorbing the nanomagnetic material on the surface of the core particle Step (iv) of bonding a silane coupling agent having a radical polymerization initiator or chain transfer agent: dispersing the particles bound with the silane coupling agent in a solvent, and dispersing the particles selected from hydroxyl groups and polyethylene glycol groups in the solvent. step (v) of forming a polymer layer on the surface of the particles by living radical polymerization of a monomer having at least one hydrophilic group-containing structural unit, (v) reacting the particles with the copolymer layer formed with mercaptocarboxylic acid; process of letting

The magnetic particles for antibody binding according to one embodiment of the present invention have iron elution suppression and particle dispersibility in water, and also have a large amount of physiologically active substances such as antibodies supported, making them highly suitable for immunodiagnosis. Sensitization becomes possible.

FIG. 1 is a schematic diagram showing the structure of magnetic particles for antibody binding according to one embodiment of the present invention.

Hereinafter, a mode for carrying out the present invention (hereinafter simply referred to as "this embodiment") will be described in detail. The present embodiment below is an illustration for explaining the present invention, and is not intended to limit the present invention to the following content. The present invention can be implemented with appropriate modifications within the scope of its spirit.

Magnetic particles for antibody binding according to one aspect of the present invention include:
A magnetic particle having a core particle, a first coating layer formed on the surface of the core particle, and a second coating layer formed on the surface of the first coating layer,
The first coating layer is a crosslinked inorganic layer containing nanomagnetic material,
The second coating layer is a polymer layer having at least one hydrophilic group-containing structural unit selected from a hydroxyl group and a polyethylene glycol group, and forms a covalent bond with the first coating layer, and forms a covalent bond with the first coating layer. It has a carboxyl group at the end of the polymer chain on the opposite side from the bond.

The core particle used in the antibody-binding magnetic particle is a particle located at the center of the magnetic particle, and there are no particular limitations on the shape or material of the core particle. The shape of the core particles can be selected as appropriate, including spherical shapes that are close to true spheres, non-spherical shapes such as ellipsoids, cubes, cylinders, and polygonal columns, and particles that have irregularities with a large specific surface area such as porous shapes and those with protrusions. can be mentioned. Particles having irregularities with a large specific surface area, such as porous particles or those having protrusions, are preferable because they are suitable for coating the surface of the core particle with a large amount of nanomagnetic material.

In order to obtain magnetic particles with good magnetic responsiveness, the particle size of the core particles is preferably 0.1 μm or more, more preferably 0.5 μm or more, particularly preferably 1 μm or more, and most preferably 2 μm or more. Further, in order to obtain magnetic particles with good redispersibility in water, the particle size of the core particles is preferably 100 μm or less, more preferably 50 μm or less, particularly preferably 10 μm or less, and most preferably 5 μm or less.

Examples of the material of the core particles include organic compounds such as polymers, inorganic compounds, and the like. Although it can be appropriately selected from these, examples include polymer particles containing a styrene monomer unit, an acrylate monomer unit, or a methacrylate monomer unit, or silica particles.

Examples of the styrene monomers include styrene, α-methylstyrene, vinyltoluene, p-methylstyrene, 2-methylstyrene, 3-methylstyrene, 4-methylstyrene, 4-ethylstyrene, and 4-tert-butyl. Styrene, 3,4-dimethylstyrene, 4-methoxystyrene, 4-ethoxystyrene, 2-chlorostyrene, 3-chlorostyrene, 4-chlorostyrene, 2,4-dichlorostyrene, 2,6-dichlorostyrene, 4- Examples include chloro-3-methylstyrene, divinylbenzene, and sodium p-styrenesulfonate.

Examples of the acrylate monomer or methacrylate monomer include (meth)acrylate, methyl (meth)acrylate, ethyl (meth)acrylate, propyl (meth)acrylate, n-hexyl (meth)acrylate, 2 - (Cyclo)alkyl (meth)acrylates such as ethylhexyl (meth)acrylate and cyclohexyl (meth)acrylate; Alkoxy(cyclo)alkyl (meth)acrylates such as 2-methoxyethyl (meth)acrylate and p-methoxycyclohexyl (meth)acrylate; ) Acrylates; Polyvalent (meth)acrylates such as trimethylolpropane tri(meth)acrylate; Vinyl esters such as vinyl acetate, vinyl propionate, vinyl versatate; 2-cyanoethyl (meth)acrylate, 2-cyanopropyl Cyanoacrylates such as (meth)acrylate, 3-cyanopropyl (meth)acrylate; hydroxymethyl (meth)acrylate, 2-hydroxyethyl (meth)acrylate, 6-hydroxyhexyl (meth)acrylate, 4-hydroxycyclohexyl (meth)acrylate; ) acrylate, neopentyl glycol mono(meth)acrylate, 3-chloro-2-hydroxypropyl(meth)acrylate, 3-amino-2-hydroxypropyl(meth)acrylate; substituted hydroxy(meth)acrylates; glycidyl(meth)acrylate; ) acrylate, acrylates containing glycidyl groups such as methylglycidyl methyl acrylate, epoxidized cyclohexyl (meth)acrylate; trimethylolpropane ethoxy triacrylate, pentaerythritol ethoxytetraacrylate, trimethylolpropane propoxy triacrylate, pentaerythritol triacrylate, pentaerythritol Monomers containing (meth)acrylates such as polyfunctional (meth)acrylates such as tetraacrylate, dipentaerythritol hexaacrylate, polyethylene glycol diacrylate, polypropylene glycol diacrylate, tricyclodecane dimethanol diacrylate, and ethoxylated phenyl acrylate. Examples include polymer particles used as units. Furthermore, the acrylate may be crosslinked.

