Pigment-containing resin particles, particles for specimen testing, specimen testing reagent, target substance testing kit, method of producing pigment-containing resin particles, method of producing particles for specimen testing, and target substance detection method

Abstract

Provided are pigment-containing resin particles that make it possible to detect biological components with a high degree of sensitivity. The pigment-containing resin particles each have a core part and a shell part, and each of the core part and the shell part is crosslinked and contains a europium complex. A statistical value LogP_Eu of the LogP of the plurality of ligands in the europium complex satisfies relationship (51) and relationship (52), below. (51) LogP_Eu ≥ 5.80 (52) LogP_Eu > LogP_Core > LogP_Shell

Description

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

[0001] This disclosure relates to dye-containing resin particles, particles for specimen testing, specimen testing reagents, test kits for target substances, methods for producing dye-containing resin particles, methods for producing specimen testing particles, and methods for detecting target substances.

[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.

[0004] Japanese Patent Publication No. 2022-187791

[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.

[0006] This disclosure provides, as one embodiment, a dye-containing resin particle having a core portion and a shell portion, wherein the dye-containing resin particle contains a europium complex, and 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 (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) [wherein 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 portion, and LogP_Shell is the value of the octanol / water partition coefficient (LogP) of the main component among the monomers forming the shell portion. The present invention provides dye-containing resin particles that satisfy the following conditions, thereby solving the above problem.

[0007] This disclosure provides dye-containing resin particles in which the decrease in luminescence intensity of the europium complex is suppressed. Furthermore, by using these dye-containing resin particles, highly sensitive detection of target substances becomes possible.

[0008] This is a schematic diagram illustrating the structure of dye-containing resin particles according to the first embodiment of this disclosure. This is a schematic diagram illustrating the structure of conventional dye-containing resin particles.

[0009] Below, an example of dye-containing resin particles of the first embodiment of this disclosure will be described with reference to Figure 1. As shown in Figure 1, the dye-containing resin particles of the first embodiment of this disclosure 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 is 0.1 or less, it indicates that the particle size distribution is monodisperse.

[0012] (Fluorescence Polarization De-polarization Method) By encapsulating a europium complex that exhibits polarized emission as a luminescent dye within resin particles, it is possible to detect changes in polarized emission characteristics 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 fluorescence anisotropy.

[0013] Fluorescence anisotropy means that there is anisotropy in the transition moment (transition dipole moment). Polarized emission generally means that in the case of a luminescent dye 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 fluorescence anisotropy. It should be noted that in this disclosure, fluorescence anisotropy may also be referred to as 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) (where Q: rotational relaxation time of the material, V: volume of the material, η: viscosity of the solvent, k: Boltzmann constant, T: absolute temperature. Note that the rotational relaxation time Q of the material is the time required for the molecules 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) (where, p0: degree of polarization when the material is stationary (Q = ∞) p: degree of polarization A: constant τ: luminescence lifetime of the material Q: rotational relaxation time.)

[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 of the luminescent material and the rotational relaxation time, 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 experimentally determine the degree of polarization of the emission shown in equation (22), 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 the equation (23) below. r(t) = (I∥(t) - GI⊥(t)) / (I∥(t) + 2GI⊥(t)) (23) (where, r(t): fluorescence 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. Note that the correction value is the ratio of I⊥ / I∥ measured with excitation light whose vibration direction is 90 degrees different from the excitation light 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 particle size of luminescent materials due to antigen-antibody reactions, etc., as values ​​of fluorescence anisotropy. Fluorescence anisotropy is the value of polarization corrected for G and 2G, and the polarization value is obtained by removing G and 2G from equation (23). In actual fluorescence anisotropy measurements, a correction value for G is necessary, but if only the relative polarization value 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] (Polydispersion index) In fluorescence depolarization, high detection sensitivity is obtained by detecting changes in the motion of dye-containing resin particles. Generally, smaller particles rotate faster, and larger particles rotate slower. For example, in antigen-antibody reactions, such as particle (antibody)-antigen-particle (antibody) aggregation of particles results in larger aggregated particles, and the slower rotational motion of these aggregated particles is detected. Therefore, if small and large particles are mixed in the initial stages before the reaction in the system, the detection sensitivity may decrease. For this reason, it is necessary for the dye-containing resin particles to have a uniform particle size distribution, and specifically, it is preferable that the polydispersion index mentioned above is 0.1 or less.

