Method for producing particles for immunoturbidimetry, immunoturbidimetry particles, reagents, test kits, and detection methods

Abstract

The present invention provides a method for producing immunoturbidimetric particles with a high refractive index and good particle size distribution, immunoturbidimetric particles, reagents, a test kit, and a detection method. [Solution] A method for producing particles for immunoturbidimetry, comprising: a core particle formation step of emulsion polymerization of monomers to form core particles; a metal oxide layer formation step of dispersing the core particles in a liquid containing an alcohol-based medium and forming a metal oxide layer on the surface of the core particles by a sol-gel method using a metal alkoxide; and a crystallization step of dispersing the particles with the metal oxide layer in an aqueous medium and crystallizing the metal oxide layer; and a reagent, test kit, and detection method using the particles.

Description

[Technical Field]

[0001] This disclosure relates to a method for producing immunoturbidimetric particles, immunoturbidimetric particles, reagents, test kits, and detection methods. [Background technology]

[0002] In recent years, immunoturbidimetric testing has attracted attention as a simple and rapid immunoassay method. This method involves mixing a dispersion of particles having antibodies or antigens on their surface as ligands with a sample that may contain a target substance (antigen or antibody). If the sample contains the target substance (antibody or antigen), an agglutination reaction occurs among the particles. This agglutination reaction can be optically detected as changes in scattered light intensity, transmitted light intensity, absorbance, etc., to identify the presence or absence of disease.

[0003] Traditionally, polystyrene-based latex particles, primarily composed of polystyrene, have been used as particles for immunoturbidimetric methods because they are easy to sensitize (immobilize) with antigens or antibodies, are relatively inexpensive, and their polymerization reactions are easy to control. However, immunoturbidimetric methods using polystyrene latex particles sometimes failed to detect trace components in the low-concentration range. Therefore, there was a need to develop particles with higher sensitivity than polystyrene latex particles.

[0004] To solve the above problem, it is necessary to increase the absorbance change due to particle aggregation associated with immune complex formation. Since one of the factors that controls absorbance change is known to be the refractive index of the components that make up the particles, particle development using high refractive index materials is being pursued. For example, particle development is underway using titanium dioxide, which is well known as a high refractive index material. Patent document 1 proposes particles for immunoturbidimetry in which titanium dioxide nanoparticles are coated onto carboxylic acid-modified polystyrene particles, and a ligand is attached to these particles. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2008-241357 [Overview of the project] [Problems that the invention aims to solve]

[0006] However, the method of attaching titanium dioxide nanoparticles described in Patent Document 1 has insufficient sensitivity when used as a reagent for immunoturbidimetry, and it has been found that the coefficient of variation becomes large in measurements due to variations in interparticle characteristics such as particle size distribution and electronegativity of the particle surface. The purpose of this disclosure is to provide a method for producing immunoturbidimetry particles that can detect trace components in the low-concentration range and reduce measurement variability in immunoturbidimetry. [Means for solving the problem]

[0007] The inventors have discovered a method for producing particles for immunoturbidimetry that can detect trace components in low-concentration regions and reduce measurement variability by forming a metal oxide layer on the outside of the core particle using a metal alkoxide, and then crystallizing it, leading to this disclosure.

[0008] In other words, the first aspect of this disclosure is, A core particle formation step in which monomers are emulsion polymerized to form core particles. A metal oxide layer formation step is performed, in which the core particles are dispersed in a liquid containing an alcohol-based medium, and a metal oxide layer is formed on the surface of the core particles by a sol-gel method using a metal alkoxide, and A method for producing particles for immunoturbidimetry, characterized by comprising a crystallization step of dispersing particles having the metal oxide layer formed thereon in an aqueous medium and crystallizing the metal oxide layer.

[0009] Furthermore, a second aspect of this disclosure is: These are particles for immunoturbidimetry produced by the aforementioned manufacturing method.

[0010] and the third aspect of the present disclosure is a reagent characterized in that the particles for immunoturbidimetry are dispersed in an aqueous solution.

[0011] Furthermore, the fourth aspect of the present disclosure is an inspection kit characterized by having the reagent and a container containing the reagent.

[0012] In addition, the fifth aspect of the present disclosure is a method for detecting a target substance in a specimen by in vitro diagnosis, characterized by mixing the reagent and a specimen that may contain the target substance.

[0013] Finally, the sixth aspect of the present disclosure is a method for detecting a target substance in a specimen by in vitro diagnosis, comprising a step of mixing the reagent and a specimen that may contain the target substance to obtain a mixed solution, a step of irradiating the mixed solution with light, and a step of detecting at least one of transmitted light and scattered light from the light irradiated on the mixed solution.

