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 in which the deterioration of light emission intensity has been suppressed. The pigment-containing resin particles each have a core part and a shell part and contain a europium complex. The core part and the shell part are each crosslinked, the core part contains a polymer of hydrophobic monomers which includes a styrene monomer as the main component and another monomer, and the shell part contains a polymer of hydrophilic monomers. The other monomer satisfies relationship (B1) and relationship (15), below.　(B1) LogP_f ≥ 3.60 (15) (Mol_f) / (Mol_St) ≥ 0.05

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. 2022-70712

[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] In one embodiment of the present disclosure, to solve the above problems, a dye-containing resin particle is provided having a core portion and a shell portion, wherein the dye-containing resin particle contains a europium complex, the core portion and the shell portion are each crosslinked, the core portion has a resin containing a polymer of hydrophobic monomers that includes a styrene monomer as the main component and a first monomer of a different type from the styrene monomer, the shell portion has a resin containing a hydrophilic monomer, and the first monomer satisfies the following formulas (B1) and (15): LogP_f ≥ 3.60 (B1) (Mol_f) / (Mol_St) ≥ 0.05 (15) (wherein in formula (B1), LogP_f is the octanol / water partition coefficient of the first monomer, and in formula (15), Mol_f represents the number of Mol of the first monomer, and Mol_St represents the number of Mol of the styrene monomer.)

[0007] According to this disclosure, it is possible to obtain dye-containing resin particles in which the decrease in the luminescence intensity of the europium complex is suppressed. Furthermore, by using such 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 a dye-containing resin particle, which is a first embodiment of the present disclosure, will be described with reference to Figure 1. As shown in Figure 1, the dye-containing resin particle of this embodiment consists 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 particle of this embodiment and a fluorescence polarization depolarization method that can be used with the dye-containing resin particle of this embodiment 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 changing their position and moving due to Brownian motion.

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

[0012] (Fluorescence Polarization De-polarization Method) By encapsulating a europium complex exhibiting polarized emission as a coloring material within resin particles, even slight changes in the dispersion state of the resin particles in liquid can be detected as changes in polarized emission characteristics. 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 the value related to fluorescence anisotropy (such as a change in fluorescence anisotropy or a change in fluorescence polarization degree). In this disclosure, fluorescence anisotropy may also be referred to as polarization 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 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 fluorescence anisotropy.

[0014] The fluorescence depolarization method is based on the principle of measuring the shift in the transition moment due to the rotational motion of the light-emitting material during the time that polarized emission occurs. The rotational motion of the light-emitting material can be expressed by equation (21): Q = 3Vη / kT (21) (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 can be seen that in order to measure a large change in polarization degree, the relationship between the luminescence lifetime and rotational relaxation time of the luminescent material, i.e., the volume (particle size) of the luminescent material, is important, and the larger the particle size of the luminescent material, the longer the luminescence lifetime needs to be.

[0017] To 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] Furthermore, it is preferable that the luminescent particles of this disclosure have a fluorescence anisotropy <r> of 0.01 or higher, which can be determined by the following formula (24). (In formula (24), <r>: fluorescence anisotropy I) ＶＶ : The emission intensity of the emission component whose vibration direction is parallel to the first polarization when excited by the first polarization. ＶＨ : The emission intensity of the emission component whose vibration direction is perpendicular to the first polarization when excited by the first polarization. ＨＶ : When excited with a second polarization whose vibration direction is perpendicular to the first polarization, the emission intensity of the emission component whose vibration direction is perpendicular to the second polarization is I. ＨＨ : The emission intensity of the emission component whose vibration direction is parallel to the second polarization when excited with a second polarization whose vibration direction is perpendicular to the first polarization. (G: Correction value.)

[0020] (Polydispersion 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, and larger particles rotate slower. For example, aggregation of particles in the configuration of a particle (antibody) - antigen - particle (antibody) that has undergone an antigen-antibody reaction results in larger aggregated particles, and the slower rotational motion of these aggregated particles is detected. Therefore, if small or large particles are mixed in the initial stages before the reaction in the system, there is a concern that the detection sensitivity will decrease. For this reason, it is necessary for the dye-containing resin particles to have a uniform particle size distribution, and specifically, the polydispersion index must be 0.1 or less.

