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

Abstract

To provide a highly sensitive detection method and reagents for enzyme reaction-related substances. [Solution] A method for detecting a target substance related to an enzymatic reaction, comprising: a first step of mixing a sample that may contain the target substance with a reagent to obtain a liquid sample containing hydrogen peroxide, peroxidase, phenols, luminescent particles, and a plurality of hydrophilic polymers having binding functional groups; a second step of generating an aggregate of the hydrophilic polymers containing the luminescent particles by binding the binding functional groups of the hydrophilic polymers based on a reaction between the hydrogen peroxide, the peroxidase, and the phenols that 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 from 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 of 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 employed. 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 by utilizing the enzyme activity. Alternatively, substrates that have weak luminescence before the action of the enzyme but whose luminescence intensity increases after the action are also used.

[0004] However, when the enzyme is at a very low concentration, the amount of the fluorescent dye released from the synthetic substrate also decreases, resulting in weak luminescence from the fluorescent dye and making it difficult to measure the 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 adsorb to 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 a 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 emission intensity of the fluorescence 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 a technique for producing hydrogels using horseradish peroxidase (HRP), an oxidoreductase. Patent Document 3 addresses the challenge of encapsulating living cells in hydrogels under mild conditions using HRP, with a view to medical applications. [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] International Publication No. 2015 / 159995 [Overview of the project] [Problems that the invention aims to solve]

[0009] According to this disclosure, detecting substances related to the enzymatic reaction of peroxidases requires a complicated procedure, and it has been difficult to detect them with high sensitivity. [Means for solving the problem]

[0010] The first aspect of this disclosure is, A method for detecting target substances related to enzymatic reactions, A first step involves mixing a sample that may contain the target substance with a reagent to obtain a liquid sample containing hydrogen peroxide, peroxidase, phenols, luminescent particles, and a plurality of hydrophilic polymers having binding functional groups. A second step of generating an aggregate of the hydrophilic polymer containing the luminescent particles by bonding the binding functional groups of the hydrophilic polymer based on a reaction between the hydrogen peroxide, the peroxidase, and the phenols that occurs when the target substance is present in the liquid sample, A method for detecting a target substance, comprising: a third step of obtaining a value relating to the fluorescence anisotropy of the liquid sample after the second step;

[0011] Furthermore, 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 at least hydrogen peroxide, phenols, luminescent particles, and a hydrophilic polymer having a binding functional group.

[0012] Furthermore, 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 at least a substrate for the oxidase, peroxidase, phenols, luminescent particles, and a hydrophilic polymer having a binding functional group.

[0013] Also, a fourth aspect of the present disclosure is a reagent for detecting a target substance using measurement of a value related to fluorescence anisotropy, wherein the target substance is hydrogen peroxide, and the reagent contains at least peroxidase, phenols, luminescent particles, and a hydrophilic polymer having a binding functional group.

[0014] Also, a fifth aspect of the present disclosure is a reagent for detecting a target substance using measurement of a value related to fluorescence anisotropy, wherein the target substance is a substrate of oxidase, and the reagent contains at least the oxidase, peroxidase, phenols, luminescent particles, and a hydrophilic polymer having a binding functional group.

[0015] Also, a sixth aspect of the present disclosure is a reagent for detecting a target substance using measurement of a value related to fluorescence anisotropy, wherein the target substance is phenols, and the reagent contains at least peroxidase, hydrogen peroxide, luminescent particles, and a hydrophilic polymer having a binding functional group.

[0016] Also, a seventh aspect of the present disclosure is a reagent for detecting a target substance using measurement of a value related to fluorescence anisotropy, wherein the target substance is an inhibitor of the enzymatic reaction of peroxidase, and the reagent contains hydrogen peroxide, peroxidase, phenols, luminescent particles, and a hydrophilic polymer having a binding functional group.

Advantages of the Invention

[0017] According to the present disclosure, substances related to the enzymatic reaction of peroxidase can be measured simply and with high sensitivity. In particular, even when the amount of the enzyme is small or the enzyme activity is low, the enzyme activity can be measured with high sensitivity and quickly.

Brief Description of the Drawings

[0018] [Figure 1] It is a diagram for explaining the steps of a method for detecting a target substance according to one aspect of the present disclosure. [Figure 2] This is a diagram for explaining a method for detecting peroxidase that utilizes the formation of an aggregate of a hydrophilic polymer according to an embodiment of the present disclosure. [Figure 3] This is a diagram for explaining a method for detecting glucose oxidase that utilizes the formation of an aggregate of a hydrophilic polymer according to an embodiment of the present disclosure. [Figure 4] This is a diagram for explaining a method for detecting hydrogen peroxide concentration that utilizes an aggregate of a hydrophilic polymer according to an embodiment of the present disclosure. [Figure 5] This is a diagram for explaining a method for detecting glucose concentration that utilizes an aggregate of a hydrophilic polymer according to an embodiment of the present disclosure.

Mode for Carrying Out the Invention

[0019] 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".

[0020] (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, when 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 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 emission.

[0021] 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 are dispersed individually in a liquid solution, they rotate vigorously due to Brownian motion and exhibit a low degree of fluorescence polarization. On the other hand, when luminescent particles bind to larger carriers of other sizes, 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.

[0022] 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.

[0023] (Method for detecting target substances) The method for detecting a target substance according to this embodiment is a method for detecting a target 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 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 that may contain a target substance with a reagent to obtain a liquid sample containing hydrogen peroxide, peroxidase, phenols, luminescent particles, and multiple hydrophilic polymers having binding functional groups. (2) A second step (S1002) is performed in which, based on the reaction between hydrogen peroxide, peroxidase, and phenols that occurs when a target substance is present in the liquid sample, the binding functional groups of the hydrophilic polymer bond to it, thereby generating an aggregate of hydrophilic polymers containing luminescent particles. (3) A third step (S1003) in which the value relating to the fluorescence anisotropy of the liquid sample in the second step is measured, (4) A fourth step in which the target substance is detected based on the fluorescence anisotropy value of the liquid sample, This is a method for detecting a target substance that has [certain properties].

