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Kit for sealing beads and detecting target molecule

Foreign code F170009221
File No. AF19-01US4
Posted date Sep 13, 2017
Country United States of America
Application number 201615351522
Gazette No. 20170115284
Gazette No. 10267793
Date of filing Nov 15, 2016
Gazette Date Apr 27, 2017
Gazette Date Apr 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
Title Kit for sealing beads and 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]
1. A kit comprising:
an 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;
a hydrophobic solvent; and
a device for removing air in the plurality of receptacles while keeping the hydrophilic solvent as it is in the plurality of receptacles;
each of the plurality of receptacles being capable of storing only one of the beads, and
said kit 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; wherein step (ii) is carried out between step (i) and step (iii).

[claim2]
2. The kit as set forth in claim 1, wherein:
each of the plurality of receptacles has a hydrophilic bottom surface.

[claim3]
3. The kit as set forth in claim 1, wherein:
the upper layer section has a hydrophobic surface facing the lower layer section.

[claim4]
4. The kit as set forth in claim 2, wherein:
the upper layer section has a hydrophobic surface facing the lower layer section.

[claim5]
5. The kit as set forth in claim 1, 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.

[claim6]
6. The kit as set forth in claim 2, 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.

[claim7]
7. The kit as set forth in claim 3, 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.

[claim8]
8. The kit as set forth in claim 4, 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.
  • 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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