JP4485823B2 - Magnetic inclusion particles and immunoassay particles - Google Patents

Description

The present invention relates to magnetic inclusion particles having uniform magnetism, excellent dispersion stability, and a narrow particle size distribution, and immunoassay particles using the same.

Conventionally, as a method for producing magnetic substance-containing polymer particles, (1) a method for producing a magnetic substance by adding iron ions to the produced polymer particles, and (2) a process for polymerizing polymer particles from monomers. There are known a method of including the magnetic particles (see Patent Document 1), and (3) a method of combining separately prepared polymer particles and magnetic particles (see Patent Document 2). In addition, (4
There is a method of coating magnetic particles with a polymer or the like (see Patent Document 3).

In the method (1), since iron ions are absorbed by polymer particles, there is a problem that the magnetic material is exposed on the surface and the magnetic material is oxidized. Further, the method (2) has a problem that the magnetic particles are not uniformly incorporated into the polymer particles, and a problem that the particle size is difficult to control and the particle size distribution becomes wide. Further, the method (3) has a problem that the polymer particles are aggregated and cannot be used for particles having a small particle size. Further, in the method (4), since the coating cannot be made uniform, the floatability and dispersibility are poor, and a part of the surface of the magnetic particles may be exposed.

On the other hand, radioimmunoassay, enzyme immunoassay, fluorescent immunoassay and the like have been conventionally known as microimmunoassay methods and have already been put into practical use. These methods are methods for detecting the presence or absence of an antibody or antigen that specifically reacts with an antigen or an antibody to which an isotope, enzyme, or fluorescent substance is added as a label, respectively. In such an immunoassay, magnetic particles are used to perform B / F separation efficiently and simply. In addition, uses other than B / F separation (see Patent Document 4) and immunoassay methods (see Patent Document 5, Patent Document 6, and Patent Document 7) using magnetic inclusion particles themselves as labeling materials are disclosed.

JP-A-9-208788 JP-A-6-231957 JP-A-6-92640 JP 2000-88852 A JP-A-6-148189 JP 7-225233 A JP 2001-524675 A

In view of the above problems, an object of the present invention is to provide magnetic encapsulated particles having uniform magnetism, excellent dispersion stability, and a narrow particle size distribution, and immunoassay particles using the same.

The present invention relates to a linker containing a magnetic material having an average particle diameter of 1 to 30 nm in a dispersed state inside a particle made of an organic polymer substance, and having a functional group capable of covalently bonding to an antigen or antibody on the particle surface Is a magnetic substance-encapsulated particle.
The present invention is described in detail below.

The magnetic substance-encapsulated particles of the present invention have an average particle size of 1 to 30 nm inside particles made of an organic polymer substance
The magnetic material is contained in a dispersed state.
The organic polymer material is mainly composed of a polymer comprising a hydrophobic monomer for forming the core of the particle and a hydrophilic monomer for forming the particle shell while forming particles that are stably dispersed in water. Ingredients.

<Hydrophobic monomer>
Examples of the hydrophobic monomer include styrene monomers such as styrene, α-methylstyrene, p-methylstyrene, p-chlorostyrene, and chloromethylstyrene; vinyl chloride; vinyl esters such as vinyl acetate and vinyl propionate; Unsaturated nitriles such as acrylonitrile; methyl (meth) acrylate, ethyl (meth) acrylate, butyl (meth) acrylate, 2-ethylhexyl (meth) acrylate, stearyl (meth) acrylate, ethylene glycol (meth) acrylate, trifluoroethyl Acrylic monomers such as (meth) acrylate, pentafluoropropyl (meth) acrylate, cyclohexyl (meth) acrylate, glycidyl methacrylate, and tetrahydrofurfuryl (meth) acrylate It is. These hydrophobic monomers may be used alone or in combination of two or more.

