Method for detecting and measuring target substances based on measurement of polarization anisotropy and particles therefor

Abstract

To provide a measurement method and a kit for a target having high detection sensitivity.SOLUTION: A method for measuring at least one of the presence or absence and the concentration of a target in a sample solution includes: (a) a step of preparing first particles that specifically bind to the target and include a rare earth complex, and second particles that have a larger average particle diameter than that of the first particles and specifically bind to the target; (b) a step of mixing the sample solution, the first particles, and the second particles to obtain a mixed solution; and (c) a step of determining at least one of the presence or absence and the concentration of the target from the polarization anisotropy of the mixed solution obtained in (b).SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for detecting and measuring a target substance in a test sample and to particles used in the method. [Background technology]

[0002] In the fields of medicine and clinical testing, highly sensitive detection of trace amounts of biological components from blood, harvested organ parts, etc. is necessary to investigate the causes of disease. Immunoassay is a widely used method for detecting biological components. One type of immunoassay is the latex agglutination method, which utilizes an antigen-antibody reaction. Latex agglutination is a method for detecting and quantifying antigens in liquid samples such as biological samples by mixing the liquid sample with latex particles carrying antibodies that specifically bind to the antigens and measuring the degree of latex agglutination. In latex agglutination, an antigen is captured by an antibody bound to latex, and multiple latex particles are cross-linked via the captured antigen, resulting in agglutination. In other words, the amount of antigen in a liquid sample, such as a biological sample, can be quantified by evaluating the degree of latex agglutination. The degree of agglutination can be quantified by evaluating the change in the amount of light transmitted or scattered through the liquid sample.

[0003] While the latex agglutination method allows for simple and rapid quantitative evaluation of antigens, it has the problem that it cannot detect antigens when the amount of antigen in a liquid sample such as a biological sample is small. In order to improve detection sensitivity, it is conceivable to replace the system that scatters transmitted light with a method that detects luminescence characteristics with higher sensitivity. Specifically, a specimen testing method using fluorescence depolarization has been proposed (Patent Documents 1 to 3).

[0004] Patent Document 1 proposes improving a fluorescence polarization depolarization device for clinical use. Fluorescence polarization depolarization does not require the prior separation of the analyte from unreacted luminescent substances, i.e., a washing process called B / F (Bound / Free) separation, which is required in general fluorometric assays. Therefore, sample testing is as simple as latex agglutination. Furthermore, it is believed that measurement can be performed using a testing system similar to latex agglutination by simply mixing a luminescent substance that specifically reacts with the analyte. However, Patent Document 1 proposes using a single molecule, such as fluorescein, as the luminescent material, which, in principle, could only be applied to drugs, low-molecular-weight antigens, etc.

[0005] Patent Document 2 proposes the use of latex particles with a dye adsorbed to them that has a long luminescence lifetime, enabling the detection of high-molecular-weight substances such as proteins, which was difficult to achieve with Patent Document 1. Based on the principles of fluorescence depolarization, Patent Document 2 proposes the measurement of high-molecular-weight substances by balancing the decrease in rotational Brownian motion of substances in liquid that occurs with increasing particle size with the length of the luminescence lifetime. However, because the particle surface is supported by the biomolecule bovine serum albumin (BSA) to suppress nonspecific adsorption, there is a possibility that the particle size distribution will broaden and that the BSA, which is a protein, will cause lot-to-lot variation. As a result, it is difficult to say that antigen detection with extremely high sensitivity has been achieved.

[0006] Patent Document 3 proposes a measurement method in which a fluorescent label and a target antigen are competitively adsorbed onto large particles through an antigen-antibody reaction. However, with this method, one antibody reacts with one fluorescent label, making it difficult to capture changes in the degree of polarization with high sensitivity. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Special Publication No. 3-52575 [Patent Document 2] Patent No. 2893772 [Patent Document 3] Japanese Patent Application Publication No. 3-103765 Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention has been made in view of the above background art, and aims to provide a highly sensitive measurement method and test kit based on the change in polarization anisotropy, particularly a method for detecting reactions more sensitively by increasing the change in polarization anisotropy. [Means for solving the problem]

[0009] The present invention provides a method for determining at least one of the presence or absence and concentration of a target substance in a sample solution, the method comprising the following steps: (a) preparing first particles that specifically bind to a target substance and contain a rare earth complex, and second particles that have an average particle size larger than the first particles and specifically bind to the target substance; (b) A step of mixing the sample liquid, the first particles, and the second particles to obtain a mixed liquid. (c) A step of determining at least one of the presence or absence and concentration of the target substance from the polarization anisotropy of the mixture obtained in (b).

[0010] The present invention also provides a test kit including the first particles and the second particles, and a method for producing the same. [Effects of the Invention]

[0011] According to the present invention, particle aggregation in a liquid can be detected with high sensitivity by measurement using polarized light, and a target substance can be measured with high sensitivity. In particular, the reaction can be captured more sensitively from the change in polarization anisotropy detected when particles aggregate in a liquid.

[0012] Furthermore, we propose particles that are highly effective in suppressing nonspecific adsorption by forming a hydrophilic layer on the particle surface, without using BSA, etc. Therefore, the particles of the present invention can be used more advantageously in measurements based on polarization anisotropy, enabling even more sensitive measurements. [Brief explanation of the drawings]

[0013] [Figure 1] 1A to 1C are diagrams illustrating a measurement method according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0014] Preferred embodiments of the present invention will be described in detail below, but the scope of the present invention is not limited thereto.

[0015] As one embodiment, the present invention provides a method for determining at least one of the presence or absence and concentration of a target substance in a sample solution, the method comprising the following steps: (a) preparing first particles that specifically bind to a target substance and contain a rare earth complex, and second particles that have an average particle size larger than the first particles and specifically bind to the target substance; (b) A step of mixing the sample liquid, the first particles, and the second particles to obtain a mixed liquid. (c) A step of determining at least one of the presence or absence and concentration of the target substance from the polarization anisotropy of the mixture obtained in (b).

[0016] Polarization anisotropy is an index that indicates the tendency of the light emitted by an object irradiated with polarized light to maintain the component of the azimuth of the irradiated polarized light. Specifically, polarization anisotropy is expressed by the following formula 1: <r>It is shown as follows.

number

[0017] By placing a polarizer on the incident side of the light, the target is excited with the first polarized light. If a polarizer is placed on the detection side parallel to the polarizer on the incident side and the intensity of the emitted light is measured, the emission intensity of the emission component whose vibration direction is parallel to the first polarized light, i.e., I VV On the other hand, if a polarizer is set on the detection side in a direction perpendicular to the polarizer on the incident side and the emission intensity is measured, the emission intensity of the emission component whose vibration direction is perpendicular to the first polarization, i.e., I VH can be measured.

[0018] On the incident side of the light, a polarizer is placed in a direction perpendicular to the direction when the first polarized light is obtained, i.e., the object is excited with the second polarized light. On the detection side, a polarizer is placed in a direction parallel to the polarizer on the incident side, and the intensity of the emitted light is measured. The emission intensity of the emitted light component whose vibration direction is parallel to the second polarized light, i.e., I HV On the other hand, if a polarizer is set on the detection side in a direction perpendicular to the polarizer on the incident side and the emission intensity is measured, the emission intensity of the emission component whose vibration direction is perpendicular to the second polarized light, i.e., I HH can be measured.

[0019] Furthermore, as one embodiment, the present invention provides a test kit for determining at least one of the presence or absence and concentration of a target substance. The test kit includes first particles and second particles, the first particles specifically binding to a target substance and including a rare earth complex, and the second particles specifically binding to the target substance.

[0020] Specifically binding to a target substance refers to having the property of specifically interacting with the target substance, and particularly preferably refers to having the ability to specifically bind to the target substance. Examples of binding mechanisms include electrostatic interactions, van der Waals interactions, and hydrogen bonding interactions. The sites and mechanisms by which the first particle and the second particle bind to the target substance may be the same or different.