[0020] <First Embodiment> (Resin Particle Structure, Core-Shell Structure) The dye-containing resin particles of this embodiment have a core and a shell. First, the resin material of the core is preferably low in specific gravity. Dye-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. If the specific gravity of the particles is high, the dye-containing resin particles will settle in the container, requiring stirring and redispersion before use, which can make the operation complicated. Therefore, in order to suppress the settling of dye-containing resin particles, the core portion, which occupies the majority of the volume of one dye-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-to-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, as the dye to be contained, an europium complex showing fluorescence anisotropy is used because the wavelength and intensity of the emission are hardly affected by the surroundings and the emission has a long lifetime. The europium complex is composed of an europium element and ligands, and its structure is represented by the following formula (P): Eu(S)a(T)b(U)c (P) [However, in formula (P), Eu is europium, (S), (T), and (U) are ligands, and a, b, and c represent the number of each ligand coordinated.]. Considering the emission lifetime, visible emission wavelength region, etc., the use of the europium complex is essential. Europium generally has an emission lifetime of 0.1 to 1.0 ms. It is necessary to appropriately adjust this emission lifetime and the rotational relaxation time obtained from formula (21). In the case of europium in the aqueous dispersion, a particle size with a diameter of 80 nm or more and 200 nm or less is preferable because the fluorescence anisotropy represented by formula (23) changes greatly before and after the antigen-antibody reaction.

[0024] Among the ligands constituting the europium complex, at least one is a ligand having a light-harvesting function. The light-harvesting function is the action of exciting at a specific wavelength and exciting the central metal of the complex by energy transfer. Further, it is preferable that ligands such as β-diketone are present in the ligands constituting the europium complex to prevent the coordination of water molecules. Ligands such as β-diketone coordinated to rare earth ions suppress the deactivation process due to the transfer of energy to solvent molecules, etc., and strong fluorescence emission can be obtained.

[0025] The europium complex may be a polynuclear complex as long as it shows fluorescence anisotropy. The fluorescence anisotropy of the europium complex is represented by formula (23). When it can be considered that the Brownian rotational motion of the europium complex in the medium has stopped, it is desirable that the fluorescence anisotropy is 0.08 or more. The state where the Brownian rotational motion can be considered to have stopped indicates a state where the rotational relaxation time of the particles is sufficiently longer than the emission lifetime of the europium complex.

[0026] It is preferable that the europium complex be incorporated more into the core part of the resin particles having a core-shell structure, because the luminescence intensity per particle becomes stronger. Specifically, the content of the europium complex contained per 1 g of the dye-containing resin particles is preferably 0.001 g or more. The content of the europium complex can be calculated by quantifying europium by high-frequency inductively coupled plasma (ICP) emission analysis.

[0027] On the other hand, if the europium complex aggregates within the core part, it affects the excitation efficiency of the europium complex due to the interaction between the ligands, etc., making it difficult to measure fluorescence anisotropy with reproducibility. Whether the europium complex 3 shows a non-aggregated luminescence behavior within the core part can be determined by taking the excitation spectrum of the sample.

[0028] Luminescent particles with strong luminescence intensity not only enable simple highly sensitive measurement, but also maintain strong luminescence even when the particle size is reduced, making it possible to increase the biochemical reaction rate. Since the diffusion coefficient of Brownian motion in the liquid becomes larger when the particle size is smaller, it becomes possible to detect the reaction in a shorter time.

[0029] (Method for calculating LogP of ligand) 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 the present embodiment, is calculated in the following two steps. (1) First, the structural formula of each ligand is described in the "SMILES notation", which is one of the "linear notation methods" for expressing the structural formula as a one-line character string. (2) Next, the LogP of each ligand is calculated by describing the character string in the SMILES notation in the program "ALOGPS2.1" provided by the Virtual Computational Chemistry Laboratory VCCLAB, and the average weighted by each coordination number is taken (https: / / vcclab.org / lab / ).

