Analysis method, analysis device, and analysis kit for calculating concentration of target substance

Abstract

The present invention relates to an analysis method, an analysis apparatus, and an analysis kit for calculating the concentration of a target substance. Provided is 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 using a reagent that reacts with the target substance, the analysis method comprising: an input step for inputting the target substance; putting a sample containing the target substance, a hydrophilic polymer which reacts with the silanol group of the glass container, and a reagent into the glass container; a reaction step of reacting the target substance and the reagent with each other to provide a reaction solution; and an analysis step of determining at least any one of the presence or absence of the target substance and the concentration of the target substance in the reaction liquid, in which the inputting step includes inputting the reagent after or at the same time as the inputting of the hydrophilic polymer.

Description

Technical Field

[0001] The present invention relates to an analysis method, an analysis device and an analysis kit for calculating the concentration of a target substance. Background Art

[0002] In the field of medicine and clinical trials, for example, it is necessary to detect or quantify with high sensitivity trace biological components from the collected parts such as blood or organs to study the causes and presence or absence of diseases, for example. In the test technology of biological components, immunoassays are widely used. In many immunoassays, a washing step called binding / free (B / F) separation is required. As an immunoassay that does not require B / F separation, there is a latex agglutination method utilizing antigen-antibody reaction. In the latex agglutination method, latex particles each loaded with antibodies such as antibodies specifically bound 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 an antibody that is bound to latex particles and is specific to the target substance, and a plurality of latex particles are cross-linked via the captured target substance, resulting in agglutination of the latex particles. That is, the amount of the target substance in a liquid sample such as a biological sample can be quantified by evaluating the degree of agglutination of the latex particles. The degree of agglutination can be quantified by measuring and evaluating the change in the amount of light transmitted or scattered by the liquid sample.

[0004] The latex agglutination method can detect / quantitatively evaluate an antigen as a target substance in a simple and rapid manner. Meanwhile, the latex agglutination method involves a problem of detection limit, that is, the antigen cannot be detected when the amount of the antigen in a liquid sample such as a biological sample is small.

[0005] In order to improve the detection sensitivity of the target substance, it is required to measure the degree of agglutination with higher sensitivity. That is, it is conceivable to replace the system for measuring the change in the amount of light transmitted or scattered by the liquid sample with a detection / quantification method using luminescence characteristics with higher sensitivity. Specifically, for example, a sample testing method using a fluorescence depolarization method has been proposed (Japanese Patent Publication No. H03-52575 and Japanese Patent No. 2893772). In Japanese Patent Publication No. H03-52575, it is proposed to improve the equipment used for the fluorescence depolarization method for clinical use.

[0006] In the fluorescence depolarization method, the B / F separation required by the general fluorescence determination method is not required. Therefore, the use of the fluorescence depolarization method can perform simple sample testing like the latex agglutination method. In addition, it is envisaged that the fluorescence depolarization method can be used to measure by the same test system as the latex agglutination method by mixing only the luminescent substance that specifically reacts with the target substance during the measurement process. At the same time, in Japanese Patent Publication No. H03-52575, it is proposed to use a single molecule such as fluorescein as a luminescent material, which is only applicable to drugs, low molecular weight antigens, etc. in principle.

[0007] Japanese Patent No. 2893772 solves the problem of Japanese Patent Publication No. H03-52575, that is, the problem that the fluorescence depolarization method is only applicable to drugs and low molecular weight antigens. That is, in Japanese Patent No. 2893772, with the purpose of applying the fluorescence depolarization method to macromolecules such as proteins, it is proposed to use a material obtained by adsorbing a dye having a long-life luminescence characteristic to latex particles as a luminescent material. In Japanese Patent No. 2893772, it is proposed to quantify high molecular weight substances by balancing the reduction of the rotational Brownian motion of the substance in the liquid due to the increase in particle diameter and the length of the luminescence life based on the principle of the fluorescence depolarization method. In the fluorescence depolarization method using such particles, it is necessary to reduce signal noise by surface treating the particles to make the surface of the particles hydrophilic, thereby suppressing nonspecific adsorption.

[0008] When high-sensitivity measurement is performed by using measurement based on the fluorescence depolarization method, it is required to measure the minute difference in the amount of light emitted between the polarized light components with high accuracy. Therefore, it is necessary to remove the factor of signal noise for performing high-sensitivity measurement.

[0009] One of such signal noises is a problem that occurs when reusing an optical cell for measuring polarized light components. Generally, when an optical cell made of glass is used for measurement, the optical cell can be reused by washing after use. Therefore, the use of an optical cell made of glass is suitable in terms of cost. However, when the washing of the optical cell after the measurement of a sample is insufficient, and the luminescent material used for measurement remains on the wall of the cell even in a trace amount, accurate spectroscopic measurement cannot be performed when measuring the next sample.

[0010] In particular, in the measurement based on the fluorescence depolarization method aiming at high-sensitivity measurement, high-precision washing may even become a factor for determining the sensitivity limit of the measurement method.

[0011] In particular, the affinity between each particle whose surface is modified to be hydrophilic and the silanol group present on the glass surface can be increased, with the result that the particles each have a high probability of being attached to the wall surface of the cell. Summary of the invention

[0012] Provided is an analytical method capable of reducing noise when calculating the concentration of a target substance by using a reagent that reacts with the target substance, particularly when performing high-sensitivity measurements by using a small amount of luminescent particles, by suppressing nonspecific adsorption of the reagent on a glass container, such as a method based on polarization anisotropy.

[0013] As an embodiment, the present invention provides an analytical method for determining at least any one of the presence or absence of a target substance and the concentration of the target substance by using a reagent that reacts with the target substance, the analytical method comprising: an introduction step of introducing a sample containing the target substance, a hydrophilic polymer that reacts with a silanol group of a glass container, and a reagent into a glass container; a reaction step of causing the target substance and the reagent to react with each other to provide a reaction liquid; and an analytical step of determining at least any one of the presence or absence of the target substance and the concentration of the target substance in the reaction liquid, wherein the reagent comprises a luminescent reagent having a hydrophilic surface, and the luminescent reagent comprises luminescent particles, and wherein the introduction step comprises introducing the reagent after or simultaneously with introducing the hydrophilic polymer.

[0014] In addition, as an embodiment, the present invention provides an analytical device for determining at least any one of the presence or absence of a target substance and the concentration of the target substance; the analytical device includes: a glass container for accommodating a reaction liquid including a sample containing the target substance and a reagent reacting with the target substance; a unit configured to introduce the sample containing the target substance into the glass container; a unit configured to introduce a hydrophilic polymer that reacts with the silanol group of the glass container into the glass container; a unit configured to introduce the reagent into the glass container after or simultaneously with the introduction of the hydrophilic polymer; an irradiation unit configured to irradiate the glass container with light that is linearly polarized light as irradiation light; a measuring unit configured to measure the fluorescence intensity of fluorescence of two polarized light components (two polarized light components) in parallel and orthogonal directions relative to the linearly polarized light; and a processing unit configured to process the results obtained by the measuring unit to thereby determine at least any one of the presence or absence of the target substance and the concentration of the target substance in the reaction liquid, wherein the reagent includes a luminescent reagent having a hydrophilic surface, and the luminescent reagent includes luminescent particles.

[0015] In addition, as an embodiment, the present invention provides an analytical kit for use in an analytical method for determining at least any one of the presence or absence of a target substance and the concentration of the target substance based on polarization anisotropy, the analytical kit comprising: a reagent that reacts with the target substance; a glass container for containing a reaction solution including a sample containing the target substance and the reagent; and a hydrophilic polymer that reacts with a silanol group of the glass container, wherein the reagent includes a luminescent reagent having a hydrophilic surface, and the luminescent reagent includes luminescent particles.

[0016] Further features of the present invention will become apparent from the following description of exemplary embodiments with reference to the attached drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is a schematic diagram for explaining an analysis method according to one embodiment of the present invention.

[0018] Figure 2 is a schematic diagram for explaining an analysis apparatus according to one embodiment of the present invention.

[0019] Figure 3 is a schematic diagram for explaining a part of an analysis apparatus according to one embodiment of the present invention.

[0020] Figure 4 is a schematic diagram for illustrating a luminescent reagent used in one embodiment of the present invention. DETAILED DESCRIPTION

[0021] Exemplary embodiments of the present invention are described in detail below. However, these embodiments are not intended to limit the scope of the present invention.

[0022] The present invention provides the following analytical method as one embodiment.

[0023] An analysis method according to one embodiment of the present invention is an analysis method for determining at least any one of the presence or absence of a target substance and the concentration of the target substance by using a reagent that reacts with the target substance, the analysis method comprising: an introduction step of introducing a sample containing the target substance, a hydrophilic polymer that reacts with a silanol group of a glass container, and a reagent into a glass container; a reaction step of causing the target substance and the reagent to react with each other to provide a reaction liquid; and an analysis step of determining at least any one of the presence or absence of the target substance and the concentration of the target substance in the reaction liquid, wherein the reagent comprises a luminescent reagent having a hydrophilic surface, and the luminescent reagent comprises luminescent particles, and wherein the introduction step comprises introducing the reagent after or simultaneously with introducing the hydrophilic polymer.

[0024] Herein, a polymer having a pyrrolidone ring such as polyvinylpyrrolidone is sometimes abbreviated as "PVP" hereinafter.

[0025] You can refer to Figure 1 An analysis method according to one embodiment of the present invention is described. That is, the analysis method according to one embodiment of the present invention includes an input step 11, a reaction step 12, and an analysis step 13, and may further include a removal step 14 and a washing step 15. These steps are described below.

[0026] (About investment steps)

[0027] The analysis method according to one embodiment of the present invention comprises a step of introducing a sample containing a target substance, a hydrophilic polymer that reacts with a silanol group of a glass container, and a reagent into a glass container. In the step of introducing, the reagent is introduced after or simultaneously with the introduction of the hydrophilic polymer.

[0028] As long as the reagent is not put into the glass container before the hydrophilic polymer, the hydrophilic polymer, the sample and the reagent can all be put into at the same time, or can be put into separately from each other. In addition, as long as the reagent is not put into the glass container before the hydrophilic polymer, two of the hydrophilic polymer, the sample and the reagent can be put into at the same time, and the other can be put into at different times. Specifically, it is given: it involves first putting into the hydrophilic polymer, and then putting into the sample and the reagent at the same time or separately from each other; and it involves first putting into the sample, and then putting into the hydrophilic polymer and the reagent at the same time or after putting into the hydrophilic polymer. In addition, it is given: it involves putting into the method of hydrophilic polymer and reagent at the same time, and then putting into the sample; and it involves putting into the method of hydrophilic polymer and sample at the same time, and then putting into the reagent. When putting into the sample and the reagent at the same time, the sample and the reagent can be put into another container after reacting with each other.

