Methods for forming lipid bilayers on biochips

Patent metadata

Field	Value
Publication number	US-10246743-B2
Application number	US-201615385779-A
Country	US
Kind code	B2
Filing date	Dec 20, 2016
Priority date	Oct 23, 2013
Publication date	Apr 2, 2019
Grant date	Apr 2, 2019

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.

First claim

What is claimed is:

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.

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 1 , wherein the chip comprises wells, and wherein the sensing electrodes are in the wells.

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 said 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.

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.

12. The method of claim 6 or 11 , 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.

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

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

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

16. The method of claim 1 or 8 , wherein said lipid comprises an organic solvent.

17. The method of claim 1 or 8 , wherein said bubble is a vapor bubble.

18. The method of claim 1 or 8 , wherein said lipid is selected from the group consisting of diphytanoyl-phosphatidylcholine (DPhPC), 1,2-diphytanoyl-sn-glycero-3 phosphocholine, 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.

19. The method of claim 8 , wherein the chip comprises wells, and wherein the sensing electrodes are in the wells.

20. 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 said 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 said chip, said 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.

21. The system of claim 20 , wherein the chip comprises wells, and wherein the sensing electrodes are in the wells.

22. The system of claim 20 , wherein the control system is external to said chip.

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

24. The system of claim 20 , further comprising a fluid flow system operably coupled to said control system and said chip, wherein said fluid flow system is configured to direct the flow of said lipid solution and said bubble.

Assignees

• Genia Tech Inc

Inventors

• Davis Randall W
• Liu Edward Shian
• Harada Eric Takeshi
• Aguirre Anne
• Trans Andrew
• Pollard James
• Cech Cynthia

Classifications

• G01N33/48721

  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) · CPC title

• G01N27/44791

  Microapparatus (sample containers with integrated microfluidic structures B01L3/5027) · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12M1/34

  Measuring or testing with condition measuring or sensing means, e.g. colony counters · CPC title

• G01N27/3277

  being a redox reaction, e.g. detection by cyclic voltammetry (voltammetry per se G01N27/42, G01N27/48) · CPC title