Resin particle and affinity particle comprising the same

Abstract

To provide a resin particle that is resistant to non-specific adsorption when used for detecting a target substance such as an antigen, in particular, to provide a resin particle exhibiting high sensitivity when detecting the target substance using a fluorescence depolarization method.SOLUTION: A resin particle has a europium complex, wherein the resin particle has a core-shell structure, wherein the resin particle has a core portion that contains a specific copolymer, and the resin particle has a shell portion that contains a specific polymer, wherein at least one of the core portion and the shell portion has a crosslinked structure.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to resin particles and affinity particles having the same.

Background Art

[0002] In recent years, in the fields of medicine and clinical examinations, it has become possible to highly sensitively detect trace amounts of biological components from blood, a part of a collected organ, and the like. Patent Document 1 discloses resin fine particles, and it is described that molecular recognition bodies such as antigens and antibodies are fixed to the particles and used in the fields of analytical reagents and diagnostic agents.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] In the prior art, there was room for improvement in non-specific adsorption.

Means for Solving the Problems

[0005] According to the resin particles of the present invention, the resin particles have a europium complex, the resin particles have a core-shell structure, the core part of the resin particles contains a copolymer having a unit represented by the following formula (CORE_1), and the shell part of the resin particles contains a polymer having a unit represented by the following formula (SHELL_1) and a polymer having a unit represented by the following formula (SHELL_2), and at least one of the core part and the shell part contains a crosslinked structure.

[0006]

Chemical Formula

[0007] In the above formula (CORE_1), X1 is H or CH3, m1 and m2 are independently integers of 1 or more, and m1 and m2 are represented by 10 < m1 / m2.

[0008]

Chemical formula

[0009] In the above formulas (SHELL_1) and (SHELL_2), X2 and X3 are each independently H or CH3, Y1 is OH or OCH3, Y2 is the following formula (SHELL_3) or CH2CH2OH, m3 and m4 are integers of 1 or more, and n is an integer of 1 or more and 40 or less.

[0010]

Chemical formula

[0011] According to the resin particles of the present invention, when used for detecting a target substance such as an antigen, resin particles in which non-specific adsorption hardly occurs can be provided. In particular, when detecting a target substance using the fluorescence polarization decay method, resin particles with high sensitivity can be provided.

Brief Description of the Drawings

[0012]

Figure 1

Figure 2

Modes for Carrying Out the Invention

[0013] Hereinafter, the present invention will be described in detail with reference to embodiments, but the present invention is not limited to these embodiments.

[0014] The resin particles according to this embodiment will be described in detail with reference to FIG. 1.

[0015] <Problems Solved by Resin Particles According to the Present Embodiment> As a result of investigations by the present inventors, it was found that when the resin particles disclosed in Patent Document 1 are used, problems of non-specific adsorption may occur. The problem of non-specific adsorption refers to a problem in which, between the resin particles and the component to be inspected, non-specific adsorption occurs not between the particles and the substance to be detected (target substance such as an antigen), but between the particles and contaminants. That is, an object of the present embodiment is to provide resin particles in which non-specific adsorption hardly occurs when used for detecting a target substance such as an antigen. In particular, an object is to provide resin particles with high sensitivity when detecting a target substance using the fluorescence polarization decay method.

[0016] As shown in FIG. 1, the resin particles according to the present embodiment contain a europium complex 3 and have a core-shell structure. The core-shell structure includes a core portion 1 and a shell portion 2 covering the surface thereof. It can also be said that the resin particles according to the present embodiment are resin particles including a first layer and a second layer in this order. The first layer may be particles, and it can also be said that in the aforementioned resin particles, the first layer is the core portion and the aforementioned shell portion is the second layer. Note that, between the first layer and the second layer in the present embodiment, and further outside the second layer (on the surface side of the resin particles), there may be a layer different from the second layer. Hereinafter, regarding the resin particles according to the present embodiment that include a first layer and a second layer in this order, a configuration in which the first layer is the core portion and the second layer is the shell portion will be described as an example.

[0017] In FIG. 1, D1 is the radius of the core portion, and D2 is the thickness of the shell portion. In the present embodiment, the core portion contains a copolymer represented by the following formula (CORE_1). Here, the copolymer is typically a random copolymer, but may be a block copolymer as long as the effects of the present invention can be obtained.

[0018]

Chemical formula

[0019] In the above formula (CORE_1), X1 is H or CH3, m1 and m2 are independently integers of 1 or more, and m1 and m2 are represented by 10 < m1 / m2.

[0020] In this embodiment, it is preferable that the core part contains a copolymer represented by the above formula (CORE_1) as a main material.

[0021] The shell part in this embodiment includes a polymer having a unit represented by the following formula (SHELL_1) and a polymer having a unit represented by the following formula (SHELL_2).

[0022]

Chemical formula

[0023] In the above formulas (SHELL_1) and (SHELL_2), X2 and X3 are each independently H or CH3, Y1 is OH or OCH3, Y2 is the following formula (SHELL_3) or CH2CH2OH, m3 and m4 are integers of 1 or more, and n is an integer of 1 or more and 40 or less.

[0024]

Chemical formula

[0025] Moreover, it is preferable that the shell part contains a polymer having a unit represented by the formula (SHELL_1) and a polymer having a unit represented by the above formula (SHELL_2) as main materials.

[0026] In addition, in this embodiment, at least one of the core part and the shell part contains a crosslinked structure. Note that both the core part and the shell part may have a crosslinked structure in this embodiment.

[0027] Furthermore, the polydispersity index of the resin particles according to this embodiment may be 0.1.

[0028] When the resin particles according to this embodiment having such a configuration are used for detecting a target substance such as an antigen, nonspecific adsorption hardly occurs. In particular, when detecting a target substance using the fluorescence polarization decay method, the sensitivity is high.

[0029] The shell part in this embodiment contains, for example, at least one selected from the group consisting of polymers of hydroxyethyl methacrylate, polymers of polyethylene glycol monomethyl ether methacrylate, polymers of 2-methoxyethyl acrylate, polymers of 2-methoxyethyl methacrylate, and polymers of glycidyl methacrylate. The crosslinked structure contained in the shell part is formed using, for example, trimethylolpropane trimethacrylate.

[0030] In addition, since the core part in this embodiment has a crosslinked structure, the europium complex tends to stay in the core part and is less likely to leak to the outside of the resin particles. The crosslinked structure contained in the core part is formed using, for example, divinylbenzene.

[0031] In addition, since the core part contains a copolymer having a unit represented by the above formula (CORE_1) and the polydispersity index is 0.1 or less, the sensitivity is high especially when detecting a target substance using the fluorescence polarization decay method. The reasons will be described in detail below.

