Analysis method and analyzer by measurement based on polarization anisotropy

Abstract

To perform a reaction of a target substance and a luminescent reagent in a short time and perform measurement based on polarization anisotropy with high sensitivity.SOLUTION: An analysis method is to measure a value (R) related to polarization anisotropy by using a luminescent reagent reacting with a target substance, thereby determining at least one of the presence or absence of the target substance and the concentration of the target substance, and the method includes: a reaction step of mixing a sample including the target substance with the luminescent reagent and reacting the mixed solution to obtain a reaction solution; a dilution step of diluting the reaction solution to obtain a diluted solution; and a measuring step of measuring the R of the diluted solution. The luminescent reagent includes luminous particles.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This invention relates to an analysis method and an analysis apparatus based on polarization anisotropy measurement. [Background technology]

[0002] In the fields of medicine and clinical testing, the highly sensitive detection or quantification of trace amounts of biological components from blood or collected organ tissue is necessary for investigating the causes and presence of diseases. Among the various methods for testing biological components, immunoassay is widely used. Many immunoassays require a washing step called B / F (Bound / Free) separation. One immunoassay that does not require B / F separation is the latex agglutination method, which utilizes antigen-antibody reactions. In the latex agglutination method, latex particles carrying antibodies that specifically bind to a target substance are mixed with a liquid that may contain the target substance, and the degree of agglutination of the latex particles is measured.

[0003] In the latex agglutination method, the target substance is captured by antibodies specific to the target substance bound to latex particles. Multiple latex particles then crosslink through the captured target substance, resulting in the aggregation of latex particles. In other words, the amount of the target substance in a liquid sample, such as a biological sample, can be quantified by evaluating the degree of latex particle aggregation. This degree of aggregation can be quantified by measuring and evaluating the change in the amount of light transmitted or scattered through the liquid sample.

[0004] While the latex agglutination method allows for the simple and rapid detection and quantitative evaluation of target substances, such as antigens, it has a limitation in its detection limit, meaning it cannot detect antigens in small amounts in liquid samples such as biological samples.

[0005] To improve the detection sensitivity of target substances, it is necessary to measure the degree of aggregation with higher sensitivity. In other words, it is conceivable to replace systems that measure changes in the amount of light transmitted or scattered through a liquid sample with methods that utilize more sensitive luminescence characteristics for detection and quantification. Specifically, sample testing methods using fluorescence depolarization have been proposed (Patent Documents 1 and 2).

[0006] Patent Document 1 proposes improvements to a fluorescence polarization depolarization apparatus for clinical use. The fluorescence polarization depolarization method does not require B / F separation, which is necessary in general fluorescence measurement methods. Therefore, using the fluorescence depolarization method allows for simple sample testing, similar to the latex agglutination method. Furthermore, it is believed that using the fluorescence depolarization method allows for measurement using the same testing system as the latex agglutination method, simply by mixing a luminescent substance that specifically reacts with the target substance. On the other hand, Patent Document 1 proposes using single molecules such as fluorescein as the luminescent material, and in principle, this could only be applied to drugs or low-molecular-weight antigens.

[0007] Patent Document 2 addresses the problem in Patent Document 1, which was that the depolarization method of fluorescence could only be applied to drugs and low-molecular-weight antigens. Specifically, Patent Document 2 aims to apply the depolarization method of fluorescence to macromolecules such as proteins, and proposes using a material in which a dye with long-life luminescence properties is adsorbed onto latex particles as the luminescent material. Patent Document 2 proposes the quantitative determination of macromolecules by balancing the decrease in rotational Brownian motion of the substance in the liquid, which occurs as the particle size increases, with the length of the luminescence lifetime, based on the principle of the depolarization method of fluorescence. However, in Patent Document 2, since the fluorescent substance is supported on the particles after the latex particle synthesis, it is difficult to stably determine the polarization anisotropy of the inspection particles due to interactions between fluorescent substances adsorbed near the particle surface. Furthermore, in Patent Document 2, in order to suppress non-specific adsorption, the particles are supported with bovine serum albumin (BSA), a biomolecule, on their surface, resulting in a wide particle size distribution and the possibility of variations between lots due to the protein BSA. Therefore, the concentration of the target substance is measured on the order of μg / mL, and there is no significant difference in measurement sensitivity compared to the latex method. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Special Publication No. 3-52575 [Patent Document 2] Patent No. 2893772 [Overview of the project] [Problems that the invention aims to solve]

[0009] Measurements based on depolarization fluorescence require sufficient reaction between the target substance and the luminescent substance. However, the reaction can take a long time, for example, if the affinity between the target substance and the luminescent substance is weak. A challenge in measurements using depolarization fluorescence has been to perform measurements in the shortest possible time while maintaining high sensitivity. [Means for solving the problem]

[0010] As one embodiment of the present invention, the present invention provides an analytical method for determining at least one of the presence or absence of a target substance and the concentration of a target substance by measuring a value (R) related to polarization anisotropy using a luminescent reagent that reacts with the target substance. A reaction step in which a sample containing the target substance and the luminescent reagent are mixed and reacted to obtain a reaction solution, A dilution step to obtain a diluted solution by diluting the reaction solution, and A measurement step of measuring the R of the diluent, It has, and further, The present invention provides an analytical method characterized in that the luminescent reagent contains luminescent particles.

[0011] In addition, as one embodiment, the present invention provides an analytical apparatus that measures at least one of the presence or absence of a target substance and the concentration of the target substance by measuring a value (R) related to polarization anisotropy using a luminescent reagent that reacts with the target substance, a reaction unit that mixes and reacts a sample containing the target substance and the luminescent reagent to obtain a reaction solution, a dilution unit that dilutes the reaction solution to obtain a diluted solution a measurement unit that measures the R of the diluted solution, and a control unit and is characterized in that the luminescent reagent contains luminescent particles. An analytical apparatus is provided.

Effects of the Invention

[0012] According to the analytical method according to the embodiment of the present invention, in the reaction step, since the sample containing the target substance and the luminescent reagent react in a concentrated state, the reaction time can be shortened. On the other hand, since the reaction solution is diluted in the dilution step, in the measurement step, the scattering of the luminescent reagent is suppressed, and highly sensitive measurement becomes possible. Also, when the viscosity of the solution during measurement is too high, the value related to polarization anisotropy becomes high, and the change in R cannot be captured significantly. However, this problem is also solved by providing a dilution step. Further, it has been found that by shortening the optical path length of the optical system when measuring R, the influence of multiple scattering can be further suppressed.

Brief Description of the Drawings

[0013] [Figure 1] It is a schematic diagram for explaining the analytical method according to the embodiment of the present invention. [Figure 2] It is a schematic diagram for explaining the apparatus according to the embodiment of the present invention. [Figure 3] It is a schematic diagram for explaining the luminescent reagent used in the embodiment of the present invention. [Figure 4] It is a diagram for explaining the result of quantifying the CRP antigen concentration using the analytical method according to the embodiment of the present invention. [Figure 5]This is a diagram for explaining the result of quantifying the CRP antigen concentration using the analysis method according to an embodiment of the present invention. [Figure 6] This is a diagram for explaining the result of quantifying the TSH antigen concentration using the analysis method according to an embodiment of the present invention.

Mode for Carrying Out the Invention

[0014] Hereinafter, preferred embodiments of the present invention will be described in detail, but the scope of the present invention is not limited thereby. As one of the embodiments, the present invention provides the following analysis method. An analysis method for determining at least one of the presence or absence of a target substance and the concentration of the target substance by measuring a value (R) related to polarization anisotropy using a luminescent reagent that reacts with the target substance, A reaction step of mixing a sample containing the target substance and the luminescent reagent and reacting them to obtain a reaction solution, A dilution step of diluting the reaction solution to obtain a diluted solution, and A measurement step of measuring the R of the diluted solution, which has, and further, The analysis method characterized in that the luminescent reagent contains luminescent particles.