[0024] (Measurement principle 1: Measurement of peroxidase activity) The measurement principle of this disclosure will be explained using Figure 2, illustrating a method for highly sensitively measuring the activity of peroxidase, an example of an oxidoreductase, using crosslinking of hydrophilic polymers. Peroxidase 40, the target substance, can be crosslinked by multiple hydrophilic polymers 30 having binding functional groups (functional groups with binding ability) in the presence of a specified amount of hydrogen peroxide and phenols to form an aggregate 50 of hydrophilic polymers 30 (which can also be called a polymer crosslinked body or a hydrophilic polymer crosslinked structure). The generated aggregate 50 of hydrophilic polymers 30 grows to an appropriate size. Here, sodium alginate into which thiol groups have been introduced is used as an example of a hydrophilic polymer 30 having binding functional groups. Luminescent particles 10 are kept in this solution as probe molecules for fluorescence polarization measurement. If, for example, thiol groups have been introduced as binding functional groups 20 in these luminescent particles 10, a compound is formed between the aggregate 50 of hydrophilic polymers 30 made of alginic acid and the luminescent particles 10 based on the bonding of the thiol groups. 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, aggregates 50 of the hydrophilic polymer 30 are generated in proportion to its activity and bind to the luminescent particles 10, significantly reducing the mobility of the luminescent particles 10. This decrease in mobility is observed as an increase in the fluorescence polarization degree of the luminescence emitted by the luminescent particles 10. Therefore, the activity of peroxidase 40 in the sample can be determined by measuring the fluorescence polarization degree of the luminescent particles 10. 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.

[0025] (Measurement principle 2: Measurement of glucose oxidase activity) In addition to peroxidase, the enzymatic activity of other oxidases, which are substances involved in the enzymatic reactions of other peroxidases, can also be measured in a similar manner. For example, glucose oxidase is used as an example (Figure 3). The glucose 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 rapidly crosslink multiple hydrophilic polymers 30 having binding functional groups to form aggregates 50 in the presence of a specified amount of glucose, peroxidase 40, and phenols. This principle is the same as measurement principle 1 used for the formation of aggregates 50 of hydrophilic polymers 30 made of alginic acid by peroxidase 40, except that glucose is converted to hydrogen peroxide depending on the activity of glucose oxidase 60. Hydrogen peroxide activates peroxidase 40, and aggregates 50 of hydrophilic polymers 30 are generated. Similar to the measurement principle 1 described above, by coexisting luminescent particles 10 as probe molecules for fluorescence polarization measurement in this solution, the motility of the luminescent particles 10 will change depending on the activity of glucose oxidase 60. By obtaining a calibration curve of fluorescence polarization degree 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 degree of the solution.

[0026] (Measurement principle 3: Measurement of hydrogen peroxide concentration) This disclosure also shows that the concentration of hydrogen peroxide, an example of an enzyme reaction-related substance, can be measured by a similar method (Figure 4). For example, hydrogen peroxide, as the target substance, can rapidly form aggregates 50 of hydrophilic polymers 30 in the presence of a specified amount of peroxidase 40 and phenols. Luminescent particles 10 are kept in this solution as probe molecules for fluorescence polarization measurement. The activity of peroxidase 40 changes depending on the amount of hydrogen peroxide, the target substance. By measuring the formation of the aggregates 50 of hydrophilic polymers 30 and the change in the mobility of the luminescent particles 10 in response to this change using fluorescence polarization, the hydrogen peroxide concentration can be measured. A calibration curve of fluorescence polarization degree and hydrogen peroxide concentration can be obtained in advance using hydrogen peroxide of known concentration, and the hydrogen peroxide concentration can be determined from the measurement result of the fluorescence polarization degree of the solution.

[0027] (Measurement principle 4: Measurement of glucose concentration) This disclosure also shows that glucose concentration, an example of an enzyme reaction-related substance, can be measured by a similar method (Figure 5). For example, glucose, as the target substance, can rapidly form aggregates 50 of hydrophilic polymers 30 (gel) in the presence of a specified amount of glucose oxidase 60, peroxidase 40, and phenols. Similar to the measurement principle of glucose oxidase activity described in Measurement Principle 2, glucose concentration can be measured by utilizing the cascade reaction of glucose oxidase 60 and peroxidase 40. Luminescent particles 10 are coexisting in this solution as probe molecules for fluorescence polarization measurement. Depending on the amount of glucose, the target substance, the activity of peroxidase 40 changes, and by measuring the formation of aggregates 50 of hydrophilic polymers 30 and the change in the mobility of luminescent particles 10 using fluorescence polarization, it becomes possible to measure the glucose concentration. A calibration curve of fluorescence polarization degree and glucose concentration can be obtained in advance using glucose of a known concentration, and the glucose concentration can be determined from the measurement result of the fluorescence polarization degree of the solution.

[0028] (Measurement principle 5: Measurement of phenol concentration) This disclosure also shows that the concentration of phenols, which are an example of enzyme reaction-related substances, can be measured by a similar method. For example, phenols, which are the target substances, can rapidly form aggregates 50 of hydrophilic polymers 30 by bonding the binding functional groups of the hydrophilic polymers 30 together in the presence of a specified amount of peroxidase 40 and hydrogen peroxide. Luminescent particles 10 are kept in this solution as probe molecules for fluorescence polarization measurement. At this time, the luminescent particles 10 are incorporated into the formation of aggregates 50 of hydrophilic polymers 30. Depending on the amount of phenols, which are the target substances, the degree (e.g., size) of the formation of aggregates 50 of hydrophilic polymers 30 by peroxidase 40 changes, and by measuring the change in the motility of the luminescent particles 10 that accompanies this change using fluorescence polarization, the concentration of phenols can be measured. A calibration curve of fluorescence polarization degree and phenol concentration can be obtained in advance using phenols of known concentration, and the concentration of phenols can be determined from the measurement result of the fluorescence polarization degree of the solution.

[0029] By forming an aggregate 50 of hydrophilic polymers 30 using an enzymatic reaction in this way, it becomes possible to measure the target substance, which is the substance involved in the enzymatic reaction, with high sensitivity and speed, even when the amount of the substance involved in the enzymatic reaction of the target substance is small or when there are many impurities.