If necessary, the core particle may contain nanomagnetic material. Further, as a pretreatment for providing the first coating layer, the surface of the core particle may be coated with a substance having a positive charge or a negative charge. Further, a paramagnetic thin film coating layer may be provided on the surface of the core particle. When providing a paramagnetic coating layer, the thickness is preferably 0.001 to 1 μm, more preferably 0.001 to 0.1 μm, since it is suitable for reducing residual magnetization of particles and suppressing agglomeration of particles. , 0.001 to 0.01 μm is particularly preferred, and 0.001 to 0.005 μm is most preferred. There are no particular limitations on the method of coating the surface of the core particle with a substance having a positive or negative charge, but methods include forming a polymer layer on the surface of the core particle using a monomer having a charge on the side chain; Examples include a method in which the surface of the core particle is coated with a charged polymer by a layer-by-layer method.

The first coating layer formed on the surface of the core particle is a crosslinked inorganic layer containing nanomagnetic material. The nanomagnetic material is a particle having a smaller particle size than the core particle, and further has magnetic responsiveness. Since it is preferable to have superparamagnetism, the particle size of the nanomagnetic material is preferably 0.001 to 1 μm, more preferably 0.001 to 0.5 μm, particularly preferably 0.001 to 0.1 μm, Most preferably 0.001 to 0.05 μm. Examples of the material include iron oxide. The nanomagnetic material may contain an inorganic surface modifier such as a lipid or a silane coupling agent on the surface. Examples of the silane coupling agent include vinyltrimethoxysilane, vinyltriethoxysilane, 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane, 3-glycidoxypropyltrimethoxysilane, and p-styryltrimethoxysilane. Silane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltriethoxysilane, 3-acryloxypropyltrimethoxysilane, N- 2-(aminoethyl)-3-aminopropylmethyldimethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, tris-(trimethoxysilylpropyl)isocyanurate, 3-ureidopropyltrialkoxysilane, Examples include 3-mercaptopropylmethyldimethoxysilane, bis(triethoxysilylpropyl)tetrasulfide, and 3-isocyanatepropyltriethoxysilane.

The nanomagnetic material is preferably dispersed in the crosslinked inorganic layer. By dispersing the nanomagnetic material in the crosslinked inorganic layer, the surfaces of all the nanomagnetic materials can be uniformly coated with the crosslinked inorganic layer, and the acid resistance of the magnetic particles can be improved.

The content of nanomagnetic material is preferably 0.1 g/g or more, and 0.2 g as the content of nanomagnetic material per 1 g of magnetic particles, since it is suitable for forming magnetic particles with good magnetic response. /g or more is more preferable, 0.5 g/g or more is particularly preferable, and 0.7 g/g or more is most preferable. In addition, since it is suitable for increasing the dispersibility of magnetic particles in water, the content of nanomagnetic material per gram of magnetic particles is preferably 0.9 g/g or less, and more preferably 0.8 g/g or less. It is preferably 0.7 g/g or less, particularly preferably 0.6 g/g or less, and most preferably 0.6 g/g or less.

The crosslinked inorganic layer is formed by forming a crosslinked inorganic layer on the core particle. Examples of crosslinked inorganic substances include silica, glass, oxides such as magnesium oxide and alumina oxide, and ceramics such as zirconia and hydroxyapatite. Among these, silica is preferred because it is particularly suitable for increasing acid resistance. By providing a crosslinked inorganic layer, it is possible to increase acid resistance while being hydrophilic, and therefore it is possible to impart contradictory properties of suppressing elution of iron and dispersibility of particles in water. In addition, the crosslinked inorganic layer is coated along the shape of the magnetic material on the particle surface and does not completely bury the magnetic material, making it possible to obtain magnetic particles with a large specific surface area and reducing the amount of physiologically active substances such as antibodies carried. can be increased. The thickness of the crosslinked inorganic layer is preferably 0.001 μm or more, more preferably 0.005 μm or more, particularly preferably 0.01 μm or more, and most preferably 0.05 μm or more, since it is suitable for increasing acid resistance. . Further, since it is suitable for forming magnetic particles with a large specific surface area, the thickness of the crosslinked inorganic layer is preferably 1 μm or less, more preferably 0.5 μm or less, particularly preferably 0.1 μm or less, and most preferably 0.01 μm or less. .