[0015] The analysis method according to the present embodiment solves the problem of performing a measurement based on polarization anisotropy capable of performing a reaction between a target substance and a luminescent reagent in a short time and enabling highly sensitive measurement. The reaction between the target substance and the luminescent reagent can be exemplified by the binding of the target substance and a substance (for example, a ligand described later) that can bind to the target substance among the substances contained in the luminescent reagent. The inventors of the present application focused on the fact that in order to shorten the reaction time, it is preferable that the sample containing the target substance and the luminescent reagent react in a concentrated state, particularly when the amount of the target substance is small or when the affinity between the target substance and the luminescent reagent is low. Here, by including luminescent particles containing multiple fluorescent substances in the luminescent reagent, the change in the size of the luminescent reagent after the reaction can be greatly increased. As a result, the change in the value (R) based on polarization anisotropy can be captured more significantly, enabling highly sensitive measurements. Furthermore, by including luminescent particles in the luminescent reagent and making them particle-shaped, it becomes possible to support a large number of ligands capable of capturing the target substance per particle of the luminescent reagent, thereby increasing the reactivity to the target substance. As a result, highly sensitive measurements become possible. On the other hand, when measuring R, if the concentration of the luminescent reagent is high, multiple scattering occurs, making it difficult to capture significant changes in R. In other words, when multiple scattering occurs, the R value also increases for luminescent reagents that have not reacted with the target substance and have not aggregated, making it difficult to capture changes in R before and after reaction with the target substance. The inventors of this application have found that by concentrating the target substance and luminescent reagent in the above reaction step, and then going through a dilution step, it is possible to realize an analytical method based on polarization anisotropy that has a short reaction time and high sensitivity. In other words, the objective is to provide an analysis method and an analysis apparatus that can determine, in a short time and with high sensitivity, the presence or absence of a target substance and the concentration of the target substance based on polarization anisotropy, using the analysis method according to the present embodiment described above.

[0016] (Values ​​related to polarization anisotropy) In this embodiment, the value relating to polarization anisotropy (sometimes denoted as R) is defined as follows. That is, for light emitted when a light-emitting material is excited by irradiating it with polarized light, R is a value that shows the relationship between the light emission intensity of the polarization component parallel to the irradiated polarization and the light emission intensity of the polarization component perpendicular to the irradiated polarization. More specifically, R is a value calculated by determining the light emission intensity of the light emission component whose direction of vibration is parallel to the polarization when the light-emitting material is excited with a certain polarization. Furthermore, R is a value that shows the ratio of the difference between the light emission intensity of the light emission component whose direction of vibration is parallel to the first polarization when excited with the first polarization, and the light emission intensity of the light emission component whose direction of vibration is perpendicular to the first polarization when excited with the first polarization, and the sum of these two values. However, R may be corrected by the ratio of the light emission intensity of the light emission component whose direction of vibration is perpendicular to the second polarization when excited with a second polarization whose direction of vibration is perpendicular to the first polarization, and the light emission intensity of the light emission component whose direction of vibration is parallel to the second polarization when excited with a second polarization whose direction of vibration is perpendicular to the first polarization, and other constants. Values ​​related to polarization anisotropy include values ​​referred to as polarization anisotropy, degree of polarization, etc.

[0017] More specifically, for example, R can be the r in equation (1) below.

number

[0018] Furthermore, R can be represented as r' in equation (2) below.

number

[0019] The conditions for measuring R are preferably, for example, in a liquid at a temperature of 0°C to 50°C, with a viscosity of 0.5 mPa·s to 50 mPa·s. If the luminescent reagent is a particle containing a europium complex, it is preferable to measure the concentration of the luminescent reagent at 0.001 mg / ml to 0.1 mg / ml, and the measurement wavelength (excitation wavelength) is preferably 500 nm to 700 nm. Furthermore, for R0, which is measured for the luminescent reagent that is not mixed with the target substance, it is preferable that R0 ≥ 0.001 is satisfied.

[0020] (Regarding the reaction process) Figure 1 can be used to refer to each step. In the reaction step, a sample containing the target substance and a luminescent reagent are mixed to form a mixture, and the target substance and the luminescent reagent are reacted. The mixture is a liquid containing the luminescent reagent and the target substance, and may also contain other additives. The reaction is preferably carried out at a pH of 3.0 to 11.0. The mixing temperature is in the range of 20°C to 50°C. The reaction time is set considering the concentration of the target substance in the sample and the affinity between the target substance and the luminescent reagent, but is preferably 5 minutes to 24 hours, and more preferably 5 minutes to 1 hour. The target substance and luminescent reagent will be described later.

[0021] (Regarding the dilution process) In the dilution step, the reaction solution is diluted to obtain a diluent. The dilution step is performed after the reaction step. By including a dilution step, it becomes possible to react the luminescent reagent and the target substance at a sufficiently high concentration, and a sufficient reaction can be carried out in a short time. The reaction rate between the target substance and the ligand is determined by their binding constants, and the binding constants depend on the diffusion constants of the target substance and the ligand. Therefore, if the concentration of either the target substance or the ligand in the reaction system is low, the reaction rate will also be slow. For example, in antigen-antibody reactions, the dissociation rate constant is often slower than the binding rate, and it is an equilibrium reaction, but once bound, it is difficult to dissociate. Therefore, the analysis method of this embodiment, in which the concentrations of the target substance and ligand in the system are kept high when the reaction is carried out and then diluted when measurement is performed, is effective.

[0022] The analytical method in this embodiment exhibits a strong effect because the luminescent reagent contains luminescent particles. The inclusion of luminescent particles in the luminescent reagent allows for highly sensitive detection of changes in the anisotropy of polarized emission, corresponding to their aggregation and dispersion behavior. In other words, by using particles for the luminescent reagent, changes in R can be captured more significantly, enabling highly sensitive measurements. On the other hand, when measuring R, if the concentration of the luminescent reagent is high, multiple scattering occurs, making it difficult to capture significant changes in R. In other words, when multiple scattering occurs, R0, which is the R value when measuring particles that have not reacted with the target substance and have not aggregated, becomes high, making it difficult to capture the change in R before and after the reaction with the target substance. However, if the concentration of the luminescent reagent is low during the reaction, the reaction between the target substance and the luminescent reagent will take a long time. By diluting after the reaction, multiple scattering during measurement can be suppressed, and the concentration of the luminescent reagent can be increased during the reaction process, thereby shortening the reaction time. Also, if the viscosity of the solution is too high during measurement, R0 will be high, making it difficult to capture a large change in R. This point can also be resolved by including a dilution step.

[0023] On the other hand, when measuring R in a liquid containing high concentrations of particles, the polarization component of the emission is eliminated due to scattering by the particles, resulting in a high R value even when the target substance is not present. Therefore, it is necessary to appropriately dilute the reaction solution through a dilution process before measurement. Furthermore, the viscosity of the substance being measured greatly affects R, but R stabilizes when the viscosity becomes constant after dilution in the dilution process. The dilution ratio is not particularly specified as long as R can be measured sufficiently, but it is preferably 2 times or more, more preferably between 2 times and 1000 times, and even more preferably between 10 times and 100 times. Alternatively, it is preferable to dilute it so that the luminescence reagent is 0.05 mg / ml or less. Furthermore, it is preferable to dilute it so that the viscosity is 25 mPa·s or less, more preferably 2.5 mPa·s or less.