[0021] <First Embodiment> (Resin Particle Structure, Core-Shell Structure) The dye-containing resin particles of the first embodiment of this disclosure have their functions divided into a core portion and a shell portion. First, it is important that the resin material of the core portion has a low specific gravity. The 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. Therefore, 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 makes the operation complicated. To suppress the settling of the dye-containing resin particles, the core portion, which occupies the majority of the volume of one dye-containing resin particle, has a low specific gravity, more specifically 1.10 g / cm³. ３ It is preferable to use the following materials. Specifically, polystyrene is preferred.

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

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

[0024] (Europium Complex) In the dye-containing resin particles of this embodiment, a europium complex exhibiting fluorescence anisotropy is used as the colorant because its emission wavelength and intensity are less affected by the surroundings, and its emission has a long lifespan. The europium complex is composed of a europium element and ligands, and its structure can be represented by the following formula (P): Eu(S)a(T)b(U)c (P) [In formula (P), Eu is europium, (S), (T), and (U) are ligands, and a, b, and c indicate the number of ligands coordinating.]

[0025] 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 fluorescence anisotropy represented by equation (23) changes significantly before and after the antigen-antibody reaction.

[0026] 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.

[0027] If the europium complex exhibits fluorescence anisotropy, it may be a polynuclear complex. The fluorescence anisotropy of the europium complex is represented by Equation (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 luminescence lifetime of the europium complex.

[0028] It is preferable that the europium complex is incorporated more in 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. On the other hand, if the europium complex aggregates in the core part, it affects the excitation efficiency of the europium complex due to the interaction between the ligands, etc., and it becomes difficult to measure the fluorescence anisotropy with reproducibility. Whether the europium complex 3 shows non-aggregated luminescence behavior in the core part can be determined by taking the excitation spectrum of the sample.

[0029] Emissive particles with strong luminescence intensity not only enable simple highly sensitive measurement, but also maintain strong luminescence even when the particle size is reduced, so it becomes 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.

[0030] 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 ２is a group independently selected from an alkyl group, a thienoyl phenyl group, and a thienyl group, each of which may have a substituent. Also, R ３ is a hydrogen atom or a methyl group. Also, R ４ and R ５ are groups independently selected from an alkyl group and a phenyl group, each of which may have a substituent. Also, R ６ is a group selected from an alkyl group, a phenyl group, and a triphenylene group, each of which may have a substituent. Also, R ７ and R ８ are groups independently selected from an alkyl group or a phenyl group, each of which may have a substituent. Here, R １ , R ２ , R ４ to R ６ The substituents in are groups independently selected from a methyl group, a fluoro group, a chloro group, and a bromo group, and the alkyl groups in R １ , R ２ , R ４ to R ８ have 2 to 12 carbon atoms and may be different groups or the same group. Also, 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).)]

[0031] (Core part) The radical polymerizable monomer that is a raw material for the polymer of the core part preferably consists of a hydrophobic monomer containing a styrene-based monomer as a main component monomer. Here, the main component monomer is the raw material monomer having the largest blending amount among the raw material monomers when forming the core part (which can also be referred to as the main constituent monomer).

[0032] When styrene monomer is used alone as the monomer to form the polymer in the core, the particle size exceeds 500 nm, which may be undesirable for use as dye-containing resin particles in 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.

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

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

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

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

[0037] In order for the europium complex to not leak out of the resin particles, it is preferable that the hydrophobic monomer forming the core portion contains a styrene monomer, which is the main component, and a first monomer of a different type from the styrene monomer, and that the hydrophobic monomer is polymerized in a state where the first monomer satisfies the following formulas (B1) and (15): LogP_f ≥ 3.60 (B1) (Mol_f) / (Mol_St) ≥ 0.05 (15) (wherein in formula (B1), LogP_f is the octanol / water partition coefficient of the first monomer, and in formula (15), Mol_f is the number of moles of the first monomer, and Mol_St is the number of moles of the styrene monomer.) Furthermore, it is even more preferable that the octanol / water partition coefficient of all ligands in the europium complex is 2.0 or higher.

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

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

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

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

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

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

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

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

[0046] Furthermore, as the shell material, a resin containing at least one of the structures represented by the following formulas (12) and (13) is preferred. (In equations (12) and (13), X2 and X3 are, independently, H and CH ３ Selected from, Y1 is either OH or OCH ３ Therefore, Y2 is given by the following formula (14) or CH ２ CH ２ OH, m3 and m4 are integers greater than or equal to 1, and n is an integer between 1 and 40 (inclusive).