Advantages of the Invention

[0014] According to the present disclosure, it is possible to produce particles for immunoturbidimetry that can detect trace components in a low-concentration region and reduce measurement variations. In addition, reagents and kits using the particles can be provided.

Brief Description of the Drawings

[0015] [Figure 1] Measurement result of X-ray diffraction pattern of metal oxide layer-forming particles 1 [Figure 2] Measurement result of X-ray diffraction pattern of particles 1

Embodiments for Carrying Out the Invention

[0016] Hereinafter, embodiments of the present disclosure will be described in detail, but the technical scope of the present disclosure is not limited to these embodiments. The method for producing particles for immunoturbidimetry of the present disclosure is as follows: (1) A core particle forming step of emulsion polymerization of a monomer to form core particles; (2) A metal oxide layer forming step of dispersing the core particles in a liquid containing an alcohol-based medium and forming a metal oxide layer on the surface of the core particles by a sol-gel method using a metal alkoxide, and (3) A crystallization step of dispersing the particles having a metal oxide layer in an aqueous medium and crystallizing the metal oxide layer. It is characterized by having the above steps.

[0017] In immunoturbidimetry, in order to detect trace components in the low concentration region, it is necessary to increase the change in absorbance due to particle aggregation accompanying the formation of immune complexes. The inventors of the present invention conceived that by forming a titanium oxide layer on the core particles and further increasing the refractive index of the titanium oxide layer by crystallization, it is possible to detect trace components in the low concentration region and reduce measurement variations, and thus arrived at the present disclosure.

[0018] The core particles of the present disclosure can be obtained by general methods for producing polymer particles such as emulsion polymerization method, soap-free polymerization method, dispersion polymerization method, suspension polymerization method, phase inversion emulsification method, wet grinding method, and dry grinding method. Although not particularly limited, particles obtained by the emulsion polymerization method and the soap-free polymerization method are preferable because they can obtain a desired volume average particle diameter and a uniform particle size distribution as particles for immunoturbidimetry.

[0019] Examples of the monomer are not particularly limited, but it is preferably a high refractive index. Preferred specific structures therefor include a styrene structure, a fluorene structure, a dinaphthothiophene structure, a naphthalene structure, an anthracene structure, and a phenanthrene structure. Specifically, it contains a repeating unit represented by formula (1).

Chemical formula

[0020] The form of the immunoturbidimetric particles produced by the manufacturing method of this disclosure is characterized by containing an organic polymer and a metal oxide, having a first layer (core layer) having a first organic polymer and a second layer (metal oxide layer) having a metal oxide, wherein the second layer is located outside the first layer.

[0021] The first layer having the first organic polymer in the immunoturbidimetric particles of this disclosure preferably further has a crosslinked structure. The crosslinked structure is obtained by polymerization using a crosslinkable radical polymerizable monomer, and is a monomer having two or more radical polymerizable unsaturated bonds in one molecule. Examples of such crosslinkable monomers include polyfunctional (meth)acrylates such as ethylene glycol diacrylate, ethylene glycol dimethacrylate, trimethylolpropane triacrylate, trimethylolpropane trimethacrylate, pentaerythritol triacrylate, pentaerythritol trimethacrylate, dipentaerythritol hexaacrylate, and dipentaerythritol hexamethacrylate, conjugated diolefins such as butadiene and isoprene, divinylbenzene, diallyl phthalate, allyl acrylate, and allyl methacrylate. Alternatively, two or more crosslinkable radical polymerizable monomers may be used. The crosslinked structure is more preferably the structure represented by formula (2). The cross-linked structure makes the particles physically stronger, eliminating concerns about cracking or chipping even when repeated centrifugation is performed during purification. [ka] (Z represents a substituted or unsubstituted phenylene group or naphthalene group; in the case of substitution, the substituent is a methyl group or an ethyl group. Z may differ for each structural unit.)

[0022] Examples of crosslinkable radical polymerizable monomers used to form the crosslinkable structure of formula (2) include 1,2-divinylbenzene, 1,3-divinylbenzene, 1,4-divinylbenzene, 2,6-diethynylnaphthalene, and 2,7-diethynylnaphthalene. These may be used individually or in combination. Among the crosslinkable radical polymerizable monomers exemplified, divinylbenzene is preferred. Although the reason is unclear, when divinylbenzene is used, it exhibits excellent handling properties during radical polymerization reactions and improves the monomer conversion rate during particle formation.

[0023] The metal oxide layer of this disclosure can be obtained by a sol-gel method for hydrolyzing a metal oxide precursor. That is, it can be obtained by carrying out a hydrolysis reaction in the presence of a metal oxide precursor, an alcohol-based medium, and water.