[0029] In the step of adding, the hydrophilic polymer may be added to the glass container simultaneously with the diluent after being dissolved in the diluent in advance. The diluent may be used as a solvent when the hydrophilic polymer is added to the glass container, and may contain a sample, a reagent, or any other substance.

[0030] When the concentration of the hydrophilic polymer relative to the diluent is sufficient, the reaction between the silanol groups present on the inner wall of the glass container and the hydrophilic polymer begins immediately after the injection, and the inner wall of the glass container is coated with the hydrophilic polymer. The reaction between the hydrophilic polymer and the silanol group is, for example, hydrogen bonding or adsorption based on electrostatic interaction. Even when the hydrophilic polymer and the reagent are injected at the same time, the luminescent reagent can be prevented from being adsorbed on the wall of the glass container. It is assumed that when the concentration of the hydrophilic polymer is set to be higher than the concentration of the luminescent reagent, the hydrophilic polymer reacts with the silanol groups on the wall of the glass container significantly exceeding the luminescent reagent. It is only necessary that the concentration of the hydrophilic polymer is 0.001 mass % or more and 2.0 mass % or less relative to the reaction solution in the analysis step described later. It is more appropriate to use a hydrophilic polymer concentration of 0.03 mass % or more and 0.3 mass % or less relative to the reaction solution in the analysis step.

[0031] The reagent can also be put into the glass container without putting the target substance into the glass container. The hydrophilic polymer that reacts with the silanol group has been adsorbed on the wall of the glass container before the luminescent reagent reacts with the silanol group on the wall, so the luminescent reagent can be prevented from being adsorbed on the wall of the glass container. When the luminescent reagent is adsorbed on the wall of the glass container, its rotational motion is suppressed, which leads to an increase in the value (R) of polarization anisotropy described later, for example, and thus becomes a factor in the occurrence of measurement noise. Therefore, when the adsorption of the luminescent reagent on the wall of the glass container is suppressed, the possibility of measurement noise can be reduced.

[0032] The hydrophilic polymer, target substance, reagent and diluent are described below.

[0033] (About reaction steps)

[0034] The analysis method according to one embodiment of the present invention includes a reaction step in which a target substance and a reagent react with each other to provide a reaction solution. The reaction solution is a liquid including a sample containing the target substance and a reagent, and is used as an object for determining at least one of the presence or absence of the target substance and the concentration of the target substance in the analysis step. In addition to the sample and reagent containing the target substance, the reaction solution may include an additive, etc., or may include a hydrophilic polymer. The input step may include inputting a sample and a reagent containing the target substance into a glass container to mix the materials, so that the target substance and the reagent react with each other in the glass container to provide a reaction solution. When the target substance and the reagent react with each other by inputting a sample and a reagent containing the target substance into a glass container in the input step and mixing the materials with each other, the input step may be considered to include a reaction step. The reaction is preferably carried out in a state in which the pH of the reaction solution is in the range of more than 3.0 and less than 11.0. In addition, the reaction temperature may fall within the range of 20°C to 50°C. The reaction time is set in consideration of, for example, the concentration of the target substance in the sample or the affinity between the target substance and the reagent, but is preferably about 5 minutes to about 24 hours, more preferably about 5 minutes to about 1 hour.

[0035] (About analysis steps)

[0036] An analysis method according to one embodiment of the present invention includes an analysis step of determining at least any one of the presence or absence of a target substance and the concentration of the target substance in a reaction liquid.

[0037] In the analysis step, at least one of the presence or absence of the target substance and the concentration of the target substance in the reaction liquid can be determined by measuring the value of polarization anisotropy (R) of the reaction liquid. At this time, ΔR can be obtained by measuring the R of the reaction liquid immediately after mixing the sample containing the target substance with the reagent, and subtracting the obtained R from the R of the reaction liquid measured after a certain period of reaction. The presence or absence of the target substance in the reaction liquid or its concentration can be obtained by comparing the size of the obtained ΔR with the ΔR measured in advance in the same manner for a liquid with a known concentration of the target substance.

[0038] Alternatively, after a sample containing a target substance is mixed with a reagent, R of the reaction solution may be measured at regular intervals, R may be plotted against the number of measurements, and the slopes may be compared to quantitatively evaluate the target substance.

[0039] Alternatively, the target substance may be quantitatively evaluated by comparing R of a reaction solution after a certain period of time has passed since a sample containing the target substance and a reagent were mixed.

[0040] Alternatively, the target substance can be quantitatively evaluated by presetting the endpoint R and comparing the time required to reach R.

[0041] In the analysis method according to one embodiment of the present invention, the presence or absence of the target substance can also be determined by comparing the concentration of the target substance with a predetermined threshold value. For example, it can be determined in a manner that when the concentration of the target substance is equal to or greater than the predetermined threshold value, the target substance is determined to be present, and when the concentration of the target substance is lower than the predetermined threshold value, the target substance is determined to be absent.

[0042] The measurement conditions are preferably as follows: for example, in the analysis step, the temperature of the reaction solution is 0°C or higher and 50°C or lower, and the viscosity of the reaction solution is 0.5mPa·s or higher and 50mPa·s or lower. In the analysis step, the concentration of the luminescent reagent in the reaction solution is preferably 0.0001mg / ml or higher and 0.1mg / ml or lower, and the detection wavelength is preferably 500nm or higher and 700nm or lower. In order to suppress the influence of multiple scattering, the optical path of the optical system can be shortened. For example, the optical path, which is usually set to 10mm in many cases, can be set to 5mm or lower.

[0043] (About removal steps)

[0044] The analysis method according to one embodiment of the present invention may include a removal step of removing the analyzed reaction solution from the glass container. The method for removing the reaction solution is not particularly limited, but a suction method is appropriately used. In addition, after removing the reaction solution, it is preferably quickly transferred to the washing step described later before the residual liquid that has not been removed dries.

[0045] (About washing steps)

[0046] The analysis method according to one embodiment of the present invention may include a washing step of washing the glass container after the analysis. In the washing step, the residual reaction solution can be completely removed by rinsing the glass container with a washing liquid. Pure water can be used as the washing liquid. A solution in which the pH is adjusted by adding an acidic or alkaline substance to the pure water can also be used. In addition, washing can be performed by repeatedly using pure water, an acidic liquid, and an alkaline liquid in sequence. A detergent such as a surfactant that improves the washing effect can also be added to the washing liquid. Acid, alkali, and detergent components can also be removed by repeatedly rinsing the glass container with pure water at the end of the washing step.

[0047] In order to improve the washing effect, the wall surface of the glass container may be rubbed with a sponge or the like which does not damage the glass container during the rinsing operation.

[0048] In order to improve the washing effect, the wall surface of the glass container may be vibrated by irradiating ultrasonic waves or the like to the wall surface of the glass container, thereby generating bubbles in the liquid during the rinsing operation.

[0049] The hydrophilic polymer of the silanol group adsorbed on the wall surface of the glass container does not need to be completely removed by washing. It can be expected that when the next sample is analyzed, the hydrophilic polymer will play an effect of preventing the luminescent reagent from attaching together with the newly supplied hydrophilic polymer.

[0050] In an analysis method according to one embodiment of the present invention, the luminescent reagent includes particles, so that the target substance whose concentration will be measured can be detected in response to the aggregation / dispersion behavior of the particles. For example, in an analysis method for determining the presence or absence of a target substance or the concentration of a target substance based on polarization anisotropy, when the luminescent reagent includes particles, the change in the value (R) of polarization anisotropy can be captured to a large extent, so that high-sensitivity measurement can be performed. At the same time, for example, when the luminescent reagent is non-specifically adsorbed on the wall of a glass container during the measurement of R, a numerical value higher than the appropriate value of R that is expected to be measured is measured. That is, the adsorbed luminescent reagent becomes measurement noise. The luminescent reagent preferably has a hydrophilic surface property to prevent it from reacting non-specifically with impurities in the liquid. However, due to the hydrophilicity of the luminescent reagent, the luminescent reagent can form hydrogen bonds with the silanol groups on the surface of the glass container.

[0051] The inventors of the present invention studied reducing the noise of the luminescent signal in the reaction between the target substance and the reagent when the glass container is reused, thereby performing highly sensitive measurement, such as measurement based on polarization anisotropy.

[0052] In order to reduce the luminescence signal noise, it is only necessary to prevent the luminescence reagent having a level equal to or higher than its measurement sensitivity from being adsorbed onto the wall surface of the glass container.

[0053] That is, it is only necessary to suppress the adsorption reaction between the luminescent reagent and the silanol group of the glass container due to the luminescent reagent having a hydrophilic surface.

[0054] Especially when high-sensitivity measurement using a small amount of luminescent reagent is performed, the adsorption phenomenon of the luminescent reagent on the wall surface of the glass container is prominently manifested as noise. For example, when an analysis based on polarization anisotropy is performed, the ratio of the aggregated luminescent reagent is observed before and after the reaction by using the value (R) of polarization anisotropy. Therefore, as the concentration of the luminescent reagent is set lower, the change of R observed with the aggregation of the luminescent reagent increases. That is, when high-sensitivity measurement is performed, it is preferred to set the concentration of the luminescent reagent to be lower. However, when the concentration of the luminescent reagent is low, even when the luminescent reagent is adsorbed on the wall surface of the glass container, R also changes (increases), which becomes noise during measurement. Therefore, suppressing the adsorption of the luminescent reagent on the inner wall surface of the glass container significantly contributes to the stability of high-sensitivity measurement.

[0055] In the analysis method, analysis device and analysis kit according to one embodiment of the present invention, the surface of the glass container is coated with a hydrophilic polymer before the luminescent reagent reacts with the silanol groups on the wall surface of the glass container. Thus, the luminescent reagent having a hydrophilic surface can be inhibited from being adsorbed onto the surface of the glass container via the silanol groups. As a result, noise components such as an increase in the polarization anisotropy value (R) that occur due to the adsorption of the luminescent reagent onto the wall surface of the glass container can be prevented.

[0056] In addition, according to the above method, apparatus, and kit, it was found that when washing the glass container after measurement, the luminescent reagent can be sufficiently removed by a simple washing method.