[0024] (Regarding the measurement process) In the measurement process, the R of the reaction solution is measured. The measurement conditions are preferably such that the solution is in a solution at a temperature of 0 to 50°C, and the viscosity of the solution is between 0.5 mPa·s and 50 mPa·s. It is preferable that the concentration of the luminescent reagent is between 0.001 mg / ml and 0.1 mg / ml, and that the measurement wavelength (excitation wavelength) is between 500 nm and 700 nm. To suppress the effects of multiple scattering, the optical path of the optical system can be shortened. For example, the optical path is generally set to 10 mm, but this can be reduced to 5 mm or less.

[0025] (Target substance) Examples of target substances include antigens, antibodies, small molecule 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 product-derived substances, and all kinds of small molecule compounds. Nucleic acids include DNA, RNA, cDNA derived from bacteria, viruses, cells, etc., parts or fragments thereof, synthetic nucleic acids, primers, probes, etc. Small molecule compounds include cytokines, hormones, neurotransmitters, signaling molecules, membrane proteins, etc., and their receptors, etc. The analytical method according to this embodiment can determine at least one of the presence or absence of these target substances and the concentration of the target substances. The presence or absence of a target substance can be determined by comparing the concentration of the target substance with a predetermined threshold. For example, if the concentration of the target substance is above a predetermined threshold, it can be determined that the target substance is present, and if it is below a predetermined threshold, it can be determined that the target substance is absent.

[0026] (Luminescent reagent) In this embodiment, the luminescent reagent is a reagent that produces light, and in particular, a reagent that emits light when excited by light irradiation, excluding those that produce light through chemical reactions, such as luminol. The light emission includes phosphorescence and fluorescence, but phosphorescence is preferred. More preferably, in this embodiment, the luminescent reagent contains a europium complex. Furthermore, more preferably, in this embodiment, the luminescent reagent contains particles. Most preferably, in this embodiment, the luminescent reagent contains particles containing a europium complex. It is also preferable that the luminescent reagent has a ligand specific to the target substance. Having a ligand enables the detection and quantification of the target substance based on polarization anisotropy in the luminescent reagent of this embodiment. In this embodiment, a ligand is a compound that specifically binds to a particular target substance.

[0027] Any ligand can be used as long as it exhibits affinity to a specific substance. Examples of ligand-target substance or target substance-ligand combinations include: antigens and antibodies, small molecules and their receptors, enzymes and substrates, and complementary nucleic acids. Furthermore, antibodies and their specific counterparts include allergens, bacteria, viruses, cells, cell membrane components, cancer markers, various disease markers, antibodies, blood-derived substances, food-derived substances, natural product-derived substances, and any small molecule compounds. Furthermore, receptors and their specific counterparts include small molecule compounds, cytokines, hormones, neurotransmitters, signaling molecules, and membrane proteins. Furthermore, DNA, RNA, cDNA, parts or fragments thereof, synthetic nucleic acids, primers, probes, etc. derived from bacteria, viruses, cells, etc., and nucleic acids that are complementary to them. In addition to the above, any combination known to exhibit affinity can be used as a target substance-ligand combination. In this embodiment, the ligand is typically one of antibodies, antigens, or nucleic acids. Furthermore, the combination of antigen and antibody, as a target substance and ligand, has been clearly demonstrated in the following examples and is considered a particularly preferred example.

[0028] Figure 3 is a schematic diagram showing an example of a luminescent reagent used in this embodiment. The luminescent reagent 4 used in this embodiment has a particle substrate 1 containing, for example, a europium complex 3 as a luminescent molecule. Furthermore, the luminescent reagent 4 may have a hydrophilic layer 2 covering its surface. The diameter of the particles in Figure 3 is between 25 nm and 500 nm.

[0029] The diameter of a particle can be determined by dynamic light scattering. When a laser beam is shone on particles dispersed in a solution and the scattered light is observed with a photon detector, the intensity distribution due to the interference of the scattered light is constantly fluctuating because the particles are constantly moving due to Brownian motion. Dynamic light scattering is a measurement method that observes Brownian motion as fluctuations in scattered light intensity. The fluctuations in scattered light with respect to time are represented by an autocorrelation function, and the translational diffusion coefficient is determined. From the determined diffusion coefficient, the Stokes diameter can be calculated, allowing the particle size dispersed in the solution to be derived.

[0030] From the viewpoint of maintaining particle uniformity and monodispersity, it is desirable that nothing be applied to the surface of the luminescent reagent particles. However, in order to use it in the analytical method according to this embodiment, it is necessary to prevent nonspecific adsorption of substances other than the target substance onto the particles, so it is preferable that the surface of the luminescent reagent has a hydrophilic layer to maintain hydrophilicity.

[0031] A commonly used method for maintaining hydrophilicity is to support BSA on the surface of the particles, but this method can result in lot-to-lot variations. Therefore, it is preferable that the luminescent reagent contains a hydrophilic layer made of a hydrophilic polymer. The concentration of the luminescent reagent in the mixture is preferably 0.000001% by mass or more and 1% by mass or less, more preferably 0.00001% by mass or more and 0.001% by mass or less.

[0032] The luminescent reagent used in this embodiment contains a europium complex, which enables long-lived emission. Preferably, the luminescent reagent used in this embodiment has an average particle diameter of 25 nm to 500 nm, and more preferably, an average particle diameter of 50 nm to 300 nm. If the average particle diameter exceeds 500 nm, the R (R0) before aggregation becomes high, and the difference with R after the aggregation reaction becomes small. Also, if the average particle diameter is less than 25 nm, the change in size before and after aggregation becomes small, making it difficult to detect the change in R by depolarizing the emission.

[0033] By reducing the particle size distribution of the luminescent reagent and introducing a europium complex as the luminescent molecule, it is possible to detect changes in polarized emission characteristics even if there are slight changes in the dispersion state of the particles in the liquid. Specifically, even if the concentration of the target substance in the solution is on the order of nanograms to picograms per 1 mL, for example between 1 picogram and 100 picograms, when the luminescent reagent aggregates via the target substance, the change in the rotational Brownian motion of the luminescent reagent can be detected as a change in polarization anisotropy.

[0034] Polarized emission refers to the phenomenon in light-emitting materials with anisotropic transition moments (transition dipole moments) where, if the excitation light is polarized along the transition moment, the emitted light will also be polarized along the transition moment. Europium complexes exhibit fluorescence emission based on energy transfer from ligands to the central metal ion, resulting in a complex transition moment. However, the red emission around 610 nm, originating from the electron transition from the lowest excited state 5D0 to 7F2, is polarized emission.

[0035] The principle of polarization anisotropy involves measuring the shift in the transition moment due to the rotational motion of the light-emitting material during the time that polarized light emission occurs. The rotational motion of the light-emitting material can be expressed by equation (3). Q = 3Vη / kT···(3) Here, Q: Material rotation relaxation time V: Volume of material η: viscosity of the solvent k: Boltzmann constant T: Absolute temperature That is the case. The rotational relaxation time of a material is the time required for the numerator to rotate by an angle θ (68.5°) such that cosθ = 1 / e.

[0036] From equation (3), 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 size if the luminescent material is in particle form. On the other hand, the relationship between the luminescence lifetime of the luminescent material and the degree of polarization, which is a value related to polarization anisotropy, can be expressed by equation (4). p0 / p=1+A(τ / Q)···(4) Here, p0: Polarization degree when the material is stationary (Q=∞) p: Polarization degree A: Constant τ: Luminous lifetime of the material Q: Rotation relaxation time That is the case. From equations (3) and (4), it can be seen that the degree of polarization is influenced by the luminescence lifetime and rotational relaxation time of the luminescent material, i.e., the volume (particle size) of the luminescent material, and that is, the balance between the particle size and luminescence lifetime of the luminescent material has an influence.