[0024] Examples of metal oxide precursors include metal chlorides, metal acetates, metal alkoxides, and metal hydroxides. Among these, metal alkoxides, metal acetates, and metal hydroxides are preferred from the viewpoint of by-product impurities (e.g., chlorides). In particular, metal alkoxides represented by the chemical formula Mx(OR)y (where M represents a metal element, R represents an alkyl group, and x and y are each independently integers between 1 and 4), metal hydroxides represented by the chemical formula Mx(OH)y·nH2O (where M represents a metal element, x and y are each independently integers between 1 and 4, and n is an integer of 1 or more), and compounds containing the aforementioned metal alkoxides and / or metal hydroxides are preferred.

[0025] In this disclosure, titanium alkoxides are more preferred. Specifically, examples include titanium methoxide, titanium ethoxide, titanium-diisopropoxide bis(2,4-pentanedione), titanium-diisopropoxide bis(ethylacetoacetate), titanium-n-butoxide, titanium isopropoxide, titanium methoxypropoxide, titanium-n-nonyloxide, titanium-n-propoxide, titanium stearyl oxide, titanium triisostearyl isopropoxide, titanium trimethylsiloxide, and the like.

[0026] Examples of alcohol-based media include ethanol, 1-propanol, 2-propanol, and butanol. Furthermore, it is preferable to include a nonionic water-soluble polymer in the alcohol-based medium during the metal oxide layer formation process. By adding a nonionic water-soluble polymer and forming a metal oxide layer, it is possible to suppress particle aggregation during metal oxide layer formation, thereby reducing measurement variability. Examples of nonionic water-soluble polymers include polyvinylpyrrolidone, polyethyleneimine ethoxylate, and polyvinyl alcohol.

[0027] In this disclosure, crystallization refers to the formation of a crystal lattice, which is an arrangement in which the unit cell arrangement of the metal crystal is repeated, in a metal oxide. By performing X-ray diffraction on the metal crystal, the crystal structure can be determined by the presence or absence of peaks, and the proportion of crystallized metal oxide (also referred to as the degree of crystallinity in this disclosure) can be calculated from the size of the peak area. In the crystallization process of the metal oxide layer in this disclosure, it is preferable to raise the temperature of the aqueous medium to 50°C or higher. By keeping the temperature of the aqueous medium within this range, titanium oxide can be partially crystallized from an amorphous state. This process improves the refractive index of the particles, thereby improving the detection sensitivity in immunoturbidimetry.

[0028] In the crystallization process of the metal oxide layer according to this disclosure, the pH of the aqueous medium is preferably 6 to 9, and more preferably it contains a salt in a concentration range of 1 mM to 100 mM. This suppresses pH fluctuations due to temperature changes in the metal oxide layer-forming particle dispersion and suppresses aggregation of metal oxide layer-forming particles in the crystallization process, thereby reducing measurement variability.

[0029] The immunoturbidimetric particles of this disclosure may have a third layer (organic layer) containing a second organic polymer outside the metal oxide layer. By forming an organic layer, the adsorption rate of antibodies that serve as biosensors is improved, and higher sensitivity can be achieved. The structure of the resin of the organic layer of this disclosure is not particularly limited as long as it contains repeating units represented by formula (3). [ka] (R3 represents a hydrogen atom or a methyl group. R4 represents a group having an epoxy group, a hydroxyl group, or a carboxyl group. 3 and R 4 (These may differ for each structural unit.)

[0030] The structure represented by formula (3-A) is preferable to the structure represented by formula (3-A). The structure represented by formula (3-A) has either a hydroxyl group or a carboxyl group. Therefore, its ability to suppress nonspecific adsorption is equivalent to or better than that of the structure having an epoxy group, which is preferable. Furthermore, it is thought that having either a hydroxyl group or a carboxyl group allows it to form hydrogen bonds with titanium dioxide and interact well. [ka] (R 31 and R 32 One of them represents a hydroxyl group, and the other represents a hydroxyl group or the group represented by formula (3-B). [ka] (R33 represents a single bond or a methylene group. R 34 , R 35 , R 36 represents a hydrogen atom, a methyl group, a hydroxy group, a carboxy group, a hydroxymethyl group, or a carboxymethyl group, and one or more of R 34 , R 35 , and R 36 contain a hydroxy group or a carboxy group. Y1 represents a sulfur atom or an imino group. *1 represents the bonding position with the structure represented by formula (3-A).)

[0031] By including a vinyl polymer as represented by formula (3), it is considered that there is no exposure of titanium oxide and uniform coating can be achieved. That is, the coating resin can interact well with titanium oxide and can uniformly coat without exposure of titanium oxide. Therefore, detachment of the resin can be suppressed and a decrease in the aqueous dispersion stability of the particles for immunoturbidimetry can be suppressed. Examples of the specific structure of formula (3) are shown in the following (3-A-1) to (3-A-12), but are not limited thereto.