[0030] (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 that may contain the target substance is mixed with a reagent to obtain a liquid sample containing hydrogen peroxide, peroxidase, phenols, luminescent particles, and multiple hydrophilic polymers having binding functional groups. (2) A second step in which an aggregate of hydrophilic polymers containing luminescent particles is generated by the bonding of the binding functional groups of the hydrophilic polymers based on the reaction between hydrogen peroxide, peroxidase, and phenols that 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.

[0031] The following provides a more detailed explanation of each step.

[0032] (First step) In this disclosure, the first step involves mixing a sample that may contain a target substance with a reagent to obtain a liquid sample containing hydrogen peroxide, peroxidase, phenols, luminescent particles, and a plurality of hydrophilic polymers having a first binding functional group.

[0033] (Second step) In this disclosure, the second step involves forming an aggregate of hydrophilic polymers containing luminescent particles by an enzymatic reaction that occurs in the presence of a target substance in a liquid sample. For example, an aggregate of hydrophilic polymers containing luminescent particles is obtained by reacting an oxidoreductase (the target substance) with phenols, hydrogen peroxide, luminescent particles, and a hydrophilic polymer having a binding functional group. The order of the reactions is not particularly limited. That is, an aggregate of hydrophilic polymers containing luminescent particles may be obtained by simultaneously reacting the oxidoreductase (the target substance) with phenols, a hydrophilic polymer, hydrogen peroxide, and luminescent particles, or an aggregate of hydrophilic polymers may be formed by first reacting the oxidoreductase (the target substance) with phenols, a hydrophilic polymer having a binding functional group, and hydrogen peroxide, and then reacting the luminescent particles with the aggregate of hydrophilic polymers to obtain an aggregate of hydrophilic polymers containing luminescent particles. Furthermore, the aggregate of hydrophilic polymers containing luminescent particles may be a combination of luminescent particles and an aggregate of hydrophilic polymers.

[0034] When the target substance is hydrogen peroxide, which is an example of an enzyme reaction-related substance, the hydrogen peroxide, which is the target substance, is reacted with a solution containing peroxidase, phenols, a hydrophilic polymer having a binding functional group, and luminescent particles to obtain an aggregate of hydrophilic polymers containing luminescent particles. Here, the order of the reaction is not particularly limited, but since the aggregate of hydrophilic polymers is generated with hydrogen peroxide as a trigger, it is preferable to add the target substance, hydrogen peroxide, last. Furthermore, the "aggregate of hydrophilic polymers containing luminescent particles" includes both cases in which the aggregate of hydrophilic polymers and luminescent particles are bound together, such as when the binding functional group (first binding functional group) of a hydrophilic polymer in the aggregate of hydrophilic polymers is bound to the binding functional group (second binding functional group) of a luminescent particle, and cases in which luminescent particles are incorporated when the hydrophilic polymer forms an aggregate, thereby forming an aggregate of hydrophilic polymers containing luminescent particles.

[0035] If the target substance is glucose, which is an example of an enzyme reaction-related substance, the target substance glucose may be reacted with a solution containing glucose oxidase, peroxidase, phenols, a hydrophilic polymer having a binding functional group, and luminescent particles to obtain an aggregate of hydrophilic polymers containing luminescent particles. Here, the order of the reaction is not particularly limited, but since glucose acts as a trigger to generate the aggregate of hydrophilic polymers, it is preferable to add the target substance glucose last.

[0036] When the target substance is a phenol, which is an example of an enzyme reaction-related substance, a solution containing peroxidase, a hydrophilic polymer having a binding functional group, and luminescent particles is reacted with the target substance (phenol) and hydrogen peroxide to obtain an aggregate of hydrophilic polymer containing luminescent particles. Here, the order of the reaction is not particularly limited, but since the aggregate of hydrophilic polymer is generated with hydrogen peroxide as a trigger, it is preferable to add hydrogen peroxide last.

[0037] (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).

[0038] In this disclosure, the target substance is a substance related to an enzymatic reaction. Samples include, for example, bodily fluids such as blood, urine, and saliva containing the target substance, buffer solutions containing the target substance, culture media or tissue extracts 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 may also contain hydrogen peroxide. Hydrogen peroxide promotes the peroxidase reaction, but various measurements can be performed by appropriately adjusting the concentrations and mixing ratios of peroxidase, phenols, luminescent particles, etc., and the conditions for fluorescence polarization measurement.

[0039] (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.

[0040] (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, in such cases, the substance related to the enzymatic reaction of peroxidase is any substance that affects the activity of peroxidase, and may also be an inhibitor of the enzymatic reaction of peroxidase. It may be a substance that produces peroxidase through an enzymatic reaction, or a substance that causes the enzymatic reaction of peroxidase. More specifically, these 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, so the quantity and activity of the inhibitor can also be measured.

[0041] (Luminous particles) The luminescent particles used in this disclosure are nanoparticles containing a luminescent substance, and are luminescent particles whose degree of fluorescence polarization can be measured by fluorescence polarization. Furthermore, the luminescent particles according to this disclosure may be bound to or included in aggregates of hydrophilic polymers produced from hydrophilic polymers having a bonding functional group (which may be called a first bonding functional group), but it is preferable that the luminescent particles are bound to aggregates of hydrophilic polymers. When the luminescent particles are bound to aggregates of hydrophilic polymers, the mode of binding may be a chemical bond such as a covalent bond, an ionic bond, or a coordination bond, or it may be physical adsorption. When the mode of binding is a chemical bond, it is preferable that the luminescent particles have a bonding functional group (which may be called a second bonding functional group) such as a thiol group, an amino group, a carboxyl group, or a maleimide group, as will be described later. A thiol group is particularly preferable because the reaction proceeds mildly and rapidly.

[0042] The luminescent particles of this disclosure are nanoparticles, and the average particle diameter, which is the average of the particle diameters, is between 1 nm and 1000 nm, preferably between 25 nm and 500 nm, and more preferably between 50 nm and 300 nm. If the average particle diameter exceeds 500 nm, the fluorescence polarization degree of the luminescent particles themselves will be high before they interact with the hydrophilic polymer, and the change in fluorescence polarization degree after the reaction (after interaction with the hydrophilic polymer) may become small. Also, if the average particle diameter is less than 25 nm, the content of the luminescent substance in the nanoparticles will be low, resulting in a low emission intensity per particle, which may reduce the measurement sensitivity and accuracy. Therefore, luminescent particles of 50 nm or larger are preferred. The size of the luminescent particles can be determined by dynamic light scattering (DLS) measurement.

