Dye-containing resin particles, particles for specimen testing, specimen testing reagents, test kits for target substances, method for manufacturing dye-containing resin particles, method for manufacturing specimen testing particles, method for detecting target substances

Abstract

This invention provides dye-containing resin particles with suppressed reduction in luminescence intensity and particles for sample testing that can detect target substances with high sensitivity. [Solution] Dye-containing resin particles having a core portion and a shell portion and containing a europium complex. The core portion and the shell portion are each crosslinked, the core portion contains a polymer of hydrophobic monomers including a styrene monomer as the main component and another monomer, and the shell portion contains a polymer of hydrophilic monomers. The other monomer satisfies the following formulas (B1) and (15). LogP_f≧3.60 (B1) (Mol_f) / (Mol_St) ≧ 0.05 (15)

Description

[Technical Field]

[0001] The present invention relates to dye-containing resin particles, particles for specimen testing, specimen testing reagents, a test kit for target substances, a method for producing dye-containing resin particles, a method for producing specimen testing particles, and a method for detecting target substances. [Background technology]

[0002] In recent years, in the fields of medicine and clinical testing, there has been a need to detect trace amounts of biological components from blood or parts of collected organs with high sensitivity, and immunoassay, which utilizes antigen-antibody reactions, has become widely used as a means to achieve this. Among immunoassays, latex agglutination (LA) and fluorescence depolarization (FPIA), which do not require the separation of unreacted antibodies or antigens, are suitable for use in medical and clinical testing settings because they allow for rapid quantification with simple procedures.

[0003] Patent Document 1 describes resin particles for use in fluorescence polarization depolarization, which contain a europium complex and have a hydrophilic polymer on their surface. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2022-70712 [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] However, when the resin particles described in Patent Document 1 are used, the luminescence intensity of the europium complex contained in the resin particles decreases over time when mixed with the sample solution, which may result in insufficient detection sensitivity. [Means for solving the problem]

[0006] In one embodiment of the present disclosure, in order to solve the above problems, there are provided pigment-containing resin particles having a core part and a shell part, wherein the pigment-containing resin particles contain a europium complex, the core part and the shell part are each crosslinked, the core part has a resin containing a polymer of a hydrophobic monomer including a styrene monomer as a main component and a first monomer of a different type from the styrene monomer, the shell part has a resin containing a hydrophilic monomer, and the first monomer satisfies the following formula (B1) and the following formula (15). LogP_f≧3.60 (B1) (Mol_f) / (Mol_St) ≧ 0.05 (15) (However, in formula (B1), LogP_f is the octanol / water partition coefficient of the first monomer, and in formula (15), Mol_f represents the number of moles of the first monomer, and Mol_St represents the number of moles of the styrene monomer.) [[Effect of the Invention]]

[0007] According to the present disclosure, it is possible to obtain pigment-containing resin particles in which a decrease in the emission intensity of the europium complex is suppressed. Further, by using such pigment-containing resin particles, highly sensitive detection of a target substance becomes possible. [[Brief Description of the Drawings]]

[0008] [Figure 1] It is a schematic diagram for explaining the structure of pigment-containing resin particles according to one embodiment of the present invention. [Figure 2] It is a schematic diagram for explaining the structure of conventional pigment-containing resin particles. [[Mode for Carrying Out the Invention]]

[0009] An example of one embodiment of the present disclosure will be described below with reference to Figure 1. As shown in Figure 1, the particles of this embodiment consist of a core portion 1 containing a europium complex 3, which is a luminescent dye, and a shell portion 2 that covers its surface. First, a method for measuring the particle size of the dye-containing resin particles of this embodiment and a fluorescence polarization depolarization method that can be used with the dye-containing resin particles of this embodiment will be described.

[0010] (Measurement of particle size) The diameter of resin particles can be determined by dynamic light scattering. When particles dispersed in a solution are irradiated with laser light and the scattered light is observed with a photon detector, the intensity distribution due to the interference of scattered light is constantly fluctuating because the particles are constantly changing their position and moving due to Brownian motion.

[0011] Dynamic light scattering is a measurement method that observes Brownian motion as fluctuations in scattered light intensity. The fluctuations in scattered light with respect to time are represented by an autocorrelation function, and the translational diffusion coefficient is determined. From the determined diffusion coefficient, the Stokes diameter can be found, and the particle size of the particles dispersed in the solution can be derived. In addition, the polydispersity index (PDI) is calculated through measurement to represent the width of the particle size distribution. If the polydispersity index value is 0.1 or less, it indicates that the particle size distribution is monodisperse.

[0012] (Fluorescence polarization depolarization method) By encapsulating a europium complex that exhibits polarized emission as a coloring material within the resin particles, it is possible to detect changes in polarized emission properties even if there are slight changes in the dispersion state of the resin particles in liquid. Specifically, when an antigen-antibody reaction occurs and particles aggregate via the antigen, changes in the rotational Brownian motion of the particles can be detected as changes in polarization anisotropy or fluorescence polarization degree.

[0013] Polarization anisotropy means that there is anisotropy in the transition moment (transition dipole moment). Polarized emission generally means that in the case of luminescent dyes with anisotropic transition moments, if the excitation light is polarized along that transition moment, the emitted light will also be polarized along that transition moment. In the case of europium complexes, since fluorescence emission is based on energy transfer from ligands to the central metal ion, the transition moment of polarized emission is complex, but the red emission around 610 nm, which originates from the electron transition from the lowest excited state 5D0 to 7F2, exhibits polarization anisotropy.

[0014] The fluorescence depolarization method is based on the principle of measuring the shift in the transition moment due to the rotational motion of the light-emitting material during the time that polarized emission occurs. The rotational motion of the light-emitting material can be expressed by equation (21). Q = 3Vη / kT (21) (Here, Q: Material rotation relaxation time V: Volume of material η: viscosity of the solvent k: Boltzmann constant T: Absolute temperature The rotational relaxation time Q of the material is the time required for the numerator to rotate by an angle θ (68.5°) such that cosθ = 1 / e.

[0015] From equation (21), it can be seen that the rotational relaxation time of the luminescent material is proportional to the volume of the material, that is, the cube of the particle radius. On the other hand, the relationship between the luminescence lifetime and the degree of polarization of the material in fluorescence depolarization can be expressed by equation (22). p0 / p=1+A(τ / Q) (22) (Here, p0: Polarization degree when the material is stationary (Q=∞) p: Polarization degree A: Constant τ: Luminous lifetime of the material Q: Rotation relaxation time (That is the case.)

[0016] From equations (21) and (22), it can be seen that in order to measure a large change in polarization degree, the relationship between the luminescence lifetime and rotational relaxation time of the luminescent material, i.e., the volume (particle size) of the luminescent material, is important, and the larger the particle size of the luminescent material, the longer the luminescence lifetime needs to be.

[0017] To determine the degree of polarization of the emission shown in equation (22) experimentally, polarized light should be incident on the sample, and the emission should be detected at a 90-degree angle to the direction of propagation and vibration of the excitation light. At this time, the detected light should be separated into polarization components parallel and perpendicular to the polarization of the incident light, and the degree of polarization should be evaluated using equation (23) below. r(t)= (I∥(t)−GI⊥(t)) / (I∥(t)+2GI⊥(t)) (23) (Here, r(t): Polarization anisotropy at time t I∥(t): Emission intensity of the emission component parallel to the excitation light at time t I⊥(t): Emission intensity of the emission component perpendicular to the excitation light at time t. G: Correction value The correction value is the ratio of I⊥ / I∥ measured with excitation light whose vibration direction is 90 degrees different from that used for the sample measurement.

[0018] In other words, within the appropriate particle size and luminescence lifetime range, it is possible to sensitively read changes in the particle size of luminescent materials due to antigen-antibody reactions, etc., as values ​​of polarization anisotropy. Polarization anisotropy is the value of the degree of polarization corrected for G and 2G, and the degree of polarization is obtained by removing G and 2G from equation (23). In actual measurement of polarization anisotropy, a correction value for G is necessary, but if only the relative degree of polarization needs to be determined, it is possible to evaluate it using the formula obtained by removing the correction values ​​for G and 2G from equation 23.

[0019] Furthermore, the luminescent particles of the present invention have polarization anisotropy determined by the following formula (24). <r>It is preferable that it is 0.01 or higher.

number

[0020] (polydispersity index) In fluorescence depolarization, high detection sensitivity is achieved by detecting changes in the motion of dye-containing resin particles. Generally, smaller particles rotate faster, while larger particles rotate slower. For example, when an antigen-antibody reaction occurs... Particle (antibody) - antigen - particle (antibody) The detection method works by detecting the aggregation of particles with this composition, which causes the aggregated particles to become larger and slower in rotational motion. Therefore, there is a concern that the detection sensitivity will decrease if small or large particles are mixed in the initial stages before the reaction in the system. For this reason, the pigment-containing resin particles must have a uniform particle size distribution, and specifically, the polydispersity index must be 0.1 or less.

