JPH1083902A - Metal oxide coated particles and method for producing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide metal-oxide coated particles, which are valuable in various fields including a medial care and consists of substrate particles with the surfaces covered evenly with a metal-oxide layer, and a method of manufacturing the coated particles. SOLUTION: In these metal-oxide coated particles, the surfaces of substrate particles of a mean particle diameter of 5 to 500nm are coated with a metal oxide layer and the amount of the saturation magnetization of the coated particles in the intensity of magnetic field of 10kOe is 40 to 90emu/g. The above particles are obtained by a method wherein a metallic component is added to a substrate particle dispersion liquid and the metal oxide layer is made to form on the surfaces of the substrate particles under ultrasonic irradiation.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to metal oxide-coated particles having a substrate particle surface uniformly covered with a metal oxide, and a method for producing the same, which is useful in various fields including medical treatment.

[0002]

2. Description of the Related Art Magnetic iron oxide-coated particles, in which a core such as a polymer is used as substrate particles and the surface of which is coated with magnetic iron oxide, are used as carriers in the fields of diagnosis and treatment because of their properties against magnetic fields.

Heretofore, the above-mentioned magnetic iron oxide-coated particles have been produced by a so-called mechanical stirring method using mechanical stirring. However, 0.5 μm
When a particle having a small particle diameter of less than m (500 nm) is used, it is difficult to uniformly adhere magnetic iron oxide to the surface of the base particles by the conventional mechanical stirring method, and as shown in FIG. Iron oxide adheres in particulate form. For this reason, the surface magnetic iron oxide layer of the prepared magnetic iron oxide-coated particles has a state in which the surface of the ferrite and the surface of the core particles are mixed, and is nonuniform and discontinuous, and the surface of the base particles is partially exposed. When such magnetic iron oxide-coated particles are used as a carrier for diagnosis, uniform surface treatment is difficult, and the amount of magnetic iron oxide attached is insufficient, so that a satisfactory amount of magnetization cannot be obtained. The magnetic effect was also inadequate.

[0004] In order to produce magnetic iron oxide-coated particles of a small particle size having a uniform and continuous surface magnetic iron oxide layer, an attempt was made to increase the amount of magnetic iron oxide used in the treatment. Depending on the stirring method, the magnetic iron oxide was peeled off even once adhered to the base particles, and magnetic iron oxide-coated particles having a uniform and continuous magnetic iron oxide layer could not be obtained.

Further, Japanese Patent Application Laid-Open No. Hei 3-237019 discloses a ferrite coating method for controlling the saturation magnetization of a ferrite coating by regulating the pH and oxidation-reduction potential at the time of coating ferrite on particles or fibrous materials. Is done. In JP-A-6-231957, the supply rate ratio between the oxidizing agent and the ferrous ion during ferrite coating is adjusted,
A method for improving the coverage of ferrite by reducing the particle size of the ferrite particles coated on the surface is described.

However, there have been no magnetic iron oxide-coated particles having a high saturation magnetization using base particles having a relatively small average particle size.

Also, when using base particles having a relatively large particle size (0.5 μm or more), the magnetic iron oxide may locally become particulate or peel off.

When magnetic iron oxide-coated particles are used as a carrier, the smaller the particle size, the larger the surface area even with the same weight, so that the reactivity is good, but the recovery becomes difficult. for that reason,
There is a demand for magnetic iron oxide-coated particles having a small particle size and a high magnetization.

[0009]

DISCLOSURE OF THE INVENTION The present invention relates to metal oxide coated particles having a substrate particle surface uniformly and continuously covered with a metal oxide, which is useful in various fields including medical treatment, and production thereof. Provide a way.

[0010]

Means for Solving the Problems The present inventor has conducted intensive research and has succeeded in using ultrasonic treatment to obtain a metal oxide covered with a uniform and continuous metal oxide layer regardless of the particle size of the substrate particles. It was found that product-coated particles were obtained, and the present invention was completed.