[0043] As the particle material (matrix material) for the luminescent particles, 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.

[0044] 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, it is preferable that the luminescent particles are bound to an aggregate of hydrophilic polymers. To promote the bonding between luminescent particles and the hydrophilic polymer aggregate, if the bonding functional group of the hydrophilic polymer is designated as the first bonding functional group, a second bonding functional group may be introduced on the surface of the luminescent particles as appropriate. The first and second bonding functional groups may be the same type of functional group or different types of functional groups. For example, by having a second bonding functional group such as a thiol group, amino group, carboxyl group, or maleimide group on the surface of the luminescent particles, bonding with the hydrophilic polymer aggregate having the first bonding functional group such as a thiol group, carboxyl group, or amino group can be promoted. This is because electrostatic bonding, disulfide bonding, and bonding between thiol groups and maleimide groups occur between the luminescent particles and the hydrophilic polymer aggregate. Therefore, a preferred example of a luminescent particle surface is one that is coated with a hydrophilic polymer such as polyvinylpyrrolidone and further has thiol groups introduced.

[0045] The luminescent particles used in this disclosure can be configured not to specifically bind to the target substance. That is, for an enzyme, which is an example of a target substance in this disclosure, the luminescent particles do not serve as a substrate for the enzyme. With this configuration, the luminescent particles can function independently as probes in fluorescence polarization. This independence has the advantage of making it possible to set the optimal conditions for the luminescent particles or the enzyme separately, thus simplifying the design of measurement conditions. To detect enzymes as target substances, immunoassays using antibody-conjugated luminescent particles, in which an antibody against the enzyme is bound to the luminescent particles, 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 with 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.

[0046] 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.

[0047] 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).

[0048] 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.

[0049] (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.

[0050] (Hydrophilic polymer having binding functional groups) In this disclosure, the hydrophilic polymer having a binding functional group is a molecule that serves as a raw material for generating an aggregate of hydrophilic polymers, and includes a skeletal molecule and a binding functional group. Considering that the aggregate of hydrophilic polymers is a gel-like substance, the hydrophilic polymer having a binding functional group can also be called a gel precursor. The hydrophilic polymer having a binding functional group is suitable to be a substance with a molecular weight in the range of approximately 1,000 to approximately 5,000,000, more preferably approximately 10,000 to 1,000,000. In one example of the measurement method in this disclosure, it is necessary to include luminescent particles in the generated aggregate of hydrophilic polymers and significantly reduce the mobility of the luminescent particles. On the other hand, before measuring the target substance, from the viewpoint of handling the solution, it is preferable that the molecular weight of the hydrophilic polymer having a binding functional group be small. It is also preferable that it does not interfere with the measurement of the target substance. Although a small molecular weight is preferable, it is required to have a large number of binding functional groups such as phenol groups and thiol groups necessary for the formation of the aggregate of hydrophilic polymers. It is required to be water-soluble, not interact with enzymes, and not have excessively high viscosity. Based on these requirements, alginic acid, hyaluronic acid, dextran, albumin, gelatin, and polyethylene glycol are cited as examples of hydrophilic polymer backbone molecules.

[0051] In this disclosure, HRP may be used to rapidly crosslink multiple hydrophilic polymers having bonding functional groups to form an aggregate of hydrophilic polymers. Phenol groups and thiol groups are known substrates for HRP catalytic reactions. Therefore, the hydrophilic polymers having bonding functional groups in this disclosure are water-soluble molecules having many of these bonding functional groups, and upon oxidation by HRP, the bonding functional groups of different hydrophilic polymers bond (covalently), thereby crosslinking the hydrophilic polymers and forming an aggregate of hydrophilic polymers (i.e., gelling). In this case, the bonding of bonding functional groups may be between different types of bonding functional groups, or it may be between the same type of bonding functional groups. That is, the hydrophilic polymers may have different types of bonding functional groups, and an aggregate of hydrophilic polymers may be formed by the bonding of different types of bonding functional groups, or an aggregate of hydrophilic polymers may be formed by the bonding of the same type of bonding functional groups.

[0052] Examples of hydrophilic polymers having phenolic or thiol groups as binding functional groups include sodium alginate with phenolic groups, hyaluronic acid with phenolic groups, gelatin with phenolic groups, albumin with phenolic groups, sodium alginate with thiol groups, hyaluronic acid with thiol groups, gelatin with thiol groups, and albumin with thiol groups.

[0053] One method for introducing a phenol group into a water-soluble molecule is to introduce tyramine to the carboxyl group of sodium alginate using a coupling agent. Instead of tyramine, the amino acid tyrosine may be used. Another method for introducing a thiol group into a water-soluble molecule is to introduce aminoethanethiol to the carboxyl group of sodium alginate using a coupling agent. Instead of aminoethanethiol, the amino acid cysteine ​​may be used. Alternatively, a thiol group may be generated by reducing albumin, thereby reducing intramolecular disulfide bonds.

[0054] In the case of a hydrophilic polymer into which a phenol group has been introduced, HRP catalyzes the oxidative coupling reaction of the phenol group in the presence of hydrogen peroxide, thereby allowing the hydrophilic polymer to gel (formation of an aggregate of hydrophilic polymers). In this case, it is preferable that a phenol group, which is the same functional group as the first bonding functional group, is introduced as a second bonding functional group on the surface of the luminescent particles.

[0055] The size of the hydrophilic polymer aggregate, which significantly changes the fluorescence polarization degree of the luminescent particles in response to enzyme activity, can be observed by dynamic light scattering (DLS) measurements. For example, if the average particle diameter of the luminescent particles is about 100 nm, it is preferable that the hydrophilic polymer aggregate be about 1000 nm in size. When luminescent particles bind to a hydrophilic polymer aggregate that is about 10 times larger than their average particle diameter, the volume change increases by about 1000 times, significantly reducing the mobility of the luminescent particles and making it possible to obtain a large signal change using fluorescence polarization. The increase in average particle diameter should be at least 1.5 times, preferably 5 times or more, and particularly preferably 10 times or more.

