Methods for forming lipid bilayers on biochips

• US 9,567,630 B2
• Filed: 10/22/2014
• Issued: 02/14/2017
• Est. Priority Date: 10/23/2013
• Status: Active Grant

• Current Assignee: Roche Sequencing Solutions, Inc. (Roche Holding AG)
• Original Assignee: Genia Technologies, Inc. (Roche Holding AG)
• Inventors: Davis, Randall W., Liu, Edward Shian, Harada, Eric Takeshi, Aguirre, Anne, Trans, Andrew, Pollard, James, Cech, Cynthia
• Primary Examiner(s): Ball, J. Christopher
• Application Number:
  US14/521,425
• Publication Number:
  US 20150152494A1
• Time in Patent Office: 846 Days
• US Class Current: 1/1
• CPC Class Codes: C12M 1/34 Measuring or testing with condition measuring or sensing means, e.g. colony counters
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: G01N 27/3277 being a redox reaction, e.g. detection by cyclic voltammetry voltammetry per se G01N27/42, G01N27/48
• CPC Class Codes: G01N 27/3278 involving nanosized elements, e.g. nanogaps or nanoparticles nanopores G01N33/48721; magnetic beads G01N27/745
• CPC Class Codes: G01N 27/44791 Microapparatus sample containers with integrated microfluidic structures B01L3/5027
• 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

This disclosure provides a biochip comprising a plurality of wells. The biochip includes a membrane that is disposed in or adjacent to an individual well of the plurality of wells. The membrane comprises a nanopore, and the individual well comprises an electrode that detects a signal upon ionic flow through the pore in response to a species passing through or adjacent to the nanopore. The electrode can be a non-sacrificial electrode. A lipid bilayer can be formed over the plurality of wells using a bubble.

Claims

1. A method for forming a lipid bilayer for use in a nanopore sensing device, comprising:

(a) providing a primed chip comprising a fluid flow path in fluid communication with a plurality of sensing electrodes;

(b) flowing a lipid solution into the fluid flow path; and

(c) flowing at least one bubble onto the fluid flow path, thereby forming a lipid bilayer adjacent to the sensing electrodes, wherein the bubble spans the plurality of sensing electrodes, and wherein each of the sensing electrodes is in a well disposed on the trans side of the bilayer and opposite to the cis side of the bilayer wherein the top electrode is disposed; and

wherein the primed chip does not comprise pre-deposited lipid molecules and/or pore proteins.

2. The method of claim 1, wherein the bubble is adjacent to the sensing electrodes for at least about 5 milliseconds.

3. The method of claim 1, wherein the bubble is adjacent to the sensing electrodes for at least about 30 seconds.

4. The method of claim 1, wherein the bubble is adjacent to the sensing electrodes for at most about 5 minutes.

5. The method of claim 1, wherein a lipid bilayer is formed over at least 25% of the sensing electrodes.

6. The method of claim 1, further comprising inserting a nanopore into the lipid bilayers adjacent to each of the sensing electrodes.

7. The method of claim 6 or 1, wherein the nanopore is Mycobacterium smegmatis porin A (MspA), alpha-hemolysin, any protein having at least 70% homology to at least one of MspA or alpha-hemolysin, or any combination thereof.

12. The method of claim 1 or 8, wherein the lipid bilayer exhibits a resistance greater than about 1 GΩ

.

13. The method of claim 1, wherein the lipid comprises an organic solvent.

14. The method of claim 1 or 8, wherein the bubble is a vapor bubble.

15. The method of claim 1 or 8, wherein the lipid is selected from the group consisting of diphytanoyl-phosphatidylcholine (DPhPC), 1,2-diphytanoyl-sn-glycero-3phosphocholine, 1,2-Di-O-Phytanyl-sn-Glycero-3-phosphocholine (DoPhPC), palmitoyl-oleoyl-phosphatidyl-choline (POPC), dioleoyl-phosphatidyl-methylester (DOPME), dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylinositol, phosphatidylglycerol, sphingomyelin, 1,2-di-O-phytanyl-sn-glycerol;

1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-350];

1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-550];

1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-750];

1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000];

1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000];

1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lactosyl;

GM1 Ganglioside, Lysophosphatidylcholine (LPC) or any combination thereof.

8. A method for forming a lipid bilayer for use in a nanopore sensing device, comprising:

(a) providing a primed chip comprising a fluid flow path in fluid communication with a plurality of sensing electrodes;

(b) flowing at least one bubble into the fluid flow path and adjacent to the plurality of sensing electrodes such that the bubble spans the plurality of sensing electrodes; and

(c) contacting the periphery of the bubble with a lipid, wherein the lipid diffuses under the bubble and onto the fluid flow path, thereby forming a lipid bilayer adjacent to the sensing electrodes, and wherein each of the sensing electrodes is in a well disposed on the trans side of the bilayer and opposite to the cis side of the bilayer wherein the top electrode is disposed; and

wherein the primed chip does not comprise pre-deposited lipid molecules and/or pore proteins.

9. The method of claim 8, wherein the bubble is contacted with the lipid for at least about 5 milliseconds to about 10 minutes.

10. The method of claim 8, wherein a lipid bilayer is formed over at least 50% of the sensing electrodes.

11. The method of claim 8, further comprising inserting a nanopore into the lipid bilayers adjacent to each of the sensing electrodes.

16. The method of claim 6 or 11, wherein inserting the nanopore comprises applying a sequentially increasing electrical stimulus through the electrode to facilitate the insertion of the nanopore in the lipid bilayer.

17. The method of claim 6 or 11, wherein the lipid bilayer and the nanopore protein together exhibit a resistance of about 1 GΩ

or less.

18. A nanopore sensing system, comprising:

(a) a chip comprising a fluid flow path in fluid communication with a plurality of sensing electrodes, wherein each of the sensing electrodes is configured to detect an ionic current or change in resistance, conductance, charge, or voltage upon a nucleic acid incorporation or capture event; and

(b) a control system coupled to the chip, the control system programmed to;

i. flow an ionic solution across the chip followed by flowing a lipid solution into the fluid flow path;

ii. flow at least one bubble into the fluid flow path and adjacent to the sensing electrodes for a time period of at least about 0.5 second, wherein the bubble spans the plurality of sensing electrodes, and wherein the flow of the bubble into the fluid flow path forms a lipid bilayer adjacent to the sensing electrodes, and wherein each of the sensing electrodes is in a well disposed on the trans side of the bilayer and opposite to the cis side of the bilayer wherein the top electrode is disposed; and

wherein the chip does not comprise pre-deposited lipid molecules and/or pore proteins.

19. The system of claim 18, wherein the control system is external to the chip.

20. The system of claim 18, wherein the control system comprises a computer processor.

21. The system of claim 18, further comprising a fluid flow system operably coupled to the control system and the chip, wherein the fluid flow system is configured to direct the flow of the lipid solution and the bubble.