# US 10,246,743 B2 · Methods for forming lipid bilayers on biochips

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- Number: US 10,246,743 B2
- Document ID: US10246743B2
- Status: Granted
- Kind: B2
- Application number: 15/385,779
- Filed: 2016-12-20
- Published: 2017-07-13
- Granted: 2019-04-02
- Earliest claimed: 2013-10-23
- Assignees: Genia Technologies, Inc.
- Applicants: Genia Technologies, Inc.
- Inventors: Randall W. Davis; Edward Shian Liu; Eric Takeshi Harada; Anne Aguirre; Andrew Trans; James Pollard; Cynthia Cech
- Web page: https://patentgrep.com/patent/US10246743B2

## CPC

- C12Q 1/6869: Methods for sequencing
- C12M 1/34: Measuring or testing with condition measuring or sensing means, e.g. colony counters
- G01N 27/3277: {being a redox reaction, e.g. detection by cyclic voltammetry (voltammetry per se G01N27/42, G01N27/48)}
- G01N 27/3278: {involving nanosized elements, e.g. nanogaps or nanoparticles (nanopores G01N33/48721; magnetic beads G01N27/745)}
- G01N 27/44791: {Microapparatus (sample containers with integrated microfluidic structures B01L3/5027)}
- 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)}
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS}
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## 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

### Claim 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.

### Claim 2 (depends on 1)

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

### Claim 3 (depends on 1)

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

### Claim 4 (depends on 1)

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

### Claim 5 (depends on 1)

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

### Claim 6 (depends on 1)

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

### Claim 7 (depends on 1)

The method of claim 1, wherein the chip comprises wells, and wherein the sensing electrodes are in the wells.

### Claim 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.

### Claim 9 (depends on 8)

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

### Claim 10 (depends on 8)

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

### Claim 11 (depends on 8)

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

### Claim 12 (depends on 6, 11)

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.

### Claim 13 (depends on 6, 11)

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.

### Claim 14 (depends on 1, 8)

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

### Claim 15 (depends on 6, 11)

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.

### Claim 16 (depends on 1, 8)

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

### Claim 17 (depends on 1, 8)

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

### Claim 18 (depends on 1, 8)

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.

### Claim 19 (depends on 8)

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

### Claim 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.

### Claim 21 (depends on 20)

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

### Claim 22 (depends on 20)

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

### Claim 23 (depends on 20)

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

### Claim 24 (depends on 20)

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.