阅读说明：本技术 磁性粒子以及检测试剂 (Magnetic particle and detection reagent ) 是由 大日方秀平 胁屋武司 稻叶祐也 于 2020-03-26 设计创作，主要内容包括：本发明提供一种能够提高集磁性,并且能够保持较高集磁性的磁性粒子。本发明涉及的磁性粒子是用于与目标物质进行特异性相互作用的磁性粒子,所述磁性粒子具备：树脂粒子、配置于所述树脂粒子的外表面上并且包含与铁氧体不同的磁性体的磁性层、以及担载于所述磁性层的外表面侧并且与所述目标物质进行特异性相互作用的物质,所述磁性层为连续层。(The invention provides a magnetic particle capable of improving magnetism collection and keeping high magnetism collection. The magnetic particle according to the present invention is a magnetic particle for specifically interacting with a target substance, and includes: the magnetic particle comprises a resin particle, a magnetic layer which is arranged on the outer surface of the resin particle and contains a magnetic body different from ferrite, and a substance which is carried on the outer surface side of the magnetic layer and specifically interacts with the target substance, wherein the magnetic layer is a continuous layer.)

1. A magnetic particle for specifically interacting with a target substance, the magnetic particle comprising:

resin particles,

A magnetic layer disposed on an outer surface of the resin particle and containing a magnetic material different from ferrite, and

a substance that is carried on the outer surface side of the magnetic layer and that specifically interacts with the target substance,

the magnetic layer is a continuous layer.

2. The magnetic particle according to claim 1,

the magnetic body is metal.

3. The magnetic particle according to claim 1 or 2, comprising:

a shell layer disposed on an outer surface of the magnetic layer,

the shell layer is made of inorganic oxide, organic polymer or nonmagnetic metal,

the shell layer binds to the substance.

4. The magnetic particle according to claim 3,

the shell layer includes a1 st shell layer disposed on an outer surface of the magnetic layer and a 2 nd shell layer disposed on an outer surface of the 1 st shell layer,

the material of the 1 st shell layer is inorganic oxide or nonmagnetic metal,

the material of the 2 nd shell layer is inorganic oxide or organic polymer,

the 2 nd shell binds to the substance.

5. The magnetic particle according to claim 4,

the inorganic oxide or the nonmagnetic metal in the material of the 1 st shell layer includes: copper, silver, gold, palladium-silver alloy, platinum-silver alloy, indium-silver alloy, tin-silver alloy, bismuth-silver alloy, tungsten-silver alloy, molybdenum-silver alloy, ruthenium-silver alloy, rhodium-silver alloy, iridium-silver alloy, phosphorus or boron.

6. The magnetic particle according to claim 4 or 5,

the material of the 2 nd shell layer is the inorganic oxide,

the inorganic oxide is an inorganic oxide having a silicon atom, a germanium atom, a titanium atom or a zirconium atom.

7. The magnetic particle according to any of claims 1 to 6,

the substance is an antigen or an antibody.

8. The magnetic particle according to any of claims 1 to 6,

the substance is avidin or streptavidin.

9. The magnetic particle according to any of claims 1 to 8,

the content of the magnetic material is 2 vol% or more and 60 vol% or less in 100 vol% of the magnetic particles.

10. Magnetic particles according to any one of claims 1 to 9 for use as a detection reagent.

11. A detection reagent comprising the magnetic particle of any one of claims 1 to 9.

Technical Field

The present invention relates to magnetic particles containing a magnetic body. The present invention also relates to a detection reagent using the magnetic particle.

Background

In the fields of research and development of pharmaceuticals, clinical examinations, and the like, magnetic particles are used to measure the concentration of a target substance in a sample. For example, magnetic particles having an antibody, an antigen, or the like on the surface thereof are widely used in immunoassays such as chemiluminescence immunoassay (CLIA method). The magnetic particles are generally bound to an antigen or an antibody as a target substance, and then are magnetically collected by a magnet or the like.

Patent document 1 discloses magnetic polymer particles in which a plurality of magnetic fine particles are aggregated on the outer periphery of polymer particles, and a polymer coating film is further formed on the outer periphery of the polymer particles, wherein the mass ratio of the magnetic fine particles in the magnetic polymer particles is 20 to 70% by mass. The magnetic polymer particles can be used as a detection reagent.

Patent document 2 discloses a metal-containing resin particle for immunoassay having a normal magnetism, which has a core particle having an agglomerate of fine particles of a metal and/or a metal oxide formed on the surface thereof and an organic polymer resin layer formed on the outermost surface thereof, wherein the resin particle has an average particle diameter of 0.05 to 0.5 μm and a CV value of the particle diameter of 10% or less.

Documents of the prior art

Patent document

Patent document 1: japanese patent laid-open publication No. 2003-282310

Patent document 2: japanese laid-open patent publication No. 2005-098974

Disclosure of Invention

Technical problem to be solved by the invention

Conventionally, as described in patent documents 1 and 2, magnetic particles having a magnetic layer formed by aggregating magnetic fine particles and forming a layer have been generally used. However, in the magnetic layer formed by aggregating fine particles of a magnetic substance and forming the layer, the surface area of the magnetic layer is increased, and oxygen easily reaches the inside of the magnetic layer, so that the magnetic substance is easily oxidized. If the magnetic substance in the magnetic layer is oxidized, the magnetism collection property is lowered.

When magnetic particles having low magnetic concentration are used, the measurement accuracy and measurement sensitivity may be deteriorated when the concentration of a target substance such as an antigen or an antibody is measured.

The invention aims to provide a magnetic particle which can improve magnetism collection and can keep higher magnetism collection. It is also a limiting object of the present invention to provide magnetic particles that can maintain a high magnetic concentration for a long period of time. In addition, the present invention aims to provide a detection reagent using the magnetic particle.

Means for solving the problems

According to a broad aspect of the present invention, there is provided a magnetic particle for specific interaction with a target substance, the magnetic particle comprising: the magnetic particle comprises a resin particle, a magnetic layer which is arranged on the outer surface of the resin particle and contains a magnetic body different from ferrite, and a substance which is carried on the outer surface side of the magnetic layer and specifically interacts with the target substance, wherein the magnetic layer is a continuous layer.

In a specific embodiment of the magnetic particle according to the present invention, the magnetic material is a metal.

In one specific aspect of the magnetic particle according to the present invention, the magnetic particle includes a shell layer disposed on an outer surface of the magnetic layer, the shell layer is made of an inorganic oxide, an organic polymer, or a nonmagnetic metal, and the shell layer is bonded to the substance.

In one specific aspect of the magnetic particle according to the present invention, the shell layer includes a1 st shell layer disposed on an outer surface of the magnetic layer and a 2 nd shell layer disposed on an outer surface of the 1 st shell layer, a material of the 1 st shell layer is an inorganic oxide or a nonmagnetic metal, a material of the 2 nd shell layer is an inorganic oxide or an organic polymer, and the 2 nd shell layer is bonded to the substance.

In a specific embodiment of the magnetic particle according to the present invention, the inorganic oxide or the nonmagnetic metal in the material of the 1 st shell layer includes: copper, silver, gold, palladium-silver alloy, platinum-silver alloy, indium-silver alloy, tin-silver alloy, bismuth-silver alloy, tungsten-silver alloy, molybdenum-silver alloy, ruthenium-silver alloy, rhodium-silver alloy, iridium-silver alloy, phosphorus or boron.

In a specific embodiment of the magnetic particle according to the present invention, the material of the 2 nd shell is the inorganic oxide, and the inorganic oxide is an inorganic oxide having a silicon atom, a germanium atom, a titanium atom, or a zirconium atom.

In a specific embodiment of the magnetic particle of the present invention, the substance is an antigen or an antibody.