The hydrophobic monomer is preferably methyl (meth) acrylate, ethyl (meth) acrylate, butyl (meth) acrylate, 2-ethylhexyl (meth) acrylate, stearyl (meth) acrylate, ethylene glycol (meth) acrylate, Fluoroethyl (meth) acrylate, pentafluoropropyl (meth) acrylate,
Acrylic monomers such as cyclohexyl (meth) acrylate, glycidyl methacrylate, and tetrahydrofurfuryl (meth) acrylate are used.

As the hydrophobic monomer, more preferably, an acrylic monomer having a glycidyl group having an excellent ability to take in metal ions at a high concentration during particle polymerization is used because particle formation and magnetic substance formation by polymerization proceed simultaneously. It is done. Among the acrylic monomers, glycidyl methacrylate (GMA) is particularly preferably used because of its high affinity with iron ions and magnetite.

A crosslinkable monomer can also be used as the hydrophobic monomer. Examples of the crosslinkable monomer include divinylbenzene, divinylbiphenyl, divinylnaphthalene, ethylene glycol di (meth) acrylate, 1,6-hexanediol di (meth) acrylate,
Neopentyl glycol di (meth) acrylate, trimethylolpropane tri (meth)
Examples include acrylate, tetramethylol methane tri (meth) acrylate, tetramethylol propane tetra (meth) acrylate, diallyl phthalate and its isomer, triallyl isocyanurate and its derivative. These crosslinkable monomers may be used independently and may use 2 or more types together.
Among these, ethylene glycol di (meth) acrylate is preferably used because of its high affinity with iron ions and magnetite.

<Hydrophilic monomer>
Examples of the hydrophilic monomer include carboxylic acids having a polymerizable unsaturated bond such as acrylic acid, methacrylic acid, itaconic acid, fumaric acid, and maleic acid; phosphate esters having a polymerizable unsaturated bond; A sulfonic acid ester having a bond; having a cationic group such as a salt of an amine having an acryloyl group such as a quaternary salt of dimethylaminoethyl methacrylate or a quaternary salt of diethylaminoethyl methacrylate or a salt of a nitrogen-containing compound having a vinyl group such as vinylpyridine Vinyl monomers: Nonionic vinyl monomers such as 2-hydroxyethyl methacrylate, polyethylene glycol (meth) acrylate, (meth) acrylamide, methylolacrylamide, glycerol methacrylate (GLM), and the like.
These hydrophilic monomers may be used independently and may use 2 or more types together. Among these, polyethylene glycol (meth) acrylate represented by the following general formula is preferably used because it has a high ability to stably disperse particles in water and does not hinder the formation of a magnetic substance.
_{CH 2 = CR-COO- (CH} 2 -CH 2 -O) n-H
In the formula, R represents H or CH ₃ , and n represents an integer of 2 to 20. The preferable lower limit of n is 2, and the preferable upper limit is 10.

The magnetic material is preferably formed by oxidation of metal ions inside the particles in the polymerization process for forming the particles.
<Metal ion>
Although the said metal ion will not be specifically limited if it forms a magnetic body, Preferably, they are an iron ion, cobalt ion, nickel ion, etc., More preferably, it is an iron ion.
Magnetite, which is a magnetic substance, is obtained by oxidizing ferric chloride with an oxidizing agent or the like.

The average particle diameter of the magnetic material is 1 to 30 nm. When the thickness is less than 1 nm, the magnetic response characteristics of the magnetic material are reduced. Moreover, when it exceeds 30 nm, the dispersibility in particle | grains will fall. A preferred lower limit is 2 nm, a preferred upper limit is 20 nm, and a more preferred upper limit is 10 nm.

The magnetic substance-encapsulated particles of the present invention contain the magnetic substance in a dispersed state inside the particles made of the organic polymer substance. That is, in the magnetic substance-encapsulated particle of the present invention, the magnetic substance is present in a dispersed state inside the particle without being exposed on the particle surface.

