WO2020196786A1 - Particules magnétiques et agent de test - Google Patents

Particules magnétiques et agent de test Download PDF

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Publication number
WO2020196786A1
WO2020196786A1 PCT/JP2020/013814 JP2020013814W WO2020196786A1 WO 2020196786 A1 WO2020196786 A1 WO 2020196786A1 JP 2020013814 W JP2020013814 W JP 2020013814W WO 2020196786 A1 WO2020196786 A1 WO 2020196786A1
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WIPO (PCT)
Prior art keywords
magnetic
particles
substance
resin particles
layer
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PCT/JP2020/013814
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English (en)
Japanese (ja)
Inventor
秀平 大日方
脇屋　武司
祐也 稲葉
Original Assignee
積水化学工業株式会社
積水メディカル株式会社
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Application filed by 積水化学工業株式会社, 積水メディカル株式会社 filed Critical 積水化学工業株式会社
Priority to CN202080023454.4A priority Critical patent/CN113614530A/zh
Priority to JP2021509614A priority patent/JP7513345B2/ja
Publication of WO2020196786A1 publication Critical patent/WO2020196786A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/553—Metal or metal coated

Definitions

  • the present invention relates to magnetic particles containing a magnetic material.
  • the present invention also relates to a test agent using the above magnetic particles.
  • Magnetic particles are used to measure the concentration of target substances in samples in fields such as pharmaceutical research and development and clinical tests.
  • immunoassays such as chemiluminescence immunoassay (CLIA method)
  • magnetic particles having an antibody or antigen on the surface are widely used.
  • these magnetic particles are magnetized by a magnet or the like after being bound to an antigen or an antibody which is a target substance.
  • magnetic particles magnetic particles having a magnetic substance inside the resin particles (see, for example, Patent Documents 1 and 2) and magnetic particles having a magnetic layer formed on the outer surface of the resin particles (for example, Patent Documents). 3) is used.
  • the conventional magnetic particles may have low magnetic collection.
  • the magnetic material may oxidize and the magnetic collection may decrease over time.
  • conventional magnetic particles may have low dispersibility.
  • the number of magnetic particles that cannot be completely collected by the magnet increases, and the measurement accuracy and measurement sensitivity deteriorate when measuring the concentration of the target substance such as an antibody or antigen.
  • the magnetic particles may not be sufficiently redispersed after magnetic separation, and the measurement accuracy, measurement sensitivity and measurement reproducibility may decrease.
  • An object of the present invention is to provide magnetic particles capable of enhancing magnetic collection and dispersibility, and maintaining high magnetic collection. Further, a limited object of the present invention is to provide magnetic particles capable of maintaining high magnetic collection for a long period of time. Another object of the present invention is to provide a test agent using the above magnetic particles.
  • magnetic particles used for specifically interacting with a target substance the magnetic inclusion resin particles containing the first magnetic substance inside, and the magnetic inclusion resin particles.
  • a magnetism having a magnetic layer arranged on the outer surface of the magnetic layer and containing a second magnetic substance, and a substance supported on the outer surface side of the magnetic layer and specifically interacting with the target substance. Particles are provided.
  • the first magnetic material is a metal or a metal oxide
  • the second magnetic material is a metal or a metal oxide
  • a shell layer is further provided on the outer surface of the magnetic layer, the material of the shell layer containing an inorganic oxide or an organic polymer, and the shell layer. , The substance is bound.
  • the material of the shell layer is an inorganic oxide containing the inorganic oxide, wherein the inorganic oxide has a silicon atom, a germanium atom, a titanium atom or a zirconium atom. is there.
  • the substance is an antigen or antibody.
  • the substance is avidin or streptavidin.
  • the total content of the first magnetic substance and the second magnetic substance in 100% by volume of the magnetic particles is 10% by weight or more and 95% by weight or less. ..
  • the content of the first magnetic substance is 10% by weight in a total of 100% by weight of the contents of the first magnetic substance and the second magnetic substance. More than 90% by weight or less.
  • the content of the first magnetic substance in 100% by volume of the region from the outer surface to the center of the magnetic inclusion resin particles up to 1/3 of the thickness is 0.8 or more and 4.0 or less. is there.
  • the magnetic particles are used as a test agent.
  • a test agent containing the above-mentioned magnetic particles is provided.
  • the magnetic particles according to the present invention are used to specifically interact with a target substance.
  • the magnetic particles according to the present invention include magnetic encapsulating resin particles containing a first magnetic substance inside, and a magnetic layer arranged on the outer surface of the magnetic encapsulating resin particles and containing a second magnetic substance. It includes a substance that is supported on the outer surface side of the magnetic layer and that specifically interacts with the target substance. Since the magnetic particles according to the present invention have the above-mentioned structure, the magnetic collection and dispersibility can be enhanced, and the magnetic collection can be maintained high.
  • FIG. 1 is a cross-sectional view schematically showing magnetic particles according to the first embodiment of the present invention.
  • FIG. 2 is a cross-sectional view schematically showing magnetic particles according to a second embodiment of the present invention.
  • FIG. 3 is a cross-sectional view schematically showing the magnetic particles according to the third embodiment of the present invention.
  • FIG. 4 is a cross-sectional view schematically showing the magnetic particles according to the fourth embodiment of the present invention.
  • FIG. 5 is a schematic diagram for explaining each region for obtaining the content of the first magnetic substance in the magnetic inclusion resin particles.
  • the magnetic particles according to the present invention are used to specifically interact with a target substance.
  • the magnetic particles according to the present invention are magnetic particles that can specifically interact with a target substance.
  • the magnetic particles according to the present invention include magnetic encapsulating resin particles containing a first magnetic substance inside, and a magnetic layer arranged on the outer surface of the magnetic encapsulating resin particles and containing a second magnetic substance. It includes a substance that is supported on the outer surface side of the magnetic layer and that specifically interacts with the target substance.
  • the magnetic particles according to the present invention have the above-mentioned structure, the magnetic collection and dispersibility can be enhanced, and the magnetic collection can be maintained high. In the present invention, the magnetism collection can be maintained high for a long period of time. In the magnetic particles according to the present invention, in the measurement of the target substance using the magnetic particles, the amount of binding to the target substance per unit weight of the magnetic particles can be increased, and therefore the measurement sensitivity can be increased.
  • Conventional magnetic particles may have low magnetic collection or dispersibility.
  • the conventional magnetic particles having a magnetic substance inside the resin particles it is difficult to sufficiently increase the content of the magnetic substance because the space inside the resin particles is limited. Therefore, it is difficult to improve the magnetic collection with the conventional magnetic particles.
  • the conventional magnetic particles having a magnetic layer on the outer surface of the resin particles when the content of the magnetic substance contained in the magnetic layer is increased, the magnetic collection can be increased to some extent, but the particles of the magnetic particles. This is not preferable because the diameter becomes large and the amount of the target substance bonded per unit area of the magnetic particles decreases. Further, when the magnetic layer on the outer surface of the resin particles is thickened, the repulsive force between the magnetic particles is lowered, and the dispersibility may be lowered.
  • the smaller the particle diameter the lower the content of the magnetic substance.
  • the magnetic collection may decrease, and as a result, the measurement sensitivity may decrease.
  • the magnetic substance is oxidized and the magnetic collection property is lowered with time. There is.
  • the content of the magnetic substance can be increased and the magnetic collection can be enhanced while keeping the particle diameter of the magnetic particles small.
  • the magnetic particles according to the present invention can enhance the dispersibility.
  • high magnetic collection can be maintained even if the magnetic material deteriorates to some extent.
  • the magnetic particles according to the present invention even if the specific magnetic layer containing the second magnetic material is deteriorated to some extent, the magnetic particles contain the first magnetic material, so that the magnetic collection property can be maintained high.
  • the measurement accuracy, measurement sensitivity and measurement reproducibility can be improved in the measurement of the target substance using the magnetic particles.
  • the average particle size of the magnetic particles is preferably 0.5 ⁇ m or more, more preferably 1.0 ⁇ m or more, preferably 10 ⁇ m or less, more preferably 7 ⁇ m or less, still more preferably 5 ⁇ m or less, particularly preferably 4 ⁇ m or less, most preferably. It is preferably 3.5 ⁇ m or less.
