Composite particles, labeling substance, immunological measurement method, reagent for immunological measurement, and method for manufacturing composite particles

Abstract

Provided are composite particles having excellent rigidity and visibility.　According to the present invention, provided are composite particles comprising resin particles and metal particles. The resin particles each include a core portion and a shell layer that covers a surface of the core portion. The glass transition temperature of the core portion is 50 to 200°C. The glass transition temperature of the shell layer is -100 to 0°C. With respect to 100 vol% of the total of the metal particles, 80 to 100 vol% of the metal particles are included in the shell layer or present on a surface of the shell layer.

Description

Composite particles, labeled substances, immunoassays, immunoassay reagents, and methods for producing composite particles

[0001] The present invention relates to composite particles that can be used in immunoassays, labeling substances using the same, immunoassays, and reagents for immunoassays, as well as methods for producing composite particles.

[0002] Analytical techniques for detecting specific substances from mixtures containing a wide variety of substances are utilized in many industries. One such analytical technique is immunoassay, which is a measurement method that utilizes highly selective antigen-antibody reactions. For example, by binding a labeling substance to an antigen, antibody, or a complex of these, and detecting changes in the concentration of the labeling substance, qualitative and quantitative measurement becomes possible. Therefore, by using a labeling substance with excellent labeling ability, the substance to be analyzed can be easily detected and measured with higher accuracy.

[0003] JP 2011-117906 A JP 2009-192270 A JP 2017-120242 A JP 2012-242162 A

[0004] When plasmonic nanoparticles such as gold nanoparticles are used as labeling substances, detection can be confirmed visually without the use of special analytical equipment. However, because the color of plasmonic nanoparticles is determined by their particle size and preparation conditions, it is difficult to obtain the desired vivid and deep color, resulting in insufficient visibility, which is particularly noticeable when the antigen concentration is low.

[0005] Prior Art Documents 1 and 2 disclose a method for enhancing the visibility of labeled substances by further modifying the labeled substances with other metals after the reaction between the antibody (labeled antibody) bound to the labeled substance and the antigen, thereby amplifying the detection sensitivity. However, this method requires specialized equipment and is limited in its usable environment. Prior Art Documents 3 and 4 disclose composite particles in which gold nanoparticles are bound to resin particles. However, because the three-dimensional arrangement and particle size distribution of the gold nanoparticles are not controlled, the optical properties of surface plasmon resonance are not fully exhibited, resulting in insufficient color clarity and luminescence intensity. Furthermore, the gold nanoparticles bound to the surface of the resin particles are easily detached by external forces, posing issues with sensitivity stability.

[0006] The present invention has been made in view of the above circumstances, and aims to provide composite particles that are excellent in rigidity and visibility.

[0007] The composite particles of the present invention have excellent rigidity due to the high glass transition temperature of the core portion, and excellent visibility due to the localization of metal particles near the surface of the resin particles. Specifically, the present invention relates to the following: [1] Composite particles comprising resin particles and metal particles, wherein the resin particles comprise a core portion and a shell layer covering the surface of the core portion, the core portion having a glass transition temperature of 50 to 200°C, the shell layer having a glass transition temperature of -100 to 0°C, and 80 to 100% by volume of the metal particles, relative to 100% by volume of the total metal particles, being encapsulated in the shell layer or present on the surface of the shell layer. [2] The composite particle according to [1], wherein 80 to 100% by volume of the metal particles, relative to 100% by volume of the total metal particles, are encapsulated in the shell layer. [3] The composite particle according to [1] or [2], wherein the average particle diameter is 200 to 1500 nm. [4] The composite particle according to any one of [1] to [3], wherein the metal particles are gold nanoparticles having an average particle diameter of 5 to 80 nm and a particle diameter polydispersity index (PDI) of 0.2 or less as determined by dynamic light scattering. [5] The composite particle according to any one of [1] to [4], wherein, in an image of the composite particle observed with a field emission scanning electron microscope (FE-SEM) at a magnification of 100,000 to 500,000 and an accelerating voltage of 50 kV, the metal particle coverage (ΣAm / A) is obtained by dividing the total area (ΣAm) of the metal particles within a square inscribed on the circumference of the composite particle by the area (A) of the square, and is 20 to 80%. [6] The composite particle according to any one of [1] to [5], wherein the shell layer contains an amino group-containing polymer. [7] The composite particle according to any one of [1] to [6], wherein the core portion contains a styrene-based polymer. [8] A labeled substance comprising the composite particle according to any one of [1] to [7]. [9] The labeled substance according to [8], having an antigen or an antibody on the surface of the composite particle.

[10] An immunoassay method using the labeled substance according to [8].

[11] A reagent for immunoassay, having the labeled substance according to [8].

[12] A method for producing composite particles, comprising a supporting step, in which metal particles are supported on resin particles, the resin particles comprising a core portion and a shell layer covering a surface of the core portion, the core portion having a glass transition temperature of 50 to 200°C, and the shell layer having a glass transition temperature of -100 to 0°C.

[0008] 1 is a cross-sectional schematic diagram of a composite particle 100 according to one embodiment of the present invention, which comprises encapsulated particles 21, partially exposed particles 22, and surface-adsorbed particles 23 as metal particles 20. FIG. 1 is a cross-sectional schematic diagram of a composite particle 100 according to one embodiment of the present invention, which comprises only encapsulated particles 21 as metal particles 20. FIG. 1 is a schematic diagram showing an example in which a circumference 30 of composite particle 100 and a square 40 inscribed in circumference 30 are defined using image analysis software in a top view image of composite particle 100 comprising metal particles 20 including surface-adsorbed particles 23, obtained with a field emission scanning electron microscope (FE-SEM) at a magnification of 100,000 to 500,000 and an acceleration voltage of 50 kV, in order to calculate the metal particle coverage (ΣAm / A).