The magnetic substance content of the magnetic substance-encapsulated particles of the present invention is preferably adjusted in the range of 0.1 to 40% by weight depending on the polymerization composition. If it is less than 0.1% by weight, the magnetic response characteristics of the magnetic substance-encapsulated particles are reduced, and the measurement sensitivity when used for immunoassay is lowered. If it exceeds 40% by weight,
The polymerization operability of the particles is lowered, and it becomes difficult to take in metal ions during the particle polymerization. A preferred lower limit is 1% by weight and a preferred upper limit is 30% by weight.

The magnetic substance-encapsulated particles of the present invention are particles in which a linker having a functional group capable of covalently bonding to an antigen or an antibody is bonded to the particle surface made of the organic polymer substance.
<Linker>
The above-mentioned linker is a chemical substance that exists between a particle made of an organic polymer substance and a compound such as an antigen or an antibody when the magnetic substance-encapsulated particle is used for immunoassay. The linker is a compound that does not cause steric hindrance and is difficult to cause nonspecific adsorption.
Before binding to organic polymer particles and compounds such as antigens and antibodies, it can be covalently bound to antigens or antibodies such as amino groups, carboxyl groups, epoxy groups, tosyl groups, thiol groups, etc. at the molecular ends. It preferably has a functional group. As a linker in the present invention, an epoxy group derived from a glycidyl group-containing monomer or a hydroxyl group, an amino group, etc. generated by ring-opening of an epoxy group and an antigen, an antibody, etc. can be bonded to the particle surface at an appropriate distance. If there is no particular limitation. Preferably, a compound having an epoxy group at a plurality of terminals, for example,
Examples include polyethylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, neopentyl glycol diglycidyl ether, and trimethylolpropane polyglycidyl ether. More preferably, polyethylene glycol diglycidyl ether is used.

The average particle diameter of the magnetic substance-encapsulated particles of the present invention is preferably adjusted in the range of 0.05 to 1 μm depending on the polymerization conditions. When it is less than 0.05 μm or exceeds 1 μm, it is difficult to control the particle shape of the magnetic substance-encapsulated particles and to perform the polymerization operation. A preferable lower limit is 0.07 μm, and a preferable upper limit is 0.8 μm.

The magnetic substance-encapsulated particles of the present invention can be produced by producing particles made of an organic polymer substance containing a magnetic substance in a dispersed state therein, and introducing a linker on the particle surface.
Particles made of an organic polymer substance containing a magnetic substance in a dispersed state include a step of polymerizing a hydrophobic monomer and / or a hydrophilic monomer in an aqueous solvent to form particles, and metal ions in the particles. It is manufactured by a method in which the metal ions are oxidized to form a magnetic material while being incorporated, and the step of forming the particles and the step of forming the magnetic material proceed simultaneously.
In the step of polymerizing a hydrophobic monomer and / or hydrophilic monomer in an aqueous solvent to form particles, a polymerization initiator is used.

<Polymerization initiator>
The polymerization initiator is not particularly limited, and examples thereof include water-soluble organic azo compounds, inorganic peroxides, and organic peroxides.
Preferable examples of the polymerization initiator include potassium persulfate (hereinafter referred to as “KPS”; polymerization temperature 70 ° C.), azobisamidinopropane hydrochloride (polymerization temperature 70 ° C.), 2,2-azobis [2
-(2-Imidazolin-2-yl) propane] dihydrochloride (polymerization temperature 60 ° C.)
Is mentioned. Among these, KPS, which is a peroxide-based polymerization initiator, is expected to contribute to the oxidation of divalent iron ions at the start of polymerization, and when KPS is used, polymerization with monomers and iron ions and magnetite formation occur. It is assumed that they will proceed simultaneously. Azobisamidinopropane hydrochloride and 2,2-azobis [2- (2-imidazolin-2-yl) propane]
Dihydrochloride has a weak oxidizing power and becomes a polymerization initiator involved in the mild oxidation reaction of divalent iron ions. As azobisamidinopropane hydrochloride, trade name “V-50”
As a 2,2-azobis [2- (2-imidazolin-2-yl) propane] dihydrochloride (manufactured by Wako Pure Chemical Industries, Ltd.), the trade name “VA-044” (manufactured by Wako Pure Chemical Industries, Ltd.) Is commercially available.
The polymerization initiator may be consumed by oxidation of Fe ²⁺ or may lose radical activity due to Fe ³⁺ , and therefore it is effective to add it later in the particle growth process for the purpose of promoting particle growth. In this case, new secondary particles are not formed, and the particle surface is coated with the polymer.