  • the average particle size of the magnetic particles is at least the above lower limit, the magnetism collection can be further enhanced.
  • the average particle size 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 binding amount of the target substance can be increased. it can.
  • the average particle size of the magnetic particles is a number average particle size.
  • the particle size of the magnetic particles is determined, for example, by observing 50 arbitrary magnetic particles with an electron microscope or an optical microscope and calculating the average value of the particle sizes of each magnetic particle. 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 size of the magnetic particles is preferably 10% or less, more preferably 5% or less.
  • the coefficient of variation of the average particle size of the magnetic particles is not more than the above 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 measured as follows.
  • CV value (%) ( ⁇ / Dn) ⁇ 100 ⁇ : Standard deviation of the particle size of the magnetic particles Dn: Average value of the particle size of the magnetic particles
  • FIG. 1 is a cross-sectional view schematically showing magnetic particles according to the first embodiment of the present invention.
  • the magnetic particle 1 shown in FIG. 1 is used to specifically interact with the target substance.
  • the magnetic particles 1 include magnetic inclusion resin particles 2, a magnetic layer 3, a shell layer 4, and a substance 5 that specifically interacts with a target substance.
  • the substance 5 is, for example, a physiologically active substance such as avidin, streptavidin, an antigen and an antibody.
  • the magnetic inclusion resin particles 2 have resin particles 21 and a first magnetic material 22.
  • the magnetic inclusion resin particles 2 contain a first magnetic substance 22 inside.
  • the resin particles 21 contain a first magnetic material 22 inside.
  • the first magnetic material 22 is dispersed inside the magnetic inclusion resin particles 2.
  • the first magnetic material 22 is dispersed inside the resin particles 21.
  • the first magnetic material 22 is uniformly distributed inside the magnetic inclusion resin particles 2.
  • the first magnetic material does not have to be uniformly distributed inside the magnetic inclusion resin particles.
  • the first magnetic material may be arranged from the center of the magnetic inclusion resin particles to the outer surface so that the content of the first magnetic material increases.
  • the magnetic layer 3 is arranged on the outer surface of the magnetic inclusion resin particles 2.
  • the magnetic layer 3 is arranged on the outer surface of the resin particles 21 in the magnetic inclusion resin particles 2.
  • the magnetic layer 3 contains a second magnetic material.
  • the shell layer 4 is arranged on the outer surface of the magnetic layer 3.
  • the substance 5 is supported on the outer surface side of the magnetic layer 3.
  • the substance 5 is supported on the outer surface of the shell layer 4.
  • the shell layer 4 and the substance 5 are bonded to each other.
  • the substance 5 is present on the surface of the magnetic particles 1.
  • the magnetic layer 3 is a single magnetic layer.
  • the magnetic layer 3 covers the entire outer surface of the magnetic inclusion resin particles 2.
  • the magnetic layer may cover the entire outer surface of the magnetic inclusion resin particles, or the magnetic layer may cover a part of the surface of the magnetic inclusion resin particles.
  • the magnetic layer may be a single magnetic layer or a multi-layered magnetic layer composed of two 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.
  • the shell layer may cover the entire outer surface of the magnetic layer, or the shell layer may cover a part of the surface of the magnetic layer.
  • FIG. 2 is a cross-sectional view schematically showing the magnetic particles according to the second embodiment of the present invention.
  • the magnetic particles 1A shown in FIG. 2 are used to specifically interact with the target substance.
  • the magnetic particles 1A include magnetic inclusion resin particles 2A, a magnetic layer 3A, and a substance 5A that specifically interacts with a target substance.
  • the substance 5A is, for example, avidin, streptavidin, an antigen, an antibody, or the like.
  • the magnetic particles 1A do not have the shell layer.
  • the magnetic inclusion resin particles 2A have resin particles 21A and a first magnetic body 22A.
  • the distribution state of the first magnetic substance is different between the magnetic encapsulating resin particles 2 shown in FIG. 1 and the magnetic encapsulating resin particles 2A shown in FIG.
  • the first magnetic substance 22A is not uniformly distributed inside the magnetic inclusion resin particles 2A. From the center of the magnetic inclusion resin particles 2A to the outer surface, the first magnetic body 22A is arranged so that the content of the first magnetic body 22A increases.
  • the first magnetic material may be uniformly distributed inside the magnetic inclusion resin particles.
  • the magnetic layer 3A is arranged on the outer surface of the magnetic inclusion resin particles 2A.
  • the magnetic layer 3A contains a second magnetic material.
  • the substance 5A is supported on the outer surface of the magnetic layer 3A.
  • the substance 5A is present on the surface of the magnetic particles 1A.
  • the magnetic layer 3A is a single magnetic layer.
  • the magnetic layer 3A covers the entire outer surface of the magnetic inclusion resin particles 2A.
  • the magnetic layer may be a single magnetic layer or a multi-layered magnetic layer composed of two or more layers.
  • FIG. 3 is a cross-sectional view schematically showing the magnetic particles according to the third embodiment of the present invention.
  • the magnetic particles 1B shown in FIG. 3 are used to specifically interact with the target substance.
  • the magnetic particles 1B include magnetic inclusion resin particles 2B, a magnetic layer 3B, a shell layer 4B, and a substance 5B that specifically interacts with a target substance.
  • the substance 5B is, for example, a bioactive substance such as avidin, streptavidin, an antigen and an antibody.
  • the magnetic inclusion resin particles 2B have resin particles 211B, a first magnetic body 22B, and a resin layer 212B.
  • the magnetic inclusion resin particles 2B contain a first magnetic substance 22B inside.
  • the first magnetic substance 22B is contained in a layered manner.
  • the magnetic inclusion resin particles 2B have a magnetic layer containing the first magnetic body 22B inside.
  • the resin particles 211B do not contain the first magnetic material 22B.
  • the magnetic inclusion resin particles 2B contains the first magnetic body 22B inside.
  • a magnetic layer containing the first magnetic substance 22B is arranged on the outer surface of the resin particles 211B, and the resin layer 212B is arranged on the outer surface of the magnetic layer.
  • the magnetic layer 3B is arranged on the outer surface of the magnetic inclusion resin particles 2B.
  • the magnetic layer 3B contains a second magnetic material.
  • the shell layer 4B is arranged on the outer surface of the magnetic layer 3B.
  • the substance 5B is supported on the outer surface side of the magnetic layer 3B.
  • the substance 5B is supported on the outer surface of the shell layer 4B.
  • the shell layer 4B and the substance 5B are bonded.
  • the substance 5B exists on the surface of the magnetic particles 1B.
  • the magnetic layer 3B is a single magnetic layer.
  • the magnetic layer 3B covers the entire outer surface of the magnetic inclusion resin particles 2B.
  • the magnetic layer may be a single magnetic layer or a multi-layered magnetic layer composed of two or more layers.
  • the shell layer 4B is a single-layer shell layer.
  • the shell layer 4B covers the entire outer surface of the magnetic layer 3B.
  • FIG. 4 is a cross-sectional view schematically showing the magnetic particles according to the fourth embodiment of the present invention.
  • the magnetic particles 1C shown in FIG. 4 are used to specifically interact with the target substance.
  • the magnetic particles 1C include magnetic inclusion resin particles 2C, a magnetic layer 3C, a shell layer 4C, and a substance 5C that specifically interacts with a target substance.
  • the substance 5C is, for example, a bioactive substance such as avidin, streptavidin, an antigen and an antibody.
  • the magnetic inclusion resin particles 2C have resin particles 211C, first magnetic materials 221C and 222C, and a resin layer 212C.
  • the magnetic inclusion resin particles 2C contain the first magnetic substances 221C and 222C inside.
  • the first magnetic body 221C is dispersed inside the magnetic inclusion resin particles 2C.
  • the first magnetic material 221C is dispersed inside the resin particles 211C.
  • the first magnetic body 221C is dispersed inside the resin particles 211C.
  • the first magnetic substance 222C is contained in a layered manner.
  • the magnetic inclusion resin particles 2C have a magnetic layer containing the first magnetic substance 222C inside.
