Nanopipette device and method for subcellular analysis

Patent metadata

Field	Value
Publication number	US-10696962-B2
Application number	US-201414775168-A
Country	US
Kind code	B2
Filing date	Mar 13, 2014
Priority date	Mar 14, 2013
Publication date	Jun 30, 2020
Grant date	Jun 30, 2020

Abstract

Described herein are devices and methods for extracting cellular material from living cells and then depositing them into to a receptacle in a nanoliter scale. Using a nanopipette integrated into a scanning ion conductance microscope (SICM), extraction of mitochondrial DNA from human BJ fibroblasts and Green Fluorescent Protein (GFP) transcripts from HeLa/GFP cells was achieved with minimal disruption to the cellular milieu and without chemical treatment prior to obtaining the isolated sample. Success of the extraction was confirmed by fluorescence microscopy and PCR analysis of the extracted material. The method and apparatus may be applied to many different cell types and intracellular targets, allowing not only single cell analysis, but single subcellular compartment analysis of materials extracted in their native state.

First claim

What is claimed is:

1. A system for extracting target intracellular content from a single cell, comprising:
(a) a nanopipette mounted on an xyz controller;
(b) a first electrode positioned within the interior of the nanopipette;
(c) a second electrode positioned outside of the nanopipette;
(d) a current detecting circuit and a voltage control circuit, the current detecting circuit detecting ionic current between the first electrode and the second electrode and the voltage control circuit controlling flow of liquid into and out of the nanopipette; and
(e) a computer comprising programming that:
instructs the xyz controller to move the nanopipette in response to changes in ionic current sensed by the current detecting circuit; and
position the nanopipette tip at a fixed distance from surface of the single cell and move the nanopipette at a speed of 100 μm/sec to penetrate the cell membrane of the single cell; and
instructs the voltage control circuit to:
(i) apply a first bias voltage that holds a first electrolyte solution in the nanopipette;
(ii) apply a second bias voltage that causes influx of target intracellular content from within the single cell when the nanopipette is positioned at a location of the target intracellular content within the single cell; and
(iii) apply a third bias voltage that causes efflux of the target intracellular content from the nanopipette.

2. The system of claim 1 , wherein
said xyz controller is attached to the nanopipette for effecting mechanical movements of the nanopipette in submicron x and y steps, and effecting movement of said nanopipette in a z direction towards or away from the single cell, and,
said xyz controller further having electronic controls for controlling said mechanical movements according to user defined control and
said voltage control is configured to reverse voltage in said voltage control circuit when said xyz controller has positioned the nanopipette tip within the single cell.

3. The system of claim 1 , where the first electrode comprises a silver material.

4. The system of claim 1 , wherein the first electrode comprises the first electrolyte solution, wherein the first electrolyte solution comprises a hydrophobic liquid and an ionic material comprising a borate.

5. The system of claim 1 , wherein the nanopipette is quartz.

6. A method for extracting a target intracellular content from an individual cell, comprising:
(a) preparing a solution containing at least one cell having a target intracellular content for extraction;
(b) providing the system of claim 1 and operating the system for:
i. manipulating the nanopipette to approach said one cell by sensing a drop in ionic current through the tip of the nanopipette;
ii. generating a positive voltage between the first electrode and the second electrode to prevent liquid from entering in the nanopipette during insertion into the cell;
iii. positioning the nanopipette to penetrate a predetermined cellular location and inserting it into the cell at a speed of 100 μm/sec;
iv. adjusting voltage between the first electrode and the second electrode in order to cause nanopipette influx and extract target intracellular content;
v. further adjusting the voltage in order to prevent extracted target intracellular content from leaving the nanopipette; and
vi. further adjusting the voltage to cause an efflux of the extracted target intracellular content to a sample container.