[0021] Any substance that interacts with other substances can be a target substance. Examples of target substances include antigens, antibodies, low-molecular-weight compounds, various receptors, enzymes, substrates, nucleic acids, cytokines, hormones, neurotransmitters, signaling molecules, and membrane proteins. Antigens include allergens, bacteria, viruses, cells, cell membrane components, cancer markers, various disease markers, antibodies, blood-derived substances, food-derived substances, natural products, and all low-molecular-weight compounds. Nucleic acids include DNA, RNA, cDNA, parts or fragments thereof, synthetic nucleic acids, primers, probes, and the like derived from bacteria, viruses, and cells. Low-molecular-weight compounds include cytokines, hormones, neurotransmitters, signaling molecules, membrane proteins, and their receptors.

[0022] The first particles contain a rare earth complex and are luminescent. That is, they exhibit the property of luminescence when irradiated with an excitation wavelength. The rare earth complex of the first particles is a luminescent rare earth complex. Preferred examples thereof include complexes of europium, terbium, neodymium, erbium, yttrium, lanthanum, cerium, samarium, gadolinium, dysprosium, thulium, ytterbium, and scandium. The luminescence of rare earth complexes has a long lifetime. Since polarization anisotropy depends on changes in the rotational motion of the luminescent material during the luminescence period, it is preferable to use a rare earth complex with a long luminescence lifetime as the luminescent material.

[0023] The first particles and the second particles form aggregates via the target substance. Before mixing with the sample liquid, the first particles preferably have a relatively low polarization anisotropy, i.e., the polarization anisotropy to be measured is low, and fluorescence polarization is depleted.

[0024] On the other hand, when particles aggregate with each other via the target substance, the polarization anisotropy becomes relatively high. In other words, it is desirable to cancel out part (or all) of the depolarization of fluorescence polarization.

[0025] The first particles and the second particles form aggregates in the dispersion medium via the target substance. Aggregation between first particles and aggregation between second particles may also occur. The polarization anisotropy exhibited by the aggregates in the dispersion medium is preferably increased compared to the polarization anisotropy exhibited by non-aggregated first particles in the dispersion medium. The increase is preferably 0.004 or more, more preferably 0.01 or more, and even more preferably 0.02 or more.

[0026] The first particles preferably exhibit a polarization anisotropy of 0.2 or less, more preferably 0.15 or less, and even more preferably 0.12 or less in a dispersion medium. Furthermore, the aggregates exhibit a polarization anisotropy in a dispersion medium of preferably 0.05 or more, more preferably 0.09 or more, and even more preferably 0.13 or more.

[0027] Furthermore, the total amount of the first particles and second particles used in the measurement in the dispersion medium is preferably 0.000001% by mass to 1% by mass, and the weight ratio thereof is preferably in the range of 1:9 to 9:1.

[0028] When measuring to determine polarization anisotropy, polarized excitation light (first polarization) can be irradiated by placing a polarizer such as a polarizing filter on the incident side of the light. If a polarizing filter is placed perpendicular to this, a second polarization whose vibration direction is perpendicular to the first polarization can be irradiated. If a polarizer is placed parallel to the incident side polarizer on the detection side, the emission intensity of the emission component whose vibration direction is parallel to the excitation side can be measured. If a polarizer is placed perpendicular to the incident side polarizer on the detection side, the emission intensity of the emission component whose vibration direction is perpendicular to the excitation light can be measured. The emission intensity can be measured using a spectrophotometer, etc.

[0029] The measurement is preferably carried out in the dispersion solution at a temperature ranging from 0° C. to 100° C., more preferably from 4° C. to 50° C. The solution is preferably an aqueous solvent, such as a buffer solution, physiological saline, or water. Measurement conditions can be set appropriately by those skilled in the art, and in doing so, reference can be made to the examples and the like described later in this specification, but the conditions are not limited thereto.

[0030] To ensure sufficient change in polarization anisotropy before and after particle aggregation, it is desirable that the first particles are not too large. On the other hand, if the first particles are too small, problems such as insufficient aggregation via the target substance or insufficient luminescence, as well as manufacturing problems, may occur. The average particle size of the first particles is preferably 10 nm or more and 1 μm or less. That is, the first particles are preferably 1 μm (micron) or less, more preferably 500 nm or less, even more preferably 400 nm or less, and even more preferably 250 nm or less. The average particle size of the first particles is preferably 10 nm or more, more preferably 20 nm or more, and even more preferably 50 nm or more. Note that the average particle size in this specification is the number average particle size, and the average particle size can be measured by dynamic light scattering.

[0031] The first particles preferably have a small particle size distribution, with a PDI of 0.1 or less. The first particles may contain polystyrene and a polymer having a siloxane bond, and may include a hydrophilic polymer on the particle surface. The first particles may have a layer on the particle surface containing a hydrophilic polymer containing an ether, betaine, or pyrrolidone ring. By including a hydrophilic polymer on the particle surface, nonspecific adsorption is suppressed. In this way, by suppressing nonspecific adsorption using a hydrophilic polymer rather than a protein, the particle size distribution is suppressed, making the particles more advantageous for use in measurements based on polarization anisotropy.

[0032] The second particles are particles having a larger average particle size than the first particles and having affinity for a target substance. The second particles preferably have an average particle size of 100 nm or more and 5 μm or less. That is, the lower limit of the average particle size of the second particles is 100 nm or more, more preferably 150 nm, in order to sufficiently increase the polarization anisotropy of the aggregates. The upper limit is preferably 5 μm or less, more preferably 1 μm or less, from the viewpoint of particle dispersion stability, etc.

[0033] Furthermore, the second particles preferably have a small particle size distribution, with a PDI of 0.1 or less. The second particles contain polystyrene and a polymer having a siloxane bond, and may contain a hydrophilic polymer on the particle surface. The second particles may also have a layer on the particle surface containing a hydrophilic polymer containing an ether, betaine, or pyrrolidone ring. By containing a hydrophilic polymer on the particle surface, nonspecific adsorption is suppressed. In this way, by suppressing nonspecific adsorption using a hydrophilic polymer rather than a protein or the like, the particle size distribution is suppressed, making the particles more advantageous for use in measurements based on polarization anisotropy.

[0034] The excitation wavelength of the first particle may be different from the excitation wavelength of the second particle, or the emission wavelength of the first particle may be different from the emission wavelength of the second particle. Alternatively, the excitation wavelength of the first particle may be different from the excitation wavelength of the second particle, and the emission wavelength of the first particle may be different from the emission wavelength of the second particle. That is, the second particle can emit light at an excitation wavelength different from the excitation wavelength of the first particle, and can also emit light at an emission wavelength different from the emission wavelength of the first particle. By providing the second particle with emission characteristics different from those of the first particle, multidimensional measurements such as excitation at multiple wavelengths or observing emission at multiple wavelengths are possible.

[0035] On the other hand, it is preferable that the second particles are not excited by excitation light having a wavelength suitable for exciting the first particles, specifically, the second particles are not excited by excitation light having a wavelength of 300 nm or more and 450 nm or less.

[0036] Furthermore, it is preferable that the second particles do not emit light that overlaps with that of the first particles, specifically, the second particles do not have an emission maximum in the wavelength region of 550 nm or more and 650 nm or less.

[0037] The first particle and the second particle can have a ligand to have affinity for the target substance. Any ligand can be used as long as it shows affinity for a specific substance. Examples of combinations of a ligand and a target compound or a target compound and a ligand include: antigens and antibodies, low molecular weight compounds and their receptors, enzymes and substrates, and complementary nucleic acids. Further examples include antibodies and their specific allergens, bacteria, viruses, cells, cell membrane components, cancer markers, various disease markers, antibodies, blood-derived substances, food-derived substances, natural product-derived substances, and all low molecular weight compounds. Further examples include receptors and their specific low molecular weight compounds, cytokines, hormones, neurotransmitters, signaling substances, membrane proteins, etc. Further examples include DNA, RNA, cDNA, parts or fragments thereof, synthetic nucleic acids, primers, probes, etc. derived from bacteria, viruses, cells, etc., and nucleic acids complementary thereto. Other combinations known to have affinity can also be used as target substance and ligand combinations. Representative examples of the ligand include antigens, antibodies, and nucleic acids.