The second coating layer formed on the surface of the first coating layer is a polymer layer having at least one hydrophilic group-containing structural unit selected from a hydroxyl group and a polyethylene glycol group; It forms a covalent bond with the layer and has a carboxyl group at the end of the polymer chain on the opposite side from the covalent bond.

Particles with a first coating layer formed on the surface of the core particles do not have functional groups for binding physiologically active substances such as antibodies to the particles, and therefore cannot support physiologically active substances, so crosslinking is not possible. By providing the second coating layer on the surface of the particles provided with the inorganic layer, it becomes possible to carry antibodies while suppressing non-specific adsorption, and it becomes possible to increase the sensitivity of immunodiagnosis. Carboxyl group-containing structural units alone result in high nonspecific adsorption, making it impossible to increase the sensitivity of immunodiagnosis. By having two structural units, a carboxyl group-containing structural unit and a hydrophilic group-containing structural unit, it becomes possible to increase the sensitivity of immunodiagnosis.

The second coating layer is formed by polymerizing a monomer having at least one hydrophilic group-containing structural unit selected from a hydroxyl group and a polyethylene glycol group. The monomer is preferably a hydroxyl group-containing acrylate or a hydroxyl group-containing methacrylate, or a polyethylene glycol group-containing acrylate or a polyethylene glycol group-containing methacrylate because the polymerization rate is fast and it is suitable for increasing the mass productivity of magnetic particles. . Moreover, polyethylene glycol group-containing acrylate or polyethylene glycol group-containing methacrylate is more preferable because it is particularly suitable for increasing the sensitivity of immunodiagnosis. Examples of the monomer having at least one hydrophilic group-containing structural unit selected from a hydroxyl group and a polyethylene glycol group include hydroxyethyl acrylate, hydroxyethyl methacrylate, N-(2-hydroxyethyl)acrylamide, and 2-hydroxyphenyl. Acrylate, 2-hydroxyphenyl methacrylate, 3-hydroxyphenyl acrylate, 3-hydroxyphenyl methacrylate, 4-hydroxyphenyl acrylate, 4-hydroxyphenyl methacrylate, polyethylene glycol monoacrylate, polyethylene glycol monomethacrylate, polypropylene glycol monoacrylate, methoxypolyethylene glycol Examples include monoacrylate, methoxypolyethylene glycol monomethacrylate, diethylene glycol monomethyl ether acrylate, diethylene glycol monomethyl ether methacrylate, diethylene glycol monoethyl ether acrylate, diethylene glycol monoethyl ether methacrylate, polyethylene glycol methyl ether acrylate, polyethylene glycol methyl ether methacrylate, and the like.

Further, the second coating layer may be formed by copolymerizing a monomer other than the monomer having a hydrophilic group-containing structural unit. For example, at least one hydrophobic group-containing structural unit selected from long chain and cyclic alkyl groups having 1 to 6 carbon atoms is suitable for imparting hydrophobicity for adsorbing or bonding antibodies to particle surfaces. It is preferable to have. This hydrophobic group-containing structural unit is a monomer having at least one hydrophilic group-containing structural unit selected from a hydroxyl group and a polyethylene glycol group, and a long chain and cyclic alkyl group having 1 to 6 carbon atoms. It can be introduced by copolymerizing a monomer having at least one hydrophobic group-containing structural unit. The monomer having a hydrophobic group-containing structural unit is preferably a hydrophobic group-containing acrylate or a hydrophobic group-containing methacrylate, such as methyl acrylate, methyl methacrylate, ethyl acrylate, ethyl methacrylate, propyl acrylate, propyl methacrylate, n- Butyl acrylate, n-butyl methacrylate, t-butyl acrylate, t-butyl methacrylate, n-pentanoyl acrylate, n-pentanoyl methacrylate, n-hexyltyl acrylate, n-hexyl methacrylate, cyclopropyl acrylate, cyclopropyl methacrylate, cyclohexyl Acrylate, cyclohexyl methacrylate, phenyl acrylate, phenyl methacrylate, etc. can be mentioned. Furthermore, examples of monomers other than the hydrophobic group-containing monomer include styrene.

Further, as other monomers, monomers having two or more polymerizable functional groups may be used in combination as a crosslinking agent. Examples of the crosslinking agent include divinylbenzene, ethylene glycol diacrylate, ethylene glycol dimethacrylate, trimethylolpropane triacrylate, trimethylolpropane trimethacrylate, pentaerythritol triacrylate, ditrimethylolpropane tetraacrylate, pentaerythritol tetraacrylate, and the like. can be mentioned. By using a crosslinking agent in combination, it is possible to form a crosslinked structure in which polymer chains are connected to each other at multiple bonding points in the second coating layer, thereby increasing the solvent resistance, heat resistance, and mechanical strength of the particles. can.