[0037] To experimentally determine the polarization degree of a light-emitting material as shown in equation (4), polarized light should be incident on the light-emitting material, and the emission should be detected at a 90-degree angle to the direction of propagation and vibration of the excitation light. At this time, the detected light should be separated into polarization components parallel and perpendicular to the polarization of the incident light, and the polarization anisotropy shown in equation (5) can be used as a value related to polarization anisotropy. r(t)=(I∥(t)-GI⊥(t)) / (I∥(t)+2GI⊥(t))...(5) Here, r(t): Polarization anisotropy at time t I∥(t): Emission intensity of the emission component parallel to the excitation light at time t I⊥(t): Emission intensity of the emission component perpendicular to the excitation light at time t. G: Correction value, the ratio of I⊥ / I∥ measured with excitation light that has a vibration direction 90 degrees different from the excitation light used for sample measurement. That is the case.

[0038] In other words, within the appropriate particle size and luminescence lifetime range, changes in the size of the luminescent material due to reactions with the target substance can be sensitively detected as changes in polarization anisotropy. Specifically, the r(t) of non-aggregated luminescent materials is observed to be low, while the r(t) of aggregated luminescent materials is observed to be high. This is the principle of polarization anisotropy.

[0039] Furthermore, the values ​​related to polarization anisotropy may be corrected using G and 2G, or they may be values ​​without G and 2G.

[0040] (particle matrix 1) The shapes of the luminescent reagent 4 and particle substrate 1 used in this embodiment are not limited. The particle substrate 1 is not particularly specified as long as it is a material that can stably incorporate the europium complex, but it is preferably a polymer containing styrene units and organosilane units, and in particular a polymer obtained by polymerizing a composition containing radically polymerizable organosilane with styrene as the main component is preferably used. By including styrene as the main component in the composition, it is possible to produce particles with a very uniform particle size distribution by the emulsion polymerization method described later, and by using a polymer containing organosilane units, silanol groups (Si-OH) are generated in the polymer in an aqueous solvent, forming siloxane bonds (Si-O-Si) with each other on the surface of the particle substrate, and a hydrophilic layer and ligand described later can be attached via this. The particles in this embodiment preferably have ligand-binding functional groups on the outside of the particle substrate that can bind ligands.

[0041] (Hydrophilic layer 2) The hydrophilic layer 2 can be composed of a hydrophilic polymer or hydrophilic molecule on the outside of the particle substrate 1. A hydrophilic polymer or hydrophilic molecule is a polymer or molecule containing a hydrophilic group, and specific examples of hydrophilic groups include molecules and polymers having hydroxyl groups, ethers, pyrrolidones, betaine structures, etc. Specific examples of hydrophilic polymers include polyethylene glycol, polyvinylpyrrolidone, sulfobetaine polymers, phosphobetaine polymers, and polyglycidyl methacrylic acid, which has a glycidyl group opened and a hydroxyl group modified at the end of the molecule. These can be the main components of the hydrophilic layer 2. Alternatively, the hydrophilic layer 2 may be formed by directly attaching a single molecule having a hydrophilic group to the surface of the particle substrate 1 using a silane coupling agent or the like. There is no limit to the thickness of the hydrophilic layer 2, but it does not need to be thicker than the thickness required to exhibit hydrophilicity. If the hydrophilic layer 2 is too thick, it may become like a hydrogel, and the thickness of the hydrophilic layer may become unstable due to hydration caused by ions in the solvent. The thickness of the hydrophilic layer 2 is preferably between 1 nm and 15 nm.

[0042] (Europium complex 3) The luminescent reagent used in this embodiment may include europium complex 3 as the luminescent dye. Europium complex 3 has the characteristics that the wavelength and intensity of its emission are less affected by the surroundings, and that its emission has a long lifetime. Europium complex 3 is composed of europium element and ligand. Considering the emission lifetime and the visible emission wavelength range, europium complex is preferred as the luminescent dye. Europium generally has an emission lifetime of 0.1 ms to 1.0 ms. It is necessary to appropriately adjust this emission lifetime and the rotational relaxation time obtained from formula (1). In the case of europium in an aqueous dispersion, if the diameter of the luminescent reagent is between 50 nm and 300 nm, R changes significantly before and after aggregation.

[0043] At least one of the ligands constituting europium complex 3 has a light-harvesting function. Light-harvesting function refers to the action of exciting the central metal of the complex by energy transfer when excited at a specific wavelength. Furthermore, it is preferable that the ligands constituting europium complex 3 include ligands such as β-diketones to prevent coordination of water molecules. Ligands such as β-diketones that coordinate to europium ions suppress the deactivation process due to energy transfer to solvent molecules, etc., and strong fluorescence emission is obtained.

[0044] Europium complex 3 may be a multinuclear complex. Furthermore, specific examples of europium complexes include [Tris(2-thenoyltrifluoroacetone)bis(triphenylphosphineoxide)europium(III)], [Tris(2-thenoyltrifluoroacetone)(triphenylphosphineoxide)(dibenzylsulfoxide)europium(III)], and [Tris(2-thenoyltrifluoroacetone)(phenanthroline)europium(III)].

[0045] When the Brownian rotation of europium complex 3 can be considered to have stopped in the medium, it is desirable that R, represented by equation (3), be 0.08 or greater. The state in which Brownian rotation can be considered to have stopped means that the rotational relaxation time of the particles is sufficiently longer than the luminescence lifetime of europium complex 3.

[0046] It is preferable for europium complex 3 to be incorporated in large quantities into the particle substrate 1, as this increases the luminescence intensity per particle. On the other hand, if europium complex 3 aggregates in the particle substrate 1, the interaction between ligands affects the excitation efficiency of europium complex 3, making it difficult to measure R reproducibly. Whether europium complex 3 exhibits non-aggregated luminescence behavior in the particle substrate 1 can be determined from the excitation spectrum of the sample.

[0047] Particles that emit strong light not only enable highly sensitive measurements, but also maintain their luminescence even when the particle size is small, thus accelerating biochemical reaction rates. Smaller particle sizes result in a larger diffusion coefficient of Brownian motion in liquids, allowing for faster reaction detection.

[0048] By using a liquid containing dispersed particles in the analytical method according to this embodiment, it is possible to detect changes in the anisotropy of polarized emission in response to the aggregation and dispersion behavior of the particles with high sensitivity. Such a dispersion of particles in an aqueous solvent can be used as a highly sensitive diagnostic reagent using polarization anisotropy. A buffer solution may be used as the aqueous solvent. Furthermore, surfactants, preservatives, sensitizers, etc., may be added to the aqueous solvent to increase the stability of the liquid containing dispersed particles.

[0049] (Method for manufacturing luminescent reagents) Next, an example of a method for producing the luminescent reagent used in this embodiment will be described. A method for producing a luminescent reagent comprises a first step of preparing an emulsion by mixing a radical polymerizable monomer containing at least styrene and a radical polymerizable organosilane, a radical polymerization initiator, a polarizing luminescent europium complex, and a hydrophilic polymer with an aqueous medium.

[0050] Furthermore, the method for producing the luminescent reagent includes a step (second step) of heating the emulsion to polymerize the radical polymerizable monomer.

[0051] A method for producing a luminescent reagent may include a step (third step) of imparting a ligand-binding functional group, as described later, to the surface of the luminescent reagent. Here, the ligand-binding functional group is a functional group capable of binding a ligand, and specifically, any of the following can be used: a carboxyl group, an amino group, a thiol group, an epoxy group, a maleimide group, a succinimidyl group, or an alkoxysilyl group (silicone alkoxide structure).