[0038] The ligand can be introduced into the first particle or the second particle as follows. That is, after forming the particle substrate or the layer that forms the surface thereof, the ligand can be bound via the functional group that the particle possesses. Alternatively, the ligand can be incorporated when forming the particle substrate or the layer that forms the surface thereof. The ligand may be a modified version of a ligand used in latex agglutination or the like.

[0039] In yet another embodiment, the present invention provides a test kit comprising first particles and second particles dispersed in a dispersion medium. The present invention also provides a method for producing the test kit, comprising the step of dispersing the first particles and the second particles in the dispersion medium. The test kit may be enclosed in a housing.

[0040] In one embodiment, the present invention provides a first method for producing particles, comprising at least a first step and a second step. In the first step, a rare earth complex, a radical polymerizable monomer containing styrene, a radical polymerization initiator, and a polymer containing a unit having a pyrrolidone ring are mixed with an aqueous medium to prepare an emulsion. In the second step, the emulsion obtained in the first step is stirred to polymerize the radical polymerizable monomer.

[0041] Particles that bind to a target substance are sometimes referred to as affinity particles, particles that emit light are sometimes referred to as luminescent particles, and particles with a relatively large average particle size are sometimes referred to as large particle sizes. Particles that are luminescent and bind to a target substance are sometimes referred to as luminescent affinity particles. Particles that have a relatively large average particle size and bind to a target substance are sometimes referred to as large affinity particles. In the present invention, the first particles correspond to luminescent affinity particles, and the second particles correspond to large affinity particles.

[0042] By narrowing the particle size distribution and incorporating luminescent rare earth complexes into the particles, minute changes in the particle dispersion state in the liquid can be detected as changes in the polarized luminescence characteristics. Specifically, particles with affinity for the target substance sandwich the target substance and aggregate (a sandwich reaction occurs), causing a change in the rotational Brownian motion of the particles. This can be detected as a change in polarization anisotropy. This method makes it possible to detect target substances in nanograms to picograms per mL of solution.

[0043] Rare earth complexes can emit long-lived polarized phosphorescence. Polarization anisotropy is related to the anisotropy of the transition moment (transition dipole moment) of the luminescent dye. In the case of a luminescent dye with an anisotropic transition moment, when polarized light aligned with the transition moment is used as excitation light, the emitted light will also be polarized along the transition moment. In the case of rare earth complexes, when the anisotropic ligand is excited, fluorescence emission is exhibited based on energy transfer from the ligand to the central metal ion, so exciting the ligand with polarized light will emit polarized light.

[0044] The principle of fluorescence depolarization is to measure the shift in transition moment due to the rotational motion of the luminescent material during the time when polarized light emission occurs. The rotational motion of the luminescent material can be expressed by Equation 2. Q=3Vη / kT (Formula 2) Q: Rotational relaxation time of the material V: Volume of material η: viscosity of the solvent k: Boltzmann constant T: absolute temperature The rotational relaxation time of a material is the time it takes for a molecule to rotate through an angle θ (68.5°) such that cosθ=1 / e.

[0045] Equation 2 shows that the rotational relaxation time of the luminescent material is proportional to the volume of the material, i.e., the cube of the particle size of the luminescent material. On the other hand, the relationship between the luminescence lifetime of the material and the degree of polarization in fluorescence depolarization can be expressed by Equation 3. p0 / p=1+A(τ / Q)...(Formula 3) 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

[0046] From Equations 2 and 3, in order to measure large changes in the degree of polarization, 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; the larger the particle size of the luminescent material, the longer the luminescence lifetime must be.

[0047] To experimentally determine the degree of polarization of luminescence shown in Equation 3, polarized light is incident on the sample and the luminescence is detected at a 90-degree angle to the propagation and vibration direction of the excitation light. At this time, the detected light is separated into polarization components parallel and perpendicular to the polarization of the incident light, and evaluated according to Equation 4. r(t)=(I∥(t)−GI⊥(t)) / (I∥(t)+2GI⊥(t)) (Equation 4) r(t): Polarization anisotropy at time t I∥(t): Emission intensity of the emission component parallel to the excitation light at time t I⊥(t): Emission intensity of the emission component perpendicular to the excitation light at time t G: Correction value, the ratio of I⊥ / I∥ measured with excitation light whose vibration direction differs by 90 degrees from the excitation light used in sample measurement

[0048] In other words, if the particle (or aggregate) size and luminescence lifetime are within an appropriate range, it is possible to sensitively read changes in the size of the luminescent material due to antigen-antibody reactions, etc., as a value of polarization anisotropy. Note that polarization anisotropy is a value corrected by G and 2G, and the degree of polarization is the value obtained by removing G and 2G from Equation 4. In actual measurements, a correction value for G is required, so polarization anisotropy is calculated.

[0049] In terms of material design, it is suitable for the first particles to be small and for the particle size to increase to the point where the polarization anisotropy is saturated upon reaction with the target substance. However, if a sandwich-type antigen-antibody reaction is carried out using only one type of particle, the size of the particles formed after the reaction is limited. If the size of the first particles is increased in order to increase the polarization anisotropy, the anisotropy before the reaction will increase, and ultimately the change in polarization anisotropy cannot be increased.

[0050] Therefore, we focused on the idea that if a first particle can react with a second particle with a larger average particle size than the first particle in a sandwich-type reaction, the difference in polarization anisotropy before and after the reaction would be large.

[0051] (Detailed description of particles) An example of the particle according to this embodiment will be described in detail with reference to the drawings.

[0052] (First Particle 1) The first particles (luminescent affinity particles) 1 according to this embodiment have a narrow particle size distribution, and the particle surfaces are coated with a hydrophilic material. Luminescent rare earth complexes are present inside the particles.

[0053] FIG. 1 is a schematic diagram showing an example of a first particle and a second particle according to this embodiment. FIG. 1 shows an example in which the particles are spherical. The diameter (average particle size) of the first particles is preferably 10 nm or more, or 20 nm or more, more preferably 25 nm or more, and is preferably 1 μm or less, and more preferably 400 nm or less. It is particularly preferable that the average particle size of the first particles in this embodiment is 20 nm or more and 400 nm or less. A more uniform particle size distribution is preferable. For example, particles having a polydispersity index (hereinafter abbreviated as PDI) of 0.1 or less when measured by dynamic light scattering can be suitably used.

[0054] PDI can be determined by irradiating particles dispersed in a solution with a laser beam and observing the scattered light with a photon detector. Because particles are constantly moving due to Brownian motion, the intensity distribution due to the interference of scattered light also fluctuates constantly. Dynamic light scattering is a particle measurement method that observes Brownian motion as fluctuations in scattered light intensity. The fluctuations in scattered light over time are expressed as an autocorrelation function to determine the translational diffusion coefficient. The Stokes diameter can be calculated from the determined diffusion coefficient, and the size of particles dispersed in solution can be derived. In addition, cumulant diameter and PDI can be determined by cumulant analysis of the autocorrelation function. PDI indicates the degree of polydispersity of particle size distribution; the smaller this value, the more uniform the particle size distribution. Generally, particles with a PDI of 0.1 or less are considered to be monodisperse in the solvent.

[0055] The first particle 1 according to this embodiment preferably has a pdi of 0.1 or less. To stably maintain a pdi of 0.1 or less, it is desirable not to apply a nonspecific adsorption inhibitor to the particle surface. On the other hand, for the purposes of the present invention, it is necessary to prevent substances other than the target substance from nonspecifically adsorbing to the particle, and therefore a coating is required to maintain the hydrophilicity of the particle surface.

[0056] However, with the commonly used BSA loading method, it is difficult to maintain a stable pdi of 0.1 or less. Furthermore, BSA loading can result in lot-to-lot variations. Therefore, a hydrophilic polymer coating is required to achieve a pdi of 0.1 or less relative to particle size.

