Method for detecting enzymatic reaction product
| 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 |
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| 発明の名称 （英語） |
Method for detecting enzymatic reaction product
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| 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. |
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