Method for detecting a target substance, and reagent for detecting a target substance

Abstract

To provide a highly sensitive method for measuring enzyme reaction-related substances and the corresponding measuring reagents. [Solution] A method for detecting a target substance which is a substance related to an enzymatic reaction, comprising: a first step of mixing a sample solution which may contain the target substance with a reagent to obtain a liquid sample which contains hydrogen peroxide, peroxidase, phenols, and a plurality of luminescent particles which have binding functional groups; a second step in which the luminescent particles aggregate as the binding functional groups of the plurality of luminescent particles bind based on a reaction between the hydrogen peroxide, the peroxidase, and the phenols in the liquid sample which occurs due to the presence of the target substance in the liquid sample; and a third step of obtaining a value relating to the fluorescence anisotropy of the liquid sample in the second step.

Description

Technical Field

[0001] The present disclosure relates to a method for detecting a target substance and a reagent for detecting a target substance.

Background Art

[0002] Enzymes play a role in catalyzing chemical reactions in vivo. Measuring enzyme activity is very important not only in medical research on enzymes, but also in enzyme tests, clinical tests using enzymes as reagents, and the field of substance production using enzymes.

[0003] Conventionally, enzyme activity has been measured by various methods. Generally, a method using a natural or synthetic substrate for an individual enzyme is performed. In the method using a synthetic substrate, enzyme activity can be measured by optically detecting and measuring a dye released from the synthetic substrate by the action of the enzyme using the enzyme activity. Alternatively, substrates such as those with weak luminescence before the action of the enzyme but increased luminescence intensity after the action are also used.

[0004] However, when the enzyme is at a very low concentration, the amount of fluorescent dye released from the synthetic substrate also decreases, resulting in weak luminescence from the fluorescent dye and making it difficult to measure enzyme activity or reducing the measurement accuracy. In addition, fluorescent dyes are easily affected by the surrounding environment. For example, in a liquid sample containing many contaminants such as blood, they are adsorbed by proteins and lipids, etc. which are contaminants, and the fluorescence characteristics of the substrate change, making it difficult to perform highly sensitive measurements. Furthermore, in order to eliminate the influence of contaminants, it is possible to separate the enzyme, which is the target substance, or the contaminants, but the separation operation is complicated and the measurement time may be long.

[0005] One known method for measuring enzyme activity is fluorescence polarization. Fluorescence polarization, which utilizes fluorescence polarization, is a method for measuring the rotational motion of a fluorescent substance. When the fluorescent substance is excited, the polarization of its fluorescence changes according to its rotational motion. That is, when the fluorescent substance does not rotate, polarized emission is observed, whereas when the fluorescent substance rotates freely, fluorescence is emitted in all planes, and the polarization is eliminated. A characteristic of fluorescence polarization is that, in the assay, the fluorescence emission intensity is not used as an indicator, but rather a value indicating fluorescence anisotropy. Another characteristic is that separation and washing operations are not required. In other words, a homogeneous assay is possible, and the sample can be added to the solution and measured directly, eliminating the need for complicated separation work and allowing for measurement in a short time.

[0006] To date, methods for measuring enzyme activity using fluorescence polarization have been disclosed. Patent Document 1 discloses the measurement of the activity of proteases, which are proteolytic enzymes. This method utilizes the fact that when a substrate molecule containing a luminescent substance is cleaved by a protease, the size of the luminescent substance decreases, and the motility of the luminescent substance changes. Patent Document 2 discloses the measurement of the activity of enzymes that catalyze phosphate modification, consisting of kinases, phosphatases, cyclases, and phosphodiesterases.

[0007] Patent Document 3 discloses an immunoassay technique using fluorescence polarization. In Patent Document 3, a highly sensitive immunoassay is constructed by using luminescent particles, agglutinating the luminescent particles via an antigen-antibody reaction, and observing the change in fluorescence polarization before and after agglutination. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Japanese Patent Application Publication No. 10-099097 [Patent Document 2] Japanese Patent Publication No. 2008-148698 [Patent Document 3] Japanese Patent Publication No. 2022-187791 [Overview of the project] [Problems that the invention aims to solve]

[0009] However, it has been difficult to detect substances related to the enzymatic reaction of peroxidase in liquid samples in a simple, rapid, and highly sensitive manner. [Means for solving the problem]

[0010] The present inventors, after diligent research to solve the above problems, have discovered that by introducing a mechanism in which multiple luminescent particles are aggregated by the action of oxidoreductase in a liquid sample, and the mobility of the luminescent particles is greatly reduced, it becomes possible to measure the fluorescence polarization of enzyme activity, enzyme amount, or amount of enzyme reaction-related substances in a short time and with high sensitivity, and have completed the invention of this disclosure. In other words, this disclosure provides a method for measuring the mobility of luminescent particles in a liquid sample using the measurement of a value related to fluorescence anisotropy.

[0011] The first aspect of this disclosure is, A method for detecting a target substance that is related to an enzymatic reaction, A first step involves mixing a sample solution that may contain the target substance with a reagent to obtain a liquid sample containing hydrogen peroxide, peroxidase, phenols, and a plurality of luminescent particles having binding functional groups. A second step in which the luminescent particles aggregate due to the bonding functional groups of the plurality of luminescent particles bonding together based on the reaction between the hydrogen peroxide, the peroxidase, and the phenols in the liquid sample, which occurs when the target substance is present in the liquid sample. A third step involves obtaining a value relating to the fluorescence anisotropy of the liquid sample in the second step, This is a method for detecting a target substance that has [certain properties].

[0012] A second aspect of this disclosure is, A reagent for detecting a target substance using the measurement of fluorescence anisotropy values, wherein the target substance is a peroxidase, and the reagent comprises hydrogen peroxide, phenols, and a plurality of luminescent particles having binding functional groups.

[0013] A third aspect of this disclosure is: A reagent for detecting a target substance using the measurement of fluorescence anisotropy values, wherein the target substance is an oxidase, and the reagent comprises an oxidase substrate, peroxidase, phenols, and a plurality of luminescent particles having binding functional groups.

[0014] A fourth aspect of this disclosure is, A reagent for detecting a target substance using the measurement of fluorescence anisotropy values, wherein the target substance is hydrogen peroxide, and the reagent comprises a peroxidase, phenols, and a plurality of luminescent particles having binding functional groups.

[0015] The fifth aspect of this disclosure is, A reagent for detecting a target substance using the measurement of fluorescence anisotropy values, wherein the target substance is a substrate for oxidase, and the reagent comprises oxidase, peroxidase, phenols, and a plurality of luminescent particles having binding functional groups.

[0016] The sixth aspect of this disclosure is A reagent for detecting a target substance using the measurement of fluorescence anisotropy values, wherein the target substance is a phenol, and the reagent comprises peroxidase, hydrogen peroxide, and a plurality of luminescent particles having binding functional groups.

[0017] The seventh aspect of this disclosure is A reagent for detecting a target substance using the measurement of fluorescence anisotropy values, wherein the target substance is an inhibitor of the enzymatic reaction of peroxidase, and the reagent comprises hydrogen peroxide, peroxidase, phenols, and a plurality of luminescent particles having binding functional groups. [Effects of the Invention]

[0018] According to the present disclosure, for example, even when the amount of an enzyme is small or the enzyme activity is low, substances related to the enzyme reaction can be detected simply and with high sensitivity.

Brief Description of Drawings

[0019] [Figure 1] It is a diagram for explaining the steps of a method for detecting a target substance according to an aspect of the present disclosure. [Figure 2] It is a diagram for explaining a method for detecting peroxidase according to an embodiment of the present disclosure. [Figure 3] It is a diagram for explaining a method for detecting oxidase according to an embodiment of the present disclosure. [Figure 4] It is a diagram for explaining a method for detecting hydrogen peroxide concentration according to an embodiment of the present disclosure. [Figure 5] It is a diagram for explaining a method for detecting glucose concentration according to an embodiment of the present disclosure.