[0056] (hydrogen peroxide) This disclosure uses hydrogen peroxide for the crosslinking reaction of hydrophilic polymers having bonding functional groups with HRP. The mechanism of the gelation reaction by peroxidase used in this disclosure is that when phenols are reacted with HRP, a radical transfer reaction occurs from phenoxy radicals generated in the reaction solution to thiol groups, and the thiol radicals rapidly undergo disulfide bonding reactions with other thiol groups. Hydrogen peroxide is also generated during the disulfide bonding reaction, and it has been reported that the HRP catalytic cycle 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 bonding 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, and specifically, it is necessary to generate an aggregate of hydrophilic polymers that can significantly change the fluorescence polarization degree of the luminescent particles used as probes within a reaction time of several minutes. Under the conditions shown in the prior art (Comparative Example 2) that do not use hydrogen peroxide, a sufficient gel to change the fluorescence polarization degree of the luminescent particles could not be generated. Therefore, it is considered that the addition of hydrogen peroxide is essential for the measurement method disclosed in this disclosure to work. Note that hydrogen peroxide is a peroxide and functions as an oxidizing agent.

[0057] 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 gelation reaction and obtain a stronger signal.

[0058] (Third step) In this disclosure, the third step is to obtain a value relating to the fluorescence anisotropy of the liquid sample from the second step described above. Known methods can be used to obtain the value relating to fluorescence anisotropy in the fluorescence polarization method, and various commercially available measuring devices can be used for this purpose. The fluorescence polarization method is also called the fluorescence depolarization method, but in this specification, the fluorescence polarization method and the fluorescence depolarization method are synonymous. Here, 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) have the relationship shown in the following equation (1). P = 3r / (2+r)···(1)

[0059] The degree of fluorescence polarization can be measured using milli P (hereinafter abbreviated as mp), which indicates the change in plane polarization, and is defined by the following equation (2). Here, I H and I V These represent the intensity of the fluorescence signal polarized perpendicular to the polarization direction of the excitation light, respectively. mp={(I H -I V ) / (I H +I V )} × 1000···(2)

[0060] In this disclosure, it is preferable to measure the target substance's enzyme or enzyme reaction-related substance using fluorescence polarization, with the mobility of luminescent particles in the sample solution as an indicator. That is, depending on the enzyme activity, enzyme amount, or amount of enzyme reaction-related substance, aggregates of hydrophilic polymers are formed in the sample solution, and luminescent particles are included in these aggregates of hydrophilic polymers (luminescent particles bind to the aggregates of hydrophilic polymers or are incorporated into the aggregates of hydrophilic polymers), resulting in a decrease in the mobility of the luminescent particles. In this disclosure, the mobility of the luminescent particles is measured by fluorescence polarization. In fluorescence polarization, for example, the degree of fluorescence polarization (mp) can be used as an indicator.

[0061] This disclosure describes the use of luminescent particles as probes, which contain a europium complex exhibiting polarized emission as a luminescent material within the particles. Slight changes in the rotational motion of these luminescent particles in a liquid sample can be detected as changes in their polarized emission properties. Specifically, when the luminescent particles are immobilized in a gel with a larger size than the particles themselves, and the particle's mobility decreases significantly, the decrease in the particle's rotational Brownian motion can be detected with high sensitivity as a change in the fluorescence anisotropy value.

[0062] The timing for obtaining values ​​related to fluorescence anisotropy can be set as appropriate. For example, values ​​related to fluorescence anisotropy may be obtained after the second step. Alternatively, obtaining values ​​related to fluorescence anisotropy when the reaction is complete is a convenient and preferred method. For example, values ​​related to fluorescence anisotropy can be obtained 5 minutes after adding the target substance to the solution.

[0063] Furthermore, before reacting the target substance (enzyme or enzyme reaction-related substance) in the second step, for example, at the stage when the first liquid sample is obtained, the fluorescence polarization degree of the sample solution can be measured as a value relating to the fluorescence anisotropy, and this can be taken as the initial fluorescence polarization degree (mp(0)). Then, the reaction can proceed, and the fluorescence polarization degree (mp(t)) at a certain time point can be measured again when the luminescent particles bind to the aggregate of hydrophilic polymers that is produced, or it can be measured continuously at regular time intervals. That is, by measuring the change in fluorescence polarization degree over time, the change in fluorescence polarization degree (Δmp) can 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).

[0064] The measurement conditions for the fluorescence polarization degree 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 can be appropriately selected depending on the type of target substance, the amount of hydrogen peroxide, etc. It is preferable to measure the fluorescence polarization degree 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 degree in the range of 0.001 mg / mL to 0.1 mg / mL.

[0065] (Fourth step) In this disclosure, the fourth step is to detect a target substance in a liquid sample based on the fluorescence anisotropy value of 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 amount of enzyme reaction-related substance.

[0066] In the third step, the degree of decrease in the mobility of the luminescent particles in response to their interaction with the hydrophilic polymer aggregate is detected as a value related to the fluorescence anisotropy of the liquid sample. 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-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-related substances, the activity and amount of oxidoreductase in the liquid sample, or the amount of enzyme-related substances can be measured. For example, a large fluorescence anisotropy value obtained in the third step means that the luminescent particles are strongly bound to the hydrophilic polymer aggregate, which can be associated with high oxidoreductase activity.

[0067] Furthermore, the term "detection 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 fluorescence anisotropy value and the activity or amount of oxidoreductase, or the amount of enzyme reaction-related substances, may be either quantitative or qualitative.

[0068] Quantitative correlation 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 determine the enzyme activity of a target substance from the fluorescence anisotropy value obtained by measuring the target substance. By establishing such a correlation, enzyme activity in a liquid sample can be measured. The enzyme activity measured here can also be determined as enzyme concentration, mass, number of molecules, etc. The fluorescence anisotropy value obtained may be the change in fluorescence anisotropy value of the liquid sample before and after performing the second step.