The second coating layer forms a covalent bond with the first coating layer and has a carboxyl group at the end of the polymer chain opposite to the covalent bond. The carboxyl group-containing structural unit is not particularly limited as long as it has a carboxyl group. As a method for forming a covalent bond with the first coating layer in the second coating layer, a silane coupling agent having an initiator or chain transfer agent for living radical polymerization is covalently bonded to the first coating layer. This can be done by forming the second coating layer by living radical polymerization after the reaction. Further, as a method for providing a carboxyl group at the end of the polymer chain opposite to the covalent bond, an organic reaction reagent that reacts with the initiator or chain transfer agent for living radical polymerization is used. This is possible by introducing a carboxyl group into the chain transfer agent.

The carboxyl group-containing structural unit is introduced into the polymer layer by reacting mercaptocarboxylic acid with particles forming a polymer layer having at least one hydrophilic group-containing structural unit selected from a hydroxyl group and a polyethylene glycol group. can do. A carboxyl group may be introduced onto the surface of the crosslinked inorganic layer. Examples of the mercaptocarboxylic acid include mercaptoacetic acid, mercaptopropionic acid, mercaptooxalic acid, mercaptosuccinic acid, mercaptomaleic acid, mercaptophthalic acid, mercaptobenzoic acid, and the like.

The second coating layer, a copolymer layer, is covalently bonded to the first coating layer. By being covalently bonded, acid resistance and solvent resistance can be improved. The presence of covalent bonds can be confirmed by the fact that the polymer does not disappear even after washing with a solvent that dissolves the polymer. If it is not covalently bonded, almost all of the coated polymer will dissolve.

By covalently bonding the second coating layer to the first coating layer, it is suitable for increasing the acid resistance and solvent resistance of the magnetic particles. It is preferable to have a silane coupling agent having (and residues). Examples of the silane coupling agent include vinyltrimethoxysilane, vinyltriethoxysilane, 2-(3,4-epoxycyclohexyl)ethyltrimethoxysilane, 3-glycidoxypropyltrimethoxysilane, and p-styryltrimethoxysilane. Silane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltriethoxysilane, 3-acryloxypropyltrimethoxysilane, etc. be able to.

There is no particular limitation on the amount of polymer coated in the second coating layer, but since it is suitable for producing magnetic particles with a large specific surface area, the amount of polymer coated per 1 g of magnetic particles is 1 g/g or less. is preferable, 0.5 g/g or less is more preferable, 0.1 g/g or less is particularly preferable, and 0.05 g/g or less is most preferable. In addition, since it is suitable for increasing acid resistance, the amount of polymer coating per gram of magnetic particles is preferably 0.001 g/g or more, more preferably 0.01 g/g or more, and 0.1 g/g or more. Particularly preferred, and most preferably 0.5 g/g or more. The amount of polymer coated can be measured by measuring the TG-DTA of the particles before and after coating with the polymer, and determining the difference in thermogravimetric loss before and after coating with the polymer. It can also be calculated by determining the thickness of the polymer from a scanning electron microscope image of a particle cross section or a transmission electron microscope image of the particles.

The amount of carboxyl groups in the magnetic particles is preferably 5 μmol/g or more, more preferably 10 μmol/g or more, since the magnetic particles are suitable for binding many antibodies. , 20 μmol/g or more is particularly preferable, and 50 μmol/g or more is most preferable. In addition, the amount of carboxyl groups per 1 g of magnetic particles is preferably 200 μmol/g or less because it is suitable for suppressing non-specific adsorption of substances other than antibodies to magnetic particles and measurement noise in immunodiagnosis. , more preferably 100 μmol/g or less, particularly preferably 50 μmol/g or less, and most preferably 20 μmol/g or less. Examples of methods for measuring the amount of carboxyl groups include a method of mixing pyrenyldiazomethane and magnetic particles and quantifying the amount of pyrenyldiazomethane that has disappeared through the reaction, or a method of directly quantifying the amount of carboxyl groups on the surface of the magnetic particles by potentiometric titration. I can do it.