[0032] (Measurement of particle size) In this embodiment, the particle size (diameter) of the resin particles can be determined by the dynamic light scattering method. When laser light is irradiated onto particles dispersed in a liquid and the scattered light is observed with a photon detector, since the particles are constantly moving their positions due to Brownian motion, the intensity distribution due to the interference of the scattered light is constantly fluctuating.

[0033] Here, the dynamic light scattering method is a measurement method that observes the state of this Brownian motion as the fluctuation of the scattered light intensity. The fluctuation of the scattered light with respect to time is represented by an autocorrelation function, and the translational diffusion coefficient is determined. From the determined diffusion coefficient, the Stokes diameter is obtained, and the particle size of the particles dispersed in the solution can be derived. In addition, in order to represent the width of the particle size distribution, a polydispersity index (PDI) is calculated by measurement. When the value of the polydispersity index is greater than 0 and less than or equal to 0.1, it indicates that the sample has a monodisperse particle size distribution.

[0034] Here, the fluorescence depolarization method obtains high detection sensitivity by detecting changes in the movement of resin particles. Generally, small particles have a fast rotational motion, and large particles have a slow rotational motion. In this case, the state in which the aggregated particles become larger and their rotational motion becomes slower due to the aggregation of particles such as, for example, particle (antibody)-antigen-particle (antibody) generated by the antigen-antibody reaction is detected.

[0035] Therefore, if small particles and large particles are mixed at the initial stage before the reaction in the system, there is a concern that the detection sensitivity will decrease. Therefore, it is necessary for the resin particles to have a uniform particle size distribution. Specifically, it is required that the aforementioned polydispersity index is 0.1 or less.

[0036] Hereinafter, the details of the resin particles according to this embodiment will be described.

[0037] (Structure of resin particles, core-shell structure) In this embodiment, the functions of the resin particles are divided into a core part and a shell part. First, it is important for the resin material of the core part to have a low specific gravity. The resin particles are assumed to be used, for example, after being left standing and stored for a long period of several months. Therefore, when the specific gravity of the particles is high, the resin particles will settle in the container, and a stirring and redispersion process will be required before use, making the operation complicated. Therefore, in order to suppress the sedimentation of the resin particles, it is preferable to use a material with a low specific gravity, more specifically, a material with a specific gravity of 1.10 g / cm^3 or less, for the core part that occupies most of the volume of one resin particle. Specifically, polystyrene is preferable.

[0038] Next, for the resin material of the shell part, it is important to suppress aggregation between particles and, in addition, suppress adsorption between the particles and the container. Originally, in the inspection using the fluorescence polarization decay method, the purpose is to detect the aggregation of particles that have undergone an antigen-antibody reaction, for example, particles (antibody)-antigen-particles (antibody). Therefore, simple particle-particle aggregation without an antigen or aggregation between particles with antibodies and a specimen that does not interact with the antibody has an adverse effect of reducing the accuracy of the inspection. This effect is called non-specific adsorption and is one of the causes of reduced detection sensitivity. Non-specific aggregation between particles and particles (or specimen) may be caused by the hydrophobic sites on the adjacent particle surfaces binding to each other through hydrophobic interaction. Also, when the particle surface has long hydrophilic groups, particles and particles (or specimen) may aggregate due to the entanglement of the hydrophilic groups.

[0039] Therefore, the resin material of the shell part in this embodiment is provided with a function of adsorbing water molecules on the surface at a short distance from the particle surface by hydrogen bonding. Thereby, even when particles - particles (or specimen) are close to each other, water molecules are coordinated in the gaps between the particles, suppressing direct contact between the particle surfaces or suppressing long-distance entanglement, and thus preventing aggregation between the particles and particles (or specimen). Also, similarly, when a hydrophobic container and particles are close to each other, by coordinating water molecules between the particles and the container, adsorption of the particles to the container can be prevented. Also, it is more preferable that the relationship between the radius (D1) of the core part and the film thickness (D2) of the shell part shown in FIG. 1 satisfies the following formula (RA_1). 50 > D1 / D2 > 5 / 3 (RA_1) If the crosslinked shell part is thin, it is difficult to obtain the effect of reducing non-specific adsorption. On the other hand, when the shell part becomes thick, the volume of the shell part in one resin particle increases, resulting in an increase in the specific gravity of the resin particle. As a result, the sedimentation rate of the particle when the resin particle is stored increases, which is not preferable.

[0040] (Europium complex) Since the wavelength and intensity of the luminescence are less affected by the surroundings and the luminescence has a long lifetime, this case uses a europium complex that exhibits polarization anisotropy. The europium complex 3 is composed of a europium element and ligands. When used in the fluorescence depolarization method, considering the luminescence lifetime, visible luminescence wavelength region, etc., it is preferable to use a europium complex. Europium generally has a luminescence lifetime of 0.1 ms or more and 1.0 ms or less. It is necessary to appropriately adjust this luminescence lifetime and the rotational relaxation time obtained from the formula (A1) described later. 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 polarization anisotropy represented by the formula (A3) described later changes greatly before and after the antigen-antibody reaction.

[0041] Among the ligands constituting the europium complex 3, at least one is a ligand having a light-harvesting function. The light-harvesting function refers to 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 3 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.

[0042] The europium complex 3 may be a polynuclear complex as long as it exhibits polarization anisotropy. The polarization anisotropy of the europium complex 3 is shown by the formula (A3) described later. When it can be considered that the Brownian rotational motion of the europium complex 3 in the medium has stopped, it is desirable that the polarization 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 3.

[0043] It is preferable that the europium complex 3 is incorporated more into the core part 1 because the luminescence intensity per particle becomes stronger. Specifically, the content of the europium complex contained per 1 g of the resin particles is preferably 0.001 g or more.

[0044] In addition, the content of the europium complex can be calculated from the quantification of europium by high-frequency inductively coupled plasma (ICP) emission analysis.

[0045] The europium complex in the resin particles according to this embodiment is represented by, for example, the following formula (COMP_1). Eu(A) x (B) y (C) z ···(COMP_1) However, in formula (COMP_1), (A) is a ligand represented by the following formula (COMP_2), (B) is a ligand represented by the following formula (COMP_3) or (COMP_4), and (C) is a ligand represented by the following formula (COMP_5).

[0046]

Chemical formula

[0047] In the above formulas (COMP_2) to (COMP_5), R 1 , R 2 are each independently an alkyl group, a perfluoroalkyl group, a phenyl group or a thiophene group which may each have a substituent, R 3 is a hydrogen atom or a methyl group, R 4 , R 5 are each independently an alkyl group or a phenyl group which may each have a substituent, R 6 is an alkyl group, a phenyl group or a triphenylene group which may each have a substituent, R 7 , R 8 are each independently an alkyl group or a phenyl group which may each have a substituent. In the above formula (COMP_4), the bond indicated by the dotted line may or may not be present, and the substituents are each independently any one of a methyl group, a fluoro group, a chloro group and a bromo group, and the number of carbon atoms of the alkyl group is each independently any one of 2 or more and 12 or less.