<PH adjuster>
In the present invention, in order to produce a magnetic substance simultaneously with polymerization, the pH in the polymerization system is preferably adjusted to basic. The system using KPS as the polymerization initiator has the advantage that monodisperse particles with good dispersion stability in water and a narrow particle size distribution can be obtained, but the oxidation power cannot be controlled and the inside of the polymerization system becomes acidic. Therefore, there is a demerit that the particles are weakly attracted to the magnet. On the other hand, 2,2-azobis [2- (not having an oxidizing power as the polymerization initiator).
The system using 2-imidazolin-2-yl) propane] dihydrochloride has the advantage that the pH in the polymerization system is almost neutral.
In order to keep the pH in the polymerization system weakly basic, a general base can be used.
Preferably NH ₄ OH is used as a pH adjuster. The pH adjuster can be added several times as necessary.

<Polymerization method>
The particles made of the organic polymer substance can be produced by a particle polymerization method such as suspension polymerization, dispersion polymerization, emulsion polymerization, soap-free emulsion polymerization, etc., but the Cv value of the magnetically encapsulated particles finally obtained is 5
%, It is preferably produced by soap-free emulsion polymerization excellent in control of particle size distribution.
Hereinafter, although the manufacturing method of the particle | grains by soap free emulsion polymerization is illustrated, it is not limited to this method.

A typical polymerization composition is as follows.
Monomer composition comprising hydrophilic monomer / hydrophobic monomer: 3 g
H ₂ O: 100 g
The monomer composition and water are weighed into a four-necked flask. A stir bar and a reflux condenser are attached to each port. Next, the system is put into a thermostat and the inside of the system is replaced with nitrogen while stirring. The thermostat is preferably adjusted to the polymerization temperature of the polymerization initiator to be added. For example, when KPS or azobisamidinopropane hydrochloride is used as the polymerization initiator, the temperature is preferably set to 70 ° C., and 2 as the polymerization initiator. , 2-Azobis [2- (2-imidazolin-2-yl) propane]
When using dihydrochloride, it is preferable to set it as 50-60 degreeC. Thereafter, a polymerization initiator dissolved in water is injected into the system with a syringe. The polymerization is started at this time, and an aqueous solution of FeCl ₂ .4H ₂ O serving as a magnetic source is injected using a syringe after a predetermined time. FeCl ₂ · 4H
₂ O uses what melt | dissolved 1 to 3 times mole of the polymerization initiator in 5 g of water. That is, particles are produced by initiating polymerization of the above-described monomer with a polymerization initiator and oxidizing (magnetizing) divalent iron ions.
The polymerization is preferably performed for 2 to 24 hours from the start. In order to obtain an appropriate oxidizing power, NH ₄ OH may be added during the polymerization, and further, a polymerization initiator may be added during the polymerization in order to promote particle growth by the polymerization. In this way, particles made of an organic polymer substance containing a magnetic substance in a dispersed state can be obtained.

A reactive emulsifier may be added as a copolymerization monomer to the monomer composition.
<Reactive emulsifier>
Examples of the reactive emulsifier include reactive emulsifiers represented by the following general formula.

These reactive emulsifiers may be used alone or in combination of two or more.
The produced particles are purified by repeated centrifugation and redispersion with distilled water to remove residual monomers, polymerization initiators, unreacted iron ions, and the like. After centrifugation, the supernatant is discarded by decantation, distilled water is added, and redispersion is performed with a glass rod. After purification, the obtained particles are transferred to a glass vial, sealed and stored with a lid / parafilm.