That is, in the first embodiment of the present invention, an average particle size of 5
The present invention provides metal oxide-coated particles in which the surface of base particles having a thickness of about 500 nm is coated with a metal oxide layer and has a saturation magnetization of 40 to 90 emu / g at a magnetic field strength of 10 kOe. In particular, the present invention provides metal oxide-coated particles in which the base particles are synthetic polymer particles or natural polymer particles, or metal oxide-coated particles in which the metal oxide is ferrite. A second aspect of the present invention provides a method for producing metal oxide-coated particles in which a metal component is added to a substrate particle dispersion and a metal oxide layer is formed on the surface of the substrate particles under ultrasonic irradiation. . In particular, the present invention provides a production method in which the base particles are synthetic polymer particles or natural polymer particles, a production method in which the metal oxide is ferrite, or a production method in which the base particles have an average particle size of 5 to 100,000 nm. .

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. The base particles used as a nucleus for attaching the metal oxide in the present invention are not particularly limited as long as they can take a particle state in an aqueous solvent, and may be any of organic particles and inorganic particles. . Examples of the organic polymer particles include synthetic polymer particles and natural polymer particles. Examples of the synthetic polymer particles include polystyrene particles, styrene-butadiene copolymer particles, and styrene- (meta).
There are acrylate copolymer particles, which can be obtained as a latex by emulsion polymerization.

The monomer used for forming the (meth) acrylate copolymer particles is, for example, (meth)
2-hydroxyethyl acrylate, 2-hydroxypropyl (meth) acrylate, (meth) acrylic acid-1
-Methyl-2-hydroxyethyl, glycerol monomethacrylate, 2-acrylamido-2-methylpropanesulfonic acid, 2-sulfoethyl methacrylate, acid phosphoxyethyl methacrylate, methacrylic acid-3-
Chloro-2-acid phosphoxypropyl, acid phosphoxypropyl methacrylate, ethyl (meth) acrylate, n-butyl (meth) acrylate, i-butyl (meth) acrylate, (meth) acrylic acid-2 -Ethylhexyl, lauryl methacrylate, cyclohexyl methacrylate, (meth) acrylamide, N-methylolacrylamide, N-butoxymethylacrylamide,
Examples include glycidyl (meth) acrylate and methyl glycidyl (meth) acrylate.

As natural polymer particles, gelatin particles,
Suitable examples include polypeptides, proteins, and polysaccharides. Examples of inorganic particles include metal particles and ceramic particles. In addition, depending on the application,
Porous polymer particles, whiskers, dextran and the like are also preferably used.

Although the size of the base particles is not particularly limited,
Any size can be used as long as it can be uniformly dispersed in the reaction system (in an aqueous solvent) during the reaction. Particle size is 100 μm or less, especially particle size is 5 to 100000 nm
When ferrite is formed as a continuous film on the surface, particles having a high saturation magnetization can be obtained because the surface area is large with respect to the mass. In particular, when the average particle size of the base particles is 5 to 5
Metal oxide coated particles having a saturation magnetization of 40 to 90 emu / g at a magnetic field strength of 10 kOe and a magnetic field intensity of 10 nm have not been known so far, and can be produced only by using the production method of the present invention. Was. The metal oxide coated particles having such a small particle size and a high saturation magnetization can be suitably used in various fields. For example, if the base particle diameter is 500 nm or less, a metal oxide-coated particle suitable for a contrast agent, a drug delivery system, etc., and if the base particle diameter is 10,000 to 100,000 nm, a metal oxide-coated particle suitable for an electrophotographic carrier or the like. Is obtained.
For use in a diagnostic kit, the base particle size should be 300-5
000 nm metal oxide coated particles are preferred.

As the metal oxide coating the surface of the base particles, so-called ferrite is preferably mentioned. Preferred ferrites include magnetite containing only iron as a metal element, and other, zinc, cobalt, nickel, manganese, copper, vanadium, antimony, lithium, molybdenum, titanium, rubidium, aluminum,
Those containing other metals and metal ion species such as silicon, chromium, tin, calcium, cadmium, and indium are also preferably used. It may contain one or more metals other than iron.

