Methods of non-destructive nanostraw intracellular sampling for longitudinal cell monitoring

Patent metadata

Field	Value
Publication number	US-2025382603-A1
Application number	US-202519296495-A
Country	US
Kind code	A1
Filing date	Aug 11, 2025
Priority date	Sep 13, 2016
Publication date	Dec 18, 2025
Grant date	—

Abstract

Methods and apparatuses to non-destructively and periodically sample a small quantity of intracellular proteins and mRNA from the same single cell or cells for an extended period of time. Specifically, describe herein are non-perturbative methods for time-resolved, longitudinal extraction and quantitative measurement of intracellular proteins and nucleic acids from a variety of cell types using systems including nanostraws.

First claim

We claim:

1 . A method of nondestructive sampling of intracellular sample material from within a cell at one or more time points, the method comprising:
introducing a cell to a cell culture chamber in contact with and on top of a nanostraw; applying a voltage of between an upper electrode and a lower electrode through the nanostraw to open one or more pores in a portion of the cell membrane extending over an opening of the nanostraw; capturing a sample material released from within the cell and into the nanostraw in a sample collector beneath the nanostraw; stopping the application of voltage between the upper and lower electrodes and allowing the cell membrane to recover for a minimum recovery time before reapplying the voltage and capturing additional sample material.

2 . The method of claim 1 , wherein the voltage is between 1 and 100 V.

3 . The method of claim 1 , wherein the capturing comprises immobilizing the sample material onto a capture substrate or capturing the sample in at least one sample collector beneath the nanostraw.

4 . The method of claim 1 , wherein the application of the voltage is stopped before more than 15% of the sample material within the cell is released and the cell membrane is allowed to recover before reapplying the voltage and capturing additional sample material.

5 . The method of claim 1 , wherein the nanostraw comprises a plurality of nanostraws, with one or more nanostraw in each of a plurality of sample regions.

6 . The method of claim 5 , wherein the capturing comprises immobilizing the sample material onto a capture substrate or capturing the sample in a plurality of sample collectors, each sample collector comprising the capture substrate and corresponding to each of the plurality of sample regions.

7 . The method of claim 6 , further comprising analyzing the captured sample material for one or more biomarker.

8 . The method of claim 1 , wherein each nanostraw is tubular in shape and has an outer diameter between about 20 nm to about 5 microns and is configured to transfer a pulsed voltage.

9 . The method of claim 1 , wherein the voltage is applied as a pulsed voltage through the nanostraw of between about 1 V and 100V, a pulse width of between about 10 microseconds and 50 milliseconds for a duration of between 1 second and 300 seconds.

10 . The method of claim 1 , wherein an outer diameter of the nanostraw is configured to support a cell without penetrating the cell's cell membrane.

11 . The method of claim 1 , wherein the introducing comprises introducing cell media and media-free buffer into the cell culture chamber and media-free buffer and/or cell media is introduced to the cell culture chamber after the application of voltage.

12 . A method of nondestructive sampling of intracellular sample material from within a cell at one or more time points, the method comprising:
applying a voltage of between 1 and 100 V between an upper electrode and a lower electrode through a nanostraw to open one or more pores in a portion of the cell membrane extending over an opening of the nanostraw; capturing a sample material released from within the cell and into the nanostraw in a sample collector beneath the nanostraw, wherein capturing comprises immobilizing the sample material onto a capture substrate; stopping the application of voltage between the upper and lower electrodes and allowing the cell membrane to recover before more than 15% of the sample material within the cell is released; and allowing the cell to recover for a minimum recovery time of at least 1 hour before reapplying the voltage and capturing additional sample material.

13 . A method of nondestructive sampling of intracellular sample material from within a cell at one or more time points, the method comprising:
applying a voltage between an upper electrode and a lower electrode through at least one nanostraw in each of a plurality of sample regions of a nanostraw substrate to open one or more pores through a cell membrane extending over an opening of each nanostraw; capturing sample material at each of the plurality of sample regions, wherein the sample material is released into the nanostraws to a plurality of sample collectors beneath the at least one nanostraw corresponding to each of the plurality of sample regions; stopping the application of voltage between the upper and lower electrodes; allowing the cell to recover for a minimum recovery time of at least 1 hour before reapplying the voltage and capturing additional sample material at each of the plurality of sample regions; and identifying a different biomarker from the captured sample material for each of the plurality of sample regions at different times.

Assignees

• Univ Leland Stanford Junior

Inventors

• Melosh Nicholas A
• Cao Yuhong
• Hjort Karl Martin
• Jonsson Amanda

Classifications

• C12Q1/24

  Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms · CPC title

• C12N13/00

  Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves · CPC title

• C12Q2523/307

  Denaturation or renaturation by electric current/voltage · CPC title

• C12M41/38

  of metabolites or enzymes in the cells · CPC title

• C12M35/02

  Electrical or electromagnetic means, e.g. for electroporation or for cell fusion · CPC title