[0048] x, y, z satisfy the following formulas (COMP_6), (COMP_7), (COMP_8), (COMP_9). x = 3, (COMP_6) y = 1 or 2, (COMP_7) z = 0 or 1, (COMP_8) x + y + z = 4 or 5 (COMP_9) In addition, as the europium complex in the present embodiment, a structure represented by the following formula (COMP_10) or the following formula (COMP_11) is exemplified.

[0049]

Chemical formula

[0050]

Chemical formula

[0051] The structure represented by the formula (COMP_10) can be expressed as Eu(TTA)3(TPPO)2 (resin particles 1 to 5, 7 to 15 in the examples and comparative examples described later).

[0052] The structure represented by the formula (COMP_11) can be expressed as Eu(TTA)3(TPPO)(DBSO) (resin particle 6 in the examples described later).

[0053] On the other hand, if the europium complex 3 aggregates in the core part 1, the excitation efficiency of the europium complex 3 is affected by the interaction between the ligands, making it difficult to measure the reproducible polarization anisotropy. A method for determining whether the europium complex 3 shows non-aggregated luminescence behavior in the core part 1 can be determined by taking the excitation spectrum of the sample.

[0054] Luminescent particles with strong luminescence intensity not only enable simple high-sensitivity 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 is larger for smaller particle sizes, it becomes possible to detect reactions in a shorter time (manufacturing method of resin particles).

[0055] (Core part · Radical polymerizable monomer) The radical polymerizable monomer as the material of the core part is characterized by containing at least a styrene-based monomer and a copolymer of styrene sulfonic acid as the main material. In this embodiment, the main material refers to the material with the largest blending amount among the resin materials constituting the core part.

[0056] (Core part) When using styrene alone as the main material of the core part, the particle size exceeds 500 nm, which is inappropriate as the resin particles used in the fluorescence polarization decay method. Therefore, by containing a charged material during particle synthesis, the repulsion of charges between particles can be utilized to suppress the coalescence and aggregation of particles during particle synthesis. Specifically, styrene sulfonic acid is useful as the charged material.

[0057] In this case, a copolymer with styrene sulfonic acid having a charge is required. However, the ratio of styrene to styrene sulfonic acid also changes depending on the selection of the polymerization initiator described later, so it cannot be determined unconditionally. At least, the amount of styrene monomer needs to be more than 10 times the amount of styrene sulfonic acid monomer. When styrene sulfonic acid is added excessively, fine particles are generated, the polydispersity index deteriorates, and as a result, there is a concern about a decrease in detection sensitivity.

[0058] In addition to the main material, a monomer selected from the group consisting of acrylate-based monomers and methacrylate-based monomers may be included. For example, butadiene, vinyl acetate, vinyl chloride, acrylonitrile, methyl methacrylate, methacrylonitrile, methyl acrylate, etc. can be mentioned. These monomers can be used alone or in combination of multiple types.

[0059] In addition, the resin material of the core part needs to be cross-linked with a monomer having two or more double bonds in one molecule, such as divinylbenzene, trimethylolpropane trimethacrylate, ethylene glycol dimethacrylate, etc. The principle of the inspection using fluorescence depolarization cancellation is to detect the change in the movement of resin particles with a europium complex. Therefore, when the core part is not cross-linked, the europium complex rotates inside the resin particles. As a result, it becomes difficult for the movement of the resin particles and the movement of the europium complex to be linked, and there is a concern that the detection sensitivity will decrease. On the other hand, when the core part is cross-linked, the movement of the europium complex in the resin particles can be suppressed, so that the detection sensitivity can be increased. In addition, the luminescence intensity of the europium complex is obtained by a ligand. Therefore, there is a concern that when the ligand is exposed to the external environment of the particles and the ligand is detached, the luminescence intensity will decrease. In this case, by cross-linking the core part, the europium complex can be retained in the core part, and it is also possible to suppress the decrease in the luminescence intensity due to the detachment of the ligand caused by exposure.

[0060] As the cross-linking agent for the core part, divinylbenzene is particularly preferable. Since divinylbenzene is a compound similar to styrene, which is the structure of the main component of the core part, it can spread uniformly in the core part without being localized in the core part. Therefore, uniform cross-linking within the core part becomes possible, and as a result, the rotational movement of the europium complex due to cross-linking and the exposure of the europium complex to the outside of the resin particle system can be suppressed.

[0061] Regarding the presence or absence of cross-linking, it can be determined by the following method.

[0062] After dispersing the resin particles in pyridine at a concentration of 5 wt%, shake them at 50 °C for 3 hours. Before and after this operation, measure the particle size by the above-mentioned dynamic light scattering method. When it is not cross-linked, the resin particles dissolve and it becomes difficult to measure. Or, the particles coagulate with each other and become more than twice the particle size before the operation, and the shape of the particles cannot be maintained.

[0063] (Shell part) The main material of the shell part is a resin represented by the following formulas (SHELL_1) and (SHELL_2).

[0064]

Chem.

[0065] In the above formulas (SHELL_1) and (SHELL_2), X2 and X3 are H or CH3, Y1 is OH or OCH3, Y2 is the following formula (SHELL_3) or CH2CH2OH, m3 and m4 are integers of 1 or more, and n is an integer of 1 or more and 40 or less.

[0066]

Chem.

[0067] Here, the main material refers to the material with the largest blending amount among the resin materials constituting the shell part. The material of the shell part has a hydrogen bonding site arranged near the main chain of the side chain. The hydrogen bonding site can form a hydrogen bond with water molecules in the system. These water molecules are immobilized near the shell. Due to these water molecules, when other resin particles, a sample that does not cause an antigen-antibody reaction, a test container, etc. are close to the resin particles, the water molecules can coordinate in the gaps between the particles, preventing the direct contact between the particle surfaces. By this action, it becomes possible to suppress the non-specific adsorption between the resin particles and between the resin particles and the sample.

[0068] As the material of the shell part, preferably, specifically, hydroxyethyl methacrylate, polyethylene glycol monomethyl ether methacrylate, 2-methoxyethyl acrylate, 2-methoxyethyl methacrylate, and glycidyl methacrylate represented by the following formulas (SHELL_4), (SHELL_5), and (SHELL_6) are used as polymerizable monomers and polymerized outside the core part to form the shell part.

[0069]

Chem.