[0021] <First Embodiment> (Resin particle structure, core-shell structure) The pigment-containing resin particles of this embodiment are functionally divided into a core portion and a shell portion. First, it is important that the resin material of the core portion has a low specific gravity. Pigment-containing resin particles are expected to be used after being left to stand and stored for a long period of time, for example, several months. Therefore, if the specific gravity of the particles is high, the pigment-containing resin particles will settle in the container, requiring stirring and redispersion before use, which makes the operation complicated. To suppress the settling of the pigment-containing resin particles, the core portion, which occupies the majority of the volume of each pigment-containing resin particle, has a low specific gravity, more specifically 1.10 g / cm³. 3 It is preferable to use the following materials. Specifically, polystyrene is preferred.

[0022] Next, regarding the resin material of the shell, it is important to suppress aggregation between particles and, in addition, to suppress adsorption between particles and the container. Originally, the purpose of testing using fluorescence polarization depolarization is to detect aggregation of particles that have undergone an antigen-antibody reaction, for example, particle (antibody)-antigen-particle (antibody) type aggregation. Therefore, simple particle-particle aggregation that does not involve an antigen, or aggregation of antibody-contaminated particles with a sample that does not react with the antibody, is called nonspecific adsorption and is one of the causes of decreased accuracy and detection sensitivity of the test. Nonspecific aggregation between particles (or samples) may be caused by hydrophobic interactions between hydrophobic sites on the surfaces of adjacent particles. Also, if the particle surface has long hydrophilic groups, aggregation between particles (or samples) may occur due to entanglement of the hydrophilic groups.

[0023] Therefore, in the dye-containing resin particles of this embodiment, the resin material used for the shell portion has the function of adsorbing water molecules to the particle surface by hydrogen bonding. As a result, even when particles (or a sample) are in close proximity, water molecules coordinate to the gaps between the particles, suppressing direct contact between the particle surfaces, and also suppressing entanglement between long hydrophilic groups, thereby suppressing aggregation between particles (or a sample). Similarly, when a hydrophobic container is in close proximity to the particles, water molecules coordinate between the particles and the container, preventing the particles from adsorbing to the container.

[0024] (Europium complex) In the dye-containing resin particles of this embodiment, a europium complex exhibiting polarization anisotropy is used as the colorant because its emission wavelength and intensity are less affected by the surroundings, and its emission has a long lifespan. The europium complex is composed of a europium element and a ligand, and its structure can be represented by the following formula (1). Eu(A)x(B)y(C)z (1) Considering the luminescence lifetime and the visible emission wavelength range, the use of europium complexes is essential. Europium generally has a luminescence lifetime of 0.1 to 1.0 ms. It is necessary to appropriately adjust this luminescence lifetime and the rotational relaxation time obtained from equation (21). In the case of europium in an aqueous dispersion, a particle size with a diameter of 80 nm to 200 nm is preferable because the polarization anisotropy represented by equation (23) changes significantly before and after the antigen-antibody reaction.

[0025] At least one of the ligands constituting the europium complex has a light-harvesting function. Light-harvesting function refers to the action of exciting the central metal of the complex by energy transfer when excited at a specific wavelength. Furthermore, it is preferable that the ligands constituting the europium complex include ligands such as β-diketones to prevent coordination of water molecules. Ligands such as β-diketones that coordinate to rare earth ions suppress the deactivation process due to energy transfer to solvent molecules, etc., and strong fluorescence emission is obtained.

[0026] The europium complex may be a multinuclear complex as long as it exhibits polarization anisotropy. The polarization anisotropy of the europium complex is given by equation (23). It is desirable that the polarization anisotropy be 0.08 or higher when the Brownian rotation motion of the europium complex can be considered to have stopped in the medium. The state in which the Brownian rotation motion can be considered to have stopped means that the rotational relaxation time of the particles is sufficiently longer than the luminescence lifetime of the europium complex.

[0027] It is preferable that the europium complex is incorporated in large quantities into the core of the resin particles having a core-shell structure, as this increases the luminescence intensity per particle. Specifically, it is preferable that the europium complex content per gram of dye-containing resin particles be 0.001 g or more. Furthermore, the content of the europium complex can be calculated by quantifying europium using high-frequency inductively coupled plasma (ICP) emission spectrometry.

[0028] On the other hand, if the europium complex aggregates within the core, the interactions between ligands can affect the excitation efficiency of the europium complex, making it difficult to measure reproducible polarization anisotropy. Whether europium complex 3 exhibits non-aggregated luminescence behavior within the core can be determined by taking the excitation spectrum of the sample.

[0029] Highly luminescent particles not only enable high-sensitivity measurements, but also maintain strong luminescence even with small particle sizes, thus accelerating biochemical reaction rates. Smaller particle sizes result in a larger diffusion coefficient for Brownian motion in liquids, allowing for faster reaction detection.

[0030] Preferably, the europium complex contained in the dye-containing resin particles of this embodiment is represented by the following formula (1). Eu(A)x(B)y(C)z (1) [However, in formula (1), Eu is europium, (A) is the ligand represented by formula (2) below, (B) is the ligand represented by any of the following formulas (3), (41), (42), or (43), and (C) is the ligand represented by formula (5) below. [ka] (In equations (2), (3), (41), (42), (43), and (5), R 1 and R 2 R is a group that may have substituents and is independently selected from an alkyl group, a thienolphenyl group, and a thienyl group. 3 is a hydrogen atom or a methyl group. Also, R 4 and R 5 are groups independently selected from an alkyl group and a phenyl group, each of which may have a substituent. Also, R 6 is a group selected from an alkyl group, a phenyl group, and a triphenylene group, each of which may have a substituent. Also, R 7 and R 8 are groups independently selected from an alkyl group or a phenyl group, each of which may have a substituent. Here, the substituents in R<00000​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​When styrene monomer is used alone as the monomer to form the polymer in the core, the particle size exceeds 500 nm, which may be undesirable for use as dye-containing resin particles in fluorescence polarization depolarization methods. Therefore, it is preferable to include an electrically charged material in the suspension medium during resin particle synthesis (polymerization) to suppress coalescence and association of suspended particles during resin particle synthesis by utilizing the repulsion of charges between suspended particles. Specifically, it is preferable to include styrene sulfonic acid as the electrically charged material.

[0033] Furthermore, the monomer may contain other monomers in addition to the main material (main component monomer). Examples of radical polymerizable monomers other than styrene include butadiene, vinyl acetate, vinyl chloride, acrylonitrile, methyl methacrylate, methacrylonitrile, and methyl acrylate. Monomers selected from these monomers can be used individually or in combination.

[0034] (Method for calculating the LogP of monomers forming the core) The octanol / water partition coefficient (LogP) of the monomers forming the core portion of the dye-containing resin particles in this embodiment is calculated in the following two steps. (1) First, the monomer's structural formula is written using "SMILES notation," which is one of the "linear notations" for representing a single line of text. (2) Next, enter the aforementioned SMILES notation string into "ALOGPS2.1", a program provided by the Virtual Computational Chemistry Laboratory VCCLAB, and have it calculate LogP (https: / / vcclab.org / lab / ).

[0035] To give a specific example, for dodecyl methacrylate, shown in formula (A1) below, the SMILES notation is "CCCCCCCCCCCCOC(=O)C(C)=C". Calculating LogP from this SMILES notation using "ALOGPS2.1" yields "6.32". [ka]

[0036] The phenomenon of the luminescence intensity of europium complexes contained in dye-containing resin particles decreasing over time is thought to be due to a concentration gradient of europium complexes between the inside and outside of the resin particles. In other words, it is thought that the high concentration of europium complexes present inside the resin particles gradually diffuses to the outside of the particles where the europium concentration is low (almost zero), and leaks out. As a result, it is thought that some of the ligands of the europium complexes that leak out to the outside of the resin particles are replaced by water, and they are not excited even when exposed to excitation light, leading to a decrease in luminescence intensity.

[0037] In order to prevent the europium complex from leaking out of the resin particles, it is preferable that the hydrophobic monomer forming the core portion contains a styrene monomer, which is the main component, and a first monomer of a different type from the styrene monomer, and that the hydrophobic monomer is polymerized in a state where the first monomer satisfies the following formulas (B1) and (15). LogP_f≧3.60 (B1) (Mol_f) / (Mol_St) ≧ 0.05 (15) (However, in equation (B1), LogP_f is the octanol / water partition coefficient of the first monomer, and in equation (15), Mol_f is the number of moles of the first monomer, and Mol_St is the number of moles of styrene monomer.) Furthermore, it is more preferable that the octanol / water partition coefficient of all ligands in the europium complex is 2.0 or higher.

[0038] Here, a hydrophobic monomer is a monomer with low solubility in water, for example, a monomer with a LogP of 1.0 or higher. Furthermore, the above formulas (B1) and (15) that the first monomer satisfies only need to be satisfied in the step of forming the core or a sub-step constituting that step, regardless of whether the method of producing the dye-containing particles described later is a synthesis method (a method of forming a core containing a europium complex and then forming a shell around the core) or an immersion method (a method of forming resin particles having a core-shell structure and then immersing the resin particles in a europium complex solution to impregnate the resin particles with the europium complex).