[0009] Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. Various features shown in the following embodiments can be combined with each other. Furthermore, each feature can be an invention independently.

[0010] 1. Composite Particle 100 FIG. 1 is a cross-sectional schematic diagram of a composite particle 100 according to one embodiment of the present invention, which includes encapsulated particles 21, partially exposed particles 22, and surface-adsorbed particles 23 as metal particles 20. FIG. 2 is a cross-sectional schematic diagram of a composite particle 100 according to one embodiment of the present invention, which includes only encapsulated particles 21 as metal particles 20. As shown in FIGS. 1 and 2, the composite particle 100 includes a resin particle 10 having a core 11 and a shell layer 12 covering the core 11 and a plurality of metal particles 20 supported on the resin particle 10. The metal particles 20 can be classified into encapsulated particles 21, which are metal particles completely encapsulated in the resin particle 10; partially exposed particles 22, which are metal particles having a portion embedded in the resin particle 10 and a portion exposed outside the resin particle 10; and surface-adsorbed particles 23, which are metal particles adsorbed on the surface of the resin particle 10. As shown in FIGS. 1 and 2, the particle diameter D of the entire composite particle 10 is １and the particle diameter D of the resin particles 10 ２ and the particle diameter D of the metal particles 20 ３ The relationship between １ ≧D ２ >D ３ As shown in FIG. 2, when all the metal particles 20 are encapsulated particles 21, D １ =D ２ is.

[0011] The average particle diameter of the composite particles 100, i.e., the particle diameter D in FIGS. １ The average of the particle diameters is preferably 200 to 1500 nm. The average particle diameter of the composite particle 100 is, for example, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 nm, and may be within a range between any two of the values ​​exemplified here. In this specification, the "average particle diameter" of the composite particle 100, the resin particle 10, and the metal particle 20 all refer to the particle diameter (median diameter) at an integrated value of 50% in the particle size distribution determined by dynamic light scattering. If the average particle diameter of the composite particles 100 is too small, the amount of metal particles 20 carried will be small, and visibility will tend to be insufficient, while if the average particle diameter is too large, when used as a labeling substance for immunoassay (hereinafter also simply referred to as "labeling substance") or a reagent for immunoassay (hereinafter also simply referred to as "reagent"), the particles will tend to clog the pores of a chromatographic medium such as a membrane filter and will tend to have reduced dispersibility. The average particle diameter of the composite particles 10 can be measured by dynamic light scattering.

[0012] 2. Resin Particles 10 The resin particles 10 are relatively larger than the metal particles 20 and include a core 11 and a shell layer 12 covering the core 11's surface. The core 11 and shell layer 12 are composed of polymers with different glass transition temperatures (Tg), with the core 11 having a Tg of 50 to 200°C and the shell layer 12 having a Tg of -100 to 0°C. In other words, the core 11 is harder than the shell layer 12, making it difficult for the metal particles 20 to penetrate into the core 12 during the process of supporting the metal particles 20 on the resin particles 10 (supporting process), as described below. The glass transition temperature of the polymer is determined using the value described in the Polymer Handbook, Second Edition. If the glass transition temperature of the polymer is not described in the above literature, values ​​described in other literature or product catalogs, or values ​​measured by producing the polymer, can be used.

[0013] The polymer constituting the core portion 11 is a hydrophobic polymer having a Tg of 50 to 200° C. Specific examples of the Tg of the polymer constituting the core portion 11 include 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200° C., and may be within a range between any two of the values ​​exemplified here. Examples of polymers constituting the core portion 11 include styrene-based polymers obtained by polymerizing raw material monomers including styrene-based monomers such as styrene, methylstyrene, dimethylstyrene, trimethylstyrene, ethylstyrene, and diethylstyrene, as well as polyesters, polyolefins, poly(meth)acrylates, polycarbonates, polyphenylene sulfide, polyphenylene oxide, polyethersulfone, polyamides, polyamideimides, polytetrafluoroethylene, polyacrylonitrile, and copolymers of the monomers constituting these polymers. Among these, from the viewpoint of ensuring the mechanical strength of the resin particles 10 and dispersibility in a dispersion medium such as water, it is preferable to use styrene-based polymers, which have sufficient rigidity for practical use and a relatively low specific gravity. The raw material monomers for the styrene-based polymer may be solely the styrene-based monomers described above, or may contain other monomers in addition to the styrene-based monomers. The styrene-based monomers may be used alone or in combination of two or more. The proportion of the styrene-based monomer in the raw material monomers is, for example, 60 to 100% by mass, specifically, for example, 60, 65, 70, 75, 80, 85, 90, 95, or 100% by mass, and may be within a range between any two of the values ​​exemplified here. By using such a polymer to form the core portion 11, the mechanical strength of the resin particle 10 is increased, preventing the composite particle 100 from being destroyed by external forces applied during transportation or during the manufacture of reagents such as test kits. The structure of the polymer forming the core portion 11 may be a random polymer, a block polymer, or a graft polymer.

