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 that enable highly sensitive detection of biological components. [Solution] The device has a core portion and a shell portion, the core portion and the shell portion are each crosslinked, and contains a europium complex, wherein the statistical value LogP_Eu of multiple ligands of the europium complex is given by the following formula (51) LogPEu ≥ 5.80 (51) and the following formula (52) LogP_Eu > LogP_Core > LogP_Shell (52) Dye-containing resin particles characterized by satisfying the following conditions.

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. 2022-187791 [Overview of the project] [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] One embodiment of the present disclosure is a dye-containing resin particle having a core part and a shell part, wherein the dye-containing resin particle contains a europium complex, and the core part and the shell part are each cross-linked, and LogP_Eu, which is a statistical value of the octanol / water partition coefficient (LogP) of the ligand of the europium complex, satisfies the following formula (51) LogP_Eu ≧ 5.80 (51) and the following formula (52) LogP_Eu > LogP_Core > LogP_Shell (52) [However, in formula (52), LogP_Core is the value of the octanol / water partition coefficient (LogP) of the main component among the monomers forming the core part, and LogP_Shell is the value of the octanol / water partition coefficient (LogP) of the main component among the monomers forming the shell part.] A dye-containing resin particle is provided which satisfies the above, thereby solving the above problems.

Advantages of the Invention

[0007] According to the present disclosure, dye-containing resin particles in which a decrease in the emission intensity of a europium complex is suppressed are provided. Further, by using the above dye-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 the dye-containing resin particle according to one embodiment of the present disclosure. [Figure 2] It is a schematic diagram for explaining the structure of a conventional dye-containing resin particle.

Modes 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 the first embodiment, which will be described later, 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 and a fluorescence depolarization method that can be used with the dye-containing resin particles will be described.

[0010] (Measurement of particle size) The diameter of resin particles can be determined by dynamic light scattering. When laser light is shone on particles dispersed in a solution 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 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 luminescent dye 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, the change in the rotational Brownian motion of the particles can be detected as a change in polarization anisotropy.

[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 principle of the fluorescence depolarization method is to measure 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, i.e., 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 is clear 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] (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, in antigen-antibody reactions, such as particle (antibody)-antigen-particle (antibody) aggregation occurs, causing the aggregated particles to become larger and slower in rotation, which is then detected. Therefore, if small and large particles are mixed in the initial stages before the reaction in the system, there is a concern that the detection sensitivity will decrease. Thus, it is necessary for the dye-containing resin particles to have a uniform particle size distribution, and specifically, the polydispersity index mentioned above must be 0.1 or less.

[0020] <First Embodiment> (Resin particle structure, core-shell structure) The pigment-containing resin particles of this embodiment have a core portion and a shell portion. First, the resin material of the core portion is preferably low in 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 can make 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, should have a low specific gravity, more specifically, 1.10 g / cm³. 3 It is preferable to use the following materials. Specifically, it is preferable that the core part contains polystyrene, and in particular that it be the main component.

[0021] Next, it is important that the resin material of the shell suppresses aggregation between particles, and in addition, suppresses 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.

[0022] Therefore, in the dye-containing resin particles of this embodiment, it is preferable to use a resin material for the shell portion that has the function of adsorbing water molecules on the particle surface by hydrogen bonding. This suppresses aggregation of particles (or samples) by allowing water molecules to coordinate in the gaps between particles even when particles (or samples) are in close proximity, preventing direct contact between particle surfaces and suppressing entanglement between long hydrophilic groups. Similarly, when a hydrophobic container is used and the container and particles are in close proximity, water molecules coordinate between the particles and the container, preventing the particles from adsorbing to the container.

[0023] (Europium complex) In the dye-containing resin particles of this embodiment, a europium complex exhibiting polarization anisotropy is used as the dye, due to its characteristics of having a wavelength and intensity of emission that is less affected by the surroundings and having a long emission lifespan. The europium complex is composed of a europium element and a ligand, and its structure is shown in formula (1) below. Eu(A)x(B)y(C)z (1) It can be expressed as follows. 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 expressed by equation (23) changes significantly before and after the antigen-antibody reaction.

[0024] 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.

[0025] 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.

[0026] 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. The europium complex content can be calculated by quantifying europium using radiofrequency inductively coupled plasma (ICP) emission spectrometry.

[0027] On the other hand, if the europium complex aggregates within the core, the interactions between ligands will affect the excitation efficiency of the europium complex, making it difficult to measure polarization anisotropy reproducibly. Whether europium complex 3 exhibits non-aggregated luminescence behavior within the core can be determined by taking the excitation spectrum of the sample.

[0028] Highly luminescent particles not only enable high-sensitivity measurements, but also maintain strong luminescence even with reduced particle size, thus accelerating biochemical reaction rates. Smaller particle sizes result in a larger diffusion coefficient for Brownian motion in liquids, allowing for faster reaction detection.

[0029] (Method for calculating the LogP of ligands) The LogP_Eu, which is the statistical value of the octanol / water partition coefficient (LogP) of the ligand of the europium complex contained in the dye-containing resin particles of this embodiment, is calculated in the following two steps. (1) First, the structural formula of each ligand 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, to calculate the LogP of each ligand, and then take the weighted average by the respective coordination number (https: / / vcclab.org / lab / ).