[0057] The first particles according to this embodiment preferably have a diameter of 10 nm or more and 1 μm or less. More preferably, the diameter is 20 nm or more and 500 nm or less. Even more preferably, the diameter is 50 nm or more and 400 nm or less, and even more preferably, the diameter is 50 nm or more and 250 nm or less. If the average particle size is greater than 1 μm, the polarization anisotropy before aggregation increases, and the difference with the polarization anisotropy after the aggregation reaction decreases. Furthermore, if the average particle size is less than 10 nm, the volume per particle decreases, the amount of rare earth complex that can be contained decreases, and the luminescence intensity required for measurement may not be obtained.

[0058] (Particle matrix 2) The particle substrate 2 refers to the core portion of the first particle 1. The particle substrate 2 is preferably spherical or nearly spherical. There are no particular restrictions on the material of the particle substrate 2, as long as it can stably contain the rare earth complex. Polystyrene particles polymerized primarily from styrene monomers, or particles in which the polystyrene particles contain a polymer containing siloxane bonds, can be suitably used as the particle substrate. Polystyrene particles can be produced as particles with a very uniform particle size distribution by the emulsion polymerization method described below. Furthermore, the silanols present in the polymer containing siloxane bonds can be used to provide a hydrophilic coating or ligand, as described below.

[0059] (Hydrophilic layer 3) The hydrophilic layer 3 is formed on the outer surface of the particle substrate 2 and is composed of molecules or polymers containing hydrophilic groups. Molecules containing hydrophilic groups refer to molecules or polymers containing hydroxyl groups, ethers, pyrrolidones, betaine structures, etc. Specifically, the hydrophilic layer 3 can be primarily composed of polyethylene glycol, polyvinylpyrrolidone, sulfobetaine, phosphobetaine polymers, or polyglycidyl methacrylate, in which glycidyl groups are ring-opened and hydroxyl groups are attached to the ends of the molecules. The hydrophilic layer 3 can be formed by attaching monomolecules containing hydrophilic groups to the surface of the silica particles 1 using a silane coupling agent or the like, or by forming the hydrophilic layer simultaneously with the synthesis of the particle substrate 2, as described below. The thickness of the hydrophilic layer 3 is not limited as long as it exhibits hydrophilicity. However, if the hydrophilic layer is too thick, it may become hydrogel-like and hydrate due to the influence of ions in the solvent, causing the thickness of the hydrophilic layer to change and become unstable. The thickness of the hydrophilic layer is preferably between 1 nm and 50 nm.

[0060] (Rare Earth Complex 4) The luminescent dye of the present invention preferably uses a luminescent rare earth complex because the wavelength and intensity of the emitted light are not easily affected by the surroundings and the emitted light has a long lifetime. The rare earth complex 4 is composed of a rare earth element and a ligand. The rare earth element is selected from europium, terbium, neodymium, erbium, yttrium, lanthanum, cerium, samarium, gadolinium, dysprosium, thulium, ytterbium, scandium, and the like. Considering the luminescence lifetime and the visible emission wavelength range, europium and terbium are particularly preferred. For example, europium generally has a luminescence lifetime of 0.1 to 1.0 ms. It is preferable to appropriately adjust the luminescence lifetime and the rotational relaxation time obtained from Equation 2. In the case of europium in an aqueous dispersion, if the first particles have a diameter of approximately 50 to 300 nm, the polarization anisotropy represented by Equation 4 changes significantly before and after aggregate formation.

[0061] At least one of the ligands constituting the rare earth complex 4 must have a light-harvesting function. The light-harvesting function refers to the ability to be excited by a specific wavelength and excite the central metal of the complex through energy transfer. Furthermore, the light-harvesting ligand is preferably a molecule with an anisotropic transition moment, such as phenanthroline, but is not limited to this. Furthermore, it is preferable that the ligands constituting the rare earth complex 4 contain a ligand such as a β-diketone, which prevents the coordination of water molecules. The β-diketone or other ligand coordinated to the rare earth ion suppresses the deactivation process due to energy transfer to solvent molecules, etc., resulting in strong fluorescence.

[0062] The rare earth complex 4 may be a polynuclear complex as long as it has an anisotropic transition moment.

[0063] It is desirable that the polarization anisotropy be 0.1 or more when the Brownian rotation of the rare-earth complex 4 in the medium can be considered to have stopped. The state in which the Brownian rotation can be considered to have stopped means that the rotational relaxation time of the particle is sufficiently longer than the luminescence lifetime of the rare-earth complex 4.

[0064] (Ligand 5) In this embodiment, a ligand refers to a compound that specifically binds to a target substance or a portion thereof. The ligand has a selective or specific affinity for the target substance. Examples of combinations of a target substance and a ligand, or a ligand and a target substance, include, but are not limited to, antigens and antibodies, enzyme proteins and their substrates, signal substances such as hormones or neurotransmitters and their receptors, and nucleic acids and nucleic acids. A representative example of a nucleic acid is deoxyribonucleic acid. In FIG. 1, the ligand 5 is contained in the second particle 7 and the first particle 1. The ligand 5 can be bound via a binding functional group present on the particle substrate 2 or the hydrophilic layer 3, or the ligand can be incorporated during the preparation of the particle substrate or the formation of the hydrophilic layer. Furthermore, a known ligand used in the latex agglutination method can be referred to and a similar ligand can be used as the ligand 5. Any ligand that causes agglutination in the latex agglutination method is likely to be effectively used in this embodiment. Representative examples of the ligand in this embodiment include antibodies, antigens, and nucleic acids.

[0065] (Target substance 6) The target substance 6 in Figure 1 is the substance to be measured, which exhibits affinity for and specifically adsorbs to the ligand 5. The target substance 6 can be any substance as long as the corresponding ligand is available. Examples of target substances include antibodies, antigens, nucleic acids, low-molecular-weight compounds, various receptors, enzymes, and substrates. Furthermore, allergens, cytokines, hormones, bacteria, viruses, DNA, RNA, cDNA, cells, cell membrane components, cancer markers, various disease markers, antibodies, blood-derived substances, food-derived substances, and natural product-derived substances are also included. The target substance 6 reacts with the ligands 5 of the first particle 1 and the second particle 7. This results in the formation of an aggregate 8 via the target substance 6. Note that Figure 1 shows a schematic example of one second particle 7, one target substance 6, and one first particle 1. In reality, both the first particle 1 and the second particle 7 have a large number of ligands, bind to a large number of target substances 6, and through these bind to a large number of further first particles 1 and / or second particles to form aggregates 8.

[0066] (Second Particle 7) The second particles (large affinity particles) 7 according to this embodiment have the same particle substrate 2, hydrophilic layer 3, and ligand 5 as the first particles 1, but do not necessarily contain the rare earth complex 4. The particles can have the same properties as the first particles except that they do not contain the rare earth complex 4 and have a different particle size. The larger the second particles 7, the greater the effect of causing a change in polarization anisotropy due to the formation of aggregates. On the other hand, if the particle size is too large, the dispersion stability in the liquid decreases, and the particles may settle. Furthermore, if the particles are too large, the light scattering per particle increases, and polarization may be lost. Therefore, the average particle size of the second particles 7 is preferably 100 nm or more and 5 μm or less. The second particles 7 preferably do not emit or absorb light to such an extent as to interfere with measurement in the wavelength range for measuring polarization anisotropy or in the wavelength range that excites the rare earth complex 4 that exhibits polarization anisotropy.

[0067] When a liquid in which the first particles 1 and the second particles 7 according to this embodiment are dispersed is used, the anisotropy of polarized luminescence in response to the aggregation and dispersion behavior of the particles can be detected with high sensitivity. Therefore, a colloidal liquid in which the first particles 1 and the second particles 7 are dispersed in an aqueous solvent can be used as a highly sensitive test reagent using the fluorescence depolarization method. A buffer solution can also be used as the aqueous solvent. Furthermore, to increase the stability of the dispersion, a surfactant, a preservative, a sensitizer, etc. may be added to the aqueous solvent.

