Systems for forming a nanopore in a lipid bilayer

• US 10,371,692 B2
• Filed: 04/14/2017
• Issued: 08/06/2019
• 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 A
• Application Number:
  US15/488,432
• Publication Number:
  US 20170322195A1
• Time in Patent Office: 844 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 system for forming a nanopore in a lipid bilayer, comprising:

a chamber containing a nanopore forming solution, wherein the nanopore forming solution is deposited over a lipid bilayer, the nanopore forming solution having a concentration level and a corresponding activity level of pore molecules such that nanopores are preferentially inserted via an active mechanism that depends on a voltage stimulus waveform applied across the lipid bilayer over a passive mechanism that depends on a self-assembly process; and

a processor programmed to;

control a voltage source to initiate formation of a nanopore in the lipid bilayer, wherein initiating formation of a nanopore comprises applying the voltage stimulus waveform having a predetermined duration to the lipid bilayer.

2. The system 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 in a self-assembly process 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 therein.

3. The system of claim 1, wherein the processor is further programmed to:

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

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

controlling the voltage source to increase the amplitude of the voltage stimulus waveform applied to the lipid bilayer; and

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

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

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

7. The system 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 system 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 system 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 system of claim 9, wherein the processor is further programmed to control the voltage source to apply an erasing voltage stimulus waveform level to erase the lipid bilayer when it is determined that more than one nanopore are formed in the lipid bilayer.

11. The system of claim 1, wherein the processor is further programmed to control the voltage source to apply a reverse oxidation stimulus level to the lipid bilayer after the voltage stimulus waveform is applied.

12. The system of claim 11, wherein the voltage stimulus waveform and the reverse oxidation stimulus level comprise a positive voltage stimulus level and a negative voltage stimulus level.

13. The system of claim 1, wherein the processor is further programmed to:

after the voltage stimulus waveform is applied, control the voltage source to apply a measuring voltage stimulus to the lipid bilayer to determine that a nanopore has been formed based on a measurement in response to the measuring voltage stimulus, wherein an absolute magnitude of the voltage stimulus waveform amplitude is different from an absolute magnitude of the measuring voltage stimulus amplitude.

14. The system of claim 1, wherein the predetermined duration is selected such that only a single nanopore is formed in the lipid bilayer.

15. The system of claim 14, wherein the predetermined duration is between 50 milliseconds and one second.

18. The system of claim 1, wherein the voltage stimulus waveform has a voltage amplitude that increases over time to a predetermined magnitude.

16. A system for forming a nanopore in a lipid bilayer, comprising:

a chamber containing a nanopore forming solution, wherein the nanopore forming solution is deposited over a lipid bilayer; and

a processor programmed to;

control a source to initiate formation of a nanopore in the lipid bilayer, wherein initiating formation of a nanopore comprises applying an agitation stimulus level of a predetermined duration to the lipid bilayer; and

after the agitation stimulus level, control the source to apply 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.

17. The system of claim 16, wherein the voltage stimulus waveform has a voltage amplitude that increases over time to a predetermined magnitude.

19. A system for forming a nanopore in a lipid bilayer, comprising:

a chamber containing a nanopore forming solution, wherein the nanopore forming solution is deposited over a lipid bilayer, the nanopore forming solution having a concentration level and a corresponding activity level of pore molecules such that nanopores are preferentially inserted via an active mechanism that depends on a voltage stimulus waveform applied across the lipid bilayer over a passive mechanism that depends on a self-assembly process; and

a processor programmed to;

control a voltage source to initiate formation of a nanopore in the lipid bilayer, wherein initiating formation of a nanopore comprises applying the voltage stimulus waveform to the lipid bilayer;

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

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

controlling the voltage source to increase an amplitude of the voltage stimulus waveform applied to the lipid bilayer; and

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

20. The system of claim 19, wherein the amplitude of the voltage stimulus waveform does not exceed a predetermined amplitude.