Modes for Carrying Out the Invention

[0020] Hereinafter, the present disclosure will be described in more detail. In the present disclosure, "detection" refers not only to the presence or absence of a target substance, but also to knowing the content, content concentration, or activity. It is used in the meaning including both qualitative and quantitative cases, and "detection" may also be referred to as "measurement".

[0021] (Fluorescence polarization degree determined by fluorescence polarization method) Fluorescence polarization method is used to analyze the motility of fluorescent molecules in a solution. The principle of the fluorescence polarization method will be explained by taking the case of the luminescent particles of the present disclosure as an example. When luminescent particles in a liquid (the particles contain luminescent molecules showing fluorescence anisotropy) are excited by plane-polarized light, polarized fluorescence is emitted in the same plane. However, if the luminescent particles rotate by Brownian motion during the excited state, fluorescence is emitted in a plane different from the excitation plane, so the fluorescence polarization degree is canceled. That is, the fluorescence polarization degree represents the degree of rotational motion of the luminescent particles from the time of excitation until fluorescence is emitted.

[0022] The detection method for luminescent particles in this embodiment is a method for measuring the mobility of luminescent particles in a liquid sample. When luminescent particles in a liquid are dispersed individually in the solution, they rotate vigorously due to Brownian motion and exhibit a low degree of fluorescence polarization. On the other hand, when luminescent particles aggregate and their apparent size increases, Brownian motion in the solution decreases, and the degree of fluorescence polarization increases. Therefore, in the fluorescence polarization method, the mobility of luminescent particles in the solution is analyzed using the change in the degree of fluorescence polarization as an indicator. The degree of fluorescence polarization can be measured using milli P (hereinafter abbreviated as mp), which indicates a change in plane polarization.

[0023] Changes in the motility of luminescent particles can be detected as changes in fluorescence polarization, meaning that by controlling the motility, it can be used to detect various target substances in liquid samples. In this embodiment and this embodiment, the motility of luminescent particles is evaluated using changes in fluorescence polarization as an indicator, but in this disclosure, the motility of luminescent particles can be evaluated using changes in values ​​related to fluorescence anisotropy as an indicator, or fluorescence anisotropy itself may be used. In other words, the value related to fluorescence anisotropy may be either fluorescence polarization or fluorescence anisotropy itself.

[0024] (Method for detecting target substances) The method for detecting a target substance according to this embodiment is a method for detecting a target substance including a substance related to an enzymatic reaction, and as shown in Figure 1, it comprises the following steps (1) to (3). Here, when detecting the target substance, the method may also include a step (4) in which the target substance is detected using the fluorescence anisotropy value obtained in step (3), as shown in Figure 1. (1) A first step (S1001) involves mixing a sample solution that may contain the target substance with a reagent to obtain a liquid sample containing hydrogen peroxide, peroxidase, phenols, and multiple luminescent particles having binding functional groups. (2) A second step (S1002) in which the luminescent particles aggregate due to the bonding functional groups of multiple luminescent particles bonding together based on the reaction between hydrogen peroxide, peroxidase, and phenols in the liquid sample, which occurs when the target substance is present in the liquid sample. (3) A third step (S1003) to obtain values ​​related to the fluorescence anisotropy of the liquid sample from the second step. (4) A fourth step in which the target substance is detected based on the fluorescence anisotropy value.

[0025] (Measurement principle 1: Measurement of peroxidase activity) The measurement principle of this disclosure will be explained using Figure 2, illustrating a method for measuring the activity of peroxidase as an example of an oxidoreductase. The target substance, peroxidase 40, catalyzes crosslinking by oxidation in the presence of a specified amount of hydrogen peroxide and phenols, through the binding of binding functional groups 20 (functional groups with binding ability) on the luminescent particles 10. In other words, the binding functional group 20 of the first luminescent particle among the luminescent particles 10 binds to the binding functional group of a second luminescent particle, which is different from the first luminescent particle, based on the reaction with hydrogen peroxide, peroxidase 40, and phenols. As a result, multiple luminescent particles 10 aggregate via the binding functional groups 20. Note that the binding functional groups of the first luminescent particle and the second particle that bind here may be of the same type or different types, as long as they bind based on the reaction with hydrogen peroxide, peroxidase 40, and phenols. When the binding functional groups are of the same type, for example, a thiol group is preferred. Before the addition of peroxidase 40, the luminescent particles 10 are dispersed in the solution and have high mobility. However, when peroxidase 40 is added, depending on its activity, a cross-linking (also called aggregation) structure is formed between the luminescent particles 10 by their binding functional groups, and the mobility of the luminescent particles 10 decreases. This decrease in mobility is observed as an increase in the fluorescence polarization degree of the luminescent particles 10. Therefore, by measuring the fluorescence polarization degree of the luminescent particles 10, the activity of peroxidase 40 in the sample solution can be determined.

[0026] In reality, a large number of luminescent particles 10 exist in the solution, and the proportion of aggregated luminescent particles 10 increases in accordance with the activity of the peroxidase 40 in the sample solution. Here, the fluorescence polarization degree obtained by fluorescence polarization is the average value of the entire solution, i.e., the entire amount of luminescent particles 10, so there is a correlation between the proportion of aggregated luminescent particles 10 and the fluorescence polarization degree. Therefore, for example, a calibration curve of fluorescence polarization degree and activity can be obtained in advance using a peroxidase of known activity, and the peroxidase activity in the sample solution can be determined from the measurement results of the fluorescence polarization degree of the solution.

[0027] (Measurement principle 2: Measurement of glucose oxidase activity) In addition to peroxidase, the enzymatic activity of other oxidases can also be measured by a similar method. For example, this will be explained using glucose oxidase (Figure 3). The oxidase activity measurement in this disclosure utilizes a cascade reaction of two enzymes: glucose oxidase 60 and peroxidase 40. The target substance, glucose oxidase 60, can aggregate luminescent particles 10 in the presence of a specified amount of glucose, peroxidase 40, and phenols.

[0028] This principle is the same as peroxidase activity measurement principle 1 described above, except that glucose is converted to hydrogen peroxide depending on the activity of glucose oxidase 60. That is, glucose is converted to hydrogen peroxide depending on the activity of glucose oxidase 60. Hydrogen peroxide activates peroxidase 40, causing the luminescent particles 10 to aggregate. Therefore, the motility of the luminescent particles 10 changes depending on the activity of glucose oxidase 60. By obtaining a calibration curve of fluorescence polarization and activity in advance using glucose oxidase of known activity, the glucose oxidase activity in the sample solution can be determined from the measurement results of the fluorescence polarization of the solution.

[0029] (Measurement principle 3: Measurement of hydrogen peroxide concentration) In this disclosure, the concentration of hydrogen peroxide, which is an example of a substance involved in the enzymatic reaction of peroxidase, can be measured by a similar method (Figure 4). For example, hydrogen peroxide, which is the target substance, can aggregate luminescent particles 10 in the presence of a specified amount of peroxidase 40 and phenols.

[0030] The activity of peroxidase 40 changes depending on the amount of the target substance, hydrogen peroxide. By measuring the resulting change in the motility of the luminescent particles 10 using fluorescence polarization, it becomes possible to measure the hydrogen peroxide concentration. By obtaining a calibration curve of fluorescence polarization degree and hydrogen peroxide concentration using hydrogen peroxide of known concentration beforehand, the hydrogen peroxide concentration can be determined from the measurement results of the fluorescence polarization degree of the solution.

[0031] (Measurement principle 4: Measurement of glucose concentration) In this disclosure, glucose concentration, which is an example of a substance involved in the enzymatic reaction of peroxidase, can also be measured by the same method (Figure 5). For example, glucose, as the target substance, can cause the luminescent particles 10 to aggregate in the presence of a specified amount of glucose oxidase 60, peroxidase 40, and phenols.