[0039] By adding the first monomer to the styrene monomer, the main component forming the core, in a mixing ratio of 5 mol% or more, the decrease in luminescence intensity can be suppressed. Therefore, when the mixing ratio of the first monomer to the styrene monomer, the main component forming the core, is 5 mol% or more, the LogP (i.e., LogP_st) of the styrene monomer, the main component of the hydrophobic monomer forming the core, is 2.92. If the LogP (i.e., LogP_f) of the first monomer is 3.60 or more, the overall LogP of the core can be increased. As a result, the europium complex, which has low affinity for water, is more likely to remain in the core region, which is a more hydrophobic region, i.e., a region with high LogP. Consequently, the europium complex is less likely to leak out of the resin particles, and the decrease in luminescence intensity is suppressed.

[0040] In particular, the first monomer is preferably a monomer represented by the following formula (11). [ka] (In formula (11), X4 is H or CH3, Y3 is H or an alkyl group having 3 or fewer carbon atoms, and m5 is an integer between 3 and 9.)

[0041] For example, it is preferable to use dodecyl methacrylate represented by the above formula (A1), 2-ethylhexyl methacrylate represented by the following formula (A2), or hexyl methacrylate represented by the following formula (A3) as the first monomer and copolymerize it with styrene in the core. [ka] [ka]

[0042] In addition, the core resin material must be crosslinked with monomers that have two or more double bonds in a single molecule, such as divinylbenzene (LogP=3.58), trimethylolpropane trimethacrylate (LogP=3.00), and ethylene glycol dimethacrylate (LogP=1.74). This crosslinking must be sufficient. The fluorescence depolarization method uses a measurement principle that detects changes in the movement of dye-containing resin particles using europium complexes. However, if the core is not (sufficiently) crosslinked, the europium complexes will rotate within the dye-containing resin particles. This makes it difficult for the movement of the dye-containing resin particles and the movement of the europium complexes to be coordinated, raising concerns about a decrease in detection sensitivity. On the other hand, if the core is (sufficiently) crosslinked, the movement of the europium complexes within the dye-containing resin particles is suppressed, making it possible to increase detection sensitivity. In this embodiment and in this embodiment, the resin material is said to be "crosslinked" to the extent that the movement of europium complexes within the dye-containing resin particles is effectively suppressed (sufficiently). For example, when the main monomer component of the hydrophobic monomer forming the core is styrene and the crosslinking agent is divinylbenzene, the weight content (degree of crosslinking) of divinylbenzene is preferably about 5% to 25%, and more preferably about 10% to 20%. The method for determining whether or not the core is crosslinked will be described later.

[0043] When styrene is used as the main material for the core, divinylbenzene is particularly preferred as the crosslinking agent. Divinylbenzene is a compound with a structure similar to styrene, which is the main component of the hydrophobic monomer that forms the core, and therefore can spread uniformly within the core without localizing. As a result, uniform crosslinking is possible within the core.

[0044] Here, in the embodiments and examples of the present disclosure, it is possible to determine whether or not the "core portion is crosslinked" by the following method. First, the dye-containing resin particles are dispersed in pyridine at a concentration of 5 wt%, and then shaken at 50°C for 3 hours. The particle size is measured before and after this operation by the dynamic light scattering method described above. If the "core portion is not crosslinked," the dye-containing resin particles will dissolve, making measurement difficult, or the melted particles will aggregate, making it impossible to maintain the particle size before the operation. Specifically, if the following formulas (25) and (26) are satisfied, it can be determined that the "core portion is crosslinked." (Dv_After) / (Dv_Before)<1.5 (25) (Dn_After) / (Dn_Before)<1.5 (26) (In equations 25 and 26, the volume-average particle size obtained by dynamic light scattering before the aforementioned pyridine treatment is denoted as Dv_Before (unit: [nm]), and the number-average particle size is denoted as Dn_Before (unit: [nm]). Similarly, the volume-average particle size obtained after the aforementioned pyridine treatment is denoted as Dv_After (unit: [nm]), and the number-average particle size is denoted as Dn_After (unit: [nm]).)

[0045] (Shell part) The main material of the shell portion is preferably a hydrophilic monomer having hydrogen bonding sites near the main chain of the side chains. Here, the main material of the shell portion (main component monomer) refers to the raw material monomer that is present in the largest amount among the raw material monomers used to form the shell portion. The hydrogen bonding site is a substituent that can form hydrogen bonds with water molecules in the system, specifically an amino group, carbonyl group, carboxyl group, hydroxyl group, or thiol group. Water molecules are immobilized near the shell by hydrogen bonding. As a result, even when other dye-containing resin particles, samples that do not produce antigen-antibody reactions, or test containers are in close proximity to the dye-containing resin particles, water molecules can coordinate to the gaps between the particles, preventing direct contact between the particle surfaces. This action suppresses non-specific adsorption between dye-containing resin particles and between dye-containing resin particles and samples, thereby improving detection sensitivity. A hydrophilic monomer is a monomer with high solubility in water, for example, a monomer with a LogP value less than 1.0.

[0046] Furthermore, as the shell material, a resin containing at least one of the structures represented by the following formulas (12) and (13) is preferred. [ka] (In equations (12) and (13), X2 and X3 are independently selected from H and CH3, Y1 is OH or OCH3, and Y2 is the following equation (14) or CH2CH2OH.) [ka] m3 and m4 are integers greater than or equal to 1, and n is an integer between 1 and 40 (inclusive).

[0047] As a material for the shell portion, particularly preferred are hydroxyethyl methacrylate, polyethylene glycol monomethyl ether methacrylate, glycidyl methacrylate, 2-methoxyethyl acrylate, 2-methoxyethyl methacrylate, polyethylene glycol monomethacrylate, and 3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propane-1-sulfonic acid], represented by the following formulas (31), (32), (33), (34), (35), (36), and (37), which are polymerizable monomers and used to construct the shell portion by polymerization on the outside of the core portion. [ka] [ka] (n=1 or more and 40 or less) [ka] [ka] [ka] [ka] (n=1 or more and 40 or less) [ka]

[0048] In addition, the resin material of the shell must be crosslinked with monomers that have two or more double bonds in a single molecule, such as divinylbenzene, trimethylolpropane trimethacrylate, or ethylene glycol dimethacrylate. Here, "crosslinked" means that the resin material is crosslinked to a sufficient extent to exert the following crosslinking effects. For example, if the monomer that constitutes the resin material of the shell is hydroxyethyl methacrylate and the crosslinking agent is trimethylolpropane trimethacrylate, the weight content (degree of crosslinking) of trimethylolpropane trimethacrylate is preferably about 5% to 30%.

[0049] Figure 2 is a schematic diagram of resin particles in which a conventional uncrosslinked hydrophilic polymer 20 is adsorbed onto the surface of the core portion 1. Suppression of nonspecific adsorption on the surface of the resin particles can also be reduced by adsorbing an uncrosslinked hydrophilic polymer 20 onto the surface of the resin particles, but in that case, the following problems may occur. The core portion 1 contains a europium complex 3. In Figure 2, the surface of the core portion 1 has a mixture of regions E2 in which the hydrophilic polymer 20 covers the surface of the core portion and regions E1 in which the surface of the core portion is exposed. This is thought to be due to the fact that the surface composition of the core portion has both hydrophobic and hydrophilic regions in minute areas, so the hydrophilic polymer is easily adsorbed onto the hydrophilic core portion surface, while the hydrophilic polymer is not easily adsorbed onto the hydrophobic core portion surface.

[0050] When conventional dye-containing resin particles are placed in a test container, hydrophobic interactions between the non-hydrophilic surface of the test container and the hydrophobic region (region E1 in Figure 2) of the dye-containing resin particles can cause the particles to adsorb to the surface of the test container. Since the Brownian motion of the dye-containing resin particles adsorbed to the container is restricted, this can ultimately lead to a decrease in the sensitivity of the fluorescence depolarization method.

[0051] In contrast, in the dye-containing resin particles of this embodiment, as shown in Figure 1, the resin of the shell portion is crosslinked, so that the shell portion material is forcibly fixed to the particle surface even against the hydrophobic surface of the core portion. As a result, the particle surface of the core portion can be uniformly coated with the shell portion material.

[0052] In addition, crosslinking the shell prevents the hydrogen bonding sites located in the side chains of the shell material from extending widely toward the solvent, water. If the shell is not crosslinked, the hydrophilic polymer will spread toward water, raising concerns about interaction with the hydrophilic sites of other nearby particles (or target substances). On the other hand, in this case, crosslinking the shell component prevents the shell material from spreading away from the dye-containing resin particles. Therefore, interaction with other nearby particles (or samples) becomes less likely, suppressing particle-to-particle adsorption, reducing non-specific adsorption, and consequently improving detection sensitivity.