[0030] Furthermore, in the dye-containing resin particles of this embodiment, the calculated LogP_Eu is given by the following formula (51) LogP_Eu ≥ 5.80 (51) and the following formula (52) LogP_Eu > LogP_Core > LogP_Shell (52) [However, in equation (52), LogP_Core is the value of the octanol / water partition coefficient of the main component monomer of the core part, and LogP_Shell is the value of the octanol / water partition coefficient of the main component monomer of the shell part.]

[0031] Preferably, the europium complex contained in the dye-containing resin particles of the present 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 a ligand represented by the following formula (2), (B) is a ligand represented by any one of the following formulas (3), (41), (42), or (43), and (C) is a ligand represented by the following formula (5). [Chemical formula] (In formulas (2), (3), (41), (42), (43), and (5), R 1 and R 2 are each independently a group selected from an alkyl group, a thienoyl phenyl group, and a thienyl group, which may optionally have a substituent. R 3 is a hydrogen atom or a methyl group. R 4 and R 5 are each independently a group selected from an alkyl group and a phenyl group, which may optionally have a substituent. R 6 is a group selected from an alkyl group, a phenyl group, and a triphenylene group, which may optionally have a substituent. Also, R 7 and R 8 S are each independently a group selected from an alkyl group or a phenyl group, which may optionally have a substituent. Here, the substituents that R 1 , R 2 , R 4 ~R 6 may optionally have are each independently a group selected from a methyl group, a fluoro group, a chloro group, and a bromo group. The alkyl groups in R 1 , R 2 , R 4 ~R 6 each independently have 2 to 12 carbon atoms and may be different or the same groups.) And x, y, z are the following formulas (y), (7), (8), and (9): x = 3 (6) y = 1 or 2 (7) z = 0 or 1 (8) x+y+z=4 or 5 (9) The following satisfies the condition. Represented by ], the statistical value LogP_Eu of the ligand possessed by the europium complex is given by the following formula (50) LogP_Eu=(LogP_B×y+LogP_C×z) / (y+z) (50) [However, in equation (50), LogP_B is the value of the octanol / water partition coefficient of ligand (B) in equation (1), and LogP_C is the value of the octanol / water partition coefficient of ligand (C) in equation (1).] This is defined as follows.

[0032] To give a specific example, in the europium complex Eu(TTA)3(TOPO)2 shown in formula (A1) below, the structure corresponding to (B) in the aforementioned formula (1) is TOPO [TTA: 2-thienoyltrifluoroacetone, TOPO: triocthylphosphine oxide]. [ka] The SMILES notation for TOPO is "CCCCCCCCP(=O)(CCCCCCCC)CCCCCCCC". Calculating LogP from this SMILES notation using the program "ALOGPS2.1" yields "8.44". Therefore, substituting LogP (LogP_B in equation (50)) corresponding to ligand (B) and the coordination number y=2 into the aforementioned equation (50) to calculate LogP_Eu, we get: LogP_Eu=((Log_B)×y) / (y) = (8.44 × 2) / (2) =8.44 It can be calculated as follows.

[0033] To give another example, in the europium complex Eu(TTA)3(TPPO)(DBSO) shown in formula (A2) below, the structure corresponding to ligand (B) in formula (1) above is TPPO, and the structure corresponding to ligand (C) is DBSO [TPPO: triphenylphosphine oxide, DBSO: dibenzyl sulfoxide]. [ka] The SMILES notation for TPPO is "O=P(C1=CC=CC=C1)(C1=CC=CC=C1)C1=CC=CC=C1", and the LogP for TPPO is calculated as "4.04" by the program "ALOGPS2.1". On the other hand, the SMILES notation for DBSO is "O=S(CC1=CC=CC=C1)CC1=CC=CC=C1", and the LogP for DBSO is calculated as "2.28" by "ALOGPS2.1". Therefore, if we substitute the LogP corresponding to ligand (B) and ligand (C) and their respective coordination numbers y=z=1 into the above equation (50), LogP_Eu=((Log_B)×y+(Log_C)×z) / (y+z) =(4.04×1+2.28×1) / (1+1) =3.16 It can be calculated as follows.

[0034] The phenomenon of the luminescence intensity of europium complexes contained in 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, resulting in a decrease in luminescence intensity.

[0035] In order for the europium complex to not leak out of the resin particles, it is necessary to satisfy the aforementioned equation (51). Specifically, if the calculated LogP_Eu is 5.8 or higher, the ligand of the europium complex has low affinity for water and is highly hydrophobic. As a result, the europium complex will not easily diffuse into water and will remain inside the particles, thus suppressing the decrease in luminescence intensity.

[0036] In particular, it is preferable that at least one of the ligands of the europium complex contains an alkyl group. By using an alkyl group as a ligand, high hydrophobicity can be obtained with a small amount of alkyl group, allowing more europium complex to be retained within the particle. As a result, the luminescence intensity becomes higher, and the detection sensitivity can be increased.

[0037] (Core part) The radical polymerizable monomer used as the raw material for the core is preferably a hydrophobic monomer containing a styrene-based monomer as the main component monomer. Here, the main component monomer is the raw material monomer that is present in the largest amount among the raw material monomers of the polymer contained in the resin forming the core (this can also be called the main constituent monomer).

[0038] When styrene monomer is used alone as the raw material monomer for the core, the particle size exceeds 500 nm, which may make it unsuitable as a dye-containing resin particle for use in the fluorescence polarization depolarization method. 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.

[0039] In addition to the main monomer component, other monomers may also be included. Examples of radical polymerizable monomers other than styrene-based monomers 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.