[0032] Similar to the glucose oxidase activity measurement principle described in Measurement Principle 2, glucose concentration can be measured by utilizing the cascade reaction between glucose oxidase 60 and peroxidase 40. Luminescent particles 10 are coexisting in this solution as probe molecules for fluorescence polarization measurement. The activity of peroxidase 40 changes depending on the amount of glucose, the target substance. By measuring the resulting change in the motility of luminescent particles 10 using fluorescence polarization, glucose concentration can be measured. A calibration curve between fluorescence polarization degree and glucose concentration can be obtained beforehand using glucose of known concentration, and the glucose concentration can be determined from the measurement results of the fluorescence polarization degree of the solution.

[0033] (Measurement principle 5: Measurement of phenol concentration) This disclosure also shows that the concentration of phenols, which are examples of substances involved in the enzymatic reaction of peroxidase, can be measured by a similar method. For example, phenols used as target substances can cause luminescent particles 10 to aggregate in the presence of a specified amount of peroxidase 40 and hydrogen peroxide.

[0034] The degree of aggregation of luminescent particles 10 by peroxidase 40 changes depending on the amount of the target substance, phenols. By detecting the resulting change in the motility of the luminescent particles 10 using fluorescence polarization measurement, it becomes possible to measure the concentration of phenols. By obtaining a calibration curve of fluorescence polarization and phenol concentration in advance using phenols of known concentrations, the concentration of phenols can be determined from the measurement results of the fluorescence polarization of the solution.

[0035] (Detailed explanation of the measurement method) As described above, one example of this disclosure includes the following steps (1) to (3). Here, when detecting the target substance, there may be a step (4) in which the target substance is detected using the fluorescence anisotropy value obtained in step (3), as shown in Figure 1. (1) A first step in which a sample solution that may contain the target substance is mixed with a reagent to obtain a liquid sample containing hydrogen peroxide, peroxidase, phenols, and multiple luminescent particles having binding functional groups. (2) A second step in which the luminescent particles aggregate due to the bonding functional groups of multiple luminescent particles bonding together based on the reaction between hydrogen peroxide, peroxidase, and phenols in the liquid sample, which occurs when the target substance is present in the liquid sample. (3) A third step to obtain values ​​related to the fluorescence anisotropy of the liquid sample from the second step. (4) A fourth step in which the target substance is detected based on the fluorescence anisotropy value.

[0036] The following provides a more detailed explanation of each step.

[0037] (First step) In this disclosure, the first step is to mix a sample solution that may contain a target substance with a reagent to obtain a liquid sample containing hydrogen peroxide, peroxidase, phenols, and a plurality of luminescent particles having binding functional groups. The method of mixing the sample solution that may contain a target substance with the reagent may be to add the reagent to the sample solution, to add the sample solution to the reagent, or to extract and mix from each of the sample solution and the reagent.

[0038] (Second step) In this disclosure, the second step is to form a crosslinking (also called aggregation) structure between multiple luminescent particles in a liquid sample using an enzyme. For example, when an oxidoreductase, which is the target substance, reacts with phenols and hydrogen peroxide, the binding functional groups of the multiple luminescent particles bond together, forming a crosslinking structure between the multiple luminescent particles.

[0039] When the target substance is hydrogen peroxide, which is an example of an enzyme reaction-related substance, the hydrogen peroxide is reacted with a solution containing peroxidase, phenols, and luminescent particles having binding functional groups to cause the binding functional groups of the particles to bind and the luminescent particles to aggregate. Here, the order of the reaction is not particularly limited, but since interparticle aggregation proceeds with hydrogen peroxide as a trigger, it is preferable to add the target substance, hydrogen peroxide, last.

[0040] When the target substance is glucose, which is an example of an enzyme reaction-related substance, the glucose is reacted with a solution containing glucose oxidase, peroxidase, phenols, and luminescent particles having binding functional groups to carry out an aggregation reaction of the luminescent particles. Here, the order of the reaction is not particularly limited, but since glucose acts as a trigger to form aggregation between the luminescent particles, it is preferable to add the target substance, glucose, last.

[0041] When the target substance is a phenol, which is an example of an enzyme reaction-related substance, the target substance (phenol) and hydrogen peroxide are reacted in a solution containing peroxidase and luminescent particles to carry out the aggregation reaction of the luminescent particles. Here, the order of the reaction is not particularly limited, but since interparticle aggregation is generated with hydrogen peroxide as a trigger, it is preferable to add hydrogen peroxide last.

[0042] (Liquid sample) The liquid sample that can be used in this disclosure may be any liquid obtained by mixing a reagent containing components for measuring the activity or concentration of a target substance with a liquid that may contain the target substance (also called a sample solution).

[0043] In this disclosure, the target substance is a substance involved in the enzymatic reaction of peroxidase. The sample solution is, for example, a bodily fluid such as blood, urine, or saliva containing the target substance, a buffer solution containing the target substance, a culture medium or tissue extract of cells or microorganisms containing the target substance, drinking water containing the target substance, river water containing the target substance, or wastewater containing the target substance. The sample solution may also contain hydrogen peroxide. Hydrogen peroxide promotes the reaction of peroxidase, but various measurements can be performed by appropriately adjusting the concentration and mixing ratio of peroxidase, phenols, luminescent particles, etc., and the conditions for measuring the degree of fluorescence polarization.

[0044] (oxidoreductase) In this disclosure, oxidoreductases are preferred as the enzymes used in the reaction. Oxidoreductases are enzymes that catalyze redox reactions. Examples of oxidoreductases include oxidases, dehydrogenases, reductases, and oxygenases. Specific examples include peroxidases, various oxidases, catalase, glucose dehydrogenase, lactate dehydrogenase, pyruvate dehydrogenase, alcohol dehydrogenase, and thioredoxin reductase. Furthermore, examples of peroxidases include horseradish peroxidase (HRP), myeloperoxidase, cytochrome P450, and manganese peroxidase. Examples of oxidases include glucose oxidase, lactate oxidase, pyruvate oxidase, cholesterol oxidase, alcohol oxidase, amino acid oxidase, and choline oxidase.

[0045] (Enzyme reaction-related substances) The enzyme reaction-related substances in this disclosure are substances related to the enzymatic reaction of peroxidase, and any substance whose quantity and activity can be measured by the measurement method of this disclosure is acceptable. In other words, the enzyme reaction-related substances are any substances that affect the activity of peroxidase, and may also be inhibitors of the enzymatic reaction of peroxidase. Examples include substrates of oxidoreductases, hydrogen peroxide, phenols, and sodium azide, which is an inhibitor of peroxidase activity. The quantity and activity of these enzyme reaction-related substances change the degree of fluorescence polarization obtained by the fluorescence polarization measurement of this disclosure. Examples of substrates of oxidoreductases include glucose, lactic acid, pyruvate, and cholesterol. Furthermore, the presence of an inhibitor of the enzymatic reaction of peroxidase in the sample solution makes it less likely for the luminescent particles to aggregate due to the binding of the binding functional groups of the luminescent particles to occur, so the quantity and activity of the inhibitor can also be measured.

[0046] (Luminous particles) The luminescent particles having bonding functional groups used in this disclosure are nanoparticles containing a luminescent substance, and the degree of fluorescence polarization of their emission can be measured by fluorescence polarization. Furthermore, the luminescent particles according to this disclosure are capable of forming crosslinks between themselves by the action of an enzymatic reaction. The crosslinking mode between the luminescent particles may be a chemical bond such as a covalent bond, an ionic bond, or a coordination bond. For chemical bonding, the luminescent particles have bonding functional groups such as thiol groups, amino groups, carboxyl groups, and maleimide groups. Among the bonding functional groups, thiol groups are particularly preferred because the reaction proceeds mildly and rapidly.

[0047] The luminescent particles of this disclosure are nanoparticles, and the average particle diameter, which is the average of the particle diameters, is 1 nm to 1000 nm, preferably 25 nm to 500 nm, and more preferably 50 nm to 300 nm. If the average particle diameter exceeds 500 nm, the degree of fluorescence polarization before crosslinking between particles becomes high, and the amount of change in the degree of fluorescence polarization after interparticle aggregation may become small. Also, if the average particle diameter is less than 25 nm, the content of the luminescent substance in the nanoparticles becomes low, the luminescence intensity per particle becomes low, and as a result, the measurement sensitivity and accuracy may decrease, so luminescent particles of 50 nm or more are preferred. The size of the luminescent particles can be determined by dynamic light scattering (DLS) measurement.