[0070] [Chemical formula]

[0071] [Chemical formula]

[0072] [Chemical formula]

[0073] [Chemical formula]

[0074] (Shell part crosslinking) In addition, the resin material of the shell part needs to be crosslinked with a monomer having two or more double bonds in one molecule, such as divinylbenzene, trimethylolpropane trimethacrylate, ethylene glycol dimethacrylate, etc.

[0075] Figure 2 is a schematic diagram of resin particles in which a conventional non-crosslinked hydrophilic polymer 20 is adsorbed on the surface of the core part 1. Non-specific adsorption of the resin particles can also be reduced by adsorbing the non-crosslinked hydrophilic polymer 20 on the surface of the resin particles, but the following problems occur. The core part 1 contains the europium complex 3. In the figure, on the surface of the core part 1, a region E2 where the hydrophilic polymer 20 covers the surface of the core part and a region E1 where the surface of the core part is exposed are mixed. This is considered to be due to the fact that the surface composition of the core part has minute hydrophobic regions and minute hydrophilic regions, so the hydrophilic polymer is likely to be adsorbed on the hydrophilic core part surface, while it is difficult for the hydrophilic polymer to be adsorbed on the hydrophobic core part surface.

[0076] When conventional resin particles are put into a test container, due to the hydrophobic interaction between the surface of the test container, which is not hydrophilic, and the hydrophobic part of the resin particle surface (Fig. E1 in the same figure), the resin particles adsorb onto the surface of the test container. The resin particles adsorbed on the container have their movement restricted, resulting in a problem that the sensitivity of the fluorescence polarization extinction method decreases as a result.

[0077] On the other hand, in this case, as shown in Fig. 1, by crosslinking the resin of the shell part, the shell part material is forcibly immobilized on the surface of the core part even for the hydrophobic surface of the core part. As a result, the surface of the core part particles can be uniformly coated with the shell part material.

[0078] In addition, by crosslinking the shell part, it is possible to prevent the hydrogen bond sites arranged in the side chains of the shell part material from extending widely toward water, which is the solvent. When the shell part is not crosslinked, the hydrophilic polymer spreads toward water, and there is concern about the interaction with the hydrophilic sites of another particle (or specimen) approaching. On the other hand, in this case, due to the crosslinking of the shell member, the material of the shell part does not spread away from the resin particles. Therefore, since the interaction with another approaching particle (or specimen) is unlikely to occur, the adsorption between particles is suppressed, and the non-specific adsorption property can be improved.

[0079] As the crosslinking agent for the shell part, trimethylolpropane trimethacrylate is particularly preferable. Since trimethylolpropane trimethacrylate is a compound similar to the structure of the main component of the shell part, it can spread uniformly in the shell part without being localized within the shell part. Therefore, uniform crosslinking is possible for the entire shell part, and as a result, the non-specific adsorption property can be improved.

[0080] Regarding the presence or absence of crosslinking, it can be judged by the following method in the same manner as the core part described above.

[0081] After dispersing resin particles in pyridine at a concentration of 5 wt%, shake them at 50 °C for 3 hours. Before and after this operation, measure the particle size by the dynamic light scattering method described above. In the case of uncrosslinked particles, the resin particles dissolve, making measurement difficult. Or, the particles coagulate with each other and become more than twice the particle size before the operation, and the shape of the particles cannot be maintained.

[0082] (Radical polymerization initiator) As the radical polymerization initiator, azo compounds, organic peroxides, etc. can be widely used. Specifically, 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), potassium persulfate (KPS), etc. can be mentioned.

[0083] (Aqueous medium) A buffer solution can also be used as the aqueous solvent. Further, in order to increase the stability of the liquid in which the resin particles according to this embodiment are dispersed, a surfactant, a preservative, a sensitizer, etc. may be added to the aqueous solvent.

[0084] When using the liquid in which the resin particles according to this embodiment are dispersed, the anisotropy of polarized luminescence can be detected with high sensitivity with respect to the aggregation and dispersion behavior of the particles. Therefore, the colloidal solution in which the resin particles according to this embodiment are dispersed in an aqueous solvent can be used as a highly sensitive test reagent using the fluorescence polarization decay method.

[0085] (Ligand·affinity particles) In this embodiment, affinity particles having the resin particles according to this embodiment and a ligand can be provided. In this case, the resin particles have at least one reactive functional group selected from the group consisting of a carboxy group, an amino group, a thiol group, an epoxy group, a maleimide group, and a succinimidyl group in the shell portion. The ligand is bonded to this functional group. These reactive functional groups are on the surface side of the resin particles, that is, on the side opposite to the central side.

[0086] In this embodiment, a ligand is a compound that specifically binds to a specific target substance. The site where the ligand binds to the target substance is determined and has selective or specific high affinity. For example, an antigen and an antibody, an enzyme protein and its substrate, a signal substance such as a hormone or a neurotransmitter and its receptor, a nucleic acid, etc. are exemplified, but the ligand in this embodiment is not limited to these. Examples of the nucleic acid include deoxyribonucleic acid. The affinity particles in this embodiment are particles having selective or specific high affinity (affinity) for the target substance. The ligand in this embodiment is preferably any one of an antibody, an antigen, and a nucleic acid.

[0087] In this embodiment, as the chemical reaction for chemically bonding the reactive functional group of the resin particles according to this embodiment and the ligand, a conventionally known method can be applied within the range capable of achieving the object of the present invention. When the ligand is to be amide-bonded, a catalyst such as 1-[3-(dimethylaminopropyl)-3-ethylcarbodiimide] can be appropriately used. The resin particles according to this embodiment can also immobilize the ligand by physical adsorption.

[0088] When the affinity particles in this embodiment use an antibody (antigen) as the ligand and an antigen (antibody) as the target substance, they can be preferably applied to an immunolatex agglutination method or a fluorescence polarization decay method.

[0089] (In vitro diagnostic test reagent) The test reagent for in vitro diagnosis in this embodiment, that is, the test reagent for detecting a target substance in a specimen by in vitro diagnosis, has the affinity particles according to this embodiment and a dispersion medium for dispersing the affinity particles. The amount of the affinity particles according to this embodiment contained in the test reagent in this embodiment is preferably from 0.000001% by mass to 20% by mass, and more preferably from 0.0001% by mass to 1% by mass. The test reagent according to this embodiment may contain a third substance such as a solvent or a blocking agent in addition to the affinity particles according to this embodiment within the range capable of achieving the object of the present invention. Two or more kinds of third substances such as a solvent and a blocking agent may be combined and contained. Examples of the solvent used in this embodiment include various buffer solutions such as phosphate buffer, glycine buffer, Good buffer, Tris buffer, and ammonia buffer, but the solvent contained in the test reagent in this embodiment is not limited thereto.

[0090] When the test reagent in this embodiment is used for detecting an antigen or an antibody in a specimen, an antibody or an antigen can be used as the ligand.