[0014] The polymer constituting the shell layer 12 has a Tg of -100 to 0°C and is capable of electrostatic interaction with the metal particles 20. Specific examples of the Tg of the polymer constituting the shell layer 12 include -100, -95, -90, -85, -80, -75, -70, -65, -60, -55, -50, -45, -40, -35, -30, -25, -20, -15, -10, -5, and 0°C, and may be within a range between any two of the values ​​exemplified here. In one embodiment, the polymer constituting the shell layer 12 is preferably an amino group-containing polymer. The amino group-containing polymer is, for example, a polymer obtained by copolymerizing a monomer component containing a polymerizable monomer having an amino group, or a polymer obtained by modifying a base polymer with an amino group. Examples of the base polymer include polybutadiene, polyisoprene, polyisobutylene, styrene-butadiene copolymer (SBR), isobutylene-isoprene copolymer, acrylonitrile-butadiene copolymer, ethylene-propylene copolymer, polychloroprene, polyoxymethylene, polyethylene oxide, polypropylene oxide, polyvinylidene fluoride, and polybutoxyacrylate. The method for modifying the base polymer with amino groups is not particularly limited, and conventionally known methods such as a substitution reaction with ammonia (ammonolysis) and reductive amination can be used. Examples of such amino group-containing polymers include aminated polybutadiene (PB-NH ２ ), aminated styrene-butadiene copolymer (SBR-NH ２ ), aminated polyisoprene, aminated polybutyl acrylate, etc. are examples. The content of amino groups in the amino group-containing polymer is preferably 0.01 to 2.0% by mass relative to 100% by mass of the amino group-containing polymer. Specific examples of the content of amino groups in the amino group-containing polymer are 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 1.5, and 2.0% by mass, and may be within a range between any two of the values ​​exemplified here. The structure of the polymer constituting the shell layer 12 may be a random polymer, a block polymer, or a graft polymer.

[0015] In one embodiment, the amino group-containing polymer is aminated polybutadiene (PB-NH ２ The content of amino groups in the aminated polybutadiene is preferably 0.01 to 2.0% by mass relative to 100% by mass of the aminated polybutadiene. Specific examples of the content of amino groups in the aminated polybutadiene are 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1.0, 1.5, and 2.0% by mass, and may be within a range between any two of the values ​​exemplified here.

[0016] The polymer constituting the shell layer 12 contains amino groups, which allows the composite particle 100 to easily bind to antibodies having carboxy groups. Furthermore, because the amino-group-containing polymer has a positive surface potential, when the metal particles 20 have a negative surface potential, they can bond through electrostatic interaction. Therefore, when the metal particles 20 are supported on the resin particle 10, the metal particles 20 tend to penetrate and be encapsulated in the shell layer 12, and the proportion of metal particles present in the core portion 11 of the encapsulated particles 21 is 0 to approximately 0% by mass. Thus, the presence of amino groups in the shell layer 12 reduces the proportion of partially exposed particles 22 and surface-adsorbed particles 23, allowing the metal particles 20 to be concentratedly distributed within the shell layer 12. This allows the composite particle 100 to achieve excellent visibility and stable sensitivity when used in immunoassays.

[0017] The average particle diameter of the resin particles 10, i.e., the particle diameter D in FIGS. ２The average is preferably 200 to 1500 nm. The average particle size of the resin particles 10 is, for example, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 nm, and may be within a range between any two of the values ​​exemplified here. If the average particle size of the resin particles 10 is too small, the amount of metal particles 20 supported tends to be small, resulting in insufficient visibility. Furthermore, if the average particle size is too large, when used as a labeling substance or reagent, the resin particles 10 tend to clog the pores of chromatographic media such as membrane filters and have reduced dispersibility. The average particle size of the resin particles 10 can be measured by dynamic light scattering.

[0018] 3. Metal Particles 20 Examples of the metal particles 20 include particles of gold, silver, copper, aluminum, platinum, palladium, nickel, etc. In particular, it is preferable to use gold nanoparticles because they have a negative surface potential and provide excellent visibility. The average particle diameter of the metal particles 20, i.e., the particle diameter D in FIGS. 1 and 2, is ３ The average of the metal particles 20 is preferably 5 to 80 nm. Specifically, the average particle diameter of the metal particles 20 may be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 nm, and may be within a range between any two of the values ​​exemplified here. If the average particle diameter of the metal particles 20 is too small or too large, localized surface plasmon resonance and light energy absorption due to electronic transition are less likely to occur, which tends to reduce detection sensitivity. When the metal particles 20 are gold nanoparticles, the average particle diameter of the metal particles 20 is preferably 5 to 80 nm, and more preferably 5 to 50 nm.

[0019] Particle diameter D of metal particles 20 ３can be measured by dynamic light scattering. When gold nanoparticles are used as the metal particles 20, the polydispersity index (PDI) of the particle size of the gold nanoparticles determined by dynamic light scattering is preferably 0.2 or less. The PDI is an index that represents the width of the particle size distribution, and its value ranges from 0 to 1. The smaller the PDI value, the narrower the width of the particle size distribution. The PDI of the particle size of gold nanoparticles determined by dynamic light scattering is, for example, 0, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, or 0.20, and may be within a range between any two of the values ​​exemplified here. By setting the PDI value of the particle diameter of the gold nanoparticles to 0.2 or less, the plasmon absorption band becomes sharper, and the desired clear and deep color tone can be obtained, resulting in excellent visibility when used as a labeling substance. Note that, if nominal values ​​are provided by the manufacturer or the like for the average particle diameter and PDI of the metal particles 20, those nominal values ​​may be used.