[0069] In this disclosure, similarly, a relationship between the concentration of a hydrogen peroxide solution of known concentration or a glucose solution of known concentration and the value relating to fluorescence polarization can be determined in advance. The concentration of the target substance can be determined from the fluorescence polarization obtained by measuring the target substance.

[0070] Furthermore, in the fourth step, the change in the value related to the fluorescence anisotropy of the liquid sample can also be treated relatively. That is, enzyme activity, enzyme amount, 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 group of enzymes of the same concentration.

[0071] (Reagents for detecting target substances using measurement of fluorescence anisotropy values) According to this disclosure, the above measurement system may be provided as reagents as listed in (1) to (3) below.

[0072] (1) A reagent for use in a method for measuring peroxidase enzymatic reaction-related substances in this disclosure, comprising luminescent particles, phenols, hydrophilic polymers having binding functional groups, and hydrogen peroxide. Luminescent particles, phenols, and hydrophilic polymers having binding functional groups can be provided in solution, dry, frozen, or freeze-dried states. Hydrogen peroxide can be provided in solution. Conventional stabilizers for hydrogen peroxide, such as salicylic acid, can be included.

[0073] 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, the second solution contains phenols and hydrophilic polymers having binding functional groups, and the third solution contains hydrogen peroxide. Furthermore, the reagents of this disclosure can be separated into solution state and dry state, and the dry reagent can be used for various measurements by converting it to a solution state with an accompanying dissolving solution before measurement.

[0074] (2) Reagents for use in the method for measuring hydrogen peroxide of a target substance according to the present disclosure, comprising an oxidoreductase, luminescent particles, phenols, and a hydrophilic polymer having a binding functional group. Oxidoreductases, luminescent particles, phenols, and hydrophilic polymers having binding functional groups 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 contains oxidoreductases, the second solution contains luminescent particles, and the third solution contains phenols and hydrophilic polymers having binding functional groups.

[0075] (3) A reagent for detecting glucose, which is the target substance according to the present disclosure, comprising glucose oxidase, peroxidase, luminescent particles, phenols, and a hydrophilic polymer having a binding functional group. Glucose oxidase, peroxidase, luminescent particles, phenols, and hydrophilic polymers having binding functional groups 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, 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, and the fourth solution may contain phenols and hydrophilic polymers having binding functional groups. [Examples]

[0076] 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 methods for measuring enzyme activity, enzyme quantity, or enzyme reaction-related substance quantity according to this disclosure are examples of particles having a second binding functional group on their surface that can bind to an aggregate of hydrophilic polymers. Here, we show an example of the synthesis of luminescent particles having a thiol group as the second binding functional group on their surface.

[0077] 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.

[0078] A dispersion of luminescent particles obtained by emulsion polymerization was taken and added to an aqueous solution containing 1% by mass of Tween20 (manufactured by Kishida Chemical Co., Ltd.). After stirring for 10 minutes, the silane coupling agent X12-1135 (manufactured by Shin-Etsu Chemical Co., Ltd.) was added and stirred overnight (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.

[0079] 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. To the MES buffer containing the dispersed particles, 0.5% by mass of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide and N-hydroxysulfosuccinimide sodium were added, 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 (hereinafter referred to as luminescent particle T1). The aqueous solution containing luminescent particle T1 is called luminescent particle T1 solution. Note that thiol-modified luminescent particles can also be called luminescent particles having a second binding functional group.

[0080] 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.

[0081] (2: Preparation of thiol group-introduced sodium alginate A1) The hydrophilic polymer having a first binding functional group used in the enzyme activity measurement method according to this disclosure is a hydrophilic polymer having a first binding functional group that crosslinks with an oxidoreductase to form an aggregate of hydrophilic polymers. Here, sodium alginate having a thiol group was synthesized.

[0082] First, sodium alginate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was dissolved in deionized water to prepare a 1.0% by mass solution. 0.25 mL of this solution was added to 1.6 mL of pH 6.0 MES buffer. 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide was added to the resulting sodium alginate MES buffer to a final concentration of 0.5% by mass, and then 2-aminoethanethiol hydrochloride was added. The mixture was reacted at 25°C for 2 hours to introduce 2-aminoethanethiol into the sodium alginate. After the reaction, the unreacted 2-aminoethanethiol was removed by dialysis with water. Finally, a 1.0% by mass solution of thiol-introduced sodium alginate was obtained. Hereafter, thiol-introduced sodium alginate will be referred to as thiol-containing sodium alginate A1. An aqueous solution of thiol-containing sodium alginate A1 will be referred to as thiol-containing sodium alginate A1 solution. In this embodiment, both the first and second bonding functional groups are thiol groups.

[0083] (3: Preparation of phenol solutions) Tyramine hydrochloride was dissolved in phosphate buffer (containing 0.01% by mass of Tween 20) to prepare a phosphate buffer solution of tyramine hydrochloride with a final concentration of 40 mM.

[0084] (4: Preparation of hydrogen peroxide solution) A 3.5% by mass hydrogen peroxide aqueous solution was prepared by diluting 35% by mass hydrogen peroxide solution with water.

[0085] (5: 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).

[0086] 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.

[0087] 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 (second binding functional groups 20 on the luminescent particles) are added with 2-aminoethanethiol hydrochloride. The sodium alginate (hydrophilic polymer 30 having a first binding functional group) to which thiol groups have been added with 2-aminoethanethiol hydrochloride forms a disulfide-crosslinked sodium alginate gel (aggregate of hydrophilic polymers 50) with HRP (peroxidase 40), tyramine hydrochloride (phenols), and hydrogen peroxide. The aggregation of the hydrophilic polymers and the luminescent particles binds via thiol groups, changing the mobility of the luminescent particles, which is detected as a change in fluorescence polarization degree. Furthermore, in Example 6 described later, a measurement example using glucose oxidase (glucose oxidase 60) as the source of hydrogen peroxide is shown.

[0088] (Example 1: Fluorescence polarization measurement of a sample containing peroxidase) Water, luminescent particle T1 solution, tyramine hydrochloride, and hydrophilic polymer A1 having a thiol group 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, 3.5% by mass of hydrogen peroxide aqueous solution was added to the solution in the wells and thoroughly mixed, and then the microwell plate was 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

[0089] (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.