  • the magnetic inclusion resin particles 2C contains the first magnetic material 222C inside.
  • a magnetic layer containing the first magnetic substance 222C is arranged on the outer surface of the resin particles 211C, and the resin layer 212C is arranged on the outer surface of the magnetic layer.
  • the magnetic layer 3C is arranged on the outer surface of the magnetic inclusion resin particles 2C.
  • the magnetic layer 3C contains a second magnetic material.
  • the shell layer 4C is arranged on the outer surface of the magnetic layer 3C.
  • the substance 5C is supported on the outer surface side of the magnetic layer 3C.
  • the substance 5C is supported on the outer surface of the shell layer 4C.
  • the shell layer 4C and the substance 5C are bonded.
  • the substance 5C is present on the surface of the magnetic particles 1C.
  • the magnetic layer 3C is a single magnetic layer.
  • the magnetic layer 3C covers the entire outer surface of the magnetic inclusion resin particles 2C.
  • the magnetic layer may be a single magnetic layer or a multi-layered magnetic layer composed of two or more layers.
  • the shell layer 4C is a single-layer shell layer.
  • the shell layer 4C covers the entire outer surface of the magnetic layer 3C.
  • the magnetic inclusion resin particles contain a resin.
  • the magnetic inclusion resin particles include resin particles.
  • the magnetic encapsulating resin particles contain a first magnetic substance inside. It is preferable that the magnetic encapsulating resin particles contain the first magnetic substance inside by having the following constitution (1) or (2). (1) It contains resin particles and a first magnetic substance, and the first magnetic substance is dispersed inside the resin particles. (2) The resin particles, the first magnetic material, and the resin layer are included, and the first magnetic material is contained on the outer surface of the resin particles from the center of the magnetic inclusion resin particles toward the outer surface. The magnetic layer and the resin layer are arranged alternately.
  • the magnetic inclusion resin particles may have only the configuration (1), may have only the configuration (2), or may have the configuration (1) and the configuration (2).
  • the magnetic encapsulating resin particles have the configuration (2), it is preferable that a magnetic layer containing the first magnetic substance is arranged on the outer surface of the resin particles, and the magnetic encapsulating resin particles The outermost layer is preferably a resin layer.
  • the magnetic encapsulating resin particles having the above configuration (1) are, for example, the magnetic encapsulating resin particles shown in FIGS. 1 and 2.
  • the magnetic encapsulating resin particles having the above configuration (2) are, for example, the magnetic encapsulating resin particles shown in FIG.
  • the magnetic encapsulating resin particles having the above-mentioned configuration (1) and the above-mentioned configuration (2) are, for example, the magnetic encapsulating resin particles shown in FIG.
  • the magnetic encapsulating resin particles may have one resin layer and one magnetic layer containing the first magnetic material. Often, it may have two or more resin layers and two or more magnetic layers including the first magnetic material.
  • Resin particles and resin layer examples include polyolefin resins such as polyethylene, polypropylene, polystyrene, polyvinyl chloride, polyvinylidene chloride, polyisobutylene and polybutadiene; acrylic resins such as polymethylmethacrylate and polymethylacrylate; polycarbonate and polyamide.
  • divinylbenzene copolymer and the like examples include a divinylbenzene-styrene copolymer and a divinylbenzene- (meth) acrylic acid ester copolymer.
  • the material of the resin particles and the resin layer only one kind may be used, or two or more kinds may be used in combination. Further, when the magnetic inclusion resin particles have the above configuration (2), the material of the resin particles and the material of the resin layer may be the same or different.
  • the resin particles preferably have a porous structure.
  • the resin particles in the magnetic inclusion resin particles having the above configuration (1) preferably have a porous structure.
  • the resin particles in the magnetic inclusion resin particles having the above configuration (2) may or may not have a porous structure.
  • the BET specific surface area of the resin particles is preferably 20 m 2 / g or more, more preferably 40 m 2 / g or more, further preferably 100 m 2 / g or more, preferably 800 m 2 / g or less, and more preferably 700 m 2 It is / g or less, more preferably 650 m 2 / g or less.
  • the BET specific surface area is not less than the above lower limit and not more than the above upper limit, the content of the first magnetic substance contained in the magnetic inclusion resin particles can be increased, and the magnetic collection can be further enhanced.
  • the average pore diameter of the resin particles is preferably 0.5 nm or more, more preferably 1 nm or more, preferably 30 nm or less, and more preferably 10 nm or less.
  • the average pore diameter is equal to or greater than the above lower limit and equal to or less than the above upper limit, the magnetic substance can be more easily contained inside the resin particles, and the content of the first magnetic substance contained in the magnetic inclusion resin particles can be contained. The amount can be increased, and the magnetic collection can be further enhanced.
  • the BET specific surface area and the average pore diameter of the resin particles can be measured from the adsorption isotherm of nitrogen in accordance with the BJH method.
  • Examples of the measuring device for measuring the BET specific surface area and the average pore diameter of the resin particles include "NOVA4200e" manufactured by Cantachrome Instruments.
  • Resin particles satisfying the preferable ranges such as the BET specific surface area and the average pore diameter can be obtained, for example, by a method for producing resin particles including the following steps.
  • the polymerizable monomer include a monofunctional monomer and a polyfunctional monomer.
  • the organic solvent that does not react with the polymerizable monomer is not particularly limited as long as it is incompatible with a polar solvent such as water, which is a polymerization medium.
  • the organic solvent include cyclohexane, toluene, xylene, ethyl acetate, butyl acetate, allyl acetate, propyl acetate, chloroform, methylcyclohexane, methyl ethyl ketone and the like.
  • the amount of the organic solvent added is preferably 1 part to 80 parts by weight, more preferably 20 parts by weight to 60 parts by weight, based on 100 parts by weight of the polymerizable monomer component.
  • the amount of the organic solvent added is in the above-mentioned preferable range, the BET specific surface area, the above-mentioned average pore diameter, and the like can be controlled in a more suitable range, and it becomes easy to obtain dense pores inside the particles.
  • the average particle size of the resin particles is preferably 0.5 ⁇ m or more, more preferably 1 ⁇ m or more, preferably 10 ⁇ m or less, more preferably 7 ⁇ m or less, still more preferably 5 ⁇ m or less, particularly preferably 4 ⁇ m or less, and most preferably 3. It is 5 ⁇ m or less.
  • the average particle size of the resin particles is at least the above lower limit, the magnetism collection can be further enhanced.
  • the average particle size of the resin particles is not more than the above upper limit, the sedimentation of the magnetic particles can be effectively suppressed, the dispersibility can be further improved, and the particle size of the magnetic particles can be reduced.
  • the target substance can be effectively interacted with each other.
  • the average particle size of the resin particles is a number average particle size.
  • the average particle size of the resin particles is obtained by observing 50 arbitrary resin particles with an electron microscope or an optical microscope and calculating the average value of the particle size 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 first magnetic material is preferably a metal or a metal oxide, and more preferably a ferromagnetic material or a paramagnetic material.
  • the first magnetic material examples include iron, cobalt, nickel, ruthenium, lanthanoids, ferrite and the like.
  • the ferrite in chromite to mug ( ⁇ Fe 2 O 3) and MFe compounds represented by 2 O 4 (MFe 2 O 4 , M is, Co, Ni, Mn, Zn , Mg, Cu, Fe, Li 0 .5 Fe 0.5 etc.) and the like.
  • the ferrite is preferably ferric tetroxide (Fe 3 O 4 ).
  • the first magnetic material may be an alloy. Examples of the alloy include nickel-cobalt alloy, cobalt-tungsten alloy, iron-platinum alloy, iron-cobalt alloy and the like.
  • the said metal may be a metal ion.
  • the first magnetic material only one kind may be used, or two or more kinds may be used in combination.
  • the first magnetic material is preferably cobalt or ferrite, and more preferably cobalt or triiron tetroxide. It is preferable, and cobalt is more preferable.
  • the content of the first magnetic substance in 100% by volume of the magnetic particles is preferably 10% by volume or more, more preferably 20% by volume or more, preferably 80% by volume or less, and more preferably 70% by volume or less. ..
