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Method for detecting enzymatic reaction product NEW

外国特許コード F210010334
整理番号 IP14P005
掲載日 2021年2月2日
出願国 アメリカ合衆国
出願番号 201916976399
公報番号 20200407769
出願日 平成31年3月4日(2019.3.4)
公報発行日 令和2年12月31日(2020.12.31)
国際出願番号 JP2019008411
国際公開番号 WO2019168200
国際出願日 平成31年3月4日(2019.3.4)
国際公開日 令和元年9月6日(2019.9.6)
優先権データ
  • 特願2018-037085 (2018.3.2) JP
発明の名称 (英語) Method for detecting enzymatic reaction product NEW
発明の概要(英語) Provided is a technique that can be used to detect a pathogenic microorganism such as influenza virus with high sensitivity, and specifically, a method for detecting a reaction product, including reacting an enzyme with a substance serving as a substrate for a reaction involving the enzyme in a hydrophilic solvent that is in interfacial contact with a hydrophobic solvent, wherein the reaction product is produced directly and released from the substrate, and wherein the hydrophilic solvent contains at least one of a buffering substance or trimethylamine oxide (TMAO).
従来技術、競合技術の概要(英語) BACKGROUND ART
In recent years, a simple influenza virus test kit using immunochromatography has been developed (see Patent Literature 1). Since a method using immunochromatography can detect influenza virus for several minutes to several tens of minutes, the method is utilized for diagnosis, treatment or the like of infection.
Heretofore, a technique of optically detecting influenza virus based on a reaction between neuraminidase as an enzyme contained in influenza virus and a chromogenic substrate has been known (see Patent Literatures 2 and 3). Examples of the chromogenic substrate to be used include 4-methylumbelliferyl-N-acetyl-α-D-neuraminic acid (4MU-NANA, see Patent Literature 2) and a derivative of 4-alkoxy-N-acetylneuraminic acid or 4,7-dialkoxy-N-acetylneuraminic acid (see Patent Document 3). For example, in the method using 4MU-NANA as the chromogenic substrate, 4-methylumbelliferone, which is a fluorescent substance, is produced by the decomposition of 4MU-NANA by neuraminidase. An enzymatic activity value of neuraminidase can be calculated based on the amount of 4-methylumbelliferone produced, and the number of particles of influenza virus can also be quantified based on the enzymatic activity value.
In the context of the present invention, Non-Patent Literature 1 discloses a method for carrying out a single-molecule enzyme assay by use of a femtoliter droplet array accessible directly from the outside, wherein the droplet is covered with oil.
特許請求の範囲(英語) [claim1]
1. A method for detecting a reaction product, comprising reacting an enzyme with a substance serving as a substrate for a reaction involving the enzyme in a hydrophilic solvent that is in interfacial contact with a hydrophobic solvent, wherein the reaction product is produced directly and released from the substrate, and wherein the hydrophilic solvent contains at least one of a buffering substance or trimethylamine oxide (TMAO).

[claim2]
2. The method according to claim 1, wherein the concentrations of the buffering substance and trimethylamine oxide in the hydrophilic solvent are 1 M or more.

[claim3]
3. The method according to claim 1, wherein the buffering substance is DEA (diethanolamine) or HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid).

[claim4]
4. The method according to claim 1, wherein the hydrophilic solvent has a pH value greater than an acid dissociation constant (pKa) of the reaction product.

[claim5]
5. The method according to claim 1, wherein:
the hydrophilic solvent contains a pathogenic microorganism;
the enzyme is an enzyme present on a surface of the pathogenic microorganism or in the pathogenic microorganism and having substrate cleavage activity;
the substance is a chromogenic substrate; and
the reaction product produced by cleavage of the chromogenic substrate involving the enzyme is optically detected.

[claim6]
6. The method according to claim 5,
wherein:
the pathogenic microorganism is influenza virus;
the enzyme is neuraminidase; and
the chromogenic substrate and the reaction product are 4-methylumbelliferyl-N-acetyl-α-D-neuraminic acid and 4-methylumbelliferone, respectively, or a derivative containing fluorescein and fluorescein, respectively.

[claim7]
7. The method according to claim 5, wherein the hydrophilic solvent contains a biological sample separated from a subject infected with the pathogenic microorganism or a subject suspected to be infected with the pathogenic microorganism.

[claim8]
8. A method for detecting a reaction product, comprising reacting an enzyme with a substance serving as a substrate for a reaction involving the enzyme in a hydrophilic solvent that is in interfacial contact with a hydrophobic solvent, wherein the reaction product is produced directly and released from the substrate, the method comprising:
a step of adding at least one of a buffering substance or trimethylamine oxide (TMAO) to the hydrophilic solvent to suppress migration of the reaction product to the hydrophobic solvent from the hydrophilic solvent.

[claim9]
9. The method according to claim 2, wherein the buffering substance is DEA (diethanolamine) or HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid).

[claim10]
10. The method according to claim 6, wherein the hydrophilic solvent contains a biological sample separated from a subject infected with the pathogenic microorganism or a subject suspected to be infected with the pathogenic microorganism.
  • 発明者/出願人(英語)
  • TABATA KAZUHITO
  • MINAGAWA YOSHIHIRO
  • NOJI HIROYUKI
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
国際特許分類(IPC)
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