[0057] (Value of polarization anisotropy)

[0058] In an analysis method according to one embodiment of the present invention, in the analysis step, at least one of the presence or absence of a target substance and the concentration of the target substance in a reaction solution can be determined by measuring the value (R) of the polarization anisotropy of the reaction solution. In one embodiment of the present invention, the value (R) of the polarization anisotropy can be defined as follows. That is, for the luminescence generated by exciting a luminescent substance by irradiation with polarized light, R can be set to a value showing the relationship between the luminescence intensity of a polarized light component in a parallel direction relative to the polarized light as the incident light and the luminescence intensity of a polarized light component in a perpendicular direction relative to the polarized light as the incident light. More specifically, R can be set to a value calculated from the luminescence intensity of a luminescent component having a vibration direction parallel to the vibration direction of a given polarized light and a luminescent component having a vibration direction orthogonal (perpendicular) to the vibration direction of a given polarized light, each luminescence intensity being obtained when the luminescent substance is excited by polarized light. In addition, R can be set to a value representing the ratio of the difference between the luminous intensity of the luminous component whose vibration direction is parallel to the first polarized beam when excited by the first polarized beam and the luminous intensity of the luminous component whose vibration direction is orthogonal to the first polarized beam when excited by the first polarized beam, relative to the sum of the luminous intensities.

[0059] R can be corrected by: the ratio of the luminous intensity of the luminous component whose vibration direction is orthogonal to the vibration direction of the second polarized beam when excited by the second polarized beam whose vibration direction is orthogonal to the vibration direction of the first polarized beam and the luminous intensity of the luminous component whose vibration direction is parallel to the vibration direction of the second polarized beam when excited by the second polarized beam whose vibration direction is orthogonal to the vibration direction of the first polarized beam; and other constants. The value of polarization anisotropy includes values ​​called "polarization anisotropy property" and "polarization degree".

[0060] More specifically, for example, R may be "r" in the following formula (1):

[0061]

[0062] In formula (1), I VV represents the luminous intensity of the luminous component whose vibration direction is parallel to the vibration direction of the first polarized light beam when excited by the first polarized light beam, I VH I represents the luminous intensity of the luminous component whose vibration direction is orthogonal to the vibration direction of the first polarized light beam when excited by the first polarized light beam, HV I represents the luminous intensity of the luminous component whose vibration direction is orthogonal to the vibration direction of the second polarized light beam when excited by the second polarized light beam whose vibration direction is orthogonal to the vibration direction of the first polarized light beam, HH represents the luminous intensity of the luminous component whose vibration direction is parallel to the vibration direction of the second polarized beam when excited by the second polarized beam whose vibration direction is orthogonal to the vibration direction of the first polarized beam, and G represents the correction value.

[0063] In addition, R may be r' in the following formula (2):

[0064]

[0065] In formula (2), each symbol is the same as that in formula (1).

[0066] The measurement conditions of R are as follows. For example, when measuring, the temperature of the reaction liquid is preferably 0° C. or higher and 50° C. or lower, and the viscosity of the reaction liquid is preferably 0.5 mPa·s or higher and 50 mPa·s or lower. When the luminescent reagent includes luminescent particles containing a europium complex, the concentration of the luminescent reagent in the reaction liquid is preferably 0.0001 mg / ml or higher and 0.1 mg / ml or lower, and the measurement wavelength is preferably 500 nm or higher and 700 nm or lower.

[0067] In addition, R0, which represents R measured for a reagent that has not reacted with a target substance, preferably satisfies the formula: R0 ≥ 0.01.

[0068] (Diluent)

[0069] The diluent may contain an aqueous solvent as a main component. The pH of the diluent may be adjusted to promote the reaction between the target substance and the reagent. The pH may be adjusted from 1.0 to 14.0 by using a buffer solution. The pH of the diluent may be appropriately used to be 5.0 to 9.0. In addition, in order to suppress nonspecific adsorption and promote the reaction between the target substance and the reagent, an additive may also be added thereto. However, it is necessary to select and use an additive that does not cause interaction with each of the target substance and the reagent.

[0070] (Hydrophilic polymer reactive with silanol groups)

[0071] Polymers with such physical properties can be used, i.e., polymers react with the silanol groups present on the wall of the glass container and do not non-specifically adsorb luminescent agents, as hydrophilic polymers reacting with silanol groups.Silanol groups represent structures in which hydroxyl groups are directly bonded to silicon.Silanol groups are also hydrophilic, and in aqueous solution, silanol groups and water molecules usually form weak bonds via hydrogen bonds in many cases.However, the reactivity of silanol groups is high, and according to conditions, hydrogen bonds can be formed with molecules with functional groups other than water.This property causes luminescent agents to be non-specifically adsorbed to the silanol groups on the wall of the glass container.In order to prevent this phenomenon, it is effective to make hydrophilic polymers adsorbed in advance to the silanol groups on the wall of the glass container.

[0072] For example, a hydrophilic polymer that forms hydrogen bonds with silanol groups of the glass container can be used as the hydrophilic polymer. In one embodiment of the present invention, the hydrophilic polymer that forms hydrogen bonds with silanol groups is required to have a hydrogen bonding ability that is equal to or stronger than the affinity between the luminescent agent and the silanol groups.

[0073] Specifically, for example, polyvinyl pyrrolidone or polyethylene glycol can be used appropriately.

[0074] In addition, a hydrophilic polymer adsorbed on a silanol group by electrostatic interaction can be used as a hydrophilic polymer. The silanol group is positively charged or negatively charged according to the pH conditions used, so the hydrophilic polymer ion can also be adsorbed thereto. In this case, the hydrophilic polymer reacted with the silanol group can have a carboxyl or amine ion functional group. Specifically, for example, polyethyleneimine can be used.

[0075] The hydrophilic polymer reacting with the silanol group in one embodiment of the present invention preferably has a molecular weight and concentration that allows the hydrophilic polymer to be completely dissolved in the diluent and does not affect the viscosity of the diluent. Specifically, the weight average molecular weight of the hydrophilic polymer is preferably 100 or more and 100,000 or less. The concentration of the hydrophilic polymer can be set to 0.001 mass % or more and 2.0 mass % or less relative to the reaction solution in the analysis step, and is preferably 0.03 mass % or more and 0.3 mass % or less.

[0076] (Target substance)

[0077] Examples of target substances may include antigens, antibodies, low molecular weight compounds, various receptors, enzymes, substrates, nucleic acids, cytokines, hormones, neurotransmitters, transmitters and membrane proteins. Examples of antigens include allergens, bacteria, viruses, cells, cell membrane components, cancer markers, various disease markers, antibodies, blood-derived substances, food-derived substances, substances derived from natural products and any low molecular weight compounds. Examples of nucleic acids include DNA, RNA or cDNA derived from bacteria, viruses or cells, parts or fragments thereof, synthetic nucleic acids, primers and probes. Examples of low molecular weight compounds include cytokines, hormones, neurotransmitters, transmitters, membrane proteins and their receptors.

[0078] (Reagents)

[0079] The analytical method, analytical equipment and analytical kit according to one embodiment of the present invention can be used for sample testing or in vitro diagnosis. Reagents for such purposes include luminescent reagents with hydrophilic surfaces, and may include a dispersion medium for dispersing the luminescent reagent. The amount of the luminescent reagent introduced into the reagent is preferably 0.000001% by mass or more and 20% by mass or less, more preferably 0.0001% by mass or more and 1% by mass or less. In the scope of being able to achieve the purpose of the present invention, in addition to the luminescent reagent, the reagent may also include a third substance such as an additive or a sealing agent. The reagent may include a combination of two or more third substances such as an additive and a sealing agent. Examples of dispersion media include various buffer solutions, such as phosphate buffer solution, glycine buffer solution, Good's buffer solution, Tris buffer solution and ammonia buffer solution, but the dispersion medium is not limited thereto.

[0080] The reagent may include a ligand, and when the reagent is used to detect an antigen or an antibody in a test sample, the antibody or the antigen may be used as the ligand.

[0081] (Luminescent Reagent)

[0082] The luminescent reagent in one embodiment of the present invention has a hydrophilic surface and comprises luminescent particles. In one embodiment of the present invention, the luminescent reagent is a reagent that produces luminescence, particularly a reagent that is excited and emits light when irradiated with light, and does not include reagents based on luminescence produced by chemical reactions, such as luminol. Luminescence includes phosphorescence and fluorescence, but phosphorescence is preferred. In one embodiment of the present invention, the luminescent particles more preferably include europium complexes. In addition, the luminescent reagent preferably includes a ligand specific to the target substance. That is, the luminescent reagent preferably includes a ligand that binds to the target substance. The introduction of the ligand enables the luminescent reagent to detect / quantify the target substance based on polarization anisotropy. In one embodiment of the present invention, "ligand" refers to a compound that specifically binds to a specific target substance.

[0083] Any compound that shows affinity for a specific substance can be used as a ligand. Examples of combinations of ligands and target substances, or target substances and ligands may include the following. That is, examples may include: antigens and antibodies; low molecular weight compounds and their receptors; enzymes and substrates; and nucleic acids that are complementary to each other. In addition, examples may include antibodies and any of the following substances specific to them: allergens, bacteria, viruses, cells, cell membrane components, cancer markers, various disease markers, antibodies, blood-derived substances, food-derived substances, substances from natural products, and any low molecular weight compounds. In addition, examples may include receptors and any of the following substances specific to them: low molecular weight compounds, cytokines, hormones, neurotransmitters, transmitters, and membrane proteins. In addition, examples may include DNA, RNA or cDNA derived from bacteria, viruses or cells, parts or fragments thereof, synthetic nucleic acids, primers or probes, and nucleic acids complementary thereto. In addition to the above, any combination known to have affinity is used as a combination of target substances and ligands. Typical examples of ligands are any of antibodies, antigens or nucleic acids. In addition, it is particularly preferred that the ligand is an antibody and the target substance is an antigen.

[0084] Figure 4 4 is a schematic diagram for illustrating a luminescent agent used in one embodiment of the present invention. A luminescent agent 44 used in one embodiment of the present invention, for example, includes a particle matrix 41 used as a luminescent particle containing a europium complex 43 as a luminescent molecule. In addition, the luminescent agent 44 has a hydrophilic layer 42 covering its surface. The luminescent agent used in one embodiment of the present invention can emit long-life phosphorescence due to the europium complex. In the luminescent agent used in one embodiment of the present invention, the average particle size as the average value of the particle size of the luminescent agent containing a hydrophilic layer is preferably 25 nm or more and 500 nm or less, more preferably 50 nm or more and 300 nm or less. When the average particle size is greater than 500 nm, R (R0) before aggregation increases, with the result that the difference between R0 and R after the aggregation reaction decreases. In addition, when the average particle size is less than 25 nm, the size change before and after aggregation decreases, with the result that, for example, in the measurement of polarization anisotropy based on the use of phosphorescence, it is difficult to capture the change in R.

[0085] The particle size of the luminescent reagent can be obtained by dynamic light scattering. When the particles dispersed in the solution are irradiated with a laser and the resulting scattered light is observed with a photon detector, the intensity distribution caused by the interference of the scattered light fluctuates continuously because the particles constantly move their positions through Brownian motion. The dynamic light scattering method is a state measurement method for observing Brownian motion as a fluctuation of the intensity of the scattered light. The fluctuation of the scattered light relative to time is expressed as an autocorrelation function, and the translational diffusion coefficient is determined. The Stokes diameter is determined from the determined diffusion coefficient, and the size of each particle dispersed in the solution can be derived.

