Nanopipette apparatus for manipulating cells

Patent metadata

Field	Value
Publication number	US-10513434-B2
Application number	US-201715463860-A
Country	US
Kind code	B2
Filing date	Mar 20, 2017
Priority date	Mar 3, 2011
Publication date	Dec 24, 2019
Grant date	Dec 24, 2019

Abstract

Disclosed herein are methods and systems for controlled ejection of desired material onto surfaces including in single cells using nanopipettes, as well as ejection onto and into cells. Some embodiments are directed to a method and system comprising nanopipettes combined with an xyz controller for depositing a user defined pattern on an arbitrary substrate for the purpose of controlled cell adhesion and growth. Alternate embodiments are directed to a method and system comprising nanopipettes combined with an xyz controller and electronic control of a voltage differential in a bore of the nanopipette electroosmotically injecting material into a cell in a high-throughput manner and with minimal damage to the cell. Yet other embodiments are directed to method and system comprising functionalized nanopipettes combined with scanning ion conductance microscopy for studying molecular interactions and detection of biomolecules inside a single living cell.

First claim

What is claimed is:

1. A method of detecting an analyte in a single living cell, comprising:
positioning a tip of a nanopipette within 50 to 200 nm of the cell membrane of the living cell, wherein the nanopipette comprises a working electrode disposed in the nanopipette in contact with a solution in the nanopipette, wherein the nanopipette is functionalized with an analyte-binding reagent, wherein the nanopipette is biased at a positive voltage by a circuit connected to the working electrode and to a reference electrode positioned in a liquid contacting the single living cell;
inserting the tip of the nanopipette to a defined depth into the single living cell, wherein the positioning and inserting is controlled via an xyz controller
monitoring current through the tip of the nanopipette;
detecting the analyte in the single living cell by detecting a reduction in current through the tip of the nanopipette.

2. The method according to claim 1 , wherein the inserting is at a speed of from 50 to 200 μm/s.

3. The method according to claim 2 , wherein the inserting is at a speed of 80 to 150 μm/s.

4. The method according to claim 1 , wherein the analyte-binding reagent is immobilized on an interior surface of the nanopipette at or near the tip of the nanopipette.

5. The method according to claim 4 , wherein the analyte-binding reagent is linked by sulfo-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) to a poly-1-lysine (PLL) coating on the interior surface.

6. The method according to claim 1 , wherein the tip of the nanopipette has a diameter of from 37 to 82 nm.

7. The method according to claim 1 , wherein the analyte-binding reagent is an antibody specific for the analyte.

8. The method according to claim 1 , wherein the analyte-binding reagent is an aptamer specific for the analyte.

9. The method according to claim 1 , wherein the analyte is a receptor and the analyte-binding reagent is a ligand of the receptor.

10. The method according to claim 1 , wherein the analyte is a ligand and the analyte-binding reagent is a receptor of the ligand.

11. The method according to claim 1 , wherein the analyte is an oncoprotein.

12. The method according to claim 1 , further comprising, prior to the inserting, immobilizing the single living cell on a substrate.

13. The method according to claim 12 , wherein immobilizing the single living cell on the substrate comprises placing the cell in a cavity in the substrate, the cavity sized to hold only a single cell.

14. The method according to claim 12 , comprising applying a pressure differential across the cavity to aid in immobilizing the single living cell.

15. The method according to claim 13 , wherein the substrate comprises one or more through-holes for applying negative pressure to immobilize the single living cell in the cavity.

16. The method according to claim 1 , wherein the single living cell is a bacterial cell or a fungal cell.

17. The method according to claim 1 , wherein the single living cell is an animal cell.

18. The method according to claim 17 , wherein the animal cell is a mammalian cell.

19. The method according to claim 18 , wherein the mammalian cell is a HeLa cell.

20. The method according to claim 1 , wherein the predefined depth is between 1 μm to 2 μm.

Assignees

• Univ California

Inventors

• Seger R Adam
• Actis Paolo
• Vilozny Boaz
• Pourmand Nader

Classifications

• B82Y15/00

  Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors · CPC title

• C12M41/48

  Automatic or computerized control (automatic analysis G01N35/00) · CPC title

• B01L3/50273

  characterised by the means or forces applied to move the fluids · CPC title

• B01J2219/00371

  comprising electrodes · CPC title

• C12M41/00

  Means for regulation, monitoring, measurement or control, e.g. flow regulation (controlling or regulating chemical, physical or physicochemical processes B01J19/0006; heating or cooling apparatus for laboratory use B01L7/00; electro optical investigation of individual particles, flow cytometers G01N15/14; automatic analysis G01N35/00; controlling or regulating in general G06N) · CPC title