[0040] In addition, the resin material of the core must be crosslinked with monomers having two or more double bonds in a single molecule, such as divinylbenzene, trimethylolpropane trimethacrylate, or ethylene glycol dimethacrylate. This crosslinking must be sufficient. The principle of the fluorescence depolarization method is to detect changes in the movement of dye-containing resin particles using europium complexes. However, if the core is not 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, potentially reducing the detection sensitivity. On the other hand, if the core is crosslinked, the movement of the europium complexes within the dye-containing resin particles is suppressed, making it possible to increase the detection sensitivity. In the embodiments and examples of this disclosure, the resin material being "crosslinked" means that it is crosslinked to an extent that effectively suppresses the movement of the europium complexes within the dye-containing resin particles. For example, if the main monomer component of the core is styrene and the crosslinking agent is divinylbenzene, the weight content (degree of crosslinking) of divinylbenzene is preferably 5% to 30% by weight, and more preferably 10% to 20%. The method for determining whether or not the core is crosslinked will be described later.

[0041] When styrene is used as the main monomer component of the core, divinylbenzene is particularly preferred as the crosslinking agent. Since divinylbenzene is a compound with a structure similar to styrene, the main material of the core, it can spread uniformly within the core without localizing. Therefore, uniform crosslinking is possible within the core.

[0042] Here, whether or not the "core portion is crosslinked" in the embodiments and examples of this disclosure can be determined 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, the following formulas (25) and (26) (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 Dv_Before (unit: [nm]), the number-average particle size is Dn_Before (unit: [nm]), and the volume-average particle size obtained after the aforementioned pyridine treatment is Dv_After (unit: [nm]), and the number-average particle size is Dn_After (unit: [nm]).) If the above conditions are met, it can be determined that "the core is cross-linked."

[0043] (Shell part) The shell portion preferably contains a polymer of hydrophilic monomers having hydrogen bonding sites near the main chain of the side chains as its main material. Here, the main material of the shell portion refers to the raw material monomer (which can also be called the main component monomer) that is present in the largest amount among the raw material monomers of the polymer contained in the resin forming the shell portion. The hydrogen bonding sites are substituents that can form hydrogen bonds with water molecules in the system, and specifically, these are amino groups, carbonyl groups, carboxyl groups, hydroxyl groups, and thiol groups. 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, making it possible to improve detection sensitivity.

[0044] Furthermore, the shell material is given by the following formulas (12) and (13) [ka] (X2 and X3 are H or CH3, Y1 is OH or OCH3, and Y2 is the following formula (14)) [ka] Alternatively, a resin containing at least one of the following structures is preferred: CH2CH2OH, where m3 and m4 are integers of 1 or more, and n is an integer between 1 and 40.

[0045] 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]

[0046] In addition, the resin material of the shell portion needs to be crosslinked with monomers having 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 portion is hydroxyethyl methacrylate and the crosslinking agent is trimethylolpropane trimethacrylate, it is preferable that the weight content (degree of crosslinking) of trimethylolpropane trimethacrylate be 5% by weight or more and 30% by weight or less.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] In addition, by crosslinking the shell portion, it is possible to prevent the hydrogen bonding sites located in the side chains of the shell portion material from extending widely toward the solvent, water. If the shell portion is not crosslinked, the hydrophilic polymer will spread toward water, raising concerns about interaction with the hydrophilic sites of other particles (or samples) that come into contact with it. On the other hand, in this case, by crosslinking the shell component, the shell portion material does not spread away from the dye-containing resin particles, making interaction with other particles (or samples) that come into contact with it less likely to occur. As a result, adsorption between particles is suppressed, non-specific adsorption is suppressed, and consequently, detection sensitivity is improved.

[0051] 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.

[0052] 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, 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 "shell 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. More specifically, the following formulas (25) and (26) (Dv_After) / (Dv_Before)<1.5 (25) (Dn_After) / (Dn_Before)<1.5 (26) If the following conditions are met in equations (25) and (26), then it can be determined that "the shell portion is cross-linked." (Dv_Before (unit: [nm]) is the volume-average particle size measured by the dynamic light scattering method before the pyridine treatment described above, Dn_Before (unit: [nm]) is the number-average particle size, and Dv_After (unit: [nm]) is the volume-average particle size after the pyridine treatment described above, Dn_After (unit: [nm]) is the number-average particle size.)

[0053] (Relationship between the LogP values ​​of the europium complex, core, and shell) Furthermore, in the dye-containing resin particles of this embodiment, the LogP values ​​of the main component monomer of the core and the main component monomer of the shell are calculated in the same manner as the method for calculating the LogP of the europium complex, and as described above, the value is given by the following formula (52) LogP_Eu > LogP_Core > LogP_Shell (52) [In equation (52), the LogP of the main component monomer of the core is denoted as LogP_Core, and the LogP of the main component monomer of the shell is denoted as LogP_Shell.] This must be satisfied.

[0054] Specifically, when Eu(TTA)3(TOPO)2 shown in formula (A2) above is used as the europium complex, styrene monomer is used as the main monomer component of the core, and hydroxyethyl methacrylate shown in formula (31) above is used as the main monomer component of the shell, the LogP values ​​for each material are as follows. LogP_Eu=8.44, LogP_Core=2.92, LogP_Shell=0.27 In this case, satisfying equation (52) makes it difficult for the europium complex to diffuse into water, and the europium complex remains inside the particle, thus suppressing the decrease in luminescence intensity.