[0053] Trimethylolpropane trimethacrylate is particularly preferred as the crosslinking agent for the shell portion. Since trimethylolpropane trimethacrylate is a compound with a structure similar to the main component of the shell portion, it can spread uniformly throughout the shell portion without localization. As a result, uniform crosslinking is possible throughout the entire shell portion, which in turn improves non-specific adsorption and consequently improves detection sensitivity.

[0054] In the embodiments and examples of this disclosure, whether or not the "shell portion is crosslinked" can be determined by the following method, similar to the core portion described above. First, pigment-containing resin particles are dispersed in pyridine at a concentration of 5 wt%, and then shaken at 50°C for 3 hours. The particle size is measured before and after this operation by the dynamic light scattering method described above. If the "shell portion is not crosslinked," the pigment-containing resin particles will dissolve, making measurement difficult, or the melted particles will aggregate, making it impossible to maintain the particle size before the operation. More specifically, if the following equations (25) and (26) are satisfied, it can be determined that the "shell portion is crosslinked." (Dv_After) / (Dv_Before)<1.5 (25) (Dn_After) / (Dn_Before)<1.5 (26) (In equations (25) and (26), Dv_Before (unit: [nm]) is the volume-average particle size obtained by dynamic light scattering before pyridine treatment, and Dn_Before (unit: [nm]) is the number-average particle size. After pyridine treatment, Dv_After (unit: [nm]) is the volume-average particle size, and Dn_After (unit: [nm]) is the number-average particle size.)

[0055] The dye-containing resin particles of this embodiment are defined by the LogP values ​​calculated for each of the hydrophobic monomers that form the resin contained in the core portion: styrene (a monomer mainly composed of hydrophobic monomers) and the first monomer. These LogP values ​​can be calculated by analyzing the manufactured dye-containing resin particles using a known method to identify the monomer species that are the raw materials and to quantify the respective composition ratios.

[0056] (Radical polymerization initiator) When synthesizing resin particles, a wide range of radical polymerization initiators can be used, selected from azo compounds, organic peroxides, and the like. Specifically, examples include 2,2'-azobis(isobutyronitrile), 2,2'-azobis(2,4-dimethylvaleronitrile), 2,2'-azobis(2-methylbutyronitrile), 4,4'-azobis(4-cyanovaleric acid), 2,2'-azobis(2-methylpropionamidine) dihydrochloride, 2,2'-azobis(2-methylpropionic acid) dimethyl, tert-butyl hydroperoxide, benzoyl peroxide, ammonium persulfate (APS), sodium persulfate (NPS), and potassium persulfate (KPS).

[0057] (dispersion medium) Generally, water or an aqueous medium (aqueous solvent) is used as the dispersion medium when synthesizing resin particles. A buffer solution can also be used as the aqueous medium. Furthermore, surfactants, preservatives, sensitizers, etc., may be added to the dispersion medium to increase the stability of the dispersion containing the resin particles.

[0058] (Method for encapsulating europium complex) Methods for arranging europium complexes within resin particles (in other words, methods for producing resin particles containing europium complexes) include a "synthesis method" consisting of sub-steps 1 and 2, in which a solution is obtained by adding a europium complex to a synthesis system (solution) containing hydrophobic monomers, and a core portion containing the europium complex is formed by polymerization and crosslinking of the hydrophobic monomers (sub-step 1), and then a shell portion is formed around the core portion by polymerization and crosslinking of hydrophilic monomers in the presence of the core portion (sub-step 2); and an "immersion method" which includes a step of forming resin particles having a core-shell structure by polymerization in a synthesis system that does not contain raw material monomers, and then a step of immersing the resin particles in a europium complex solution to impregnate the resin particles with the europium complex.

[0059] In the synthesis method, the polymerization reaction of the raw material monomers must proceed while the europium complex is efficiently incorporated into the core within the synthesis system. Therefore, the amount of europium complex incorporated into the resin particles is easily affected by the solubility of the europium complex in the liquid medium (generally an aqueous medium) in the synthesis system. In contrast, the immersion method allows for the dissolution of the europium complex at high concentrations using an organic solvent, thus enabling higher concentration impregnation of the resin particles with the europium complex. As a result, the luminescence intensity per pigment-containing resin particle increases, and detection sensitivity can be further improved.

[0060] On the other hand, the organic solvent used to dissolve the europium complex in the immersion method should be one that causes little change in the particle size of the resin particles during immersion, can dissolve the europium complex at a high concentration, and is miscible with water. Examples of such organic solvents include acetone, pyridine, ethanol, tetrahydrofuran, N,N-dimethylformamide, and N-methyl-2-pyrrolidone.

[0061] <Second Embodiment> (Particles for specimen testing) The dye-containing resin particles of the first embodiment can be used as particles for sample testing (which may also be called affinity particles) by providing a site that specifically reacts with a target substance. In this case, the resin particles have at least one reactive functional group selected from the group consisting of carboxyl groups, amino groups, thiol groups, epoxy groups, maleimide groups, and succinimidyl groups in the shell portion, and a ligand that specifically reacts with the target substance is bound to this functional group. These reactive functional groups are located on the surface side of the resin particles, that is, on the side opposite the center.

[0062] Here, the site that specifically reacts with the target substance can be formed, for example, by binding a compound (in other words, a ligand) that specifically binds to a particular target substance (or can be said to capture a particular target substance) to the dye-containing resin particle. The site where the ligand binds to the target substance is fixed and has a specificly high affinity. Examples of ligand-target substance combinations include antigens and antibodies, enzyme proteins and their substrates, signaling substances such as hormones and neurotransmitters and their receptors, and complementary single-stranded nucleic acids, but ligands are not limited to these. In other words, the particles for sample testing are particles that have a specificly high affinity for the target substance. Furthermore, the ligand in the particles for sample testing is preferably an antibody, an antigen, or a nucleic acid.

[0063] To obtain the particles for sample testing in this embodiment, the method for bonding the reactive functional group of the dye-containing resin particles to the ligand can be conventionally known methods to the extent that the objectives of the present invention can be achieved. When the ligand is bonded via an amide linkage, a catalyst such as 1-[3-(dimethylaminopropyl)-3-ethylcarbodiimide] can be used as appropriate. The resin particles according to this embodiment can also have the ligand immobilized by physical adsorption.

[0064] (Applications of particles for specimen testing - Fluorescence polarization depolarization method) When the pigment-containing resin particles of the present invention are used to test specimens, and these particles have an antibody (antigen) as a ligand, they can be preferably applied to fluorescence depolarization methods for detecting the antigen (antibody) as a target substance.

[0065] <Third Embodiment> (Diagnostic reagents for in vitro diagnostics) The dye-containing resin particles of this embodiment can be used for the detection of target substances in a sample by in vitro diagnostics. Such an in vitro diagnostic reagent comprises the sample testing particles described in the second embodiment and a dispersion medium for dispersing the sample testing particles. The amount of sample testing particles contained in the reagent is preferably 0.000001% to 20% by mass, and more preferably 0.0001% to 1% by mass. Such a reagent may also contain substances such as solvents and blocking agents in addition to the sample testing particles, to the extent that the objectives of the present invention can be achieved. Two or more types of solvents and blocking agents may be included in combination. Examples of solvents to be used include various buffers such as phosphate buffer, glycine buffer, Good's buffer, Tris buffer, and ammonia buffer, but the solvents contained in the reagent are not limited to these. When the reagent is used for the detection of an antigen or antibody in a sample, an antibody or antigen can be used as a ligand.

[0066] <Fourth Embodiment> (Test kit) The specimen testing reagent of the third embodiment can be used in a test kit for detecting a target substance in a specimen by in vitro diagnostics. Such a test kit comprises the test reagent and a housing that encloses the test reagent. The test kit may contain a sensitizer that promotes particle aggregation during the antigen-antibody reaction. Examples of sensitizers include, but are not limited to, polyvinyl alcohol, polyvinylpyrrolidone, and sodium alginate. The test kit may also include a positive control, a negative control, and a serum diluent. As media for the positive and negative controls, serum, physiological saline, or a solvent that does not contain the target substance that can be measured may be used. Such a test kit can be used to detect a target substance in the same manner as kits used for detecting a target substance in a specimen by conventional in vitro diagnostics. The concentration of the target substance can also be measured by conventionally known methods. It is particularly suitable for use in detecting a target substance in a specimen by immunolatex agglutination or fluorescence depolarization.

[0067] (Detection method) A method for detecting a target substance in a sample by in vitro diagnostics using the sample testing particles described in the second embodiment can be a detection method comprising the steps of: mixing a sample solution that may contain the target substance with the sample testing particles described in the second embodiment to obtain a mixed solution; and irradiating the mixed solution with light to obtain a value relating to the fluorescence polarization of the mixed solution. That is, by optically detecting the agglutination reaction that occurs in the mixed solution, the presence or amount of the target substance in the sample solution can be detected, and furthermore, the concentration of the target substance can also be measured. Here, "obtaining a value relating to the fluorescence polarization of the mixed solution" may refer to obtaining a measurement of the polarization anisotropy of the mixed solution, or it may refer to measuring the degree of fluorescence polarization.