  • the content of the first magnetic substance is at least the above lower limit, the magnetism collection can be further enhanced.
  • the content of the first magnetic substance is not more than the above upper limit, the dispersibility can be further enhanced.
  • the content of the first magnetic material is preferably 10% by weight or more, more preferably 20% by weight or more, and preferably 90% by weight in the total of 100% by weight of the first magnetic material and the second magnetic material. % Or less, more preferably 85% by weight or less.
  • the content of the first magnetic substance is not less than the above lower limit and not more than the above upper limit, the magnetic collection can be further enhanced and the high magnetic collection can be maintained for a longer period of time.
  • the content of the first magnetic substance (hereinafter referred to as the content (1)) in 100% by volume of the region (R1) from the outer surface to the center of the magnetic inclusion resin particles to the thickness of 1/3 is described.
  • the content (1) is at least the above lower limit and at least the above upper limit, the magnetism collection can be further enhanced. Further, when the content (1) is not more than the above upper limit, the resin skeleton is well retained and the particle shape can be well maintained.
  • the region (R1) is a region outside the broken line L1 of the magnetic inclusion resin particles 2 in FIG.
  • the content of the first magnetic substance (hereinafter referred to as the content (2)) in 100% by volume of the region (R2) having a thickness of 2/3 from the center of the magnetic inclusion resin particles to the outer surface is described.
  • the region (R2) is a region inside the broken line L1 of the magnetic inclusion resin particles 2 in FIG.
  • the ratio of the content (1) to the content (2) is preferably 0.8 or more, more preferably 1.0 or more, and preferably 4 It is 0.0 or less, more preferably 3.0 or less.
  • the above ratio (content (1) / content (2)) is at least the above lower limit and at least the above upper limit, the magnetism collection and dispersibility can be further enhanced.
  • the absolute value of the difference between the content (1) and the content (2) is preferably 25% by volume or less, and more preferably 10% by volume or less.
  • the first magnetic material is preferably uniformly present inside the magnetic inclusion resin particles, and is preferably uniformly contained inside the magnetic inclusion resin particles.
  • the above-mentioned content (1) and the above-mentioned content (2) can be measured as follows.
  • the above-mentioned content (1) and the above-mentioned content (2) can be calculated.
  • the above-mentioned content (1) and the above-mentioned content (2) are averages calculated by arithmetically averaging the contents (1) and the contents (2) of 20 arbitrarily selected magnetic inclusion resin particles.
  • the content is preferably.
  • the method for producing the magnetic encapsulating resin particles is not particularly limited. For example, by mixing the resin particles having a porous structure and the first magnetic material and introducing the first magnetic material into the resin particles, a magnetic body having the above configuration (1) is provided. Encapsulating resin particles can be obtained. Further, for example, the resin particles having a solid structure and the first magnetic material are mixed, the outer surface of the resin particles is coated with the first magnetic material, and then the outer surface of the first magnetic material is coated. By coating the above with a resin, magnetic inclusion resin particles having the above configuration (2) can be obtained.
  • the magnetic particles according to the present invention include a magnetic layer containing a second magnetic material.
  • the magnetic layer containing the second magnetic material is arranged on the outer surface of the magnetic inclusion resin particles.
  • the first magnetic material and the second magnetic material may be the same or different.
  • the second magnetic material is preferably a metal or a metal oxide, and more preferably a ferromagnetic material or a paramagnetic material.
  • the second magnetic material examples include iron, cobalt, nickel, ruthenium, lanthanoids, ferrite and the like.
  • the ferrite in chromite to mug ( ⁇ Fe 2 O 3) and MFe compounds represented by 2 O 4 (MFe 2 O 4 , M is, Co, Ni, Mn, Zn , Mg, Cu, Fe, Li 0 .5 Fe 0.5 etc.) and the like.
  • the ferrite is preferably ferric tetroxide (Fe 3 O 4 ).
  • the second magnetic material may be an alloy. Examples of the alloy include nickel-cobalt alloy, cobalt-tungsten alloy, iron-platinum alloy, iron-cobalt alloy and the like.
  • the said metal may be a metal ion.
  • the second magnetic material only one kind may be used, or two or more kinds may be used in combination.
  • the second magnetic material is preferably ferrite, and more preferably triiron tetroxide.
  • the content of the second magnetic material is preferably 10% by weight or more, more preferably 30% by weight or more, preferably 90% by weight, based on 100% by weight of the total of the first magnetic material and the second magnetic material. % Or less, more preferably 70% by weight or less.
  • the content of the second magnetic substance is not less than the above lower limit and not more than the above upper limit, the magnetic collection can be further enhanced and the high magnetic collection can be maintained for a longer period of time.
  • the content of the second magnetic substance in 100% by volume of the magnetic particles is preferably 4% by volume or more, more preferably 8% by volume or more, preferably 40% by volume or less, and more preferably 25% by volume or less. ..
  • the content of the second magnetic substance is at least the above lower limit, the magnetism collection can be further enhanced.
  • the content of the second magnetic substance is not more than the above upper limit, the dispersibility can be further enhanced.
  • the total content of the first magnetic substance and the second magnetic substance in 100% by weight of the magnetic particles is preferably 10% by weight or more, more preferably 15% by weight or more, and preferably 95% by weight or less. , More preferably 80% by weight or less.
  • the total content is at least the above lower limit, the magnetism collection can be further enhanced.
  • the dispersibility can be further enhanced.
  • the total content of the first magnetic substance and the second magnetic substance in 100% by volume of the magnetic particles is preferably 20% by volume or more, more preferably 30% by volume or more, and preferably 80% by volume or less. , More preferably 70% by volume or less.
  • the total content is at least the above lower limit, the magnetism collection can be further enhanced.
  • the dispersibility can be further enhanced.
  • the surface area covered by the magnetic layer containing the second magnetic substance is preferably 70% or more, more preferably 80% or more, still more preferably. It is 95% or more, most preferably 100%.
  • the surface area is at least the above lower limit, the magnetic collection can be further enhanced, the high magnetic collection can be maintained for a longer period of time, and the dispersibility can be further enhanced.
  • the thickness of the magnetic layer is preferably 20 nm or more, more preferably 50 nm or more, preferably 1000 nm or less, and more preferably 200 nm or less.
  • the thickness of the magnetic layer is the thickness of the entire magnetic layer when the magnetic layer is multi-layered.
  • the thickness of the magnetic layer is at least the above lower limit, the magnetic collection can be further enhanced, and the high magnetic collection can be maintained for a longer period of time.
  • the thickness of the magnetic layer is not more than the above upper limit, the sedimentation of the magnetic particles can be effectively suppressed, the dispersibility can be further improved, and the particle size of the magnetic particles can be reduced, so that the magnetic particles can be reduced.
  • the amount of binding of the target substance per unit weight of the above can be increased.
  • the thickness of the magnetic layer can be measured by observing the cross section of the magnetic particles, for example, using a transmission electron microscope (TEM). Regarding the thickness of the magnetic layer, it is preferable to calculate the average value of five thicknesses of any magnetic layer as the thickness of the magnetic layer of one magnetic particle, and the average value of the thickness of the entire magnetic layer is one magnetic. It is more preferable to calculate as the thickness of the magnetic layer of the particles.
  • the thickness of the magnetic layer is preferably obtained by calculating the average value of the thickness of the magnetic layer of each magnetic particle for 10 arbitrary magnetic particles.
  • the magnetic layer may be a continuous layer, or may be a layer formed by particles in which the second magnetic material is aggregated in the form of particles. Further, the magnetic layer may be entirely covered on the outer surface of the resin particles, or may be partially covered. Further, the magnetic layer may have a sea-island structure. From the viewpoint of effectively suppressing the decrease in magnetic collection, the magnetic layer is preferably a continuous layer.
  • the continuous layer has a structure having few seams or no seams, unlike a shape in which innumerable seams (for example, 1000 or more per magnetic particle) exist in the magnetic layer such as an aggregate of fine particles. Point to. From the viewpoint of further enhancing the magnetic collection, the magnetic layer is preferably a layer formed by particles in which the second magnetic material is aggregated in the form of particles.