The antibody-binding magnetic particles according to one aspect of the present invention can be obtained by a manufacturing method including the following steps (i) to (v).
(i) A step of physically adsorbing the nanomagnetic material on the surface of the core particle (ii) A step of forming a crosslinked inorganic layer on the surface of the particle to which the nanomagnetic material is adsorbed (iii) A step of physically adsorbing the nanomagnetic material on the surface of the core particle Step (iv) of bonding a silane coupling agent having a radical polymerization initiator or chain transfer agent: dispersing the particles bound with the silane coupling agent in a solvent, and dispersing the particles selected from hydroxyl groups and polyethylene glycol groups in the solvent. step (v) of forming a polymer layer on the surface of the particles by living radical polymerization of a monomer having at least one hydrophilic group-containing structural unit, (v) reacting the particles with the copolymer layer formed with mercaptocarboxylic acid; In the step (i), a method of contacting a nanomagnetic material having a charge opposite to the surface charge of the core particle, a method of contacting a hydrophobic nanomagnetic material with a hydrophobic core particle, or a method of contacting a hydrophobic nanomagnetic material with a core particle having paramagnetism. By bringing the nanomagnetic material into contact with the particles, the nanomagnetic material is physically adsorbed onto the surface of the core particle. There is no particular limitation on the method of bringing the core particles into contact with the nanomagnetic material, and it is possible to do so by stirring in a liquid phase or a gas phase.

In step (ii), a crosslinked inorganic layer is formed on the surface of the particles to which the nanomagnetic material was adsorbed in step (i). There is no particular limitation on the method of forming the crosslinked inorganic layer, but particles are dispersed in a solution containing a reagent and a reaction catalyst that are raw materials for the crosslinked inorganic layer, and the reaction is allowed to proceed to form a crosslinked inorganic layer on the surface of the particles. Examples include a method of coating the particle surface with the crosslinked inorganic layer or its raw material.

In step (iii), a silane coupling agent having a living radical polymerization initiator or chain transfer agent is bonded to the surface of the crosslinked inorganic layer formed in step (ii). There are no particular limitations on the method of bonding the silane coupling agent to the surface of the crosslinked inorganic layer, and methods include methods of dispersing particles in a solvent and adding the silane coupling agent, and methods of directly coating the particle surface with the silane coupling agent. Selectable. Furthermore, an acidic or basic aqueous solution serving as a reaction catalyst may be used in combination, if necessary.

In step (iv), the particles forming the crosslinked inorganic layer in step (ii) are dispersed in a solvent, and a monomer having at least one hydrophilic group-containing structural unit selected from a hydroxyl group and a polyethylene glycol group is added in the solvent. A polymer layer is formed on the surface of the particles by living radical polymerization. There are no particular limitations on the polymerization method other than living radical polymerization, and there are dispersion polymerization methods in which polymerization is performed with all components other than particles dissolved in a solvent, and monomers that are incompatible with the solvent are used to form particles. Examples include seed polymerization methods in which polymerization is carried out with monomers adsorbed on the polymer. Further, the seed polymerization method can be appropriately selected from soap-free polymerization that does not use an emulsifier and emulsion polymerization that uses an emulsifier. As the method of living radical polymerization, atom transfer radical polymerization (ATRP) or reversible addition-fragmentation chain transfer (RAFT) polymerization is preferred. In the case of atom transfer radical polymerization, a silane coupling agent having a halogen group such as a bromo group is bonded to particles, and polymerization is performed in the presence of a transition metal complex as a catalyst. In the case of reversible addition-fragmentation chain transfer polymerization, a silane coupling agent having a chain transfer agent such as a dithioester is bonded to particles, and polymerization is performed in the presence of a radical generator such as an az compound.

In step (iv), a monomer having at least one hydrophilic group-containing structural unit selected from a hydroxyl group and a polyethylene glycol group is subjected to living radical polymerization to form a polymer layer, and then step (v) ) may be reacted with mercaptocarboxylic acid to introduce a carboxyl group onto the particle surface. The reaction method between the particle surface and the carboxylic acid anhydride is not particularly limited, and can be achieved by dispersing the particles in a water-free solvent and adding an excess amount of mercaptocarboxylic acid.

The antibody-binding magnetic particles according to one embodiment of the present invention have antibodies immobilized on the surface of the particles, and can be used in immunodiagnosis devices. As for the binding method of the antibody, it is preferable to bind through the carboxyl group on the particle surface. For example, the carboxyl group is activated by reacting a condensing agent such as carbodiimide, and the antibody has the activated carboxyl group. Can be bonded by reacting with an amino group.