The production method of the present invention is characterized in that ultrasonic waves are applied when coating the surface of the base particles with the metal oxide.

The ultrasonic wave to be irradiated means a sound wave having a frequency of 15 kHz or more and up to MHz, GHz and THz, and preferably has a frequency of 15 to 30 kHz. The intensity of the ultrasonic wave used in the present invention is not particularly limited, but it is preferable to use an ultrasonic wave having an output of 200 W or more for a 500 cc reaction solution. By using a frequency in this range, uniform complete coverage can be obtained.

The base particles are dispersed in a dispersion medium and coated with a metal oxide as a base particle dispersion. A water-based dispersion medium is preferable. The pH of the aqueous solution is appropriately selected and controlled depending on the types of anions and metal ions present in the aqueous solution, but is preferably 6 to 11, more preferably 7 to 1
1 range. By performing the reaction under these conditions, a favorable oxidation reaction can be performed. A buffer such as ammonium acetate or a salt having a buffering effect may be added to stabilize the pH. In order to carry out the production method of the present invention, the temperature of the reaction system may be lower than or equal to the boiling point of the aqueous solution. Also, the reaction is carried out essentially in a deoxygenated atmosphere. An excessive amount of oxygen is not preferable because an unnecessary oxidation reaction proceeds. Specifically, the reaction is preferably performed under a nitrogen atmosphere. At this time, oxygen is also removed from the solvent (aqueous solution) that causes the reaction to proceed to obtain a deoxygenated aqueous solution.

According to the method of the present invention, the surface of the base particles can be coated with the metal oxide by an adsorption reaction. Preferably, ferrous ions or other metal ions are reacted with a reaction system (reaction solvent). ), And oxidizing while supplying it. Ferrous ions or other metal ions are provided in the solvent in the form of hydrochloride, sulfate, acetate, and the like. Preferably, it is distributed in the form of hydrochloride or sulfate. Examples of the oxidizing agent to be added include nitrites such as alkali metals and alkaline earth metals, nitrates, hydrogen peroxide, organic oxides, perchloric acid, and dissolved oxygen water. Preferred are nitrite and hydrogen peroxide.

The formation of the metal oxide coating layer starts by adding an oxidizing agent solution to a deoxygenated aqueous solution in which ferrous ions or other metal ions and base particles are diffused. As a method of adding the oxidizing agent, it is preferable to drop an aqueous solution of the oxidizing agent into the solution in a fixed amount as in a titration method in analytical chemistry. As described above, when the droplets are dropped by a fixed amount, the thickness of the coating film can be easily adjusted.

Although the production method of the present invention using the titration method has been described above, in addition to the above, a chelating ferrite plating method (Journal of the Japan Society of Applied Magnetics, pp. 485-487 (199)
6)) may be used to carry out the production method of the present invention.
Also in this case, the frequency and intensity of the ultrasonic wave to be irradiated are the same as in the case of the titration method. Irrespective of the method used, a uniform continuous film is formed by irradiating ultrasonic waves when forming the metal oxide coating film, regardless of the size of the base particles.

In a preferred embodiment of the method of the present invention, first, the base particles are dispersed in deoxygenated water, and if necessary, an additive such as a surfactant may be added so that the base particles are well dispersed. . Then, if necessary, a pH buffer or the like is added for adjusting the pH of the solvent, and ferrous ion is further added in the form of a salt. Also, if necessary, other metal ions may be
It is added simultaneously with iron ions. After all the components have been added, the reaction is allowed to proceed by adding the oxidizing solution to the solution by titration as described above. At this time, the reaction is performed while continuously irradiating the solution with ultrasonic waves from the addition of the ferrous ion to the end of the formation of the coating.