  • the average particle size of the particles is preferably 1 nm or more, more preferably 2 nm or more, preferably 2 nm or more. Is 50 nm or less, more preferably 20 nm or less.
  • the average particle size of the granules is at least the above lower limit, the magnetism collection can be further enhanced.
  • the second magnetic substance can be satisfactorily arranged on the outer surface of the magnetic inclusion resin particles, and the magnetic layer can be satisfactorily formed.
  • the method for forming the magnetic layer is not particularly limited. For example, by mixing the magnetic inclusion resin particles and the particles of the second magnetic substance, a magnetic layer can be formed on the outer surface of the magnetic inclusion resin particles.
  • the magnetic particles according to the present invention preferably include a shell layer.
  • the material of the shell layer contains an inorganic oxide or an organic polymer.
  • the shell layer is an inorganic oxide shell layer containing the inorganic oxide as the material of the shell layer, or an organic polymer shell layer containing the organic polymer as the material of the shell layer.
  • the shell layer is preferably arranged on the outer surface of the magnetic layer containing the second magnetic material.
  • the magnetic particles include a shell layer, it is possible to strongly prevent the elution of impurities from the magnetic inclusion resin particles and the like, the elution of the magnetic material, and the elution of impurities from the magnetic layer. Therefore, when the magnetic particles include the shell layer, they can be suitably used as a test agent.
  • the shell layer may or may not contain a magnetic material. It is more preferable that the shell layer does not contain a magnetic material.
  • the shell layer is more preferably a non-magnetic layer containing no magnetic material.
  • the material of the shell layer preferably contains the above-mentioned inorganic oxide, and more preferably an inorganic oxide shell layer containing the inorganic oxide.
  • the inorganic oxide means a compound having at least a metal element or a metalloid element and an oxygen atom.
  • the inorganic oxide is not particularly limited. Only one kind of the above-mentioned inorganic oxide may be used, or two or more kinds may be used.
  • the inorganic oxide is preferably an inorganic oxide having a silicon atom, a germanium atom, a titanium atom or a zirconium atom. Further, the inorganic oxide preferably has a functional group capable of reacting with the outer surface of the magnetic layer.
  • the method for reacting the magnetic surface with the non-magnetic layer is not particularly limited, and examples thereof include covalent bonds and coordination bonds.
  • the inorganic oxide examples include an alkoxysilane such as tetraethyl orthosilicate and a silane compound typified by a hydrolyzate thereof, and an alkoxygermanium such as germanium tetraethoxydo and a germanium compound typified by the hydrolyzate thereof.
  • an alkoxysilane such as tetraethyl orthosilicate and a silane compound typified by a hydrolyzate thereof
  • an alkoxygermanium such as germanium tetraethoxydo and a germanium compound typified by the hydrolyzate thereof.
  • Titanium compounds typified by alkoxytitanium such as titanium tetraethoxydo and its hydrolyzate
  • zirconium compounds such as zirconium tetrabutoxide and zirconium compounds typified by its hydrolyzate.
  • the inorganic oxide is preferably a compound having a small specific gravity, and among the above examples, the above silane compound is most preferable.
  • silane compound examples include tetraethyl orthosilicate; a vinyl group-containing silane compound such as vinyltrimethoxysilane, vinyltriethoxysilane, and 7-octenyltrimethoxysilane; 2- (3,4-epoxycyclohexyl) ethyltrimethoxy.
  • Silane 3-glycidoxypropylmethyldimethoxysilane, 3-glycidoxypropyltrimethoxysilane, 3-glycidoxypropylmethyldiethoxysilane, 3-glycidoxypropyltriethoxysilane, 8-glycidoxyoctyltri Epoxy group-containing silane compounds such as methoxysilane; styryl group-containing silane compounds such as p-styryltrimethoxysilane; 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropylmethyldiethoxy Methacrylic group-containing silane compounds such as silane, 3-methacryloxypropyltriethoxysilane, 8-methacryloxyoctyltrimethoxysilane; acrylic group-containing silane compounds such as 3-acryloxypropyltrimethoxysilane; N-2- (aminoethyl)
  • an inorganic oxide shell layer having a silicon atom can be formed on the outer surface of the magnetic layer.
  • the content of the inorganic oxide in 100% by weight of the inorganic oxide shell layer is preferably 70% by weight or more, more preferably 80% by weight or more.
  • Organic polymer is not particularly limited, but is preferably a vinyl polymer.
  • vinyl-based monomer used as the material for the vinyl-based polymer examples include styrene monomers such as styrene, ⁇ -methylstyrene, chlorostyrene, and divinylbenzene; vinyl ether compounds such as methylvinyl ether, ethylvinyl ether, and propylvinyl ether; vinyl acetate, Acid vinyl ester compounds such as vinyl butyrate, vinyl laurate, vinyl stearate; halogen-containing monomers such as vinyl chloride and vinyl fluoride; (meth) acrylic acid; methyl (meth) acrylate, ethyl (meth) acrylate, propyl (Meta) acrylate, butyl (meth) acrylate, 2-ethylhexyl (meth) acrylate, lauryl (meth) acrylate, cetyl (meth) acrylate, stearyl (meth) acrylate, cyclohexyl (meth)
  • Halogen-containing (meth) acrylate compound such as trifluoromethyl (meth) acrylate and pentafluoroethyl (meth) acrylate; vinyl trimethoxysilane, vinyl triethoxysilane, dimethoxymethyl vinyl cysilane, dimethoxyethyl vinyl silane, diethoxy Methylvinylsilane, diethoxyethylvinylsilane, ethylmethyldivinylsilane, methylvinyldimethoxysilane, ethylvinyldimethoxysilane, methylvinyldiethoxysilane, ethylvinyldiethoxysilane, p-styryltrimethoxysilane, 3-methacryloxypropylmethyldimethoxysilane , 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-meth
  • the vinyl-based polymer may be a homopolymer obtained by polymerizing one kind of vinyl-based monomer, or may be a copolymer obtained by polymerizing two or more kinds of vinyl-based monomers.
  • a copolymerization monomer such as a vinyl monomer as a main raw material and, if necessary, a polymerization initiator, an emulsifier, a dispersant, a surfactant, an electrolyte, a cross-linking agent, and a molecular weight as auxiliary raw materials.
  • An organic polymer shell layer can be formed on the outer surface of the magnetic layer by adding a modifier or the like and polymerizing in a liquid.
  • the content of the organic polymer in 100% by weight of the organic polymer shell layer is preferably 70% by weight or more, more preferably 80% by weight or more.
  • the shell layer has a carboxyl group, a hydroxyl group, an epoxy group, an amino group, a tosyl group, a thiol group, a triethylammonium group, a dimethylamino group and a sulfonic acid before binding to the substance that specifically interacts with the target substance. It is preferable to have a functional group such as a group.
  • the substance that specifically interacts with the target substance can be well supported on the outer surface of the shell layer, and the substance can be supported on the surface of the magnetic particles. It can be arranged well.
  • the shell layer may have a linker portion on the outer surface.
  • the functional group which is a bonding point with the substance that specifically interacts with the target substance, is arranged at a position farther from the outermost surface of the shell layer.
  • the functional group and the substance can come into contact with each other at a position where there are less steric obstacles. Therefore, the substance is easily bonded, and the substance can be satisfactorily arranged on the surface of the magnetic particles.
  • the linker portion can be covalently bonded to a target substance such as a carboxyl group, a hydroxyl group, an epoxy group, an amino group, a tosyl group, or a thiol group at the terminal before binding to the above substance that specifically interacts with the target substance. It is preferable to have a functional group. By reacting the functional group with the functional group of the substance that specifically interacts with the target substance, the shell layer and the substance can be chemically bonded.
  • the epoxy group may be an epoxy group derived from a glycidyl group-containing monomer.
  • the hydroxyl group may be a hydroxyl group generated by ring-opening of the epoxy group.
  • the material of the linker portion is preferably an epoxy compound having a plurality of epoxy groups at the ends.
  • the epoxy compound having a plurality of epoxy groups at the end is preferably polyethylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, neopentyl glycol diglycidyl ether, or trimethylol propanepolyglycidyl ether.