[0055] The dye-containing resin particles of this embodiment are defined by the statistical value of the octanol / water partition coefficient (LogP_Eu) of the ligand of the europium complex, which is the raw material for the dye-containing resin particles, the octanol / water partition coefficient (LogP_Core) calculated for the main component monomer of the core, and the octanol / water partition coefficient (LogP_Shell) calculated for the main component monomer of the shell. These octanol / water partition coefficient values ​​can be calculated by identifying the structural formula of the ligand of the europium complex by analyzing the manufactured dye-containing resin particles using a known method, or by identifying the monomer species that are the raw materials for the core and shell, quantifying the respective composition ratios of the ligands, and then taking the average of the LogP values ​​of each component weighted by their 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) There are two methods for arranging europium complexes within resin particles: a "synthesis method" in which the europium complex is added to a synthesis system containing raw material monomers, polymerization is carried out to form a core containing the europium complex (sub-step 1), and then a shell is formed on the outside of the core (sub-step 2); and an "immersion method" in which resin particles having a core-shell structure are formed by polymerization in a synthesis system that does not contain raw material monomers, and then the resin particles are immersed in a europium complex solution to impregnate them with the europium complex.

[0059] To produce the dye-containing resin particles of this embodiment, it is preferable to use an immersion method, in which resin particles having a core-shell structure are immersed in a europium complex solution, obtained by dissolving the europium complex in an organic solvent, to impregnate the resin particles with the europium complex in the solution.

[0060] 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 high-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.

[0061] The organic solvent used to dissolve the europium complex in the immersion method should be one that causes minimal change in the particle size of the resin particles during immersion, can dissolve the europium complex at high concentrations, and is miscible with water. Examples of such organic solvents include acetone, pyridine, ethanol, tetrahydrofuran, N,N-dimethylformamide, and N-methyl-2-pyrrolidone.

[0062] <Second Embodiment> (Particles for specimen testing) The dye-containing resin particles of the first embodiment can be used as particles for sample testing (affinity particles) by providing a site that specifically reacts with a target substance. In this case, the resin particles have a shell portion that contains 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, and the site that specifically reacts with the target substance is bonded 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.

[0063] 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 (captures a particular target substance) to the dye-containing resin particle. The site among the ligands that specifically reacts with the target substance is predetermined and has a selective or specific 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 mutually complementary single-stranded nucleic acids, but ligands are not limited to these. In other words, particles for sample testing are particles that have a specificly high affinity for the target substance. The ligand in particles for sample testing is preferably an antibody, an antigen, or a nucleic acid.

[0064] 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.

[0065] (Applications of particles for specimen testing - Fluorescence polarization depolarization method) When the sample testing particles of this embodiment have an antibody (antigen) as a site that specifically reacts with the target substance, they can be preferably applied to a fluorescence polarization depolarization method for detecting the antigen (antibody) which is the target substance.

[0066] <Third Embodiment> (In vitro diagnostic specimen testing reagents) The specimen testing particles described in the second embodiment can be used in a test reagent for detecting a target substance in a specimen solution by in vitro diagnostics. Such an in vitro diagnostic test reagent comprises the specimen testing particles described in the second embodiment and a dispersion medium for dispersing the specimen testing particles. The amount of specimen testing particles contained in the test reagent is preferably 0.000001% to 20% by mass, and more preferably 0.0001% to 1% by mass. Such a test reagent may also contain substances such as solvents and blocking agents in addition to the specimen testing particles, to the extent that the objective of this embodiment 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 test reagent are not limited to these. When the test reagent is used for detecting an antigen or antibody in a specimen solution, an antibody or antigen can be used as a ligand.

[0067] <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.

[0068] (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 mean obtaining the polarization anisotropy of the mixed solution, or obtaining the degree of fluorescence polarization.

[0069] The mixing of the sample testing particles and the sample solution is preferably carried out within 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 solution using fluorescence polarization depolarization. [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.), and 0.13 g of divinylbenzene (hereinafter referred to as "DVB") (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 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(trioctylphosphine oxide)europium(III) (hereinafter referred to as "Eu(TTA)3(TOPO)2" or "E420") (manufactured by Central Techno Co., Ltd., LogP=8.44), 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 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.

[0081] [Example 8] Dye-containing resin particles 8 were prepared in the same manner as in Example 1, except that tris(2-cenoyltrifluoroacetonato)(bis[2-(diphenylphosphino)phenyl]ether oxide)europium(III) (hereinafter referred to as "Eu(TTA)3(DPEPO)" or "E450") (manufactured by Central Techno Co., Ltd., LogP=6.05) was used as the europium complex instead of Eu(TTA)3(TOPO)2 (E420, LogP=8.44) used in Example 1.

[0082] [Example 9] Dye-containing resin particles 9 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 crosslinking agent for forming the core, instead of DVB used in Example 1.

[0083] [Example 10] 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 10 were produced using the same procedure as in Example 1. Specifically, the dye-containing resin particles were produced as follows.

[0084] 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(TOPO)2(E420) (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.), and 0.13 g of DVB 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.

[0085] 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 100K with approximately 4 L of deionized water to wash the product, and the obtained suspension was centrifuged to purify the suspended particles. These were then redispersed in MES buffer at pH 7 to obtain dye-containing resin particles 10.

[0086] [Comparative Example 1] Dye-containing resin particles 11 were prepared by synthesis in the same manner as in Example 10, except that the step of forming a shell portion on the surface of the particles constituting the core portion was omitted.