[0052] (Radical polymerizable monomer) The luminescent reagent is manufactured by polymerizing a radically polymerizable monomer, which includes at least styrene and a radically polymerizable organosilane. The radically polymerizable monomer may further include monomers selected from the group consisting of acrylate monomers and methacrylate monomers. Examples of monomers include butadiene, vinyl acetate, vinyl chloride, acrylonitrile, methyl methacrylate, methacrylonitrile, methyl acrylate, and mixtures thereof. That is, one or more of these monomers can be used in addition to styrene and a radically polymerizable organosilane. A monomer having two or more double bonds in a single molecule, such as divinylbenzene, may also be used as a crosslinking agent.

[0053] By including radically polymerizable organic silanes in the radically polymerizable monomer, siloxane bonds are conferred to the particle substrate 1. Examples of radically polymerizable organic silanes include vinyltrimethoxysilane, vinyltriethoxysilane, p-styryltrimethoxysilane, 3-methacryloxypropylmethyldimethoxysilane, 3-methacryloxypropyltrimethoxysilane, 3-methacryloxypropylmethyldiethoxysilane, 3-methacryloxypropyltriethoxysilane, 3-acryloxypropyltrimethoxysilane, or combinations thereof. By using radically polymerizable organic silanes, an inorganic oxide skeleton is formed within the particle substrate 1, which improves the physical and chemical stability of the luminescent reagent. Furthermore, by using radically polymerizable organic silanes, the affinity between the particle substrate 1 and the hydrophilic layer 2 and ligand-binding functional groups is increased.

[0054] Furthermore, the inclusion of a radically polymerizable organic silane in the radically polymerizable monomer imparts silanol groups to the surface of the particle substrate 1. These silanol groups form hydrogen bonds with hydrophilic polymers, such as PVP. As a result, hydrophilic polymers such as PVP are more strongly adsorbed onto the surface of the particle substrate 1.

[0055] (Radical polymerization initiator) As radical polymerization initiators, a wide range of azo compounds and organic peroxides can be used. Specifically, examples include 2,2'-azobis(isobutyronitrile), 2,2'-azobis(2,4-dimethylvaleronitrile), 2,2'-azobis(2-methylbutyronitrile), 4,4'-azobis(4-cyanovaleric acid), 2,2'-azobis(2-methylpropionamidine) dihydrochloride, 2,2'-azobis(2-methylpropionic acid) dimethyl, tert-butyl hydroperoxide, benzoyl peroxide, ammonium persulfate (APS), sodium persulfate (NPS), potassium persulfate (KPS), and the like.

[0056] (Hydrophilic polymer) The luminescent reagent may include a hydrophilic polymer as a hydrophilic layer. The hydrophilic polymer preferably suppresses nonspecific adsorption. Examples of hydrophilic polymers include ethers, betaines, and hydrophilic polymers containing units having pyrrolidone rings. The hydrophilic layer is contained in the synthesized luminescent reagent and preferably exists mainly on the outer particle surface of the particle substrate. In this specification, polymers having pyrrolidone rings may be abbreviated as "PVP". By adding PVP during the synthesis of the luminescent reagent, it is possible to simultaneously impart nonspecific adsorption suppression ability and ligand binding ability to the luminescent reagent. Since the PVP added during synthesis is more hydrophilic than the radical polymerizable monomer, it exists at the interface between the solvent and the particle substrate during polymerization. The particle substrate adsorbs PVP to its outer surface by partially incorporating PVP during polymerization or by physical and chemical adsorption such as the interaction between the pyrrolidone ring and styrene (a radical polymerizable monomer).

[0057] The molecular weight of PVP is preferably between 10,000 and 100,000, and more preferably between 40,000 and 70,000. If the molecular weight is less than 10,000, the hydrophilicity of the surface of the luminescent reagent is weak, making it prone to nonspecific adsorption. If the molecular weight is greater than 100,000, the hydrophilic layer becomes too thick, causing gelation and making it difficult to handle.

[0058] In addition to PVP, a hydrophilic polymer may be added as a protective colloid during particle substrate synthesis.

[0059] Furthermore, the luminescent reagent preferably satisfies the condition A2-A1≦0.1. A1 and A2 are defined as follows: A1 is the absorbance of a mixture prepared by adding 30 μL of a 0.1 wt% dispersion of a luminescent reagent to 60 μL of buffer containing 16 μL of 15-fold diluted human serum, immediately after the addition, and A2 is the absorbance after standing at 37°C for 5 minutes after the addition. The absorbance is measured at a light path of 10 mm and a wavelength of 572 nm. Particles with an A2-A1 ratio of 0.1 or less are preferable because they exhibit less nonspecific adsorption of impurities in serum.

[0060] (aqueous medium) The aqueous medium (aqueous solution) used in the above-described method for producing the luminescent reagent preferably contains 80% to 100% by weight of water. The aqueous solvent is preferably water or a water-soluble organic solvent, and examples include a solution of methanol, ethanol, isopropyl alcohol, or acetone mixed with water. If the content of an organic solvent other than water exceeds 20% by weight, dissolution of polymerizable monomers may occur during particle production.

[0061] Furthermore, it is preferable that the above-mentioned aqueous medium is pre-adjusted to a pH of 6 to 9. If the pH is less than 6 or greater than 9, the alkoxide or silanol groups of the radically polymerizable organic silane may undergo condensation polymerization or react with other functional groups before polymer formation, potentially causing the resulting particles to aggregate. In this embodiment, condensation polymerization of the alkoxide is not intentionally performed before polymerization.

[0062] The pH adjustment described above is preferably done using a pH buffer, but it may also be done with an acid or a base.

[0063] In addition, surfactants, defoamers, salts, thickeners, etc. may be added to the aqueous medium in amounts of 10% or less.

[0064] When manufacturing the luminescent reagent, it is preferable to first dissolve PVP in an aqueous medium adjusted to a pH of 6 to 9. The PVP content is preferably 0.01% to 10% by weight relative to the aqueous medium, and more preferably 0.03% to 5% by weight. If the content is less than 0.01% by weight, the amount adsorbed to the particle substrate will be small and the effect will not be exhibited. If the content is more than 10% by weight, the viscosity of the aqueous medium will increase, which may prevent sufficient stirring.

[0065] Next, a radical polymerizable monomer containing styrene (A) and a radical polymerizable organic silane (B) is added to the aqueous medium to form an emulsion. The weight ratio of styrene (A) to radical polymerizable organic silane (B) is 6:4 to 100:1. Furthermore, the europium complex is mixed into the prepared emulsion. At this time, if the solubility of the europium complex is low, a water-insoluble organic solvent may be added. The weight ratio of the europium complex to the radical polymerizable monomer is 1:1000 to 1:10.

[0066] If the weight ratio of styrene (A) to radically polymerizable organic silane (B) is less than 6:4, the overall specific gravity of the particles will increase, potentially leading to significant particle sedimentation. Furthermore, to improve the adhesion between PVP and luminescent particles, it is desirable to have a weight ratio of styrene (A) to radically polymerizable organic silane (B) of 100:1 or higher.

[0067] The weight ratio of the aqueous medium to the total amount of radically polymerizable monomers is preferably between 5:5 and 9.5:0.5. If the weight ratio is less than 5:5, the aggregated particles produced may become significant. If the weight ratio is greater than 9.5:0.5, particle formation will not be a problem, but the amount produced may be reduced.