[0068] (An example of a method for producing luminescent particles) Next, an example of a method for producing phosphor particles according to this embodiment will be described. The luminescent particles according to this embodiment are prepared by mixing a radical polymerizable monomer, a radical polymerization initiator, a rare earth complex, and a hydrophilic polymer with an aqueous medium to prepare an emulsion (first step). Furthermore, the emulsion is heated to polymerize the monomers (second step). The luminescent particles according to this embodiment are obtained by copolymerizing a compound having a radically polymerizable double bond. Furthermore, if necessary, a step (third step) of providing the surface of the light-emitting particle with a functional group capable of binding a ligand can be included. Here, the functional group capable of binding a ligand can be any of a carboxyl group, an amino group, a thiol group, an epoxy group, a maleimide group, a succinimidyl group, and a silicon alkoxide group.

[0069] (1. Radical Polymerizable Monomer) The radical polymerizable monomer may include a styrene-based monomer. Furthermore, a monomer selected from the group consisting of an acrylate-based monomer and a methacrylate-based monomer may be included. Examples of such monomers include butadiene, vinyl acetate, vinyl chloride, acrylonitrile, methyl methacrylate, methacrylonitrile, and methyl acrylate. In addition to the styrene-based monomer, at least one selected from the above group of monomers may be used. That is, these monomers may be used alone or in combination. Furthermore, a monomer having two or more double bonds in one molecule, such as divinylbenzene, may be used as a crosslinking agent.

[0070] Furthermore, a radically polymerizable monomer containing an organosilane may be further used to incorporate siloxane bonds into the luminescent particles. Examples of organosilane compounds include vinyltrimethoxysilane, vinyltriethoxysilane, p-styryltrimethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltriethoxysilane, and 3-acryloxypropyltrimethoxysilane. At least one selected from these monomer groups can be used. These organosilane compounds may be used alone or in combination. The radically polymerizable monomer containing an organosilane forms an inorganic oxide skeleton within the synthesized luminescent particles, improving the physical and chemical stability of the luminescent particles. Furthermore, the radically polymerizable monomer containing an organosilane enhances the affinity between the units having functional groups capable of bonding to ligands and the hydrophilic polymer on the surface of the luminescent particles and the skeleton material of the styrene-containing polymer microparticles.

[0071] When siloxane bonds are incorporated into the light-emitting particles, the ratio of units having siloxane bonds to styrene monomers is preferably 40% by mass or less.

[0072] Furthermore, by using radically polymerizable monomers containing organosilanes, silanol groups can be attached to the surface of the luminescent particles. Hydrogen bonding between the silanol groups and hydrophilic polymers, such as PV polyvinylpyrrolidone, allows the polyvinylpyrrolidone to adhere more firmly to the particle surface.

[0073] (2. Radical Polymerization Initiator) A wide range of radical polymerization initiators can be used, including azo compounds, organic peroxides, etc. Specific examples of radical polymerization initiators 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-methylpropionate) dimethyl, tert-butyl hydroperoxide, benzoyl peroxide, ammonium persulfate (APS), sodium persulfate (NPS), and potassium persulfate (KPS).

[0074] (3. Hydrophilic Polymers) Examples of hydrophilic polymers for suppressing nonspecific adsorption include hydrophilic polymers containing units having ether, betaine, or pyrrolidone rings. The hydrophilic polymers are contained in the synthesized luminescent particles and can be present mainly on the particle surface. As a preferred example, we will explain the use of a polymer having a pyrrolidone ring (hereinafter sometimes abbreviated as PVP).

[0075] PVP can be added during particle synthesis, and adding it simultaneously imparts nonspecific adsorption suppression and ligand binding capabilities to particles. Because PVP is more hydrophilic than radically polymerizable monomers, it exists at the interface between the solvent and the particles being formed during synthesis. PVP is adsorbed onto the particle surface by engulfing part of the PVP polymer chain during polymerization or by physical or chemical adsorption, such as by interactions between the pyrrolidone ring and styrene (radical polymerizable monomer).

[0076] The molecular weight of PVP is preferably 10,000 or more and 100,000 or less, and more preferably 40,000 or more and 70,000 or less. If the molecular weight is less than 10,000, the hydrophilicity of the surface of the luminescent particles is weak, making them prone to nonspecific adsorption. If the molecular weight is more than 100,000, the hydrophilic layer on the surface of the luminescent particles becomes too thick, causing the luminescent particles to gel and become difficult to handle.

[0077] In addition to PVP or separately from PVP, a further hydrophilic polymer may be added as a protective colloid during the synthesis of luminescent particles.

[0078] In one example of the light-emitting particles prepared as described above, 30 μL of a 0.1% by mass particle dispersion was added to 60 μL of buffer solution mixed with 16 μL of serum, and the mixture was left at 37°C for 5 minutes. The difference in absorption spectrum at a wavelength of 572 nm at an optical path of 10 mm before and after the addition was 0.1 or less. This indicates that nonspecific adsorption of impurities in serum was sufficiently small.

[0079] (4.Aqueous medium) The aqueous solution (aqueous medium) used in the synthesis preferably contains 80% to 100% water. Examples of aqueous solutions include water and solutions prepared by mixing water with a water-soluble organic solvent, such as methanol, ethanol, isopropyl alcohol, or acetone. If the solution contains more than 20% of an organic solvent other than water, the polymerizable monomer may dissolve during the production of luminescent particles.

[0080] Furthermore, when a radically polymerizable monomer containing an organic silane is used, the aqueous solution (aqueous medium) is preferably adjusted in advance to a pH of 6 or more and 9 or less. If the pH is less than 6 or more than 9, the alkoxide groups and silanol groups in the organic silane compound may undergo condensation polymerization or react with other functional groups before the formation of luminescent particles, which may cause the resulting particles to aggregate. In this embodiment, the alkoxide is not intentionally condensed before the formation of particles.

[0081] The pH is preferably adjusted using a pH buffer, but may also be adjusted using an acid or a base. In addition, surfactants, antifoaming agents, salts, thickeners, etc. may also be added in an amount of 10% or less relative to the aqueous medium.

[0082] In the production of polymer particles according to this embodiment, it is preferable to first dissolve PVP in an aqueous medium whose pH has been adjusted to 6 to 9. PVP is added in an amount of 0.01 to 10% by mass, preferably 0.03 to 5% by mass, relative to the aqueous medium. If the amount is less than 0.01% by mass, the amount adsorbed to the polymer particles is small, and the effect is not achieved. If the amount is more than 10% by mass, the viscosity of the aqueous medium increases, which may prevent sufficient stirring.

[0083] Next, a radical polymerizable monomer (A) is added to the aqueous medium to form an emulsion. At this time, a radical polymerizable monomer (B) containing an organosilane may also be added to the aqueous medium in addition to (A) to form an emulsion. The mass ratio of (A) to (B) is 6:4 to 10:0. Furthermore, a luminescent rare earth complex is mixed with the prepared monomer solution. If the solubility of the luminescent rare earth complex is low, a water-insoluble organic solvent may be added. The mass ratio of the luminescent rare earth complex to the monomer is 1:1000 to 1:10.

[0084] If (A) is less than 60%, the specific gravity of the particles as a whole increases, which may result in significant particle settling. Although (B) is not necessary, it is more preferable to add (B) in order to improve the adhesion between the PVP and the luminescent particles.

[0085] The mass ratio of the aqueous medium to the total amount of (A) and (B) is preferably 5:5 to 9.5:0.5. If the ratio of the aqueous medium is 50% or less, nonspecific aggregation of the generated particles may become a problem. Even if the ratio is 95% or more, there is no problem with the generation of luminescent particles, but the amount generated may be low.

[0086] The radical polymerization initiator is used by dissolving it in water, a buffer, etc. The radical polymerization initiator can be used in an amount of 0.5 to 10% by mass relative to the total mass of (A) and (B).

[0087] The emulsion is heated at any temperature between 50°C and 80°C for a period of time between 2 and 24 hours. The emulsion is heated to polymerize the monomers.