[0087] [Comparative Example 2] Dye-containing resin particles 12 were prepared in the same manner as in Example 1, except that the crosslinking agent DVB was not added when forming the core portion, and the crosslinking agent TMP was not added when forming the shell portion.

[0088] [Comparative Example 3] Dye-containing resin particles 13 were prepared in the same manner as in Example 1, except that the crosslinking agent TMP was not added when forming the shell portion.

[0089] [Comparative Example 4] Dye-containing resin particles 14 were prepared in the same manner as in Example 1, except that the crosslinking agent DVB was not added when forming the core.

[0090] [Comparative Example 5] Dye-containing resin particles 15 were prepared in the same manner as in Example 1, except that tris(2-cenoyltrifluoroacetonato)(1,10-phenanthroline)europium(III) (hereinafter referred to as "Eu(TTA)3(Phen)" or "E460") (manufactured by Central Techno Co., Ltd., LogP=2.31) was used as the europium complex instead of Eu(TTA)3(TOPO)2(E420) used in Example 1.

[0091] [Comparative Example 6] Dye-containing resin particles 16 were prepared in the same manner as in Example 1, except that 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., LogP=4.04) was used as the europium complex instead of Eu(TTA)3(TOPO)2 (E420, LogP=8.44) used in Example 1.

[0092] The amounts of each component used to produce the dye-containing resin particles 1-16 obtained in Examples 1-10 and Comparative Examples 1-6 are shown in Table 1 below. [Table 1]

[0093] <Preparation of particles for specimen testing> The aqueous dispersion of dye-containing resin particles 1 prepared in Example 1 was replaced with pyridine, and then succinic anhydride was added to confer carboxylic acid groups to a portion of the HEMA. 0.25 mL of this dispersion of dye-containing resin particles 1 (1.2 wt%) with carboxylic acid groups was taken, and the solvent was replaced with 1.6 mL of pH 6.0 MES buffer to obtain an MES buffer for dispersing dye-containing resin particles 1. 0.5 wt% of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide and N-hydroxysulfosuccinimide sodium were added to this, and the mixture was reacted at 25°C for 1 hour. The resulting dispersion was washed with pH 5.0 MES buffer, and 100 μg / mL of anti-CRP antibody was added and the mixture was reacted at 25°C for 2 hours to conjugate the anti-CRP antibody to the dye-containing resin particles 1. The antibody-conjugated dye-containing resin particles 1 were then washed with pH 8 Tris buffer. After the reaction, the dye-containing resin particles 1 to which the antibody had been bound were washed with phosphate buffer to obtain particles for sample testing modified with 0.3 wt% anti-CRP antibody. The binding of the antibody to the dye-containing resin particles was confirmed by measuring the decrease in antibody concentration in the buffer to which the antibody had been added using a BCA assay.

[0094] <Evaluation of pigment-containing resin particles> The following evaluations were performed using aqueous dispersions of dye-containing resin particles 1-16 prepared in Examples 1-10 and Comparative Examples 1-6. The evaluation results are shown in Table 2.

[0095] (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.

[0096] (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).

[0097] (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.

[0098] 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.

[0099] (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. Results A or B were considered good, and C was considered unacceptable.

[0100] [Table 2]

[0101] 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, cross-linking of the core and shell portions, having a LogP(LogP_Eu) ligand of the europium complex of 5.80 or higher, and having LogP_Eu > LogP_Core > LogP_Shell suppresses the decrease in luminescence intensity over time and improves the retention rate of luminescence intensity.

[0102] <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]

[0103] 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 could be detected even at a low concentration of 0.001 mg / ml for the sample testing particles, it was confirmed that the sample testing particles exhibited strong luminescence.