Hereinafter, the present invention will be described in detail by citing embodiments for carrying out the present invention, but these are merely examples for explaining the present invention, and are not intended to limit the present invention to the following contents. Further, the present invention can be modified and implemented as appropriate within the scope of the gist of the present invention. In addition, unless otherwise specified, commercially available reagents were used.
[Measurement of polymer coverage]
TG-DTA measurements were performed on the particles before and after coating with the polymer, and the thermogravimetric loss from 100°C to 560°C was determined. When the thermal weight loss in the particles before polymer coating is ΔTG1, and the thermal weight loss in the particles after polymer coating is ΔTG2, ΔTG2−ΔTG1 is defined as the polymer coating amount.
[Measurement of carboxyl group amount]
Add 2 mL of ethyl acetate to 10 mg of magnetic particles, and stir at room temperature for 20 minutes. 2 mL of ethyl acetate in which 0.12 mg of pyrenyldiazomethane was dissolved was added to this particle dispersion, and the mixture was stirred at room temperature for 50 minutes. After removing the particles, the supernatant liquid was diluted 10 times with methanol, the absorbance at 392 nm was measured, and this absorbance was designated as A1, and the absorbance of the solution when the same operation was performed without using particles was designated as A0. At that time, the amount of carboxyl groups was determined from the following formula.
138×(A0-A1)μmol/g
[Evaluation of water dispersibility]
0.1 g of dried powdered magnetic particles was added to 10 g of ion-exchanged water, and the mixture was shaken and stirred by hand for evaluation.
○: Uniformly dispersible in water ×: Not dispersible in water [Evaluation of iron ion elution]
Add 10 mg of magnetic particles to 1 mL of pH 3.5 citrate buffer and stir at 37°C for 17 hours. A solution obtained by removing particles from the solution was used as a measurement sample, and the amount of iron ions eluted was evaluated by measuring the absorbance of the solution at a wavelength of 460 nm.
[Preparation of diluted solid phase suspension and labeled antibody solution for detection]
An excess amount of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was added to the magnetic particles dispersed in a buffer solution (pH 6.0), and the mixture was incubated at 37°C for 1 hour. Anti-thyroid stimulating hormone (TSH) antibodies were added in a buffer solution (pH 3.5) and incubated at 37°C for 3 hours to bind the anti-TSH antibodies to the magnetic particles. Next, the magnetic particles were subjected to blocking treatment by incubation with a blocking agent solution for 17 hours to obtain anti-TSH antibody-immobilized magnetic particles (A). Furthermore, (A) was diluted with a buffer solution (pH 6.0) containing 8% collagen peptide to prepare a diluted solid phase suspension (B). Further, a conjugate of anti-TSH antibody and alkaline phosphatase was diluted with a buffer solution (pH 6.2) containing 5% bovine serum albumin to prepare a labeled antibody solution for detection (C).
[Preparation of samples for NSB (non-specific adsorption) and positive count evaluation]
A 0.03 mol/L Tris-HCl buffer (pH 8.0) containing 5% collagen peptide was prepared and used as a sample (D) for NSB (non-specific adsorption) evaluation. In addition, thyroid stimulating hormone (TSH) was added to 0.03 mol/L Tris-HCl buffer (pH 8.0) containing 5% collagen peptide to prepare a positive count evaluation sample (E).
[Measurement of NSB (non-specific adsorption) and positive count, evaluation of sensitivity]
Measurement of NSB and positive counts and evaluation of sensitivity were performed using measurement reagents (lyophilized product containing diluted solid phase suspension (B) and labeled antibody solution for detection (C)) NSB measurement was performed using the following procedure. Ta.

After dissolving the lyophilized diluted solid phase suspension (B) in pure water containing a surfactant and the NSB evaluation sample (D), immunoreaction was performed at 37°C for 5 minutes, and the first B/ F separation was performed. Next, a labeled antibody solution for detection (C) dissolved in pure water containing a surfactant was added, and an immunoreaction was performed at 37° C. for 3 minutes, followed by a second B/F separation. Thereafter, a chemiluminescent substrate for alkaline phosphatase (DIFURAT, 3-(5-tert-butyl-4,4-dimethyl-2,6,7-trioxabicyclo[3,2,0]hept-1-yl)phenylphosphorus Acid ester disodium salt) and chemiluminescence auxiliary reagent were added, and the luminescence intensity (count/sec (cps)) was measured and determined as NSB.

The positive count was measured using the following procedure.

After dissolving the lyophilized diluted solid phase suspension (B) in pure water containing a surfactant and a positive count evaluation sample (E), an immunoreaction was performed at 37°C for 5 minutes, and the first B /F separation was performed. Next, a labeled antibody solution for detection (C) dissolved in pure water containing a surfactant was added, and an immunoreaction was performed at 37° C. for 3 minutes, followed by a second B/F separation. Thereafter, a chemiluminescent substrate for alkaline phosphatase (DIFURAT, 3-(5-tert-butyl-4,4-dimethyl-2,6,7-trioxabicyclo[3,2,0]hept-1-yl)phenylphosphorus Acid ester disodium salt) and a chemiluminescence auxiliary reagent were added, and the luminescence intensity (count/sec (cps)) was measured as a positive count.

