US 10,696,962 B2Granted

Nanopipette device and method for subcellular analysis

AssigneeThe Regents of the University of California

InventorsPaolo Actis, Michelle M. Maalouf, Nader Pourmand

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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.

Claims45

Dependent claims

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.

12 more elements

Dependencies are inferred from the claim text.

Facts

Application: 14/775,168Filed 13 Mar 2014 · us provisional application
Priority: 14 Mar 2013
Published: US 2016/0032275 A14 Feb 2016
Granted: 30 Jun 2020
Term ends: Not estimatedThe source records don’t support an estimate.

Classification17 codes

C12N 15/1003
Processes for the isolation, preparation or purification of DNA or RNA: extracting or separating nucleic acids from biological samples, e.g. pure…
C12M 47/06
Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass: hydrolysis; Cell lysis;…
C12Q 1/6806
Involving nucleic acids: preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
C12Q 1/686
Nucleic acid amplification reactions: polymerase chain reaction [PCR]