[0030] And in the dye-containing resin particles of the present embodiment, the calculated LogP_Eu 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 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). (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 are each independently a group selected from an alkyl group or a phenyl group, which may optionally have a substituent. Here, the above-mentioned substituents that R １ , R ２ , R ４ to 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, and R １ , R ２ , R ４ to R６ The alkyl groups in each of the above formulas each independently have 2 to 12 carbon atoms, and each group may be different or the same.) where x, y, and z satisfy 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 statistical value LogP_Eu of the ligands in the europium complex is expressed as follows: LogP_Eu = (LogP_B × y + LogP_C × z) / (y + z) (50) [wherein 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).]

[0032] A concrete example is the europium complex Eu(TTA) represented by the following formula (A1). ３ (TOPO) ２ In the aforementioned formula (1), the structure corresponding to (B) is TOPO [TTA: 2-thienoyltrifluoroacetone, TOPO: trioctylphosphine oxide]. The SMILES notation for TOPO is "CCCCCCCCCP(=O)(CCCCCCCCCC)CCCCCCCCCC". Calculating LogP from this SMILES notation using the program "ALOGPS2.1" yields "8.44". Therefore, substituting the LogP corresponding to ligand (B) (LogP_B in equation (50)) and the coordination number y=2 into the aforementioned equation (50), we can calculate LogP_Eu as follows: LogP_Eu = ((Log_B) × y) / (y) = (8.44 × 2) / (2) = 8.44.

[0033] Another example is the europium complex Eu(TTA) represented by the following formula (A2). ３In (TPPO)(DBSO), 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]. 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, substituting LogP corresponding to ligand (B) and ligand (C) and their respective coordination numbers y = z = 1 into the above equation (50), we can calculate that LogP_Eu = ((Log_B) × y + (Log_C) × z) / (y + z) = (4.04 × 1 + 2.28 × 1) / (1 + 1) = 3.16.

[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 becomes less likely to diffuse into water and remains inside the particles, which is thought to suppress 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) 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 fluorescence polarization depolarization. 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 the present 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, if the following equations (25) and (26) are satisfied: (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 the aforementioned pyridine treatment, and Dn_Before (unit: [nm]) is the number-average particle size, and Dv_After (unit: [nm]) is the volume-average particle size obtained after the aforementioned pyridine treatment, and Dn_After (unit: [nm]) is the number-average particle size.) then it can be determined that "the core is cross-linked".

[0043] (Shell portion) 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 that forms the shell portion. The hydrogen bonding site is a substituent that can form hydrogen bonds with water molecules in the system, and specifically refers to an amino group, a carbonyl group, a carboxyl group, a hydroxyl group, or a 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, making it possible to improve detection sensitivity.

[0044] Furthermore, the shell material is given by the following formulas (12) and (13) (X２ , X ３ is H or CH ３ Therefore, Y1 is either OH or OCH. ３ And Y2 is given by the following equation (14) Or, CH ２ CH ２ A resin containing at least one of the following structures is preferred: OH, 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 on the outside of the core portion. (n=1 or more and 40 or less) (n=1 or more and 40 or less)

[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, crosslinking the shell portion prevents the hydrogen bonding sites located in the side chains of the shell 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 nearby particles (or samples). On the other hand, in this case, crosslinking the shell component prevents the shell material from spreading away from the dye-containing resin particles, making interaction with other nearby particles (or samples) less likely. As a result, particle-to-particle adsorption is suppressed, non-specific adsorption is reduced, 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, if the following equations (25) and (26) are satisfied, then it can be determined that "the shell portion is cross-linked". (25) (Dn_After) / (Dn_Before) < 1.5 (26) (In equations (25) and (26), Dv_Before (unit: [nm]) is the volume-average particle size measured by the dynamic light scattering method before the pyridine treatment mentioned 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 mentioned above, Dn_After (unit: [nm]) is the number-average particle size.)

[0053] (Relationship between LogP values ​​of Europium complex, core, and shell) In addition, 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, it is necessary that the following equation (52) LogP_Eu > LogP_Core > LogP_Shell (52) [In equation (52), LogP_Core is the LogP of the main component monomer of the core, and LogP_Shell is the LogP of the main component monomer of the shell.] is satisfied.