[0086] From the viewpoint of maintaining the uniformity and monodispersity of the particles, the luminescent agent is preferably not disposed on the surface of the luminescent particles. However, the luminescent agent used in one embodiment of the present invention has a hydrophilic surface, and preferably has a hydrophilic layer on the surface of the luminescent particles to suppress the nonspecific adsorption of substances other than the target substance to the luminescent agent.

[0087] A method involving loading bovine serum albumin (BSA) on the particle surface is widely used as a technique for maintaining hydrophilicity, but this method may lead to differences between multiple batches. Therefore, the luminescent reagent preferably includes a hydrophilic layer comprising a hydrophilic polymer for the hydrophilic layer. The hydrophilic layer is described below.

[0088] Generally, in an analysis for calculating the concentration of a target substance with high sensitivity, particularly in an analysis based on polarization anisotropy, measurement is performed in a state where the amount of the target substance and the reagent that reacts with the target substance is small. In this case, the concentration of the luminescent reagent in the reaction solution is preferably 0.000001 mass % to 1 mass %, and more preferably 0.00001 mass % to 0.001 mass %.

[0089] By reducing the particle size distribution of the luminescent reagent and introducing a europium complex as a luminescent molecule, for example, even when the dispersion state of the particles in the liquid changes slightly, it is easy to capture the change in the value of polarization anisotropy (R). Specifically, even when the concentration of the target substance in the reaction liquid is about a few picograms to about a few nanograms per milliliter, when the luminescent reagent agglomerates via the target substance, the change in the rotational Brownian motion of the luminescent reagent can be captured as a change in the value of polarization anisotropy (R), for example.

[0090] "Polarized luminescence" refers to the following phenomenon: when a luminescent material having anisotropy of transition moment (transition dipole moment) uses polarized light along its transition moment as excitation light, its luminescence is also polarized light along the transition moment. Europium complexes show fluorescent luminescence based on energy transfer from ligands to central metal ions, so the transition moment is complex, but red luminescence near 610nm originating from the electron transition from the lowest excited state 5D0 to 7F2 is emitted as polarized light.

[0091] The principle of polarization anisotropy is to measure the shift in transition moment caused by the rotational motion of the luminescent material during polarized luminescence. The rotational motion of the luminescent material can be expressed by equation (3):

[0092] Q=3Vη / kT……(3)

[0093] where Q represents the rotational relaxation time of the material, V represents the volume of the material, η represents the viscosity of the solvent, "k" represents the Boltzmann constant, and T represents the absolute temperature.

[0094] The rotational relaxation time of a material is the time required for a molecule to rotate through an angle θ (68.5°) of cosθ = 1 / e.

[0095] It can be seen from formula (3) that the rotational relaxation time of the luminescent material is proportional to the volume of the material, that is, when the luminescent material is in the form of particles, it is the cube of the particle size. At the same time, the relationship between the luminescence lifetime of the luminescent material and the degree of polarization (which is the value of polarization anisotropy) can be expressed by formula (4):

[0096] p0 / p=1+A(τ / Q)····(4)

[0097] Wherein, p0 represents the degree of polarization when the material is stationary (Q=∞), "p" represents the degree of polarization, A is a constant, τ represents the luminescence lifetime of the material, and Q represents the rotational relaxation time.

[0098] It can be seen from equations (3) and (4) that the polarization degree is affected by the luminescence lifetime and rotational relaxation time of the luminescent material, that is, it is affected by the volume (particle size) of the luminescent material. In other words, it is affected by the balance between the particle size and luminescence lifetime of the luminescent material.

[0099] When the polarization degree of the luminescent material represented by the formula (4) is obtained by experiment, it is appropriate to allow polarized light to be incident on the luminescent material and detect the luminescence in a direction of 90° with respect to the traveling direction and the vibration direction of the excitation light. In this case, it is appropriate to detect by dividing the detection light into polarized light components in a parallel direction and a perpendicular direction with respect to the polarized light as the incident light, and to set the polarization anisotropy represented by the formula (5), for example, as the value of the polarization anisotropy:

[0100] r(t)=(I ∥ (t)-GI ⊥ (t)) / (I ∥ (t)+2GI ⊥ (t)) …… (5)

[0101] Where r(t) represents the polarization anisotropy at time “t”, I ∥I(t) represents the luminous intensity of the luminescent component parallel to the excitation light at time “t”, I⊥(t) represents the luminous intensity of the luminescent component perpendicular to the excitation light at time “t”, and G represents the correction value, i.e., I measured with the excitation light whose vibration direction is 90° different from the excitation light used for sample measurement. ⊥ / I ∥ Ratio.

[0102] That is, when the particle size and luminescence lifetime fall within an appropriate range, the size change of the luminescent material, for example due to a reaction with a target substance, can be sensitively read as a change in polarization anisotropy. That is, the r(t) of the unaggregated luminescent material is observed to be low, and the r(t) of the aggregated luminescent material is observed to be high. This is the principle of polarization anisotropy.

[0103] The value of polarization anisotropy may be corrected with G and 2G, or may be a value obtained by removing G and 2G.

[0104] (Luminous particles)

[0105] The overall shape of the luminescent agent used in one embodiment of the present invention and the luminescent particles ( Figure 4 The shape of the particle matrix 41 in is not particularly limited. As long as the material of the luminescent particles can stably introduce europium complexes, the luminescent particles are not particularly specified, but preferably are polymers comprising styrene units and organosilane units. In particular, for example, a polymer obtained by polymerizing a composition comprising styrene as a main component with a free radical polymerizable organosilane is preferred. When the composition comprises styrene as a main component, particles having an extremely uniform particle size distribution can be produced by the emulsion polymerization method described later. In addition, when a polymer comprising an organosilane unit is used, in an aqueous solvent, silanol groups (Si-OH) are generated in the polymer, and the silanol groups form siloxane bonds (Si-O-Si) with each other on the surface of the luminescent particles, thereby being able to provide a hydrophilic layer or ligand described later. The luminescent agent preferably has a ligand binding functional group capable of binding a ligand to the outside of the luminescent particle.

[0106] (Hydrophilic layer)

[0107] Hydrophilic layer ( Figure 4The hydrophilic layer 42 in the luminescent particle can be formed by introducing a hydrophilic polymer or a hydrophilic molecule for the hydrophilic layer on the outside of the luminescent particle. The hydrophilic polymer or the hydrophilic molecule for the hydrophilic layer is a polymer or molecule containing a hydrophilic group, and specific examples of the hydrophilic group include molecules or polymers each having a hydroxyl, ether, pyrrolidone or betaine structure. Specific examples of hydrophilic polymers for the hydrophilic layer include polyethylene glycol, polyvinyl pyrrolidone, polymers of sulfobetaine, polymers of phosphobetaine, and polymethacrylate glycidyl esters whose molecules have ends modified by hydroxyl groups by opening the glycidyl ring. These hydrophilic polymers can each be used as the main component of the hydrophilic layer. Alternatively, the hydrophilic layer can be formed by directly providing a single molecule with a hydrophilic group on the surface of the luminescent particle using a silane coupling agent or the like. There is no limit to the thickness of the hydrophilic layer, but it does not need to be set to be larger than the thickness that can show hydrophilicity. When the hydrophilic layer is too thick, the hydrophilic layer may become hydrogel-like and may be hydrated by the influence of ions in the solvent, thereby making its thickness unstable. The thickness of the hydrophilic layer is preferably 1 nm or more and 15 nm or less.

[0108] (Europium Complex)

[0109] The luminescent agent used in one embodiment of the present invention may comprise a europium complex ( Figure 4 The europium complex 43 in is used as a luminescent dye. In the luminescent agent of one embodiment of the present invention, the luminescent particles preferably include a europium complex. The europium complex has the characteristics that the wavelength and intensity of its luminescence are almost unaffected by the surrounding environment, so the luminescence has a long life. The europium complex includes a europium element and a ligand. Taking into account the luminescence lifetime and the visible luminescence wavelength region, etc., the luminescent dye is preferably a europium complex. Europium generally has a luminescence lifetime of 0.1ms to 1.0ms. The luminescence lifetime and rotational relaxation time obtained by formula (3) need to be appropriately adjusted. In the case of europium in an aqueous dispersion, for example, when the diameter of the luminescent agent is about 50nm to about 300nm, the value of polarization anisotropy (R) changes significantly before and after aggregation.

[0110] At least one of the constituent ligands of the europium complex is a ligand having a light-collecting function. "Light-collecting function" refers to the action of being excited at a specific wavelength to excite the central metal of the complex by energy transfer. In addition, it is preferred that the constituent ligands of the europium complex include ligands such as β-diketone to prevent the coordination of water molecules. Ligands such as β-diketone coordinated to europium ions suppress the deactivation process caused by energy transfer to solvent molecules, etc., thereby providing strong fluorescence luminescence.

[0111] The europium complex may be a polynuclear complex.

[0112] In addition, specific examples of europium complexes include [tris(2-thenoyltrifluoroacetone)(bis(triphenylphosphine oxide))europium(III)], [tris(2-thenoyltrifluoroacetone)(triphenylphosphine oxide)(dibenzyl sulfoxide)europium(III)], and [tris(2-thenoyltrifluoroacetone)(phenanthroline)europium(III)].

[0113] In the state where the Brownian rotational motion of the europium complex can be regarded as being stationary in the medium, "r" represented by formula (1) is preferably 0.08 or more. The state where the Brownian rotational motion can be regarded as being stationary refers to a state where the rotational relaxation time of the particles is sufficiently longer than the luminescence lifetime of the europium complex.

[0114] Since the luminescence intensity of each particle becomes stronger, it is preferred to introduce the europium complex into the luminescent particles in as much amount as possible. At the same time, when the molecules of the europium complex are aggregated in the luminescent particles, the interaction between the ligands affects the excitation efficiency of the europium complex, making it difficult to measure R, etc. while maintaining reproducibility. Whether the europium complex shows non-aggregated luminescence behavior in the luminescent particles can be judged by the excitation spectrum.

[0115] Particles with strong luminescence not only enable highly sensitive measurements, but also increase the speed of biochemical reactions by maintaining luminescence even when their particle size decreases. As the particle size becomes smaller, the diffusion coefficient of Brownian motion in the liquid becomes larger, so the reaction can be detected in a shorter time.

[0116] In one embodiment of the present invention, when a liquid in which such particles are dispersed is used in an analysis, for example, a change in the value (R) of polarization anisotropy can be detected with high sensitivity in response to the aggregation / dispersion behavior of the particles. The dispersion obtained by dispersing such particles in an aqueous solvent can be used as a highly sensitive reagent using polarization anisotropy, for example. A buffer solution can be used as an aqueous solvent. In addition, in order to improve the stability of the liquid in which the particles are dispersed, a surfactant, a preservative or a sensitizer, etc. can be added to the aqueous solvent.