Chemical formula

[0033] The organic layer of this disclosure, like the core particles, can be obtained by general polymer particle manufacturing methods such as emulsion polymerization, soap-free polymerization, dispersion polymerization, suspension polymerization, phase inversion emulsion, wet pulverization, and dry pulverization. Although not particularly limited, particles obtained by emulsion polymerization and soap-free polymerization are preferred because they provide a uniform particle size distribution for use in immunoturbidimetry.

[0034] (Method for measuring the titanium dioxide content in particles) This disclosure describes a method for measuring the titanium dioxide content in particles. The titanium dioxide content in particles is measured using a thermomass spectrometer. For example, NEXTA® STA200 (Hitachi High-Tech Science Corporation) is used. 5 to 10 mg of the cured material was weighed into an aluminum pan and measured under a nitrogen atmosphere. The temperature conditions were: held at 30°C for 30 minutes, then the temperature was increased from 30°C to 500°C at a rate of 10°C / min, followed by holding at 500°C for 10 minutes. For evaluation, the titanium dioxide content in the particles was measured from the mass loss of the resin component at 300°C.

[0035] (Method for measuring particle crystallization) Particle crystallization is measured using X'Pert-Pro (Malvern Panalytical). X-ray diffraction patterns are measured in the range of 20°≦2θ≦60° under conditions of X-ray output: 45kV, 40mA. The degree of crystallinity is calculated and evaluated as the ratio of the peak area indicating the crystalline component to the total measured peak area.

[0036] (Method for measuring volume-average particle size and particle size distribution in an aqueous dispersion of particles) This disclosure describes the method for measuring the volume-average particle size (Dv) of particles. The Dv of particles present in an aqueous dispersion is measured by dynamic light scattering. For example, a Zetasizer (Zetasizer Ultra: Malvern Panalytical) is used, and the measurement is performed at 25°C. Furthermore, the particle size distribution in this disclosure is calculated by measuring the number-average particle size (Dn) using the dynamic light scattering method described above, and then using the ratio of Dv to Dn (Dv / Dn).

[0037] (Particles for immunoturbidimetry) The manufacturing method disclosed herein can be used to produce particles for immunoturbidimetry. The particles for immunoturbidimetry may also be affinity particles having a ligand on their surface for detecting a target substance.

[0038] (A substance (ligand) that specifically binds to a target substance) A ligand is a compound that specifically binds to a receptor on a particular target substance. The binding site of a ligand to the target substance is fixed, and it has a selective or specific high affinity. Examples of ligands include, but are not limited to, antigens and antibodies, enzyme proteins and their substrates, signaling molecules such as hormones and neurotransmitters and their receptors, nucleic acids, avidin and biotin, etc. Specific examples of ligands include antigens, antibodies, antigen-binding fragments (e.g., Fab, F(ab')2, F(ab'), Fv, scFv, etc.), naturally occurring nucleic acids, artificial nucleic acids, aptamers, peptide aptamers, oligopeptides, enzymes, coenzymes, etc.

[0039] The ligand in the immunoturbidimetric particles of this disclosure is preferably an antibody or a virus-derived antigen. The ligand being an antibody or virus-derived antigen enables highly sensitive detection of target substances that bind to the antibody or antigen. In this disclosure, the method for immobilizing the ligand on the particles can be any known method, and the ligand can be immobilized by physically or chemically binding it to the particles. Examples of chemical binding methods include carbodiimide-mediated reactions, NHS ester activation reactions, and methods in which avidin is attached to a carboxyl group and then a biotin-modified ligand is attached.

[0040] (reagent) The immunoturbidimetric particles of this embodiment can also be used as a reagent for detecting a target substance via a ligand. While the form of the reagent is not limited, it is preferable to use a reagent in which the immunoturbidimetric particles of this disclosure are dispersed in an aqueous solution.

[0041] (Test kit) The reagent containing the immunoturbidimetry particles of this embodiment can also be used as a test kit for detecting a target substance via a ligand. The form of the test kit is not limited, but it preferably comprises the reagent containing the immunoturbidimetry particles of this disclosure and a container containing the reagent. The composition of the reagent is not particularly limited, but as an example, a preferred form is one in which the first reagent includes a buffer and a surfactant, and the second reagent includes a buffer, a surfactant, and particles for immunoturbidimetry.