[0068] The mixing of the sample testing particles and the sample is preferably carried out in a pH range of 3.0 to 11.0. Typically, the mixing temperature is in the range of 20°C to 50°C, and the mixing time is in the range of 1 minute to 60 minutes. It is also preferable to use a solvent during mixing. The concentration of the sample testing particles in the reaction system is preferably 0.000001% to 1% by mass, more preferably 0.00001% to 0.005% by mass. For the detection of the target substance in the sample, it is preferable to detect the agglutination reaction resulting from the mixing of the sample testing particles and the sample using fluorescence polarization depolarization.

[0069] Specifically, the method for detecting a target substance in a sample by in vitro diagnostics using affinity particles containing dye-containing resin particles according to the present invention comprises the steps of: mixing the sample with a test reagent to obtain a mixture; irradiating the mixture with polarized light; and separating and detecting the polarized component of the emission of affinity particles in the mixture. In other words, by optically detecting the agglutination reaction that occurs in the mixture, the target substance in the sample can be detected, and furthermore, the concentration of the target substance can also be measured. [Examples]

[0070] The present invention will be specifically described below with reference to examples. However, the present invention is not limited to these examples.

[0071] <Manufacturing of pigment-containing resin particles> [Example 1] (1) Preparation of resin particles In a four-necked flask equipped with a mechanical stirrer, 100 g of pH 7 2-morpholinoethanesulfonic acid buffer (hereinafter referred to as "MES buffer") (manufactured by Kishida Chemical Co., Ltd.), 1.00 g of styrene monomer (manufactured by Kishida Chemical Co., Ltd.), 0.10 g of sodium styrenesulfonate (styrenesulfonic acid monomer) (manufactured by Tokyo Chemical Industry Co., Ltd.), 0.13 g of divinylbenzene (hereinafter referred to as "DVB") (manufactured by Tokyo Chemical Industry Co., Ltd.), and 0.012 g of dodecyl methacrylate (manufactured by Tokyo Chemical Industry Co., Ltd.) were added. The mixture was stirred at room temperature for 15 minutes under nitrogen flow conditions with the mechanical stirrer set to 300 rpm. Then, the four-necked flask was immersed in an oil bath set to 70°C and stirred for a further 15 minutes under nitrogen flow conditions. To the resulting mixture, 0.01 g of 2,2'-azobis(2-methylpropionamidine) dihydrochloride (hereinafter referred to as "V50") (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and emulsion polymerization was carried out for 6 hours to prepare a suspension containing particles that constitute the core of the desired resin particles.

[0072] Next, to the obtained suspension, 0.20 g of hydroxyethyl methacrylate (hereinafter referred to as "HEMA") (manufactured by Tokyo Chemical Industry Co., Ltd.), 0.05 g of trimethylolpropane trimethacrylate (hereinafter referred to as "TMP") (manufactured by Tokyo Chemical Industry Co., Ltd.), and 0.01 g of V50 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were added as materials for the shell portion, and emulsion polymerization was carried out for 10 hours to form a shell portion on the surface of the particles constituting the core portion, thereby preparing a suspension containing the desired resin particles.

[0073] Subsequently, the obtained suspension was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 100K with approximately 4 L of deionized water to wash the product and obtain a dispersion of the desired resin particles having a core-shell structure.

[0074] (2) Preparation of dye-containing resin particles 1 A europium complex solution was prepared by mixing 1.00 g of N-methyl-2-pyrrolidone (manufactured by Kishida Chemical Co., Ltd.) with 0.04 g of tris(2-cenoyltrifluoroacetonato)bis(triphenylphosphine oxide) europium(III) (hereinafter referred to as "Eu(TTA)3(TPPO)2" or "E400") (manufactured by Central Techno Co., Ltd.), which is a polarizing luminescent europium complex. The resin particles obtained in (1) above were mixed with the europium complex solution prepared here, and the suspension was shaken and stirred at 55°C for 3 hours. The suspension was then purified by centrifugation, and the suspended particles were redispersed in MES buffer at pH 7 to obtain dye-containing resin particles 1.

[0075] [Example 2] Dye-containing resin particles 2 were prepared in the same manner as in Example 1, except that polyethylene glycol monomethyl ether methacrylate (hereinafter referred to as "PEG") (manufactured by Sigma-Aldrich Japan LLC) was used as the monomer material for the shell portion instead of HEMA, as used in Example 1.

[0076] [Example 3] Dye-containing resin particles 3 were prepared in the same manner as in Example 1, except that glycidyl methacrylate (hereinafter referred to as "GMA") (manufactured by Tokyo Chemical Industry Co., Ltd.) was used as the monomer material for the shell portion instead of HEMA, as in Example 1.

[0077] [Example 4] Dye-containing resin particles 4 were prepared in the same manner as in Example 1, except that 2-methoxyethyl acrylate (hereinafter abbreviated as "MEA") (manufactured by Tokyo Chemical Industry Co., Ltd.) was used as the monomer material for the shell portion instead of HEMA, as used in Example 1.

[0078] [Example 5] Dye-containing resin particles 5 were prepared in the same manner as in Example 1, except that 2-methoxyethyl methacrylate (hereinafter abbreviated as "MEMe") (manufactured by Tokyo Chemical Industry Co., Ltd.) was used as the monomer material for the shell portion instead of HEMA, as used in Example 1.

[0079] [Example 6] Dye-containing resin particles 6 were prepared in the same manner as in Example 1, except that polyethylene glycol monomethacrylate (manufactured by Sigma-Aldrich Japan LLC) was used as the monomer material for the shell portion instead of HEMA, as in Example 1.

[0080] [Example 7] Dye-containing resin particles 7 were prepared in the same manner as in Example 1, except that tris(2-cenoyltrifluoroacetonato)bis(trioctylphosphine oxide)europium(III) (hereinafter referred to as "Eu(TTA)3(TOPO)2" or "E420") (manufactured by Central Techno Co., Ltd.) was used as the europium complex instead of "Eu(TTA)3(TOPO)2" (E400) used in Example 1.

[0081] [Example 8] Dye-containing resin particles 8 were prepared in the same manner as in Example 1, except that 0.008 g of hexyl methacrylate (manufactured by Tokyo Chemical Industry Co., Ltd.) was used as the first monomer added when forming the core, instead of 0.012 g of dodecyl methacrylate used in Example 1.

[0082] [Example 9] Dye-containing resin particles 9 were prepared in the same manner as in Example 1, except that 0.010 g of 2-ethylhexyl methacrylate (manufactured by Tokyo Chemical Industry Co., Ltd.) was used as the first monomer added when forming the core, instead of 0.012 g of dodecyl methacrylate used in Example 1.

[0083] [Example 10] Dye-containing resin particles 10 were prepared in the same manner as in Example 1, except that 3-[[2-(methacryloyloxy)ethyl]dimethylammonio]propane-1-sulfonic acid (manufactured by Tokyo Chemical Industry Co., Ltd.) was used as the monomer material for the shell portion instead of HEMA used in Example 1.

[0084] [Example 11] Dye-containing resin particles 11 were prepared in the same manner as in Example 1, except that 0.009 g of 1-ethylcyclohexyl methacrylate (manufactured by Tokyo Chemical Industry Co., Ltd.) was used as the first monomer added when forming the core, instead of 0.012 g of dodecyl methacrylate used in Example 1.

[0085] [Example 12] Dye-containing resin particles 12 were prepared in the same manner as in Example 1, except that ethylene glycol dimethacrylate (manufactured by Tokyo Chemical Industry Co., Ltd.) was used as the core crosslinking agent instead of DVB used in Example 1, and ethylene glycol dimethacrylate (manufactured by Tokyo Chemical Industry Co., Ltd.) was used as the shell crosslinking agent instead of TMP used in Example 1.

[0086] [Example 13] In Example 1, an immersion method was used to produce the dye-containing resin particles. In this example, however, a synthesis method was used instead of the immersion method, and the dye-containing resin particles 13 were produced using the same procedure as in Example 1. Specifically, the dye-containing resin particles were produced as follows.

[0087] In a four-necked flask equipped with a mechanical stirrer, 100 g of MES buffer solution at pH 7 (manufactured by Kishida Chemical Co., Ltd.), 0.04 g of the polarizing luminescent europium complex Eu(TTA)3(TPPO)2(E400) (manufactured by Central Techno Co., Ltd.), 1.00 g of styrene monomer (manufactured by Kishida Chemical Co., Ltd.), 0.10 g of sodium styrene sulfonate (styrene sulfonate monomer) (manufactured by Tokyo Chemical Industry Co., Ltd.), 0.13 g of DVB, and 0.012 g of dodecyl methacrylate (manufactured by Tokyo Chemical Industry Co., Ltd.) were added. The mixture was stirred at room temperature for 15 minutes under nitrogen flow conditions with the mechanical stirrer set to 300 rpm. Then, the four-necked flask was immersed in an oil bath set to 70°C and stirred for another 15 minutes under nitrogen flow conditions. 0.01 g of V50 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to the resulting mixture, and emulsion polymerization was carried out for 6 hours to prepare a suspension containing particles that constitute the core of the desired resin particles.

