METHOD AND DEVICE TO PROBE A MEMBRANE BY APPLYING AN IN-PLANE ELECTRIC FIELD

• US 20080102464A1
• Filed: 09/07/2007
• Published: 05/01/2008
• Est. Priority Date: 09/07/2006
• Status: Active Grant

• Current Assignee: Kent State University
• Original Assignee: Thusara Sugat Chandra Abeygunaratne
• Inventors: ABEYGUNARATNE, Thusara Sugat Chandra
• Granted Patent: US 7,803,588 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: G01N 33/5438 Electrodes

Abstract

The present invention disposes a membrane, for example, a biological cell membrane including amphiphilic lipids, cholesterol and proteins, between two electrical conductive walls having a height at least as great as the thickness of the membrane. The conductive walls are fabricated on an electrically insulative chip base. Electrical conductors extend from each of the conductive walls on the base. An electric field is propagated between the conductive walls in the plane of the membrane. The chip base has one or more through hole between the electrically conducting walls where the membrane is deposed. The chip is placed inside a container that has a base and can have a surrounding wall. The container base has a well below the through hole of the electrically insulative base when it is placed inside the container. At least one passageway extends from the well to the periphery of the container. A third electrode can be fabricated on the bottom of the well. This invention probes changes of the membrane as an in-plane electric field is applied between the conductive walls, using electric current measurements, impedance gain phase analysis, raster scanning by atomic force microscope, and observation with confocal, fluorescence or other microscopes, but not limited to these tools. The invention probes cellular processes such as structural changes of proteins, channeling mechanisms, protein-protein interactions and interaction of proteins with other molecules in an environment that mimics that of an actual living cell membrane. The well, i.e., the interior of the cell, may include various ions, proteins, RNA, DNA, and various cellular components in a suitable medium while extracellular materials such as ions, ligands and other molecules in a suitable medium can be placed in contact with the top of the membrane. Movement of the electrically charged particles through the membrane can be monitored by probing the voltage change across the third electrode and another electrode, for example the AFM tip, placed in contact with the membrane in the exterior fluid. The passageways are used to introduce substances into and out of the well.

Claims

1. A device for probing changes in a membrane, comprising:

a chip including a base comprised of insulating material, electrically conductive walls spaced apart from each other and disposed on said chip base, electrically conducting pads extending from said conductive walls toward a periphery of said chip base, and a through hole formed in said chip base between said conductive walls; and

a container for supporting said chip, said container including a base comprised of insulating material, said chip being placed in contact with said container base, a well being formed in said container base below said through hole, an electrode fabricated on the bottom of the said well, and a passageway formed in said base extending from said well to a periphery of said container.

2. The device of claim 1 said container comprising an electrical conducting pad extending from the said electrode in said well and being disposed on said container base.

3. The device of claim 1 said container including a wall at a periphery thereof comprising an electrical conducting pad, wherein electrical contact is made between said electrical conducting pad on said container wall and one of said electrical conducting pads on said chip base when said chip contacts said container.

4. The device of claim 1 said container comprising a conduit disposed in said passageway.

5. The device of claim 1 said container comprising two said passageways each including a conduit disposed therein, one said conduit being an inlet for inserting components into said well and the other said conduit being an outlet for removing components from said well.

6. The device of claim 1 comprising

a membrane supported on said chip base between said conductive walls and over said through hole;

and

a first material disposed over said membrane and a second material disposed in said well.

7. The device of claim 6 wherein said second material comprises components selected from the group consisting of RNA, DNA, proteins, ions, cell organelles and combinations thereof.

8. The device of claim 6 wherein said first material is selected from the group consisting of ions, ligands and combinations thereof.

9. The device of claim 1 wherein the height of said conductive walls approximates a thickness of said membrane.

10. The method of claim 1 wherein said conductive walls have a height above said base of not more than 10 nanometers.

11. An apparatus comprising said device of claim 1, a power source for applying an electric field between said conductive walls, and an instrument adapted to probe or image said membrane.

12. The apparatus of claim 11 wherein said electric field is provided by AC voltage pulses.

13. The apparatus of claim 11 wherein said electric field is provided by DC voltage pulses.

14. The apparatus of claim 11 wherein said instrument is selected from the group consisting of:

atomic force microscope, confocal microscope, laser scanning confocal microscope, scanning electron microscope, transmission electron microscope, phase contrast microscope, differential-interference-contrast microscope, dark-field microscope, bright-field microscope, electron microscope tomography, fluorescence microscope, X-ray spectrometer, impedance gain phase analyzer and combinations thereof.

15. The apparatus of claim 11 comprising means to determine properties of said membrane selected from the group consisting of:

topographical, electrical, viscous, elastic, and combinations thereof.

16. A method for probing changes of a membrane by applying an electric field, comprising:

providing a chip including a base comprised of insulating material, electrically conductive walls spaced apart from each other and disposed on said chip base, electrically conducting pads extending from said conductive walls toward a periphery of said chip base, and a through hole formed in said chip base between said conductive walls;

providing a container for supporting said chip, said container including a base comprised of insulating material, said chip being disposed in contact with said container base, a well formed below said through hole, and a passageway extending from a periphery of said container to said well;

positioning a membrane between said conductive walls in contact with said chip base and over said through hole;

adding a first material on top of said cell membrane;

adding a second material through said passageway into said well; and

applying an electric field between said conductive walls along the plane of said membrane.

17. The method of claim 16 wherein said first material is selected from the group consisting of ions, ligands and combinations thereof.

18. The method of claim 16 wherein said second material comprises components selected from the group consisting of RNA, DNA, proteins, ions, cell organelles and combinations thereof.

19. The method of claim 16 wherein said membrane is selected from the group consisting of:

a monolayer or bilayer comprised of amphipathic lipids, a cell membrane, at least one biological cell, a molecular monolayer, a molecular bilayer, and combinations thereof.

20. The method of claim 16 comprising monitoring an effect of said electric field on a protein in contact with said membrane.

21. The method of claim 16 comprising probing or imaging said membrane using an atomic force microscope, comprising contacting said membrane with an electrically conductive tip of said atomic force microscope, providing a third electrode on a lower surface of said well and along said container base, and establishing electrical conduction between said conductive tip and the said third electrode.

22. The method of claim 16 comprising probing or imaging said membrane using an instrument selected from the group consisting of:

atomic force microscope, confocal microscope, laser scanning confocal microscope, scanning electron microscope, transmission electron microscope, phase contrast microscope, differential-interference-contrast microscope, dark-field microscope, bright-field microscope, electron microscope tomography, fluorescence microscope, X-ray spectrometer, impedance gain phase analyzer and combinations thereof.

23. The method of claim 16 comprising measuring properties of said membrane selected from the group consisting of:

topographical, electrical, viscous, elastic, and combinations thereof.

24. The method of claim 16 comprising applying said electric field to said membrane as voltage pulses.

25. The method of claim 16 wherein said membrane includes constituents selected from the group consisting of glycolipids, phospholipids, cholesterol, proteins, and combinations thereof.