[0042] (Detection method) The reagent containing the immunoturbidimetric particles of this embodiment can detect a target substance in a sample for in vitro diagnostic purposes. In this disclosure, "detection" may refer to both qualitative and quantitative detection of the target substance. While the method for detecting a target substance using the reagent containing the immunoturbidimetric particles of this embodiment is not particularly limited, a preferred method, as an example, comprises the following three steps. (1) A step of obtaining a mixture by mixing a sample that may contain a target substance with a reagent containing the immunoturbidimetry particles of this embodiment. (2) Step of irradiating the mixture with light (3) A step of detecting at least one of the transmitted light and scattered light from the light irradiated onto the mixture. [Examples]

[0043] The present disclosure will be described in detail below with reference to examples, but the present disclosure is not limited to these examples. [Particle Production Example 1] (Process 1 / Core Particle Formation Process) A mixture was prepared by weighing 12.68 g of styrene (St: Kishida Chemical Co., Ltd.), 0.23 g of divinylbenzene (DVB: Kishida Chemical Co., Ltd.), and 1512.02 g of deionized water into a 2 L four-neck separable flask. This mixture was maintained at 70°C while stirring at 140 rpm, and the inside of the four-neck separable flask was deoxygenated by flowing nitrogen at a flow rate of 200 mL / min. Next, a solution prepared separately by dissolving 0.55 g of V-50 (Fujifilm Wako Pure Chemical Corporation) in 20 g of deionized water was added to the mixture to initiate soap-free emulsion polymerization. After 23 hours of reaction from the start of polymerization, a dispersion of core particle 1 consisting of a copolymer of St and DVB was obtained. A portion of this dispersion was taken and evaluated using dynamic light scattering (Zetasizer Ultra: Malvern Panalytical), and the volume-average particle size was found to be 190 nm.

[0044] (Process-2 / Metal oxide layer formation process) A dispersion of core particles 1 with a solid content concentration of 0.6% by mass was prepared using deionized water to a concentration of 1 g. This dispersion was mixed with 20.94 g of ethanol (Kishida Chemical Co., Ltd.) containing 0.2% by mass of polyvinylpyrrolidone K-30 (PVP K-30: Kishida Chemical Co., Ltd.), and the mixture was maintained at 70°C while stirring at 800 rpm. Next, a solution of 125 μL of Titanium(IV)n-butoxide, monomer (TBOT: Kishida Chemical Co., Ltd.), prepared separately, mixed with 9.88 g of ethanol was added to the above mixture to initiate the sol-gel reaction. The reaction was allowed to proceed for 6 hours from the start of the sol-gel reaction, and the metal oxide layer-forming particles 1 were separated from the mixture by centrifugation and redispersed in ethanol. Furthermore, the metal oxide layer-forming particles 1 were separated from the dispersion using a centrifuge, and the process of redispersing the particles 1 in ion-exchanged water was repeated twice to purify the metal oxide layer-forming particles 1. The final aqueous dispersion was prepared to contain 5.0% by mass of metal oxide layer-forming particles 1 and stored. A portion of this dispersion was taken, and the dynamic light scattering of the metal oxide layer-forming particles 1 was evaluated, revealing a volume-average particle size of 200 nm. In addition, the metal oxide content was evaluated using differential thermal-thermogravimetric analysis (NEXTA® STA200RV: Hitachi High-Tech Corporation), which revealed it to be 43% by mass of the particle mass. Furthermore, when the crystallinity was evaluated by X-ray diffraction, as shown in Figure 1, no crystallization peak was observed in the metal oxide layer-forming particles 1.

[0045] (Process-3 / Metal oxide layer crystallization process) A 10 g dispersion of metal oxide layer-forming particle 1 with a solid content concentration of 0.5% by mass was prepared using a pre-prepared N-(2-Hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES: Kishida Chemical Co., Ltd.) buffer. The HEPES buffer used was a 10 mM HEPES buffer adjusted to pH 7.86 using a 1N sodium hydroxide aqueous solution. The above metal oxide layer-forming particle 1 dispersion was maintained at 70°C while being stirred at 800 rpm to initiate the crystallization of the metal oxide layer. After 24 hours of reaction from the start of crystallization, particles 1 with a crystallized metal oxide layer were obtained. A portion of these particles was taken, and the dynamic light scattering of particle 1 was evaluated, revealing a volume-average particle size of 200 nm. Furthermore, when the crystallinity was evaluated by X-ray diffraction, a peak was observed as shown in Figure 2. This indicates that a portion of the titanium oxide layer on the surface of particle 1 has crystallized and adopts an anatase-type crystalline structure.

[0046] [Particle production example 2] (Process 1 / Core Particle Formation Process) Core particles 2 were prepared by step 1 described in Example 1. (Process-2 / Metal oxide layer formation process) Metal oxide layer-forming particles 2 were prepared by step 2 described in Example 1.