[0020] The thickness T of the shell layer 12 and the particle diameter D of the metal particle 20 ３ The ratio T / D ３ is at least 1, preferably 1 to 5, and more preferably 1 to 3. ３ Specifically, T / D is, for example, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0, and may be within a range between any two of the values ​​exemplified here. ３ By setting the thickness of the resin particles 10 within this range, the metal particles 20 can be encapsulated in the shell layer 12, and the metal particles 20 can be distributed in a concentrated manner near the surface of the resin particles 10. This is preferable because it can maximize the occurrence of light energy absorption due to electron transition in addition to localized surface plasmon resonance.

[0021] As described above, the metal particles 20 are classified into encapsulated particles 21, partially exposed particles 22, and surface-adsorbed particles 23. The encapsulated particles 21 are particles whose entire surface is covered with the polymer that constitutes the resin particles 10. The partially exposed particles 22 are particles whose surface area is 5% or more but less than 100% covered with the polymer that constitutes the resin particles 10. The surface-adsorbed particles 23 are particles whose surface area is more than 0% but less than 5% covered with the polymer that constitutes the resin particles 10. The encapsulated particles 21, partially exposed particles 22, and surface-adsorbed particles 23 all exhibit localized surface plasmon resonance and light energy absorption due to electronic transition, thereby contributing to improved visibility of labeled substances, reagents, etc. Therefore, from the perspective of visibility, the metal particles 20 may be any of the encapsulated particles 21, partially exposed particles 22, and surface-adsorbed particles 23. On the other hand, the encapsulated particles 21 have a larger contact area with the resin particles 10 than the partially exposed particles 22 and the surface-adsorbed particles 23, and in addition, the anchoring effect due to the embedded state is more pronounced, so that the encapsulated particles 21 are less likely to detach from the resin particles 10. Therefore, from the viewpoint of the sensitivity stability and durability of the labeling substance or reagent using the composite particles 100, it is preferable to increase the abundance ratio of the encapsulated particles 21 in the metal particles 20.

[0022] In one embodiment, it is preferable that 80 to 100% by volume of the metal particles 20, relative to a total of 100% by volume of the metal particles 20 in the composite particle 100, are encapsulated in the shell layer 12 as encapsulated particles 21, or are present on the surface of the shell layer 12 as partially exposed particles 22 and / or surface-adsorbed particles 23. In one embodiment, it is more preferable that 80 to 100% by volume of the metal particles 20, relative to a total of 100% by volume of the metal particles 20 in the composite particle 100, are encapsulated in the shell layer 12 as encapsulated particles 21. The volume ratio of the encapsulated particles 21 in the shell layer 12 to the total 100 volume% of the metal particles 20 in the composite particle 100 is specifically, for example, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 volume%, and may be within a range between any two of the values ​​exemplified here. Note that the volume ratios (metal particle abundance ratios) of the partially exposed particles 22 and surface-adsorbed particles 23, the encapsulated particles 21 in the shell layer 12, and the encapsulated particles 21 in the core portion 11 to the total 100 volume% of the metal particles 20 can be calculated, for example, using a transmission electron microscope (TEM) by a method described in the Examples.

[0023] The metal particles 20 are preferably located near the surface of the composite particle 100. Specifically, from an image of the composite particle 100 observed with a field emission scanning electron microscope (FE-SEM) under conditions of a magnification of 100,000 to 500,000 times and an acceleration voltage of 50 kV, the metal particle coverage (ΣAm / A) obtained by dividing the total area (ΣAm) of the metal particles 20 within a square inscribed on the circumference of the composite particle 100 by the area (A) of the square is preferably 20 to 80%. Specific examples of the metal particle coverage (ΣAm / A) are 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, and 80, and may be within a range between any two of the numerical values ​​exemplified here.

[0024] The circumference of the composite particle 100 is the particle diameter D １ When all of the metal particles 20 in the composite particle 100 are encapsulated particles 21, the particle diameter D ２is the circumference of the circle relating to the above. In addition, the total area (ΣAm) of the metal particles 20 and the area (A) of the square can be calculated from the SEM image of the obtained composite particle 100 using conventionally known image analysis software (Image J, etc.). FIG. 3 is a schematic diagram showing an example in which a circumference 30 of the composite particle 100 and a square 40 inscribed in the circumference 30 are provided using image analysis software in a top view image of the composite particle 100 comprising the metal particles 20 including the surface-adsorbed particles 23, obtained by FE-SEM under conditions of a magnification of 100,000 to 500,000 times and an acceleration voltage of 50 kV, in order to calculate the metal particle coverage (ΣAm / A). As shown in FIG. 3, when photographed under the above conditions, it is possible to image the metal particles 20 located at a depth of several tens of nanometers from the surface of the resin particle 10, so it is possible to determine the extent to which the vicinity of the surface of the composite particle 100 is covered with the metal particles 20. When the metal particle coverage (ΣAm / A) is within the above range, the plasmon absorption band becomes sharper, and the desired clear and deep color tone can be obtained, resulting in excellent visibility when used as a labeling substance.

[0025] 4. Method for Producing Resin Particles 10 The method for producing resin particles 10 is not particularly limited, and for example, a conventionally known method for producing core-shell structures can be used. Specifically, for example, a relatively simple method disclosed in Japanese Patent No. 5239004 (Method for Producing Anisotropic Polymer Microparticles) can be employed. In this method, a good solvent solution is prepared by dissolving two polymers having different solubility parameters in a good solvent, a poor solvent for the polymers that is compatible with the good solvent is added, and then the good solvent is removed, thereby producing approximately spherical microparticles having multiple phases formed by aggregation or aggregation of the respective polymers.