[0117] (Production method of luminescent reagent)

[0118] Next, an example of a production method of a luminescent reagent used in one embodiment of the present invention is described.

[0119] The method for producing the luminescent agent may include the step of mixing a radical polymerizable monomer including styrene and a radical polymerizable organosilane, a radical polymerization initiator, a polarized luminescent europium complex, and a hydrophilic polymer for a hydrophilic layer with an aqueous medium to prepare an emulsion (first step).

[0120] Furthermore, the method for producing a luminescent agent may include a step of heating the emulsion to polymerize the radical polymerizable monomer (second step).

[0121] The production method of the luminescent reagent can include the step (third step) of arranging the ligand binding functional group described later on the surface of the luminescent reagent. In this article, the ligand binding functional group refers to a functional group that can bind to a ligand. Specifically, a carboxyl group, an amino group, a thiol group, an epoxy group, a maleimide group, a succinimide group or an alkoxysilyl group (silicon alkoxylate structure) can be used.

[0122] (Free radical polymerizable monomer)

[0123] The production of the luminescent reagent is carried out by polymerizing a free radical polymerizable monomer, and the free radical polymerizable monomer includes at least styrene and a free radical polymerizable organosilane. The free radical polymerizable monomer may further include a monomer selected from the group consisting of: an acrylate monomer; and a methacrylate monomer. Examples of monomers may include butadiene, vinyl acetate, vinyl chloride, acrylonitrile, methyl methacrylate, methacrylonitrile, methyl acrylate, and mixtures thereof. That is, in addition to styrene and a free radical polymerizable organosilane, one or more of these monomers may also be used. In addition, a monomer having more than two double bonds per molecule such as divinylbenzene may be used as a crosslinking agent.

[0124] The introduction of free radical polymerizable organosilane in free radical polymerizable monomer provides siloxane bond with luminescent particles. The example of free radical polymerizable organosilane can include vinyl trimethoxy silane, vinyl triethoxy silane, p-phenylene trimethoxy silane, 3-methacryloxy propyl methyl dimethoxy silane, 3-methacryloxy propyl trimethoxy silane, 3-methacryloxy propyl methyl diethoxy silane, 3-methacryloxy propyl triethoxy silane, 3-acryloxy propyl trimethoxy silane, and combination thereof. The use of free radical polymerizable organosilane is used to form the skeleton of inorganic oxide in luminescent particles, to improve the physical and chemical stability of luminescent reagent. In addition, the use of free radical polymerizable organosilane improves the affinity between luminescent particles and each hydrophilic layer and ligand binding functional group.

[0125] In addition, the introduction of free radical polymerizable organosilane in the free radical polymerizable monomer provides silanol groups to the surface of the luminescent particles. The silanol groups and the hydrophilic polymers for the hydrophilic layer such as PVP form hydrogen bonds. Therefore, the hydrophilic polymers for the hydrophilic layer such as PVP are more strongly adsorbed to the surface of the luminescent particles, thereby forming a hydrophilic layer.

[0126] (Free Radical Polymerization Initiator)

[0127] As the radical polymerization initiator, a wide range of compounds selected from, for example, azo compounds and organic peroxides can be used. Specific examples thereof may 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 ester, tert-butyl hydroperoxide, benzoyl peroxide, ammonium persulfate (APS), sodium persulfate (NPS), and potassium persulfate (KPS).

[0128] (Hydrophilic polymer for hydrophilic layer)

[0129] The luminescent agent may include a hydrophilic polymer for a hydrophilic layer as a hydrophilic layer. The hydrophilic polymer for the hydrophilic layer is preferably capable of inhibiting nonspecific adsorption. Examples of hydrophilic polymers for the hydrophilic layer include hydrophilic polymers for the hydrophilic layer each containing a unit having an ether, betaine or pyrrolidone ring. Preferably, the hydrophilic layer is contained in the synthesized luminescent agent and is mainly present on the particle surface outside the luminescent particle. For example, when PVP is added during the synthesis of the luminescent agent, the luminescent agent can be simultaneously given nonspecific adsorption inhibition ability and ligand binding ability. The PVP added during the synthesis has a higher hydrophilicity than each free radical polymerizable monomer, and therefore exists at the interface between the solvent and the luminescent particles polymerized during the synthesis. The luminescent particles can adsorb PVP etc. on the outside of the luminescent particles by participating in a part of the PVP during polymerization, or by physical / chemical adsorption such as the interaction between the pyrrolidone ring and styrene (free radical polymerizable monomer).

[0130] The weight average molecular weight of PVP is preferably 10,000 or more and 100,000 or less, more preferably 40,000 or more and 70,000 or less. When the weight average molecular weight is less than 10,000, the hydrophilicity of the surface of the luminescent reagent is weak, so nonspecific adsorption is easily caused. When the weight average molecular weight is greater than 100,000, the hydrophilic layer becomes so thick that it gels, thereby becoming difficult to handle.

[0131] When synthesizing the luminescent reagent, in addition to PVP, other hydrophilic polymers for the hydrophilic layer may be added as protective colloids.

[0132] In addition, the luminescent reagent preferably satisfies A2-A1≤0.1.

[0133] A1 and A2 are defined as follows. That is, with respect to a mixture obtained by adding 30 μl of a 0.1 mass % luminescent reagent dispersion to 60 μl of a buffer solution mixed with 16 μl of human serum diluted 15 times, the absorbance of the mixture immediately after addition is represented by A1, and the absorbance of the mixture after standing at 37° C. for 5 minutes after addition is represented by A2. The absorbance is measured at an optical path of 10 mm and a wavelength of 572 nm.

[0134] Particles having an A2-A1 of 0.1 or less are preferred because they have little nonspecific adsorption of impurities in serum.

[0135] (Water-based media)

[0136] The aqueous medium (aqueous solution) used for the production method of the above-mentioned luminescent reagent preferably contains water of more than 80 mass % and less than 100 mass % in the medium. The aqueous medium is preferably water or a water-soluble organic solvent, and examples thereof include solutions obtained by mixing water with methanol, ethanol, isopropanol, or acetone. When an organic solvent other than water is introduced with more than 20 mass %, dissolution of polymerizable monomers may occur during the production of the luminescent reagent.

[0137] In addition, the aqueous medium is preferably adjusted in advance to a pH of 6 or more and 9 or less. When the pH value is less than 6 or greater than 9, the alkoxy or silanol group of the radical polymerizable organosilane may undergo polycondensation or reaction with other functional groups before forming a polymer, resulting in aggregation of the obtained particles. In this example, polycondensation of the alkoxylate is not intentionally performed before polymerization.

[0138] The above pH is preferably adjusted with a pH buffer, but may be adjusted with an acid or a base.

[0139] In addition to the above, a surfactant, a defoaming agent, a salt, a thickener, or the like may be added and used in a ratio of 10% by mass or less relative to the aqueous medium.

[0140] In the production of the luminescent reagent, it is preferred that, first, PVP is dissolved in an aqueous medium whose pH has been adjusted to 6 to 9. The content of PVP is preferably 0.01% by mass or more and 10% by mass or less, and more preferably 0.03% by mass or more and 5% by mass or less relative to the aqueous medium. When the content is less than 0.01% by mass, the amount of adsorption on the luminescent particles is small and the effect is not exhibited. In addition, when the content exceeds 10% by mass, the viscosity of the aqueous medium may increase and hinder sufficient stirring.

[0141] Subsequently, a radical polymerizable monomer comprising styrene (A) and a radical polymerizable organosilane (B) is added to the above-mentioned aqueous medium to prepare an emulsion. The weight ratio between styrene (A) and the radical polymerizable organosilane (B) is 6:4 to 100:1. In addition, the prepared emulsion is mixed with a europium complex. At this time, when the solubility of the europium complex is low, a water-insoluble organic solvent may be added. The weight ratio between the europium complex and the radical polymerizable monomer is 1:1,000 to 1:10.

[0142] When the weight ratio of styrene (A) to free radical polymerizable organosilane (B) is less than 6 / 4, the specific gravity of the particles as a whole will increase, resulting in significant sedimentation of the particles. In addition, in order to improve the adhesion between PVP and the luminescent particles, it is preferred that the weight ratio of styrene (A) to free radical polymerizable organosilane (B) is set to 100 / 1 or less.

[0143] The weight ratio between the weight of the aqueous medium and the total amount of the radical polymerizable monomers is preferably 5:5 to 9.5:0.5. When the weight ratio between the weight of the aqueous medium and the total amount of the radical polymerizable monomers is less than 5 / 5, significant aggregation of the generated particles may occur. In addition, when the weight ratio between the weight of the aqueous medium and the total amount of the radical polymerizable monomers is greater than 9.5 / 0.5, although there is no problem in the production of particles, the amount thereof may be reduced.

[0144] The radical polymerization initiator is used by being dissolved in water, a buffer solution, etc. The amount of the radical polymerization initiator in the emulsion can be set to 0.5 mass % or more and 10 mass % or less relative to the total amount of styrene (A) and the radical polymerizable organosilane (B).

[0145] In the step of heating the emulsion, it is only necessary to uniformly heat the entire emulsion. The heating temperature can be arbitrarily set between 50° C. and 80° C., and the heating time can be arbitrarily set between 2 hours and 24 hours. By heating the emulsion, the free radical polymerizable monomer is polymerized.

[0146] The luminescent agent may have a ligand-binding functional group on its surface. The ligand-binding functional group may be provided to the hydrophilic layer or luminescent particles of the luminescent agent. The ligand-binding functional group is not particularly limited as long as the functional group can bind to an antibody, an antigen or an enzyme, etc. However, for example, the functional group may be a carboxyl group, an amino group, a thiol group, an epoxy group, a maleimide group, a succinimide group or a silane oxide group, or a group containing any of these functional groups. For example, a silane coupling agent having a ligand-binding functional group may be mixed with the synthesized particles to provide a functional group on the particle surface. Specifically, an aqueous solution of a silane coupling agent having a carboxyl group may be prepared and mixed with a dispersion of the synthesized particles to provide a carboxyl group to the particle surface. At this point, a dispersant such as Tween 20 may be added to the reaction solution. The reaction temperature may be arbitrarily set between 0°C and 80°C, and the reaction time may be arbitrarily set between 1 hour and 24 hours. In order to suppress the abrupt condensation reaction of the silane coupling agent, it is appropriate to set the temperature to be about 25° C. or lower than room temperature, and the reaction time to be about 3 hours to about 14 hours.