Sensitivity was evaluated by determining the ratio of positive counts/NSB.
[Example 1]
(Preparation of magnetized fine particles)
5.0 g of polydivinylbenzene particles (particle size 2.5 μm), 1.2 g of polyvinylpyrrolidone, and 6 mL of a 1N hydrochloric acid aqueous solution were dispersed in 160 mL of pure water at a rotation speed of 180 rpm. After raising the temperature to 80° C. in a nitrogen atmosphere, 6 g of urea, 2 g of iron (II) chloride tetrahydrate, and 3 g of iron (III) chloride hexahydrate were added and reacted for 5 hours. After this particle dispersion liquid was filtered to separate the particles and the reaction liquid, the particles were again dispersed in 200 mL of pure water at a rotation speed of 180 rpm. After raising the temperature to 80° C. under a nitrogen atmosphere, a 0.2N aqueous sodium hydroxide solution was added dropwise and the mixture was allowed to react for 15 hours. After washing with pure water, 5.75 g of particles coated with iron oxide were obtained.

1 g of the obtained iron oxide-coated particles was dispersed in 50 mL of pure water at room temperature and at a rotation speed of 100 rpm. To this particle dispersion, 1.0 g of a magnetic material having a cationic dispersant on the surface (material: magnetite, particle size: 10 nm, product name: EMG607, manufactured by Ferrotech) was added, and the mixture was heated at room temperature and at a rotation speed of 100 rpm. The reaction was allowed to proceed for 2 hours. By washing with pure water and further drying at 50° C. for 15 hours, 1.93 g of magnetized particles were obtained. The content of magnetic material per gram of magnetic particles was 0.32 g/g.
(Silica coating on magnetized particles)
1 g of the magnetized fine particles were dispersed in 400 mL of ethanol, 3 g of tetraethyl orthosilicate was added, and the mixture was stirred at room temperature at 300 rpm. 21 g of 25% ammonia water was added, and the mixture was reacted for 4 hours at 35° C. and a rotational speed of 300 rpm. It was washed with pure water and ethanol, and dried under reduced pressure. The thickness of the silica layer confirmed by a transmission electron microscope was about 20 nm.
(Introduction of ATRP initiator into silica-coated particles)
Disperse 1 g of silica-coated particles in 75 mL of methanol, add 0.5 g of 3-(trimethoxysilylpropyl)-2-bromo-2-methylpropionic acid and 15 g of 25% ammonia water, and rotate at 300 rpm at 60°C. The mixture was allowed to react for 3 hours. It was washed with methanol and pure water, and dried under reduced pressure.
(Polymer coating on ATRP initiator-introduced particles)
Disperse 0.1 g of the ATRP initiator-introduced particles in 18 mL of methanol, add 0.4 g of a mixture of 2-hydroxyethyl methacrylate:trimethylolpropane triacrylate = 60:40, 0.0022 g of copper(II) bromide, and ascorbin. After adding 0.02 g of sodium chloride and 0.015 g of 2,2'-bipyridyl and degassing, the mixture was reacted at 40° C. for 3 hours under a nitrogen atmosphere. It was washed with pure water and methanol and dried under reduced pressure.
(Introduction of carboxyl group)
0.1 g of the polymer-coated particles were dispersed in 20 mL of a mixed solvent of pure water: methanol = 50:50, 0.074 g of lithium carbonate and 0.092 g of thioglycolic acid were added, and the mixture was reacted at 37° C. for 12 hours. Ta. It was washed with pure water and methanol and dried under reduced pressure.
(Evaluation results)
Polymer coverage was 0.090 g/g. The amount of carboxyl groups was 32 μmol/g.

The prepared magnetic particles had excellent water dispersibility and little elution of iron ions. Further, the NSB was 67, the positive count was 953,269, and the sensitivity was 14,228, indicating excellent sensitivity.
[Example 2]
Particles were produced in the same manner as in Example 1 except that trimethylolpropane trimethacrylate was used instead of trimethylolpropane triacrylate as the monomer for the polymer coating in Example 1.
(Evaluation results)
Polymer coverage was 0.106 g/g. The amount of carboxyl groups was 30 μmol/g.

The prepared magnetic particles had excellent water dispersibility and little elution of iron ions. Further, the NSB was 59, the positive count was 1018909, and the sensitivity was 17270, indicating excellent sensitivity.
[Example 3]
Particles were produced in the same manner as in Example 1 except that polyethylene glycol methyl ether acrylate (Mn=300) was used instead of 2-hydroxyethyl methacrylate as the monomer for the polymer coating in Example 1.
(Evaluation results)
Polymer coverage was 0.096 g/g. The amount of carboxyl groups was 29 μmol/g.

The prepared magnetic particles had excellent water dispersibility and little elution of iron ions. Further, the NSB was 48, the positive count was 959,202, and the sensitivity was 19,983, indicating excellent sensitivity.
[Example 4]
As the monomer for the polymer coating in Example 1, polyethylene glycol methyl ether acrylate (Mn=300) was used instead of 2-hydroxyethyl methacrylate, and trimethylolpropane trimethacrylate was used instead of trimethylolpropane triacrylate. Particles were produced in the same manner as in Example 1 except for the following.
(Evaluation results)
Polymer coverage was 0.102 g/g. The amount of carboxyl groups was 34 μmol/g.