In a specific embodiment of the magnetic particle according to the present invention, the substance is avidin or streptavidin.

In one specific embodiment of the magnetic particle according to the present invention, the content of the magnetic material is 2 vol% or more and 60 vol% or less in 100 vol% of the magnetic particle.

In a specific embodiment of the magnetic particle according to the present invention, the magnetic particle is used as a detection reagent.

According to a broad aspect of the present invention, there is provided a detection reagent comprising the above-described magnetic particles.

ADVANTAGEOUS EFFECTS OF INVENTION

The present invention relates to magnetic particles for specific interaction with a target substance. The magnetic particle according to the present invention includes: the magnetic layer is a continuous layer and is supported on the outer surface side of the magnetic layer and specifically interacts with the target substance. The magnetic particle according to the present invention has the above-described configuration, and therefore can improve magnetic collection properties and maintain high magnetic collection properties.

Drawings

Fig. 1 is a cross-sectional view schematically showing a magnetic particle according to embodiment 1 of the present invention.

Fig. 2 is a cross-sectional view schematically showing magnetic particles according to embodiment 2 of the present invention.

Detailed description of the invention

The present invention will be described in detail below.

(magnetic particle)

The present invention relates to magnetic particles for specific interaction with a target substance. The magnetic particles according to the present invention are those that can specifically interact with a target substance. The magnetic particle according to the present invention includes: the magnetic layer is a continuous layer and is supported on the outer surface side of the magnetic layer and specifically interacts with the target substance.

The magnetic particle according to the present invention has the above-described configuration, and therefore can improve magnetic collection properties and maintain high magnetic collection properties. The invention can keep higher magnetism collection for a long time. The magnetic particle according to the present invention can suppress deterioration of magnetism collection with time. In addition, the magnetic particles according to the present invention can improve the dispersibility of the magnetic particles. In addition, the magnetic particles according to the present invention can increase the amount of binding to a target substance in measurement of the target substance using the magnetic particles, and therefore can improve measurement accuracy and measurement sensitivity.

In the present invention, a magnetic material having high saturation magnetization can be used as a magnetic material different from ferrite, and the magnetic collection property can be further improved. Further, by using a magnetic material having high saturation magnetization, the content of the magnetic material can be reduced, and the specific gravity of the magnetic particles can be reduced. Therefore, the dispersibility of the magnetic particles can be further improved.

The average particle diameter of the magnetic particles is preferably 0.5 μm or more, more preferably 1 μm or more, preferably 10 μm or less, more preferably 7 μm or less, further preferably 5 μm or less, particularly preferably 4.5 μm or less, and most preferably 4 μm or less. If the average particle diameter of the magnetic particles is not less than the lower limit, the magnetic collection property can be further improved. If the average particle diameter of the magnetic particles is not more than the upper limit, the content of the substance that specifically interacts with the target substance per unit weight can be increased, and the amount of the target substance bound can be increased.

The average particle diameter of the magnetic particles is a number average particle diameter. The particle size of the magnetic particles can be determined, for example, by observing arbitrary 50 magnetic particles with an electron microscope or an optical microscope and calculating the average value of the particle sizes of the respective magnetic particles. It is preferable to prepare a sample obtained by drying the magnetic particles and observe the obtained sample with an electron microscope or an optical microscope.

The coefficient of variation (CV value) of the average particle diameter of the magnetic particles is preferably 10% or less, and more preferably 5% or less. When the coefficient of variation of the average particle diameter of the magnetic particles is equal to or less than the upper limit, the measurement accuracy can be further improved in the measurement of the target substance using the magnetic particles.

The coefficient of variation (CV value) can be determined as follows.

CV value (%) - (ρ/Dn) × 100

ρ: standard deviation of particle size of magnetic particles

Dn: average value of particle diameter of magnetic particles

The present invention will be described in detail below with reference to the accompanying drawings.

Fig. 1 is a cross-sectional view schematically showing a magnetic particle according to embodiment 1 of the present invention.

The magnetic particles 1 shown in fig. 1 are used for interacting with a target substance. The magnetic particle 1 includes: resin particle 2, magnetic layer 3, 1 st shell layer 41, 2 nd shell layer 42, and substance 5 that interacts with the target substance. The substance 5 is, for example, a physiologically active substance such as avidin, streptavidin, an antigen, or an antibody.

The magnetic layer 3 is a magnetic layer containing a magnetic substance different from ferrite. The magnetic layer 3 is a continuous layer. The magnetic layer 3 is, for example, a layer different from a layer formed by aggregating fine particles of a magnetic substance and forming the layer.

The magnetic layer 3 is disposed on the outer surface of the resin particle 2. The 1 st shell layer 41 is disposed on the outer surface of the magnetic layer 3. The 2 nd shell layer 42 is disposed on the outer surface of the 1 st shell layer 41. The substance 5 is carried on the outer surface side of the magnetic layer 3. Substance 5 is carried on the outer surface of second shell layer 42. The 2 nd shell 42 is combined with the substance 5. The substance 5 is present on the surface of the magnetic particles 1.

The magnetic layer 3 is a single-layer magnetic layer. The magnetic layer 3 covers the entire outer surface of the resin particle 2. In the magnetic particle, the magnetic layer may cover the entire outer surface of the resin particle, or the magnetic layer may cover a part of the surface of the resin particle. In the magnetic particle, the magnetic layer may be a single-layer magnetic layer or a multilayer magnetic layer including 2 or more layers.

The 1 st shell layer 41 is a single-layer shell layer. The 1 st shell layer 41 covers the entire outer surface of the magnetic layer 3. In the magnetic particle, the 1 st shell layer may cover the entire outer surface of the magnetic layer, and the 1 st shell layer may cover a part of the surface of the magnetic layer.

The 2 nd shell 42 is a single-layer shell. The 2 nd shell layer 42 covers the entire outer surface of the magnetic layer 3. In the magnetic particle, the 2 nd shell layer may cover the entire outer surface of the 1 st shell layer, and the 2 nd shell layer may cover a part of the surface of the 1 st shell layer.

Fig. 2 is a cross-sectional view schematically showing a magnetic particle according to embodiment 2 of the present invention.

The magnetic particles 1A shown in fig. 2 are used for interaction with a target substance. The magnetic particles 1A include: resin particles 2, magnetic layer 3, shell layer 4, and substance 5 that interacts with the target substance. The substance 5 is, for example, avidin, streptavidin, an antigen, an antibody, or the like.

The magnetic layer 3 is a magnetic layer containing a magnetic substance different from ferrite. The magnetic layer 3 is a continuous layer. The magnetic layer 3 is, for example, a layer different from a layer formed by aggregating fine particles of a magnetic substance and forming the layer.

The magnetic layer 3 is disposed on the outer surface of the resin particle 2. The shell layer 4 is disposed on the outer surface of the magnetic layer 3. On the outer surface side of the magnetic layer 3, a substance 5 is carried. The substance 5 is carried on the outer surface of the shell layer 4. Shell 4 binds to substance 5. The substance 5 is present at the surface of the magnetic particles 1.

The magnetic layer 3 is a single-layer magnetic layer. The magnetic layer 3 covers the entire outer surface of the resin particle 2. In the magnetic particle, the magnetic layer may cover the entire outer surface of the resin particle, or the magnetic layer may cover a part of the surface of the resin particle. In the magnetic particle, the magnetic layer may be a single-layer magnetic layer or a multi-layer magnetic layer including 2 or more layers.

The shell layer 4 is a single-layer shell layer. The shell layer 4 covers the entire outer surface of the magnetic layer 3. In the magnetic particle, the shell layer may cover the entire outer surface of the magnetic layer, or may cover a part of the surface of the magnetic layer.