  • the epoxy compound having a plurality of epoxy groups at the end is more preferably polyethylene glycol diglycidyl ether.
  • the shell layer and the substance that specifically interacts with the target substance are preferably bonded, and more preferably chemically bonded.
  • the surface area covered by the shell layer is preferably 95% or more, more preferably 99% or more, and most preferably 100%. is there.
  • the surface area is equal to or greater than the above lower limit, the content of the substance that specifically interacts with the target substance can be increased, and as a result, the measurement accuracy and measurement in the measurement of the target substance using magnetic particles The sensitivity can be increased.
  • the thickness of the shell layer is preferably 20 nm or more, more preferably 40 nm or more, preferably 500 nm or less, and more preferably 300 nm or less.
  • the thickness of the shell layer is not less than the above lower limit and not more than the above upper limit, the sedimentation of the magnetic particles can be effectively suppressed, the dispersibility can be further improved, and the particle diameter of the magnetic particles can be reduced. Therefore, the target substance can be effectively bound.
  • the thickness of the shell layer can be measured by observing the cross section of the magnetic particles, for example, using a transmission electron microscope (TEM). Regarding the thickness of the shell layer, it is preferable to calculate the average value of the thickness of any shell layer at five points as the thickness of the shell layer of one magnetic particle, and the average value of the thickness of the entire shell layer is one magnetism. It is more preferable to calculate as the thickness of the shell layer of particles.
  • the thickness of the shell layer is preferably obtained by calculating the average value of the thickness of the shell layer of each magnetic particle for 10 arbitrary magnetic particles.
  • Substances that specifically interact with the target substance On the surface of the magnetic particles according to the present invention, there is a substance that specifically interacts with the target substance.
  • the substance include sugar chains, peptide chains, proteins, antigens, nucleotide chains and the like.
  • the substance can be appropriately changed depending on the type of the target substance. Only one type of the above substance may be used, or two or more types may be used in combination.
  • 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 interaction between the substance and the target substance, or may be a covalent bond between the substance and the target substance.
  • Examples of the non-covalent interaction include hydrophobic interaction, electrostatic interaction, van der Waals force, hydrogen bond, coordination bond, ionic bond and the like.
  • the substance that specifically interacts with the target substance is preferably a substance capable of non-covalent interaction with the target substance.
  • the combination of the above-mentioned substance that specifically interacts with the above-mentioned target substance and the target substance includes a combination of an antibody and an antigen, a combination of a sugar chain and a protein such as a lectin, and a combination of a protein such as an enzyme and an inhibitor.
  • Examples thereof include 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.
  • the substance that specifically interacts with the target substance is preferably an antigen or an antibody.
  • the above antibody may be a polyclonal antibody or a monoclonal antibody.
  • the antibody may be treated with a proteolytic enzyme such as papain and pepsin, or may be a Fab and F (ab') 2 fragment or the like.
  • the method of arranging the substance that specifically interacts with the target substance is not particularly limited.
  • the substance can be arranged on the surface of the magnetic particles by mixing the substance with the magnetic particles before the substance is supported.
  • the target substance is a substance that specifically interacts with the above-mentioned substance.
  • target substance examples include proteins, nucleic acids, hormones, cancer markers, respiratory-related markers, heart disease markers, drugs and the like.
  • lipid proteins such as high specific gravity lipoprotein (HDL), low specific gravity lipoprotein (LDL), and ultralow specific gravity lipoprotein; alkaline phosphatase, amylase, acidic phosphatase, ⁇ -glutamyltransferase ( ⁇ -GTP), lipase.
  • HDL high specific gravity lipoprotein
  • LDL low specific gravity lipoprotein
  • ⁇ -GTP ultralow specific gravity lipoprotein
  • CK Cleatin kinase
  • LDH lactic acid dehydrogenase
  • GAT glutamate oxaloacetate transaminase
  • GPT glutamate pyruvate transaminase
  • PK protein kinase
  • Antibodies such as streptidine O antibody, anti-human hepatitis B virus surface antigen antibody (HBs antigen), anti-human hepatitis C virus antibody, anti-rheumatic factor; albumin, hemoglobin, myoglobin, transferase, protein A, C reactive protein ( CRP) and
  • nucleic acid examples include DNA and RNA.
  • hormone examples include thyroid stimulating hormone (TSH), thyroid hormone (FT3, FT4, T3, T4), parathyroid hormone (PTH), and human chorionic gonadotropin (hCG) estradiol (E2).
  • TSH thyroid stimulating hormone
  • FT3, FT4, T3, T4 thyroid hormone
  • PTH parathyroid hormone
  • hCG human chorionic gonadotropin estradiol
  • cancer marker examples include ⁇ -fetoprotein (AFP), PIVKA-II, carcinoembryonic antigen (CEA), CA19-9, and prostate-specific antigen (PSA).
  • AFP ⁇ -fetoprotein
  • PIVKA-II carcinoembryonic antigen
  • CA19-9 carcinoembryonic antigen
  • PSA prostate-specific antigen
  • Examples of the respiratory-related marker include KL-6 and the like.
  • Examples of the above-mentioned heart disease marker include troponin T (TnT), human brain natriuretic peptide precursor N-terminal fragment (NT-proBNP), and the like.
  • TnT troponin T
  • NT-proBNP human brain natriuretic peptide precursor N-terminal fragment
  • Examples of the above-mentioned drugs include antiepileptic drugs, antibiotics, theophylline and the like.
  • the magnetic particles are used for radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), chemiluminescence immunoassay (ECLIA), chemiluminescence immunoassay (CLIA and CLEIA), and absorbance measurement. , And in the measurement of surface plasmon resonance and the like, it is preferably used.
  • the magnetic particles are preferably used in the measurement of the sandwich method and the competitive method.
  • the magnetic particles are suitably used for measuring the concentration of the target substance in a sample.
  • the magnetic particles are preferably used as a test agent.
  • the magnetic particles are preferably magnetic particles that can be used as a test agent.
  • the concentration of the target substance can be measured using the magnetic particles as follows, for example.
  • a liquid containing the above magnetic particles (for example, a test agent described later) and a sample containing the target substance are mixed to obtain a mixed liquid.
  • the obtained mixed solution is heated or the like to obtain a reaction solution in which the substance that specifically interacts with the target substance in the magnetic particles and the target substance in the sample are bound to each other (first reaction step).
  • a magnetic force is applied to the reaction solution by a magnet or the like to collect magnetic particles (magnetic collection step).
  • a washing solution is added and mixed (washing step).
  • the magnetic collection step and the cleaning step may be repeated a plurality of times.
  • the target substance and the labeling substance are reacted to measure the concentration of the target substance (second reaction step).
  • labeling substance examples include alkaline phosphatase, ⁇ -galactosidase, peroxidase, microperoxidase, glucose oxidase, glucose-6-phosphate dehydrogenase, malic acid dehydrogenase, which are preferably used in enzyme immunoassay (EIA).
  • Enzymes such as luciferase, tyrosinase, acidic phosphatase; radioactive isotopes such as 99mTc, 131I, 125I, 14C, 3H, 32P preferably used in radioimmunosassay (RIA); suitable in fluorescence immunoassay (FIA), for example.
  • Fluorescent substances such as fluorescein, dancil, fluorescamine, coumarin, naphthylamine and derivatives thereof, and fluorescent substances such as green fluorescent protein (GFP) used in, for example, luciferin, isolminol, luminol, bis (2,4,6-trifluorophenyl).
  • GFP green fluorescent protein
  • Luminescent substances such as oxalate, such as phenol, naphthol, anthracene and derivatives thereof that have ultraviolet absorption, such as 4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl, 3 -Amino-2,2,5,5-tetramethylpyrrolidin-1-oxyl, 2,6-di-t-butyl- ⁇ - (3,5-di-t-butyl-4-oxo-2,5-)
  • spin labeling agents such as compounds having an oxyl group such as cyclohexadien-1-iriden) -p-trioxyl.
  • the labeling substance is preferably an enzyme or a fluorescent substance, more preferably alkaline phosphatase, peroxidase or glucose oxidase, and even more preferably peroxidase.