[0088] Next, 0.20 g of HEMA (manufactured by Tokyo Chemical Industry Co., Ltd.), 0.05 g of TMP (manufactured by Tokyo Chemical Industry Co., Ltd.), and 0.01 g of V50 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were added to the obtained suspension as shell material, and emulsion polymerization was carried out for 10 hours to form a shell on the surface of the particles constituting the core, thereby preparing a suspension containing the desired resin particles. Subsequently, the obtained suspension was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 100 K with approximately 4 L of deionized water to wash the product, and the dispersion of dye-containing resin particles 13 and the obtained suspension were centrifuged to purify the suspended particles. These were then redispersed in MES buffer at pH 7 to obtain dye-containing resin particles 10.

[0089] [Example 14] In Example 1, 0.012 g of dodecyl methacrylate was used as the first monomer added when forming the core. In this example, however, 0.037 g of dodecyl methacrylate was used, and the dye-containing resin particles 14 were prepared in the same manner as in Example 1.

[0090] [Comparative Example 1] Dye-containing resin particles 15 were prepared by synthesis in the same manner as in Example 13, except that the step of forming a shell portion on the surface of the particles constituting the core portion was omitted.

[0091] [Comparative Example 2] Dye-containing resin particles 16 were prepared in the same manner as in Example 1, except that core crosslinking agents and shell crosslinking agents were not added when forming the core and shell portions, respectively.

[0092] [Comparative Example 3] Dye-containing resin particles 17 were prepared in the same manner as in Example 1, except that a shell crosslinking agent was not added when forming the shell portion.

[0093] [Comparative Example 4] Dye-containing resin particles 18 were prepared in the same manner as in Example 1, except that a core crosslinking agent was not added when forming the core.

[0094] [Comparative Example 5] Dye-containing resin particles 19 were prepared in the same manner as in Example 1, except that the same amount of benzyl methacrylate (manufactured by Tokyo Chemical Industry Co., Ltd.) was used as the first monomer added when forming the core, instead of the 0.012 g of dodecyl methacrylate used in Example 1.

[0095] [Comparative Example 6] In Example 1, 0.012 g of dodecyl methacrylate was used as the first monomer added when forming the core. In this example, however, 0.007 g of dodecyl methacrylate was used, and the dye-containing resin particles 20 were prepared in the same manner as in Example 1.

[0096] The amounts of each component used to produce the dye-containing resin particles 1-20 obtained in Examples 1-14 and Comparative Examples 1-6 are shown in Table 1 below. [Table 1]

[0097] <Preparation of particles for specimen testing> A dispersion of synthesized dye-containing resin particles 1 was substituted with pyridine, and then succinic anhydride was added to conjugate a carboxylic acid group to a portion of the HEMA. 0.25 mL of the dispersion of dye-containing resin particles 1 (1.2 wt%) with carboxylic acid conjugation was taken, and the solvent was replaced with 1.6 mL of pH 6.0 MES buffer. 0.5 wt% of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide and N-hydroxysulfosuccinimide sodium were added to the MES buffer containing dye-containing resin particles 1, and the mixture was reacted at 25°C for 1 hour. After the reaction, the dispersion was washed with pH 5.0 MES buffer, 100 μg / mL of anti-CRP antibody was added, and the mixture was allowed to bind to the dye-containing resin particles 1 at 25°C for 2 hours. After binding, the antibody-conjugated dye-containing resin particles 1 were washed with pH 8 Tris buffer. After the reaction, the dye-containing resin particles 1 to which the antibody was bound were washed with phosphate buffer to obtain particles for sample testing modified with 0.3 wt% anti-CRP antibody (hereinafter sometimes referred to as "particles for sample testing"). The binding of antibodies to the resin particles was confirmed by measuring the decrease in antibody concentration in the buffer solution to which the antibodies were added using a BCA assay.

[0098] <Evaluation of pigment-containing resin particles> The following evaluations were performed using aqueous dispersions of dye-containing resin particles 1-20 prepared in Examples 1-14 and Comparative Examples 1-6. The evaluation results are shown in Table 2. Here, in Table 2, "Core St Mol ratio" indicates the number of styrene monomers in the hydrophobic monomer adjusted to Mol 100, and "Mol ratio of the first monomer Mol_f" is the number of moles of the first monomer in the hydrophobic monomer when the number of moles of styrene monomer is set to 100. In addition, "LogP_f of the first monomer" indicates the octanol / water partition coefficient of the first monomer.

[0099] (particle size, polydispersity index) The shape of the dye-containing resin particles was evaluated using an electron microscope (Hitachi High-Technologies S5500). The particle size of the dye-containing resin particles was evaluated using dynamic light scattering (Malvern Zetasizer Nano S) at a resin particle concentration of 0.1 mg / mL. The polydispersity index can also be obtained using this measurement.

[0100] (Dye-containing resin particle concentration) The particle concentration in a suspension containing dispersed dye-containing resin particles was evaluated using a gravimetric analyzer (Rigaku ThermoPlus TG8120).

[0101] (Emission intensity retention rate, initial emission intensity) The fluorescence intensity of the dye-containing resin particles was measured using an excitation light wavelength of 340 nm and an observation light peak wavelength of 614 nm. A Shimadzu RF-6000 fluorescence spectrophotometer was used. 30 μl of each dye-containing resin particle dispersion (0.01 mg / mL) was mixed with 1500 μl of pure water, and the fluorescence intensity was measured immediately. Fluorescence intensity was measured once every minute, for a total of 21 measurements from the start of measurement until 20 minutes later.

[0102] The initial luminescence intensity was denoted as Lum_Ini, and the luminescence intensity after 20 minutes was denoted as Lum_Fin. The luminescence intensity was substituted into the following equation (60) to calculate the luminescence intensity retention rate, Lum%,. Lum% = (Lum_Fin) / (Lum_Ini) (60) For the luminescence intensity retention rate (Lum%), a value of 0.80 or higher was rated A, 0.70 or higher and less than 0.80 was rated B, and less than 0.70 was rated C. An evaluation result of A or B was considered good, and C was considered unacceptable. Similarly, for the initial luminescence intensity (Lum_Ini), a value of 6000 or higher was rated A, 5000 or higher and less than 6000 was rated B, and less than 5000 was rated C. An evaluation result of A or B was considered good, and C was considered unacceptable.

[0103] (Particle size stored at 40℃) A dispersion of the prepared dye-containing resin particles was diluted with pure water to 1 mg / mL and prepared. 8 mL of this dispersion was placed in a 10 mL sealed container and stored at 40°C for 3 months. The change in particle size before and after storage was measured by dynamic light scattering. A change in particle size of 7 nm or less was classified as A, a change greater than 7 nm and less than or equal to 10 nm as B, and a change greater than 10 nm as C. An evaluation result of A or B was considered good, and C was considered unacceptable.

[0104] [Table 2]

[0105] As shown in the results in Table 2, as demonstrated by the comparison between the comparative example and the example, it was found that having a core-shell structure, with the core and shell portions being cross-linked, and containing a first monomer with a LogP of 3.6 or higher in the core at a Mol ratio of 0.05 or higher with respect to the St monomer, can suppress the decrease in luminescence intensity over time and improve the retention rate of luminescence intensity.

[0106] <Evaluation of particles for specimen testing> The polarization anisotropy of the obtained sample particles was measured before and after mixing with CRP (antigen). The sample particles were fixed at 0.001 mg / mL, and the CRP concentration was investigated from 0 to 10000 pg / mL. The evaluation was performed using a microplate reader (Nivo, PerkinElmer). A filter with a center wavelength of 355 nm and a full width at half maximum of 40 nm was used for the excitation light, a filter with a center wavelength of 615 nm and a full width at half maximum of 8 nm was used for the emission light, and a D400 dichroic mirror was used. The measurement time was set to 1 second, and the polarization anisotropy r was measured for 30 minutes from the start of the reaction, and the change in polarization anisotropy r, Δr, during that time was calculated. The measurement temperature was fixed at 37°C. The evaluation results are shown in Table 3. [Table 3]

[0107] As shown in Table 3, the change in polarization anisotropy (Δr) before and after mixing with CRP was confirmed to change depending on the concentration of CRP, enabling highly sensitive detection of CRP. Furthermore, since the change in polarization anisotropy was detected even at a low concentration of 0.001 mg / ml of affinity particles, it was confirmed that these particles exhibit strong luminescence and are suitable for sample testing.