[0026] The two polymers used in the above method are selected from the polymers listed above, and the difference in solubility parameter between the polymers is 0.1 MPa or less. １／２ More than 20 MPa １／２ For example, when polymer A and polymer B are used as two polymers, the difference in solubility parameter between polymer A and polymer B is 0.1 MPa. １／２ More than 20 MPa １／２The following combinations are selected: The solubility parameter of each polymer is calculated by the Fedors calculation method "Robert F. Fedors, Polymer Engineering and Science, 14, 147-154 (1974)."

[0027] For example, the resin particles 10 according to one embodiment of the present invention are made of polystyrene (PSt, solubility parameter: 18 MPa １／２ ) and aminated polybutadiene (PB-NH ２ The solubility parameter of non-aminated polybutadiene (PB) is 17.5 MPa. １／２ Since the solubility parameter does not change significantly due to amination, the difference in solubility parameter between polystyrene and aminated polybutadiene is 0.1 MPa. １／２ More than 20 MPa １／２ The results are as follows. In addition, polystyrene and aminated styrene-butadiene copolymer (SBR-NH ２ The resin particles 10 can also be produced by using a combination of aminated polybutadiene and aminated styrene-butadiene copolymer, or a combination of aminated polybutadiene and aminated styrene-butadiene copolymer.

[0028] The good solvent and poor solvent used in the above method are solvents that are mutually compatible but have significantly different dissolving powers for the two or more polymers. A solvent with a strong dissolving power for the polymer is called a good solvent, and a solvent with a weak dissolving power or almost no dissolving power is called a poor solvent. They are appropriately selected and used in combination. A good solvent is a solvent whose difference in solubility parameter with respect to each of the two or more polymers is 5.0 MPa or less. １／２ It is preferable to use a good solvent having a solubility parameter of 5.0 MPa or less. １／２ Super 30MPa １／２ It is preferable to select a poor solvent having a viscosity of 10.0 MPa or less. １／２ Super 30MPa １／２ It is preferred to select an anti-solvent that is:

[0029] Examples of solvents that can be used in the above method include tetrahydrofuran (THF), dimethyl ether, benzene, toluene, hexane, chloroform, acetone, methanol, ethanol, water, dimethylformamide, dimethyl sulfoxide, dioxane, acetonitrile, 1-propanol, and isopropanol. From these, good solvents and poor solvents having the above-described properties can be selected depending on the two or more polymers used. In this case, it is desirable to collect or confirm known data such as solubility parameter data for the polymer used in various solvents, compatibility data between solvents, and boiling points, and take these into consideration. The solubility parameter of the solvent is calculated using the Fedors calculation method.

[0030] For example, the resin particles 10 according to one embodiment of the present invention are prepared by dissolving the resin particles 10 in tetrahydrofuran (THF, solubility parameter: 18.6 MPa) as a good solvent. １／２ ), and water as a poor solvent (solubility parameter: 47.9 MPa １／２ ) can be produced by the above method.

[0031] In the above method, multiphase polymer microparticles (corresponding to resin particles 10 in the present invention) can be formed by adding a poor solvent to a good solvent solution and then removing the good solvent. The particle size of the multiphase polymer microparticles can be controlled by adjusting the concentration of two or more polymers in the good solvent solution and the amount of poor solvent added (ratio to the amount of good solvent). The good solvent can also be removed by utilizing the difference in boiling point between the good solvent and the poor solvent. For example, resin particles 10 according to one embodiment of the present invention can be produced by a relatively simple procedure of mixing a poor solvent with a good solvent solution in which two or more polymers are dissolved, and then rapidly removing the good solvent under reduced pressure. The degree of pressure reduction can be, for example, 10 －３ Pa to 10 ４ Pa, preferably 10 Pa to 10 ３ The degree of pressure reduction is, for example, 10 －３ , 10 －２ , 10 －１ , 1, 10, 10 ２ , 10 ３ , 10４ The pressure in the solvent is preferably 0.05 Pa, and may be within a range between any two of the values ​​exemplified here. Such reduced pressure can be achieved, for example, by using a rotary evaporator, a vacuum pump, or other general pressure reducing equipment. Alternatively, the good solvent can be removed by evaporation at room temperature and normal pressure without reducing the pressure.

[0032] 5. Manufacturing Method of Composite Particle 100 The manufacturing method of the composite particle 100 of the present invention includes a supporting step. The supporting step is a step of supporting metal particles 20 on resin particles 10. In the supporting step, a dispersion of metal particles 20 is brought into contact with the resin particles 10, and the metal particles 20 are impregnated into the resin particles 10. A specific example of the supporting step is mixing the dispersion of resin particles 10 and the dispersion of metal particles 20, and then allowing the mixture to stand for a predetermined period of time.

[0033] The composite particle 100 according to one embodiment of the present invention can be produced by adding a gold nanoparticle colloidal solution to an aqueous dispersion of resin particles 10 and then allowing the mixture to stand for a predetermined period of time. For example, when water is used as a poor solvent in the above-described production process of resin particles 10, the aqueous dispersion of resin particles 10 can be obtained by removing the good solvent. Alternatively, a commercially available gold nanoparticle colloidal solution can be used. It is particularly preferable to use a gold nanoparticle colloidal solution having an average particle size of 5 to 80 nm and a particle size PDI of 0.2 or less as determined by dynamic light scattering.