The magnetic particles to which the present invention relates may be such that: the magnetic material includes the magnetic layer including a magnetic material different from ferrite as a1 st magnetic layer, a non-magnetic layer on an outer surface of the 1 st magnetic layer, and a 2 nd magnetic layer including a magnetic material on an outer surface of the non-magnetic layer. The nonmagnetic layer is a layer formed of a nonmagnetic substance. Examples of the nonmagnetic layer include: organic polymer layers, inorganic oxide layers, and nonmagnetic metal layers. In this case, the 1 st magnetic layer is preferably a continuous layer, and the 2 nd magnetic layer is preferably a continuous layer. In this case, even if the 2 nd magnetic layer is oxidized to some extent, the 1 st magnetic layer located inside the 2 nd magnetic layer in the magnetic particles is less likely to be further oxidized, and therefore, the decrease in magnetic concentration with time can be more effectively suppressed, and a high magnetic concentration can be maintained.

Furthermore, the magnetic particles to which the present invention relates may be such that: the magnetic particle comprises a1 st magnetic layer including a magnetic body different from ferrite, wherein 2 or more nonmagnetic layers and 2 or more magnetic layers are arranged on an outer surface of the 1 st magnetic layer, and the nonmagnetic layers and the magnetic layers are alternately arranged on the outer surface of the 1 st magnetic layer in a direction from a center of the magnetic particle to an outer surface. The nonmagnetic layer is a layer formed of a nonmagnetic substance. Examples of the nonmagnetic layer include: organic polymer layers, inorganic oxide layers, and nonmagnetic metal layers. In this case, the 1 st magnetic layer is preferably a continuous layer, and the magnetic layers of 2 or more layers are preferably continuous layers. By providing the plurality of magnetic layers, the proportion of the oxidized magnetic layer can be reduced, and the decrease of the magnetism collection property with the lapse of time can be further effectively suppressed, and a high magnetism collection property can be maintained.

Further details of the magnetic particles will be explained below.

(resin particles)

Examples of the material of the resin particles include: polyolefin resins such as polyethylene, polypropylene, polystyrene, polyvinyl chloride, polyvinylidene chloride, polyisobutylene, and polybutadiene; acrylic resins such as polymethyl methacrylate and polymethyl acrylate; polycarbonate, polyamide, phenol-formaldehyde resin, melamine-formaldehyde resin, benzoguanamine-formaldehyde resin, urea-formaldehyde resin, phenol-formaldehyde resin, melamine resin, benzoguanamine resin, urea-formaldehyde resin, epoxy resin, unsaturated polyester resin, saturated polyester resin, polyethylene terephthalate, polysulfone, polyphenylene oxide, polyacetal, polyimide, polyamideimide, polyether ether ketone, polyether sulfone, divinylbenzene polymer, divinylbenzene copolymer, and the like. Examples of the divinylbenzene copolymer include: divinylbenzene-styrene copolymers and divinylbenzene- (meth) acrylate copolymers. The resin particles may be made of only one kind of material, or 2 or more kinds of materials may be used in combination.

The average particle diameter of the resin particles is preferably 0.5 μm or more, more preferably 1 μm or more, preferably 10 μm or less, more preferably 7 μm or less, further preferably 5 μm or less, particularly preferably 4.5 μm or less, and most preferably 4 μm or less. When the average particle diameter of the resin particles is not less than the lower limit, the magnetic collection property can be further improved. If the average particle diameter of the resin particles is not more than the upper limit, sedimentation of the magnetic particles can be effectively suppressed, and dispersibility can be further improved, and the particle diameter of the magnetic particles can be reduced, so that the resin particles can effectively interact with a target substance.

The average particle diameter of the resin particles is a number average particle diameter. The average particle diameter of the resin particles is determined by observing 50 arbitrary resin particles with an electron microscope or an optical microscope and calculating the average particle diameter of each resin particle. It is preferable to prepare a sample obtained by drying the magnetic particles or the resin particles and observe the obtained sample with an electron microscope or an optical microscope.

The resin particles may or may not have a porous structure. The resin particle may have a porous structure in a surface layer portion and a non-porous structure in a central portion. The magnetic layer can be made to reach the inside of the resin particle by making the surface layer portion of the resin particle have a porous structure.

(magnetic layer)

The magnetic particles according to the present invention include a magnetic layer containing a magnetic material different from ferrite. The magnetic layer containing a magnetic body is disposed on the outer surface of the resin particle.

The magnetic layer is a continuous layer. The magnetic layer may cover the entire outer surface of the resin particle, or may cover a part thereof. The continuous layer may cover the entire outer surface of the resin particle, or may cover a part of the outer surface. The magnetic layer is, for example, a layer different from a layer (aggregate layer) in which fine particles of a magnetic material are aggregated and formed into a layer. The continuous layer is, for example, a layer having a structure with few or no seams, which is different from a shape in which numerous seams (for example, 1000 or more magnetic particles per 1) are present on a magnetic layer, such as an aggregate of fine particles. The magnetic layer is preferably a plated layer. The magnetic layer may also have an island-in-sea structure.

The magnetic body is a different magnetic body from ferrite. The ferrite may be maghemite (γ Fe)₂O₃) And with MFe₂O₄The compound (MFe)₂O₄In the formula, M is Co, Ni, Mn, Zn, Mg, Cu, Fe, Li_0.5Fe_0.5Etc.) and so on. The magnetic body is preferably a metal, and more preferably a ferromagnetic body.

Examples of the magnetic material include: cobalt, nickel, iron, holmium, dysprosium, terbium and the like. The magnetic body may be an alloy. Examples of the alloy include: nickel-cobalt alloys, cobalt-tungsten alloys, iron-platinum alloys, iron-cobalt alloys, samarium-cobalt alloys, aluminum-nickel-cobalt alloys, and neodymium-iron-boron alloys, and the like. Further, the metal may be a metal ion. The magnetic substance may be used alone, or 2 or more kinds may be used in combination.

The magnetic material may be a magnetic material containing no iron atom.

From the viewpoints of further improving the magnetic concentration, further effectively suppressing the decrease in the magnetic concentration with the passage of time, and further maintaining a higher magnetic concentration for a long period of time, the magnetic body is preferably a nickel-cobalt alloy, cobalt, or nickel, more preferably a nickel-cobalt alloy.

The content of the magnetic substance in 100 vol% of the magnetic particles is preferably 1 vol% or more, more preferably 2 vol% or more, preferably 70 vol% or less, and more preferably 60 vol% or less. When the content of the magnetic substance is not less than the lower limit, the magnetism collection property can be further improved. When the content of the magnetic substance is not more than the upper limit, the dispersibility can be further improved.

The surface area of the outer surface of the resin particle, which is coated with the magnetic layer, is preferably 90% or more, more preferably 95% or more, even more preferably 99% or more, and most preferably 100% of the total surface area of the outer surface of the resin particle. When the surface area is not less than the lower limit, the magnetic collection property can be further improved, the decrease of the magnetic collection property with time can be further effectively suppressed, and the high magnetic collection property can be further maintained for a long period of time.

The thickness of the magnetic layer is preferably 50nm or more, more preferably 100nm or more, preferably 500nm or less, and more preferably 300nm or less. The thickness of the magnetic layer is the thickness of the entire magnetic layer when the magnetic layer is a plurality of layers. If the thickness of the magnetic layer is equal to or greater than the lower limit, the magnetic concentration can be further increased, the decrease in magnetic concentration with time can be further effectively suppressed, and the high magnetic concentration can be further maintained for a long period of time. If the thickness of the magnetic layer is equal to or less than the upper limit, sedimentation of the magnetic particles can be effectively suppressed, dispersibility can be further improved, and the particle diameter of the magnetic particles can be reduced, so that the amount of the target substance bonded per unit weight of the magnetic particles can be increased.