[0108] The present invention includes embodiments comprising the following configurations. (Composition 1) A dye-containing resin particle having a core portion and a shell portion, wherein the dye-containing resin particle contains a europium complex, The core portion and the shell portion are each bridged, The core portion has a resin containing a polymer of hydrophobic monomers, which includes a styrene monomer as the main component and a first monomer of a different type from the styrene monomer. The shell portion has a resin containing a polymer of hydrophilic monomers, Dye-containing resin particles characterized in that the first monomer satisfies the following formulas (B1) and (15). LogP_f≧3.60 (B1) (Mol_f) / (Mol_St) ≧ 0.05 (15) (However, in equation (B1), LogP_f is the octanol / water partition coefficient of the first monomer, and in equation (15), Mol_f represents the number of moles of the first monomer, and Mol_St represents the number of moles of the styrene monomer.) (Configuration 2) The dye-containing resin particles of the configuration 1 are characterized in that the octanol / water partition coefficient of all ligands in the europium complex is 2.0 or higher. (Composition 3) The dye-containing resin particles of configuration 1 or configuration 2 are characterized in that the europium complex is represented by the following formula (1). Eu(A)x(B)y(C)z (1) [However, in formula (1), Eu is europium, (A) is a ligand represented by formula (2) below, (B) is a ligand represented by any of the following formulas (3), (41), (42), or (43), and (C) is a ligand represented by formula (5) below.] [ka] (In equations (2), (3), (41), (42), (43), and (5), R 1 and R 2 R is a group that may have substituents and is independently selected from an alkyl group, a thienolphenyl group, and a thienyl group. 3 R is a hydrogen atom or a methyl group. 4 and R 5 R is a group that may have substituents and is independently selected from alkyl groups and phenyl groups. 6 R is a group selected from alkyl groups, phenyl groups, and triphenylene groups, which may have substituents. 7 and R 8 R is a group that may have substituents and is independently selected from alkyl groups or phenyl groups. 1 , R 2 , R 4 ~R 6 The substituents in are groups independently selected from methyl, fluoro, chloro, and bromo groups, and R 1 , R 2 , R 4 ~R 8 The alkyl group in this expression has 2 to 12 carbon atoms, and each atom may be a different group or the same group. Furthermore, x, y, and z in equation (1) satisfy equations (6), (7), (8), and (9) below. x=3 (6) y=1 or 2 (7) z=0 or 1 (8) x+y+z=4 or 5 (9)] (Composition 4) Dye-containing resin particles having any of configurations 1 to 3, characterized in that the resin contained in the shell portion includes at least one of the structures of the following formulas (12) and (13). [ka] (In equations (12) and (13), X2 and X3 are H or CH3, Y1 is OH or OCH3, Y2 is the group represented by the following equation (14) or CH2CH2OH, and m3 and m4 are integers greater than or equal to 1.) [ka] n is an integer between 1 and 40 (inclusive). (Composition 5) Dye-containing resin particles having any of configurations 1 to 4, characterized in that the first monomer is a compound represented by the following formula (11). [ka] (In formula (11), X4 is H or CH3, Y3 is H or an alkyl group having 3 or fewer carbon atoms, and m5 is an integer between 3 and 9.) (Composition 6) Dye-containing resin particles having any of configurations 1 to 5, characterized in that the core portion contains a resin formed using a crosslinking agent containing divinylbenzene, and the shell portion contains a resin formed using a crosslinking agent containing trimethylolpropanetrimethacrylate. (Composition 7) A method for producing dye-containing resin particles containing a europium complex, A process for obtaining resin particles having a core and a shell, The process includes a step of mixing a solution containing a europium complex with resin particles having a core portion and a shell portion. The process of obtaining the resin particles comprises a sub-step 1 for forming the core portion and a sub-step 2 for forming the shell portion. Sub-step 1 is a sub-step in which a hydrophobic monomer containing styrene, which is the main component, and a first monomer is polymerized and crosslinked to form a resin, and sub-step 2 is a sub-step in which a hydrophilic monomer is polymerized and crosslinked to form a resin. A method for producing dye-containing particles, characterized in that the first monomer in the sub-step 1 satisfies the following formulas (B1) and (15). LogP_f≧3.60 (B1) (Mol_f) / (Mol_St) ≧ 0.05 (15) (However, in equation (B1), LogP_f is the octanol / water partition coefficient of the first monomer, in equation (15), Mol_f is the number of moles of the first monomer, and Mol_St is the number of moles of the styrene monomer.) (Composition 8) A method for producing dye-containing resin particles having a core portion and a shell portion, which contain a europium complex, The process comprises the steps of: polymerizing and crosslinking the hydrophobic monomer in a solution containing styrene as the main component, a hydrophobic monomer containing a first monomer, and a europium complex to form a core; and polymerizing and crosslinking the hydrophilic monomer in the presence of the core to form the shell around the core. A method for producing dye-containing particles, characterized in that the step of forming the core portion is a step of polymerizing and crosslinking the hydrophobic monomer in the solution in which the first monomer satisfies the following formulas (B1) and (15). LogP_f≧3.60(B1) (Mol_f) / (Mol_St) ≧ 0.05 (15) (However, in equation (B1), LogP_f is the octanol / water partition coefficient of the first monomer, in equation (15), Mol_f is the number of moles of the first monomer, and Mol_St is the number of moles of the styrene monomer.) (Composition 9) A method for producing dye-containing resin particles of configuration 7 or configuration 8, characterized in that the europium complex is represented by the following formula (1). Eu(A)x(B)y(C)z (1) [However, in formula (1), Eu is europium, (A) is the ligand represented by formula (2) below, (B) is the ligand represented by any of the following formulas (3), (41), (42), or (43), and (C) is the ligand represented by formula (5) below. [ka] (In equations (2), (3), (41), (42), (43), and (5), R 1 and R 2 Each of these is independently an alkyl group, a thienoylphenyl group, or a thienyl group, which may each have a substituent. 3 R is a hydrogen atom or a methyl group, 4 and R 5 Each is independently an alkyl group or phenyl group which may each have substituents, and R 6 R is an alkyl group, a phenyl group, or a triphenylene group, which may each have substituents. 7 and R 8 Each of these is independently an alkyl group or phenyl group which may have substituents, and each substituent is independently one of a methyl group, a fluoro group, a chloro group, or a bromo group, and each of the alkyl groups independently has 2 to 12 carbon atoms. Furthermore, x, y, and z are given by the following equations (6), (7), (8), and (9). x=3 (6) y=1 or 2 (7) z=0 or 1 (8) x+y+z=4 or 5 (9) [Satisfies the condition.] (Composition 10) A method for producing dye-containing resin particles of configuration 7 or configuration 8, characterized in that the resin formed by polymerizing and crosslinking the hydrophilic monomer contains at least one of the structures represented by the following formula (12) and the following formula (13). [ka] (X2 and X3 are H or CH3, Y1 is OH or OCH3, and Y2 is the following formula (14) [ka] The group is represented by or CH2CH2OH, where m3 and m4 are integers greater than or equal to 1, and n is an integer between 1 and 40. (Composition 11) A method for producing dye-containing resin particles of configuration 7 or configuration 8, characterized in that the hydrophilic monomer is a compound represented by the following formula (11). [ka] (In formula (11), X4 is H or CH3, Y3 is H or an alkyl group having 3 or fewer carbon atoms, and m5 is an integer between 3 and 9.) (Composition 12) A method for producing dye-containing resin particles of configuration 7, characterized in that sub-step 1 is a sub-step in which the hydrophobic monomer is polymerized using a crosslinking agent containing divinylbenzene, and sub-step 2 is a sub-step in which the hydrophilic monomer is polymerized using a crosslinking agent containing trimethylolpropane trimethacrylate. (Composition 13) A method for producing dye-containing resin particles of configuration 8, characterized in that the step of forming the core portion is a step of polymerizing the hydrophobic monomer using a crosslinking agent containing divinylbenzene, and the step of forming the shell portion is a step of polymerizing the hydrophilic monomer using a crosslinking agent containing trimethylolpropane trimethacrylate. (Composition 14) A method for producing particles for specimen testing, characterized by having a step of forming a site that specifically reacts with a target substance in dye-containing resin particles obtained by a method for producing dye-containing resin particles having one of the configurations 7 to 13. (Composition 15) Particles for specimen testing, characterized by having dye-containing resin particles having one of the configurations 1 to 6, and a site on the surface of the dye-containing resin particles that specifically reacts with a target substance. (Composition 16) Particles for sample testing having a configuration of 15, characterized in that they are used for detecting the target substance using fluorescence depolarization. (Composition 17) A specimen testing reagent characterized by containing 15 particles for specimen testing. (Composition 18) A test kit for a target substance, characterized by containing 17 sample test reagents. (Composition 19) A method for detecting a target substance, which uses fluorescence depolarization to detect the presence or amount of the target substance, A method for detecting a target substance, comprising the steps of: mixing a sample solution that may contain a target substance with sample testing particles of component 15 to obtain a mixed solution; and irradiating the mixed solution with light to obtain a value relating to the fluorescence polarization of the mixed solution. (Composition 20) A method for detecting a target substance according to configuration 19, characterized in that the sample solution contains an aqueous solvent. [Explanation of Symbols]

[0109] 1. Core 2 Shell section 3 Europium complex 20 Hydrophilic polymers< / r> < / r>

Claims

1. A dye-containing resin particle having a core portion and a shell portion, wherein the dye-containing resin particle contains a europium complex, The core portion and the shell portion are each bridged, The core portion has a resin containing a polymer of hydrophobic monomers, which includes a styrene monomer as the main component and a first monomer of a different type from the styrene monomer. The shell portion has a resin containing a polymer of hydrophilic monomers, Dye-containing resin particles characterized in that the first monomer satisfies the following formula (B1) and the following formula (15). LogP_f≧3.60 (B1) (Mol_f) / (Mol_St) ≧ 0.05 (15) (However, in equation (B1), LogP_f is the octanol / water partition coefficient of the first monomer, and in equation (15), Mol_f represents the number of moles of the first monomer, and Mol_St represents the number of moles of the styrene monomer.)