[0104] This disclosure includes embodiments comprising the following configurations. (Composition 1) A dye-containing resin particle having a core portion and a shell portion, wherein the core portion and the shell portion are each crosslinked, and the LogP_Eu, which is the statistical value of the octanol / water partition coefficient of the ligand of the europium complex, is given by the following formula (51) LogP_Eu ≥ 5.80 (51) and the following formula (52) LogP_Eu > LogP_Core > LogP_Shell (52) A dye-containing resin particle characterized by satisfying the following conditions: [In formula (52), LogP_Core is the value of the octanol / water partition coefficient of the main component among the monomers forming the core, and LogP_Shell is the value of the octanol / water partition coefficient of the main component among the monomers forming the shell.] (Configuration 2) The dye-containing resin particles of composition 1, wherein LogP_Eu, the statistical value of the octanol / water partition coefficient of the ligands of the europium complex, is the average of the octanol / water partition coefficients of each ligand, weighted by the coordination number of each ligand. (Composition 3) Dye-containing resin particles having a composition of 1 or 2, wherein at least one of the ligands of the europium complex contains an alkyl group. (Composition 4) The europium complex is given 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 Each of these groups is independently selected from an alkyl group, a thienolphenyl group, and a thienyl group, which may optionally have substituents. 3R is a hydrogen atom or a methyl group. 4 and R 5 Each of these groups is independently selected from alkyl groups and phenyl groups, which may optionally have substituents. 6 R is a group selected from alkyl groups, phenyl groups, and triphenylene groups, which may optionally have substituents. 7 and R 8 R is a group selected from alkyl groups and phenyl groups, each independently and optionally having substituents. 1 , R 2 , R 4 ~R 6 The substituents that may be optionally selected in R are each independently selected from a methyl group, a fluoro group, a chloro group, and a bromo group, 1 , R 2 , R 4 ~R 6 The alkyl groups in each of the above formulas each independently have 2 to 12 carbon atoms, and each may be a different group or the same group.) and x, y, and z in formula (1) are the same as those in the following formulas (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) The following satisfies the condition. The LogP_Eu, which is the statistical value of the octanol / water partition coefficient of the ligand of the europium complex, is given by the following formula (50): LogP_Eu=(LogP_B×y+LogP_C×z) / (y+z) (50) [However, in equation (50), LogP_B is the value of the octanol / water partition coefficient of ligand (B) in equation (1), and LogP_C is the value of the octanol / water partition coefficient of ligand (C) in equation (1).] Dye-containing resin particles of composition 1 to 3 that satisfy this condition. (Composition 5) Dye-containing resin particles comprising 1 to 4, wherein the core portion comprises a resin having a polymer of hydrophobic monomers including styrene, and the shell portion comprises a resin having a polymer of hydrophilic monomers having side chains having hydrogen bonding sites. (Composition 6) The aforementioned hydrogen bonding site is a group selected from an amino group, a carbonyl group, a carboxyl group, a hydroxyl group, and a thiol group, and is a pigment-containing resin particle of composition 5. (Composition 7) The polymer of the hydrophilic monomer is given by formulas (12) and (13) [ka] (In equations (12) and (13), X2 and X3 are independently selected from H and CH3, respectively, Y1 is OH or OCH3, and Y2 is as shown in equation (14) below.) [ka] A pigment-containing resin particle having at least one of the following structures: a group represented by or CH2CH2OH, where m3 and m4 are integers of 1 or more, and n is an integer between 1 and 40. (Composition 8) Dye-containing resin particles comprising 5 to 7 components, wherein the degree of crosslinking of the polymer of hydrophobic monomers containing styrene contained in the core portion is 5% by weight or more and 30% by weight or less. (Composition 9) Dye-containing resin particles comprising 5 to 8 components, wherein the degree of crosslinking of the polymer of a hydrophilic monomer having side chains with hydrogen bonding sites contained in the shell portion is 5% by weight or more and 30% by weight or less. (Composition 10) A method for producing dye-containing resin particles containing a europium complex, The process includes impregnating resin particles having a core and a shell with a solution containing a europium complex. The core portion and the shell portion are each cross-linked, and the LogP_Eu, which is the statistical value of the octanol / water partition coefficient of the ligand of the europium complex, is given by the following formula (51) LogP_Eu ≥ 5.80 (51) and the following formula (52) LogP_Eu > LogP_Core > LogP_Shell (52) A method for producing dye-containing resin particles, characterized in that [In formula (52), LogP_Core is the value of the octanol / water partition coefficient of the main component among the monomers forming the core portion, and LogP_Shell is the value of the octanol / water partition coefficient of the main component among the monomers forming the shell portion.] (Composition 11) A method for producing dye-containing resin particles of the configuration 1, wherein at least one of the ligands of the europium complex contains an alkyl group. (Composition 12) The europium complex is given 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 Each of these groups is independently selected from an alkyl group, a thienolphenyl group, and a thienyl group, which may optionally have substituents. 3 R is a hydrogen atom or a methyl group. 4 and R 5 These are groups independently selected from alkyl groups and phenyl groups, which may optionally have substituents. 6 R is a group selected from alkyl groups, phenyl groups, and triphenylene groups, which may optionally have substituents. 7 and R 8 Each of these groups is independently selected from alkyl groups or phenyl groups, which may optionally have substituents. Here, R 1, R 2 , R 4 ~R 6 The substituents that may be optionally present in R are each independently selected from a methyl group, a fluoro group, a chloro group, and a bromo group, 1 , R 2 , R 4 ~R 6 The alkyl groups in each of the above formulas each independently have 2 to 12 carbon atoms, and each may be a different group or the same group.) and x, y, and z in formula (1) are the same as those in the following formulas (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) The following satisfies the condition. The LogP_Eu, which is the statistical value of the octanol / water partition coefficient of the ligand of the europium complex, is given by the following formula (50): LogP_Eu=(LogP_B×y+LogP_C×z) / (y+z) (50) A method for producing pigment-containing resin particles of composition 10 or 11, satisfying the following condition: [In equation (50), LogP_B is the value of the octanol / water partition coefficient of ligand (B) in equation (1), and LogP_C is the value of the octanol / water partition coefficient of ligand (C) in equation (1).] (Composition 13) A method for producing dye-containing resin particles having 10 to 12 components, wherein the core portion comprises a resin having a polymer of hydrophobic monomers including styrene, and the shell portion comprises a resin having a polymer of hydrophilic monomers having side chains with hydrogen bonding sites. (Composition 14) The polymer of the hydrophilic monomer is given by formulas (12) and (13) [ka] (X2 and X3 are H or CH3, Y1 is OH or OCH3, and Y2 is the following formula (14) [ka] A method for producing 10 to 13 pigment-containing resin particles having at least one of the following structures: a group represented by or CH2CH2OH, where m3 and m4 are integers of 1 or more, and n is an integer between 1 and 40. (Composition 15) The process further includes a step for producing the aforementioned resin particles, A method for producing dye-containing resin particles having 10 to 14 components, comprising a sub-step 1 of polymerizing a hydrophobic monomer containing styrene to form a core, and a sub-step 2 of polymerizing a hydrophilic monomer having side chains with hydrogen bonding sites to form a shell. (Composition 16) A method for producing pigment-containing resin particles having a composition of 15, wherein the hydrogen bonding site is a group selected from an amino group, a carbonyl group, a carboxyl group, a hydroxyl group, and a thiol group. (Composition 17) A method for producing dye-containing resin particles of configuration 15 or 16, wherein sub-step 1 is a step of polymerizing the hydrophobic monomer with divinylbenzene as a crosslinking agent, and sub-step 2 is a step of polymerizing the hydrophilic monomer with trimethylolpropane trimethacrylate as a crosslinking agent. (Composition 18) A method for producing dye-containing resin particles having a configuration of 17, wherein the sub-step 1 is a sub-step in which divinylbenzene is included in the hydrophobic monomer at a content of 5% by weight or more and 30% by weight or less, and the sub-step 2 is a sub-step in which trimethylolpropane trimethacrylate is included in the hydrophilic monomer at a content of 5% by weight or more and 30% by weight or less. (Composition 19) A method for producing particles for specimen testing, comprising the 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 of composition 10 to 18. (Composition 20) Particles for specimen testing, comprising pigment-containing resin particles with components 1 to 9, each having a site that specifically reacts with a target substance. (Composition 21) A set of 20 particles for sample testing, used for detecting target substances by depolarization fluorescence. (Composition 22) A sample testing reagent comprising 20 or 21 particles for sample testing. (Composition 23) A test kit for target substances, containing 22 sample test reagents. (Composition 24) A method for detecting a target substance, which uses fluorescence depolarization to detect the presence or amount of the target substance, A step of obtaining a mixed solution by mixing a sample solution that may contain a target substance with the sample testing particles described in configuration 20 or 21, A method for detecting a target substance, comprising the step of irradiating the mixture with light to obtain a value relating to the fluorescence polarization of the mixture. (Composition 25) A method for detecting a target substance comprising 24 components, wherein the sample solution contains an aqueous solvent. [Explanation of symbols]