The thickness of the magnetic layer can be measured, for example, by observing the cross section of the magnetic particle using a Transmission Electron Microscope (TEM). The thickness of the magnetic layer is preferably calculated as the thickness of the magnetic layer of 1 magnetic particle from the average thickness of the magnetic layer at any 5 locations, and more preferably calculated as the thickness of the magnetic layer of 1 magnetic particle from the average thickness of the entire magnetic layer. The thickness of the magnetic layer is preferably determined by calculating an average value of the thicknesses of the magnetic layers of the respective magnetic particles for any 10 magnetic particles.

Examples of a method for forming a magnetic layer on the outer surface of the resin particle include: and a method of plating the surface of the resin particle with a metal ion that is reduced to a magnetic substance.

(Shell layer)

The magnetic particle according to the present invention preferably has a shell layer. The material of the shell layer comprises: inorganic oxides, organic polymers or non-magnetic metals. The shell layer is an inorganic oxide shell layer containing the inorganic oxide as a material of the shell layer, or an organic polymer shell layer containing the organic polymer as a material of the shell layer, or a metal layer containing the nonmagnetic metal as a material of the shell layer. The material of the shell layer is preferably inorganic oxide, organic polymer or nonmagnetic metal. The shell layer is preferably disposed on an outer surface of the magnetic layer. The magnetic particle having the shell layer can favorably arrange the substance that specifically interacts with the target substance on the surface of the magnetic particle, and can improve the dispersibility of the magnetic particle.

The shell layer may or may not contain a magnetic material. The shell layer more preferably contains no magnetic substance.

The shell layer preferably has: a1 st shell layer disposed on an outer surface of the magnetic layer, and a 2 nd shell layer disposed on an outer surface of the 1 st shell layer. In this case, it is preferable that the material of the 1 st shell layer is an inorganic oxide or a non-magnetic metal, the material of the 2 nd shell layer is an inorganic oxide or an organic polymer, and the 2 nd shell layer is bonded to the substance that specifically interacts with the target substance.

The inorganic oxide in the 1 st shell is preferably different from the inorganic oxide in the 2 nd shell.

From the viewpoint of further improving the dispersibility of the magnetic particles, the material of the shell layer or the material of the 2 nd shell layer is preferably the inorganic oxide, and is preferably an inorganic oxide shell layer.

< first Shell layer >

The 1 st shell layer is a layer disposed on an outer surface of the magnetic layer. When the magnetic particle does not have the 2 nd shell layer, the 1 st shell layer is the shell layer. When the magnetic particle does not have the 2 nd shell layer, the 1 st shell layer is preferably bonded to the substance.

The material of the 1 st shell layer is preferably an inorganic oxide or a non-magnetic metal. The inorganic oxide means a compound having at least a metal element or a semimetal element and an oxygen atom. The material of the 1 st shell layer may be used in only 1 kind, or may be used in combination of 2 or more kinds.

The inorganic oxide or the nonmagnetic metal in the material of the 1 st shell layer preferably includes: copper, silver, gold, palladium-silver alloy, platinum-silver alloy, indium-silver alloy, tin-silver alloy, bismuth-silver alloy, tungsten-silver alloy, molybdenum-silver alloy, ruthenium-silver alloy, rhodium-silver alloy, iridium-silver alloy, phosphorus, or boron. In this case, oxidation of the magnetic layer can be effectively suppressed, and a high magnetic concentration can be further maintained for a long period of time.

The 1 st shell layer may be a plated layer.

< second Shell layer >

The 2 nd shell layer is a layer disposed on an outer surface of the 1 st shell layer. When the magnetic particle does not have the 1 st shell layer, the 2 nd shell layer is the shell layer. When the magnetic particle does not have the 1 st shell layer, the 2 nd shell layer is preferably bonded to the substance.

The material of the 2 nd shell layer is preferably inorganic oxide or organic polymer. The inorganic oxide means a compound having at least a metal element or a semimetal element and an oxygen atom. The material of the 1 st shell layer may be used in only 1 kind, or may be used in combination of 2 or more kinds.

The inorganic oxide means a compound having at least a metal element or a semimetal element and an oxygen atom. The inorganic oxide is not particularly limited. The inorganic oxide may be used alone in 1 kind, or may be used in 2 or more kinds.

The inorganic oxide is preferably an inorganic oxide having a silicon atom, a germanium atom, a titanium atom or a zirconium atom. In addition, the inorganic oxide preferably has a functional group that can react with the outer surface of the magnetic layer.

Specific examples of the inorganic oxide include: silane compounds represented by alkoxysilane such as tetraethyl orthosilicate and hydrolysates thereof, germanium compounds represented by alkoxygermanium such as tetraethoxygermanium and hydrolysates thereof, titanium compounds represented by alkoxytitanium such as tetraethoxytitanium and hydrolysates thereof, zirconium compounds represented by alkoxyzirconium such as tetrabutoxyzirconium and hydrolysates thereof, and the like.

The inorganic oxide is preferably a compound having a small specific gravity from the viewpoint of maintaining high dispersibility of the magnetic particles, and in the above example, the silane compound is most preferable.

Examples of the silane compound include: tetraethyl orthosilicate; vinyl group-containing silane compounds such as vinyltrimethoxysilane, vinyltriethoxysilane and 7-octenyltrimethoxysilane; epoxy group-containing silane compounds such as 2- (3, 4-epoxycyclohexyl) ethyltrimethoxysilane, 3-glycidoxypropylmethyldimethoxysilane, 3-glycidoxypropyltrimethoxysilane, 3-glycidoxypropylmethyldiethoxysilane, 3-glycidoxypropyltriethoxysilane, and 8-glycidoxyoctyltrimethoxysilane; styryl group-containing silane compounds such as p-styryl trimethoxysilane; methacryloyl group-containing silane compounds such as 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltriethoxysilane, and 8-methacryloxyoctyltrimethoxysilane; acryl-containing silane compounds such as 3-acryloxypropyltrimethoxysilane; amino group-containing silane compounds such as N-2- (aminoethyl) -3-aminopropylmethyldimethoxysilane, N-2- (aminoethyl) -3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3-triethoxysilyl-N- (1, 3-dimethyl-butylidene) propylamine, N-phenyl-3-aminopropyltrimethoxysilane, N- (vinylbenzyl) -2-aminoethyl-3-aminopropyltrimethoxysilane hydrochloride, and N-2- (aminoethyl) -8-aminooctyltrimethoxysilane; isocyanurate group-containing silane compounds such as tris- (trimethoxysilylpropyl) isocyanurate; ureido-containing silane compounds such as 3-ureidopropyltrialkoxysilane; mercapto group-containing silane compounds such as 3-mercaptopropylmethyldimethoxysilane and 3-mercaptopropyltrimethoxysilane; isocyanate group-containing silane compounds such as 3-isocyanatopropyltriethoxysilane; a silane compound containing a carboxylic acid anhydride such as 3-trimethoxysilylpropylsuccinic anhydride; and carboxylic acid-containing silane compounds such as hydrolysis products of 3-trimethoxysilylpropylsuccinic anhydride.

For example, an inorganic oxide shell layer having a silicon atom may be formed on the outer surface of the magnetic layer by treating the surface of the magnetic layer with a silane coupling agent having various functional groups. Further, similarly, an inorganic oxide shell layer having a silicon atom may be formed on the outer surface of the 1 st shell layer.

The content of the inorganic oxide in 100 wt% of the inorganic oxide shell layer is preferably 70 wt% or more, and more preferably 80 wt% or more.

The organic polymer is not particularly limited, and is preferably a vinyl polymer.

