Method for detecting target molecule

Patent metadata

Field	Value
Publication number	US-12509737-B2
Application number	US-202117531983-A
Country	US
Kind code	B2
Filing date	Nov 22, 2021
Priority date	May 21, 2019
Publication date	Dec 30, 2025
Grant date	Dec 30, 2025

Abstract

A method for detecting a target molecule of a structure body, including contacting a dispersion liquid including a structure body with a well array including wells such that the structure body is introduced into the wells, bringing a sealing solution into contact with the well array such that the structure body is sealed in at least one of the wells, extracting a content of the structure body in the well, detecting a surface target molecule present on a surface of the structure body in the well, and detecting an internal target molecule present inside the structure body in the well.

First claim

What is claimed is:

1 . A method for detecting a target molecule of a structure body, the method comprising:
contacting a dispersion liquid comprising a structure body, reagents for detecting a surface target molecule and an internal target molecule of the structure body, and optionally other reagents, with a fluid device comprising a substrate containing a well array including a plurality of wells such that the structure body and the dispersion liquid are introduced into the wells; wherein in the contacting of the dispersion liquid with the well array one or less structure body per well is introduced; wherein the fluid device further comprises a cover member on top the substrate and a flow path between the substrate and the cover member, wherein the cover member comprises an inlet port on one end of the flow pass of the fluid device and an outlet port on the other end, wherein the contacting comprises introducing the dispersion liquid to the flow path between the substrate having the well array on it and the cover member through the inlet port such that the dispersion liquid flows downward from the inlet through the flow path over the surface of the well array, covers the surface of the well array, and enters each well, binding a capture substance, capable of capturing the structure body, to the structure body, comprising bringing the structure body into contact with the capture substance before introducing the structure body into the wells, or adding the capture substance into the dispersing liquid containing the structure body, or introducing the capture substance into each well and then introducing the dispersing liquid containing the structure body such that the capture substance and the structure body are brought into contact with each other in the wells and form a complex, bringing a sealing solution into contact with the well array such that the structure body is sealed in at least one of the wells; wherein the sealing solution is introduced through the inlet port, replaces the dispersion liquid that is not accommodated in the wells from the dispersion liquid supplied to the flow path, and expels the dispersion liquid through the outlet port, and wherein the sealing solution individually seals each of the wells which accommodate the dispersion liquid containing the structure body, extracting a content of the structure body in the wells; detecting the presence of the surface target molecule present on a surface of the structure body in the wells, and detecting the presence of the internal target molecule inside the structure body in the wells, wherein the detecting the surface target molecule and the detecting the internal target molecule are conducted under same conditions in the same well, wherein the surface target molecule and the internal target molecule emit different signals, and the detecting comprises amplification of the signals by an invasive cleavage assay (ICA) reaction, and wherein the other reagents in the dispersion liquid comprise reagents for the ICA reaction, and wherein the surface target molecule is a protein, and the internal target molecule is a nucleic acid.

2 . The method according to claim 1 , wherein the extracting, the detecting of the surface target molecule, and the detecting of the internal target molecule are conducted after the bringing of the sealing solution into contact.

3 . The method according to claim 1 , wherein the detecting of the surface target molecule and the detecting of the internal target molecule are conducted after the extracting.

4 . The method according to claim 1 , wherein the detecting of the surface target molecule and the detecting of the internal target molecule are conducted simultaneously.

5 . The method according to claim 1 , wherein the structure body comprises at least one selected from the group consisting of a virus, an exosome, a cell, and an endoplasmic reticulum.

6 . The method according to claim 1 , wherein the structure body forms a complex with the capture substance, and the capture substance is a bound body of a solid phase and a specific binding substance to the structure body.

7 . The method according to claim 6 , wherein the specific binding substance comprises an antibody.

8 . The method according to claim 1 , wherein the extracting comprises heating the structure body in a liquid including a surfactant.

9 . A method for detecting analyzing a structure body, the method comprising:
detecting a presence or absence of the surface target molecule and the internal target molecule in each of the wells by the method of claim 1 , and analyzing the structure body based on a result of the detecting of the presence or absence of the surface target molecule and the internal target molecule.

Assignees

• Toppan Inc

Inventors

• Hirase Takumi
• Makino Yoichi

Classifications

• G01N33/56983

  Viruses · CPC title

• B01L2300/0851

  Bottom walls · CPC title

• B01L2300/0861

  Configuration of multiple channels and/or chambers in a single devices · CPC title

• B01L2200/0689

  Sealing · CPC title

• B01L3/502

  with fluid transport, e.g. in multi-compartment structures · CPC title