[0048] As the particle material for the luminescent particles (which can also be called the matrix material or main component), for example, nanoparticles of synthetic polymers such as polystyrene and polymethacrylate, inorganic nanoparticles such as silica, titanium dioxide, and iron oxide, and natural polymers such as dextran and casein can be used. Among these, synthetic polymers are preferred as the particle material for the luminescent particles according to this disclosure because they have a specific gravity that allows them to be dispersed in water, are stable, and have high productivity, and polystyrene particles are particularly preferred because they also have excellent particle size controllability.

[0049] Furthermore, it is preferable that the particle surface is hydrophilic. This is because hydrophilic particle surfaces can suppress nonspecific aggregation between particles and nonspecific adsorption between particles and the measurement container (plastic cuvette, quartz glass, etc.). In the measurement method using the fluorescence polarization method of this disclosure, if the mobility of the luminescent particles changes independently of enzyme activity, enzyme amount, and the concentration of enzyme reaction-related substances, it will reduce the accuracy and precision of the measurement of the target substance. Therefore, it is preferable to avoid nonspecific aggregation between particles and nonspecific adsorption of particles to the container. For this purpose, it is preferable to coat the particle surface with a hydrophilic polymer. For example, polymers such as polyvinylpyrrolidone, polyethylene glycol, polyhydroxymethacrylate, dextran, or bovine serum albumin are suitably used. Also, as mentioned above, luminescent particles crosslink between multiple particles. To promote crosslinking between luminescent particles, binding functional groups may be introduced on the surface of the luminescent particles as appropriate. For example, by having bonding functional groups such as thiol groups, amino groups, carboxyl groups, and maleimide groups on the surface of the luminescent particles, bonding between luminescent particles having thiol groups, carboxyl groups, amino groups, etc., can be promoted. This is because electrostatic bonding, disulfide bonding, and bonding between thiol groups and maleimide groups occur between the luminescent particles. These bonds can occur, for example, through the promotion of oxidation reactions to bond thiol groups together (disulfide bonding), or, when using phenols having thiol groups or maleimide groups, oligomers having many thiol groups or maleimide groups are generated, and these act as binders to bond luminescent particles having thiol groups or maleimide groups together. Therefore, a preferred example of a surface for luminescent particles is one that is coated with a hydrophilic polymer such as polyvinylpyrrolidone and further incorporates thiol groups.

[0050] The luminescent particles used in this disclosure do not specifically bind to target substances. That is, for enzymes, which are an example of target substances in this disclosure, the luminescent particles do not act as substrates for the enzyme. Similarly, enzyme reaction-related substances (e.g., glucose or hydrogen peroxide), which are also examples of target substances in this disclosure, do not specifically bind to the luminescent particles. This method allows the luminescent particles to function independently as probes in fluorescence polarization. To detect enzymes as target substances, immunoassays using antibody-conjugated luminescent particles, on which antibodies against the enzyme are bound, are known. However, measuring minute amounts of enzyme depends on the binding affinity of the antibody, and constructing a highly sensitive assay is a complicated process. Furthermore, it is extremely difficult to evaluate enzyme activity using immunoassays. On the other hand, in the method of this disclosure, by introducing sufficient substrate and luminescent particles, the measurement of enzyme amount and enzyme activity can be made highly sensitive depending on the reaction time.

[0051] The luminescent particles used in this disclosure contain a luminescent substance. Here, the luminescent substance only needs to be something that can be contained in the particles and can be measured by fluorescence polarization. The luminescent substance may be contained in the particles on the surface or inside the particles, preferably inside the particles. This is because on the particle surface, interactions occur between the luminescent substance and impurities and water molecules in the solution, which can affect the dispersibility of the luminescent particles and the measurement of fluorescence polarization. Examples of luminescent substances include fluorescent dyes such as fluorescein derivatives, rhodamine derivatives, and cyanine derivatives, as well as rare-earth luminescent complexes such as europium complexes and terbium complexes. Europium complexes are suitable as luminescent substances to be contained in the luminescent particles according to this disclosure because their emission wavelength and intensity are less affected by the surroundings, and their emission has a long lifetime.

[0052] Specific examples of europium complexes include tris(2-thenoyltrifluoroacetone)bis(triphenylphosphineoxide)europium(III), tris(2-thenoyltrifluoroacetone)(triphenylphosphineoxide)(dibenzylsulfoxide)europium(III), and tris(2-thenoyltrifluoroacetone)(phenanthroline)europium(III).

[0053] A higher content of luminescent material in the luminescent particles is preferable. This is because it increases the luminescence intensity per particle, which in turn contributes to increased sensitivity or improved measurement accuracy in the measurement of enzyme activity, enzyme quantity, and enzyme reaction-related substance concentrations as described in this disclosure. The content of rare earth luminescent complexes in the luminescent particles is preferably, for example, 0.001 g or more per gram of particle. With this content, a sufficiently high and stable luminescence can be obtained per particle. Furthermore, the content of luminescent material can be measured by elemental analysis using the elemental characteristics of the luminescent material as an indicator. For example, the content of rare earth luminescent complexes can be calculated from the quantification of the rare earth luminescent complexes using inductively coupled plasma (ICP) emission spectroscopy.

[0054] (Phenols) The phenols used in this disclosure are not particularly limited as long as they are phenol compounds having phenolic hydroxyl groups and capable of generating phenoxy radicals by the action of peroxidase. The phenols may include phenol and phenol derivatives having the above functions. Examples of phenols include low molecular weight phenol compounds with a molecular weight of 1000 or less and having at least one phenolic hydroxyl group. Preferably, the phenols are low molecular weight phenol compounds with a molecular weight of 500 or less and having one to six phenolic hydroxyl groups. Examples include phenol, catechol, resorcinol, hydroquinone, tyramine and its hydrochloride salts, serotonin, N-glycyl-L-tyrosine (Gly-Tyr), 3-(4-hydroxyphenyl)propionic acid, methyl 3-(4-hydroxyphenyl)propionate, 4-hydroxyphenylacetic acid, 3-hydroxyphenylacetic acid, p-coumaric acid, caffeic acid, dopamine, 6-hydroxydopamine, and norepinephrine. Due to their water solubility and reaction stability, tyramine, tyramine hydrochloride, phenol, glycyl-L-tyrosine, resorcinol, and serotonin are preferably used.

[0055] (hydrogen peroxide) In this disclosure, hydrogen peroxide is used in the aggregation reaction of luminescent particles by HRP. The mechanism of interparticle aggregation by peroxidase used in the method of this disclosure is presumed to be mediated by phenols produced by the peroxidase. For example, when a thiol group is used as the binding functional group of the luminescent particles, by reacting phenols with HRP, a radical transfer reaction occurs from phenoxy radicals generated in the reaction solution to thiol groups, and the thiol radicals rapidly undergo a disulfide bond reaction with other thiol groups (i.e., aggregation between luminescent particles). Furthermore, hydrogen peroxide is also generated during the disulfide bond reaction, and it has been reported that the catalytic cycle of HRP proceeds spontaneously without the addition of hydrogen peroxide from an external source. As a result, it has been reported that the oxidation of SH groups by HRP (disulfide bond reaction) is promoted under short time and low HRP concentration conditions. However, as will be described later in Example 1 and Comparative Example 2, it was found that the addition of hydrogen peroxide from an external source is essential for the fluorescence polarization method of this disclosure. This is because rapid measurement is required in this disclosure, specifically because it is necessary to significantly change the fluorescence polarization degree of the luminescent particles acting as probes within a reaction time of several minutes. Under the conditions without hydrogen peroxide as shown in the prior art (Comparative Example 2), a sufficient increase in fluorescence polarization degree was not observed. Therefore, it is considered that the addition of hydrogen peroxide is essential for the measurement method of this disclosure to work.