A diagnostic kit can be provided using the metal oxide-coated particles of the present invention or the metal oxide-coated particles provided by the production method of the present invention. The kit includes a substance that forms a specific bond with the substance to be measured (for example, one of a pair of an antibody-antigen and an enzyme-substrate), the metal oxide-coated particles of the present invention, or the present invention. Examples include metal oxide-coated particles, radioisotopes or antibodies against a substance to be measured labeled with a chromogenic enzyme, a luminescent enzyme, a luminescent enzyme, or a substrate of a luminescent enzyme, a control serum, a diluent, etc. Is done.

The substance which forms a specific bond with the substance to be measured is not limited to the metal oxide-coated particles of the present invention or the metal oxide-coated particles provided by the production method of the present invention. You don't have to. The metal oxide-coated particles of the present invention or the metal oxide-coated particles provided by the production method of the present invention can be used in kits utilizing various types of reactions other than the above examples.

As will be described in detail later, FIG. 1 shows metal oxide-coated particles produced by the method of the present invention (Example 1).
FIG. 2 shows a structure manufactured by a conventional method (Comparative Example 1). In the case of the conventional method, the metal oxide adhered to the surface of the base particles becomes particulate and forms a non-uniform and discontinuous coating layer (FIG. 2). On the other hand, the metal oxide-coated particles produced by the method of the present invention form a continuous film of metal oxide on the surface of the base particles by using ultrasonic treatment, and the surface of the base particles is completely and uniformly formed. Coated (FIG. 1).

The metal oxide-coated particles of the present invention (FIG. 1)
Is to form a continuous film of metal oxides such as ferrite adhering to the surface of the base particles (core particles) and completely coat the core particles with the continuous film so that there is no portion where the core particle surface is exposed on the surface of the coated particles and ferrite and the like It is only the metal oxide surface. Also, ferrite particles do not exist as particles on the surface,
Since it exists as a continuous film, the bonding force between the ferrite and the polymer is strong, and the film is not easily broken and peeled off.

When metal oxide-coated particles in which the metal oxide is uniformly adhered to the base particles are used as a carrier, the surface of the particles is uniform, so that the object can be fixed evenly. In addition, since the surface of the base particles is completely covered as a continuous film, non-specific adsorption of proteins and the like can be prevented, and when used as a carrier for analysis, a blank value is reduced, contributing to improvement in analysis accuracy. Furthermore, according to the present invention, since the metal oxide uniformly and completely covers the surface of the base particles, the amount of magnetization is also improved.

[0029]

The present invention will be described in more detail with reference to specific examples. However, the present invention is not limited to the following examples.

Example 1 <Synthesis of Substrate Particles> 230 parts by weight of ion-exchanged water was charged into a polymerization reactor having a stirrer, a thermometer, a monomer dropping funnel, a reflux condenser, a heating device, and a nitrogen gas inlet tube.
Styrene, 2-ethylhexyl acrylate and ethylene glycol dimethacrylate at 80 ° C.
1 part by weight of the mixed monomer (A) and 10%
10 parts by weight of an aqueous solution of ammonium persulfate was added, and then 99 parts by weight of the mixed monomer (A) was added dropwise over 3 hours to obtain a latex. Observation of the resulting base particles with an electron microscope revealed that they were substantially monodisperse and had a particle size of 300 nm.

<Preparation of Metal Oxide Coated Particles> The latex (solid content: 30 mm) was placed in a magnetic material generating apparatus having an ultrasonic horn (diameter: 30 mm), a thermometer, an oxidizing solution, a dropping funnel, a heating device, and a nitrogen gas inlet tube. %) 9 parts by weight,
N ₂ gas was introduced to degas oxygen in the emulsion of the substrate particles. Next, 100 parts by weight of a 40% ferrous chloride solution prepared in advance and 150 parts by weight of 75% ammonium acetate were charged into the apparatus, and heated to 65 ° C. with sufficient stirring and mixing. Then, while stirring, p with ammonia water
H was adjusted to 7.0. Meanwhile, 19.5kHz, 600W
And the ultrasonic irradiation was continued until the reaction was completed.
Sodium nitrite is added to this solution so that the oxidation-reduction potential becomes -500 mV. Thereafter, ammonia water and sodium nitrite are added so that the pH and the oxidation-reduction potential of the solution are kept at 7.0 and -500 mV, respectively. It was dropped. The solution after the reaction was allowed to stand for 1 hour, cooled to room temperature, and thereafter, filtration of the resulting metal oxide-coated particles and washing with ion-exchanged water were repeated. XD measurement of the particles showed that the surface composition was polycrystalline Fe ₃ O ₄ , and that the particles had a magnetization of 85 emu / g as measured by a vibrating magnetometer.

