Nanopore Platforms for Ion Channel Recordings and Single Molecule Detection and Analysis

• US 20100320094A1
• Filed: 06/30/2010
• Published: 12/23/2010
• Est. Priority Date: 05/05/2006
• Status: Active Grant

• Current Assignee: University of Utah Research Foundation (State of Utah)
• Original Assignee: University of Utah Research Foundation (State of Utah)
• Inventors: Ervin, Eric N., White, Ryan J., White, Henry S.
• Granted Patent: US 8,581,605 B2
• Time in Patent Office: Days
• US Class Current: 205/778
• CPC Class Codes: B82Y 15/00 Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• 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
• CPC Class Codes: Y02E 60/10 Energy storage using batteries
• CPC Class Codes: Y10T 428/249979 Specified thickness of void-containing component [absolute or relative] or numerical cell dimension

Abstract

A nanopore device includes a membrane having a nanopore extending there through forming a channel from a first side of the membrane to a second side of the membrane. The surface of the channel and first side of the membrane are modified with a hydrophobic coating. A first lipid monolayer is deposited on the first side of the membrane, and a second lipid monolayer is deposited on the second side of the membrane, wherein the hydrophobic coating causes spontaneous generation of a lipid bilayer across the nanopore orifice. Sensing entities, such as a protein ion channel, can be inserted and removed from the bilayer by adjusting transmembrane pressure, and adapter molecules can be electrostatically trapped in the ion channel by applying high transmembrane voltages, while resistance or current flow through the sensing entity can be measured electrically.

Claims

1. A nanopore device comprising:

a membrane having a thickness with a first, exterior, side and a second, interior, side, the first side being opposite the second side, wherein the membrane is made from a material selected from the group consisting of Si, SiO₂, Si₃N₄, alumina, nitrides, metals, polymers, and combinations thereof;

at least one nanopore extending through the membrane, thus forming at least one channel connecting the first side and the second side of the membrane, wherein the nanopore has a first opening, a second opening, and a depth, wherein the first opening opens to the first side of the membrane, and the second opening opens to the second side of the membrane, and wherein surface portions of the first side and the at least one channel connecting the first side and the second side of the membrane include modifying molecules defining hydrophobic surfaces; and

means for spanning across the first opening of the nanopore, wherein the means for spanning across the first opening of the nanopore is a supported lipid bilayer structure or a spanning lipid bilayer structure.

2. The nanopore device of claim 1, wherein the membrane is made from a glass.

3. The nanopore device of claim 1, wherein the nanopore is of a truncated conical shape and wherein the radius of the first opening of the nanopore is smaller than the radius of the second opening of the nanopore.

4. The nanopore device of claim 1, further comprising at least one sensing entity embedded within the means for spanning across the first opening of the nanopore.

5. The nanopore device of claim 1, wherein a radius of the first opening of the nanopore ranges from about 2 nm to about 500 nm, or larger.

6. The nanopore device of claim 1, further comprising a pressure apparatus for applying a transmembrane pressure between the first side and the second side of the membrane.

7. The nanopore device of claim 4, wherein the sensing entity is selected from the group consisting of a protein ion channel, a modified ion channel, an enzyme, and a biological entity that produces an electrical signal upon interacting with analytes, ions, and molecules in the solution.

8. The nanopore device of claim 7, wherein the sensing entity is a protein ion channel selected from the group consisting of α

-hemolysin, an engineered version of α

-hemolysin, and porin OmpF.

9. The nanopore device of claim 1, wherein the modifying molecules are 3-cyano-propyldimethylchlorosilane or other silanes.

10. The nanopore device of claim 1, further comprising:

means for applying an electric field across the first side and the second side of the membrane;

means for monitoring a current flow or resistance through the nanopore; and

means for processing an observed current flow or resistance to produce a useful output.

11. The nanopore device of claim 10, wherein the means for applying an electric field comprises a first electrode and a second electrode.

12. The nanopore device of claim 11, wherein first electrode is positioned on the first side of the membrane and a second electrode is positioned on the second side of the membrane.

13. The nanopore device of claim 12, wherein the first electrode and the second electrode are made of Ag/AgCl.

14. The nanopore device of claim 1, wherein the thickness of the membrane ranges from about 20 μ

m to about 75 μ

m.

15. A method for single molecule detection of an analyte using a nanopore device including a membrane having a thickness, with a first, exterior, side and a second, interior, side, the first side being opposite the second side, wherein the membrane is made from a material selected from the group consisting of glass, silicate, ceramic, Si, SiO₂, Si₃N₄, alumina, nitrides, metals, polymers, and combinations thereof;

at least one nanopore extending through the membrane, thus forming at least one channel connecting the first side and the second side of the membrane, wherein the nanopore has a first opening, a second opening, and a depth, wherein the first opening opens to the first side of the membrane, and the second opening opens to the second side of the membrane, and wherein surface portions of the first side and the at least one channel connecting the first side and the second side of the membrane include modifying molecules defining hydrophobic surfaces; and

means for spanning across the first opening of the nanopore, wherein the means for spanning across the first opening of the nanopore is a supported lipid bilayer structure or a spanning lipid bilayer structure, wherein a sensing entity is embedded in the lipid bilayer structure spanning across the first opening of the nanopore the method comprising;

providing a sample solution containing an analyte of interest;

contacting the nanopore device with the sample solution such that the first, exterior, side of the membrane is immersed in the sample solution and the at least one nanopore is filled with the sample solution;

applying an appropriate voltage across the first and second side of the membrane;

measuring the resistance or current flow through the at least one nanopore using either alternating current (AC) or direct current (DC) measurements; and

analyzing the observed resistance or current flow to produce a useful output.

16. The method according to claim 15, wherein the sensing entity of the nanopore device is a drug target or a modified drug target, the analyte of interest is a drug candidate and the useful output is the binding affinity of the drug candidate with the drug target or modified drug candidate.

17. The method according to claim 15, wherein the sensing entity of the nanopore device recognizes specific nucleotides, the analyte of interest is a nucleic acid and the useful output is sequence information of the nucleic acid.