[0047] (Process-3 / Organic layer formation process) A dispersion of metal oxide layer-forming particles 2 with a solid content of 0.2% by mass was prepared in ion-exchanged water to a volume of 149.55 g. 0.135 g of glycidyl methacrylate (GMA: Kishida Chemical Co., Ltd.) and 0.015 g of 3-Methacryloxypropyltrimethoxysillane (MPS: Shin-Etsu Chemical Co., Ltd.) were added, and the mixture was maintained at 70°C while stirring at 100 rpm. The four-neck separable flask was deoxygenated by flowing nitrogen at a flow rate of 200 mL / min. Then, a solution of 0.03 g of V-50 dissolved in 0.3 g of ion-exchanged water, which had been prepared separately, was added to the mixture to initiate shell formation. After stirring for 18 hours, a dispersion containing organic layer-forming particles 2 having a core, metal oxide layer, and shell structure was obtained. A portion of the dispersion was taken out, and its crystallinity was evaluated by X-ray diffraction, revealing peaks indicating anatase-type crystal structure.

[0048] (Process 4 / Functional group imparting process) An aqueous solution of mercaptosuccinic acid (MSA: Fujifilm Wako Pure Chemical Industries, Ltd.), prepared in advance, was added to a dispersion containing organic layer-forming particles 2. The aqueous solution was prepared so that the total number of moles of MSA was equal to the number of moles of glycidyl methacrylate. Next, triethylamine (Kishida Chemical Co., Ltd.) was added to adjust the pH to 10. Then, the mixture was heated to 70°C while stirring at 800 rpm, and maintained at this temperature for 18 hours to obtain a dispersion of particles 2 having a core, metal oxide layer, shell structure, and functional groups. Particles 2 were separated from the dispersion using a centrifuge, and the particle 2 was further purified by repeating the process of redispersing in ion-exchanged water eight times. Finally, the aqueous dispersion was prepared to contain 5.0% by mass of particles 2 and stored.

[0049] Particle 2 contained a styrene-divinylbenzene copolymer in its core, that is, a structural unit represented by formula (1) in which R1 is a hydrogen atom and R2 is a phenyl group. The organic layer also contained a structural unit represented by formula (3) in which R3 is a methyl group and R4 is a structural unit represented by any of the following formulas (3-C), (3-D), or (3-E). [ka] [ka] [ka] (*3 indicates the bonding position with the structure shown in equation (3).)

[0050] [Example of comparative particle production] (Step 1: Core particle preparation) 100 μL of carboxy-modified polystyrene microparticle dispersion (IMTEX: JSR Corporation), 900 μL of 1% bovine serum albumin-containing HEPES buffer, and 500 μL of soluble carbodiimide solution (1M aqueous solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, hereafter the same) were added to a centrifuge tube and kept at room temperature (20°C to 30°C) for 1 hour while stirring at 100 rpm. Next, the carboxy-modified polystyrene particles were separated from the dispersion by centrifugation and further redispersed in ion-exchanged water. The carboxy-modified polystyrene particles were further separated from the dispersion by centrifugation and redispersed in 500 μL of 1% bovine serum albumin-containing HEPES buffer to obtain a carboxyl-activated polystyrene microparticle dispersion.

[0051] (Process-2 / Metal oxide layer formation process) 50 μL of titanium dioxide nanoparticle dispersion and 950 μL of γ-aminopropyltriethoxysilane aqueous solution (ion-exchanged water containing 1% by mass of γ-aminopropyltriethoxysilane) were added to a centrifuge tube and kept at room temperature (20-30°C) for 1 hour while stirring at 100 rpm. Next, the titanium dioxide nanoparticles were separated from the dispersion by centrifugation, and the titanium dioxide nanoparticles were redispersed in acetate buffer (0.01 M, pH 5.0) three times. The resulting precipitate was redispersed in 500 μL of phosphate buffer (0.01 M, pH 6.0) to obtain an amino group-introduced titanium dioxide nanoparticle dispersion.

[0052] 88 μL of an amino group-introduced titanium dioxide microparticle dispersion and 500 μL of a carboxyl group-activated polystyrene microparticle dispersion were placed in a centrifuge tube and kept at room temperature (20-30°C) for 2 hours while stirring. Next, titanium dioxide-polystyrene composite microparticles were separated from the dispersions by centrifugation, and the precipitate was redispersed in 1,500 μL of phosphate buffer (0.1 M, pH 7.1) to obtain a titanium dioxide-polystyrene composite microparticle dispersion. A titanium dioxide-polystyrene composite fine particle dispersion was centrifuged using density gradient centrifugation to obtain a fraction with a density of 1.6 (containing 20% ​​by mass of titanium dioxide in the composite particles) as comparison particle 1.