[0091] (Test Kit) The test kit for detecting a target substance in a sample by in vitro diagnosis in the present embodiment has the above reagent and a housing containing the above reagent. As the kit according to the present embodiment, a sensitizer that promotes the aggregation of particles during an antigen-antibody reaction may be contained. Examples of the sensitizer include polyvinyl alcohol, polyvinylpyrrolidone, and sodium alginate, but the present invention is not limited thereto. Further, the test kit according to the present embodiment may include a positive control, a negative control, a serum diluent, and the like. As the medium for the positive control and the negative control, in addition to serum and physiological saline that do not contain a measurable target substance, a solvent may be used. The test kit according to the present embodiment can be used in the method for detecting a target substance according to the present embodiment in the same manner as a kit for detecting a target substance in a sample by ordinary in vitro diagnosis. Further, the concentration of the target substance can also be measured by a conventionally known method, and in particular, it is preferably used for detecting a target substance in a sample by an immunolatex agglutination method or a fluorescence polarization extinction method.

[0092] (Detection method) The method for detecting a target substance in a sample by in vitro diagnosis in the present embodiment has a step of mixing the affinity particles according to the present embodiment and a sample that may contain the target substance. Further, the mixing of the affinity particles according to the present embodiment and the sample is preferably performed in the range of pH 3.0 to pH 11.0. Further, 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. Further, it is preferable to use a solvent in this detection method. Further, the concentration of the affinity particles according to the present embodiment in the detection method according to the present embodiment is preferably 0.000001% by mass to 1% by mass, more preferably 0.00001% by mass to 0.001% by mass in the reaction system. The method for detecting a target substance in a sample according to the present embodiment preferably detects the agglutination reaction resulting from the mixing of the affinity particles according to the present embodiment and the sample by a fluorescence polarization extinction method. Specifically, it has a step of mixing a sample with a test reagent to obtain a mixed solution, a step of irradiating the mixed solution with polarized light, and a step of separately detecting the polarized light component of the emission of the affinity particles in the mixed solution.

[0093] By optically detecting the above-mentioned aggregation reaction occurring in the mixed solution, the target substance in the sample can be detected, and furthermore, the concentration of the target substance can also be measured.

[0094] (Fluorescence polarization decay method) By encapsulating a europium complex that exhibits polarized luminescence as a material inside the resin particles, even if there is a slight change in the dispersion state of the particles in the liquid, the change in the polarized luminescence characteristics can be detected. Specifically, when an antigen-antibody reaction occurs and the particles aggregate via the antigen, the change in the rotational Brownian motion of the particles can be detected as a change in polarization anisotropy.

[0095] Polarization anisotropy means that there is anisotropy in the transition moment (transition dipole moment). The emission of polarized light generally means that in the case of a luminescent dye with anisotropy in the transition moment, when the polarized light along the transition moment is used as the excitation light, the emitted light is also polarized along the transition moment. In the case of a europium complex, since it exhibits fluorescence emission based on the energy transfer from the ligand to the central metal ion, the transition moment of the polarized emission becomes complicated, but the red emission around 610 nm derived from the electronic transition from the lowest excited state 5D0 to 7F2 has polarization anisotropy.

[0096] The principle of the fluorescence polarization decay method is to measure the deviation of the transition moment due to the rotational motion of the luminescent material within the time when this polarized emission occurs. The rotational motion of the luminescent material can be expressed by Equation (A1). Q = 3Vη / kT ···(A1) Here, Q: Rotational relaxation time of the material V: Volume of the material η: Viscosity of the solvent k: Boltzmann constant T: Absolute temperature That is.

[0097] The rotational relaxation time of the material is the time required for the molecule to rotate by an angle θ (68.5°) at which cosθ = 1 / e.

[0098] From this formula (A1), it can be seen that the rotational relaxation time of the luminescent material is proportional to the volume of the material, that is, the cube of the particle radius. On the other hand, the relationship between the luminescence lifetime and the degree of polarization of the material in fluorescence depolarization can be expressed by formula (A2). p0 / p = 1 + A(τ / Q) ··· (A2) Here,[[]]END]] 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 That is.

[0099] From formula (A1) and formula (A2), in order to measure a large change in the degree of polarization, the relationship between the luminescence lifetime and the rotational relaxation time of the luminescent material, that is, the volume (particle size) of the luminescent material, is important. The larger the particle size of the luminescent material, the longer the luminescence lifetime needs to be.

[0100] When obtaining the degree of polarization of luminescence shown by formula (A2) from experiments, polarized light can be incident on the sample, and the luminescence can be detected in a direction 90 degrees from the traveling direction and the vibration direction of the excitation light. At this time, the detected light can be divided into polarized light components parallel and perpendicular to the polarization of the incident light for detection, and the degree of polarization can be evaluated by the mathematical formula shown in formula (A3). r(t) = (I∥(t) - GI⊥(t)) / (I∥(t) + 2GI⊥(t)) ··· (A3) Here,[[]]END]] r(t): Polarization anisotropy at time t I∥(t): Luminescence intensity of the luminescence component parallel to the excitation light at time t I⊥(t): Luminescence intensity of the luminescence component perpendicular to the excitation light at time t G: Correction value, the ratio of I⊥ / I∥ measured with excitation light whose vibration direction is 90 degrees different from that of the excitation light used for sample measurement That is.

[0101] That is, if it is within an appropriate particle size and luminescence lifetime range, it becomes possible to sensitively read a change in the particle size of a luminescent material due to an antigen-antibody reaction or the like as a value of polarization anisotropy. Note that the polarization anisotropy is the value of the degree of polarization corrected by G and 2G, and the degree of polarization is the value obtained by removing G and 2G from Equation (A3). In actual measurement, since a corrected value of G is required, the polarization anisotropy is obtained.

[0102] Furthermore, the luminescent particles in the present embodiment have a polarization anisotropy obtained by the following mathematical formula (A4) <r>It is preferably 0.01 or more.

[0103]

Number

[0104] In formula (A4), <r>···Polar anisotropy I VV ···Emission intensity of the emission component with the vibration direction parallel to the first polarization when excited with the first polarization I VH ···Emission intensity of the emission component with the vibration direction perpendicular to the first polarization when excited with the first polarization I HV ···Emission intensity of the emission component with the vibration direction perpendicular to the second polarization when excited with the second polarization whose vibration direction is perpendicular to the first polarization I HH ···Emission intensity of the emission component with the vibration direction parallel to the second polarization when excited with the second polarization whose vibration direction is perpendicular to the first polarization G···Correction value is as follows.

Example

[0105] Hereinafter, the present invention will be specifically described by way of examples. However, the present invention is not limited to such examples.