[0056] When measuring glucose oxidase activity, the addition of hydrogen peroxide from an external source is not essential because hydrogen peroxide is generated by the action of glucose oxidase. However, an appropriate amount of hydrogen peroxide may be added to accelerate the cross-linking reaction between luminescent particles and obtain a stronger signal.

[0057] (Third step) In this disclosure, the third step is to measure a value relating to the fluorescence anisotropy of the liquid sample from the second step. The fluorescence polarization method can be performed by known methods, and various commercially available measuring devices can be used for this purpose. The method for measuring the value relating to fluorescence anisotropy is also called the fluorescence polarization method or the fluorescence depolarization method, but in this specification, the fluorescence polarization method and the fluorescence depolarization method are synonymous. Here, as mentioned above, the value relating to fluorescence anisotropy is specifically the fluorescence polarization degree, but it may also be a value indicating fluorescence anisotropy that is calculated from the fluorescence polarization degree, or it may be a value that indicates fluorescence polarization degree or fluorescence anisotropy that can be obtained by calculation. The fluorescence polarization degree (p) and fluorescence anisotropy (r) are related by the following equation (1). P = 3r / (2+r)···(1)

[0058] In this disclosure, the target substance's enzyme and enzyme reaction-related substances can be measured by fluorescence polarization using the mobility of luminescent particles in a sample solution as an indicator. That is, depending on the enzyme activity, enzyme amount, or amount of enzyme reaction-related substances, crosslinking occurs between luminescent particles in the sample solution, resulting in a decrease in the mobility of the luminescent particles. In this disclosure, the mobility of the luminescent particles is measured using a value related to fluorescence anisotropy. In the fluorescence polarization method, for example, the degree of fluorescence polarization (mp) can be used as an indicator.

[0059] In this disclosure, it is preferable to use luminescent particles as probes that contain a europium complex as a luminescent material within the particles, which has a long luminescence lifetime and exhibits polarized emission. Even slight changes in the rotational motion of these luminescent particles in a liquid sample can be detected as changes in polarized emission characteristics. Specifically, when the mobility of the particles decreases significantly due to crosslinking between the luminescent particles, the decrease in the rotational Brownian motion of the particles can be detected with high sensitivity as a change in the value related to fluorescence anisotropy.

[0060] The timing of measuring the fluorescence anisotropy value can be set as appropriate. For example, the fluorescence anisotropy value may be measured after the second step. Measuring the fluorescence anisotropy value when the reaction is complete is a simple and preferred method. For example, the fluorescence anisotropy value can be measured 5 minutes after adding the target substance to the solution.

[0061] Furthermore, before reacting the target substance (enzyme or enzyme reaction-related substance) in the second step, the fluorescence polarization degree may be measured as a value related to the fluorescence anisotropy of the luminescent particles, and this may be taken as the initial fluorescence polarization degree (mp(0)). Then, the reaction may proceed, and the fluorescence polarization degree (mp(t)) at a certain time point may be measured again due to the crosslinking between the luminescent particles (after the second step), or it may be measured continuously at regular time intervals (during the second step to after the second step). In other words, by measuring the change in fluorescence polarization degree over time, the change in fluorescence polarization degree (Δmp) may be determined from the difference between the fluorescence polarization degree (mp(t)) at a certain time point and the initial fluorescence polarization degree (mp(0)). The change in fluorescence polarization degree occurs rapidly, and for example, a sufficient Δmp can be obtained in about 1 to 10 minutes. It is also possible to determine the rate of change in fluorescence polarization degree (dmp / dt).

[0062] The measurement conditions for fluorescence polarization are preferably, for example, in a liquid at a temperature of 1 to 50°C, with a viscosity of 0.5 to 50 mPa·s. If the luminescent particles are luminescent particles containing a europium complex, the concentration of the luminescent particles is not limited as long as the emission of light from the luminescent particles can be detected, and the concentration of the luminescent particles can be appropriately selected depending on the amount of the target substance, enzyme, hydrogen peroxide, phenols, etc. It is preferable to measure the fluorescence polarization in the range of 0.0001 mg / mL to 1.0 mg / mL, but in order to ensure the emission intensity from the luminescent particles and avoid the effects of scattering from the luminescent particles, it is preferable to measure the fluorescence polarization in the range of 0.001 mg / mL to 0.1 mg / mL.

[0063] (Fourth step) In this disclosure, the fourth step is to detect substances related to the enzymatic reaction in the liquid sample based on the fluorescence anisotropy value of the luminescent particles in the liquid sample obtained in the third step. More specifically, it is a step of relating the change in the fluorescence anisotropy value to enzyme activity, enzyme amount, or the amount of substances related to the enzymatic reaction.

[0064] In the third step, the degree of decrease in the mobility of the luminescent particles in response to inter-particle crosslinking is detected as a value related to fluorescence anisotropy. The degree of decrease in the mobility of the luminescent particles depends on the activity and amount of oxidoreductase present in the liquid sample, or the amount of enzyme reaction-related substances. Therefore, by relating the fluorescence anisotropy value obtained in the third step with the activity and amount of oxidoreductase in the liquid sample, or the amount of enzyme reaction-related substances, it is possible to measure the activity and amount of oxidoreductase in the liquid sample, or the amount of enzyme reaction-related substances. For example, if the fluorescence anisotropy value obtained in the third step is high, it means that the luminescent particles are aggregated and the proportion of these aggregates is large, which can be associated with high oxidoreductase activity.

[0065] Furthermore, the term "measurement method" in this disclosure includes not only quantitative measurements but also qualitative measurements (detection of the presence or absence of enzymes or enzyme reaction-related substances). Therefore, in the fourth step, the correlation between the value of fluorescence anisotropy of the luminescent particles and the activity or amount of oxidoreductase, or the amount of enzyme reaction-related substances, can be either quantitative or qualitative.

[0066] Quantitative correlations can be established, for example, by first determining a relationship between enzyme activity and fluorescence polarization using an enzyme solution with known enzyme activity. This relationship can then be used to measure the target substance and determine its enzyme activity from the resulting fluorescence polarization. By establishing such a correlation, enzyme activity in liquid samples can be measured. The measured enzyme activity can also be expressed as enzyme concentration, mass, number of molecules, etc.

[0067] In this disclosure, similarly, a relationship between the concentration and fluorescence polarization degree can be determined in advance, for example, using a hydrogen peroxide solution or a glucose solution of known concentration. The concentration of the target substance can be determined from the fluorescence polarization degree obtained by measuring the target substance.

[0068] Furthermore, in the fourth step, the change in the value related to the fluorescence anisotropy of the luminescent particles can also be treated relatively. That is, enzyme activity, enzyme quantity, or the amount of enzyme reaction-related substances may be related relatively, and for example, the value of fluorescence polarization degree (mp) obtained by fluorescence polarization measurement can be used. For example, in applications of screening highly active enzymes, screening may be performed simply based on the value of fluorescence polarization degree (mp) or the amount of change therein (Δmp) for a population of enzymes of the same concentration.

[0069] (Reagents for detecting target substances using measurement of fluorescence anisotropy values) According to this disclosure, the above measurement system may be provided as reagents (which may also be called kits for detecting target substances) as listed in (1) to (3) below.

[0070] (1) A reagent for detecting substances related to the enzymatic reaction of peroxidase in this disclosure, comprising luminescent particles having a binding functional group, phenols, and hydrogen peroxide. Luminescent particles and phenols having bonding functional groups can be provided in solution, dry, frozen, or freeze-dried state. Hydrogen peroxide can be provided in solution. Conventional stabilizers for hydrogen peroxide, such as salicylic acid, can be included.