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

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

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

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

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

[0052] In addition, by crosslinking the shell portion, it is possible to prevent 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 target substances). On the other hand, in this case, the crosslinking of the shell component prevents the shell material from spreading away from the dye-containing resin particles. Therefore, interaction with other nearby particles (or samples) becomes less likely, suppressing particle-to-particle adsorption, reducing non-specific adsorption, and consequently improving detection sensitivity.

[0053] Trimethylolpropane trimethacrylate is particularly preferred as the crosslinking agent for the shell portion (which may also be called the second crosslinking agent). 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 localizing within it. As a result, uniform crosslinking is possible throughout the entire shell portion, which in turn improves non-specific adsorption and consequently improves detection sensitivity.

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

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

[0056] (Radical polymerization 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) Methods for integrating europium complex into resin particles (in other words, methods for producing resin particles containing europium complex) include a "synthesis method" consisting of sub-steps 1 and 2, in which a solution is obtained by adding europium complex to a synthesis system (in solution) containing hydrophobic monomers, and a core portion containing europium complex is formed by polymerization and crosslinking of the hydrophobic monomers (sub-step 1), and then a shell portion is formed around the core portion by polymerization and crosslinking of hydrophilic monomers in the presence of the core portion (sub-step 2); and an "immersion method" which includes a step of forming resin particles having a core-shell structure by polymerization in a synthesis system containing raw material monomers (without the addition of europium complex), and then immersing the resin particles in a europium complex solution to impregnate the resin particles with europium complex. Here, when producing dye-containing resin particles using a synthesis method, the process includes the steps of polymerizing and crosslinking a hydrophobic monomer in a solution containing styrene as the main component, a hydrophobic monomer containing a first monomer, and a europium complex to form a core, and polymerizing and crosslinking a hydrophilic monomer in the presence of the core to form the shell around the core, wherein the step of forming the core is the step of polymerizing and crosslinking the hydrophobic monomer in the aforementioned solution in a state in which the first monomer satisfies the following formulas (B1) and (15). Furthermore, when producing dye-containing resin particles using an immersion method, as described above, the process includes the steps of forming resin particles having a crosslinked core and a crosslinked shell, and impregnating the obtained resin particles with a europium complex, wherein in the formation of the core, a hydrophobic monomer containing styrene monomer as the main component and a first monomer of a different type from the styrene monomer is polymerized, and in this case, the first monomer is a monomer that satisfies the following formulas (B1) and (15), and in the formation of the shell, a hydrophilic monomer is polymerized. LogP_f ≥ 3.60 (B1) (Mol_f) / (Mol_St) ≥ 0.05 (15) (wherein in equation (B1), LogP_f is the octanol / water partition coefficient of the first monomer, and in equation (15), Mol_f represents the number of moles of the first monomer, and Mol_St represents the number of moles of the styrene monomer.)

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

[0060] On the other hand, the organic solvent used to dissolve the europium complex in the immersion method is preferably one that causes little change in the particle size of the resin particles during immersion, can dissolve the europium complex at a high concentration, and is miscible with water. Examples of such organic solvents include acetone, pyridine, ethanol, tetrahydrofuran, N,N-dimethylformamide, and N-methyl-2-pyrrolidone. In the method for producing dye-containing resin particles, preferred materials for the europium complex, hydrophilic monomer, resin formed by polymerizing and crosslinking the hydrophilic monomer, and crosslinking agents for the core and shell portions are as described in the description of the dye-containing resin particles.

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

[0062] Here, the site that specifically 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 (it may also be said that it captures or reacts with a particular target substance) to the dye-containing resin particle. The site where the ligand binds to the target substance is fixed and has a specificly high affinity. For example, 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 are examples of ligand-target substance combinations, but ligands are not limited to these. In other words, the particles for sample testing are particles that have a specificly high affinity for the target substance. Furthermore, the ligand in the particles for sample testing is preferably an antibody, an antigen, or a nucleic acid.

