Methods for forming a nanopore in a lipid bilayer

• US 9,678,055 B2
• Filed: 01/08/2014
• Issued: 06/13/2017
• Est. Priority Date: 02/08/2010
• Status: Active Grant

• Current Assignee: Roche Sequencing Solutions, Inc. (Roche Holding AG)
• Original Assignee: Genia Technologies, Inc. (Roche Holding AG)
• Inventors: Chen, Roger J. A., Davis, Randy
• Primary Examiner(s): Schiffman, Benjamin
• Application Number:
  US14/150,322
• Publication Number:
  US 20140203464A1
• Time in Patent Office: 1,252 Days
• Field of Search: None
• CPC Class Codes: B81B 1/00 Devices without movable or flexible elements, e.g. microcapillary devices
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: G01N 15/12 by observing changes in resistance or impedance across apertures when traversed by individual particles, e.g. by using the Coulter principle
• CPC Class Codes: G01N 15/13 Details pertaining to apertures
• CPC Class Codes: G01N 33/483 Physical analysis of biological material
• CPC Class Codes: G01N 33/48721 Investigating individual macromolecules, e.g. by translocation through nanopores Coulter counters in general G01N15/12; fabrication methods for nanoscale apertures B81B1/00; sequencing of nucleic acids C12Q1/68

Abstract

A method of forming a nanopore in a lipid bilayer is disclosed. A nanopore forming solution is deposited over a lipid bilayer. The nanopore forming solution has a concentration level and a corresponding activity level of pore molecules such that nanopores are substantially not formed un-stimulated in the lipid bilayer. Formation of a nanopore in the lipid bilayer is initiated by applying an agitation stimulus level to the lipid bilayer. In some embodiments, the concentration level and the corresponding activity level of pore molecules are at levels such that less than 30 percent of a plurality of available lipid bilayers have nanopores formed un-stimulated therein.

Claims

1. A method of forming a nanopore in a lipid bilayer, comprising:

depositing a nanopore forming solution over a lipid bilayer, the nanopore forming solution having a concentration level and a corresponding activity level of pore molecules such that nanopores are substantially not formed in the lipid bilayer un-stimulated;

initiating formation of a nanopore in the lipid bilayer by a processor, comprising applying an agitation stimulus level to the lipid bilayer; and

after the agitation stimulus level, applying a measuring stimulus level to the lipid bilayer to determine that a nanopore has been formed based on a measurement in response to the measuring stimulus level, wherein an absolute magnitude of the agitation stimulus level is different from an absolute magnitude of the measuring stimulus level.

2. The method of claim 1, wherein the concentration level and the corresponding activity level of pore molecules such that nanopores are substantially not formed in the lipid bilayer un-stimulated comprise a concentration level and a corresponding activity level of pore molecules such that approximately less than 30 percent of a plurality of lipid bilayers have nanopores formed un-stimulated.

3. The method of claim 1, further comprising:

detecting whether a nanopore has been formed in the lipid bilayer; and

in the event that a nanopore is not detected, iteratively initiating formation of a nanopore in the lipid bilayer, comprising;

increasing the agitation stimulus level applied to the lipid bilayer; and

detecting whether a nanopore has been formed in the lipid bilayer.

4. The method of claim 3, wherein the iteratively initiating formation of a nanopore in the lipid bilayer is repeated until formation of a nanopore is detected.

5. The method of claim 3, wherein the iteratively initiating formation of a nanopore in the lipid bilayer is repeated until damage of the lipid bilayer is detected.

6. The method of claim 3, wherein the iteratively initiating formation of a nanopore in the lipid bilayer is repeated until the agitation stimulus level has reached a predetermined maximum threshold.

7. The method of claim 3, wherein the detection of whether a nanopore has been formed in the lipid bilayer comprises detecting a change in an electrical property of the lipid bilayer resulting from the formation of a nanopore in the lipid bilayer.

8. The method of claim 7, wherein detecting a change in the lipid bilayer electrical property comprises detecting a change in a resistance of the lipid bilayer.

9. The method of claim 7, wherein the detection of whether a nanopore has been formed in the lipid bilayer comprises determining a number of nanopores formed based on a size of change in the lipid bilayer electrical property.

10. The method of claim 9, further comprising applying an erasing stimulus level to erase the lipid bilayer when it is determined that more than one nanopore is formed in the lipid bilayer.

11. The method of claim 1, further comprising applying a reverse oxidation stimulus level to the lipid bilayer after the agitation stimulus level is applied.

12. The method of claim 11, wherein the agitation stimulus level and the reverse oxidation stimulus level comprise a negative stimulus level and a positive stimulus level.