[0054] Specifically, the europium complex is Eu(TTA) shown in formula (A2) above. ３ (TOPO) ２ When using styrene monomer as the main monomer component of the core and hydroxyethyl methacrylate shown in formula (31) above 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, by satisfying formula (52), the europium complex becomes less likely 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 part, and the octanol / water partition coefficient (LogP_Shell) calculated for the main component monomer of the shell part. 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 of the core and shell parts, 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 initiators) When synthesizing resin particles, a wide range of radical polymerization initiators can be used, selected from azo compounds, organic peroxides, etc. 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. In addition, surfactants, preservatives, sensitizers, etc. may be added to the dispersion medium to increase the stability of the dispersion of resin particles.

[0058] (Method for encapsulating europium complex) There are two methods for encapsulating europium complex within resin particles: a "synthesis method" in which polymerization is performed with the europium complex added to a synthesis system containing raw material monomers 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 containing raw material monomers (without the addition of europium complex), and then the resin particles are immersed in a europium complex solution to impregnate them with the europium complex. In the former case, sub-step 1 is a step in which a first mixture containing a hydrophobic monomer and a crosslinking agent is polymerized to form a core, and sub-step 2 is a step in which a second mixture containing part A, a hydrophilic monomer, a crosslinking agent, and a europium complex is polymerized to form a shell. In the latter case, the method includes the steps of polymerizing a mixture containing a europium complex, a hydrophobic monomer, and a first crosslinking agent to form a core, and polymerizing a second mixture containing the core, a hydrophilic monomer, and a second crosslinking agent to form a shell. In either manufacturing method, LogP_Eu, which is the statistical value of the octanol / water partition coefficient of the ligand of the europium complex, satisfies the following formula (51): LogP_Eu ≥ 5.80 (51) The hydrophobic monomer, hydrophilic monomer, and europium complex satisfy 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 of the hydrophobic monomer that forms the core, and LogP_Shell is the value of the octanol / water partition coefficient of the main component of the hydrophilic monomer that forms the shell.] Also, the first crosslinking agent and the second crosslinking agent may be the same or different.

[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 of this disclosure can be used as particles for specimen testing (affinity particles) according to the second embodiment of this disclosure by providing a site that specifically binds to a target substance. In this case, the resin particles have a shell portion which contains at least one reactive functional group selected from the group consisting of a carboxyl group, an amino group, a thiol group, an epoxy group, a maleimide group, and a succinimidyl group, and the site that specifically binds to the target substance is attached 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 binds to 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 (which can also be described as capturing or reacting with a particular target substance) to the dye-containing resin particle. The site of ligand that specifically binds to 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 sample testing particles of this embodiment, a conventionally known method can be applied to the extent that the objectives of this disclosure can be achieved, for bonding the reactive functional group of the dye-containing resin particles of the first embodiment of this disclosure to a ligand. When amide bonding the ligand, a catalyst such as 1-[3-(dimethylaminopropyl)-3-ethylcarbodiimide] can be used as appropriate. The sample testing 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 particles for specimen testing of this embodiment have an antibody (antigen) as a site that specifically binds to a 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> (Test Reagent for In Vitro Diagnostic Use) The test particles of the second embodiment of this disclosure can be used in a test reagent for detecting a target substance in a sample solution by in vitro diagnostics, which is the third embodiment of this disclosure. Such an in vitro diagnostic test reagent comprises at least the test particles of the second embodiment of this disclosure and a dispersion medium for dispersing the test particles. The amount of test particles contained in the test reagent is preferably 0.000001% by mass to 20% by mass, and more preferably 0.0001% by mass to 1% by mass. Such a test reagent may contain substances other than the test particles, such as solvents and blocking agents, to the extent that the objectives of this disclosure 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 to detect an antigen or antibody in a sample solution, an antibody or antigen can be used as a ligand.

[0067] <Fourth Embodiment> (Test Kit) The specimen test reagent of the third embodiment of this disclosure can be used in a test kit for detecting a target substance in a specimen solution for 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, a serum diluent, etc. As media for the positive control and negative control, 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 a kit used for detecting a target substance in a specimen solution for normal 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 solution by immunolatex agglutination or fluorescence depolarization.