[0105] 1. Core 2 Shell section 3 Europium complex 20 Hydrophilic polymers

Claims

1. A dye-containing resin particle having a core portion and a shell portion, wherein the core portion and the shell portion are each crosslinked, and the LogP_Eu, which is the statistical value of the octanol / water partition coefficient of the ligand of the europium complex, is given by the following formula (51) LogP_Eu ≧ 5.80 (51) and the following formula (52) LogP_Eu > LogP_Core > LogP_Shell (52) A dye-containing resin particle characterized by satisfying the following: [However, in formula (52), LogP_Core is the value of the octanol / water partition coefficient of the main component among the monomers forming the core, and LogP_Shell is the value of the octanol / water partition coefficient of the main component among the monomers forming the shell.]

2. The dye-containing resin particles according to claim 1, wherein LogP_Eu, which is the statistical value of the octanol / water partition coefficient of the ligands of the europium complex, is the average value obtained by weighting the octanol / water partition coefficient values ​​of each ligand by the coordination number of each ligand.

3. The dye-containing resin particles according to claim 1, wherein at least one of the ligands of the europium complex contains an alkyl group.

4. The europium complex is given 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 a group selected from an alkyl group, a thienoyl phenyl group and a thienyl group, which may optionally have a substituent. R ３ is a hydrogen atom or a methyl group. R ４ and R ５ are each independently a group selected from an alkyl group and a phenyl group, which may optionally have a substituent. Also, R ６ is a group selected from an alkyl group, a phenyl group and a triphenylene group, which may optionally have a substituent. Also, R ７ and R ８ are each independently a group selected from an alkyl group and a phenyl group, which may optionally have a substituent. Here, the above-mentioned substituents that may be optionally selected in R １ , R ２ , R ４ to R ６ are each independently a group selected from a methyl group, a fluoro group, a chloro group and a bromo group, and the above-mentioned alkyl groups in R １ , R ２ , R ４ to R ６ each independently have 2 to 12 carbon atoms and may be different groups or the same group.) And x, y and z in formula (1) are the following formulas (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) The following satisfies the condition. The LogP_Eu, which is the statistical value of the octanol / water partition coefficient of the ligand of the europium complex, is given by the following formula (50): LogP_Eu=(LogP_B×y+LogP_C×z) / (y+z) (50) [However, in formula (50), LogP_B is the value of the octanol / water partition coefficient of ligand (B) in formula (1), and LogP_C is the value of the octanol / water partition coefficient of ligand (C) in formula (1).] The dye-containing resin particles according to claim 1, satisfying this condition.

5. The dye-containing resin particle according to claim 1, wherein the core portion comprises a resin having a polymer of hydrophobic monomers including styrene, and the shell portion comprises a resin having a polymer of hydrophilic monomers having side chains having hydrogen bonding sites.

6. The dye-containing resin particles according to claim 5, wherein the hydrogen bonding site is a group selected from an amino group, a carbonyl group, a carboxyl group, a hydroxyl group, and a thiol group.

7. The polymer of the hydrophilic monomer is given by formulas (12) and (13) 【Chemistry 2】 (In equations (12) and (13), X ２ and X ３ H and CH ３ Each is independently selected from the above, and Y1 is either OH or OCH ３ And Y2 is given by the following equation (14) 【Transformation 3】 The group represented by or CH ２ CH ２ The dye-containing resin particle according to claim 5, having at least one of the following structures: OH, where m3 and m4 are integers of 1 or more, and n is an integer between 1 and 40.

8. The dye-containing resin particles according to claim 5, wherein the degree of crosslinking of the polymer of hydrophobic monomers containing styrene contained in the core portion is 5% by weight or more and 30% by weight or less.

