Methods of non-destructive nanostraw intracellular sampling for longitudinal cell monitoring
Inventors
Melosh, Nicholas A. • Cao, Yuhong • HJORT, Karl Martin • Jonsson, Amanda
Assignees
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Publication Number
US-12416000-B2
Publication Date
2025-09-16
Expiration 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.
Core Innovation
The invention provides a system for nondestructively sampling intracellular material from living cells over time. A cell culture chamber has a first region and a second region, and a nanostraw substrate is positioned over the second region. The nanostraw substrate comprises a plurality of sample regions, where each sample region is adapted to capture sample material, and each sample region includes a plurality of tubular nanostraws that extend through the substrate.
Each nanostraw is configured to transfer a pulsed voltage while supporting a cell, with an outer diameter configured to support cell presence without penetrating a cell’s cell membrane. Sample material collectors correspond to the sample regions and include a sample material capture substrate configured to bind to the sample material. The system includes first and second electrodes located in the first and second regions, respectively, and a controller applies the pulsed voltage through the plurality of nanostraws.
The controller is configured to apply pulsed voltage of between about 1 V and 100 V with a pulse width of between about 10 microseconds and 50 milliseconds for a duration of between 1 second and 300 seconds. The system enables repeated sampling of intracellular proteins and mRNA from living cells over days while maintaining cell viability, using an approach that supports longitudinal, time-resolved collection from living cells.
Claims Coverage
The partial content includes two independent claims, both directed to a system for nondestructively sampling intracellular material using a nanostraw substrate, electrodes in separate chamber regions, and pulsed-voltage control with specified voltage, pulse width, and duration. The key inventive features differ mainly in the nanostraw-region geometry and the specific cell-membrane interaction language, and both claims require removable or region-matched collectors with a capture substrate.
Nondestructive intracellular sampling system with first and second chamber regions
A cell culture chamber having a first region and a second region used with a nanostraw substrate positioned over the second region for nondestructively sampling intracellular material.
Nanostraw substrate with multiple sample regions and tubular nanostraws transferring pulsed voltage
A nanostraw substrate positioned over the second region comprising a plurality of sample regions adapted to capture sample material, where each sample region includes a plurality of tubular nanostraws extending through the nanostraw substrate and configured to transfer a pulsed voltage.
Cell-supporting nanostraw outer diameter constraints
Nanostraws with an outer diameter between about 20 nm to about 5 microns in one claim and between about 20 nm to about 1500 nm in the other claim, where the claim language additionally includes that the outer diameter is configured to support a cell without penetrating a cell’s cell membrane in the second independent claim.
Sample material collectors with sample material capture substrate binding to sample material
A plurality of sample material collectors, each corresponding to one sample region and comprising a sample material capture substrate configured to bind to the sample material; in the second independent claim, the collectors are removable.
Electrode placement in first and second regions with controller-applied pulsed voltage
A first electrode in the first region and a second electrode in the second region, coupled to a controller configured to apply a pulsed voltage through the plurality of nanostraws between about 1 V and 100 V with a pulse width between about 10 microseconds and 50 milliseconds for a duration between 1 second and 300 seconds.
Pattern of recessed sample regions
A nanostraw substrate comprising a pattern of recessed sample regions adapted to capture sample material in the second independent claim.
Across both independent claims, the inventive core is a nondestructive intracellular sampling system that combines a region-partitioned cell culture chamber, a nanostraw substrate with multiple sample regions, region-matched sample collectors with a capture substrate, and electrodes controlled by a controller to apply defined pulsed-voltage parameters through the nanostraws; the second independent claim further specifies recessed sample regions and explicit language that the nanostraw outer diameter supports a cell without penetrating the cell membrane, with removable collectors.
Stated Advantages
Nondestructively sampling intracellular material.
Supports sampling without penetrating a cell’s cell membrane (as stated in the second independent claim language).
Documented Applications
Longitudinal repeated sampling of intracellular proteins and mRNA from living cells over days, including hiPSC-derived cardiomyocytes and other described cell types, with agreement to lysed-cell controls.
Spatial mapping of sampled cytoplasmic regions from living cells.
Detection of non-fluorescent proteins (e.g., HSP27 via ELISA) and mRNA panels from hiPSC-derived cardiomyocytes.
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