  • Test drug The above-mentioned test agent contains the above-mentioned magnetic particles.
  • the above test agent preferably contains a buffer solution.
  • the buffer solution is preferably a buffer solution having a buffering ability at pH 5.0 or more and 9.0 or less.
  • the buffer solution include phosphate buffer solution, glycine buffer solution, veronal buffer solution, Tris buffer solution, borate buffer solution, citrate buffer solution, and Good buffer solution.
  • test agent may contain a sensitizer, a polymer compound such as a protein, an amino acid, and other components such as a surfactant.
  • the reaction between the target substance and the compound capable of binding to the target substance can be efficiently promoted, and the measurement accuracy can be improved.
  • the sensitizer include alkylated polysaccharide compounds such as methyl cellulose and ethyl cellulose, pullulan, polyvinylpyrrolidone and the like.
  • protein examples include albumin (bovine serum albumin, egg albumin, etc.), casein, gelatin, and the like.
  • the content of the magnetic particles in 100% by weight of the test agent is preferably 0.5% by weight or more, more preferably 2% by weight or more, preferably 10% by weight or less, and more preferably 5% by weight or less.
  • the measurement accuracy of the target substance can be further improved.
  • Example 1 Preparation of magnetic encapsulating resin particles: Polystyrene particles having an average particle diameter of 0.69 ⁇ m were prepared as seed particles. A mixed solution was prepared by mixing 3.9 parts by weight of the polystyrene particles, 500 parts by weight of ion-exchanged water, and 120 parts by weight of a 5.0% by weight polyvinyl alcohol aqueous solution. After the above mixed solution was dispersed by ultrasonic waves, it was placed in a separable flask and stirred uniformly.
  • the emulsion was added to the mixed solution in the separable flask in several portions and stirred for 12 hours to allow the seed particles to absorb the monomer to obtain a suspension containing the seed particles in which the monomer was swollen. ..
  • Magnetic layer (magnetic layer containing a second magnetic material): 1 part by weight of magnetic inclusion resin particles and 4 parts by weight of magnetic fluid EMG707 (aqueous dispersion of triiron tetroxide nanoparticles with an average particle diameter of about 10 nm, manufactured by Fellow Tech) (however, the content of magnetic material is about about 17% by weight) was stirred at 250 rpm for 10 minutes. The obtained particle dispersion is filtered and washed with water to form a magnetic layer containing triiron tetroxide as a second magnetic substance on the outer surface of the magnetic inclusion resin particles to form a magnetic layer. The magnetic encapsulating resin particles having were obtained.
  • EMG707 aqueous dispersion of triiron tetroxide nanoparticles with an average particle diameter of about 10 nm, manufactured by Fellow Tech
  • the obtained particle dispersion is filtered and washed with water to form a magnetic layer containing triiron tetroxide as a second magnetic substance on the outer surface of the magnetic inclusion
  • Arrangement of substances that specifically interact with the target substance 0.5 mL of an aqueous dispersion of magnetic encapsulating resin particles having a magnetic layer was added to a test tube, and the mixture was washed 3 times with a PBS solution. After removing the dispersion medium, 0.5 mL of a PBS solution (0.75 mg / mL) containing a sialylated sugar chain antigen KL-6 (hereinafter abbreviated as KL-6) antibody was added, and the mixture was stirred at 25 ° C. overnight. Then, 1.5 mL of 1.0 wt% BSA solution was added, and the mixture was stirred at 25 ° C. for 4 hours.
  • KL-6 sialylated sugar chain antigen KL-6
  • the average particle size, BET specific surface area and average pore size of the obtained resin particles, and the contents and contents (1) of the first magnetic substance and the second magnetic substance in the obtained magnetic particles are shown. Table 1 shows the ratio to the amount (2), the absolute value of the difference between the content (1) and the content (2), and the average particle size. These were obtained by the method described in the evaluation items described later.
  • Example 2 Magnetic particles were prepared in the same manner as in Example 1 except that resin particles were prepared using 80 parts by weight of toluene instead of 150 parts by weight of toluene and the composition of the magnetic particles was changed as shown in Table 1. did.
  • Example 3 Magnetic particles were prepared in the same manner as in Example 1 except that resin particles were prepared using 30 parts by weight of toluene instead of 150 parts by weight of toluene and the composition of the magnetic particles was changed as shown in Table 1. did.
  • Example 4 In the same manner as in Example 1, magnetic inclusion resin particles having a magnetic layer were obtained.
  • Shell layer formation To 1.0 part by weight of the magnetic encapsulating resin particles having the obtained magnetic layer, 400 parts by weight of ethanol and 20 parts by weight of a 28% aqueous ammonia solution (manufactured by Nacalai Tesque) were added. Then, 5.0 parts by weight of tetraethyl orthosilicate and 15 parts by weight of 8-glycidoxyoctyltrimethoxysilane were added, and the mixture was stirred for 1 hour. The obtained dispersion was filtered and then washed with water. In this way, magnetic inclusion particles having an inorganic oxide shell layer having a functional group on the surface were obtained.
  • Example 5 Magnetic particles were obtained in the same manner as in Example 4 except that the configurations of the resin particles and the magnetic particles were changed as shown in Table 1.
  • Comparative Example 1 No toluene was used, 300 parts by weight of divinylbenzene was used instead of 150 parts by weight of divinylbenzene, and 4.0 parts by weight of benzoyl peroxide was used instead of 2.0 parts by weight of benzoyl peroxide.
  • Resin particles were obtained in the same manner as in Example 1 except for the above. Further, in Comparative Example 1, the first magnetic material was not used. Using the obtained resin particles, a magnetic layer was formed on the outer surface of the resin particles by a second magnetic material in the same manner as in Example 1.
  • Shell layer formation Using the obtained resin particles having a magnetic layer, a shell layer was formed on the outer surface of the magnetic layer in the same manner as in Example 4.
  • Comparative Example 2 Resin particles were obtained in the same manner as in Comparative Example 1. Next, acetone was added to an oil-based magnetic fluid (organic solvent dispersion of triiron tetroxide nanoparticles with an average particle diameter of about 10 nm, "EXP series” manufactured by Fellow Tech) to precipitate and precipitate the particles, and then dried. By doing so, ferrite-based magnetic fine particles (average primary particle diameter: 10 nm) having a hydrophobically treated surface were obtained.
  • oil-based magnetic fluid organic solvent dispersion of triiron tetroxide nanoparticles with an average particle diameter of about 10 nm, "EXP series” manufactured by Fellow Tech
  • Shell layer formation 10 parts by weight of the obtained particles having a magnetic layer and 300 parts by weight of a 0.5% aqueous solution of a nonionic emulsifier (“Emulgen 150” manufactured by Kao Corporation) as a dispersant were put into a 1 L separable flask and stirred.
  • a nonionic emulsifier (“Emulgen 150” manufactured by Kao Corporation) as a dispersant
  • 24 parts by weight of cyclohexyl methacrylate and 60 parts by weight of 2-methacryloyloxyethyl succinic acid were used as monomers, and di (3,5,5-trimethylhexanoyl) peroxide (NOF Corporation "Perloyl”) was used as an initiator.
  • 355 1.0 part by weight was added, and the mixture was stirred at 80 ° C.
  • the organic polymer is a copolymer of cyclohexyl methacrylate and 2-methacryloyloxyethyl succinic acid.
  • Comparative Example 3 Magnetic particles were obtained in the same manner as in Comparative Example 1 except that the configurations of the resin particles and the magnetic particles were changed as shown in Table 1. The magnetic particles obtained in Comparative Example 3 do not have the first magnetic material.
  • Comparative Example 4 Magnetic particles were produced in the same manner as in Example 1 except that the configurations of the resin particles and the magnetic particles were changed as shown in Table 1. The magnetic particles obtained in Comparative Example 4 do not have a magnetic layer.
  • Comparative Example 5 Magnetic particles were obtained in the same manner as in Comparative Example 4, except that the configurations of the resin particles and the magnetic particles were changed as shown in Table 1.