[0034] The concentration of the aqueous dispersion of resin particles 10 is preferably 0.01 to 10 mg / mL. The concentration of the gold nanoparticle colloid solution is preferably 1.0×10 －３ ~1.0 x 10 ４ The amounts of the aqueous dispersion of resin particles 10 and the gold nanoparticle colloid solution can be appropriately determined depending on the desired amount of composite particles 100 to be produced.

[0035] To ensure a sufficient amount of support, the standing time after mixing the aqueous dispersion of resin particles 10 and the gold nanoparticle colloid solution is preferably 0.5 to 5 hours, more preferably 1 to 3 hours. Specific examples of the standing time are 0.5, 1, 2, 3, 4, and 5 hours, and may be within a range between any two of the values ​​exemplified here.

[0036] In order to maintain the dispersibility of the composite particles 100 in water, a dispersant such as polyethylene glycol may be added during mixing, if necessary.

[0037] In this way, by using the manufacturing method of the composite particle 100 of the present invention, metal particles 20 with uniform particle diameters can be concentrated within the shell layer 12 of the resin particle 10, and composite particles 100 can be manufactured that have excellent visibility, sensitivity stability, and durability when used as a labeling substance.

[0038] 6. Uses of the Composite Particle 100 The composite particle 100 of the present invention can be preferably used as a labeled substance in immunoassays by binding to an antigen or antibody. Furthermore, the labeled substance comprising the composite particle 100 of the present invention can be preferably used as a material for a reagent for immunoassays.

[0039] Furthermore, the composite particle 100 can be used for purposes other than immunoassay labeling substances or immunoassay reagents, such as pigments, paints, conductive materials, electrodes, etc. When used in pigments or paints, vivid, deep colors can be obtained, and when used in conductive materials or electrodes, mechanical strength is improved and conductivity due to the metal particles is exhibited.

[0040] 7. Immunoassays An immunoassay using a labeled substance comprising the composite particle 100 of the present invention also constitutes one aspect of the present invention. Examples of immunoassays include EIA, ELISA, ECLIA, CLIA, CLEIA, HA, PA, and LA, but the immunoassays of the present invention are not limited to these. Furthermore, the immunoassay of the present invention is suitable as an immunochromatographic method in which a reagent containing a labeled antibody labeled with the composite particle 100 of the present invention is reacted with a test reagent to bind the antigen contained in the test reagent to the labeled antibody, and the resulting mixture is passed through a chromatographic carrier on which another antibody is immobilized, thereby capturing the antigen in the chromatographic carrier and analyzing the captured antigen based on the label.

[0041] In the present invention, the antibody is not particularly limited, and examples thereof include polyclonal antibodies, monoclonal antibodies, antibodies obtained by genetic recombination, and antibody fragments (H chain, L chain, Fab, F(ab')2, etc.) capable of binding to antigens. Furthermore, the immunoglobulin may be any of IgG, IgM, IgA, IgE, and IgD. Specific examples of the antibody include anti-PSA antibodies, anti-AFP antibodies, anti-CEA antibodies, anti-adenovirus antibodies, anti-influenza virus antibodies, anti-HCV antibodies, anti-IgG antibodies, and anti-human IgE antibodies.

[0042] In the present invention, the measurement reagent is not particularly limited, and examples thereof include clinical specimens, food specimens, and environmental sampling specimens. Clinical specimens include, for example, body fluids such as blood, plasma, serum, lymph, urine, saliva, pancreatic juice, gastric juice, sputum, and swabs collected from mucous membranes such as the nose and throat, as well as feces. Food specimens include, for example, liquid beverages, semisolid foods, and solid foods. Environmental sampling specimens include, for example, natural samples such as soil, rivers, and seawater, specimens sampled using air samplers installed on production lines or in clean rooms within factories, and swab specimens.

[0043] When immunochromatography is used as the immunoassay, the labeled antibody is concentrated on the chromatographic carrier. Therefore, the chromatographic carrier absorbs light and develops color due to surface plasmon resonance of the labeled substance (composite particle 100). If the antigen contained in the assay reagent is at least in a certain amount, the color development due to surface plasmon resonance can be visually confirmed. Furthermore, the color development due to surface plasmon resonance can be quantitatively evaluated by measuring the optical density using an ultraviolet-visible spectrophotometer. Similarly, even when an immunoassay other than immunochromatography is used, the color development due to surface plasmon resonance can be quantitatively evaluated by measuring the optical density using an ultraviolet-visible spectrophotometer. Furthermore, the color development due to surface plasmon resonance can also be quantitatively evaluated by using image analysis software to determine the mean gray value (MGV) of an image converted to, for example, 8-bit grayscale.

[0044] The following detailed description will be given using examples, but the present invention is not limited to the following examples.

[0045] 1. Various Measurements and Evaluations The following various measurements and evaluations were carried out for the Examples and Comparative Examples.

[0046] (Measurement of Average Particle Diameter) The average particle diameters of the resin particles 10 and the composite particles 100 were measured by dynamic light scattering using a dynamic light scattering analyzer (Zetasizer nano ZS, manufactured by Malvern Panalytical). The average particle diameter and PDI of the metal particles 20 were measured using the nominal values ​​provided by the manufacturer.