[0071] The reagents of this disclosure may be a single reagent or may be composed of multiple reagents, such as a first solution, a second solution, a third solution, etc. From the viewpoint of the stability of each reagent, for example, the first solution contains luminescent particles having a crosslinkable functional group, the second solution contains phenolates, and the third solution contains hydrogen peroxide. Furthermore, the reagents of this disclosure can be separated into a solution state and a dry state, and the dry reagents can be used for various measurements by converting them to a solution state with an accompanying dissolving solution before measurement.

[0072] (2) A reagent for detecting hydrogen peroxide of a target substance according to the present disclosure, comprising an oxidoreductase, luminescent particles having a binding functional group, and phenols. Oxidoreductases, luminescent particles having binding functional groups, and phenols can be provided in solution, dry, frozen, or freeze-dried states. Furthermore, the reagents of this disclosure can be divided into multiple reagents, for example, a first solution, a second solution, and a third solution. For example, the first solution may contain oxidoreductases, the second solution may contain luminescent particles having binding functional groups, and the third solution may contain phenols.

[0073] (3) A reagent for detecting glucose, the target substance according to this disclosure, comprising glucose oxidase, peroxidase, luminescent particles having a binding functional group, and phenols. Glucose oxidase, peroxidase, luminescent particles having binding functional groups, and phenols can be provided in solution, dry, frozen, or lyophilized states. Furthermore, the reagents of this disclosure can be divided into multiple reagents, for example, a first solution, a second solution, a third solution, and a fourth solution. For example, the first solution may contain glucose oxidase, the second solution may contain peroxidase, the third solution may contain luminescent particles having binding functional groups, and the fourth solution may contain phenols. [Examples]

[0074] The present disclosure will be described in further detail below with reference to examples. (1: Preparation of thiol group-introduced luminescent particle T1) The luminescent particles used in the method for measuring enzyme activity, enzyme quantity, or the amount of enzyme reaction-related substances according to this disclosure are characterized by having binding functional groups on their surface that allow for crosslinking between the luminescent particles. Here, we show an example of the synthesis of luminescent particles having thiol groups as binding functional groups on their surface.

[0075] First, solvent A was prepared by dissolving polyvinylpyrrolidone (PVP-K30: manufactured by Tokyo Chemical Industry Co., Ltd.) in pH 7 MES (2-morpholinoethanesulfonic acid) buffer (manufactured by Kishida Chemical Co., Ltd.). Next, reaction solution B was prepared by mixing tris(2-thenoyltrifluoroacetone)bis(triphenylphosphineoxide)europium(III) (manufactured by Central Techno Co., Ltd., hereinafter abbreviated as "Eu(TTA)3(TPPO)2"), a europium complex, styrene monomer (manufactured by Kishida Chemical Co., Ltd.), and 3-methacrylateoxypropyltrimethoxysilane (manufactured by Tokyo Chemical Industry Co., Ltd., hereinafter abbreviated as "MPS"). Reaction solution B was added to a four-necked flask containing solvent A, and the mixture was stirred using a mechanical stirrer set to 300 rpm. After stirring for 15 minutes under nitrogen flow conditions, the temperature of the prepared oil bath was set to 70°C and nitrogen flow was continued for another 15 minutes. After heating and stirring the mixture, an aqueous solution of potassium persulfate (hereinafter abbreviated as "KPS") (manufactured by Aldrich) was added to the reaction solution and emulsion polymerization was carried out for 20 hours. After the polymerization reaction, the obtained suspension was ultrafiltered using an ultrafiltration membrane with a molecular weight cutoff of 100K with approximately 4 L of deionized water to wash the product and obtain a dispersion of luminescent particles.

[0076] A dispersion of luminescent particles obtained by emulsion polymerization was taken and added to an aqueous solution containing 1% by mass of Tween20 (manufactured by Kishida Chemical Co., Ltd.). After stirring for 10 minutes, the silane coupling agent X12-1135 (manufactured by Shin-Etsu Chemical Co., Ltd.) was added and stirred overnight (the mass ratio of the charged particles, pure water, and X12-1135 was 1:300:2). After stirring, the dispersion was centrifuged, the supernatant was removed, and the precipitate was redispersed in pure water. The centrifugation and redispersion process was repeated at least three times to wash the product. The precipitate after washing was redispersed in pure water. Luminescent particle 1 was obtained by the above procedure.

[0077] Next, 0.25 mL of the particle dispersion of luminescent particle 1 (particle concentration 1.2% by mass) was taken, and the solvent was replaced with 1.6 mL of pH 6.0 MES buffer. 0.5% by mass of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide and N-hydroxysulfosuccinimide sodium were added to the MES buffer containing the dispersed particles, and the mixture was reacted at 25°C for 1 hour. After the reaction, the dispersion was washed with pH 5.0 MES buffer, 2-aminoethanethiol hydrochloride was added, and the mixture was reacted at 25°C for 2 hours to introduce 2-aminoethanethiol to the particle surface. After introduction, the particles were washed with pH 8 Tris buffer. Subsequently, the particles were washed with phosphate buffer to obtain 0.3% by mass thiol-modified luminescent particles (hereafter, luminescent particle T1). The aqueous solution containing luminescent particle T1 is called luminescent particle T1 solution.

[0078] The presence of thiol groups on the particle surface was confirmed by adding Eilmann's reagent to the luminescent particle T1 solution, centrifuging the solution, and measuring the absorbance (410 nm) of the supernatant. The increase in absorbance compared to before the addition of Eilmann's reagent confirmed the presence of thiol groups on the particle surface.

[0079] (2: Preparation of phenol solutions) Tyramine hydrochloride was dissolved in phosphate buffer (containing 0.01% Tween 20) to prepare a phosphate buffer solution of tyramine hydrochloride with a final concentration of 40 mM.

[0080] (3: Preparation of hydrogen peroxide solution) A 3.5% hydrogen peroxide solution was prepared by diluting 35% hydrogen peroxide solution with water.

[0081] (4: Preparation of a sample solution containing the target substance, peroxidase) The target substance, horseradish peroxidase (HRP), was dissolved in water to prepare a sample solution with a final concentration of 2 mg / mL (0.05 mM).

[0082] Based on the sample preparation examples described above, Examples 1 to 6, described later, provide examples of fluorescence polarization measurement of luminescent particles by peroxidase enzyme reaction.

[0083] The reaction solution containing the europium complex has its surface hydrophilized with Tween 20 and a silane coupling agent (luminescent particles 10), and further thiol groups (binding functional groups 20 of the luminescent particles) are added with 2-aminoethanethiol hydrochloride. The luminescent particles form disulfide-crosslinked aggregates with HRP (peroxidase 40), tyramine hydrochloride (phenols), and hydrogen peroxide. This changes the mobility of the luminescent particles, which is detected as a change in fluorescence polarization. Furthermore, in Example 6 described later, a measurement example using glucose oxidase (glucose oxidase 60) as the source of hydrogen peroxide is shown.

[0084] (Example 1: Fluorescence polarization measurement of peroxidase) Water, luminescent particle T1 solution, and tyramine hydrochloride were mixed in the proportions shown in Table 1. The target substance (HRP) was added to this aqueous solution and thoroughly mixed. These solutions were transferred to a 96-well microplate. Next, a 3.5% hydrogen peroxide solution was added to the solutions in the wells and thoroughly mixed. The microwell plate was then placed in a fluorescence polarization analyzer (Nivo multimode microplate reader). The fluorescence polarization degree (mp) was measured after 5 minutes. The conditions for the fluorescence polarization analyzer were as follows: Mode: FP Kinetics, Excitation light: center wavelength 355 nm / width 40 nm, Emission filter: (S) Center wavelength 615nm / width 8nm, (P) Center wavelength 615nm / width 8nm, Dichroic mirror: D400, Measurement time: 1000ms Z-Focus: 5mm Measurement spot size: 2mm on the excitation side, 4mm on the emission side Flash energy: Low (10) PMT HV:1000

[0085] (Comparative Example 1: Fluorescence polarization measurement of a sample without peroxidase) The sample was prepared in the same manner as in Example 1, except that 20 μL of water was added instead of the target substance HRP. The degree of fluorescence polarization (mp) was measured in the same manner as in Example 1.