[0068] <Fifth Embodiment> (Detection Method) The fifth embodiment of the present disclosure, a method for detecting a target substance in a sample solution by in vitro diagnostics using the sample testing particles of the second embodiment of the present disclosure, can be a detection method comprising the steps of: mixing a sample solution that may contain a target substance with the sample testing particles of the second embodiment of the present disclosure to obtain a mixed solution; and irradiating the mixed solution with light to obtain a value relating to the fluorescence anisotropy of the mixed solution. That is, by optically detecting the agglutination reaction that occurs in the mixed solution, the target substance in the sample solution can be detected (this may be detection of the presence or absence of the target substance, detection of the amount of the target substance, or detection of the presence or absence and amount of the target substance), 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 fluorescence 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 solution, 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.

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

[0071] <Production of dye-containing resin particles> [Example 1] (1) Preparation of resin particles 100 g of 2-morpholinoethanesulfonic acid buffer (hereinafter referred to as "MES buffer") (manufactured by Kishida Chemical Co., Ltd.) at pH 7, 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 placed in a four-necked flask equipped with a mechanical stirrer. 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. 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 100 K 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 1.00 g of N-methyl-2-pyrrolidone (manufactured by Kishida Chemical Co., Ltd.) and tris(2-cenoyltrifluoroacetonato)bis(trioctylphosphine oxide)europium(III) (hereinafter referred to as "Eu(TTA)"), which is a polarizing luminescent europium complex. ３ (TOPO) ２0.04 g of (composed of Central Techno Co., Ltd., LogP = 8.44) was mixed with the resin particles obtained in (1) above and the europium complex solution prepared here. The suspension obtained by shaking and stirring at 55°C for 3 hours was centrifuged to purify the suspended particles, and these were then 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 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 used 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 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 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 used 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] As the europium complex, Eu(TTA) used in Example 1 ３ (TOPO) ２ Instead of (E420, LogP=8.44), use tris(2-cenoyltrifluoroacetonato)(bis[2-(diphenylphosphino)phenyl]ether oxide) europium(III) (hereinafter referred to as "Eu(TTA)"). ３ Dye-containing resin particles 8 were prepared in the same manner as in Example 1, except that (DPEPO) or "E450" was used (manufactured by Central Techno Co., Ltd., LogP = 6.05).

[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, a synthesis method was used instead of the immersion method, and the dye-containing resin particles 10 were produced in the same manner 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, add 100 g of MES buffer solution at pH 7 (manufactured by Kishida Chemical Co., Ltd.) and Eu(TTA), a polarizing luminescent europium complex. ３ (TOPO) ２0.04 g of (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 to a four-necked flask. The mixture was stirred at room temperature for 15 minutes under nitrogen flow conditions with a mechanical stirrer set to 300 rpm. The flask was then 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 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. 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 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] As the europium complex, Eu(TTA) used in Example 1 ３ (TOPO) ２ Instead of (E420), use tris(2-cenoyltrifluoroacetonato)(1,10-phenanthroline)europium(III) (hereinafter referred to as "Eu(TTA)"). ３ Dye-containing resin particles 15 were prepared in the same manner as in Example 1, except that dye (Phen) or "E460" (manufactured by Central Techno Co., Ltd., LogP = 2.31) was used.

[0091] [Comparative Example 6] As the europium complex, Eu(TTA) used in Example 1 ３ (TOPO) ２ Instead of (E420, LogP=8.44), use tris(2-cenoyltrifluoroacetonato)bis(triphenylphosphine oxide)europium(III) (hereinafter referred to as "Eu(TTA)"). ３ (TPPO) ２ Dye-containing resin particles 16 were prepared in the same manner as in Example 1, except that dye (manufactured by Central Techno Co., Ltd., LogP = 4.04) was used.

[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.

[0093] <Preparation of particles for sample 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 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 this carboxylic acid conferred 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 dispersion after the reaction was washed with pH 5.0 MES buffer, and 100 μg / mL of anti-CRP antibody was added to it 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 was 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 was added using a BCA assay.

[0094] <Evaluation of dye-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 Example 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 by this measurement.