[0053] [Examples] (Preparation of particles for immunoturbidimetry; Particle 1) Examples 1, 2, and the Comparative Example show examples of particles for immunoturbidimetry using ferritin as the target substance. For the dispersion of particle 1, 100 μL of the dispersion (1 mg as particle solids), diluted with deionized water to a solid content concentration of 1.0% by mass, was placed in a 1.5 mL microcentrifuge tube, washed by centrifugation, and then HEPES buffer at pH 7.0 was added and dispersed by sonication. To this, 24 μL of a 5.0 mg / mL dispersion of mouse monoclonal anti-ferritin antibody (isoelectric point 7.1) (0.12 mg as antibody) was added and stirred at room temperature for 1 hour to obtain immunoturbidimetric particle 1, in which the particles were sensitized with the antibody.

[0054] (Preparation of particles for immunoturbidimetry: Particle 2, comparison particle 1) For the dispersions of particle 2 and comparative particle 1, 300 μL of the dispersion (3 mg as particle solids), diluted with deionized water to a solid content concentration of 1.0% by mass, was placed in a 1.5 mL microtube. To this, 90 μL of a 5.0% by mass aqueous solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (Tokyo Chemical Industries, Ltd.) and 90 μL of a 5.0% by mass aqueous solution of N-hydroxysulfosuccinimide sodium (Tokyo Chemical Industries, Ltd.) were added, and the mixture was stirred at room temperature for 30 minutes to obtain an activated particle dispersion containing carboxyl groups (activated particle dispersion). After centrifugation, 270 μL of pH 5.5 phosphate buffer-physiological saline (PBS) was added, and the particles with activated carboxyl groups were dispersed by sonication. Then, 24 μL of a 5.0 mg / mL dispersion of mouse monoclonal anti-ferritin antibody (isoelectric point 7.1) (0.12 mg of antibody) was added, and the mixture was stirred at room temperature for 3 hours to obtain immunoturbidimetric particles 2 and immunoturbidimetric comparison particles 1, in which the particles were sensitized with the antibody.

[0055] (Preparation of the first reagent) The first reagent was prepared by dissolving 50 mM HEPES, 0.05% by mass Triton X-100, and 1.0% by mass sodium chloride (Kishida Chemical Co., Ltd.) in deionized water.

[0056] (Preparation of the second reagent) After centrifuging and washing the immunoturbidimetric particle 1, it was redispersed in 500 μL of a buffer (HEPES buffer) prepared by dissolving 10 mM HEPES, 0.01% by mass polyoxyethylene nonylphenyl ether (Triton X-100: Kishida Chemical Co., Ltd.), and 10% by mass sucrose (viscosity modifier) ​​in deionized water. Subsequently, mixing and dilution with HEPES buffer were performed so that the immunoturbidimetric particle content was 0.1% by mass to obtain the second reagent 1. The second reagents 2 and 3 were prepared using the same experimental procedure as the preparation of the second reagent 1, except that the particle type was changed from immunoturbidimetric particle 1 to immunoturbidimetric particle 2 or immunoturbidimetric comparison particle 1.

[0057] (Measurement of change in absorbance) A mixture was prepared by mixing 15 μL of a sample with a ferritin concentration of 250 ng / mL with 60 μL of the first reagent 1, and the mixture was incubated at 37°C for 290 seconds. Next, 30 μL of the second reagent 1 was mixed into the mixture, and the absorbance was measured after stirring for 42 seconds. Furthermore, this mixture was allowed to stand at 37°C for 253 seconds, and the absorbance was measured again. The difference from the absorbance after 30 seconds was defined as the absorbance change ΔAbs. Absorbance measurements were performed using a BIOSPECTROMETER (Eppendorf) spectrophotometer at a wavelength of 572 nm.

[0058] (Calculation of sensitivity index) The value of ΔAbs × 10000 was calculated and used as the ferritin sensitivity index. A higher ferritin sensitivity index is expected to indicate more sensitive detection of the target substance. The evaluation was based on the sensitivity index values ​​as follows. A: ΔAbs × 10000 was a value greater than 100. B:ΔAbs×10000 was a value less than or equal to 100. The results for each particle are shown in Table 1.

[0059] (Calculation of sensitivity variation) The change in absorbance was measured 10 times, and the coefficient of variation for all measurements was calculated and used as an indicator of sensitivity variability. A smaller coefficient of variation is expected to indicate smaller variability between measurements. The evaluation was based on the value of the coefficient of variation as follows. A: The coefficient of variation was less than 3%. B: The coefficient of variation was 3% or higher. The results for each particle are shown in Table 1.

[0060] [Table 1]

[0061] These results show that the immunoturbidimetric particles produced according to this disclosure exhibit a large change in absorbance and a small coefficient of variation at a target substance concentration of 250 ng / mL. Therefore, it was found that the particles produced according to this disclosure can detect trace components in the low-concentration range and reduce measurement variability.