The introduction of the linker to the surface of the produced particles is, for example, by dispersing the particles in an alkaline aqueous solution,
Subsequently, a chemical substance serving as a linker such as polyethylene glycol diglycidyl ether can be added and mixed for about 24 hours.

The functional group of the linker introduced on the surface of the magnetic substance-encapsulated particle of the present invention is chemically bonded to the amino group or thiol group of the antigen or antibody, and the antigen or antibody is bound to the surface of the magnetic substance-encapsulated particle. Particles for immunoassay can be produced. Such an immunoassay particle in which an antigen or an antibody is bound to the magnetic substance-encapsulated particle of the present invention is also one aspect of the present invention.

The magnetic body-encapsulated particles and the immunoassay particles of the present invention can be used in immunoassay methods.
Examples of the immunoassay include known methods such as radioimmunoassay and enzyme immunoassay using magnetic inclusion particles as a carrier, and a target antigen or antibody can be measured by a sandwich method or a competitive method. .
Further, the magnetic substance-encapsulated particles of the present invention can be used as a label in place of the isotope, enzyme, etc., which are labeling substances in the above method.

The magnetic substance-encapsulated particles and immunoassay particles of the present invention are particles having a narrow particle size distribution containing a magnetic substance uniformly dispersed therein and excellent in dispersion stability. By using the immunoassay particles for immunoassay, it is possible to carry out highly sensitive and precise measurement.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.

(Reference Examples 1-7)
Various monomers shown in Table 1 and 90 g of water were weighed in a 200 mL four-necked flask. A stirring seal, a stirring rod, a reflux condenser, and a ceramic rubber were attached to each port. The system was placed in a constant temperature bath at 70 ° C., and the inside of the system was purged with nitrogen for 30 minutes while stirring at 200 rpm. Thereafter, 0.06 g of KPS, which is a polymerization initiator dissolved in water, was dissolved in 10 g of water and injected into the system with a syringe. This time is set as the start of polymerization, and after 2 minutes, a predetermined amount of FeCl ₂ .4H ₂ is used using a syringe.
An aqueous O solution was injected. The polymerization was carried out for 20 hours from the start of polymerization. In order to obtain an appropriate oxidizing power,
During the polymerization, 0.165 g of NH ₄ OH was added.
The produced particles were purified by repeating centrifugation and redispersion four times with distilled water. At this time, centrifugation was performed at 20 ° C. and 13500 rpm. After centrifugation, the supernatant was discarded by decantation, distilled water was added, and redispersion was performed with a glass rod to obtain particles containing a magnetic substance.

The descriptions in the table are as follows.
GMA: Glycidyl methacrylate EGDM: Ethylene glycol dimethacrylate AAm: Acrylamide PE-90: Polyethylene glycol methacrylate (n = 2)
PE-350: Polyethylene glycol methacrylate (n = 8)
NE-20:

In the formula, X represents H.

SE-20:

In the formula, X represents SO ₄ NH ₄ .

About the obtained particle dispersion, the dispersion state of the particles was visually observed. The purified particles were diluted with water, deposited and fixed on a collodion film supported by a metal mesh, and the morphology of the particles was observed with a transmission electron microscope (TEM).

In Reference Example 1, a part of the agglomerates was observed, and the dispersion stability in which the particles settled with time was slightly low. Therefore, the dispersion was redispersed by ultrasonic treatment. On the other hand, in Reference Examples 2 to 5 using polyethylene glycol methacrylate as a hydrophilic monomer and Reference Examples 6 and 7 using a reactive emulsifier, no agglomerates were observed, and particles with high dispersion stability were obtained. It was. In particular, Reference Examples 6 and 7 using a reactive emulsifier were found to have a small particle size and excellent dispersion stability. In addition, it was observed that the particles of Reference Examples 1 to 7 each contained a magnetic substance in a dispersed state inside the particle, and the particle surface had a clean outline. FIG. 1 shows the T of the particles of Reference Example 2.
An EM photograph (average particle size; particles 0.21 μm, magnetic material 5 nm) was shown.