[0088] The functional group capable of binding a ligand is preferably a functional group capable of binding a protein, nucleic acid, peptide, amino acid, amino group, or the like. Examples of such functional groups include a carboxy group, an amino group, a thiol group, an epoxy group, a maleimide group, a succinimidyl group, and a silicon alkoxide group. By mixing a silane coupling agent having a functional group capable of binding a ligand with synthesized luminescent particles, the functional group can be attached to the surface of the luminescent particles. Specifically, for example, an aqueous solution of a silane coupling agent having a carboxy group can be prepared and mixed with a dispersion of synthesized luminescent particles to attach a carboxy group to the particle surface. A dispersant such as Tween 20 may be added to the reaction solution. The reaction temperature can be set between 0°C and 80°C, and the reaction time can be set between 1 hour and 24 hours. To prevent rapid condensation of the silane coupling agent, it is preferable to set the reaction temperature at room temperature (approximately 25°C) or lower and the reaction time to approximately 3 hours to 14 hours. Depending on the functional group to which the ligand can be bound, an acid or alkali catalyst may be added to promote the reaction on the particle surface.

[0089] By binding various types of ligands such as antibodies to particles synthesized by emulsion polymerization, etc., it is possible to confer affinity for target substances. The method for binding the ligand is not particularly limited, and an optimal method for binding the target antibody using functional groups present in the hydrophilic layer 3 or the particle substrate 2 may be selected.

[0090] (Method of manufacturing large particles) Large particle diameters can be obtained by the same method as in the above synthesis of luminescent particles, except for the step of introducing the rare earth complex 4. However, the large particle may contain a fluorescent dye different from the rare earth complex 4 .

[0091] (affinity particles) In this embodiment, a ligand refers to a compound that specifically binds to a specific target substance. The site at which the ligand binds to the target substance is fixed, and the ligand has a selective or specific high affinity. For example, typical examples of combinations of a target substance and a ligand, or a ligand and a target substance, include the following: antigens and antibodies, enzyme proteins and their substrates, signal substances such as hormones or neurotransmitters and their receptors, nucleic acids, etc. However, the ligands in this embodiment are not limited to these. Examples of nucleic acids include deoxyribonucleic acid, etc. The affinity particles in this embodiment have a selective or specific affinity for the target substance. Typical examples of ligands in this embodiment include antibodies, antigens, and nucleic acids. The method for chemically bonding the reactive functional group of the particle according to the present embodiment to the ligand can be any conventionally known method within the scope of achieving the object of the present invention. In addition, when bonding the ligand to an amide bond, a catalyst such as 1-[3-(dimethylaminopropyl)-3-ethylcarbodiimide] can be appropriately used. When the affinity particles of this embodiment use an antibody (antigen) as the ligand and an antigen (antibody) as the target substance, they can be preferably applied to immuno-latex agglutination assays, which are widely used in fields such as clinical testing and biochemical research.

[0092] (Test kit) The test kit according to this embodiment includes first particles and second particles. Furthermore, the test kit may include a dispersion medium. In the test kit, the first particles and the second particles may be dispersed in a dispersion medium. The test kit may also include a reagent containing the first particles, the second particles, and a dispersion medium. The amount of affinity particles according to this embodiment contained in the reagent according to this embodiment is preferably 0.000001% by mass to 20% by mass, more preferably 0.0001% by mass to 1% by mass. The reagent according to this embodiment may include a third substance, such as a solvent or a blocking agent, in addition to the affinity particles according to this embodiment, as long as the object of the present invention can be achieved. Two or more types of third substances, such as solvents or blocking agents, may be used in combination. Examples of solvents used in this embodiment include various buffer solutions, such as phosphate buffer, glycine buffer, Good's buffer, Tris buffer, and ammonia buffer, but the solvents contained in the reagent according to this embodiment are not limited to these.

[0093] The test kit may also include the reagent and a housing that contains the reagent. The kit according to the present embodiment may also contain a sensitizer that promotes particle aggregation via the target substance. Examples of sensitizers include, but are not limited to, polyvinyl alcohol, polyvinylpyrrolidone, and polyalginic acid. The kit according to the present embodiment may also include a positive control, a negative control, a serum dilution solution, and the like. As a medium for the positive control and the negative control, serum not containing the target substance, physiological saline, or a solvent may be used. The kit according to the present embodiment can be used in the target substance detection method according to the present embodiment in the same manner as a kit used for detecting a target substance in a sample by conventional in vitro diagnosis. Furthermore, the concentration of the target substance can also be measured by a conventionally known method, and is particularly suitable for use in detecting a target substance in a sample by latex agglutination.

[0094] (Detection method) The method for determining the presence or concentration of a target substance according to this embodiment includes a step of mixing first particles according to this embodiment, second particles, and a sample solution that may contain the target substance. The mixing is preferably performed at a pH range of 3.0 to 11.0. The mixing temperature is preferably 0°C to 100°C, or 4°C to 50°C, or even 20°C to 50°C, and the mixing time is preferably 1 second to 2 hours, or 1 minute to 60 minutes. This method preferably uses a solvent. In the method according to this embodiment, the concentrations of the first particles and the second particles are preferably as follows: The total amount of the first particles and the second particles in the reaction system is preferably 0.000001% by mass to 1% by mass, and more preferably 0.00001% by mass to 0.001% by mass. The detection method according to this embodiment detects the agglutination reaction that occurs as a result of mixing the first particles, the second particles, and the sample using fluorescence depolarization. Specifically, the detection method includes the steps of mixing a test reagent with a sample to obtain a mixed solution, irradiating the mixed solution with polarized light, and separating and detecting the polarized components of the light emitted by the affinity particles in the mixed solution. By optically detecting the above-mentioned agglutination reaction occurring in the mixed solution, the target substance in the specimen can be detected, and further the concentration of the target substance can also be measured. [Example]

[0095] The present invention will be specifically described below with reference to examples, although the present invention is not limited to these examples.

[0096] (1) Preparation of luminescent particles Solvent A was prepared by dissolving polyvinylpyrrolidone (PVP-K30, manufactured by Tokyo Chemical Industry Co., Ltd.) and sodium dodecyl sulfate (manufactured by Wako Pure Chemical Industries, Ltd., hereafter abbreviated as SDS) in a pH 7 MES (2-morpholinoethanesulfonic acid) buffer solution (manufactured by Kishida Chemical Co., Ltd.). The rare earth complex used was Eu(TTA)3Phen, manufactured by Central Techno Co., Ltd. Eu(TTA)3Phen is also known as [tris(2-thenoyltrifluoroacetonato)(1,10-phenanthroline)europium(III)]. Eu(TTA)3Phen is also known as [Tris(2-thenoyltrifluoroacetonato)(1,10-phenanthroline)europium(III)]. Reaction solution B was prepared by mixing Eu(TTA)3Phen, styrene monomer (Kishida Chemical Co., Ltd.), and 3-methacryloxypropyltrimethoxysilane (Tokyo Chemical Industry Co., Ltd., hereafter abbreviated as MPS). Reaction solution B was added to solvent A in a four-neck flask and stirred using a mechanical stirrer set at 300 rpm. After 30 minutes of stirring under nitrogen flow, the temperature of the prepared oil bath was set to 70°C and nitrogen flow was continued for another 30 minutes. After heating and stirring, an aqueous solution of potassium persulfate (Aldrich, KPS) was added to the reaction solution and emulsion polymerization was carried out for 10 hours. After the reaction, the resulting suspension was centrifuged to remove the supernatant, and the precipitate was redispersed in pure water. This centrifugation and redispersion process was repeated three times to wash the product. The resulting precipitate was redispersed in pure water to obtain a luminescent particle dispersion. The weight ratios of the reagents used are shown in Table 1.