7. The method of claim 6 wherein the extracted target intracellular content comprises nucleic acids or proteins.

8. The method of claim 7 wherein the nucleic acids comprise DNA or RNA.

9. The method of claim 8 wherein the DNA is mitochondrial DNA.

10. The method of claim 6 wherein the voltages are one or more of (a) −100 to −1000 mV to cause influx of target intracellular content into the nanopipette;
(b) 100-500 mV to stop influx and prevent efflux from the nanopipette; and
(c) +0.5 to 2 V to cause efflux of the extracted target intracellular content.

11. The method of claim 10 wherein the influx in step (a) lasts between 1 and 5 seconds.

12. The method of claim 6 wherein the efflux is into a sample container containing a nucleic acid storage buffer.

13. The method of claim 6 wherein the extracted target intracellular content is DNA from a single mitochondrion and further comprising the step of determining a portion of the sequence of the mitochondrial DNA.

14. The method of claim 6 , further comprising:
analyzing the extracted target intracellular content by one or both of
(i) analyzing mRNA from the extracted target intracellular content; and
(ii) analyzing one or more selected proteins from the extracted target intracellular content.

15. The method of claim 14 wherein said analyzing mRNA includes analysis of at least 500 pg of mRNA from the cell.

16. The method of claim 14 wherein the analyzing mRNA includes analyzing at least 90% of the mRNA sequences in the cell.

17. The method of claim 14 wherein said analyzing a protein comprises an ultra-sensitive assay detecting a specific protein.

18. The method of claim 17 wherein the assay comprises proximity ligation.

19. The method of claim 14 , wherein said analyzing a protein species comprises analyzing at least one of EGFR, MKK1, ERK1/2, JAK, AP1/Jun, and STAT.

20. The method of claim 14 , further comprising distinguishing a phosphorylated protein from the same protein that is not phosphorylated.

21. The method of claim 14 , wherein the analyzing is carried out multiple times on the same cell at different times.

22. The method of claim 21 further comprising distinguishing a phosphorylated protein from the same protein that is not phosphorylated.

23. The method of claim 14 , wherein analyzing a protein species from the cell comprises distinguishing a protein having post-translational modification from the same protein that is not so modified.

24. The system of claim 3 , wherein the silver material is silver tetrakis(4-chlorophenyl)borate (AgTBACl).

25. The system of claim 4 , wherein the hydrophobic liquid is 1-2 dichlorethane.

26. The system of claim 4 , wherein the ionic material comprising a borate is tetrahexylammonium tetrakis(4-chlorophenyl)borate (THATPBCl).

27. The method of claim 16 wherein said analyzing a protein species comprises analyzing at least one of EGFR, MKK1, ERK1/2, JAK, AP1/Jun, and STAT.

28. The method of claim 17 wherein said analyzing a protein species comprises analyzing at least one of EGFR, MKK1, ERK1/2, JAK, AP1/Jun, and STAT.

29. The method of claim 18 wherein said analyzing a protein species comprises analyzing at least one of EGFR, MKK1, ERK1/2, JAK, AP1/Jun, and STAT.

30. The method of claim 16 , further comprising distinguishing a phosphorylated protein from the same protein that is not phosphorylated.

31. The method of claim 17 , further comprising distinguishing a phosphorylated protein from the same protein that is not phosphorylated.

32. The method of claim 18 , further comprising distinguishing a phosphorylated protein from the same protein that is not phosphorylated.

33. The method of claim 16 , wherein the analyzing is carried out multiple times on the same cell at different times.

34. The method of claim 17 , wherein the ana

Assignees

• Univ California

Inventors

• Actis Paolo
• Maalouf Michelle M
• Pourmand Nader

Classifications

• G01Q60/44

  SICM [Scanning Ion-Conductance Microscopy] or apparatus therefor, e.g. SICM probes · CPC title

• G01N2035/1034

  Transferring microquantities of liquid · CPC title

• C12M47/06

  Hydrolysis; Cell lysis; Extraction of intracellular or cell wall material (lysis of microorganisms C12N1/06; extracting or separating nucleic acids from biological samples C12N15/1003) · CPC title

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title

• G01N2333/91

  Transferases (2.) · CPC title