[0063] To obtain the particles for sample testing according to 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 this disclosure can be achieved. When the ligand is bonded via an amide linkage, a catalyst such as 1-[3-(dimethylaminopropyl)-3-ethylcarbodiimide] can be used as appropriate. The resin particles according to this embodiment can also have the ligand immobilized by physical adsorption.

[0064] (Applications of particles for specimen testing - fluorescence polarization depolarization method) When the particles for specimen testing of this embodiment have an antibody (antigen) as a ligand, they can be preferably applied to a fluorescence polarization depolarization method for detecting the antigen (antibody) as a target substance.

[0065] <Third Embodiment> (Test Reagent for In Vitro Diagnostic Use) The dye-containing resin particles of the first embodiment of this disclosure can be used for the detection of a target substance in a sample solution by in vitro diagnostics. The test reagent for in vitro diagnostic use, which is the third embodiment of this disclosure, comprises at least the sample testing particles of the second embodiment of this disclosure and a dispersion medium for dispersing the sample testing particles. The amount of sample 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 contain substances other than sample testing 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 for the detection of an antigen or antibody in a sample solution, an antibody or antigen can be used as a ligand.

[0066] <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 sample solution for in vitro diagnostics. Such a test kit comprises the specimen test reagent of the third embodiment of this disclosure and a housing that encloses the specimen 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. The test kit of this embodiment can be used to detect a target substance in the same manner as a kit used for detecting a target substance in a sample 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 sample solution by immunolatex agglutination or fluorescence depolarization.

[0067] <Fifth Embodiment> (Detection Method) The fifth embodiment of the present disclosure, a method for detecting a target substance in a sample 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 anisotropy of the mixed solution" may mean obtaining a measured value of the fluorescence anisotropy of the mixed solution, or it may be a measurement of the degree of fluorescence polarization. It may also be a value that can be calculated by computation.

[0068] 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.

[0069] Specifically, the in vitro diagnostic method for detecting a target substance in a sample solution using affinity particles containing dye-containing resin particles according to the first embodiment of this disclosure comprises the steps of: mixing the sample with a test reagent to obtain a mixture; irradiating the mixture with polarized light; and separating and detecting the polarized component of the emission of affinity particles in the mixture. That is, by optically detecting the agglutination reaction occurring in the mixture, the target substance in the sample solution can be detected, and furthermore, the concentration of the target substance can also be measured.

[0070] The embodiments of this disclosure will be specifically described below with reference to examples. However, this disclosure is not limited to these embodiments.

[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") at pH 7 (manufactured by Kishida Chemical Co., Ltd.), 1.00 g of styrene monomer (manufactured by Kishida Chemical Co., Ltd.), 0.10 g of sodium styrenesulfonate (styrenesulfonic acid monomer) (manufactured by Tokyo Chemical Industry Co., Ltd.), 0.13 g of divinylbenzene (hereinafter referred to as "DVB") (manufactured by Tokyo Chemical Industry Co., Ltd.), and 0.012 g of dodecyl methacrylate (manufactured by Tokyo Chemical Industry Co., Ltd.) were 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 1.00 g of N-methyl-2-pyrrolidone (manufactured by Kishida Chemical Co., Ltd.) and tris(2-cenoyltrifluoroacetonato)bis(triphenylphosphine oxide)europium(III) (hereinafter referred to as "Eu(TTA)"), which is a polarizing luminescent europium complex. ３ (TPPO) ２ 0.04 g of (composed of Central Techno Co., Ltd.) 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] As the europium complex, Eu(TTA) used in Example 1 ３ (TPPO) ２ Instead of (E400), use tris(2-cenoyltrifluoroacetonato)bis(trioctylphosphine oxide)europium(III) (hereinafter referred to as "Eu(TTA)" ３ (TOPO) ２ Dye-containing resin particles 7 were prepared in the same manner as in Example 1, except that dye-containing resin particles 7 (manufactured by Central Techno Co., Ltd.) were used.

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

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

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

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

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

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

[0087] 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 (TPPO) ２ 0.04 g of (E400) (manufactured by Central Techno Co., Ltd.), 1.00 g of styrene monomer (manufactured by Kishida Chemical Co., Ltd.), 0.10 g of sodium styrene sulfonate (styrene sulfonate monomer) (manufactured by Tokyo Chemical Industry Co., Ltd.), 0.13 g of DVB, and 0.012 g of dodecyl methacrylate (manufactured by Tokyo Chemical Industry Co., Ltd.) were added 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.