[0097] [Table 1]

[0098] (2) Preparation of large particles Solvent A was prepared by dissolving polyvinylpyrrolidone (PVP-K30, manufactured by Tokyo Chemical Industry Co., Ltd.) and sodium dodecyl sulfate (SDS, manufactured by Wako Pure Chemical Industries, Ltd.) in a pH 7 MES (2-morpholinoethanesulfonic acid) buffer solution (manufactured by Kishida Chemical Co., Ltd.). Reaction solution B was prepared by mixing styrene monomer (manufactured by Kishida Chemical Co., Ltd.) and 3-methacryloxypropyltrimethoxysilane (MPS, manufactured by Tokyo Chemical Industry Co., Ltd.). Reaction solution B was added to solvent A in a four-neck flask and stirred using a mechanical stirrer set to 300 rpm. After stirring for 30 minutes under nitrogen flow, the temperature of the oil bath was set to 70 °C and nitrogen flow was continued for another 30 minutes. After heating and stirring, an aqueous solution of potassium persulfate (KPS, manufactured by Aldrich) was added to the reaction solution and emulsion polymerization was carried out for 10 hours. After the reaction, the resulting suspension was centrifuged to remove the supernatant, and the precipitate was redispersed in pure water. This centrifugation and redispersion process was repeated three times to wash the product. The resulting precipitate was redispersed in pure water to obtain a large particle dispersion. The weight ratios of the reagents used are shown in Table 2.

[0099] [Table 2]

[0100] An aliquot of the dispersion of luminescent particles and large-sized particles obtained by emulsion polymerization was added to an aqueous solution containing 1 wt% Tween 20 (Kishida Chemical Co., Ltd.). After stirring for 10 minutes, a silane coupling agent with carboxylic acid as the organic functional group, X12-1135 (Shin-Etsu Chemical Co., Ltd.), was added and stirred overnight. After stirring, the dispersion was centrifuged, the supernatant was removed, and the precipitate was redispersed in pure water. This centrifugation and redispersion process was repeated three or more times to wash the product. After washing, the precipitate was redispersed in pure water to obtain a dispersion of luminescent particles and large-sized particles. The weight ratio of particles, pure water, and X12-1135 was 1:300:2.

[0101] (3) Preparation of anti-CRP antibody-modified affinity particles A 0.25 mL aliquot of a 1.2 wt% particle dispersion of the synthesized luminescent particles and large-sized particles was taken, and the solvent was replaced with 1.6 mL of pH 6.0 MES buffer. 0.5 wt% 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide and 0.5 wt% sodium N-hydroxysulfosuccinimide were added to the particle MES buffer and allowed to react at 25°C for 1 hour. After the reaction, the dispersion was washed with pH 5.0 MES buffer, and 100 μg / mL of anti-CRP antibody was added. The anti-CRP antibody was allowed to bind to the particles for 2 hours at 25°C. After binding, the particles were washed with pH 8 Tris buffer. After the reaction, the particles were washed with phosphate buffer to obtain 0.3 wt% CRP antibody-modified affinity particles. The binding of the antibody was confirmed by measuring the decrease in the antibody concentration in the buffer solution to which the antibody was added using a BCA assay.

[0102] (4) Reaction of affinity particles with antigen solution The resulting CRP antibody-modified affinity particles were mixed with CRP antigen diluted in MES buffer, and the change in fluorescence polarization was observed before and after. The CRP antibody-modified affinity particles were fixed at 0.0001 mg / mL, and the CRP antigen concentration was varied from 0 to 1000 pg / mL. Observations were performed at room temperature. The method for measuring fluorescence polarization is described below.

[0103] Example 1 A sample corresponding to the synthesized light-emitting affinity particles-1 and a sample corresponding to the synthesized large-diameter affinity particles-1 were mixed at a weight ratio of 9:1 to prepare a dispersion. CRP antigen diluted with MES buffer was mixed with the prepared dispersion, and the change in fluorescence polarization before and after mixing was observed. The total amount of particles in the dispersion was 0.001 mg / mL, and the CRP antigen was 1 pg / mL. Measurements were performed at room temperature.

[0104] Example 2 The same study was carried out as in Example 1, except that the sample corresponding to the synthesized light-emitting affinity particles-1 and the sample corresponding to the synthesized large particle size affinity particles-1 were mixed in a weight ratio of 5:5.

[0105] Example 3 The same study was carried out as in Example 1, except that the sample corresponding to the synthesized light-emitting affinity particles-1 and the sample corresponding to the synthesized large particle size affinity particles-1 were mixed in a weight ratio of 1:9.

[0106] Example 4 A dispersion was prepared by mixing a sample corresponding to the synthesized light-emitting affinity particles-2 with a sample corresponding to the synthesized large-diameter affinity particles-2 in a weight ratio of 5:5. CRP antigen diluted with MES buffer was mixed with the prepared dispersion, and the change in fluorescence polarization before and after mixing was observed. The total amount of particles in the dispersion was 0.001 mg / mL, and the CRP antigen was 10 pg / mL. Measurements were performed at room temperature.

[0107] (Comparative Example 1) A dispersion of a sample equivalent to the synthesized luminescent affinity particle-1 was prepared. The prepared dispersion was mixed with CRP antigen diluted with MES buffer, and the change in fluorescence polarization before and after mixing was observed. The total amount of particles in the dispersion was 0.001 mg / mL, and the CRP antigen was 1 pg / mL. Measurements were performed at room temperature.

[0108] (Comparative Example 2) A dispersion of a sample equivalent to the synthesized large affinity particles-1 was prepared. CRP antigen diluted with MES buffer was mixed with the prepared dispersion, and the change in fluorescence polarization before and after mixing was observed. The total amount of particles in the dispersion was 0.001 mg / mL, and the CRP antigen was 1 pg / mL. Measurements were performed at room temperature.

[0109] (Comparative Example 3) A dispersion of a sample corresponding to the synthesized luminescent affinity particle-2 was prepared. The prepared dispersion was mixed with CRP antigen diluted with MES buffer, and the change in fluorescence polarization before and after mixing was observed. The total amount of particles in the dispersion was 0.001 mg / mL, and the CRP antigen was 1 pg / mL. The measurement was carried out at a temperature of .

[0110] (Evaluation of the product) The products in the examples and comparative examples were evaluated as follows. The shape of the product was evaluated using an electron microscope (S5500 manufactured by Hitachi High-Technologies). The average particle size of the product was evaluated using dynamic light scattering (Malvern Zetasizer Nano S). The concentration of the suspension in which the product was dispersed was evaluated using a mass spectrometer (Rigaku Thermoplus TG8120). The fluorescence spectrum of the dispersion containing the product was measured at room temperature with an excitation light of 340 nm, with polarizers inserted in the light paths on both the excitation and emission sides. The polarizer was oriented with the excitation side fixed and the emission side either parallel or perpendicular to the excitation side. The instrument used was a Hitachi High-Tech Science F-4500 spectrofluorometer. The peak wavelength of the observation light used to analyze the polarized emission was 611 nm. The polarization anisotropy r was calculated by analyzing the obtained polarized emission data using Equation 4. The evaluation of the product's inhibition of nonspecific aggregation was carried out as follows. To 30 μl of each of the luminescent affinity particle dispersions (3 mg / mL) prepared in Examples 1 to 4 and Comparative Examples 1 to 3, 60 μl of a human serum solution diluted 15-fold with a buffer solution was added, and the mixture was incubated for 5 minutes at 37° C. The absorbance at 527 nm was measured before and after incubation, and the change in absorbance before and after incubation was measured three times.

[0111] (Performance evaluation) As a result of the evaluation of nonspecific aggregation inhibition, the change in absorbance was below the specified value in all of the examples and comparative examples. That is, the change in absorbance was below the specified value of 0.01 in all of the examples 1 to 4 and comparative examples 1 to 3, confirming that the luminescent particles are capable of inhibiting nonspecific adsorption. The structures and physical properties of the particulate materials of Examples 1, 2, and 3 and Comparative Examples 1 and 2 are shown in Table 3.