Examples of the vinyl monomer used as the material of the vinyl polymer include: styrene monomers such as styrene, α -methylstyrene, chlorostyrene, and divinylbenzene; vinyl ether compounds such as methyl vinyl ether, ethyl vinyl ether and propyl vinyl ether; vinyl acid ester compounds such as vinyl acetate, vinyl butyrate, vinyl laurate and vinyl stearate; halogen-containing monomers such as vinyl chloride and vinyl fluoride; (meth) acrylic acid; alkyl (meth) acrylate compounds such as methyl (meth) acrylate, ethyl (meth) acrylate, propyl (meth) acrylate, butyl (meth) acrylate, 2-ethylhexyl (meth) acrylate, lauryl (meth) acrylate, cetyl (meth) acrylate, stearyl (meth) acrylate, cyclohexyl (meth) acrylate, isobornyl (meth) acrylate, and ethylene glycol (meth) acrylate; oxygen atom-containing (meth) acrylate compounds such as 2-hydroxyethyl (meth) acrylate, glycerol (meth) acrylate, polyoxyethylene (meth) acrylate, and glycidyl (meth) acrylate; halogen-containing (meth) acrylate compounds such as trifluoromethyl (meth) acrylate and pentafluoroethyl (meth) acrylate; and alkoxysilanes having a polymerizable double bond such as vinyltrimethoxysilane, vinyltriethoxysilane, dimethoxymethylvinylsilane, dimethoxyethylvinylsilane, diethoxymethylvinylsilane, diethoxyethylvinylsilane, ethylmethyldiethylsilane, methylvinyldimethoxysilane, ethylvinyldimethoxysilane, methylvinyldiethoxysilane, ethylvinyldiethoxysilane, p-vinyltrimethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltriethoxysilane, and 3-acryloxypropyltrimethoxysilane.

The vinyl polymer may be a homopolymer obtained by polymerizing 1 kind of vinyl monomer, or a copolymer obtained by polymerizing 2 or more kinds of vinyl monomers.

For example, a comonomer such as a vinyl monomer as a main raw material and a polymerization initiator, an emulsifier, a dispersant, a surfactant, an electrolyte, a crosslinking agent, a molecular weight modifier, and the like as auxiliary raw materials are added in the presence of the mother particles and polymerization is performed in a liquid, whereby an organic polymer shell layer can be formed on the outer surface of the magnetic layer.

The content of the organic polymer in 100 wt% of the organic polymer shell layer is preferably 70 wt% or more, and more preferably 80 wt% or more.

The shell layer preferably has a functional group such as a carboxyl group, a hydroxyl group, an epoxy group, an amino group, a tosyl group, a thiol group, a triethylammonium group, a dimethylamino group, or a sulfonic acid group before binding to the substance that specifically interacts with the target substance. When the shell layer has the functional group, the substance that specifically interacts with the target substance can be favorably supported on the outer surface of the shell layer, and the substance can be favorably arranged on the surface of the magnetic particle.

The shell may have a connection portion on an outer surface. By providing the shell layer with the connecting portion, the functional group serving as a binding site for binding to the substance can be disposed at a position further away from the outermost surface of the shell layer, and the functional group can be brought into contact with the substance, which specifically interacts with the target substance, at a position having a smaller steric hindrance. Therefore, the substance is more easily bound, and the substance can be favorably arranged on the surface of the magnetic particle.

The linker preferably has a functional group capable of forming a covalent bond with the target substance, such as a carboxyl group, a hydroxyl group, an epoxy group, an amino group, a tosyl group, or a thiol group, at an end thereof, before binding to the substance that specifically interacts with the target substance. The shell layer and the substance can be chemically bonded by reacting the functional group with a functional group of the substance that specifically interacts with the target substance. The epoxy group may be an epoxy group derived from a glycidyl group-containing monomer. The hydroxyl group may be a hydroxyl group resulting from ring opening of the epoxy group.

The material of the connecting portion is preferably an epoxy compound having a plurality of epoxy groups at the terminal. As the epoxy compound having a plurality of epoxy groups at the terminal, preferred are: polyethylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, neopentyl glycol diglycidyl ether, or trimethylolpropane polyglycidyl ether. The epoxy compound having a plurality of epoxy groups at the terminal is more preferably polyethylene glycol diglycidyl ether.

The surface area of the outer surface of the magnetic layer, which is covered with the shell layer, is preferably 95% or more, more preferably 99% or more, and most preferably 100% of the total surface area of 100%. When the surface area is not less than the lower limit, the content of the substance which specifically interacts with the target substance can be increased, and as a result, the measurement accuracy and the measurement sensitivity can be improved in the measurement of the target substance using the magnetic particles.

The thickness of the shell layer (the sum of the thickness of the 1 st shell layer and the thickness of the 2 nd shell layer) is preferably 20nm or more, more preferably 40nm or more, preferably 500nm or less, and more preferably 300nm or less. When the thickness of the shell layer is not less than the lower limit and not more than the upper limit, sedimentation of the magnetic particles can be effectively suppressed, dispersibility can be further improved, and the particle diameter of the magnetic particles can be reduced, so that the target substance can be effectively bound. When the thickness of the shell layer is equal to or greater than the lower limit, oxidation of the magnetic layer can be prevented, and further, the decrease in magnetism collection with time can be suppressed.

The thickness of the shell layer can be measured, for example, by observing a cross section of the magnetic particle using a Transmission Electron Microscope (TEM). The thickness of the shell layer is preferably calculated as the thickness of the shell layer of 1 magnetic particle from the average thickness of any 5 shell layers, and more preferably calculated as the thickness of the shell layer of 1 magnetic particle from the average thickness of the entire shell layers. The thickness of the shell layer is preferably determined by calculating an average value of the thicknesses of the shell layers of the respective magnetic particles for any 10 magnetic particles.

(substance that specifically interacts with target substance)

On the surface of the magnetic particle according to the present invention, a substance that specifically interacts with a target substance is present. Examples of the substance include: sugar chains, peptide chains, proteins, antigens, nucleotide chains, and the like. The substance may be appropriately changed depending on the kind of the target substance. The above-mentioned substances may be used alone in 1 kind, or may be used in combination in 2 or more kinds.

Examples of the interaction between the substance and the target substance include an antigen-antibody reaction, and an interaction between an enzyme and a substrate. The interaction between the substance and the target substance may be a non-covalent bond interaction between the substance and the target substance, or a covalent bond interaction between the substance and the target substance. Examples of the non-covalent bond interaction include: hydrophobic interactions, electrostatic interactions, van der waals forces, hydrogen bonding, coordination bonding, ionic bonding, and the like.

The substance that specifically interacts with the target substance is preferably a substance that can interact with the target substance in a non-covalent bond manner.

Examples of the combination of the substance which specifically interacts with the target substance and the target substance include a combination of an antibody and an antigen, a combination of a sugar chain and a protein such as a lectin, a combination of a protein such as an enzyme and an inhibitor, a combination of a peptide and a protein, a combination of a nucleotide chain and a nucleotide chain, and a combination of a nucleotide chain and a protein.

The substance that specifically interacts with the target substance is preferably a protein, more preferably avidin or streptavidin. In addition, the substance that specifically interacts with the target substance is preferably an antigen or an antibody.

The antibody may be a polyclonal antibody or a monoclonal antibody. The antibody may be treated with a protease such as papain or pepsin, or may be a Fab or F (ab') 2 fragment.

The method for disposing the substance that specifically interacts with the target substance is not particularly limited. For example, the substance may be disposed on the surface of the magnetic particle by mixing the substance and the magnetic particle before the substance is supported.

(target substance)

The target substance is a substance that specifically interacts with the aforementioned substance.

Examples of the target substance include: proteins, nucleic acids, hormones, cancer markers, ventilator-associated markers, cardiac markers, drugs, and the like.