<Preparation of Carboxylated Metal Oxide-Coated Particles> To 5 g of the metal oxide-coated particles, 50 ml of 3-aminopropyltriethoxysilane was added.
0 ml was added and reacted at 50 ° C. for 3 hours. After the reaction, centrifugation was performed, and after removing the supernatant, the resultant was suspended in distilled water and centrifuged again. This washing operation was repeated five times.
5 g of the particles after washing are taken, and 100 cc of 0.1 M
The mixture was diffused in sodium bicarbonate, and 2.85 g of glutamic anhydride was added with stirring, followed by a reaction for 15 minutes. After the completion of the reaction, centrifugation was performed, and after removing the supernatant, the suspension was suspended in distilled water and centrifuged again. This washing operation was repeated five times to obtain carboxylated metal oxide-coated particles.

[0033] Using the same substrate particles as Comparative Example 1 Example 1 to obtain a metal oxide-coated particles were similar while their mechanical stirring (200 rpm) in place of the ultrasonic irradiation. XD of this particle
The measurement showed that the surface composition was polycrystalline Fe ₃ O ₄ , and the magnetization was 40 emu /
g. Thereafter, the same treatment as in Example 1 was performed to produce carboxylated metal oxide-coated particles.

Example 2 and Comparative Example 2: To enzyme immunoassay
Use suspended <metal oxide fixed antibody to coat the particle surface> carboxylated metal oxide coated particles in deionized water, to prepare a solid content of 1% dispersion 10 ml. This solution was centrifuged (16,000 rpm, 10 min), the supernatant was removed, and a phosphate buffer (20 mM sodium phosphate, p
H5.0) was diffused into the metal oxide-coated particles to obtain a diffusion solution having a solid content of 1%. 1 ml of a 1% aqueous solution of WSC (manufactured by Dojindo Laboratories) was added to the metal oxide-coated particle diffusion solution, and the mixture was stirred well with a Vortex stirrer.
It was left at room temperature for 15 minutes. This diffusion liquid is centrifuged (16,00
After 0 rpm and 10 min, the supernatant was removed by suction, and the precipitated metal oxide-coated particles were adjusted to a 1% metal oxide-coated particle diffusion solution with a phosphate buffer (20 mM sodium phosphate, pH 7.2). ). This operation was repeated twice more. After the last centrifugation, phosphate buffer (2
9.9 ml of 0 mM sodium phosphate, 0.05% sodium azide) and anti-CRP goat serum (manufactured by ATAB) which had been treated with ammonium sulfate and then dialyzed.
1 ml was added. After stirring this diffusion liquid, it was left to cool for 3 days to fix the anti-CRP antibody on the particle surface. The diffusion solution was centrifuged (16,000 rpm, 10 min), the supernatant was removed by suction, and the precipitated metal oxide-coated particles were added to a phosphate buffer solution (20 mM sodium phosphate, 0.2 mM).
% Tween 20, 0.1% BSA, pH 7.2) to prepare a 1% metal oxide-coated particle dispersion. This antibody-immobilized metal oxide-coated particle solution is hereinafter referred to as a magnetic solution.