[0090] (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% by mass aqueous solution of hydrogen peroxide was not added. The degree of fluorescence polarization (mp) was measured in the same manner as in Example 1.

[0091] (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 phenols (tyramine hydrochloride) were not added. The degree of fluorescence polarization (mp) was measured in the same manner as in Example 1.

[0092] 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 62.9, but in the sample containing HRP (Example 1), the fluorescence polarization degree increased to 97.6. This means that the mobility of the luminescent particles in the sample solution was reduced. In other words, it is thought that aggregates of hydrophilic polymers were formed in the sample solution, and the luminescent particles bound to these aggregates, resulting in a significant decrease in the mobility of the luminescent particles.

[0093] When hydrogen peroxide was not added (Comparative Example 2), the fluorescence polarization degree was low at 63.8. From this example, it was found that the addition of hydrogen peroxide is necessary for the rapid measurement of peroxidase using fluorescence polarization. When phenols (tyramine hydrochloride) were not added (Comparative Example 3), the fluorescence polarization degree was low at 63.3. From this example, it was found that the addition of phenols (tyramine hydrochloride) is necessary for the rapid measurement of peroxidase using fluorescence polarization.

[0094] [Table 1]

[0095] (Example 2: Fluorescence polarization measurement of peroxidase) Water, luminescent particle T1 solution, tyramine hydrochloride, and sodium alginate A1 containing thiol groups were mixed in the proportions shown in Table 2. Target substances (HRP) of different concentrations (or activity levels) were added to this aqueous solution and thoroughly mixed. These solutions were transferred to a 96-well microplate. Next, 3.5% by mass aqueous hydrogen peroxide solution was added to the solutions in the wells and thoroughly mixed. The fluorescence polarization degree (mp) was then measured in the same manner as in Example 1, except that the fluorescence polarization degree was measured after 10 minutes.

[0096] The results of Example 2 are shown in Table 2. The degree of fluorescence polarization increased with increasing concentration or activity of HRP. It is thought that HRP generates aggregates of sodium alginate (a cross-linked gel structure), and the luminescent particles bind to these aggregates, significantly reducing the mobility of the luminescent particles. Using the relationship between the amount (concentration) or activity of peroxidase and the degree of fluorescence polarization (calibration curve) obtained from this example, it was found that it is possible to determine the concentration or activity of peroxidase of unknown concentration or activity by measuring its degree of fluorescence polarization.

[0097] [Table 2]

[0098] (Example 3: Fluorescence polarization measurement of peroxidase activity) Water, luminescent particle T1 solution, tyramine hydrochloride, and sodium alginate A1 containing thiol groups were mixed in the proportions shown in Table 3. Peroxidase (HRP) of the same concentration and 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 aqueous solution, the target substance and enzyme activity inhibitor. These solutions were transferred to 96-well microplates. Next, 3.5% by mass of 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.

[0099] 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.

[0100] [Table 3]

[0101] (Example 4: Measurement of hydrogen peroxide concentration by fluorescence polarization) Water, luminescent particle T1 solution, tyramine hydrochloride, HRP, and sodium alginate A1 containing thiol groups 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.

[0102] The results of Example 4 are shown in Table 4. The degree of fluorescence polarization increased with increasing hydrogen peroxide concentration. Hydrogen peroxide is thought to catalyze gel formation, and it is believed 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.

[0103] [Table 4]

[0104] (Example 5: Fluorescence polarization measurement of glucose oxidase) Water, luminescent particle T1 solution, tyramine hydrochloride, HRP, sodium alginate A1 containing thiol groups, 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 10 minutes.

[0105] The results of Example 5 are shown in Table 5. The fluorescence polarization degree increased with increasing GOX concentration. The target substance, GOX, produces hydrogen peroxide from glucose in the solution, and this hydrogen peroxide triggers gel formation. It is thought that the fluorescence polarization degree increased when luminescent particles bind to this gel. Using the relationship between GOX concentration and fluorescence polarization degree (calibration curve) obtained from this example, it was found that the concentration of GOX of unknown concentration can be determined by measuring its fluorescence polarization degree.

[0106] [Table 5]

[0107] (Example 6: Fluorescence polarization measurement of glucose) MES buffer (pH 5.0), luminescent particle T1 solution, tyramine hydrochloride, HRP, sodium alginate A1 with thiol groups, 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 1 minute.

[0108] The results of Example 6 are shown in Table 6. The fluorescence polarization degree increased with increasing glucose concentration. Hydrogen peroxide is produced from the target substance glucose by GOX, and this hydrogen peroxide triggers gel formation. It is thought that the fluorescence polarization degree increased when luminescent particles bind to this gel. Using the relationship between glucose concentration and fluorescence polarization degree (calibration curve) obtained from this example, it was found that it is possible to determine the concentration of glucose of unknown concentration by measuring its fluorescence polarization degree.

[0109] [Table 6]