[0106] (1) Preparation of resin particles 1 100 g of MES (2-morpholinoethanesulfonic acid) buffer solution (manufactured by Kikkoman Chemical Co., Ltd.) with a pH of 7, 0.04 g of a polarized light-emitting europium complex [Tris(2-thenoyltrifluoroacetonato)(Bis(triphenylphosphineoxide))europium(III)] (manufactured by Central Techno Co., Ltd., hereinafter abbreviated as "Eu(TTA)3(TPPO)2"), 1.00 g of styrene monomer (manufactured by Kikkoman Chemical Co., Ltd.), 0.10 g of sodium styrenesulfonate (styrene sulfonic acid monomer) (manufactured by Tokyo Chemical Industry Co., Ltd.), 0.13 g of divinylbenzene (hereinafter abbreviated as "DVB") (manufactured by Tokyo Chemical Industry Co., Ltd.) was placed in a four-necked flask and stirred with a mechanical stirrer set at 300 rpm. After stirring for 15 minutes under nitrogen flow conditions, the temperature of the prepared oil bath was set to 70 °C and nitrogen flow was continued for another 15 minutes. After heating and stirring the mixture, 0.01 g of 2,2'-azobis(2-methylpropionamidine) dihydrochloride (hereinafter abbreviated as "V50") (manufactured by Fujifilm Wako Pure Chemical Corporation) was added and emulsion polymerization was carried out for 6 hours to prepare the core part.

[0107] After the polymerization reaction, to the obtained suspension, as the shell part material, 0.20 g of hydroxyethyl methacrylate (hereinafter abbreviated as "HEMA") (manufactured by Tokyo Chemical Industry Co., Ltd.), 0.05 g of trimethylolpropane trimethacrylate (hereinafter abbreviated as "TMP") (manufactured by Tokyo Chemical Industry Co., Ltd.), 0.01 g of V50, were mixed and emulsion polymerization was carried out for 10 hours to prepare the shell part.

[0108] After the polymerization reaction, the obtained suspension was subjected to ultrafiltration with about 4 L of ion-exchanged water using an ultrafiltration membrane with a fractional molecular weight of 100K to wash the product and obtain a dispersion of Resin Particle 1. The amounts of each component used in the preparation of Resin Particle 1 are shown in Table 1.

[0109] (2) Preparation of Resin Particles 2 to 15 In the same procedure, the composition ratios of each reagent used in the preparation of Resin Particles 2 to 15 are shown in Table 1. Also, each material used is shown below. [Tris(2-thenoyltrifluoroacetonato)(triphenylphosphineoxide)(dibenzylsulfoxide)europium(III)] (manufactured by Central Techno Co., Ltd., hereinafter abbreviated as "Eu(TTA)3(TPPO)(DBSO"), ethylene glycol dimethacrylate (manufactured by Tokyo Chemical Industry Co., Ltd.), Polyethylene glycol monomethyl ether methacrylate (hereinafter abbreviated as "PEG") (manufactured by Sigma-Aldrich Japan LLC) Glycidyl methacrylate (hereinafter abbreviated as "GMA") (manufactured by Tokyo Chemical Industry Co., Ltd.), polyethylene glycol monomethacrylate (manufactured by Sigma-Aldrich Japan LLC) 2-Methoxyethyl acrylate (hereinafter abbreviated as "MEA") (manufactured by Tokyo Chemical Industry Co., Ltd.) 2-Methoxyethyl methacrylate (hereinafter abbreviated as "MEMe") (manufactured by Tokyo Chemical Industry Co., Ltd.)

[0110] [Table 1]

[0111] A dispersion of resin particles 1 to 15 was obtained by the above emulsion polymerization.

[0112] (Preparation of affinity particles modified with anti-CRP antibody) After substituting the dispersion of the synthesized resin particles 11 with pyridine, succinic anhydride was added to impart a carboxylic acid group to a part of HEMA.

[0113] 0.25 mL of the dispersion of resin particles 11 (1.2 wt%) having a carboxylic acid group was aliquoted and the solvent was replaced with 1.6 mL of MES buffer at pH 6.0. To the MES buffer containing resin particles 11, 0.5 wt% of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide and sodium N-hydroxysulfosuccinimide were added and reacted at 25 °C for 1 hour. After the reaction, the dispersion was washed with MES buffer at pH 5.0, 100 μg / mL of anti-CRP antibody was added, and the anti-CRP antibody was bound to resin particles 11 at 25 °C for 2 hours. After binding, the resin particles 11 to which the antibody was bound were washed with Tris buffer at pH 8. After the reaction, the resin particles 11 to which the antibody was bound were washed with phosphate buffer to obtain affinity particles modified with anti-CRP antibody at a concentration of 0.3 wt% (hereinafter sometimes abbreviated as affinity particles).

[0114] The binding of the antibody to the resin particles was confirmed by measuring the decrease in the antibody concentration in the buffer solution to which the antibody was added using a BCA assay.

[0115] <Examples and Evaluations> Each evaluation was carried out using the prepared dispersion of resin particles. The examples and their evaluation results are shown in Table 2.

[0116] (Evaluation Method) (Particle Size, Polydispersity Index, Core Part Radius, Shell Part Thickness) The shape of the resin particles was evaluated using an electron microscope (S5500 manufactured by Hitachi High-Technologies). The average particle size of the resin particles was evaluated using dynamic light scattering (Zetasizer Nano S manufactured by Malvern). Also, after the synthesis of the core part, a part of the dispersion of the resin particles was taken out and measured by dynamic light scattering to measure the particle size of the core part. Also, after the synthesis of the shell part, a part was taken out in the same manner and measured to measure the thickness of the shell part. The polydispersity index can also be obtained by the same measurement.

[0117] (Resin Particle Concentration, Eu Complex Concentration) The particle concentration in the suspension in which the resin particles were dispersed was evaluated using a gravimetric analyzer (TG8120 manufactured by Rigaku). Also, the amount of Eu complex encapsulated per 1 g of the particles was measured by quantitatively analyzing Eu using an ICP spectroscopic analyzer (PS3510 manufactured by Hitachi High-Technologies Corporation).

[0118] <Polarization Anisotropy, Nonspecific Adsorptivity> The fluorescence spectrum of the resin particles was measured with an excitation light of 340 nm, and polarizers were placed in the optical paths on the excitation side and the emission side. The direction of the polarizer was fixed on the excitation side, and the measurement was carried out with the emission side set parallel or perpendicular to the excitation side. The apparatus used was a spectrofluorophotometer F-4500 manufactured by Hitachi High-Technologies Corporation. The peak wavelength of the observation light for analyzing the polarized emission was set to 611 nm. By analyzing the obtained polarized emission data with the above mathematical formula (A4), the polarization anisotropy r was determined. 10 μL of each resin particle dispersion (0.1 mg / mL) was added to 500 μL of a human serum solution diluted 15-fold with a buffer solution, incubated at 37 °C for 10 minutes, and the change amount Δr of the value of the polarization anisotropy r before and after incubation was calculated. When the non-specific adsorption property is high, particle aggregation occurs, so Δr becomes large. Preferably, the non-specific adsorption property is preferably reduced, and therefore it is desirable that Δr is close to zero.