Examples of the protein include: lipid proteins such as high specific gravity lipoprotein (HDL), low specific gravity lipoprotein (LDL), and very low specific gravity lipoprotein; enzymes such as alkaline phosphatase, amylase, acid phosphatase, γ -glutamyltransferase (γ -GTP), lipase, Creatine Kinase (CK), Lactate Dehydrogenase (LDH), oxaloacetate transaminase Glutamate (GOT), pyruvate transaminase Glutamate (GPT), renin, Protein Kinase (PK), and tyrosine kinase; immunoglobulins such as IgG, IgM, IgA, IgD and IgE (or fragments such as Fc part, Fab part and F (ab)2 part); blood coagulation-related factors such as fibrinogen, Fibrin Degradation Products (FDP), prothrombin, thrombin and the like; antibodies such as an anti-streptolysin O antibody, an anti-human hepatitis B virus surface antigen antibody (HBs antigen), an anti-human hepatitis C virus antibody, and an anti-rheumatoid factor; albumin, hemoglobin, myoglobin, transferrin, protein A, C Reactive Protein (CRP), and the like.

Examples of the nucleic acid include DNA and RNA.

Examples of the hormone include: thyroid Stimulating Hormone (TSH), thyroid hormones (FT3, FT4, T3, T4), parathyroid hormone (PTH), and human chorionic gonadotropin (hCG) estradiol (E2), and the like.

Examples of the cancer marker include: alpha-fetoprotein (AFP), PIVKA-II, carcinoembryonic antigen (CEA), CA19-9, Prostate Specific Antigen (PSA), and the like.

Examples of the marker related to a respirator include KL-6.

Examples of the cardiac disease marker include troponin T (TnT) and an N-terminal fragment of N-terminal human brain natriuretic peptide precursor (NT-proBNP).

Examples of the drug include: antiepileptic drugs, antibiotic drugs, theophylline, etc.

(other details of magnetic particles)

The magnetic particles can be suitably used for measurement such as Radioimmunoassay (RIA), Enzyme Immunoassay (EIA), Fluorescence Immunoassay (FIA), Electrochemiluminescence (ECLIA), chemiluminescence immunoassay (CLIA and CLEIA), absorbance measurement, and surface plasmon resonance. The magnetic particles can be suitably used in the measurement by the sandwich method and the competition method.

The magnetic particles can be suitably used for measuring the concentration of the target substance in a sample.

The magnetic particles can be suitably used as a detection reagent. The magnetic particles are preferably magnetic particles that can be used as a detection reagent.

The concentration of the target substance using the magnetic particles can be measured, for example, as follows.

A solution containing the magnetic particles (e.g., a detection reagent described later) and a sample containing a target substance are mixed to obtain a mixed solution. The obtained mixed solution is heated, for example, to bind the substance that specifically interacts with the target substance in the magnetic particles to the target substance in the sample, thereby obtaining a reaction solution (1 st reaction step). Then, magnetic force is applied to the reaction solution by a magnet or the like to aggregate the magnetic particles (magnetism collecting step). After removing the unreacted sample, a washing solution is added and mixed (washing step). It should be noted that the magnetism collecting step and the washing step may be repeated many times. Then, after removing the washing solution, the target substance is reacted with the labeling substance to measure the concentration of the target substance (reaction step 2).

Examples of the labeling substance include: enzymes such as alkaline phosphatase, β -galactosidase, peroxidase, microperoxidase, glucose oxidase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, luciferase, tyrosinase, and acid phosphatase suitable for use in Enzyme Immunoassay (EIA); radioisotopes such as 99mTc, 131I, 125I, 14C, 3H, 32P and the like which are suitable for Radioimmunoassay (RIA); for example, a spin label is used for fluorescent substances such as fluorescein, dansyl, fluorescamine, coumarin, naphthylamine and derivatives thereof, fluorescent substances such as Green Fluorescent Protein (GFP), luminescent substances such as luminol, isoluminol, luminol, bis (2,4, 6-trifluorophenyl) oxalate, substances having an absorption in ultraviolet region such as phenol, naphthol, anthracene and derivatives thereof, compounds having an oxygen group such as 4-amino-2, 2,6, 6-tetramethylpiperidine-1-oxyl, 3-amino-2, 2,5, 5-tetramethylpyrrolidine-1-oxyl, 2, 6-di-tert-butyl-alpha- (3, 5-di-tert-butyl-4-oxo-2, 5-cyclohexadiene-1-ylidene) -p-trioxoxyl, and the like, which are suitable for Fluorescent Immunoassay (FIA) Compounds that characterize the labeling agent, and the like. From the viewpoint of improving the measurement sensitivity, the labeling substance is preferably an enzyme or a fluorescent substance, more preferably alkaline phosphatase, peroxidase, or glucose oxidase, and even more preferably peroxidase.

(detection reagent)

The detection reagent comprises the magnetic particles.

The detection reagent preferably comprises a buffer.

The buffer is preferably a buffer having a buffering capacity in a range of pH5.0 to 9.0. Examples of the buffer solution include: phosphate buffer, glycine buffer, Varona buffer, Tris buffer, boric acid buffer, citric acid buffer, and Good's buffer, etc.

The detection reagent may contain other components such as a sensitizer, a polymer compound such as a protein, an amino acid, and a surfactant.

The detection reagent can efficiently perform a reaction between a target substance and the substance that specifically interacts with the target substance by including the sensitizer, and can improve measurement accuracy. Examples of the sensitizer include: alkylated polysaccharide compounds such as methylcellulose and ethylcellulose, pullulan, and polyvinylpyrrolidone.

Examples of the protein include: albumin (bovine serum albumin, ovalbumin, and the like), casein, gelatin, and the like.

The content of the magnetic particles in 100 wt% of the detection reagent is preferably 0.5 wt% or more, more preferably 2 wt% or more, preferably 10 wt% or less, and more preferably 5 wt% or less. When the content of the magnetic particles is not less than the lower limit and not more than the upper limit, the measurement accuracy of the target substance can be further improved.

The present invention will be specifically described below with reference to examples and comparative examples. However, the present invention is not limited to the following examples.

(example 1)

As the resin particles, "Micropearl EX-003" manufactured by Water accumulation chemical Co., Ltd was used.

Formation of magnetic layer:

to 100 parts by weight of an alkali solution containing 5.0% by weight of a palladium catalyst solution, 10 parts by weight of Micropearl EX-003 (available from Seikagaku Kogyo Co., Ltd.) having an average particle diameter of 2.98 μm was added, and the mixture was dispersed with an ultrasonic disperser and the resulting solution was filtered to remove the resin particles.

Next, the resin particles were added to 100 parts by weight of a 1.0 wt% dimethylamine borane solution to activate the surfaces of the resin particles. The resin particles whose surfaces were activated were sufficiently washed with water, and then added to 500 parts by weight of distilled water to be dispersed, thereby obtaining a suspension.

The obtained suspension was added to a solution containing 80g/L of nickel sulfate, 10ppm of thallium nitrate, 5.0ppm of bismuth nitrate, 2.0g/L of 3 sodium citrate, and 10g/L of aqueous ammonia, to obtain a particle mixed solution.

Further, 30g/L of cobalt sulfate, 10ppm of thallium nitrate, 5.0ppm of bismuth nitrate, 2.0g/L of 3 sodium citrate, and 10g/L of ammonia water were dissolved in distilled water to prepare a cobalt plating solution.

After a 1.0 wt% solution of dimethylamine borane was added dropwise to the particle mixture adjusted to a dispersion state of 60 ℃ at a rate of 40 mL/min for 10 minutes, dropwise addition of a cobalt plating solution at a rate of 40 mL/min was started, and the surface of the resin particles was plated with a nickel-cobalt alloy. Then, the particles were collected by filtration and washed with water. In this manner, resin particles having a magnetic layer (continuous layer) containing a nickel-cobalt alloy as a magnetic body on the outer surface of the resin particles were obtained.