  • a shell layer organic polymer shell layer
  • PGMA polyglycidyl methacrylate
  • the measurement was performed to obtain the distribution results of the contents of the first magnetic substance and the second magnetic substance inside the magnetic particles. From the obtained results, the contents of the first magnetic substance and the second magnetic substance were calculated. The contents of the first magnetic material and the second magnetic material were calculated by arithmetically averaging the contents of the first magnetic material and the second magnetic material of 20 arbitrarily selected magnetic particles.
  • the content of the first magnetic substance was defined as the content (1) in 100% by volume of the region (R1) having a thickness of 1/3 from the outer surface to the inside of the magnetic particles.
  • the content of the first magnetic substance was defined as the content (2) in 100% by volume of the region (R2) having a thickness of 2/3 from the center of the magnetic particles to the outside.
  • An embedded resin body for magnetic particle inspection was prepared by adding and dispersing it to "Technobit 4000" manufactured by Kulzer so that the content of magnetic particles was 30% by weight.
  • a cross section of the magnetic particles was cut out using an ion milling device (“IM4000” manufactured by Hitachi High-Technologies Corporation) so as to pass near the center of the magnetic particles dispersed in the embedded resin body for inspection.
  • IM4000 manufactured by Hitachi High-Technologies Corporation
  • JEM-2010FEF electric field radiation type transmission electron microscope
  • EDS energy dispersive X-ray analyzer
  • the content was measured, and the distribution result of the content of the first magnetic substance in the thickness direction of the magnetic inclusion resin particles was obtained. From the obtained results, the above-mentioned content (1) and the above-mentioned content (2) were calculated.
  • the content (1) and the content (2) were calculated by arithmetically averaging the content (1) and the content (2) of 20 arbitrarily selected magnetic particles.
  • Magnetic Collection Rate As a sample solution, a solution in which magnetic particles whose absorbance at a wavelength of 550 nm was adjusted to 0.9 to 1.1 was dispersed in water was prepared. Sample solution 1. In a quartz cell installed in a spectrophotometer (“U-3900H” manufactured by Hitachi, Ltd.) with a magnet (2800G, W10mm ⁇ D10mm ⁇ H1mm) applied via a spacer (W10mm ⁇ D10mm ⁇ H4mm). 3 mL was added, and the absorbance at a wavelength of 550 nm was measured 5 to 125 seconds after the sample solution was added. The absorbance attenuation rate in 120 seconds was calculated by the following formula and used as the magnetic collection rate.
  • Magnetic collection rate (%) [ ⁇ (absorbance after 5 seconds)-(absorbance after 125 seconds) ⁇ / (absorbance after 5 seconds)] x 100
  • Dispersion rate As a sample solution, a solution in which magnetic particles whose absorbance at a wavelength of 550 nm was adjusted to 0.9 to 1.1 was dispersed in water was prepared. 1.3 mL of the sample solution was put into a quartz cell installed in a spectrophotometer (“U-3900H” manufactured by Hitachi, Ltd.), and the absorbance at a wavelength of 550 nm was measured. Next, a magnet (28000 G, W40 mm ⁇ D40 mm ⁇ H10 mm) was used to collect magnetism until the absorbance of the supernatant became zero. Then, the magnetic particles were dispersed by vortex at 2000 rpm for 5 seconds, and the absorbance at a wavelength of 550 nm was measured. From the absorbance before magnetic collection and the absorbance after magnetic collection and dispersion, the rate of change in absorbance was calculated by the following formula and used as the dispersion rate.
  • Dispersion rate (%) ⁇ (absorbance after magnetic collection and dispersion) / (absorbance before magnetic collection) ⁇ x 100
  • Dispersion rate is 95% or more ⁇ : Dispersion rate is 90% or more and less than 95% ⁇ : Dispersion rate is 85% or more and less than 90% ⁇ : Dispersion rate is less than 85%
  • Magnetic collection rate (after leaving for 1 month) The obtained magnetic particles were left at 25 ° C. for 1 month. After being left to stand, the magnetic collection coefficient was determined by the same method as in (5) magnetic collection rate.
  • ruthenium complex-labeled anti-KL-6 antibody (secondary antibody): To a polypropylene tube, 0.5 mL of a PBS-1 solution of anti-KL-6 antibody (anti-KL-6 antibody concentration 2.0 mg / mL) was added, and then 13 ⁇ L of Ru-NHS (10 mg / mL) was added. After vibrating and stirring at 25 ° C., purification was performed using a Sephadex G25 column to obtain a ruthenium complex-labeled anti-KL-6 antibody.
  • the amount of luminescence was measured as follows using an ECLIA automatic analyzer (“Picormi III” manufactured by Sekisui Medical Co., Ltd.) based on the electrochemical luminescence immunoassay method.
  • the reaction solution buffer solution containing normal rabbit serum
  • 20 ⁇ L of a solution containing 5000 U / mL KL-6 (antigen-containing solution) was added, and then 25 ⁇ L of magnetic particles were added.
  • 350 ⁇ L of Picormi BF washing solution (10 mM Tris buffer) was added, and the magnetic particles were washed three times while being trapped with a magnet.

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Abstract

L'invention concerne des particules magnétiques améliorées en termes de magnétisme et de dispersibilité, et pouvant maintenir un niveau élevé de magnétisme.　Les particules magnétiques selon la présente invention sont utilisées pour interagir spécifiquement avec une substance cible. Les particules magnétiques comprennent chacune : une particule de résine contenant un matériau magnétique contenant un premier matériau magnétique ; une couche magnétique disposée sur la surface externe de la particule de résine contenant un matériau magnétique et contenant un second matériau magnétique ; et une substance qui est supportée sur le côté de la surface extérieure de la couche magnétique et qui interagit spécifiquement avec la substance cible.
PCT/JP2020/013814 2019-03-26 2020-03-26 Particules magnétiques et agent de test WO2020196786A1 (fr)

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JPH1087711A (ja) * 1996-09-19 1998-04-07 Japan Synthetic Rubber Co Ltd 磁性ポリマー粒子の製造方法
JP2006307126A (ja) * 2005-03-31 2006-11-09 Jsr Corp 多孔質表面を有する磁性粒子およびその製造方法、ならびに生化学用担体
US20060269751A1 (en) * 2005-05-20 2006-11-30 Winstead J Magnetically-responsive microparticles with improved response times
JP2010132513A (ja) * 2008-12-08 2010-06-17 Sekisui Chem Co Ltd 磁性体内包粒子、磁性体内包粒子の製造方法、免疫測定用粒子、及び、イムノクロマトグラフィ法

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CN102194562A (zh) * 2010-02-09 2011-09-21 日立麦克赛尔株式会社 生物用磁性球的制造方法以及磁性球和磁性物
WO2016002742A1 (fr) * 2014-07-01 2016-01-07 新日鉄住金化学株式会社 Composite de résine-métal, substance de marquage, procédé d'immuno-essai, réactif d'immunoessai, procédé permettant la mesure d'un analyte, kit de mesure d'analyte et bandelette d'essai immunochromatographique sur membrane
WO2017204209A1 (fr) * 2016-05-24 2017-11-30 Jsr株式会社 Particules composites ainsi que procédé de fabrication de celles-ci, particules revêtues, support en phase solide comprenant un ligand, et procédé de détection ou séparation de substance cible dans un échantillon
JP6935644B2 (ja) * 2017-02-17 2021-09-15 国立大学法人東北大学 磁性体複合粒子およびその製造方法、並びに免疫測定用粒子

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH05158286A (ja) * 1991-12-03 1993-06-25 Mita Ind Co Ltd 磁性キャリヤ
JPH1087711A (ja) * 1996-09-19 1998-04-07 Japan Synthetic Rubber Co Ltd 磁性ポリマー粒子の製造方法
JP2006307126A (ja) * 2005-03-31 2006-11-09 Jsr Corp 多孔質表面を有する磁性粒子およびその製造方法、ならびに生化学用担体
US20060269751A1 (en) * 2005-05-20 2006-11-30 Winstead J Magnetically-responsive microparticles with improved response times
JP2010132513A (ja) * 2008-12-08 2010-06-17 Sekisui Chem Co Ltd 磁性体内包粒子、磁性体内包粒子の製造方法、免疫測定用粒子、及び、イムノクロマトグラフィ法

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