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Method for detecting enzymatic reaction product

Foreign code F210010334
File No. IP14P005
Posted date Feb 2, 2021
Country United States of America
Application number 201916976399
Gazette No. 20200407769
Date of filing Mar 4, 2019
Gazette Date Dec 31, 2020
International application number JP2019008411
International publication number WO2019168200
Date of international filing Mar 4, 2019
Date of international publication Sep 6, 2019
Priority data
  • P2018-037085 (Mar 2, 2018) JP
Title Method for detecting enzymatic reaction product
Abstract 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).
Outline of related art and contending technology 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.
Scope of claims [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.
  • Inventor, and Inventor/Applicant
  • TABATA KAZUHITO
  • MINAGAWA YOSHIHIRO
  • NOJI HIROYUKI
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
IPC(International Patent Classification)
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