[0096] (Concentration of dye-containing resin particles) 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 at 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, and a total of 21 measurements were taken 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 retention rate Lum% was calculated by substituting the luminescence intensity into the following equation (60): Lum% = (Lum_Fin) / (Lum_Ini) (60) For the luminescence intensity retention rate Lum%, a value of 0.80 or higher was designated as A, 0.70 or higher and less than 0.80 as B, and less than 0.70 as C. An evaluation result of A or B was considered good, and C was considered unacceptable. Furthermore, for the initial luminescence intensity Lum_Ini, a value of 6000 or higher was designated as A, 5000 or higher and less than 6000 as B, and less than 5000 as C. An evaluation result of A or B was considered good, and C was considered unacceptable.

[0099] (Particle size at 40°C) The dispersion of the prepared dye-containing resin particles was diluted with pure water to 1 mg / mL, and 8 mL 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, greater than 7 nm and less than or equal to 10 nm as B, and greater than 10 nm as C. Evaluation results of A or B were considered good, and C was considered unacceptable.

[0100]

[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, having the core and shell parts cross-linked, 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 fluorescence anisotropy of the obtained particles for specimen testing was measured before and after mixing with CRP (antigen). The concentration of the particles for specimen testing was fixed at 0.001 mg / mL, and the CRP concentration was examined from 0 to 10000 pg / mL. The evaluation was performed using a microplate reader (Nivo, PerkinElmer). A filter with a central wavelength of 355 nm and a full width at half maximum of 40 nm was used for the excitation light, a filter with a central 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 fluorescence anisotropy r was measured for 30 minutes from the start of the reaction, and the change in fluorescence anisotropy r Δr during that time was calculated. The measurement temperature was fixed at 37°C. The evaluation results are shown in Table 3.

[0103] As shown in Table 3, the change in fluorescence anisotropy (Δr) before and after mixing with CRP was confirmed to change depending on the CRP concentration, demonstrating highly sensitive detection of CRP. Furthermore, the fact that a change in fluorescence anisotropy could be detected even at a low concentration of 0.001 mg / ml for the sample testing particles confirmed that the sample testing particles exhibited strong luminescence.

[0104] This disclosure is not limited to the embodiments described above, and various modifications and alterations are possible without departing from the spirit and scope of this disclosure. Accordingly, the following claims are attached to make the scope of this disclosure public.

[0105] This application claims priority based on Japanese Patent Application No. 2024-223113, filed on 18 December 2024, and all of its contents are incorporated herein by reference.

[0106] 1. Core 2. Shell 3. Europium complex 20. Hydrophilic polymer

Claims

1. Dye-containing resin particles having a core portion and a shell portion, and containing a europium complex, 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, satisfies the following formulas (51) LogP_Eu ≥ 5.80 (51) and (52) LogP_Eu > LogP_Core > LogP_Shell (52) [wherein 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.].

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 or 2, wherein at least one ligand of the europium complex contains an alkyl group.

4. The europium complex is represented by the following formula (1): Eu(A)x(B)y(C)z (1) [wherein, 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). (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. Further, R ６ is a group selected from an alkyl group, a phenyl group and a triphenylene group, which may optionally have a substituent. Further, 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 which 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 R １ 、R ２ 、R ４ to R ６ The alkyl groups in each of the above formulas independently have 2 to 12 carbon atoms, and each group may be different or the same. In formula (1), x, y, and z satisfy 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 dye-containing resin particles according to any one of claims 1 to 3, wherein LogP_Eu, which is the statistical value of the octanol / water partition coefficient of the ligand in the europium complex, satisfies the following formula (50): LogP_Eu = (LogP_B × y + LogP_C × z) / (y + z) (50) [wherein 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).] 5. The dye-containing resin particle according to any one of claims 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.

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 of formula (12) and formula (13) (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) The group represented by or CH ２ CH ２ The dye-containing resin particle according to claim 5 or 6, having at least one of the structures of (where m3 and m4 are integers of 1 or more, and n is an integer of 1 or more and 40 or less).