Configuration of substances that specifically interact with the target substance:

0.5mL of the aqueous dispersion of the resin particles having the magnetic layer was added to the test tube, and washed 3 times with PBS solution. After the dispersion solvent was removed, 0.5mL of a PBS solution (0.75mg/mL) containing an antibody against sialylated glycoantigen KL-6 (hereinafter, abbreviated as KL-6) was added, and the mixture was stirred at 25 ℃ overnight. Then, 1.5mL of 1.0 wt% BSA solution was added, and the mixture was stirred at 25 ℃ for 4 hours. Subsequently, 1.5mL of a 1.0 wt% BSA solution and 1.5mL of magnetism collected on the wall surface of the test tube using a magnet were repeated 3 times to prepare a magnetic particle dispersion in which the anti-KL-6 antibody was sensitized.

(example 2)

Forming a shell layer:

to 1 part by weight of the resin particles having a magnetic layer obtained in example 1, 400 parts by weight of ethanol and 20 parts by weight of a 28% aqueous ammonia solution (manufactured by Nacalai Tesque) were added. Next, 5.0 parts by weight of tetraethyl orthosilicate and 15 parts by weight of 8-glycidoxy octyltrimethoxysilane were added, and stirring was performed for 1 hour. The obtained dispersion was filtered and washed with water. In this way, resin particles having an inorganic oxide shell layer having a functional group on the surface and a magnetic layer were obtained.

Configuration of substances that specifically interact with the target substance:

in the same manner as in example 1, magnetic particles having anti-KL-6 antibodies present on the surfaces thereof were obtained.

(example 3)

Formation of the 1 st shell layer:

1.0 part by weight of the resin particles having the magnetic layer obtained in example 1 was dispersed in 300 parts by weight of distilled water to obtain a particle mixture. As an electroless silver plating solution, a silver plating solution prepared by adjusting a mixture of 30g/L silver nitrate, 100g/L succinimide, and 20g/L formaldehyde to pH8.0 with ammonia water was gradually added dropwise to the particle mixture solution to perform electroless silver plating. Then, the particles were collected by filtration and washed with water. Thus, resin particles having a nonmagnetic silver layer (1 st shell layer) and a magnetic layer were obtained.

And 2, forming a shell layer:

an inorganic oxide shell layer (No. 2 shell layer) having a functional group was formed on the surface in the same manner as in example 2, except that resin particles having a nonmagnetic silver layer (No. 1 shell layer) and a magnetic layer were used.

Configuration of substances that specifically interact with the target substance:

in the same manner as in example 1, magnetic particles having anti-KL-6 antibodies present on the surfaces thereof were obtained.

(examples 4 to 11)

Magnetic particles were obtained in the same manner as in example 2, except that the configuration of the magnetic particles was changed as shown in tables 1 and 2.

Comparative example 1

Preparation of resin particles:

the same resin particles as in example 1 were used.

Formation of magnetic layer:

1.0 part by weight of Micropearl EX-003 (available from waterlogging chemical Co., Ltd.) having an average particle diameter of 3.00 μm and 4.0 parts by weight of a magnetic fluid (an aqueous dispersion of ferroferric oxide nanoparticles having an average particle diameter of about 10nm, "EMG 707" available from Ferrotec Co., Ltd.) were stirred at 250rpm for 10 minutes. The obtained particle dispersion was filtered and washed with water to obtain resin particles having a magnetic layer (aggregate layer) containing ferroferric oxide as a magnetic material on the outer surface of the resin particles.

Forming a shell layer:

magnetic particles having a shell layer were obtained in the same manner as in example 2.

Configuration of substances that specifically interact with the target substance:

in the same manner as in example 1, magnetic particles having anti-KL-6 antibodies present on the surface of the magnetic particles were obtained.

Comparative example 2

Preparation of resin particles:

the same resin particles as in example 1 were used.

Formation of magnetic layer:

ferrite-based magnetic fine particles (average primary particle diameter: 10nm) having a hydrophobized surface were obtained by adding acetone to an oily magnetic fluid (an organic solvent dispersion of ferroferric oxide nanoparticles having an average particle diameter of about 10nm, "EXP series" manufactured by Ferrotec corporation) to precipitate the particles, and then drying the particles.

Then, 1.0 part by weight of the resin particles and 1.0 part by weight of the ferrite-based magnetic fine particles having a hydrophobized surface were sufficiently mixed by a mixer. The obtained mixture was treated for 5 minutes at a peripheral speed of 100 m/sec (16200rpm) using a hybridization system NHS-0 type (manufactured by nean mechanical products corporation) for forming a magnetic layer of the 2 nd magnetic material on the outer surface of the resin particle.

Forming a shell layer:

10 parts by weight of the obtained particles having the magnetic layer and 300 parts by weight of a 0.5% aqueous solution of a nonionic emulsifier (manufactured by Kao corporation, "EMERGEN 150") as a dispersant were put in a 1L separable flask and stirred. Thereto, 24 parts by weight of cyclohexyl methacrylate and 60 parts by weight of 2-methacryloyloxyethylsuccinate monoester as monomers and 1.0 part by weight of bis (3,5, 5-trimethylhexanoyl) peroxide ("PEROYL 355" manufactured by Nichikoku K.K.) as an initiator were added, and the mixture was stirred at 80 ℃ and 200rpm for 8 hours. Thus, an organic polymer shell layer having a carboxyl group is formed on the outer surface of the magnetic layer. The organic polymer is a copolymer of cyclohexyl methacrylate and 2-methacryloyloxyethyl succinate monoester.

Configuration of substances that specifically interact with the target substance:

using the obtained shell layer and the resin particle having the magnetic layer, an anti-KL-6 antibody was prepared in the same manner as in example 1. Thus, magnetic particles were obtained.

Comparative example 3

10 parts by weight of nickel sulfate, 1 part by weight of sodium dodecylbenzenesulfonate and 200 parts by weight of ion-exchanged water were uniformly stirred, and the temperature of the stirred material was adjusted to 60 ℃. A5 wt% solution of dimethylamine borane was added dropwise to the stirred mixture at a rate of 4 parts by weight/min over 10 minutes to obtain an aqueous dispersion of nickel nanoparticles having an average particle diameter of about 10 nm. Next, acetone was added to the nickel nanoparticle aqueous dispersion to precipitate particles, followed by filtration and washing with ethanol. The precipitate of nickel nanoparticles on the filter paper was dispersed in an aqueous solution of 1 part by weight of sodium dodecylbenzenesulfonate to prepare a dispersion having a solid content of about 17%. This dispersion (4.0 parts by weight) and Micropearl EX-003 (average particle diameter: 2.99 μm, manufactured by Water chemical Co., Ltd.) (1.0 part by weight) were stirred at 250rpm for 10 minutes to obtain a particle dispersion. The obtained particle dispersion was filtered and washed with ethanol and water to obtain resin particles having a magnetic layer (aggregate layer) containing nickel as a magnetic body on the outer surface of the resin particles. Further, on the outer surface of the magnetic layer, a shell layer (organic polymer shell layer) containing polystyrene (PSt) was formed as a shell layer, and magnetic particles were obtained.

Comparative example 4

Resin particles having a magnetic layer (aggregate layer) containing a nickel-cobalt alloy as a magnetic body on the outer surface thereof were obtained in the same manner as in comparative example 3, except that 10 parts by weight of nickel sulfate was changed to 5 parts by weight of nickel sulfate and 5 parts by weight of cobalt sulfate. In the same manner as in comparative example 1, magnetic particles having anti-KL-6 antibodies on the shell layer and the outer surface of the shell layer were obtained.