In order to confirm that the particles produced in Reference Examples 1 to 7 are attracted to the magnet, 1.5 m
A small amount is taken into a microtube of L, diluted appropriately with distilled water, and the tube is set up on a microtube stand with magnet (manufactured by DYNAL, MPC (registered trademark) -M), and the dispersed particles are attracted to the magnet. This was confirmed visually. In particular, it was revealed that Reference Examples 2 to 5 using polyethylene glycol methacrylate as the hydrophilic monomer had a larger magnetic force than other reference examples. Table 2 shows the average particle size of the magnetic particles in each reference example.

(Reference Example 8)
Various monomers and water shown below were weighed in a 200 mL four-necked flask.
AAm / GMA / EGDM / H ₂ O = 0.15 / 2.835 / 0.015 / 90 (g)
A stirring seal, a stirring rod, a reflux condenser, and a ceramic rubber were attached to each port. 7 systems
The system was placed in a thermostatic bath at 0 ° C., and the inside of the system was purged with nitrogen for 30 minutes while stirring at 200 rpm. Thereafter, 0.06 g of KPS, which is a polymerization initiator dissolved in water, was dissolved in 10 g of water and injected into the system with a syringe. This time is set as the start of polymerization, and after a predetermined time, using a syringe, FeCl2 · 4H
A 2O aqueous solution (0.165 g of FeCl ₂ .4H ₂ O dissolved in 5 g of water) was injected. NH ₄ OH / H ₂ O = 0.165 / 5 (g) was added to obtain an appropriate oxidizing power 1 minute after the start of polymerization,
Polymerization was performed for 2 hours.

The produced particles were purified by repeating centrifugation and redispersion four times with distilled water. At this time, centrifugation was performed at 20 ° C. and 13500 rpm. After centrifugation, the supernatant was discarded by decantation, distilled water was added, and redispersion was performed with a glass rod to obtain particles containing a magnetic substance.

(Reference Example 9)
After the start of polymerization, particles containing a magnetic material were obtained in the same manner as in Reference Example 8 except that the following substances were added at a predetermined time and the polymerization time was 3 hours.
30 minutes after the start of polymerization, NH ₄ OH / H ₂ O = 0.165 / 5 (g)
60 minutes after the start of polymerization KPS / H ₂ O = 0.165 / 5 (g)

(Reference Example 10)
After the start of polymerization, particles containing a magnetic material were obtained in the same manner as in Reference Example 8 except that the following substances were added at a predetermined time and the polymerization time was 3 hours.
60 minutes after the start of polymerization KPS / H ₂ O = 0.165 / 5 (g), FeCl ₂ .4H ₂ O / H
₂ O = 0.088 / 5 (g)
120 minutes after the start of polymerization, GMA = 0.5 (g), NH ₄ OH / H ₂ O = 0.165 / 5 (g
)

(Reference Example 11)
After the start of polymerization, particles containing a magnetic material were obtained in the same manner as in Reference Example 8 except that the following substances were added at a predetermined time and the polymerization time was 4 hours.
1 minute after the start of polymerization NH ₄ OH / H ₂ O = 0.165 / 5 (g)
120 minutes after the start of polymerization KPS / H ₂ O = 0.165 / 5 (g)

Particle morphology observation by TEM was performed on the particles obtained in Reference Examples 8 to 11.
It was recognized that each of Reference Examples 9 and 10 contained more magnetic material than Reference Example 8 and the particle size was increased. Further, in Reference Example 11, it was observed that the inside of the magnetic material was almost the same as in Reference Examples 9 and 10 and the particle surface had a clean outline.
From previous experiments, it was speculated that the addition of NH ₄ OH at the early stage of particle growth is a factor inhibiting growth. On the other hand, the post-addition of the polymerization initiator has a polymerization rate of almost 100.
In addition, it was observed that secondary particles were not formed and the particle surface was covered with a polymer, which was confirmed to be an effective means. Table 3 shows the average particle size of the magnetic particles of each reference example.