[0147] Depending on the ligand binding functional group, the reaction with the particle surface can be promoted by adding acid or base catalysts.

[0148] By binding to a ligand (such as any of various antibodies), a luminescent reagent can be used as a particle for sample testing. The best technique can be selected to bind the target antibody or the like by utilizing the functional groups present on the hydrophilic layer or the luminescent particle.

[0149] (Introduction of ligand)

[0150] The methods known so far can be applied to the chemical reaction of chemically binding the ligand binding functional group to the ligand to the extent that the purpose of the present invention can be achieved. In addition, when the ligand is an amide bond, a catalyst such as 1-[3-(dimethylaminopropyl)-3-ethylcarbodiimide] can be appropriately used.

[0151] The luminescent reagent used in the embodiment of the present invention can be applied to latex immunoagglutination measurement methods widely used in the fields of clinical trials and biochemical research, but is preferably used in analytical methods based on polarization anisotropy.

[0152] (Analytical Equipment)

[0153] The present invention provides the following analysis device as one embodiment.

[0154] An analysis device according to one embodiment of the present invention is an analysis device for determining at least any one of the presence or absence of a target substance and the concentration of the target substance, and the analysis device includes: a glass container for accommodating a reaction liquid including a sample containing the target substance and a reagent reacting with the target substance; a unit configured to introduce a sample containing the target substance into the glass container; a unit configured to introduce a hydrophilic polymer reacting with a silanol group of the glass container into the glass container; a unit configured to introduce a reagent into the glass container after or simultaneously with the introduction of the hydrophilic polymer; an irradiation unit configured to irradiate the glass container with light that is linearly polarized light as irradiation light; a measuring unit configured to measure the fluorescence intensity of fluorescence of two polarized light components in parallel and orthogonal directions relative to the linearly polarized light; and a processing unit configured to process the results obtained by the measuring unit to thereby determine at least any one of the presence or absence of the target substance and the concentration of the target substance in the reaction liquid, wherein the reagent includes a luminescent reagent having a hydrophilic surface, and the luminescent reagent includes luminescent particles.

[0155] In addition, an analysis device according to one embodiment of the present invention is an analysis device for determining at least any one of the presence or absence of a target substance and the concentration of the target substance, and the analysis device includes: a glass container for accommodating a reaction liquid including a sample containing the target substance and a reagent reacting with the target substance; a unit configured to put the sample containing the target substance into the glass container; a unit configured to put a hydrophilic polymer reacting with the silanol group of the glass container into the glass container; a unit configured to put the reagent into the glass container; an irradiation unit configured to irradiate the glass container with light that is linearly polarized light as irradiation light; a measuring unit configured to measure the fluorescence intensity of fluorescence of two polarized light components in parallel and orthogonal directions relative to the linearly polarized light; and a processing unit configured to process the results obtained by the measuring unit to thereby determine at least any one of the presence or absence of the target substance and the concentration of the target substance in the reaction liquid, wherein the processing unit is configured to perform control so that the reagent is put into the glass container after or at the same time as the hydrophilic polymer is put into the glass container.

[0156] [Example of Configuration of Analytical Device]

[0157] Reference Figure 2 and Figure 3 An example of the configuration of an analysis device is described. In the analysis device, by stirring a sample such as blood or urine and a reagent in a glass container having a square tube shape to cause a reaction therebetween, and performing, for example, measurement based on polarization anisotropy, the presence or absence of a target substance or the concentration of the target substance can be determined. Figure 2As shown, the analysis device 21 includes an analysis mechanism 22 , an analysis mechanism control unit 23 , an analysis unit 24 , a display unit 25 , an operation unit 26 , a storage unit 27 , and a system control unit 28 .

[0158] The analysis mechanism 22 operates according to the control by the analysis mechanism control unit 23. The analysis mechanism 22 is arranged in the housing of the analysis device 21. For example, Figure 2 and Figure 3 As shown, the analysis mechanism 22 is equipped with a reaction disk 1, a sample disk 2, a polymer holding tank 3, a reagent tank 4, a sample arm 5-1, a sample probe 6-1, a polymer input arm 5-2, a polymer input probe 6-2, a reagent arm 5-3, a reagent probe 6-3, a stirring arm 7, a stirring rod 8, a photometric mechanism 9 and a washing mechanism 10.

[0159] like Figure 2 As shown, the reaction disk 1 holds a plurality of glass containers 101 arranged on the circumference of the reaction disk. The reaction disk 1 alternately repeats rotation and stopping at predetermined time intervals. The sample disk 2 is arranged near the reaction disk 1. The sample disk 2 holds a sample container 201 in which a sample containing a target substance is contained. The sample disk 2 rotates so that the sample container 201 in which the sample containing the target substance is contained is arranged at the sample suction position. The polymer holding tank 3 holds a plurality of polymer holding containers 301 each containing a hydrophilic polymer. The polymer holding tank 3 rotates so that the polymer holding container 301 containing the hydrophilic polymer to be dispensed is arranged at the polymer suction position. The reagent tank 4 is arranged near the reaction disk 1. The reagent tank 4 holds a plurality of reagent containers 401 each containing a reagent. The reagent tank 4 rotates so that the reagent container 401 in which the reagent to be dispensed is contained is arranged at the reagent suction position.

[0160] The sample arm 5 - 1 is disposed between the reaction disk 1 and the sample disk 2 .

[0161] The sample probe 6-1 is attached to the front end of the sample arm 5-1. The sample arm 5-1 supports the sample probe 6-1 in a manner that it can move up and down. In addition, the sample arm 5-1 supports the sample probe 6-1 in a manner that it can rotate along a pivot track in an arc shape. The pivot track of the sample probe 6-1 passes through a sample suction position above the sample disk 2 and a sample discharge position above the reaction disk 1. The sample probe 6-1 sucks the sample from the sample container 201 disposed at the sample suction position above the sample disk 2, and discharges the sample into the glass container 101 disposed at the sample discharge position above the reaction disk 1.

[0162] The polymer injection arm 5-2 is arranged near the outer periphery of the reaction disk 1. The polymer injection probe 6-2 is attached to the front end of the polymer injection arm 5-2. The polymer injection arm 5-2 supports the polymer injection probe 6-2 in a manner that can be moved up and down. In addition, the polymer injection arm 5-2 supports the polymer injection probe 6-2 in a manner that can be rotatable along a pivot track in an arc shape. The pivot track of the polymer injection probe 6-2 passes through the polymer suction position above the polymer holding tank 3 and the polymer discharge position above the reaction disk 1. The polymer injection probe 6-2 sucks the hydrophilic polymer from the polymer holding container 301 arranged at the polymer suction position above the polymer holding tank 3, and discharges the hydrophilic polymer into the glass container 101 arranged at the polymer discharge position above the reaction disk 1.

[0163] The reagent arm 5-3 is arranged between the reaction disk 1 and the reagent tank 4. The reagent probe 6-3 is attached to the front end of the reagent arm 5-3. The reagent arm 5-3 supports the reagent probe 6-3 in a movable manner up and down. In addition, the reagent arm 5-3 supports the reagent probe 6-3 in a rotatable manner along the pivot track of the arc shape. The pivot track of the reagent probe 6-3 passes through the reagent suction position above the reagent tank 4 and the reagent discharge position above the reaction disk 1. The reagent probe 6-3 inhales the reagent from the reagent container 401 arranged at the reagent suction position above the reagent tank 4, and discharges the reagent into the glass container 101 arranged at the reagent discharge position above the reaction disk 1.

[0164] The stirring arm 7 is arranged near the outer periphery of the reaction disk 1. A stirring rod 8 is attached to the front end of the stirring arm 7. The stirring arm 7 supports the stirring rod 8 in a manner that it can move up and down. In addition, the stirring arm 7 supports the stirring rod 8 in a manner that it can rotate along an arc-shaped pivot track. The stirring rod 8 stirs the liquid containing the sample and the reagent in the glass container 101 arranged at the stirring position above the reaction disk 1.

[0165] The glass container 101 is a container for containing a reaction liquid. It is desirable that at least the light incident surface and the light exit surface of the glass container 101 are optically transparent and smooth so that optical measurement can be performed by the photometric mechanism 9.

[0166] The liquid amount of the reaction liquid varies according to the amount of the sample and the reagent, or varies according to the test item. The reaction liquid is contained in the glass container 101 with a liquid amount between the minimum liquid amount and the maximum liquid amount. The maximum liquid amount is the liquid amount set for the analysis device 21, and is the maximum liquid amount of the reaction liquid that can be tested. The minimum liquid amount is the liquid amount set for the analysis device 21, and is the minimum liquid amount of the reaction liquid that can be tested.

[0167] like Figure 3As shown, the photometric mechanism 9 is arranged near the reaction disk 1. The photometric mechanism 9 operates according to the control by the analysis mechanism control unit 23. Specifically, the photometric mechanism 9 has a light source 901, a detector 902 and a polarizing plate 903. The light source 901 irradiates light toward the reaction liquid in the glass container 101 located at the photometric position in the reaction disk 1. The glass container 101 is rotated at a predetermined time interval by the reaction disk 1 so that the glass container 101 passes through the light that has been irradiated from the light source 901 and passed through the polarizing plate 903 at a substantially right angle at the photometric position. Every time the glass container 101 passes over the photometric position, the reaction liquid in the glass container 101 is measured by the photometric mechanism 9. The detector 902 is arranged at a position opposite to the light source 901 across the glass container 101 at the photometric position. The detector 902 detects the light that has passed through the polarizing plate 903 among the luminescent components from the reaction liquid in the glass container 101. As the polarizing plate 903 on the passing side (detector 902 side) of the glass container 101, two kinds of polarizing plates are prepared, namely, a polarizing plate parallel to the light polarization direction of the polarizing plate on the excitation light side (light source 901 side) and a polarizing plate perpendicular to the light polarization direction of the polarizing plate on the excitation light side (light source 901 side). In order to prepare the two kinds of polarizing plates 903 on the passing side of the glass container 101, the light emission from the sample can be divided into multiple directions by a separator. Alternatively, the light polarization direction of the polarizing plate 903 on the passing side of the glass container 101 can be switched, thereby enabling measurement. The detector 902 generates data having a measurement value corresponding to the intensity of the light that has been detected (hereinafter referred to as "photometric data"). The photometric data that has been generated is provided to the analysis unit 24.

[0168] For example, a halogen lamp, a light emitting diode (LED), or a laser generator can be used as the light source 901. The light irradiated from the light source 901 preferably includes light of a wavelength band capable of measuring the value of polarization anisotropy of the reaction liquid.

[0169] The detector 902 converts the intensity of the detected light into an electrical signal. Specifically, a photomultiplier tube, a photodiode, or an array photomultiplier tube or an array photodiode is used as the detector 902. In order to make the S / N ratio of the detection satisfactory, an optical window or a condenser can be configured between the light source 901 and the detector 902 as needed.