[0062] This embodiment includes the following configurations and methods. (Method 1) A core particle formation step in which monomers are emulsion polymerized to form core particles. A metal oxide layer formation step is performed, in which the core particles are dispersed in a liquid containing an alcohol-based medium, and a metal oxide layer is formed on the surface of the core particles by a sol-gel method using a metal alkoxide, and Crystallization step: Disperse the particles having formed the metal oxide layer in an aqueous medium and crystallize the metal oxide layer. A method for producing particles for immunoturbidimetry, characterized by having the following features. (Method 2) A method for producing particles for immunoturbidimetry according to Method 1, characterized in that the monomer in the core particle formation step is represented by the following formula (1). [ka] (R1 represents a hydrogen atom or a methyl group. R2 represents a structure containing a phenyl group or an ester bond, which may be substituted.) (Method 3) A method for producing particles for immunoturbidimetry according to Method 1, characterized in that the alcohol-based medium in the metal oxide layer formation step includes a nonionic water-soluble polymer. (Method 4) A method for producing particles for immunoturbidimetry according to any one of methods 1 to 3, characterized in that titanium alkoxide is used as the metal alkoxide in the metal oxide layer formation step. (Method 5) A method for producing particles for immunoturbidimetry according to any one of methods 1 to 4, characterized by further comprising the step of forming an organic layer on the surface of the particles on which the metal oxide layer is formed. (Method 6) A method for producing particles for immunoturbidimetry according to any one of methods 1 to 5, characterized in that, in the crystallization step, the aqueous medium in which the particles having formed the metal oxide layer are dispersed is stirred and the temperature of the aqueous medium is increased to crystallize the metal oxide layer. (Composition 7) Particles for immunoturbidimetry produced by the manufacturing method described in any one of Methods 1 to 6. (Composition 8) Particles for immunoturbidimetry according to configuration 7, characterized by having a ligand on their surface. (Composition 9) A reagent characterized in that the immunoturbidimetry particles described in component 7 are dispersed in an aqueous solution. (Composition 10) A test kit characterized by comprising the reagent described in configuration 9 and a container containing the reagent. (Method 11) A method for detecting a target substance in a specimen by in vitro diagnostics, characterized by mixing the reagent described in configuration 9 with a specimen that may contain the target substance. (Composition 12) A method for detecting a target substance in a specimen by in vitro diagnostics, comprising the steps of: mixing the reagent described in configuration 9 with a specimen potentially containing the target substance to obtain a mixed solution; irradiating the mixed solution with light; and detecting at least one of the transmitted light and scattered light from the light irradiated onto the mixed solution.

Claims

1. A core particle formation step in which monomers are emulsion polymerized to form core particles. A metal oxide layer formation step is performed, in which the core particles are dispersed in a liquid containing an alcohol-based medium, and a metal oxide layer is formed on the surface of the core particles by a sol-gel method using a metal alkoxide, and Crystallization step: Disperse the particles having formed the metal oxide layer in an aqueous medium and crystallize the metal oxide layer. A method for producing particles for immunoturbidimetry, characterized by having the following features.

2. A method for producing particles for immunoturbidimetry according to claim 1, characterized in that the monomer in the core particle formation step is represented by the following formula (1). 【Chemistry 1】 (R １ R represents a hydrogen atom and a methyl group. ２ (This indicates a structure containing a phenyl group or an ester bond, which may be substituted.)

3. The method for producing particles for immunoturbidimetry according to claim 1, characterized in that the alcohol-based medium in the metal oxide layer formation step includes a nonionic water-soluble polymer.

4. The method for producing particles for immunoturbidimetry according to claim 1, characterized in that titanium alkoxide is used as the metal alkoxide in the metal oxide layer formation step.

5. The method for producing particles for immunoturbidimetry according to claim 1, further comprising the step of forming an organic layer on the surface of the particles on which the metal oxide layer is formed.

6. The method for producing particles for immunoturbidimetry according to claim 1, characterized in that, in the crystallization step, the aqueous medium in which the particles having formed the metal oxide layer are dispersed is stirred and the temperature of the aqueous medium is increased to crystallize the metal oxide layer.

7. Particles for immunoturbidimetry produced by the manufacturing method described in any one of claims 1 to 6.

8. Particles for immunoturbidimetry according to claim 7, characterized by having a ligand on its surface.

9. A reagent characterized in that the immunoturbidimetry particles described in claim 7 are dispersed in an aqueous solution.

10. A test kit comprising the reagent described in claim 9 and a container containing the reagent.

11. A method for detecting a target substance in a specimen by in vitro diagnostics, characterized by mixing the reagent described in claim 9 with a specimen that may contain the target substance.

12. A method for detecting a target substance in a specimen by in vitro diagnostics, comprising the steps of: mixing the reagent described in claim 9 with a specimen potentially containing the target substance to obtain a mixture; irradiating the mixture with light; and detecting at least one of the transmitted light and scattered light from the light irradiated onto the mixture.

Images