[0088] Next, 0.20 g of HEMA (manufactured by Tokyo Chemical Industry Co., Ltd.), 0.05 g of TMP (manufactured by Tokyo Chemical Industry Co., Ltd.), and 0.01 g of V50 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) were added to the obtained suspension as 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 with approximately 4 L of deionized water using an ultrafiltration membrane with a molecular weight cutoff of 100 K to wash the product, and the dispersion of dye-containing resin particles 13 and the obtained suspension were centrifuged to purify the suspended particles. These were then redispersed in MES buffer at pH 7 to obtain dye-containing resin particles 13.

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

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

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

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

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

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

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

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

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

[0098] <Evaluation of Dye-Containing Resin Particles> The following evaluation was performed using aqueous dispersions of dye-containing resin particles 1-20 prepared in Examples 1-14 and Comparative Example 1-6. The evaluation results are shown in Table 2. Here, in Table 2, "Core St Mol Ratio" indicates the number of styrene monomers in the hydrophobic monomer adjusted to Mol 100, and "Mol Ratio of First Monomer Mol_f" is the number of Mol of the first monomer when the number of Mol of styrene monomer is set to 100 in the hydrophobic monomer. Also, "LogP_f of the First Monomer" indicates the octanol / water partition coefficient of the first monomer.

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

[0100] (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).

[0101] (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.

[0102] The initial luminescence intensity was denoted as Lum_Ini, and the luminescence intensity after 20 minutes was denoted as Lum_Fin. The luminescence intensity 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.

[0103] (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. An evaluation result of A or B was considered good, and C was considered unacceptable.

[0104]

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

[0106] <Evaluation of Particles for Specimen Testing> The 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.

[0107] 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, since the change in fluorescence anisotropy was detected even at a low concentration of 0.001 mg / ml of affinity particles, it was confirmed that these particles exhibit strong luminescence and are suitable for sample testing.

[0108] 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.

[0109] This application claims priority based on Japanese Patent Application No. 2024-223117, filed on 18 December 2024, and all of its contents are incorporated herein by reference.

[0110] 1. Core 2. Shell 3. Europium complex 20. Hydrophilic polymer

Claims

1. Dye-containing resin particles having a core portion and a shell portion, wherein the dye-containing resin particles contain a europium complex, the core portion and the shell portion are each crosslinked, the core portion has a resin containing a polymer of hydrophobic monomers comprising a styrene monomer as the main component and a first monomer of a different type from the styrene monomer, the shell portion has a resin containing a polymer of hydrophilic monomers, and the first monomer satisfies the following formulas (B1) and (15): LogP_f ≥ 3.60 (B1) (Mol_f) / (Mol_St) ≥ 0.05 (15) (wherein in formula (B1), LogP_f is the octanol / water partition coefficient of the first monomer, and in formula (15), Mol_f represents the number of moles of the first monomer, and Mol_St represents the number of moles of the styrene monomer.) 2. The dye-containing resin particles according to claim 1, characterized in that the octanol / water partition coefficient of all ligands in the europium complex is 2.0 or greater.

3. The pigment-containing resin particles according to claim 1 or 2, wherein the europium complex is represented by the following formula (1). Eu(A)x(B)y(C)z (1) [However, in formula (1), Eu is europium, (A) is a ligand represented by 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 groups independently selected from an alkyl group, a thienoyl phenyl group and a thienyl group, which may have a substituent. Also, R ３ is a hydrogen atom or a methyl group. Also, R ４ and R ５ are groups independently selected from an alkyl group and a phenyl group, which may have a substituent. Also, R ６ is a group selected from an alkyl group, a phenyl group and a triphenylene group, which may have a substituent. Also, R ７ and R ８ are groups independently selected from an alkyl group or a phenyl group, which may have a substituent. Here, the substituents in R １ , R ２ , R ４ to R ６ are groups independently selected from a methyl group, a fluoro group, a chloro group and a bromo group, and the alkyl groups in R １ , R ２ , R ４ to R ８ have 2 to 12 carbon atoms and may be different groups or the same group. Also, x, y, 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) (wherein they satisfy the above conditions).)] 4. The dye-containing resin particle according to any one of claims 1 to 3, characterized in that the resin contained in the shell portion contains at least one of the structures of the following formulas (12) and (13). (In equations (12) and (13), X2 and X3 are H or CH) ３ And Y1 is OH or OCH ３ Y2 is a group represented by the following formula (14) or CH ２ CH ２ OH is such that m3 and m4 are integers greater than or equal to 1. n is an integer between 1 and 40 (inclusive).