[0047] (Calculation of metal particle abundance ratio) 500 μL of the dispersion of the composite particles 100 was measured into a measurement container, dried, and then mixed with 200 mg of embedding resin (epoxy resin Epok-812 set, Wako Pure Chemical Industries), followed by thermal curing at 70° C. for 12 hours. The resulting cured product was sliced ​​into 100 nm thick sections using a microtome (UC-6, manufactured by Leica Microsystems), and then a cross-sectional photograph of the composite particles 100 was obtained using a transmission electron microscope (TEM, H-7650, manufactured by Hitachi High-Technologies). For 10 composite particles 100 randomly selected from the obtained cross-sectional photograph, the volume was calculated from the circle-equivalent diameter of the metal particles 20, and the ratio was calculated for each location of presence. In Tables 1 and 2, the abundance ratios of the partially exposed particles 22 and surface-adsorbed particles 23 in the metal particles 20, the encapsulated particles 21 in the shell layer 12, and the encapsulated particles 21 in the core portion 11 are shown as partially exposed [volume %], shell layer encapsulated [volume %], and core portion encapsulated [volume %], respectively.

[0048] (Calculation of Metal Particle Coverage) A suspension of composite particles 100 was cast onto a silicon substrate, and the composite particles 100 were observed with a field emission scanning electron microscope (FE-SEM, S-5200, manufactured by Hitachi High-Tech) at an acceleration voltage of 50 kV. As shown in Fig. 3, from the SEM image of the obtained composite particles 100, image analysis software (Image J, manufactured by the National Institutes of Health (NIH)) was used to calculate the metal particle coverage (ΣAm / A) by dividing the total area (ΣAm) of metal particles 20 within a square 40 inscribed in the circumference 30 of the composite particle 100 by the area (A) of the square 40.

[0049] 2. Examples and Comparative Examples 2-1. Example 1 (Preparation of Resin Particles 10 (P1)) Polystyrene (PSt, Mn=17000, Mw / Mn=1.03, manufactured by Polymer Source) and aminated polybutadiene (PB-NH ２ , Mn=35,000, manufactured by Polymer Source) were mixed in a weight ratio of 1:1 and dissolved in THF to prepare 1 mL of a 0.1 mg / mL solution. 1 mL of water was added to this solution at a rate of 1.0 mL / sec while stirring. After the entire amount of water was added, stirring was stopped and the THF was evaporated at room temperature and pressure to obtain a dispersion of resin particles 10 (P1). The average particle diameter of resin particles 10 (P1) was 500 nm.

[0050] (Preparation of Composite Particle 100) To 100 μL of the dispersion of resin particles 10, 500 μL of gold (Au) nanoparticle colloidal solution (A1) (average particle size: 20 nm, PDI determined by dynamic light scattering: 0.12, concentration: 52.9 mg / mL, 0.1 mM PBS, manufactured by BBI Solutions) as a dispersion of metal particles 20 and 500 μL of a polyethylene glycol aqueous solution prepared at 1 g / L as a dispersant were added, and the mixture was allowed to stand for 2 hours to obtain a dispersion of composite particles 100. The average particle size of composite particles 100 was 500 nm, and the metal particle abundance ratio was 100% by volume of particles encapsulated in the shell layer.

[0051] (Preparation of Labeled Antibody) Water was added to the resulting dispersion of composite particles 100 to a composite particle concentration of 2.5 mg / mL. 1 mL of the resulting dispersion was centrifuged to remove the supernatant, and 500 μL of coupling buffer was added using an influenza antibody set (manufactured by Nippon Shinobiological Co., Ltd.). The supernatant was then removed by centrifugation, and 300 μL of coupling buffer was added and ultrasonically dispersed. Next, the resulting dispersion was mixed with 25 μg of a 1 mg / 50 mg methanol solution of influenza antibody (Influenza A H1N1), 300 μg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) 25 mg / 50 μL coupling buffer, and 300 μg of sulfo-N-hydroxysuccinimide (Sulfo-NHS) 22.7 mg / 300 μL coupling buffer, and the mixture was stirred at room temperature for approximately 3 hours to bind the antibody to the composite particles 100. A bovine serum albumin solution was added to a final concentration of 1%, and the mixture was stirred at room temperature for 2 hours to block the surface. The mixture was centrifuged at 12,000 rpm at 4°C for 5 minutes to remove the supernatant, which was then suspended in a buffer solution containing 0.2% bovine serum albumin to obtain a 2.5 mg / mL dispersion of the labeled antibody labeled with Composite Particle 100.

[0052] (Evaluation by immunoassay) Using an ELISA kit (manufactured by Nippon Shinobiological Co., Ltd.), one well of an ELISA plate with antibodies immobilized in each well was washed, and 100 μL of antigen solution (influenza A (H1N1) virus) was added to the well, covered with a seal, and incubated at room temperature for 2 hours. After washing the well, 100 μL of the prepared labeled antibody dispersion was added and incubated at room temperature for 1 hour. Thereafter, the well was washed to prepare a sample for optical density measurement, and optical density measurement at a wavelength of 450 nm was performed using an ultraviolet-visible spectrophotometer (device name: V-670, manufactured by JASCO Corporation) to determine color development by surface plasmon resonance. Note that optical density measurements were performed when the antigen concentration of the antigen solution was 100 pg / mL and 1000 pg / mL. The measurement results are shown in Table 1.

[0053] 2-2. Example 2 Composite particles 100 were prepared and evaluated in the same manner as in Example 1, except that a gold nanoparticle colloidal solution (A2) (average particle size: 10 nm, PDI determined by dynamic light scattering: 0.15, concentration: 10 mg / mL, 0.1 mM PBS, manufactured by BBI Solutions) was used as the dispersion of metal particles 20. The measurement results are shown in Table 1.