[0068] The radical polymerization initiator is used after being dissolved in water, a buffer, etc. The amount of the radical polymerization initiator relative to the total weight of styrene (A) and radically polymerizable organosilane (B) can be used in an emulsion between 0.5% by mass and 10% by mass.

[0069] The process of heating the emulsion described above only needs to ensure that the entire emulsion is heated uniformly. The heating temperature can be arbitrarily set between 50°C and 80°C, and the heating time between 2 hours and 24 hours. By heating the emulsion, the radical polymerizable monomers are polymerized.

[0070] Luminescent reagents can have ligand-binding functional groups on their surface. The ligand-binding functional group is not particularly limited as long as it can bind antibodies, antigens, enzymes, etc., but examples include carboxyl groups, amino groups, thiol groups, epoxy groups, maleimide groups, succinimidyl groups, silicon alkoxide groups, etc., or can contain these functional groups. For example, it is possible to impart functional groups to the particle surface by mixing a silane coupling agent having ligand-binding functional groups with synthesized particles. Specifically, by preparing an aqueous solution of a silane coupling agent having carboxyl groups and mixing it with a synthesized particle dispersion, carboxyl groups can be imparted to the particle surface. At this time, a dispersant such as Tween 20 may be added to the reaction solution. The reaction temperature can be arbitrarily set between 0°C and 80°C, and the reaction time between 1 hour and 24 hours. To suppress the rapid condensation reaction of the silane coupling agent, it is preferable to set the reaction time to around 3 to 14 hours at or below room temperature of about 25°C. Depending on the ligand-binding functional group, an acid or alkali catalyst can be added to accelerate the reaction on the particle surface.

[0071] By attaching various antibodies and other ligands to the luminescent reagent, it can be used as a particle for sample testing. The optimal method for attaching the desired antibody or ligand should be selected by utilizing the functional groups present in the hydrophilic layer 2.

[0072] (Introduction of ligands) The chemical reaction for chemically bonding the ligand-binding functional group to the ligand can be carried out using conventionally known methods to the extent that the objectives of the present invention can be achieved. Furthermore, when amide bonding the ligand, catalysts such as 1-[3-(dimethylaminopropyl)-3-ethylcarbodiimide] can be used as appropriate.

[0073] The luminescent reagent used in this embodiment is preferably applicable to immunolatex agglutination assays, which are widely used in fields such as clinical testing and biochemical research.

[0074] (Analysis equipment) As one embodiment of this invention, the following analytical device is provided. An analytical device that measures at least one of the presence or absence of a target substance and the concentration of a target substance by measuring a value (R) related to polarization anisotropy using a luminescent reagent that reacts with the target substance, A reaction unit that mixes a sample containing the target substance and the luminescent reagent and reacts them to obtain a reaction solution. Dilution section for diluting the reaction solution to obtain a diluted solution A measuring unit for measuring the R of the diluent, and control unit It has, The analytical apparatus is characterized in that the luminescent reagent is a particle having a luminescent molecule. A schematic diagram of the analysis apparatus according to this embodiment is shown in Figure 2. The reaction unit is the unit that performs the reaction process. The dilution unit is the unit that performs the dilution process. The measurement unit is the unit that performs the measurement process. The control unit controls the reaction unit, the dilution unit, and the measurement unit. The control unit has the functions of a computer. For example, the control unit may be integrated with a desktop PC (Personal Computer), laptop PC, tablet PC, smartphone, etc. In order to realize the functions of a computer that performs calculations and storage, the control unit is equipped with a CPU, RAM, ROM and HDD, and may also be equipped with a communication I / F (interface), display device and input device.

[0075] (reagent) The analytical method according to this embodiment can be used for specimen testing and in vitro diagnostics. The reagent used for these purposes may include the luminescent reagent used in this embodiment and a dispersion medium for dispersing the luminescent reagent. The amount of luminescent reagent contained in the reagent is preferably 0.000001% by mass or more and 20% by mass or less, and more preferably 0.0001% by mass or more and 1% by mass or less. In addition to the luminescent reagent, the reagent may also contain third substances such as additives and blocking agents, to the extent that the objectives of the present invention can be achieved. Two or more types of third substances such as additives and blocking agents may be included in combination. Examples of dispersion media used in this embodiment include various buffers such as phosphate buffer, glycine buffer, Good's buffer, Tris buffer, and ammonia buffer, but the dispersion medium contained in the reagent in this embodiment is not limited to these. When using a reagent to detect an antigen or antibody in a sample, the ligand can be an antibody or an antigen. [Examples]

[0076] The present invention will be specifically described below with reference to examples. However, the present invention is not limited to these examples.

[0077] (1) Preparation of luminescent particles Solvent A was prepared by dissolving polyvinylpyrrolidone (PVP-K30: manufactured by Tokyo Chemical Industry Co., Ltd.) in MES (2-morpholinoethanesulfonic acid) buffer at pH 7 (manufactured by Kishida Chemical Co., Ltd.). Reaction solution B was prepared by mixing the europium complex [Tris(2-thenoyltrifluoroacetone)bis(triphenylphosphineoxide)europium(III)] (manufactured by Central Techno Co., Ltd., hereinafter abbreviated as "Eu(TTA)3(TPPO)2"), styrene monomer (manufactured by Kishida Chemical Co., Ltd.), and 3-methacrylateoxypropyltrimethoxysilane (manufactured by Tokyo Chemical Industry Co., Ltd., hereinafter abbreviated as "MPS"). Reaction solution B was added to a four-necked flask containing solvent A, and the mixture was stirred using a mechanical stirrer set to 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 performed for another 15 minutes. After heating and stirring the mixture, an aqueous solution containing potassium persulfate (hereinafter abbreviated as "KPS") (manufactured by Aldrich) was added to the reaction solution, and emulsion polymerization was carried out for 20 hours. After the polymerization reaction, the obtained suspension was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 100K with approximately 4 L of deionized water to wash the product and obtain a dispersion of luminescent particles.

[0078] A dispersion of luminescent particles obtained by emulsion polymerization was taken and added to an aqueous solution containing 1% by mass of Tween20 (manufactured by Kishida Chemical Co., Ltd.). After stirring for 10 minutes, the silane coupling agent X12-1135 (manufactured by 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. The centrifugation and redispersion process was repeated at least three times to wash the product. The precipitate after washing was redispersed in pure water. As a result, ligand-binding functional groups were introduced to particles 1-8. The mass ratio of the charged particles, pure water, and X12-1135 was 1:300:2.

[0079] (Preparation of anti-CRP antibody-modified luminescent reagents) 0.25 mL of a 1.2 wt% particle dispersion, corresponding to the synthesized luminescent particles, was taken and the solvent was replaced with 1.6 mL of pH 6.0 MES buffer. 0.5 wt% of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide and N-hydroxysulfosuccinimide sodium were added to the particle MES buffer and reacted at 25°C for 1 hour. After the reaction, the dispersion was washed with pH 5.0 MES buffer, 100 μg / mL of anti-CRP antibody was added, and the anti-CRP antibody was bound to the particles at 25°C for 2 hours. After binding, the particles were washed with pH 8 Tris buffer. After the reaction, the particles were washed with phosphate buffer to obtain a 0.3 wt% anti-CRP antibody-modified luminescent reagent (also called affinity particles).