<Preparation of CPR calibration curve by ELISA method> 1) CRP standard serum 5.0 mg / dl CRP standard serum (Towns)
With a diluent (10 mM sodium phosphate, pH 7.4,
0.1% BSA) and 5000, 20
Standard sera of 0, 20, 1, 0 μg / dl were prepared. 2) POD-labeled antibody solution A POD-labeled antibody diluent (10 mM sodium phosphate, pH 6.5,
0.1% BSA), and freeze-dried P
The OD-labeled antibody was dissolved. 3) Substrate solution TMBZ buffer (manufactured by Towns) and 1.5% aqueous hydrogen peroxide were mixed at a ratio of 100: 1 to prepare a substrate solution. 4) Assay operation CRP standard serum prepared at five different concentrations was added to a test tube in one.
The solution was dispensed in an amount of 00 μl. Add 50 magnetic solutions to each tube.
Each μl was added and reacted at 25 ° C. for 30 minutes. Thereafter, the metal oxide-coated particles were washed with a washing solution (10 mM sodium phosphate, pH 7.4, 0.005% Tween 20).
And washed three times. POD-labeled antibody solution was added to the metal oxide-coated particles in 10
0 μl was added and reacted at 25 ° C. for 30 minutes. afterwards,
The metal oxide-coated particles were washed with a washing solution (10 mM sodium phosphate, pH 7.4, 0.005% Tween 20).
Washed twice. 100 μl of the substrate solution is added to the washed metal oxide-coated particles.
Then, the mixture was reacted at 25 ° C. for 15 minutes. The reaction was stopped by adding 100 μl of a reaction stopping solution (2N sulfuric acid) to this solution. The metal oxide-coated particles were collected on the side wall of each test tube with a magnet, and 150 μl of the solution from which the metal oxide-coated particles had been removed was dispensed into a microplate by a microplate reader (manufactured by Tosoh Corporation, wavelength used: 450).
nm / 600 nm).

The metal oxide-coated particles of Example 1 and Comparative Example 1 were subjected to ELISA analysis to collect data for preparing a calibration curve. Table 1 shows the results.

[0037]

Since the surface of the metal oxide-coated particles according to the present invention prepared in Example 1 is uniformly and completely covered with the metal oxide, the metal oxide-coated particles according to Comparative Example 1 are different from the metal oxide-coated particles in blocking with BSA. Perform evenly, C
At 0 mg / dl RP, non-specific adsorption of the labeled antibody to the surface of the metal oxide-coated particles was prevented, and the blank value was successfully reduced. Similarly, even when CRP was present, nonspecific adsorption of the labeled antibody to the surface of the metal oxide-coated particles did not occur, so that the CRP concentration was accurately reflected in the data, and the analysis accuracy was improved.

[0039]

According to the present invention, it is possible to provide metal oxide-coated particles having a substrate particle surface uniformly covered with a metal oxide, and a method for producing the same, which are useful in various fields including medical treatment. .

[Brief description of the drawings]

1 is a drawing-substitute photograph showing the structure of a particle, showing a scanning electron micrograph (magnification: 50,000) of a ferrite-coated particle prepared in Example 1. FIG.

FIG. 2 is a drawing substitute photograph showing the structure of the particles, and shows a scanning electron micrograph (magnification: 50,000) of the ferrite-coated particles prepared in Comparative Example 1.

Claims (7)

[Claims] A surface of a base particle having an average particle size of 5 to 500 nm is coated with a metal oxide layer, and a magnetic field intensity of 10 kO
metal oxide-coated particles having a saturation magnetization of 40 to 90 emu / g. 2. The metal oxide-coated particles according to claim 1, wherein the base particles are synthetic polymer particles or natural polymer particles. 3. The metal oxide-coated particles according to claim 1, wherein the metal oxide is ferrite. 4. A method for producing metal oxide-coated particles, wherein a metal component is added to a substrate particle dispersion and a metal oxide layer is formed on the surface of the substrate particles under ultrasonic irradiation. 5. The method according to claim 4, wherein the base particles are synthetic polymer particles or natural polymer particles. 6. The method according to claim 4, wherein the metal oxide is ferrite. 7. The base particles having an average particle size of 5 to 10,000.
The method according to claim 4, wherein the thickness is 0 nm.