The prepared magnetic particles had excellent water dispersibility and little elution of iron ions. Further, the NSB was 66, the positive count was 1273013, and the sensitivity was 19288, indicating excellent sensitivity.
[Comparative example 1]
Particles were produced in the same manner as in Example 1 except that all of the silica coating step, ATRP initiator introduction step, polymer coating step, and carboxylic acid introduction step in Example 1 were omitted.
(Evaluation results)
The produced magnetic particles had poor water dispersibility and a large amount of iron ions were eluted.
[Comparative example 2]
All of the silica coating step, ATRP initiator introduction step, polymer coating step, and carboxylic acid introduction step in Example 1 were not performed, and instead, the magnetized particles were modified with an organic silane coupling agent by the following method. . Particles were produced in the same manner as in Example 1 in other respects.
(Modification of magnetized particles with organosilane coupling agent)
1 g of the magnetized fine particles was dispersed in 75 mL of methanol. 0.5 g of 3-methacryloxypropyltrimethoxysilane and 15 g of 25% aqueous ammonia were added, and the mixture was reacted at 60° C. and a rotational speed of 300 rpm for 3 hours. It was washed with methanol and pure water, and dried under reduced pressure.
(Evaluation results)
The produced magnetic particles had poor dispersibility in water, and a large amount of iron ions were eluted.
[Comparative example 3]
The silica coating step, the ATRP initiator introduction step, and the carboxylic acid introduction step in Example 1 were all omitted, and instead, the magnetized particles were coated with the polymer in the following manner. Particles were produced in the same manner as in Example 1 in other respects.
(Polymer coating on magnetized particles)
0.1 g of the magnetized particles were dispersed in 18 mL of pure water, 4 g of methyl methacrylate and 0.02 g of potassium persulfate were added, and after degassing, the mixture was reacted at 65° C. for 3 hours under a nitrogen atmosphere. Ta. It was washed with pure water and methanol and dried under reduced pressure.
(Evaluation results)
The prepared particles had poor dispersibility in water. The specific surface area was 1.8 m ² /g, which was small.
[Comparative example 4]
The silica coating step, the ATRP initiator introduction step, and the carboxylic acid introduction step in Example 1 were all omitted, and instead, the magnetized particles were coated with the polymer in the following manner. Particles were produced in the same manner as in Example 1 in other respects.
(Polymer coating on magnetized particles)
0.1 g of the magnetized particles were dispersed in 18 mL of pure water, 4 g of 2-hydroxyethyl methacrylate and 0.02 g of potassium persulfate were added, and after degassing, the mixture was heated at 65° C. for 3 hours under a nitrogen atmosphere. Made it react. It was washed with pure water and methanol and dried under reduced pressure.
(Evaluation results)
The prepared particles had a large amount of iron ions eluted.
[Comparative example 5]
Particles were produced in the same manner as in Example 1 except that the carboxylic acid introduction step in Example 1 was not performed.
(Evaluation results)
The produced magnetic particles had an NSB of 66, a positive count of 477,882, and a sensitivity of 7241, which were poor in sensitivity.

1 Core particle 2 Nanomagnetic material 3 Crosslinked inorganic layer 4 Polymer layer having a hydrophilic group-containing structural unit

Claims (4)

A magnetic particle having a core particle, a first coating layer formed on the surface of the core particle, and a second coating layer formed on the surface of the first coating layer,
The first coating layer is a crosslinked inorganic layer containing nanomagnetic material,
The second coating layer is a polymer layer having at least one hydrophilic group-containing structural unit selected from a hydroxyl group and a polyethylene glycol group, and forms a covalent bond with the first coating layer, and forms a covalent bond with the first coating layer. Magnetic particles for antibody binding characterized by having a carboxyl group at the end of the polymer chain on the opposite side from the binding. 2. The antibody-binding magnetic particle according to claim 1, wherein the crosslinked inorganic layer of the first coating layer contains silica. 3. The antibody-binding magnetic particle according to claim 1, wherein the polymer layer that is the second coating layer has a crosslinked structure. The method for producing antibody-binding magnetic particles according to any one of claims 1 to 3, which comprises the following steps (i) to (v).
(i) A step of physically adsorbing the nanomagnetic material on the surface of the core particle (ii) A step of forming a crosslinked inorganic layer on the surface of the particle to which the nanomagnetic material is adsorbed (iii) A step of physically adsorbing the nanomagnetic material on the surface of the core particle Step (iv) of bonding a silane coupling agent having a radical polymerization initiator or chain transfer agent: dispersing the particles bound with the silane coupling agent in a solvent, and dispersing the particles selected from hydroxyl groups and polyethylene glycol groups in the solvent. step (v) of forming a polymer layer on the surface of the particles by living radical polymerization of a monomer having at least one hydrophilic group-containing structural unit, (v) reacting the particles with the copolymer layer formed with mercaptocarboxylic acid; process of letting