[0170] The washing mechanism 10 is arranged at the outer periphery of the reaction disk 1. The washing mechanism 10 operates according to the control by the analysis mechanism control unit 23. Specifically, a washing nozzle and a drying nozzle are attached to the washing mechanism 10. The washing mechanism 10 washes the glass container 101 located at the washing position of the reaction disk 1 with the washing nozzle, and dries the glass container 101 with the drying nozzle.

[0171] The analysis mechanism control unit 23 operates the various devices and mechanisms of the analysis mechanism 22 according to the control by the system control unit 28. The analysis unit 24 calculates the value of the polarization anisotropy of the reaction liquid based on the photometric data. In addition, the analysis unit 24 quantitatively analyzes the target substance according to the test item based on the calculated value of the polarization anisotropy of the reaction liquid. The display unit 25 has a display device such as a CRT display, a liquid crystal display, an organic EL display, or a plasma display. The display unit 25 displays the analysis result obtained by the analysis unit 24. The operation unit 26 receives various instructions or information input by the operator via the input device. A pointing device such as a mouse or a trackball, a selection device such as a switch button, or an input device such as a keyboard can be appropriately used as the input device. The storage unit 27 stores, for example, the operation program of the analysis device 21. The system control unit 28 serves as the center of the analysis device 21. The system control unit 28 reads out the operation program from the storage unit 27, and controls each unit 23, 24, 25 and 27 according to the operation program.

[0172] (Analysis Kit)

[0173] The present invention provides the following analysis kit as one embodiment.

[0174] An analysis kit according to one embodiment of the present invention is an analysis kit used in an analysis method for determining at least any one of the presence or absence of a target substance and the concentration of the target substance based on polarization anisotropy, the analysis kit comprising: a reagent that reacts with the target substance; a glass container for containing a reaction liquid including a sample containing the target substance and the reagent, and a hydrophilic polymer that reacts with a silanol group of the glass container, wherein the reagent includes a luminescent reagent having a hydrophilic surface, and the luminescent reagent includes luminescent particles.

[0175] [Example]

[0176] The present invention will be specifically described below by way of examples. However, the present invention is not limited to these examples.

[0177] (1) Production of luminescent reagents

[0178] Polyvinyl pyrrolidone (PVP-K30: manufactured by Tokyo Chemical Industry Co., Ltd.) was dissolved in a 2-morpholinoethanesulfonic acid (MES) buffer solution (manufactured by Kishida Chemical Co., Ltd.) having a pH of 7 to prepare a solution A.

[0179] Next, the following materials were mixed to prepare a mixed solution B.

[0180] · [Tris(2-thenoyltrifluoroacetone)(bis(triphenylphosphineoxide))europium(III)] used as a europium complex (manufactured by Central Techno Corporation)

[0181] Styrene monomer (manufactured by Kishida Chemical Co., Ltd.)

[0182] 3-Methacryloxypropyltrimethoxysilane (manufactured by Tokyo Chemical Industry Co., Ltd., hereinafter referred to as "MPS")

[0183] In the four-necked flask that holds solution A, add mixed solution B, and stir the mixture with the mechanical stirrer that is set to 300rpm.After stirring 15 minutes under nitrogen flow condition, the temperature of the oil bath that has been prepared is set to 70 ℃, and nitrogen flows for another 15 minutes.After heating and stirring the mixture, add the aqueous solution that is dissolved with potassium persulfate (hereinafter referred to as " KPS ") (manufactured by Sigma-Aldrich) in the reaction solution, carry out emulsion polymerization 20 hours.After the polyreaction, by using the ultrafiltration membrane that molecular weight cutoff is 100K, with about 4L ion exchange water, the obtained suspension is carried out ultrafiltration, thus washing product.Thus, obtain the dispersion liquid of luminescent reagent.

[0184] Take out an aliquot of the luminescent reagent dispersion obtained by emulsion polymerization, and add it to an aqueous solution dissolved with 1 mass % Tween 20 (manufactured by Kishida Chemical Co., Ltd.). After stirring for 10 minutes, add silane coupling agent X12-1135 (manufactured by Shin-Etsu Chemical Co., Ltd.), and stir the mixture overnight. After stirring, centrifuge the dispersion, remove the supernatant, and redisperse the precipitate with pure water. Perform centrifugation and redispersion operations more than 3 times to wash the product. The washed precipitate is redispersed in pure water. Thus, the ligand binding functional group is introduced into the luminescent reagent. The mass ratio of the luminescent reagent, pure water and X12-1135 put in is set to 1:300:2.

[0185] (Production of anti-CRP antibody modified luminescent reagent)

[0186] Take out an aliquot of 0.25mL of the particle dispersion equivalent to 1.2% by mass of the synthesized luminescent reagent, and replace the solvent with 1.6mL of MES buffer solution with a pH of 6.0. Add 0.5% by mass of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide and sodium N-hydroxysulfosuccinimide to the particle MES buffer solution, and react the mixture at 25°C for 1 hour. After the reaction, wash the dispersion with a MES buffer solution with a pH of 5.0, add anti-CRP antibodies at 100 μg / mL, and bind the anti-CRP antibodies to the particles at 25°C for 2 hours. After binding, wash the particles with a Tris buffer solution with a pH of 8. After the reaction, wash the particles with a phosphate buffer solution to obtain a luminescent reagent (sometimes referred to as "affinity particles") modified with an anti-CRP antibody at a concentration of 0.3% by mass.

[0187] Binding of antibodies to the particles is identified by measuring the reduction in antibody concentration in the buffer solution to which the antibodies are added using the BCA assay.

[0188] (Preparation of Luminescent Reagent Solution)

[0189] The obtained luminescent reagent was diluted with a phosphate buffer solution (PBS) having a pH of 7.4 to a concentration of 0.1 mg / ml to prepare a luminescent reagent solution.

[0190] (Preparation of diluent)

[0191] A 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solution and a PBS buffer solution were mixed at a volume ratio of 1:1 to prepare a dilution solution.

[0192] (Example 1)

[0193] 2 mL of a liquid obtained by dissolving polyvinyl pyrrolidone (weight average molecular weight: about 40,000) in a luminescent reagent liquid at a concentration of 0.25 mass % was put into a quartz glass cell and immersed for 72 hours. After immersion, the luminescent reagent liquid was removed and the glass cell was rinsed several times with pure water. After washing, measurement was performed using the apparatus 1 described later.

[0194] (Example 2)

[0195] 2 mL of a liquid obtained by dissolving polyvinyl pyrrolidone (weight average molecular weight: about 40,000) in a luminescent reagent liquid at a concentration of 0.125 mass % was put into a quartz glass cell and immersed for 72 hours. After immersion, the luminescent reagent liquid was removed and the glass cell was rinsed several times with pure water. After washing, measurement was performed using the apparatus 1 described later.

[0196] (Example 3)

[0197] 2 mL of a liquid obtained by dissolving polyvinyl pyrrolidone (weight average molecular weight: about 40,000) at a concentration of 0.0625 mass % in a luminescent reagent liquid was put into a quartz glass cell and immersed for 72 hours. After immersion, the luminescent reagent liquid was removed and the glass cell was rinsed several times with pure water. After washing, measurement was performed by using the apparatus 1 described later.

[0198] (Example 4)

[0199] 2 mL of a liquid obtained by dissolving polyvinyl pyrrolidone (weight average molecular weight: about 40,000) in a luminescent reagent liquid at a concentration of 0.03125 mass % was put into a quartz glass cell and immersed for 72 hours. After immersion, the luminescent reagent liquid was removed and the glass cell was rinsed several times with pure water. After washing, measurement was performed by using the apparatus 1 described later.

[0200] (Comparative Example 1)

[0201] 2 mL of the luminescent reagent solution was put into a quartz glass cell and immersed for 72 hours. After immersion, the luminescent reagent solution was removed and the glass cell was rinsed several times with pure water. After washing, measurement was performed using the apparatus 1 described below.

[0202] (Comparative Example 2)

[0203] After diluting 10 times, 2 mL of the luminescent reagent solution was put into a quartz glass cell and immersed for 72 hours. After immersion, the luminescent reagent solution was removed and the glass cell was rinsed several times with pure water. After washing, measurement was performed using the apparatus 1 described below.

[0204] (Reference Example 1)

[0205] 2 mL of the luminescent reagent solution was put into the polymethyl methacrylate cell and immersed for 72 hours. After immersion, the luminescent reagent solution was removed and the glass cell was rinsed several times with pure water. After washing, measurement was performed using the apparatus 1 described below.

[0206] (Example 5)

[0207] 170 μl of the liquid obtained by adding polyvinyl pyrrolidone (weight average molecular weight: about 40,000) to the diluent at a concentration of 0.25% by mass is put into a quartz glass cell, mixed with 10 μl of CRP antigen liquid, and then mixed with 20 μl of luminescent reagent liquid, and measured. Measure "r" in formula (1). The study was conducted at CRP antigen concentrations ranging from 0 pM to 200 pM. The washing and measurement operations of the reused quartz glass unit were performed 10 times. The average value of "r" obtained by performing 10 measurements was calculated. The change in "r" relative to the concentration of CRP antigen is equivalent to the value obtained by subtracting the average value of each "r" obtained by measuring immediately after mixing from the average value of each "r" obtained by measuring 10 minutes after the reaction, divided by the time (10 minutes, dt). The measurement is performed using the device 2 described later.

[0208] (Comparative Example 3)

[0209] 170 μl of the diluent and 10 μl of the CRP antigen solution were mixed with each other, and then mixed with 20 μl of the luminescent reagent solution, and R was measured. The study was conducted at a CRP antigen concentration of 0 pM to 200 pM. The operation of washing and measuring the reused quartz cell was performed 10 times. The average value of "r" obtained by performing 10 measurements was obtained. The method for determining the amount of change in "r" is the same as in Example 5. The measurement was performed using the device 2 described later.

[0210] (Measurement)

[0211] The shape of the obtained luminescent agent was measured using an electron microscope (S-5500, manufactured by Hitachi High-Technologies Corporation).

[0212] The average particle size of the luminescent agent was measured by using dynamic light scattering (manufactured by Zetasizer Nano S, Malvern).

[0213] The concentration of the suspension in which the luminescent reagent is dispersed is measured using a gravimetric analyzer (Thermo plus TG8120, manufactured by Rigaku Corporation).

[0214] Device 1 and device 2 are used for measurements for performance evaluation. Device 1 has a structure described below and performs measurements described below.