[0080] (Preparation of anti-TSH antibody-modified luminescent reagents) 0.25 mL of a 1.2 wt% particle dispersion, corresponding to the synthesized luminescent particles, was taken and the solvent was replaced with 1.6 mL of pH 6.0 MES buffer. 0.5 wt% of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide and N-hydroxysulfosuccinimide sodium were added to the particle MES buffer and reacted at 25°C for 1 hour. After the reaction, the dispersion was washed with pH 5.0 MES buffer, 100 μg / mL of anti-TSH antibody was added, and the anti-TSH antibody was bound to the particles at 25°C for 2 hours. After binding, the particles were washed with pH 8 Tris buffer. After the reaction, the particles were washed with phosphate buffer to obtain a 1.0 wt% anti-TSH antibody-modified luminescent reagent (also called affinity particles). The anti-TSH antibody used was a monoclonal antibody, and two types of anti-TSH antibodies were used to modify the luminescent particles in order to react at least two particles with the TSH antigen, which is the substance to be measured.

[0081] The binding of antibodies to the particles was confirmed by measuring the decrease in antibody concentration in the buffer solution to which the antibodies were added using a BCA assay.

[0082] (Preparation of luminescent reagent solution) The obtained anti-CRP antibody-modified luminescent reagent was diluted in pH 7.4 phosphate (PBS) buffer to a concentration of 0.1 mg / mL to prepare a luminescent reagent solution. Similarly, the obtained anti-TSH antibody-modified luminescent reagent was diluted in pH 7.4 phosphate (PBS) buffer to a concentration of 1.0 mg / mL to prepare a luminescent reagent solution.

[0083] (Preparation of dilution additive) A diluent was prepared by mixing 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer and PBS buffer in a 1:1 volume ratio.

[0084] (Example 1) 75 μL of anti-CRP antibody-modified luminescence reagent solution and 15 μL of CRP antigen solution were mixed and allowed to stand at 37°C for 10 minutes. 1335 μL of a 37°C dilution solution was added, and R was measured. The CRP antigen concentration was examined at 200 pM. The measurement was performed using apparatus 1, described below.

[0085] (Example 2) 30 μL of anti-CRP antibody-modified luminescence reagent solution and 30 μL of CRP antigen solution were mixed and allowed to stand at 37°C for 10 minutes. 8 μL of this solution was taken and mixed with 392 μL of a dilution additive at 37°C, and R was measured. The CRP antigen concentration was investigated in the range of 0 pM to 10.24 pM. The measurement was performed using apparatus 2 described below.

[0086] (Example 3) 70 μL of anti-TSH antibody-modified luminescence reagent solution and 15 μL of TSH antigen solution were mixed and allowed to stand at 37°C for 10 minutes. 1335 μL of a 37°C diluent was added, and R was measured. The TSH antigen concentration was examined at 1600 pM. The measurement was performed using apparatus 1, described below.

[0087] (Comparative Example 1) 15 μL of CRP antigen solution was mixed with 1335 μL of diluent additive and heated at 37°C. 75 μL of anti-CRP antibody-modified luminescence reagent solution was then added, and the R value was measured. The CRP antigen concentration was examined at 200 pM. The measurements were performed using apparatus 1 described below.

[0088] (Comparative Example 2) 392 μL of diluent was mixed with 4 μL of CRP antigen solution and heated at 37°C. 4 μL of anti-CRP antibody-modified luminescence reagent solution was then added, and the R value was measured. CRP antigen concentrations were investigated in the range of 0 pM to 10.24 pM. Measurements were performed using apparatus 2, described below.

[0089] (Comparative Example 3) 15 μL of TSH antigen solution was mixed with 1335 μL of diluent additive and heated at 37°C. 70 μL of anti-TSH antibody-modified luminescence reagent solution was then added, and R was measured. The TSH antigen concentration was examined at 1600 pM. The measurements were performed using apparatus 1 described below.

[0090] (evaluation) The shape of the obtained luminescent reagent was evaluated using an electron microscope (Hitachi High-Technologies S5500).

[0091] The average particle size of the luminescent reagent was evaluated using dynamic light scattering (Malvern Zetasizer Nano S).

[0092] The concentration of the suspension containing the luminescent reagent was evaluated using a gravimetric analyzer (Rigaku ThermoPlus TG8120).

[0093] R was measured using apparatus 1 and apparatus 2. Apparatus 1 has the following configuration: An LED light source with excitation light of 340 nm was prepared, and a polarizing filter (Sigma Optical Co., Ltd., NSPFU-30C) and a short-pass filter (Edmund Optics, Ltd., 84-706) were inserted into the optical path to set up an optical system capable of irradiating a 1 cm quartz square cell. A polarizing filter (Sawlab, Ltd., PIVISC050) and a band-pass filter (Sawlab, Ltd., FB610-10) were set at a 90° angle to the incident light. Emission was 1VV and I VH To simultaneously measure in two directions, two sets were prepared with different polarizer configurations for the incident light and the 90° direction. Spectroscopic measurements were performed using Ocean Optics' QEPro for polarization detection. The sample holder was temperature-controlled to allow measurements at 37°C. Polarization anisotropy was measured with the LED light source fixed at 12mW output and the integration time set to 3 seconds. The measurement interval was 15 seconds. From the obtained fluorescence spectra of polarized emission, R was calculated by applying the emission intensity in the wavelength range of 600nm to 630nm to equation (1).

[0094] Device 2 is a device with the following configuration. An LED light source with an excitation light of 340 nm was prepared, and a polarizing filter (Sigma Koki, NSPFU-30C) and a short-pass filter (Edmund Optics, 84-706) were inserted into the optical path to set up an optical system capable of irradiating a quartz cell with an optical path length of 5 mm. The polarized emission generated from the sample was spectrally separated into two directions by setting up an excitation light cut filter (Edmund Optics, 33-910), a polarizing beam splitter (Edmund Optics, 47-127, and a polarizer (Sigma Koki, SPF-30C-32)) in that order, with the sample in between the excitation light and the optical path in a straight line, and then the polarization was separated into two directions. The spectrally separated polarized emission (in both directions) was detected using an avalanche photodiode (APD, Hamamatsu Photonics, C15522-3010SA). The sample holder was temperature-controlled to allow measurement at 37°C. Polarization anisotropy was measured with the LED light source fixed at an output of 60 mW and the integration time set to 8 milliseconds. The measurement interval was 30 seconds. The obtained polarized emission signal was measured with an oscilloscope and R was calculated by applying it to equation (1).

[0095] The non-specific aggregation suppression of the luminescent reagent was evaluated as follows. 60 μl of human serum solution, diluted 15-fold with buffer, was added to a luminescent reagent dispersion (3 mg / mL) and incubated at 37°C for 5 minutes. The absorbance at 527 nm was measured before and after incubation, and the change in absorbance before and after incubation was measured three times. Table 2 shows the average values ​​of the three measurements. A change in absorbance × 10000 of less than 1000 was considered to indicate suppression of nonspecific agglutination, while a change of 1000 or more was considered to indicate the occurrence of nonspecific agglutination.

[0096] (Performance evaluation) The synthesized luminescent reagent had a particle size of approximately 100 nm and exhibited strong red emission when excited with 340 nm light. The results of the non-specific aggregation suppression evaluation showed that the change in absorbance was below the specified value (the change in absorbance × 10000 was 1000 or less), confirming that the particles are capable of suppressing non-specific adsorption.

[0097] The results for Example 1 and Comparative Example 1 are shown in Figure 4. Figure 4 is a graph plotting reaction time on the horizontal axis and R (polarization anisotropy r) on the vertical axis. In Example 1, plotted as a circle in Figure 4, the r immediately after measurement exceeded 0.105, and then r gradually increased. On the other hand, in Comparative Example 1, plotted as an X in Figure 4, the r immediately after measurement was 0.065, and it was found that it reached a similar level of r as Example 1 after 2000 seconds. Even after subtracting the reaction time of 600 seconds in Example 1 from 2000 seconds, it was confirmed that the antigen-antibody reaction of CRP proceeded faster in Example 1, and that R showed a higher value in a shorter time.