[0086] (Comparative Example 2: Fluorescence polarization measurement of a sample without hydrogen peroxide) Samples were prepared in the same manner as in Example 1, except that a 3.5% aqueous hydrogen peroxide solution was not added. The degree of fluorescence polarization (mp) was measured in the same manner as in Example 1.

[0087] (Comparative Example 3: Fluorescence polarization measurement of a sample that does not contain phenols) Samples were prepared in the same manner as in Example 1, except that tyramine hydrochloride was not added. The degree of fluorescence polarization (mp) was measured in the same manner as in Example 1.

[0088] The results for Example 1 and Comparative Examples 1 to 3 are shown in Table 1. In the sample without HRP (Comparative Example 1), the fluorescence polarization degree was 63.0, but in the sample containing HRP, the fluorescence polarization degree increased to 80.0. This means that in Example 1, the mobility of the luminescent particles in the sample solution was reduced, indicating that aggregation of luminescent particles occurred due to the activity of HRP. From this example, it was found that the presence of peroxidase causes aggregation of luminescent particles and increases the fluorescence polarization degree.

[0089] When hydrogen peroxide was not added (Comparative Example 2), the fluorescence polarization degree was low at 63.0, similar to Comparative Example 1. It was confirmed that no aggregation of luminescent particles occurred. From this comparative example, it was found that the addition of hydrogen peroxide is necessary to measure peroxidase.

[0090] When phenols (tyramine hydrochloride) were not added (Comparative Example 3), the fluorescence polarization degree was low at 62.8. From this comparative example, it was found that the addition of phenols (tyramine hydrochloride) is necessary for the rapid measurement of peroxidase using fluorescence polarization.

[0091] [Table 1]

[0092] (Example 2: Fluorescence polarization measurement of peroxidase) Water, luminescent particle T1 solution, and tyramine hydrochloride were mixed in the proportions shown in Table 2. Target substances (HRP) of different concentrations were added to this aqueous solution and thoroughly mixed. Solutions with different HRP activity were prepared by preparing target substances (HRP) from HRP with known activity. These solutions were transferred to a 96-well microplate. Next, a 3.5% aqueous hydrogen peroxide solution was added to the solution in the wells and thoroughly mixed, and the fluorescence polarization degree (mp) was measured in the same manner as in Example 1.

[0093] The results of Example 2 are shown in Table 2. The degree of fluorescence polarization increased with increasing HRP concentration or activity. It is thought that crosslinking between luminescent particles occurred in response to HRP activity, reducing the mobility of the luminescent particles. It was found that by using the relationship between the amount or activity of peroxidase and the degree of fluorescence polarization (calibration curve) obtained from this example, it is possible to determine the activity or concentration of peroxidase at an unknown concentration by measuring its degree of fluorescence polarization.

[0094] [Table 2]

[0095] (Example 3: Fluorescence polarization measurement of peroxidase activity) Water, luminescent particle T1 solution, and tyramine hydrochloride were mixed in the proportions shown in Table 3. Peroxidase (HRP), which has the same activity, was added to this aqueous solution and thoroughly mixed. Two samples were prepared: one with water added to the mixture, and the other with sodium azide solution, the target substance and an inhibitor of peroxidase enzyme activity. These solutions were transferred to 96-well microplates. Next, a 3.5% hydrogen peroxide aqueous solution was added to the solutions in the wells and thoroughly mixed, and the fluorescence polarization degree (mp) was measured in the same manner as in Example 1.

[0096] The results of Example 3 are shown in Table 3. In the sample to which sodium azide solution was added, the degree of fluorescence polarization was lower compared to the sample without the addition. This indicates that it is possible to measure the decrease in HRP enzyme activity using fluorescence polarization.

[0097] [Table 3]

[0098] (Example 4: Measurement of hydrogen peroxide concentration by fluorescence polarization) Water, luminescent particle T1 solution, tyramine hydrochloride, and HRP were mixed in the proportions shown in Table 4. These solutions were transferred to a 96-well microplate. Next, hydrogen peroxide of various concentrations was added to the solutions in the wells as a target substance, and after thorough mixing, the degree of fluorescence polarization (mp) was measured in the same manner as in Example 1.

[0099] The results of Example 4 are shown in Table 4. The degree of fluorescence polarization increased with increasing hydrogen peroxide concentration. It appears that the mobility of the luminescent particles decreases depending on the amount of hydrogen peroxide. It was found that by using the relationship between the amount (concentration) of hydrogen peroxide and the degree of fluorescence polarization (calibration curve) obtained from this example, it is possible to determine the concentration of hydrogen peroxide solution of unknown concentration by measuring the degree of fluorescence polarization.

[0100] [Table 4]

[0101] (Example 5: Fluorescence polarization measurement of glucose oxidase) Water, luminescent particle T1 solution, tyramine hydrochloride, HRP, and glucose were mixed in the proportions shown in Table 5. These solutions were transferred to a 96-well microplate. Next, glucose oxidase (abbreviated as GOX) of various concentrations was added to the solutions in the wells as the target substance, and after thorough mixing, the fluorescence polarization degree (mp) was measured in the same manner as in Example 1, except that the fluorescence polarization degree was measured after 20 minutes.

[0102] The results of Example 5 are shown in Table 5. The fluorescence polarization degree increased with increasing GOX concentration. It is thought that the target substance, GOX, produced hydrogen peroxide from glucose in the solution, and this hydrogen peroxide triggered the activation of peroxidase, causing particle aggregation and thus increasing the fluorescence polarization degree. It was found that by using the relationship between GOX concentration and fluorescence polarization degree (calibration curve) obtained from this example, it is possible to determine the concentration of GOX of an unknown concentration by measuring its fluorescence polarization degree.

[0103] [Table 5]

[0104] (Example 6: Fluorescence polarization measurement of glucose) MES buffer (pH 5.0), luminescent particle T1 solution, tyramine hydrochloride, HRP, and GOX were mixed in the proportions shown in Table 6. These solutions were transferred to a 96-well microplate. Next, glucose of various concentrations was added as the target substance to the solution in the wells, and after thorough mixing, the fluorescence polarization (mp) was measured in the same manner as in Example 1, except that the fluorescence polarization was measured after 3 minutes.

[0105] The results of Example 6 are shown in Table 6. The fluorescence polarization degree increased with increasing glucose concentration. It is thought that hydrogen peroxide was produced from the target substance glucose by GOX, and that this hydrogen peroxide triggered the activation of peroxidase, causing particle aggregation and thus increasing the fluorescence polarization degree. It was found that by using the relationship between glucose concentration and fluorescence polarization degree (calibration curve) obtained from this example, it is possible to determine the concentration of glucose of unknown concentration by measuring its fluorescence polarization degree.

[0106] [Table 6]