2. The dye-containing resin particles according to claim 1, characterized in that the octanol / water partition coefficient of all ligands in the europium complex is 2.0 or greater.

3. The dye-containing resin particles according to claim 1, characterized in that the europium complex is represented by the following formula (1). Eu(A)x(B)y(C)z (1) [However, in formula (1), Eu is europium, (A) is a ligand represented by formula (2) below, (B) is a ligand represented by any of the following formulas (3), (41), (42), or (43), and (C) is a ligand represented by formula (5) below.] 【Chemistry 1】 (In formulas (2), (3), (41), (42), (43) and (5), R １ and R ２ are each independently selected from a group consisting of an alkyl group, a thienoyl phenyl group and a thienyl group, which may have a substituent. Further, R ３ is a hydrogen atom or a methyl group. Further, R ４ and R ５ are each independently selected from a group consisting of an alkyl group and a phenyl group, which may have a substituent. Further, R ６ is a group selected from a group consisting of an alkyl group, a phenyl group and a triphenylene group, which may have a substituent. Further, R ７ and R ８ are each independently selected from a group consisting of an alkyl group or a phenyl group, which may have a substituent. Here, the substituents in R １ , R ２ , R ４ to R ６ are each independently selected from a group consisting of a methyl group, a fluoro group, a chloro group and a bromo group, and the alkyl groups in R １ , R ２ , R ４ to R ８ have 2 to 12 carbon atoms and may be different or the same groups.) Furthermore, x, y, and z in equation (1) satisfy the following equations (6), (7), (8), and (9). x = 3 (6) y = 1 or 2 (7) z = 0 or 1 (8) x + y + z = 4 or 5 (9)

4. The dye-containing resin particle according to claim 1, characterized in that the resin contained in the shell portion contains at least one of the structures of the following formulas (12) and (13). 【Chemistry 2】 (In equations (12) and (13), X2 and X3 are H or CH) ３ And Y1 is OH or OCH ３ Y2 is a group represented by the following formula (14) or CH ２ CH ２ OH is such that m3 and m4 are integers greater than or equal to 1. 【Transformation 3】 n is an integer between 1 and 40 (inclusive).

5. The dye-containing resin particles according to claim 1, characterized in that the first monomer is a compound represented by the following formula (11). 【Chemistry 4】 (In equation (11), X4 is H or CH) ３ Y3 is H or an alkyl group having 3 or fewer carbon atoms, and m5 is an integer between 3 and 9.

6. The dye-containing resin particle according to claim 1, characterized in that the core portion comprises a resin formed using a crosslinking agent containing divinylbenzene, and the shell portion comprises a resin formed using a crosslinking agent containing trimethylolpropanetrimethacrylate.

7. A method for producing dye-containing resin particles containing a europium complex, A process for obtaining resin particles having a core and a shell, The process includes a step of mixing a solution containing a europium complex with resin particles having a core portion and a shell portion. The process of obtaining the resin particles comprises a sub-step 1 for forming the core portion and a sub-step 2 for forming the shell portion. Sub-step 1 is a sub-step in which a hydrophobic monomer containing styrene, which is the main component, and a first monomer is polymerized and crosslinked to form a resin, and sub-step 2 is a sub-step in which a hydrophilic monomer is polymerized and crosslinked to form a resin. A method for producing pigment-containing particles, characterized in that the first monomer in the sub-step 1 satisfies the following formula (B1) and the following formula (15). LogP_f≧3.60 (B1) (Mol_f) / (Mol_St) ≧ 0.05 (15) (However, in formula (B1), LogP_f is the octanol / water partition coefficient of the first monomer, in formula (15), Mol_f is the number of moles of the first monomer, and Mol_St is the number of moles of the styrene monomer.)

8. A method for producing dye-containing resin particles having a core portion and a shell portion, which contain a europium complex, The process comprises the steps of: polymerizing and crosslinking the hydrophobic monomer in a solution containing styrene as the main component, a hydrophobic monomer containing a first monomer, and a europium complex to form a core; and polymerizing and crosslinking the hydrophilic monomer in the presence of the core to form the shell around the core. A method for producing dye-containing particles, characterized in that the step of forming the core portion is a step of polymerizing and crosslinking the hydrophobic monomer in the solution in which the first monomer satisfies the following formula (B1) and the following formula (15). LogP_f≧3.60 (B1) (Mol_f) / (Mol_St) ≧ 0.05 (15) (However, in formula (B1), LogP_f is the octanol / water partition coefficient of the first monomer, in formula (15), Mol_f is the number of moles of the first monomer, and Mol_St is the number of moles of the styrene monomer.)

9. The method for producing dye-containing resin particles according to claim 7 or 8, characterized in that the europium complex is represented by the following formula (1). Eu(A)x(B)y(C)z (1) [However, in formula (1), Eu is europium, (A) is the ligand represented by formula (2) below, (B) is the ligand represented by any of the following formulas (3), (41), (42), or (43), and (C) is the ligand represented by formula (5) below. 【Transformation 5】 (In equations (2), (3), (41), (42), (43), and (5), R １ and R ２ Each of these is independently an alkyl group, a thienolphenyl group, or a thienyl group, which may each have a substituent. ３ R is a hydrogen atom or a methyl group, ４ and R ５ Each is independently an alkyl group or phenyl group which may each have substituents, and R ６ Each of these is an alkyl group, a phenyl group, or a triphenylene group, which may each have a substituent, and R ７ and R ８ Each of these is independently an alkyl group or phenyl group which may each have substituents, and each substituent is independently one of a methyl group, a fluoro group, a chloro group, or a bromo group, and each of the alkyl groups independently has 2 to 12 carbon atoms. Furthermore, x, y, and z are given by the following equations (6), (7), (8), and (9). x = 3 (6) y = 1 or 2 (7) z = 0 or 1 (8) x + y + z = 4 or 5 (9) [It satisfies the following conditions.]

10. A method for producing dye-containing resin particles according to claim 7 or 8, characterized in that the resin formed by polymerizing and crosslinking the hydrophilic monomer contains at least one of the structures represented by the following formula (12) and the following formula (13). 【Transformation 6】 (X2 and X3 are H or CH) ３ And Y1 is OH or OCH ３ And Y2 is given by the following equation (14) 【Transformation 7】 The group represented by or CH ２ CH ２ (OH is OH, m3 and m4 are integers greater than or equal to 1, and n is an integer between 1 and 40.)

11. A method for producing dye-containing resin particles according to claim 7 or 8, characterized in that the hydrophilic monomer is a compound represented by the following formula (11). 【Transformation 8】 (In equation (11), X4 is H or CH) ３ Y3 is H or an alkyl group having 3 or fewer carbon atoms, and m5 is an integer between 3 and 9.

12. The method for producing dye-containing resin particles according to claim 7, characterized in that the sub-step 1 is a sub-step in which the hydrophobic monomer is polymerized using a crosslinking agent containing divinylbenzene, and the sub-step 2 is a sub-step in which the hydrophilic monomer is polymerized using a crosslinking agent containing trimethylolpropane trimethacrylate.

13. The method for producing dye-containing resin particles according to claim 8, characterized in that the step of forming the core portion is a step of polymerizing the hydrophobic monomer using a crosslinking agent containing divinylbenzene, and the step of forming the shell portion is a step of polymerizing the hydrophilic monomer using a crosslinking agent containing trimethylolpropane trimethacrylate.

14. A method for producing particles for specimen testing, characterized by comprising the step of forming a site that specifically reacts with a target substance in the dye-containing resin particles obtained by the method for producing dye-containing resin particles described in claim 6.

15. Particles for specimen testing, characterized by having a dye-containing resin particle according to any one of claims 1 to 6, and a site present on the surface of the dye-containing resin particle that specifically reacts with a target substance.

16. Particles for sample testing according to claim 15, characterized in that they are used to detect the target substance using a fluorescence depolarization method.

17. A specimen testing reagent characterized by comprising the specimen testing particles described in claim 15.

18. A test kit for a target substance, characterized by comprising the sample test reagent described in claim 17.

19. A method for detecting a target substance, which uses fluorescence depolarization to detect the presence or amount of the target substance, A method for detecting a target substance, comprising the steps of: mixing a sample solution that may contain a target substance with the sample testing particles described in claim 15 to obtain a mixed solution; and irradiating the mixed solution with light to obtain a value relating to the fluorescence polarization of the mixed solution.

20. The method for detecting a target substance according to claim 19, characterized in that the sample solution contains an aqueous solvent.

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