EP-4737586-A1 - METHOD OF DETECTING TARGET SUBSTANCE AND REAGENT FOR INSPECTION OF TARGET SUBSTANCE

EP4737586A1EP 4737586 A1EP4737586 A1EP 4737586A1EP-4737586-A1

Abstract

Provided are a highly sensitive detection method for a target substance and a reagent for inspection thereof. Specifically, provided is a method of detecting a target substance that is an enzyme reaction-related substance, the method including: a first step of mixing a specimen and a reagent to obtain a liquid sample containing hydrogen peroxide, a peroxidase (40), a phenol derivative, a luminescent particle (10) and hydrophilic polymers (30) each having a binding functional group; a second step of generating an aggregate (50) of the hydrophilic polymers (30) including the luminescent particle (10) through the binding functional groups of the hydrophilic polymers (30) based on a reaction of the hydrogen peroxide, the peroxidase (40) and the phenol derivative, which occurs when the target substance is present in the liquid sample; and a third step of obtaining a value related to fluorescence anisotropy of the liquid sample in the second step.

Inventors

YAMAUCHI, FUMIO
KAKEGAWA, NORISHIGE
KITAGAWA, KENJI
NOMOTO, TSUYOSHI

Assignees

Canon Kabushiki Kaisha

Dates

Publication Date: 20260506
Application Date: 20251103

Claims (14)

A method of detecting a target substance that is a substance related to an enzyme reaction, the method comprising: a first step of mixing a specimen that may comprise the target substance and a reagent to obtain a liquid sample comprising hydrogen peroxide, a peroxidase (40), a phenol derivative, a luminescent particle (10) and a plurality of hydrophilic polymers (30) each having a binding functional group; a second step of generating an aggregate (50) of the hydrophilic polymers (30) comprising the luminescent particle (10) through binding of the binding functional groups of the hydrophilic polymers (30) based on a reaction of the hydrogen peroxide, the peroxidase (40) and the phenol derivative, which occurs when the target substance is present in the liquid sample; and a third step of obtaining a value related to fluorescence anisotropy of the liquid sample in the step of generating the aggregate (50) of the hydrophilic polymers (30) comprising the luminescent particle (10).
The method of detecting a target substance according to claim 1, wherein the binding functional group of each of the hydrophilic polymers is a first binding functional group, and the binding of the binding functional groups is binding of the first binding functional groups to each other.
The method of detecting a target substance according to claim 2, wherein the luminescent particle has a second binding functional group, and the first binding functional group and the second binding functional group bind to each other based on the reaction.
The method of detecting a target substance according to claim 3, wherein the first binding functional group and the second binding functional group are each selected from the group consisting of: a thiol group; a carboxy group; an amino group; and a maleimide group.
The method of detecting a target substance according to claim 4, wherein the first binding functional group and the second binding functional group are each a thiol group.
The method of detecting a target substance according to any one of claims 1 to 5, further comprising a fourth step of detecting the target substance based on the value related to fluorescence anisotropy.
The method of detecting a target substance according to any one of claims 1 to 6, wherein the hydrophilic polymers each having the binding functional group are each selected from the group consisting of: alginic acid; hyaluronic acid; gelatin; and polyethylene glycol.
The method of detecting a target substance according to claim 3, wherein the aggregate of the hydrophilic polymers comprising the luminescent particle is a conjugate of the aggregate of the hydrophilic polymers and the luminescent particle based on the binding of the first binding functional group and the second binding functional group.
The method of detecting a target substance according to any one of claims 1 to 8, wherein the phenol derivative is selected from the group consisting of: phenol; tyramine; tyramine hydrochloride; glycyl-L-tyrosine; resorcinol; and serotonin.
The method of detecting a target substance according to any one of claims 1 to 9, wherein the target substance is any one of a peroxidase, hydrogen peroxide and a phenol derivative.
The method of detecting a target substance according to any one of claims 1 to 10, wherein either the target substance is an oxidase and the liquid sample comprises a substrate for the oxidase, or the target substance is a substrate for an oxidase and the liquid sample comprises the oxidase, and wherein the hydrogen peroxide comprises a product generated by the oxidase and the substrate for the oxidase.
A reagent for inspection of a target substance using measurement of a value related to fluorescence anisotropy, wherein the target substance is a peroxidase (40), and the reagent comprises hydrogen peroxide, a phenol derivative, a luminescent particle (10) and a hydrophilic polymer (30) having a binding functional group; wherein the target substance is hydrogen peroxide, and the reagent comprises a peroxidase (40), a phenol derivative, a luminescent particle (10) and a hydrophilic polymer (30) having a binding functional group; or wherein the target substance is a phenol derivative, and the reagent comprises a peroxidase (40), hydrogen peroxide, a luminescent particle (10) and a hydrophilic polymer (30) having a binding functional group.
The reagent for inspection of a target substance according to claim 12, wherein the hydrogen peroxide comprises a product generated by the oxidase and the substrate for the oxidase.
A reagent for inspection of a target substance using measurement of a value related to fluorescence anisotropy, wherein the target substance is an oxidase, and the reagent comprises a substrate for the oxidase, a peroxidase (40), hydrogen peroxide, a phenol derivative, a luminescent particle (10) and a hydrophilic polymer (30) having a binding functional group; or wherein the target substance is a substrate for an oxidase, and the reagent comprises the oxidase, a peroxidase (40), hydrogen peroxide, a phenol derivative, a luminescent particle (10) and a hydrophilic polymer (30) having a binding functional group.

