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Method for detecting target molecule

Foreign code F190009874
File No. AF19-01US5
Posted date Jul 29, 2019
Country United States of America
Application number 201916260413
Gazette No. 20190154682
Date of filing Jan 29, 2019
Gazette Date May 23, 2019
Priority data
  • P2011-050629 (Mar 8, 2011) JP
  • 2012JP55884 (Mar 7, 2012) WO
  • 201314003509 (Sep 6, 2013) US
  • 201615082195 (Mar 28, 2016) US
  • 201615351522 (Nov 15, 2016) US
Title Method for detecting target molecule
Abstract This invention provides a technique enabling to detect target molecules of low concentration with high sensitivity. This invention includes (i) a step of introducing a hydrophilic solvent (42) containing beads (40),(41′) into a space (30) between (a) a lower layer section (10) including a plurality of receptacles (13) each of which is capable of storing only one of the beads (41),(41′) and which are separated from each other by a side wall (12) having a hydrophobic upper surface and (b) an upper layer section (20) facing a surface of the lower layer section (10) on which surface the plurality of receptacles (13) are provided; and (ii) a step of introducing a hydrophobic solvent (43) into the space (30), the step (ii) being carried out after the step (i).
Outline of related art and contending technology BACKGROUND ART
There has been known a single-molecule assay as a method for carrying out various assays by observing biomolecules such as proteins and nucleic acids in such a manner that the biomolecules are individually identified. In order to carry out the single-molecule assay, there have been known some methods.
Patent Literature 1 discloses a micro chamber for detecting single-molecule enzyme activity. This micro chamber includes a container part into which a liquid droplet can be sealed and which has capacity of storing a liquid droplet of up to 1000 fL (femtoliters). The container part is made of a recess provided in at least one of a first member and a second member which are bonded to each other. According to Patent Literature 1, an enzyme reaction is carried out in the liquid droplet. With such a configuration, the enzyme reaction can be performed with a high concentration of the reaction products, even if the number of molecules of the reaction products is quite small. Thus, it is possible to detect an activity of one molecule of enzyme.
Non-Patent Literature 1 discloses a method for carrying out a single-molecule enzyme assay by use of an array where a liquid droplet is covered with oil, in a femtoliter-order, and accessible directly from the outside. This array includes a hydrophilic region pattern made of a hydrophilic surface on which a hydrophobic region having a height of 17 nm is provided.
Non-Patent Literature 2 discloses a method for detecting a protein by a single-molecule Enzyme-Linked ImmunoSorbent Assay (ELISA). According to this method, a very small amount of proteins are captured by minute beads covered with protein-specific antibodies, and complexes of the beads and the proteins are fluorescence-labeled. Then, beads including the complexes are introduced into a reaction chamber by centrifugal force. Thereafter, the number of beads having captured the proteins is counted. In this manner, the proteins are quantitatively assayed.
Scope of claims [claim1-12]
1-12. (canceled)

[claim13]
13: A detecting method using a kit and a deaerating device,
wherein said kit comprises:
an array having a flow cell structure, said array including:
a lower layer section provided with a plurality of receptacles being separated from each other by a side wall having a hydrophobic upper surface; and
an upper layer section facing, via a space, a surface of the lower layer section on which surface the plurality of receptacles are provided;
beads;
a hydrophilic solvent; and
a hydrophobic solvent;
each of the plurality of receptacles is capable of storing only one of the beads; and
said space is used as a flow path for allowing a fluid to flow between the lower layer section and the upper layer section;
and
said deaerating device is set for removing air in the plurality of receptacles while keeping the hydrophilic solvent as it is in the plurality of receptacles;
said kit and said deaerating device being used to carry out a method for sealing the beads, the method comprising the steps of:
(i) introducing the hydrophilic solvent containing the beads into the space between the lower layer section and the upper layer section;
(ii) deaerating to remove air in the plurality of receptacles while keeping the hydrophilic solvent as it is in the plurality of receptacles; and
(iii) introducing the hydrophobic solvent into the space to flow and to displace the hydrophilic solvent;
wherein step (ii) is carried out between step (i) and step (iii).

[claim14]
14: The detecting method according to claim 13, wherein each of the plurality of receptacles has a hydrophilic bottom surface.

[claim15]
15: The detecting method according to claim 13, wherein the upper layer section has a hydrophobic surface facing the lower layer section.

[claim16]
16: The detecting method according to claim 14, wherein the upper layer section has a hydrophobic surface facing the lower layer section.

[claim17]
17: The detecting method according to claim 13, wherein at least one of the upper layer section and the lower layer section has a through-hole via which a fluid is introduced into the space.

[claim18]
18: The detecting method according to claim 14, wherein at least one of the upper layer section and the lower layer section has a through-hole via which a fluid is introduced into the space.

[claim19]
19: The detecting method according to claim 15, wherein at least one of the upper layer section and the lower layer section has a through-hole via which a fluid is introduced into the space.

[claim20]
20: The detecting method according to claim 16, wherein at least one of the upper layer section and the lower layer section has a through-hole via which a fluid is introduced into the space.

[claim21]
21: The detecting method according to claim 13, wherein each of the plurality of receptacles is made by photolithography and dry etching.

[claim22]
22: The detecting method according to claim 13, wherein a nucleic acid is captured by the bead sealed in the plurality of receptacles.

[claim23]
23: The detecting method according to claim 13, wherein said side wall is made of an amorphous fluorocarbon resin.

[claim24]
24: The detecting method according to claim 13, wherein a width of said space in the vertical direction is from 10 μm to 150 μm.
  • Inventor, and Inventor/Applicant
  • NOJI Hiroyuki
  • IINO Ryota
  • ARAKI Suguru
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
IPC(International Patent Classification)
Reference ( R and D project ) CREST Creation of Nanosystems with Novel Functions through Process Integration AREA
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