[0119] Regarding the non-specific adsorption property, Δr was defined as A when it was 0.010 or less, B when it was 0.012 or less, and C when it was greater than 0.012. The evaluation results were that A and B were good, and C was unacceptable.

[0120] <Particle size after storage at 40 °C> The prepared dispersion of resin particles was diluted to 0.1 mg / mL with pure water, 8 mL was placed in a sealed container with a volume of 10 mL, and stored at 40 °C for 3 months. The change in particle size before and after storage was measured by dynamic light scattering.

[0121] The change amount of the particle size was defined as A when it was 7 nm or less, B when it was 10 nm or less, and C when it was greater than 10 nm. The evaluation results were that A and B were good, and C was unacceptable.

[0122] <Sedimentation property, standing storage at 5 °C> The prepared dispersion of resin particles was diluted with pure water to 0.1 mg / mL, prepared, 25 mL was placed in a sealed container with a volume of 30 mL, and stored at 5 °C for 3 months. 1 mL of the dispersion was taken from the upper part of the container before storage, and the absorbance at 527 nm (UH5200 manufactured by Hitachi High-Tech Corporation) was measured. After storage, 1 mL of the dispersion was similarly taken from the upper part of the container, and the absorbance was measured. The sedimentation property of the resin particles was evaluated based on the change amount between the absorbance before storage and the absorbance after storage. When the sedimentation property of the resin particles is good, that is, when sedimentation is suppressed, the change amount of the absorbance becomes a small value.

[0123] The change amount of the absorbance of 0.02 or less was defined as A, 0.03 or less as B, and greater than 0.03 as C. The evaluation results A and B were regarded as good, and C was regarded as unacceptable.

[0124] <Evaluation of Affinity Particles> The obtained affinity particles were measured for polarization anisotropy before and after mixing with CRP (antigen). The affinity particles were fixed at 0.0001 mg / mL, and the concentration of CRP was examined at 0 to 10,000 pg / mL.

[0125] The evaluation was performed using a microplate reader (Nivo manufactured by PerkinElmer). A filter with a central wavelength of 355 nm and a half-value width of 40 nm was used for the excitation light filter, a filter with 615 nm and a half-value width of 8 nm for the emission filter, and D400 for the dichroic mirror. The measurement time was set to 1 second, the polarization anisotropy r from the start of the reaction for 30 minutes was measured, and the change amount Δr of the polarization anisotropy r during that time was calculated. The measurement temperature was fixed at 37 °C. The evaluation results are shown in Table 3.

[0126]

Table 2

[0127] According to the results shown in Table 2, as shown by the comparison between the comparative example and the example, having a shell part, the shell part having a crosslinked structure, a polydispersity index of 0.1 or less, and a specific copolymer being contained in the core part can reduce non-specific adsorption.

[0128]

Table 3

[0129] According to the results shown in Table 3, it was confirmed that the amount of change in the polarization anisotropy (Δr) before and after mixing with CRP changed according to the concentration of CRP, and highly sensitive detection of CRP was possible. In addition, since the change in polarization anisotropy could be detected even in the case of the low concentration of 0.0001 mg / ml of the affinity particles, it was confirmed that the affinity particles exhibited strong luminescence.

[0130] As is clear from this example, it is possible to provide affinity particles having high detection sensitivity for biological components and that can be used in the fluorescence polarization cancellation method.

[0131] Embodiments of the present invention include the following configurations and methods.

[0132] (Configuration 1) Resin particles having a europium complex, wherein the resin particles have a core-shell structure, the core part of the resin particles contains a copolymer having a unit represented by the following formula (CORE_1), the shell part of the resin particles contains a polymer having a unit represented by the following formula (SHELL_1) and a polymer having a unit represented by the following formula (SHELL_2), and the resin particles in which at least one of the core part and the shell part contains a crosslinked structure.

[0133]

Chemical formula

[0134] In the above formula (CORE_1), X1 is H or CH3, m1 and m2 are independently integers of 1 or more, and m1 and m2 are represented by 10 < m1 / m2.

[0135]

Chem.

[0136] In the above formulas (SHELL_1) and (SHELL_2), X2 and X3 are each independently H or CH3, Y1 is OH or OCH3, Y2 is the following formula (SHELL_3) or CH2CH2OH, m3 and m4 are integers of 1 or more, and n is an integer of 1 or more and 40 or less.

[0137]

Chem.

[0138] (Configuration 2) The resin particles according to Configuration 1, wherein the europium complex is represented by the following formula (COMP_1).

[0139] Eu(A) x (B) y (C) z ···(COMP_1) However, in the formula (COMP_1), (A) is a ligand represented by the following formula (COMP_2), (B) is a ligand represented by the following formula (COMP_3) or (COMP_4), and (C) is a ligand represented by the following formula (COMP_5).

[0140]

Chem.

[0141] In the above formulas (COMP_2) to (COMP_5), R 1 and R 2 are each independently an alkyl group, a perfluoroalkyl group, a phenyl group or a thiophene group which may each have a substituent, R 3 is a hydrogen atom or a methyl group, and R 4 , R 5 are each independently an alkyl group or a phenyl group which may each have a substituent, and R 6 is an alkyl group, a phenyl group or a triphenylene group which may each have a substituent, and R 7 and R 8 are each independently an alkyl group or a phenyl group which may each have a substituent. In the above formula (COMP_4), the bond indicated by the dotted line may or may not be present, and the substituents are each independently any one of a methyl group, a fluoro group, a chloro group and a bromo group, and the number of carbon atoms of the alkyl group is each independently any one of 2 or more and 12 or less. x, y and z satisfy the following formulas (COMP_6), (COMP_7), (COMP_8) and (COMP_9).

[0142] x = 3, (COMP_6) y = 1 or 2, (COMP_7) z = 0 or 1, (COMP_8) x + y + z = 4 or 5 (COMP_9) (Configuration 3) The resin particles according to Configuration 2, wherein the europium complex is represented by the following formula (COMP_10) or the following formula (COMP_11).

[0143] [Chemical formula]

[0144] [Chemical formula]

[0145] (Configuration 4) The resin particles according to any one of Configurations 1 to 3, wherein the content of the europium complex contained per 1 g of the resin particles is 0.001 g or more.