Description

TECHNICAL FIELD The present disclosure relates to a method of detecting a target substance and a reagent for inspection of a target substance. BACKGROUND Enzymes have a role of catalyzing chemical reactions in a living body, and measurement of enzyme activity is extremely important in the fields of not only medical research of enzymes but also inspection of enzymes, clinical inspections utilizing enzymes as reagents and substance production utilizing enzymes. Hitherto, enzyme activity has been measured by various methods. In general, a method using a natural or synthetic substrate for an individual enzyme is performed. In the method using a synthetic substrate, the enzyme activity can be measured by utilizing the enzyme activity and optically detecting and measuring a dye that is liberated from the synthetic substrate by the action of the enzyme. Alternatively, for example, a substrate is used in which luminescence is weak before the action of the enzyme, but the luminescence intensity increases after the action. However, when the enzyme concentration is extremely low, the amount of a fluorescent dye liberated from the synthetic substrate is also small, and hence the luminescence from the fluorescent dye weakens. Thus, there has been a problem in that the measurement of the enzyme activity becomes difficult, or measurement accuracy decreases. In addition, the fluorescent dye is liable to be affected by the surrounding environment, and for example, in a liquid sample containing a large amount of contaminants such as blood, the dye adsorbs to contaminants, such as proteins and lipids, and the fluorescent properties of the substrate change. As a result, high-sensitivity measurement becomes difficult in some cases. Further, in order to eliminate the influence of contaminants, it is possible to separate the enzyme that is a target substance or the contaminants, but the separation work is complicated, and the measurement time may lengthen. Fluorescence polarization measurement is known as one of the methods of measuring enzyme activity. The fluorescence polarization measurement, which utilizes fluorescence polarization, is a method of measuring the rotational motion of a fluorescent substance. When the fluorescent substance is excited, the polarization of its fluorescence (also referred to as "degree of fluorescence polarization") changes in accordance with the rotational motion of the fluorescent substance. That is, the method utilizes the fact that when the fluorescent substance does not rotate, polarized luminescence is observed, whereas when the fluorescent substance freely rotates, fluorescence is radiated in all planes, and the polarization is eliminated. A feature of the fluorescence polarization measurement is that in the assay, the degree of fluorescence polarization is used as an indicator, not the luminescence intensity of the fluorescence. Another feature is that the method does not require a separation or washing operation. That is, a homogeneous assay is possible, and a specimen can be added to a solution and measured as it is. Thus, complicated separation work is unnecessary, and measurement can be performed in a short period of time. Methods of measuring enzyme activity by the fluorescence polarization measurement have heretofore been disclosed. In Japanese Patent Laid-Open No. H10-099097, there is a disclosure of the measurement of the activity of a protease, which is a protein-degrading enzyme. The measurement utilizes the fact that when a substrate molecule having a luminescent substance is cleaved by the protease, the size of the luminescent substance decreases, and the mobility of the luminescent substance changes. In U.S. Patent Application Publication No. 2008/0145880, there is a disclosure of the measurement of the activity of an enzyme that catalyzes a phosphoric acid modification, including a kinase, a phosphatase, a cyclase and a phosphodiesterase. In U.S. Patent Application Publication No. 2017/0239356, there is a disclosure of a technology of producing a hydrogel with horseradish peroxidase (HRP), which is an oxidoreductase. U.S. Patent Application Publication No. 2017/0239356 is directed to encapsulate living cells in a hydrogel under mild conditions using HRP for medical applications. However, it has been difficult to measure a substance related to an enzyme reaction of a peroxidase in a simple manner and with high sensitivity. SUMMARY The present disclosure in its first aspect provides a method of detecting a target substance that is a substance related to an enzyme reaction, the method including: a first step of mixing a specimen that may contain the target substance and a reagent to obtain a liquid sample containing hydrogen peroxide, a peroxidase, a phenol derivative, a luminescent particle and a plurality of hydrophilic polymers each having a binding functional group; a second step of generating an aggregate of the hydrophilic polymers including the luminescent particle through