[0112] [Table 3]

[0113] The size of the luminescent affinity particles-1 in Examples 1, 2, 3, and Comparative Example 1 was 98 nm, and the PDI values ​​were 0.024, respectively, confirming that they were monodisperse particles below 0.1. The size of the large-sized particles-1 in Examples 1, 2, 3, and Comparative Example 2 was 347 nm, and the PDI values ​​were 0.082, respectively, confirming that they were monodisperse particles below 0.1. Comparing the polarization anisotropy r values ​​after the reaction and the change in polarization anisotropy Δr before and after the reaction, it was found that the Examples containing large-sized affinity particles were higher than the value in Comparative Example 1, which did not contain large-sized affinity particles. Furthermore, it was found that the polarization anisotropy r values ​​after the reaction and the change in polarization degree Δr before and after the reaction increased in the order of Examples 3, 2, and 1, which had the highest proportion of large-sized affinity particles mixed in. Comparing Example 3, which had the largest change in polarization degree, with Comparative Example 1, it was found that the Δr value was 60 times larger. On the other hand, in Comparative Example 2, which does not contain light-emitting particles, polarization anisotropy could not be detected.

[0114] The structures and physical properties of the particulate materials of Example 4 and Comparative Example 3 are shown in Table 4. [Table 4]

[0115] The size of the luminescent affinity particles-2 in Example 4 and Comparative Example 3 was 207 nm, and the pdi values ​​were both 0.039, confirming that they were monodisperse particles below 0.1. The size of the large affinity particles-2 in Example 4 was 275 nm, and the pdi values ​​were both 0.082, confirming that they were monodisperse particles below 0.1. Comparing the polarization anisotropy r after the reaction and the change in polarization anisotropy Δr before and after the reaction, it was found that Example 4, in which large affinity particles were mixed, had a higher value than Comparative Example 3, which did not contain large affinity particles. The polarization anisotropy determined by fluorescence depolarization depends on the particle size (aggregate size) and is proportional to the cube of the particle (aggregate) radius. The measurement results in the present example were obtained by examining an aggregation reaction that increases the particle (aggregate) size, and the results were reasonable and consistent with the measurement principle of fluorescence depolarization.

[0116] From the above, it was demonstrated that the measurement method according to the present invention has extremely high detection sensitivity, and that highly sensitive measurement is possible using the test kit of the present invention, or the luminescent affinity particles and large particle size affinity particles. [Explanation of symbols]

[0117] 1. First particle (luminescent affinity particle) 2 Particle matrix 3 Hydrophilic layer 4 Rare Earth Complexes 5. Ligand 6 Target substance 7 Secondary particles (large affinity particles) 8 aggregates< / r> < / r>

Claims

1. A method for determining at least one of the presence or absence of a target substance in a sample solution and the concentration of the target substance, comprising: (a) preparing first particles having an average particle size of 10 nm or more and 1 μm or less, which specifically bind to the target substance and contain a rare earth complex, and second particles having an average particle size of 100 nm or more and 5 μm or less, which have an average particle size larger than that of the first particles and specifically bind to the target substance; (b) mixing the sample liquid, the first particles, and the second particles to obtain a mixed liquid; (c) determining at least one of the presence or absence of the target substance and the concentration of the target substance from the polarization anisotropy of the mixed liquid obtained in (b).

2. The method of claim 1, wherein the polarization anisotropy is determined by Equation 1. [Equation 1] (however, <r>...Polarization anisotropy I ＶＶ ...Emission intensity of the emission component whose vibration direction is parallel to the first polarized light when excited with the first polarized light I ＶＨ ...Emission intensity of the emission component whose vibration direction is perpendicular to the first polarization when excited with the first polarization I ＨＶ ...Emission intensity of the emission component whose vibration direction is perpendicular to the second polarized light when excited with the second polarized light whose vibration direction is perpendicular to the first polarized light I ＨＨ ...Emission intensity of the emission component whose vibration direction is parallel to that of the second polarized light when excited with the second polarized light whose vibration direction is perpendicular to that of the first polarized light G: Correction value (It is).

3. A method for detecting a target substance comprising: a first particle that specifically binds to a target substance; and a second particle that specifically binds to the target substance; A test kit for determining at least one of the presence or absence of the target substance in a sample liquid and the concentration of the target substance from the polarization anisotropy of a mixed liquid obtained by mixing the sample liquid with the first particles and the second particles, comprising: the average particle size of the second particles is larger than the average particle size of the first particles; the first particles have a rare earth complex; the first particles have an average particle size of 10 nm or more and 1 μm or less; A test kit characterized in that the average particle size of the second particles is 100 nm or more and 5 μm or less.

4. The first particles are It contains polystyrene and a polymer having a siloxane bond, the first particles contain a hydrophilic polymer on the surface thereof; The first particle contains a rare earth complex therein. The test kit of claim 3.

5. The rare earth complex is a complex of a rare earth selected from europium, terbium, neodymium, erbium, yttrium, lanthanum, cerium, samarium, gadolinium, dysprosium, thulium, ytterbium, and scandium. The test kit according to claim 3 or 4.

6. The polarization anisotropy exhibited by the aggregates formed by the mixing in a dispersion medium is greater than the polarization anisotropy exhibited by the first particles in a dispersion medium by 0.004 or more. The test kit according to any one of claims 3 to 5, However, the polarization anisotropy is determined by the following formula 1. [Equation 2] (however, <r>...Polarization anisotropy I VV ... Emission intensity of the emission component whose vibration direction is parallel to that of the first polarized light when excited with the first polarized light IVH: Emission intensity of the emission component whose vibration direction is perpendicular to that of the first polarized light when excited with the first polarized light I HV : Emission intensity of the emission component whose vibration direction is perpendicular to that of the second polarized light when excited with the second polarized light whose vibration direction is perpendicular to that of the first polarized light I HH : Emission intensity of the emission component whose vibration direction is parallel to that of the second polarized light when excited with the second polarized light whose vibration direction is perpendicular to that of the first polarized light. G: Correction value (It is).

7. 7. The test kit according to claim 6, wherein the aggregate exhibits a polarization anisotropy of 0.1 or more in a dispersion medium.

8. The test kit according to any one of claims 3 to 7, characterized in that the excitation wavelength of the first particles and the excitation wavelength of the second particles are different, or the emission wavelength of the first particles and the emission wavelength of the second particles are different, or the excitation wavelength of the first particles and the excitation wavelength of the second particles are different and the emission wavelength of the first particles and the emission wavelength of the second particles are different.

9. 9. The test kit according to claim 3, wherein the second particles are not excited by excitation light having a wavelength of 300 nm or more and 450 nm or less.

10. 10. The test kit according to claim 3, wherein the second particles do not have an emission maximum in a wavelength region of 550 nm or more and 650 nm or less.

11. The average particle size of the first particles is 20 nm or more and 400 nm or less. The test kit according to any one of claims 3 to 10.

12. 12. The test kit according to claim 3, wherein the first particles and the second particles each have a ligand that specifically binds to the target substance or a portion thereof.

13. 13. The test kit of claim 12, wherein the ligand is selected from an antibody, an antigen, and a nucleic acid.

14. A test kit described in any one of claims 3 to 13, characterized in that at least one of the first particles and the second particles has a layer on the particle surface containing a hydrophilic polymer containing either an ether, a betaine, or a pyrrolidone ring.

15. 15. The test kit according to claim 3, wherein the first particles and the second particles are dispersed in a dispersion medium.

16. The test kit according to any one of claims 3 to 15, wherein the test kit is contained in a housing.

17. The ligand is an antigen or an antibody, 14. The test kit according to claim 12 or 13, characterized in that at least one of the presence or absence of the target substance in the sample solution and the concentration of the target substance is determined from a change in polarization anisotropy that occurs when the first particles and the second particles aggregate via the target substance due to an antigen-antibody reaction.

18. A test kit described in any one of claims 3 to 17, wherein the weight ratio of the first particles to the second particles is within the range of 9:1 to 1:

9.

19. A test kit described in any one of claims 3 to 18, wherein the weight ratio of the first particles to the second particles is within the range of 1:1 to 1:9.

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