[0146] (Configuration 5) The resin particles according to any one of Configurations 1 to 4, comprising at least one selected from the group consisting of a polymer of ethyl methacrylate, a polymer of polyethylene glycol monomethyl ether methacrylate, a polymer of 2-methoxyethyl acrylate, a polymer of 2-methoxyethyl methacrylate, and a polymer of glycidyl methacrylate.

[0147] (Configuration 6) The resin particles according to any one of Configurations 1 to 5, wherein the crosslinked structure contained in the shell part is formed using trimethylolpropane trimethacrylate.

[0148] (Configuration 7) The resin particles according to any one of Configurations 1 to 6, wherein the crosslinked structure contained in the core part is formed using divinylbenzene.

[0149] (Configuration 8) The resin particles according to any one of Configurations 1 to 7, wherein the relationship between the radius (D1) of the core part and the film thickness (D2) of the shell part is represented by the following formula (RA_1).

[0150] 50 > D1 / D2 > 5 / 3 (RA_1) (Configuration 9) The resin particles according to any one of Configurations 1 to 8, wherein the diameter of the resin particles is 80 nm or more and 200 nm or less.

[0151] (Configuration 10) The resin particles according to any one of Configurations 1 to 9, wherein the polydispersity index of the resin particles is 0.1 or less.

[0152] (Configuration 11) The resin particles according to any one of Configurations 1 to 10, and An affinity particle having a ligand selected from the group consisting of an antibody, an antigen, a protein, and a nucleic acid, wherein the shell portion has at least one functional group selected from the group consisting of a carboxy group, an amino group, a thiol group, an epoxy group, a maleimide group, and a succinimidyl group, and the ligand is bonded to the functional group.

[0153] (Configuration 12) The affinity particle according to Configuration 11, which is used for detecting a target substance by using a fluorescence polarization cancellation method.

[0154] (Method 1) A step of mixing the affinity particle according to Configuration 11 or 12 with a specimen that may contain a target substance to obtain a mixed solution; A step of irradiating the mixed solution with polarized light; A detection method comprising a step of separately detecting a polarization component of the emission of the affinity particle in the mixed solution.

Explanation of Reference Signs

[0155] 1 Core portion 2 Shell portion 3 Europium complex 20 Hydrophilic polymer< / r> < / r>

Claims

1. A resin particle having a europium complex, The resin particles have a core-shell structure, The core portion of the resin particle contains a copolymer having a unit represented by the following formula (CORE_1): The shell portion of the resin particle contains a polymer having a unit represented by the following formula (SHELL_1) and a polymer having a unit represented by the following formula (SHELL_2), and at least one of the core portion and the shell portion contains a crosslinked structure. 【Chemistry 1】 In the above formula (CORE_1), X １ is H or CH ３ In the formula, m1 and m2 are each independently an integer of 1 or more, and m1 and m2 are expressed as 10<m1 / m2. 【Chemistry 2】 In the above formulas (SHELL_1) and (SHELL_2), X ２ , X ３ are each independently H or CH ３ And Y １ is OH or OCH ３ And Y ２ is the following formula (SHELL_3) or CH ２ CH ２ OH, m3 and m4 are integers of 1 or more, and n is an integer of 1 or more and 40 or less. 【Chemistry 3】

2. The resin particle according to claim 1 , wherein the europium complex is represented by the following formula (COMP_1): Eu(A) ｘ (B) ｙ (C) ｚ ・・・(COMP_1) In the formula (COMP_1), (A) is a ligand represented by the following formula (COMP_2), (B) is a ligand represented by the following formula (COMP_3) or (COMP_4), and (C) is a ligand represented by the following formula (COMP_5), 【Chemistry 4】 In the above formulas (COMP_2) to (COMP_5), R １ , R ２ each independently represents an alkyl group, a perfluoroalkyl group, a phenyl group, or a thiophene group, each of which may have a substituent; R ３ is a hydrogen atom or a methyl group, R ４ , R ５ each independently represents an alkyl group or a phenyl group which may have a substituent, R ６ each represents an alkyl group, a phenyl group, or a triphenylene group which may have a substituent; R ７ , R ８ each independently represents an alkyl group or a phenyl group which may have a substituent, in the above formula (COMP_4), the bond represented by the dotted line may or may not be present, each independently represents a methyl group, a fluoro group, a chloro group, or a bromo group, and the number of carbon atoms in the alkyl group is independently any of 2 or more and 12 or less, x, y, and z satisfy the following formulas (COMP_6), (COMP_7), (COMP_8), and (COMP_9). x=3, (COMP_6) y = 1 or 2, (COMP_7) z = 0 or 1, (COMP_8) x + y + z = 4 or 5 (COMP_9)

3. The resin particle according to claim 2, wherein the europium complex is represented by the following formula (COMP_10) or the following formula (COMP_11). 【Chemistry 5】 【Chemistry 6】

4. 2. The resin particles according to claim 1, wherein the content of the europium complex per 1 g of the resin particles is 0.001 g or more.

5. 2. The resin particle according to claim 1, wherein the shell portion comprises at least one selected from the group consisting of a polymer of hydroxyethyl methacrylate, a polymer of polyethylene glycol monomethyl ether methacrylate, a polymer of 2-methoxyethyl acrylate, a polymer of 2-methoxyethyl methacrylate, and a polymer of glycidyl methacrylate.

6. The resin particle according to claim 1 , wherein the crosslinked structure contained in the shell portion is formed using trimethylolpropane trimethacrylate.

7. The resin particle according to claim 1 , wherein the crosslinked structure contained in the core portion is formed using divinylbenzene.

8. The resin particle according to claim 1 , wherein the relationship between the radius (D1) of the core portion and the film thickness (D2) of the shell portion is represented by the following formula (RA_1): 50 > D1 / D2 > 5 / 3 (RA_1)

9. 2. The resin particles according to claim 1, wherein the diameter of the resin particles is 80 nm or more and 200 nm or less.

10. 2. The resin particles according to claim 1, wherein the polydispersity index of the resin particles is 0.1 or less.

11. The resin particles according to any one of claims 1 to 10, A ligand comprising one selected from the group consisting of an antibody, an antigen, a protein, and a nucleic acid; The affinity particle has the above formula, wherein the shell portion has at least one functional group selected from the group consisting of a carboxy group, an amino group, a thiol group, an epoxy group, a maleimide group, and a succinimidyl group, and the ligand is bonded to the functional group.

12. The affinity particle according to claim 11, which is used for detecting a target substance using a fluorescence polarization method.

13. A step of mixing the affinity particles according to claim 12 with a specimen that may contain a target substance to obtain a mixed solution; irradiating the mixture with polarized light; A detection method comprising a step of detecting the polarized components of the light emitted from the affinity particles in the mixture.

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