EP-4737585-A1 - METHOD OF DETECTING TARGET SUBSTANCE AND REAGENT FOR INSPECTION OF TARGET SUBSTANCE

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 a substance related to an enzyme reaction, the method including: mixing a specimen liquid that may contain the target substance and a reagent to obtain a liquid sample containing hydrogen peroxide, a peroxidase (40), a phenol derivative and a plurality of luminescent particles (10) each having a binding functional group; aggregating the plurality of luminescent particles (10) through binding of the binding functional groups (20) of the luminescent particles (10) based on a reaction of the hydrogen peroxide, the peroxidase (40) and the phenol derivative in the liquid sample, which occurs when the target substance is present in the liquid sample; and obtaining a value related to fluorescence anisotropy of the liquid sample.

Inventors

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

Assignees

• Canon Kabushiki Kaisha

Dates

Publication Date

20260506

Application Date

20251103

Claims (14)

1. 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 liquid that may comprise the target substance and a reagent to obtain a liquid sample comprising hydrogen peroxide, a peroxidase (40), a phenol derivative and a plurality of luminescent particles (10) each having a binding functional group; a second step of aggregating the plurality of luminescent particles (10) through binding of the binding functional groups (20) of the luminescent particles (10) based on a reaction of the hydrogen peroxide, the peroxidase (40) and the phenol derivative in the liquid sample, 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.
2. The method of detecting a target substance according to claim 1, wherein the luminescent particles are nanoparticles each comprising a rare earth luminescent complex.
3. The method of detecting a target substance according to claim 1 or 2, wherein the binding functional group is selected from the group consisting of: a thiol group; a carboxy group; an amino group; and a maleimide group.
4. The method of detecting a target substance according to any one of claims 1 to 3, wherein the phenol derivative comprises any one selected from the group consisting of: tyramine; tyramine hydrochloride; phenol; glycyl-L-tyrosine; resorcinol; and serotonin.
5. The method of detecting a target substance according to any one of claims 1 to 4, wherein the target substance is a peroxidase.
6. The method of detecting a target substance according to any one of claims 1 to 4, wherein the target substance is an oxidase, wherein the liquid sample comprises a substrate for the oxidase, and wherein the hydrogen peroxide comprises a product generated by the oxidase.
7. The method of detecting a target substance according to any one of claims 1 to 4, wherein the target substance is hydrogen peroxide.
8. The method of detecting a target substance according to any one of claims 1 to 4, wherein the target substance is a substrate for an oxidase, wherein the liquid sample comprises the oxidase, and wherein the hydrogen peroxide comprises a product generated by the oxidase.
9. The method of detecting a target substance according to any one of claims 1 to 4, wherein the target substance is a phenol derivative.
10. The method of detecting a target substance according to any one of claims 1 to 9, wherein the specimen liquid comprises hydrogen peroxide.
11. 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 and a plurality of luminescent particles (10) each having a binding functional group; wherein the target substance is a hydrogen peroxide, and the reagent comprises a peroxidase (40), a phenol derivative and a plurality of luminescent particles (10) each having a binding functional group; or wherein the target substance is a phenol derivative, and the reagent comprises a peroxidase (40), hydrogen peroxide and a plurality of luminescent particles (10) each having a binding functional group.
12. 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 and a plurality of luminescent particles (10) each 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 and a plurality of luminescent particles (10) each having a binding functional group.
13. The reagent for inspection of a target substance according to claim 11 or 12, wherein the binding functional group is a thiol group.
14. The reagent for inspection of a target substance according to claim 11 or 12, wherein the luminescent particles are particles each comprising a rare earth luminescent complex.

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. 2024/0093087, there is a disclosure of an immunoassay technology using fluorescence polarization measurement. In U.S. Patent Application Publication No. 2024/0093087, a high-sensitivity immunoassay is constructed by using luminescent particles to aggregate the luminescent particles via an antigen-antibody reaction, and by observing a change in degree of fluorescence polarization before and after the aggregation. However, it has not been possible to measure a substance related to an enzyme reaction of a peroxidase in a liquid sample in a simple manner and with high sensitivity. SUMMARY As a result of intensive investigations to solve the above-mentioned problems, the inventors have found that fluorescence polarization measurement of the activity or amount of an enzyme or the amount of an enzyme reaction-related substance can be performed in a short period of time and with high sensitivity by introducing a mechanism that aggregates a plurality of luminescent particles by the action of an oxidoreductase