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

6. The dye-containing resin particle according to any one of claims 1 to 5, characterized in that the core portion comprises a resin formed using a crosslinking agent containing divinylbenzene, and the shell portion comprises a resin formed using a crosslinking agent containing trimethylolpropanetrimethacrylate.

7. A method for producing dye-containing resin particles containing a europium complex, comprising the steps of: obtaining resin particles having a core portion and a shell portion; and mixing a solution containing a europium complex with the resin particles having a core portion and a shell portion, wherein the step of obtaining the resin particles comprises a sub-step 1 for forming the core portion and a sub-step 2 for forming the shell portion, wherein the sub-step 1 is a sub-step in which a hydrophobic monomer containing styrene as a main component and a first monomer is polymerized and crosslinked to form a resin, and the sub-step 2 is a sub-step in which a hydrophilic monomer is polymerized and crosslinked to form a resin, wherein the first monomer in the sub-step 1 satisfies the following formula (B1) and the following formula (15). LogP_f ≥ 3.60 (B1) (Mol_f) / (Mol_St) ≥ 0.05 (15) (wherein in formula (B1), LogP_f is the octanol / water partition coefficient of the first monomer, in formula (15), Mol_f is the number of moles of the first monomer, and Mol_St is the number of moles of the styrene monomer.) 8. A method for producing dye-containing resin particles having a core portion and a shell portion, comprising: a step of forming a core portion by polymerizing and crosslinking a hydrophobic monomer in a solution containing styrene as a main component, a hydrophobic monomer containing a first monomer, and a europium complex; and a step of forming the shell portion around the core portion by polymerizing and crosslinking a hydrophilic monomer in the presence of the core portion, wherein the step of forming the core portion is a step of polymerizing and crosslinking the hydrophobic monomer in the solution in which the first monomer satisfies the following formula (B1) and the following formula (15). LogP_f ≥ 3.60 (B1) (Mol_f) / (Mol_St) ≥ 0.05 (15) (wherein in equation (B1), LogP_f is the octanol / water partition coefficient of the first monomer, in equation (15), Mol_f is the number of moles of the first monomer, and Mol_St is the number of moles of the styrene monomer.) 9. A method for producing dye-containing resin particles according to 7 or 8, characterized in that the europium complex is represented 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 is independently an alkyl group, a thienolphenyl group, or a thienyl group, which may each have a substituent. ３ R is a hydrogen atom or a methyl group, ４ and R ５ Each is independently an alkyl group or phenyl group which may each have substituents, and R ６ Each of these is an alkyl group, a phenyl group, or a triphenylene group, which may each have a substituent, and R ７ and R ８ Each of these is independently an alkyl group or phenyl group which may have substituents, and each substituent is independently one of a methyl group, a fluoro group, a chloro group, or a bromo group, and each alkyl group independently has 2 to 12 carbon atoms. Also, 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).

10. A method for producing dye-containing resin particles according to any one of claims 7 to 9, characterized in that the resin formed by polymerizing and crosslinking the hydrophilic monomer comprises at least one of the structures represented by the following formula (12) and the following 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 (OH is OH, m3 and m4 are integers greater than or equal to 1, and n is an integer between 1 and 40.) 11. A method for producing dye-containing resin particles according to any one of claims 7 to 9, characterized in that the hydrophilic monomer is a compound represented by the following formula (11). (In equation (11), X4 is H or CH) ３ Y3 is H or an alkyl group having 3 or fewer carbon atoms, and m5 is an integer between 3 and 9.

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

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

14. A method for producing particles for specimen testing, characterized by comprising the step of forming a site that specifically binds to a target substance in the dye-containing resin particles obtained by the method for producing dye-containing resin particles according to any one of claims 7 to 13.

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

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

17. A specimen testing reagent characterized by comprising specimen testing particles according to claim 15 or 16.

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

19. A method for detecting a target substance 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 16 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.

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

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