9. The dye-containing resin particles according to claim 5, wherein the degree of crosslinking of the polymer of a hydrophilic monomer having side chains with hydrogen bonding sites contained in the shell portion is 5% by weight or more and 30% by weight or less.

10. A method for producing dye-containing resin particles containing a europium complex, The process includes impregnating resin particles having a core and a shell with a solution containing a europium complex. The core portion and the shell portion are each cross-linked, and the LogP_Eu, which is the statistical value of the octanol / water partition coefficient of the ligand of the europium complex, is given by the following formula (51) LogP_Eu ≧ 5.80 (51) and the following formula (52) LogP_Eu > LogP_Core > LogP_Shell (52) A method for producing dye-containing resin particles, characterized in that it satisfies the following condition: [However, in formula (52), LogP_Core is the value of the octanol / water partition coefficient of the main component among the monomers forming the core, and LogP_Shell is the value of the octanol / water partition coefficient of the main component among the monomers forming the shell.]

11. A method for producing dye-containing resin particles according to claim 10, wherein at least one of the ligands of the europium complex contains an alkyl group.

12. The europium complex is given 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 4】 (In equations (2), (3), (41), (42), (43), and (5), R １ and R ２ Each of these groups is independently selected from an alkyl group, a thienolphenyl group, and a thienyl group, which may optionally have substituents. ３ R is a hydrogen atom or a methyl group. ４ and R ５ These are groups independently selected from alkyl groups and phenyl groups, which may optionally have substituents. ６ R is a group selected from alkyl groups, phenyl groups, and triphenylene groups, which may optionally have substituents. ７ and R ８ Each of these groups is independently selected from alkyl groups or phenyl groups, which may optionally have substituents. Here, R １ , R ２ , R ４ ~R ６ The substituents that may be optionally present in are each independently selected from a methyl group, a fluoro group, a chloro group, and a bromo group, R １ , R ２ , R ４ ~R ６ The alkyl groups in each of the above formulas each independently have 2 to 12 carbon atoms, and each may be a different group or the same group.) and x, y, and z in formula (1) are the same as those in formulas (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) The following satisfies the condition. The LogP_Eu, which is the statistical value of the octanol / water partition coefficient of the ligand of the europium complex, is given by the following formula (50): LogP_Eu=(LogP_B×y+LogP_C×z) / (y+z) (50) A method for producing dye-containing resin particles according to claim 10, satisfying the following: [In formula (50), LogP_B is the value of the octanol / water partition coefficient of ligand (B) in formula (1), and LogP_C is the value of the octanol / water partition coefficient of ligand (C) in formula (1).]

13. The method for producing dye-containing resin particles according to claim 10, wherein the core portion comprises a resin having a polymer of hydrophobic monomers including styrene, and the shell portion comprises a resin having a polymer of hydrophilic monomers having side chains having hydrogen bonding sites.

14. The polymer of the hydrophilic monomer is given by formulas (12) and (13) 【Transformation 5】 (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 6】 The group represented by or CH ２ CH ２ A method for producing dye-containing resin particles according to claim 13, wherein the structure is OH, m3 and m4 are integers of 1 or more, and n is an integer of 1 to 40.

15. The process further includes a step for producing the aforementioned resin particles, A method for producing dye-containing resin particles according to claim 10, wherein the step of producing the resin particles comprises a sub-step 1 of polymerizing a hydrophobic monomer containing styrene to form a core portion, and a sub-step 2 of polymerizing a hydrophilic monomer having a side chain having a hydrogen bonding site to form a shell portion.

16. The method for producing dye-containing resin particles according to claim 15, wherein the hydrogen bonding site is a group selected from an amino group, a carbonyl group, a carboxyl group, a hydroxyl group, and a thiol group.

17. The method for producing dye-containing resin particles according to claim 15, wherein the sub-step 1 is a step of polymerizing the hydrophobic monomer with divinylbenzene as a crosslinking agent, and the sub-step 2 is a step of polymerizing the hydrophilic monomer with trimethylolpropane trimethacrylate as a crosslinking agent.

18. The method for producing dye-containing resin particles according to claim 17, wherein the sub-step 1 is a sub-step in which the divinylbenzene is included in the hydrophobic monomer in a content of 5% by weight or more and 30% by weight or less, and the sub-step 2 is a sub-step in which the trimethylolpropane trimethacrylate is included in the hydrophilic monomer in a content of 5% by weight or more and 30% by weight or less.

19. A method for producing particles for specimen testing, comprising the step of forming a site that specifically reacts with a target substance in dye-containing resin particles obtained by the method for producing dye-containing resin particles according to any one of claims 10 to 18.

20. Pigment-containing resin particles according to any one of claims 1 to 9, wherein the particles have a site that specifically reacts with a target substance, for use in specimen testing.

21. Particles for sample testing according to claim 20, used for detecting a target substance by fluorescence polarization depolarization.

22. A specimen testing reagent comprising the specimen testing particles described in claim 20.

23. A test kit for a target substance comprising the sample test reagent described in claim 22.

24. A method for detecting a target substance, which uses fluorescence depolarization to detect the presence or amount of the target substance, A step of obtaining a mixed solution by mixing a sample solution that may contain a target substance with the sample testing particles described in claim 20, A method for detecting a target substance, comprising the step of irradiating the mixture with light to obtain a value relating to the fluorescence polarization of the mixture.

25. The method for detecting a target substance according to claim 24, wherein the sample solution contains an aqueous solvent.

Images