[0110] This embodiment includes the following methods and configurations. (Method 1) A method for detecting target substances related to enzymatic reactions, A first step involves mixing a sample that may contain the target substance with a reagent to obtain a liquid sample containing hydrogen peroxide, peroxidase, phenols, luminescent particles, and a plurality of hydrophilic polymers having binding functional groups. A second step of generating an aggregate of the hydrophilic polymer containing the luminescent particles by bonding the binding functional groups of the hydrophilic polymer based on a reaction between the hydrogen peroxide, the peroxidase, and the phenols that 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 having [specific characteristics]. (Method 2) A method for detecting a target substance according to Method 1, wherein the binding functional group having the hydrophilic polymer is a first binding functional group, and the bonding of the binding functional groups is bonding between the first binding functional groups. (Method 3) The method for detecting a target substance according to method 2, wherein the luminescent particle has a second bonding functional group, and the first bonding functional group and the second bonding functional group are bonded based on the reaction. (Method 4) The method for detecting a target substance according to Method 3, wherein the first binding functional group and the second binding functional group are functional groups 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 4, wherein the first binding functional group and the second binding functional group are thiol groups. (Method 6) A method for detecting a target substance according to any one of methods 1 to 5, wherein the luminescent particles are particles containing a rare earth luminescent complex. (Method 7) A method for detecting a target substance according to any one of methods 1 to 6, comprising a fourth step of detecting the target substance based on the value relating to fluorescence anisotropy. (Method 8) The method for detecting a target substance according to Method 7, wherein the fourth step is to detect the target substance based on the change in the value of the fluorescence polarization degree of the liquid sample before and after performing the second step. (Method 9) A method for detecting a target substance according to any one of methods 1 to 8, wherein the hydrophilic polymer having the binding functional group is selected from the group consisting of alginic acid, hyaluronic acid, gelatin, and polyethylene glycol. (Method 10) A method for detecting a target substance according to any one of methods 3 to 9, wherein the aggregate of the hydrophilic polymer containing the luminescent particles is a combination of the aggregate of the hydrophilic polymer and the luminescent particles based on the bonding of the first binding functional group and the second binding functional group. (Method 11) A method for detecting a target substance according to any one of methods 1 to 10, wherein the phenols are compounds selected from the group consisting of phenol, tyramine, tyramine hydrochloride, glycyl-L-tyrosine, resorcinol, and serotonin. (Method 12) A method for detecting a target substance according to any one of methods 1 to 11, wherein the target substance is a peroxidase. (Method 13) A method for detecting a target substance according to any one of methods 1 to 11, 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 14) A method for detecting a target substance according to any one of methods 1 to 11, wherein the target substance is hydrogen peroxide. (Method 15) A method for detecting a target substance according to any one of methods 1 to 11, 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 16) A method for detecting a target substance according to any one of methods 1 to 11, wherein the target substance is a phenol. (Method 17) A method for detecting a target substance according to any one of methods 1 to 11, wherein the target substance is an inhibitor of the enzymatic reaction of the peroxidase. (Composition 18) 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 at least hydrogen peroxide, phenols, luminescent particles, and a hydrophilic polymer having a binding functional group. (Composition 19) 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 at least a substrate for the oxidase, a peroxidase, phenols, luminescent particles, and a hydrophilic polymer having a binding functional group. (Composition 20) 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 at least a peroxidase, phenols, luminescent particles, and a hydrophilic polymer having a binding functional group. (Composition 21) 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 at least the oxidase, peroxidase, phenols, luminescent particles, and a hydrophilic polymer having a binding functional group. (Composition 22) 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 at least a peroxidase, hydrogen peroxide, luminescent particles, and a hydrophilic polymer having a binding functional group. (Composition 23) 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, luminescent particles, and a hydrophilic polymer having a binding functional group. (Composition 24) The reagent according to any one of configurations 18 to 23, 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 25) The reagent according to configuration 24, wherein the aforementioned binding functional group is a thiol group. (Composition 26) The reagent according to any one of configurations 18 to 25, wherein the luminescent particles are particles containing a rare earth luminescent complex. [Explanation of symbols]

[0111] 10 Luminescent particles 20 Second bonding functional group possessed by the luminescent particle 30 Hydrophilic polymer having a first bonding functional group 40 Peroxidase 50 Hydrophilic polymer aggregates 60 Glucose oxidase

Claims

1. A method for detecting target substances related to enzymatic reactions, A first step involves mixing a sample that may contain the target substance with a reagent to obtain a liquid sample containing hydrogen peroxide, peroxidase, phenols, luminescent particles, and a plurality of hydrophilic polymers having binding functional groups. A second step is to generate an aggregate of the hydrophilic polymer containing the luminescent particles by bonding the binding functional groups of the hydrophilic polymer based on a reaction between the hydrogen peroxide, the peroxidase, and the phenols that occurs when the target substance is present 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 binding functional group having the hydrophilic polymer is a first binding functional group, and the bonding of the binding functional groups is bonding between the first binding functional groups.

3. The method for detecting a target substance according to claim 2, wherein the luminescent particle has a second bonding functional group, and the first bonding functional group and the second bonding functional group bond based on the reaction.

4. The method for detecting a target substance according to claim 3, wherein the first binding functional group and the second binding functional group are functional groups 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 4, wherein the first binding functional group and the second binding functional group are thiol groups.

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

7. 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.

8. The method for detecting a target substance according to claim 7, wherein the fourth step is to detect the target substance based on the change in the value relating to the fluorescence anisotropy of the liquid sample before and after performing the second step.

9. The method for detecting a target substance according to claim 1, wherein the hydrophilic polymer having the binding functional group is selected from the group consisting of alginic acid, hyaluronic acid, gelatin, and polyethylene glycol.

10. The method for detecting a target substance according to claim 3, wherein the aggregate of hydrophilic polymers containing the luminescent particles is a combination of the aggregate of hydrophilic polymers and the luminescent particles based on the bonding of the first binding functional group and the second binding functional group.

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

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

13. A method for detecting a target substance according to any one of claims 1 to 11, wherein the target substance is an oxidase, the liquid sample contains a substrate for the oxidase, and the hydrogen peroxide contains one produced by the oxidase.

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

15. A method for detecting a target substance according to any one of claims 1 to 11, wherein the target substance is a substrate for oxidase, the liquid sample contains oxidase, and the hydrogen peroxide contains hydrogen peroxide produced by oxidase.

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

17. A method for detecting a target substance according to any one of claims 1 to 11, wherein the target substance is an inhibitor of the enzymatic reaction of the peroxidase.

18. 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 at least hydrogen peroxide, phenols, luminescent particles, and a hydrophilic polymer having a binding functional group.

19. 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 at least a substrate for the oxidase, a peroxidase, phenols, luminescent particles, and a hydrophilic polymer having a binding functional group.

20. 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 at least a peroxidase, phenols, luminescent particles, and a hydrophilic polymer having a binding functional group.

21. 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 at least the oxidase, peroxidase, phenols, luminescent particles, and a hydrophilic polymer having a binding functional group.

22. 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 at least a peroxidase, hydrogen peroxide, luminescent particles, and a hydrophilic polymer having a binding functional group.

23. 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, luminescent particles, and a hydrophilic polymer having a binding functional group.

24. The reagent according to any one of claims 18 to 23, 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.

25. The reagent according to claim 24, wherein the bonding functional group is a thiol group.

26. The reagent according to any one of claims 18 to 23, wherein the luminescent particles are particles containing a rare earth luminescent complex.

Images