Methods for creating bilayers for use with nanopore sensors

Patent metadata

Field	Value
Publication number	US-10316360-B2
Application number	US-201715809725-A
Country	US
Kind code	B2
Filing date	Nov 10, 2017
Priority date	Feb 16, 2012
Publication date	Jun 11, 2019
Grant date	Jun 11, 2019

Abstract

Provided is a method for detecting a target molecule. The method includes providing a chip, the chip including a nanopore in a membrane that is disposed adjacent to or in proximity to a sensing electrode. A nucleic acid molecule is then directed through the nanopore, the nucleic acid molecule being associated with a reporter molecule. The nucleic acid molecule also includes an address region and a probe region, the reporter molecule being associated with the nucleic acid molecule at the probe region. The reporter molecule is also coupled to a target molecule. While the nucleic acid molecule is directed through the nanopore, the address region can be sequenced to determine a nucleic acid sequence of the address region. The target molecule can then be identified, with the aid of a computer processor, based upon the nucleic acid sequence of the address region.

First claim

What is claimed is:

1. A method for detecting a target molecule, comprising:
(a) providing a chip comprising a nanopore in a membrane that is disposed adjacent or in proximity to a sensing electrode;
(b) directing a nucleic acid molecule through said nanopore, wherein said nucleic acid molecule is associated with a reporter molecule, wherein said nucleic acid molecule comprises an address region and a probe region, wherein said reporter molecule is associated with said nucleic acid molecule at said probe region, and wherein said reporter molecule is coupled to a target molecule;
(c) sequencing said address region while said nucleic acid molecule is directed through said nanopore to determine a nucleic acid sequence of said address region; and
(d) identifying, with the aid of a computer processor, said target molecule based upon the nucleic acid sequence of said address region determined in (c).

2. The method of claim 1 , wherein, the probe molecule in (b) is held in the pore by the binding of a reporter molecule to the probe region of the nucleic acid molecule.

3. The method of claim 2 , wherein up to three bases of said nucleic acid molecule are identified when the rate of progression of said nucleic acid molecule through the nanopore is reduced.

4. The method of claim 2 , wherein up to five bases of said nucleic acid molecule are identified when the rate of progression of said nucleic acid molecule through the nanopore is reduced.

5. The method of claim 2 , wherein the rate of progression of said nucleic acid molecule through said nanopore is reduced upon the interaction of said reporter molecule with said nanopore.

6. The method of claim 1 , wherein, in (b), a rate of progression of said nucleic acid molecule through said nanopore is stopped or stalled.

7. The method of claim 1 , further comprising, prior to (d), determining whether a rate of progression of said nucleic molecule through said nanopore has been reduced.

8. The method of claim 1 , wherein, in (d), said target molecule is identified if it is determined that the rate of progression of said nucleic acid molecule through said nanopore has been reduced.

9. The method of claim 1 , wherein, in (d), said target molecule is identified based upon a correlation between (i) a nucleic acid sequence of said address region and an association and (ii) a rate of progression of said nucleic acid molecule through said nanopore.

10. The method of claim 1 , wherein said nanopore is individually addressable.

11. The method of claim 1 , wherein said nucleic acid molecule is single-stranded.

12. The method of claim 1 , further comprising trapping the nucleic acid molecule in the nanopore.

13. The method of claim 12 , wherein the nucleic acid molecule is trapped in the nanopore with the aid of bulky structures formed at one or more end portions of the nucleic acid molecule.

14. The method of claim 12 , wherein the nucleic acid molecule is trapped in the nanopore with the aid of bulky structures affixed to one or more end portions of the nucleic acid molecule.

15. The method of claim 1 , further comprising reversing a direction of flow of said nucleic acid molecule through said nanopore.

16. The method of claim 15 , further comprising re-sequencing at least a portion of said address region upon reversing the direction of flow of said nucleic acid molecule.

17. The method of claim 1 , wherein said reporter molecule comprises an antibody or aptamer at an end portion of said reporter molecule, and wherein said antibody or aptamer is associated with said target molecule.

18. The method of claim 1 , wherein address region and probe region have known nucleic acid sequences.

19. The method of claim 1 , wherein said reporter molecule comprises a nucleic acid sequence that is complimentary to a nucleic acid sequence of said probe region.

20. The method of claim 1 , wherein said nucleic acid molecule is associated with said reporter molecule prior to being directed through said.

21. The method of claim 1 , wherein, prior to (b), said nucleic acid molecule is threaded through said nanopore, and wherein, in (b), said reporter molecule is associated with said nucleic acid molecule that has been threaded through said nanopore.

Assignees

• Genia Tech Inc

Inventors

• Davis Randall
• Chen Roger

Classifications

• G01N33/5432

  Liposomes or microcapsules · CPC title

• C12N2310/11

  Antisense · CPC title

• C12N15/111

  General methods applicable to biologically active non-coding nucleic acids · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Q2565/631

  being a biochannel or pore · CPC title