[0054] 2-3. Example 3 (Preparation of Resin Particle 10 (P2)) PSt (Mn=12600, manufactured by Polymer Source Co., Ltd.) and PB-NH ２ (Mn=1700, manufactured by Polymer Source) were mixed in a weight ratio of 3:7 and dissolved in THF to prepare 1 mL of a 0.1 mg / mL solution. 1 mL of water was added to this solution at a rate of 1 mL / sec while stirring. After the entire amount of water was added, stirring was stopped and the THF was evaporated at room temperature and atmospheric pressure to obtain a dispersion of resin particles 10 (P2). The average particle diameter of resin particles 10 (P2) was 1000 nm.

[0055] Composite particles 100 were prepared and evaluated in the same manner as in Example 1, except that the dispersion of resin particles 10 (P2) was used instead of the dispersion of resin particles 10. The measurement results are shown in Table 1.

[0056]

[0057] 2-4. Comparative Example 1 The optical density was evaluated in the same manner as in Example 1, except that the dispersion of Composite Particle 100 was not used, and instead a hydroxysuccinimide (NHS)-activated gold nanoparticle colloidal solution (A3) (average particle size: 20 nm, PDI determined by dynamic light scattering: 0.12, concentration: 2.5 mg / mL, manufactured by Cytodiagnostics) was used. The measurement results are shown in Table 2.

[0058] 2-5. Comparative Example 2 (Preparation of Resin Particles 10 (P3)) PSt (Mn = 17,000, Mw / Mn = 1.03, manufactured by Polymer Source) was dissolved in THF to prepare 1.0 mL of a 0.1 mg / mL solution. 1 mL of water was added to this solution at a rate of 1 mL / sec while stirring. After the entire amount of water was added, stirring was stopped and the THF was evaporated at room temperature and normal pressure to obtain a dispersion of Resin Particles 10 (P3). The average particle diameter of Resin Particles 10 (P3) was 500 nm.

[0059] An attempt was made to produce composite particles 100 in the same manner as in Example 1, except that a dispersion of resin particles 10 (P3) was used as the dispersion of resin particles 10. However, gold nanoparticles were not supported on the resin particles 10 (P3), and composite particles 100 could not be produced.

[0060] 2-6. Comparative Example 3 (Preparation of Resin Particles 10 (P4)) PB-NH ２ (Mn=35,000, manufactured by Polymer Source) was dissolved in THF to prepare 1.0 mL of a 0.1 mg / mL solution. 1 mL of water was added to this solution at a rate of 1 mL / sec while stirring. After the entire amount of water was added, stirring was stopped and the THF was evaporated at room temperature and atmospheric pressure to obtain a dispersion of resin particles 10 (P4). The average particle diameter of resin particles 10 (P4) was 500 nm.

[0061] Composite particles 100 were produced in the same manner as in Example 1, except that a dispersion of resin particles 10 (P4) was used instead of a dispersion of resin particles 10. However, there was significant aggregation between the composite particles 100, making evaluation impossible.

[0062]

[0063] Tables 1 and 2 show that the optical densities of Examples 1 to 3 are greater than those of Comparative Examples 1 to 3, and that the use of Composite Particle 100 as a labeling substance for immunoassays results in excellent visibility. Furthermore, even when the antigen concentration is as low as 100 pg / mL, an optical density comparable to that obtained when the antigen concentration is 1000 pg / mL is obtained, demonstrating that highly sensitive detection is possible even for small amounts of antigen.

[0064] 10: Resin particle, 11: Core, 12: Shell layer, 20: Metal particle, 21: Encapsulated particle, 22: Partially exposed particle, 23: Surface-adsorbed particle, 30: Circumference, 40: Square, 100: Composite particle

Claims

1. A composite particle comprising resin particles and metal particles, wherein the resin particles comprise a core part and a shell layer covering the surface of the core part, the glass transition temperature of the core part is 50 to 200 °C, the glass transition temperature of the shell layer is -100 to 0 °C, and 80 to 100% by volume of the metal particles are encapsulated in the shell layer or present on the surface of the shell layer with respect to 100% by volume of the total metal particles.

2. The composite particle according to claim 1, wherein 80 to 100% by volume of the metal particles are encapsulated in the shell layer with respect to 100% by volume of the total metal particles.

3. The composite particle according to claim 1, having an average particle diameter of 200 to 1500 nm.

4. The composite particle according to claim 1, wherein the metal particles are gold nanoparticles having an average particle diameter of 5 to 80 nm and a polydispersity index (PDI) of the particle diameter determined by the dynamic light scattering method of 0.2 or less.

5. The metal particle coverage rate (ΣAm / A) obtained by dividing the total area (ΣAm) of the metal particles within a square inscribed in the circumference of the composite particle by the area (A) of the square from an image of the composite particle observed by a field emission scanning electron microscope (FE-SEM) under the conditions of a magnification of 100,000 to 500,000 times and an acceleration voltage of 50 kV is 20 to 80%. The composite particle according to claim 1.

6. The composite particle according to claim 1, wherein the shell layer contains an amino group-containing polymer.

7. The composite particle according to claim 1, wherein the core part contains a styrene-based polymer.

8. A labeling substance comprising the composite particle according to claim 1.

9. The labeling substance according to claim 8, having an antigen or an antibody on the surface of the composite particle.

10. An immunological assay method using the labeling substance according to claim 8.

11. An immunological assay reagent having the labeling substance according to claim 8.

12. A method for producing a composite particle comprising a loading step, wherein in the loading step, metal particles are loaded onto resin particles, the resin particles comprise a core part and a shell layer covering the surface of the core part, the glass transition temperature of the core part is 50 to 200 °C, and the glass transition temperature of the shell layer is -100 to 0 °C.

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