Example 1
1.0 g of particles containing the magnetic material prepared in Reference Example 2 was put into 50 mL of 10% ammonia water and stirred at 70 ° C. for 20 hours. Next, centrifugal washing is performed 3 times with ion-exchanged water.
It was dispersed in 0 mL of ion exchange water. Subsequently, 30 g of ethylene glycol diglycidyl ether was added and mixed, and the pH was adjusted to 11 using an aqueous sodium hydroxide solution, followed by stirring at room temperature for 24 hours. After the reaction, centrifugal washing was performed three times using ion-exchanged water to obtain the magnetic substance-encapsulated particles of the present invention to which a linker having an epoxy group was bonded.

Furthermore, the immunoassay particles of the present invention were produced from the magnetic substance-encapsulated particles obtained in Example 1 by the following method.
(Preparation of immunoassay particles)
1 mL of 0.1 M borate buffer was added to 12 mg of the magnetic substance-encapsulated particles produced in Example 1, and centrifuged at 15000 rpm for 10 minutes, and the supernatant was removed. In the resulting sediment, 0
. 380 μL of 1M borate buffer, anti-α-hCG monoclonal antibody solution (5.0 m
g / mL) was added, mixed well, and stirred at room temperature for 20 hours. The reaction solution is 150
Centrifugation was performed at 00 rpm for 10 minutes to remove unreacted anti-α-hCG monoclonal antibody. The amount of the anti-α-hCG monoclonal antibody bound to the magnetic substance-encapsulated particles was confirmed to be 55% of the preparation based on the measurement of the protein concentration in the supernatant. The obtained sediment was suspended in 1 mL of 100 mM phosphate buffer (pH 7.5) and centrifuged again. The sediment is 100 mM
The bovine serum albumin was dissolved in a phosphate buffer solution (pH 7.5) in a concentration of 5% (w / v) so as to be suspended in 900 μL and stirred at 37 ° C. for 1 hour to perform a blocking treatment.
Thereafter, centrifugation was performed at 15000 RPM for 20 minutes, and 1 mL of 0.1 M borate buffer was added to the sediment and dispersed by ultrasonic waves. Subsequently, 100 mM phosphate buffer (pH 7.5)
1 ml of a solution in which bovine serum albumin and glycerol are each dissolved to a concentration of 5% (w / v), and sodium azide is further dissolved to a concentration of 0.01% (w / v).
To obtain particles for immunoassay.

Since the present invention has the above-described configuration, a magnetic substance is formed by modifying particles by forming a particle by copolymerizing a hydrophobic monomer and a hydrophilic monomer, and by incorporating metal ions into the particles. By carrying out the reaction simultaneously to obtain particles containing the magnetic substance in a dispersed state, and introducing a linker on the surface, it is possible to obtain magnetic substance-encapsulated particles having uniform magnetism and a narrow particle size distribution.

4 is a TEM photograph of particles of Reference Example 2 (average particle diameter; particles 0.21 μm, magnetic material 5 nm).

Claims (3)

A magnetic material having an average particle diameter of 1 to 30 nm is contained in a dispersed state inside particles made of an organic polymer material containing glycidyl methacrylate or ethylene glycol di (meth) acrylate and polyethylene glycol (meth) acrylate as constituent components. A magnetic substance-encapsulated particle comprising a particle surface and a linker made of polyethylene glycol diglycidyl ether bonded thereto. The magnetic substance-encapsulated particle according to claim 1, wherein the magnetic substance has an average particle diameter of 1 to 10 nm. 3. An immunoassay particle comprising the magnetic substance-encapsulated particle according to claim 1 or 2 bound to an antigen or an antibody via a linker bonded to the particle surface of the magnetic substance-encapsulated particle.