[0098] The results for Example 2 and Comparative Example 2 are shown in Figure 5. Figure 5 is a graph plotting the measured concentration of CRP antigen on the horizontal axis, the polarization anisotropy after 20 minutes (reaction time) and r on the vertical axis. Reaction time refers to the time measured with the time when the luminescent reagent and CRP antigen were mixed set to zero. In Figure 5, Example 2, plotted with circles, shows that the observed r also increased with increasing CRP concentration. On the other hand, in Comparative Example 2, plotted with crosses in Figure 5, although r increased slightly, the slope was such that it was difficult to distinguish the difference in r due to CRP concentration. Example 2 and Comparative Example 2 had a dilution ratio 10 times higher and a lower CRP measurement concentration compared to Example 1 and Comparative Example 1. Therefore, the difference in sensitivity due to the measurement method was also larger, resulting in a clearer difference.

[0099] The results of the studies for Example 2 and Comparative Example 2 are shown in Table 1. Table 1 shows that in Example 2, the change in polarization anisotropy increased to 0.095 when the CRP antigen concentration was 10.24 pM, while in Comparative Example 2 it remained at 0.045. Furthermore, the results showed that in Example 2, a calibration curve could be drawn and the lower limit of detection for concentration measurement was confirmed to be 0.16 pM, while in Comparative Example 2 the measurement range remained at approximately 10 pM. Although the type, amount, and measurement time of the reagents used were the same in Example 2 and Comparative Example 2, it became clear that there was a significant difference in measurement sensitivity depending on whether or not an appropriate dilution step was performed.

[0100] [Table 1]

[0101] The results for Example 3 and Comparative Example 3 are shown in Figure 6. Figure 6 is a plot with measurement time (i.e., reaction time between the antibody in the reagent and the antigen) on the horizontal axis and R (polarization anisotropy r) on the vertical axis. In Figure 6, ○ represents the results for Example 3 and × represents the results for Comparative Example 3. In the ○ plots in Figure 6, r exceeded 0.106 immediately after measurement and then gradually increased. On the other hand, in the × plots in Figure 6, r was 0.075 immediately after measurement and reached a level similar to that of Example 3 after 600 seconds.

[0102] From the above, it has become clear that the measurement method according to this embodiment is a method that can measure the target substance, CRP antigen, with high sensitivity and in a short time.

[0103] Therefore, using the measurement method according to this embodiment, it is possible to measure target substances quickly and with high sensitivity. It is believed that using the measurement method according to this embodiment, it is possible to realize a device that performs highly sensitive measurements for applications such as specimen testing where a large number of tests are performed in a short time.

[0104] This embodiment includes the following methods and configurations. (Method 1) An analytical method for determining at least one of the presence or absence of a target substance and the concentration of a target substance by measuring a value (R) related to polarization anisotropy using a luminescent reagent that reacts with the target substance, A reaction step in which a sample containing the target substance and the luminescent reagent are mixed and reacted to obtain a reaction solution, A dilution step to obtain a diluted solution by diluting the reaction solution, and A measurement step of measuring the R of the diluent, It has, and further, The analytical method is characterized by containing luminescent particles in the luminescent reagent. (Method 2) The analytical method according to Method 1, characterized in that the luminescent particles contain a europium complex. (Method 3) The analytical method according to Method 1 or Method 2, characterized in that the luminescent reagent contains a ligand that binds to the target substance. (Method 4) The analytical method according to method 3, characterized in that the ligand is an antibody and the target substance is an antigen. (Method 5) The analytical method according to any one of Methods 1 to 4, characterized in that when R measured with respect to the luminescent reagent that has not reacted with the target substance is defined as R0, R0 ≥ 0.001. (Method 6) The analytical method according to any one of Methods 1 to 5, characterized in that the reaction solution is diluted by more than two times in the dilution step. (Method 7) The analysis method according to any one of Methods 1 to 6, wherein in the dilution step, the luminescent reagent is diluted so as to be 0.05 mg / ml or less. (Method 8) The analysis method according to any one of Methods 1 to 7, wherein a value (R) related to polarization anisotropy is measured with an optical system having an optical path length of 5 mm or less. (Method 9) The analysis method according to any one of Methods 1 to 8, wherein the R is defined as r in the following formula (1).

Number

Explanation of Signs

[0105] 1 particle substrate 2 hydrophilic layers 3 Europium complex

Claims

1. By using a luminescent reagent that reacts with the target substance, and measuring the value related to polarization anisotropy (R), An analytical method for determining at least one of the presence or absence of the target substance and the concentration of the target substance, A reaction step in which a sample containing the target substance and the luminescent reagent are mixed and reacted to obtain a reaction solution, A dilution step to obtain a diluted solution by diluting the reaction solution, and A measurement step of measuring the R of the dilution, It has, and further, The analytical method is characterized by containing luminescent particles in the luminescent reagent.

2. The analytical method according to claim 1, characterized in that the luminescent particles contain a europium complex.

3. The analytical method according to claim 1, characterized in that the luminescent reagent contains a ligand that binds to the target substance.

4. The analytical method according to claim 3, characterized in that the ligand is an antibody and the target substance is an antigen.

5. The analytical method according to claim 1, characterized in that when R measured with respect to the luminescent reagent that has not reacted with the target substance is defined as R0, R0 ≥ 0.

001.

6. The analytical method according to claim 1, characterized in that the reaction solution is diluted by more than two times in the dilution step.

7. The analytical method according to claim 1, characterized in that the luminescent reagent is diluted in the dilution step to a concentration of 0.05 mg / ml or less.

8. The analysis method according to claim 1, characterized in that the R is measured in an optical system having an optical path length of 5 mm or less.

9. The analysis method according to claim 1, characterized in that the aforementioned R is determined by r in the following formula (1). [Math 1] (In the above formula (1), IVV is the emission intensity of the emission component whose vibration direction is parallel to the first polarization when excited with the first polarization. IVH is the emission intensity of the emission component whose vibration direction is perpendicular to the first polarization when excited with the first polarization. IHV is the emission intensity of the emission component whose vibration direction is perpendicular to the second polarization when excited with a second polarization whose vibration direction is perpendicular to the first polarization. IHH is the emission intensity of the emission component whose vibration direction is parallel to the second polarization when excited with a second polarization whose vibration direction is perpendicular to the first polarization. G... Correction value (That is the case.)

10. The analytical method according to claim 1, characterized in that the viscosity of the diluent is 25 mPa·s or less.

11. The analysis method according to claim 1, characterized in that the average particle diameter of the luminescent particles is 25 nm or more and 500 nm or less.

12. The analytical method according to claim 2, characterized in that the luminescent particles have a hydrophilic layer containing a hydrophilic polymer or hydrophilic molecules on the outside of the particle substrate containing the europium complex.

13. By using a luminescent reagent that reacts with the target substance, and measuring the value related to polarization anisotropy (R), An analytical device for determining at least one of the presence or absence of the target substance and the concentration of the target substance, A reaction unit that mixes a sample containing the target substance and the luminescent reagent and reacts them to obtain a reaction solution. A dilution unit for diluting the reaction solution to obtain a diluted solution. A measuring unit for measuring the R of the dilution solution, and control unit It has, The analytical apparatus is characterized by containing luminescent particles in the luminescent reagent.

14. The analytical apparatus according to claim 13, wherein the measuring unit measures R based on the emission intensity of a light-emitting component whose vibration direction is parallel to the first polarization when the light-emitting reagent is excited with the first polarization, and the emission intensity of a light-emitting component whose vibration direction is perpendicular to the first polarization when the light-emitting reagent is excited with the first polarization.

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