8. The dye-containing resin particles according to any one of claims 5 to 7, 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 any one of claims 5 to 8, 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, comprising the steps of: producing resin particles having a core portion and a shell portion; impregnating the resin particles having the core portion and the shell portion with a solution containing a europium complex, wherein the step of producing the resin particles comprises a sub-step 1 of polymerizing a first mixture containing a hydrophobic monomer and a crosslinking agent to form a core portion; and a sub-step 2 of polymerizing the core portion with a second mixture containing a hydrophilic monomer, a crosslinking agent and a europium complex to form the shell portion, wherein LogP_Eu, which is the statistical value of the octanol / water partition coefficient of the ligand of the europium complex, satisfies the following formula (51): LogP_Eu ≥ 5.80 (51) The first monomer, the second monomer, and the europium complex satisfy the following formula (52), characterized in that the method for producing dye-containing resin particles. 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 of the hydrophobic monomers forming the core, and LogP_Shell is the value of the octanol / water partition coefficient of the main component of the hydrophilic monomers forming the shell.] 11. A method for producing dye-containing resin particles containing a europium complex, comprising the steps of: polymerizing a mixture containing a europium complex, a hydrophobic monomer, and a first crosslinking agent to form a core; polymerizing the core, a second mixture containing a hydrophilic monomer, and a second crosslinking agent to form a shell, wherein LogP_Eu, which is the statistical value of the octanol / water partition coefficient of the ligand of the europium complex, satisfies the following formula (51): LogP_Eu ≥ 5.80 (51) The hydrophobic monomer, the hydrophilic monomer, and the europium complex satisfy the following formula (52), characterized in that the method for producing dye-containing resin particles. 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 of the hydrophobic monomers forming the core, and LogP_Shell is the value of the octanol / water partition coefficient of the main component of the hydrophilic monomers forming the shell.] 12. A method for producing dye-containing resin particles according to claim 10 or 11, wherein at least one of the ligands of the europium complex contains an alkyl group.

13. The europium complex is defined by the following formula (1): Eu(A)x(B)y(C)z (1) [wherein Eu is europium, (A) is a ligand represented by the following formula (2), (B) is a ligand represented by any of the following formulas (3), (41), (42), or (43), and (C) is a ligand represented by the following formula (5)] (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 independently have 2 to 12 carbon atoms, and each group may be different or the same. In formula (1), x, y, and z satisfy 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). A method for producing dye-containing resin particles according to any one of claims 10 to 13, wherein LogP_Eu, which is a statistical value of the octanol / water partition coefficient of the ligand possessed by the europium complex, satisfies the following formula (50): LogP_Eu = (LogP_B × y + LogP_C × z) / (y + z) (50) [wherein 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).] 14. A method for producing dye-containing resin particles according to any one of claims 10 to 13, wherein the hydrophobic monomer comprises a styrene monomer and the hydrophilic monomer has a side chain having a hydrogen bonding site.

15. The polymer of the hydrophilic monomer is of formula (12) and formula (13) (X2 and X3 are H or CH) ３ And Y1 is OH or OCH ３ And Y2 is given by the following equation (14) The group represented by or CH ２ CH ２ A method for producing dye-containing resin particles according to claim 14, wherein the structure is OH, m3 and m4 are integers of 1 or more, and n is an integer of 1 to 40.

16. The method for producing dye-containing resin particles according to claim 14 or 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. A method for producing dye-containing resin particles according to any one of claims 10 to 16, wherein divinylbenzene is used as the first crosslinking agent in the step of forming the core portion, and trimethylolpropane trimethacrylate is used as the first crosslinking agent in the step of forming the shell portion.

18. The method for producing dye-containing resin particles according to claim 17, wherein the step of forming the core portion is a step of polymerizing the divinylbenzene in a content of 5% by weight or more and 30% by weight or less relative to the hydrophobic monomer, and the step of forming the shell portion is a step of polymerizing the trimethylolpropanetrimethacrylate in a content of 5% by weight or more and 30% by weight or less relative to the hydrophilic monomer.

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

20. Particles for specimen testing, comprising dye-containing resin particles according to any one of claims 1 to 9, wherein the particles have a site that specifically binds to a target substance.

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

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 using a fluorescence depolarization method, comprising the steps of: mixing a sample solution that may contain a target substance with the sample testing particles described in claim 20 to obtain a mixed solution; and irradiating the mixed solution with light to obtain a value relating to the fluorescence anisotropy of the mixed solution.

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

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