[0107] This embodiment includes the following configurations and methods. (Method 1) A method for detecting a target substance that is related to an enzymatic reaction, A first step involves mixing a sample solution that may contain the target substance with a reagent to obtain a liquid sample containing hydrogen peroxide, peroxidase, phenols, and a plurality of luminescent particles having binding functional groups. A second step in which the luminescent particles aggregate due to the bonding functional groups of the plurality of luminescent particles bonding together based on the reaction between the hydrogen peroxide, the peroxidase, and the phenols in the liquid sample, which occurs when the target substance is present in the liquid sample. A third step involves obtaining a value relating to the fluorescence anisotropy of the liquid sample in the second step, A method for detecting a target substance, characterized by having the following features. (Method 2) The method for detecting a target substance according to Method 1, wherein the luminescent particles are nanoparticles containing a rare earth luminescent complex. (Method 3) A method for detecting a target substance according to method 1 or 2, wherein the bonding of the bonding functional group is a bonding of the same type of bonding functional group. (Method 4) A method for detecting a target substance according to any one of methods 1 to 3, wherein the bonding functional group is a functional group selected from the group consisting of a thiol group, a carboxyl group, an amino group, and a maleimide group. (Method 5) The method for detecting a target substance according to Method 3, wherein the bonding functional group is a thiol group. (Method 6) A method for detecting a target substance according to any one of methods 1 to 5, wherein the phenols are selected from the group consisting of tyramine, tyramine hydrochloride, phenol, glycyl-L-tyrosine, resorcinol, and serotonin. (Method 7) A method for detecting a target substance according to any one of methods 1 to 6, wherein the target substance is a peroxidase. (Method 8) A method for detecting a target substance according to any one of methods 1 to 6, wherein the target substance is an oxidase, the liquid sample contains a substrate for the oxidase, and the hydrogen peroxide contains hydrogen peroxide produced by the oxidase. (Method 9) A method for detecting a target substance according to any one of methods 1 to 6, wherein the target substance is hydrogen peroxide. (Method 10) A method for detecting a target substance according to any one of methods 1 to 6, wherein the target substance is a substrate for oxidase, the liquid sample contains oxidase, and the hydrogen peroxide contains hydrogen peroxide produced by oxidase. (Method 11) A method for detecting a target substance according to any one of methods 1 to 6, wherein the target substance is a phenol. (Method 12) A method for detecting a target substance according to any one of methods 1 to 6, wherein the target substance is an inhibitor of the enzymatic reaction of the peroxidase. (Method 13) A method for detecting a target substance according to any one of methods 1 to 6, wherein the sample solution contains hydrogen peroxide. (Method 14) A method for detecting a target substance according to any one of methods 1 to 13, further comprising a fourth step of detecting the target substance based on the value relating to the fluorescence anisotropy. (Composition 15) A reagent for detecting a target substance using the measurement of fluorescence anisotropy values, characterized in that the target substance is a peroxidase, and the reagent comprises hydrogen peroxide, phenols, and a plurality of luminescent particles having binding functional groups. (Composition 16) A reagent for detecting a target substance using the measurement of fluorescence anisotropy values, wherein the target substance is an oxidase, and the reagent comprises an oxidase substrate, peroxidase, phenols, and a plurality of luminescent particles having binding functional groups. (Composition 17) A reagent for detecting a target substance using the measurement of fluorescence anisotropy values, characterized in that the target substance is hydrogen peroxide, and the reagent comprises a peroxidase, phenols, and a plurality of luminescent particles having binding functional groups. (Composition 18) A reagent for detecting a target substance using the measurement of fluorescence anisotropy values, wherein the target substance is a substrate for oxidase, and the reagent comprises oxidase, peroxidase, phenols, and a plurality of luminescent particles having binding functional groups. (Composition 19) A reagent for detecting a target substance using the measurement of fluorescence anisotropy values, wherein the target substance is a phenol, and the reagent comprises peroxidase, hydrogen peroxide, and a plurality of luminescent particles having crosslinkable functional groups. (Composition 20) A reagent for detecting a target substance using the measurement of fluorescence anisotropy values, wherein the target substance is an inhibitor of the enzymatic reaction of peroxidase, and the reagent comprises hydrogen peroxide, peroxidase, phenols, and a plurality of luminescent particles having binding functional groups. (Composition 21) The reagent according to any one of configurations 15 to 20, wherein the bonding functional group is a functional group selected from the group consisting of a thiol group, a carboxyl group, an amino group, and a maleimide group. (Composition 22) The reagent according to configuration 21, wherein the bonding functional group is a thiol group. (Composition 23) The reagent according to any one of configurations 15 to 22, wherein the luminescent particles are particles containing a rare earth luminescent complex. [Explanation of symbols]

[0108] 10 Luminescent particles 20 Bonding functional groups of luminescent particles 40 Peroxidase 60 Glucose oxidase

Claims

1. A method for detecting a target substance that is related to an enzymatic reaction, A first step involves mixing a sample solution that may contain the target substance with a reagent to obtain a liquid sample containing hydrogen peroxide, peroxidase, phenols, and a plurality of luminescent particles having binding functional groups. A second step is in which the luminescent particles aggregate as a result of the bonding functional groups of the plurality of luminescent particles bonding together based on the reaction between the hydrogen peroxide, the peroxidase, and the phenols in the liquid sample, which occurs due to the presence of the target substance in the liquid sample. A third step is to obtain a value relating to the fluorescence anisotropy of the liquid sample in the second step, A method for detecting a target substance, characterized by having the following features.

2. The method for detecting a target substance according to claim 1, wherein the luminescent particles are nanoparticles containing a rare earth luminescent complex.

3. The method for detecting a target substance according to claim 1, wherein the bonding of the bonding functional groups is a bonding of the same type of bonding functional group.

4. The method for detecting a target substance according to claim 1, wherein the bonding functional group is a functional group selected from the group consisting of a thiol group, a carboxyl group, an amino group, and a maleimide group.

5. The method for detecting a target substance according to claim 3, wherein the bonding functional group is a thiol group.

6. The method for detecting a target substance according to claim 1, wherein the phenols are selected from the group consisting of tyramine, tyramine hydrochloride, phenol, glycyl-L-tyrosine, resorcinol, and serotonin.

7. The method for detecting a target substance according to any one of claims 1 to 6, wherein the target substance is a peroxidase.

8. A method for detecting a target substance according to any one of claims 1 to 6, wherein the target substance is an oxidase, the liquid sample contains a substrate for the oxidase, and the hydrogen peroxide contains hydrogen peroxide produced by the oxidase.

9. The method for detecting a target substance according to any one of claims 1 to 6, wherein the target substance is hydrogen peroxide.

10. A method for detecting a target substance according to any one of claims 1 to 6, wherein the target substance is a substrate for oxidase, the liquid sample contains oxidase, and the hydrogen peroxide contains that produced by the oxidase.

11. The method for detecting a target substance according to any one of claims 1 to 6, wherein the target substance is a phenol.

12. A method for detecting a target substance according to any one of claims 1 to 6, wherein the target substance is an inhibitor of the enzymatic reaction of the peroxidase.

13. The method for detecting a target substance according to any one of claims 1 to 6, wherein the sample solution contains hydrogen peroxide.

14. A method for detecting a target substance according to claim 1, further comprising a fourth step of detecting the target substance based on the value relating to the fluorescence anisotropy.

15. A reagent for detecting a target substance using the measurement of fluorescence anisotropy values, characterized in that the target substance is a peroxidase, and the reagent comprises hydrogen peroxide, phenols, and a plurality of luminescent particles having binding functional groups.

16. A reagent for detecting a target substance using the measurement of fluorescence anisotropy values, wherein the target substance is an oxidase, and the reagent comprises an oxidase substrate, a peroxidase, phenols, and a plurality of luminescent particles having binding functional groups.

17. A reagent for detecting a target substance using the measurement of fluorescence anisotropy values, characterized in that the target substance is hydrogen peroxide, and the reagent comprises peroxidase, phenols, and a plurality of luminescent particles having binding functional groups.

18. A reagent for detecting a target substance using the measurement of fluorescence anisotropy values, wherein the target substance is a substrate for oxidase, and the reagent comprises oxidase, peroxidase, phenols, and a plurality of luminescent particles having binding functional groups.

19. A reagent for detecting a target substance using the measurement of fluorescence anisotropy values, characterized in that the target substance is a phenol, and the reagent comprises peroxidase, hydrogen peroxide, and a plurality of luminescent particles having binding functional groups.

20. A reagent for detecting a target substance using the measurement of fluorescence anisotropy values, wherein the target substance is an inhibitor of the enzymatic reaction of peroxidase, and the reagent comprises hydrogen peroxide, peroxidase, phenols, and a plurality of luminescent particles having binding functional groups.

21. The reagent according to any one of claims 15 to 20, wherein the bonding functional group is a functional group selected from the group consisting of a thiol group, a carboxyl group, an amino group, and a maleimide group.

22. The reagent according to claim 21, wherein the bonding functional group is a thiol group.

23. The reagent according to any one of claims 15 to 20, wherein the luminescent particles are particles containing a rare earth luminescent complex.

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