(evaluation)

(1) Average particle diameter of resin particles and magnetic particles

The average particle diameters of the resin particles and the magnetic particles were calculated using a scanning electron microscope ("Regulus 8220" manufactured by HITACHIHIGH TECHNOLOGIES corporation). Specifically, the average value was determined by preparing a sample obtained by drying the obtained magnetic particles, observing the obtained sample with a scanning electron microscope, measuring the particle diameters of arbitrary 50 magnetic particles and resin particles in the magnetic particles, and calculating the average value.

(2) Content of magnetic substance in 100 vol% of magnetic particles

The cross section of the magnetic particles was observed using a Transmission Electron Microscope (TEM). In the cross section of the magnetic particle to be observed, the thickness of the magnetic layer is defined as r₁Let the radius of the resin particle be r₂The radius of the magnetic particle is defined as r₃The content (vol%) of the magnetic material was calculated by the following equation.

The content (volume%) of the magnetic material [ { (r) [ ]₁+r₂)³-(r₂)³}/(r₃)³]×100

(3) Thickness of magnetic layer and shell layer

The cross section of the magnetic particles was observed using a Transmission Electron Microscope (TEM).

The average value of the thicknesses of the magnetic layers at any 5 points was calculated as the thickness of the magnetic layer of 1 magnetic particle, and the average value of the thicknesses of the entire magnetic layers was calculated as the thickness of the magnetic layer of 1 magnetic particle. The thickness of the magnetic layer was determined by calculating the average value of the thicknesses of the magnetic layers of the respective magnetic particles for any 10 magnetic particles.

The average value of the thicknesses of the shell layers at any 5 points was calculated as the thickness of the shell layer of 1 magnetic particle, and the average value of the thicknesses of the entire shell layers was calculated as the thickness of the shell layer of 1 magnetic particle. The thickness of the shell layer is determined by calculating the average value of the thicknesses of the shell layers of the respective magnetic particles for any 10 magnetic particles.

(4) Magnetic flux concentration

A sample solution in which the absorbance at a wavelength of 550nm is adjusted to 0.9 to 1.1 is prepared by dispersing magnetic particles in water. 1.6mL of a sample solution was added to a quartz cell (cell) provided in a spectrophotometer ("U-3900H" manufactured by Hitachi, Ltd.) with a magnet (2800G, W10mm XD 10mm XH 8mm) held in contact with a spacer (W10mm XD 10mm XH 1mm), and the absorbance at a wavelength of 550nm was measured 5 to 155 seconds after the sample solution was charged. The absorbance decay rate between 150 seconds was calculated by the following equation and used as the magnetic concentration rate.

Magnetic collection ratio (%) [ { (absorbance after 5 seconds) - (absorbance after 155 seconds) }/(absorbance after 5 seconds) ] × 100

[ criterion for magnetic susceptibility determination ]

O ^ O: the magnetic collection rate is more than 90 percent

O: the magnetic collection rate is more than 60 percent and less than 90 percent

And (delta): the magnetic collection rate is more than 40 percent and less than 60 percent

X: the magnetic concentration is less than 40%

(5) Magnetic rate (after 1 month)

The magnetic particles obtained were left at 25 ℃ for 1 month. Magnetic collection densities were obtained using the magnetic particles after the standing by the same method as the magnetic collection density of (4).

[ criterion for magnetic Collection Rate (after 1 month of storage) ]

O ^ O: the magnetic collection rate is more than 90 percent

O: the magnetic collection rate is more than 60 percent and less than 90 percent

And (delta): the magnetic collection rate is more than 40 percent and less than 60 percent

X: the magnetic concentration is less than 40%

(6) Rate of dispersion

A sample solution in which the absorbance at a wavelength of 550nm is adjusted to 0.9 to 1.1 is prepared by dispersing magnetic particles in water. 1.3mL of a sample solution was added to a quartz cell (cell) provided in a spectrophotometer ("U-3900H" manufactured by Hitachi, Ltd.), and the absorbance at a wavelength of 550nm was measured. Then, using a magnet (28000G, W40mm × D40mm × H10mm), the absorbance of the supernatant was 0. Then, the magnetic particles were dispersed at 2000rpm for 5 seconds by a vortex oscillator, and the absorbance at a wavelength of 550nm was measured. From the absorbance before magnetic collection, the magnetic collection, and the absorbance after dispersion, the rate of change in absorbance was calculated as the dispersion rate by the following equation.

Dispersion ratio (%) { (absorbance after magnetic collection and dispersion)/(absorbance before magnetic collection) } × 100

[ criterion for determining Dispersion Rate ]

O ^ O: the dispersion rate is more than 95%

O: the dispersion rate is more than 90 percent and less than 95 percent

And (delta): the dispersion rate is more than 85 percent and less than 90 percent

X: the dispersion rate is less than 85 percent

(7) Immunoassay (luminescence amount per unit weight of magnetic particle)

A solution containing no KL-6 (antigen-free solution) and a solution containing 5000U/mL KL-6 (antigen-containing solution) were prepared. The binding ability between the magnetic particles and the target substance (KL-6) was evaluated by determining the difference between the amount of luminescence in the immunoassay using the antigen-free solution and the amount of luminescence in the immunoassay using the antigen-containing solution. Specifically, the evaluation was performed by the following procedure.

Preparation of ruthenium complex-labeled anti-KL-6 antibody (secondary antibody):

0.5mL of a PBS-1 solution of the anti-KL-6 antibody (anti-KL-6 antibody concentration 2.0mg/mL) was added to the polypropylene centrifuge tube, followed by 13. mu.L of Ru-NHS (10 mg/mL). After shaking and stirring at 25 ℃, purification was performed using a Sephadex G25 column to obtain a ruthenium complex-labeled anti-KL-6 antibody.

Immunoassay:

the following measurement of luminescence amount was performed using an ECLIA automatic analyzer (manufactured by SEKISUI MEDICAL Co., Ltd. "Pikorumi III") using an electrochemiluminescence immunoassay method as a measurement principle. To 200. mu.L of the reaction solution (buffer solution containing normal rabbit serum) was added 20. mu.L of a solution (antigen-containing solution) containing 5000U/mL of KL-6, and then 25. mu.L of magnetic particles was added. After reaction at 30 ℃ for 9 minutes, 350. mu.L of Pikorumi BF buffer (10mM Tris buffer) was added thereto, and the mixture was washed 3 times with magnetic particles adsorbed to a magnet. Then, 200. mu.L of a ruthenium labeled antibody-containing solution containing 1.0. mu.g/mL of a ruthenium complex labeled anti-KL-6 antibody was added, and after reaction at 30 ℃ for 9 minutes, 350. mu.L of PikorumiBF washing solution (10mM Tris buffer) was added, and the mixture was washed 3 times with magnetic particles adsorbed by a magnet. Then, 300. mu.L of Pikorumi luminescence electrolyte containing 0.1M tripropylamine was added and transported to the electrode surface, and the luminescence amount of the ruthenium complex bound to the magnetic particles was measured. The difference X between the luminescence amount in the immunoassay using the antigen-free solution and the luminescence amount in the immunoassay using the antigen-containing solution was obtained.

[ criterion for determining the amount of luminescence per unit weight of magnetic particles ]

O ^ O: the difference X is more than 150000

O: the difference X is 125000 or more and less than 150000

And (delta): the difference X is 100000 or more and less than 125000

X: the difference X is less than 100000

The composition of the magnetic particles and the results are shown in tables 1 and 2 below.

[ Table 1]

[ Table 2]

Description of the symbols

1, 1A … magnetic particles

2 … resin particles

3 … magnetic layer

4 … Shell layer

5 … substance

41 … No. 1 Shell

42 … case 2