[0215] An LED light source of 340 nm excitation light was prepared, and a polarizing filter (NSPFU-30C, manufactured by Sigmakoki Co., Ltd.) and a short-pass filter (84-706, manufactured by Edmund Optics) were inserted into the light path to set up an optical system capable of irradiating a 1 cm quartz square cell. A polarizing filter (PIVISC050, manufactured by Thorlabs Inc.) and a bandpass filter (FB610-10, manufactured by Thorlabs Inc.) were set to a direction of 90° relative to the incident light. In order to simultaneously measure the I in two directions, VV and I VH For the luminescence, two sets were prepared in which the polarizer was configured to change the direction by 90° relative to the incident light. For the detection of polarized light, the spectrum measurement was performed by using QEPro manufactured by Ocean Optics, Inc. The temperature control was set for the sample holder so that the measurement could be made at 37°C. The LED light source was fixed at an output of 12mW and the accumulation time was set to 3 seconds to perform the fluorescence measurement. Based on the fluorescence spectrum, the luminescence intensity in the wavelength range of 600nm to 630nm was determined.

[0216] The device 2 has a structure described later, and performs the measurement described later.

[0217] Prepare the LED light source of the excitation light of 340nm, and insert polarization filter (NSPFU-30C, manufactured by Sigmakoki Co., Ltd.) and short-pass filter (84-706, manufactured by Edmund Optics) into the light path, to set the optical system that can irradiate the quartz cell of 5mm light path length.For the polarized luminescence emitted from the sample, by excitation light cut-off filter (33-910, manufactured by Edmund Optics), polarization beam splitter (47-127, manufactured by Edmund Optics) and polarizer (SPF-30C-32, manufactured by Sigmakoki Co., Ltd.) are arranged in the light path of the straight line of the excitation light through the sample in the described order, polarized light is separated into two directions. Detect the polarized luminescence (each direction of two directions) separated with avalanche photodiode (APD, C15522-3010SA, manufactured by Hamamatsu Photonics KK). The temperature control of the sample holder is set to be able to measure at 37°C. The measurement of the polarized luminescent signal is carried out when the LED light source is fixed at an output of 60mW and the cumulative time is set to 8 milliseconds. The resulting signal of polarized luminescence is measured with an oscilloscope and substituted into formula (1) to obtain "r". "r" is the value of polarization anisotropy (R). Calculate the change between "r" immediately after the luminescent reagent solution is mixed with the CRP antigen solution and "r" after immersion for 10 minutes from the start of mixing.

[0218] (Performance Evaluation)

[0219] The synthesized luminescent agent has a particle size of about 100 nm and exhibits strong red luminescence under 340 nm excitation light.

[0220] According to the results of the nonspecific agglutination inhibition evaluation, the change in absorbance was less than a specific value, and thus it was confirmed that the particles were able to inhibit nonspecific adsorption.

[0221] The results of Examples 1, 2, 3 and 4, Comparative Examples 1 and 2, and Reference Example 1 are shown in Table 1. In all examples, the fluorescence intensity I in both directions VV and I VH Each is within 100 counts, which is almost at the same level as the background. That is, it is confirmed that the luminescent particles will not adhere to the wall surface of the pool even through long-term immersion, and are removed by a simple rinsing operation with pure water. In Comparative Examples 1 and 2, the fluorescence intensity exceeds 10,000 counts each, and clear luminescence is recognized. This shows that the luminescent particles are attached to the wall surface by long-term immersion. At the same time, in Reference Example 1, as in the embodiment, no fluorescence was detected, and the attachment of particles to the pool wall surface was not recognized.

[0222] In Examples 1, 2, 3 and 4, Comparative Examples 1 and 2, and Reference Example 1, when the cell after measuring the fluorescence intensity was irradiated with UV light at 360 nm, red luminescence was visually observed on the wall surface of the cell in each of Comparative Examples 1 and 2. In each of Examples 1, 2, 3 and 4 and Reference Example 1, red luminescence was not observed, and attachment of luminescent particles was not visually observed.

[0223] In Example 5, it was confirmed that "r" derived from the formula (1) and the CRP concentration were correlated with each other.

[0224] The results of "r" in Example 5 are shown in Table 2. As shown in Table 2, it is confirmed that "r" increases with the CRP concentration. Between 0pM and 100pM, it can be confirmed that the CRP concentration and the slope change (dr / dt) are particularly highly correlated with each other, and the slope change is obtained by dividing the change in "r" in 10 minutes by the time. It is confirmed that when the CRP concentration is increased to 200pM, the front zone of the change saturation of polarization anisotropy is reached, so the correlation decreases. It is confirmed that the correlation between the CRP concentration and dr / dt does not change significantly in 10 measurements, and even the 1st measurement and the 10th measurement can be performed in the same way. In addition, after the 10th measurement is completed, the washed quartz cell is subjected to fluorescence measurement. As a result, the red luminescence from the europium complex is reduced to the noise signal level, which shows that the attachment of luminescent particles to the wall surface can be suppressed.

[0225] Meanwhile, in Comparative Example 3, it was confirmed that the increase in "r" from the first evaluation was not related to the CRP antigen concentration.

[0226] At this time, no correlation was observed between the change in "r" and the CRP concentration. In addition, after the 10th measurement was completed, the washed quartz cell was subjected to fluorescence measurement. As a result, strong red luminescence from the europium complex was observed, and it was confirmed that the luminescent particles were attached to the wall surface of the cell.

[0227] As can be seen from the above results, in each of the examples in which polyvinyl pyrrolidone was added and in Reference Example 1 in which the wall of the cell was made of plastic, the formation of hydrogen bonds between the wall of the cell and the luminescent agent was not observed, and the luminescent agent was not attached to the wall of the cell. That is, it is shown that polyvinyl pyrrolidone, which is a hydrophilic polymer, forms hydrogen bonds with the silanol groups of the wall of the glass cell, thereby inhibiting the attachment of the luminescent agent.

[0228] Table 1

[0229]

[0230] Table 2

[0231]

[0232] As revealed above, the measuring method according to each embodiment of the present invention is a method capable of repeatedly measuring the concentration of CRP antigen as a target substance with high sensitivity while suppressing noise such as adhesion of light-emitting particles to a glass cell.

[0233] Therefore, when using the measuring method according to each embodiment of the present invention, the presence or absence of the target substance or its concentration can be stably measured with high sensitivity. It can be imagined that using the measuring method according to each embodiment of the present invention, it is possible to realize a device that can stably perform measurement with high sensitivity in applications such as sample testing for quality testing by repeatedly using a glass cell.

[0234] According to the present invention, an analytical method for calculating the concentration of a target substance by using a reagent that reacts with the target substance can be provided, particularly when high-sensitivity measurement is performed by using a small amount of luminescent particles, a method capable of reducing noise by suppressing nonspecific adsorption of the reagent on a glass container, such as a method based on polarization anisotropy.

[0235] While the present invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments.The scope of the following claims is to be accorded the broadest interpretation so as to encompass all such modifications and equivalent structures and functions.

Claims

1. An analytical method for determining at least one of the presence or absence of a target substance and the concentration of the target substance by using a reagent that reacts with the target substance, the analytical method comprising: An introducing step of introducing a sample containing the target substance, a hydrophilic polymer that reacts with a silanol group of the glass container, and the reagent into the glass container; A reaction step, allowing the target substance and the reagent to react with each other to provide a reaction solution; and an analysis step of determining at least one of the presence or absence of the target substance and the concentration of the target substance in the reaction solution, wherein the reagent comprises a luminescent reagent having a hydrophilic surface, and the luminescent reagent comprises luminescent particles, and The step of adding includes adding the reagent after or simultaneously with adding the hydrophilic polymer.

2. The analysis method according to claim 1, wherein The analyzing step includes determining at least any one of the presence or absence of the target substance and the concentration of the target substance in the reaction liquid by measuring a value R of polarization anisotropy of the reaction liquid. 3 . The analysis method according to claim 2 , wherein the following formula is satisfied: R0 ≥ 0.01, wherein R0 represents R measured for the reagent that has not reacted with the target substance.

4. The analysis method according to claim 2, wherein: The R is defined as "r" in the following formula (1): In formula (1), I VV represents the luminous intensity of the luminous component whose vibration direction is parallel to the vibration direction of the first polarized light beam when excited by the first polarized light beam, I VH represents the luminous intensity of the luminous component whose vibration direction is orthogonal to the vibration direction of the first polarized light beam when excited by the first polarized light beam, I HV represents the luminous intensity of the luminous component whose vibration direction is orthogonal to the vibration direction of the second polarized light beam when excited by the second polarized light beam whose vibration direction is orthogonal to the vibration direction of the first polarized light beam, I HH represents the luminous intensity of the luminous component whose vibration direction is parallel to the vibration direction of the second polarized light beam when excited by the second polarized light beam whose vibration direction is orthogonal to the vibration direction of the first polarized light beam, and G represents the correction value. The analytical method according to claim 1 , wherein the hydrophilic polymer forms a hydrogen bond with a silanol group of the glass container. The analysis method according to claim 1 , wherein the concentration of the luminescent reagent in the reaction solution is 0.0001 mg / ml or more and 0.1 mg / ml or less. The analytical method according to claim 1 , wherein the hydrophilic polymer comprises polyvinyl pyrrolidone. The analytical method according to claim 1 , wherein the luminescent particle comprises a europium complex.

9. The analysis method according to claim 1, wherein the luminescent reagent comprises a ligand that binds to the target substance. 10 . The analysis method according to claim 9 , wherein the ligand is an antibody, and the target substance is an antigen.

11. An analytical device for determining at least one of the presence or absence of a target substance and the concentration of the target substance, the analytical device comprising: a glass container for containing a reaction solution including a sample containing a target substance and a reagent that reacts with the target substance; a unit configured to put the sample containing the target substance into the glass container; a unit configured to introduce a hydrophilic polymer that reacts with a silanol group of the glass container into the glass container; A unit configured to add the reagent into the glass container after or simultaneously with the addition of the hydrophilic polymer; an irradiation unit configured to irradiate the glass container with light that is linearly polarized light as irradiation light; a measuring unit configured to measure the fluorescence intensity of fluorescence of two polarized light components in parallel and orthogonal directions with respect to the linearly polarized light; as well as a processing unit configured to process the result obtained by the measuring unit to thereby determine at least any one of the presence or absence of the target substance and the concentration of the target substance in the reaction liquid, Wherein, the reagent comprises a luminescent reagent having a hydrophilic surface, and the luminescent reagent comprises luminescent particles.

12. An analysis kit for use in an analysis method for determining at least one of the presence or absence of a target substance and the concentration of the target substance based on polarization anisotropy, the analysis kit comprising: a reagent that reacts with the target substance; a glass container for containing a reaction solution including a sample containing the target substance and the reagent; and a hydrophilic polymer reactive with the silanol groups of the glass container, The reagent comprises a luminescent reagent having a hydrophilic surface, and the luminescent reagent comprises luminescent particles.

Images