Research Article
Physiological validation of cell health upon probing with carbon nanotube endoscope and its benefit for single-cell interrogation

https://doi.org/10.1016/j.nano.2011.08.008Get rights and content

Abstract

New-generation nanoscale devices for single-cell study are intensively being developed. As has been shown, nanodevices are minimally invasive because of their order-of-magnitude smaller size in comparison to conventional glass pipettes. However, in most studies the evaluation of the nanodevice impact on cell health has not extended to their effects on cell metabolic integrity. In this work we evaluated the degree to which the insertion of a carbon-based nanotube endoscope into a cell induces mechanical and biochemical stress, and affects cellular key metabolic systems. The effects of insertion of the nanotube endoscope on cell morphological and physiological modulations were monitored and compared to those of glass micropipettes. We report that nanotube endoscope insertion does not significantly modulate the plasma membrane and actin network. The cell metabolic mechanisms such as energy production and inositol 1,4,5-trisphosphate–dependent calcium signaling remain preserved for prolonged endoscope presence within a cell.

From the Clinical Editor

In this basic science study, the effects of insertion of carbon nanotube endoscope on cell morphological and physiological modulations were monitored and compared to those of glass micropipettes. Nanotube endoscope insertion is truly minimally invasive: it does not significantly modulate the plasma membrane and actin network; the energy production and inositol 1,4,5-trisphosphate–dependent calcium signaling also remain preserved during prolonged endoscope presence within a cell.

Graphical abstract

The carbon nanotube–based endoscope with cylindrical, hollow geometry and flexible, conductive tips enables intracellular probing along with fluid transfer to and from single living cells. In this work we demonstrate that the insertion of probe tips into a cell body does not induce significant mechanical stress or affect cellular key metabolic systems in terms of their structural and functional integrity. Particularly, it is shown that the nanotube endoscope insertion does not significantly modulate the plasma membrane and actin network. The cell metabolic mechanisms such as energy production and IP3-dependent calcium signaling remain preserved for prolonged endoscope presence within a cell. The carbon nanotube endoscope opens a new realm of opportunities for single-cell and single-organelle machinery study, as well as for monitoring local communications between organelles in dynamics.

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Section snippets

Cell culture

A monolayer culture of human cervical carcinoma (HeLa) cells was grown to 85% confluence in Dulbecco's modified Eagle medium, supplemented with 10% donor horse serum and 1 mM l-glutamine, without antibiotics. Cells were maintained at 95% humidity, 37°C, and 5% CO2 atmosphere.

Measurements of cellular free calcium: confocal microscopy studies

HeLa cells seeded on MatTek glass-bottom dishes (MatTek Corp., Ashland, Massachusetts) were loaded with 2 μM Fluo-4AM cytosolic free calcium-sensitive dye (Invitrogen, Carlsbad, California) (excitation/emission wavelengths

Results

We have previously demonstrated the applicability of carbon-based nanoendoscopes for cell probing.14 No differences in the appearance of the cell membrane and modulation of cell homeostasis upon application of endoscope tips with outer diameter ranging from 50 to 200 nm have been observed. Most probably, the thickness of the nanotube wall ranging from 10 to 20 nm and its cylindrical geometry enable one to easily penetrate about 5–6 nm thickness of the plasma membrane. Additionally, the carbon

Discussion

In the present study we evaluated cellular physiological parameters as an evidence of the applicability of cylindrical nanotube endoscopes for single-cell study. Because of the carbon tip properties such as inertness and smoothness, the pipette does not become contaminated with cell constituents, and several experiments can be conducted at different locations within the cell body in one experiment. Minimal invasiveness of the endoscope makes it very convenient for navigation to different

Acknowledgments

The authors appreciate Prof. Alessandro Fatatis (Drexel University College of Medicine) for the inspiration to analyze the inositol phosphate signaling pathway and Prof. Gianluca Gallo for providing EYFP-β-actin plasmid (Drexel University College of Medicine).

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    This work was supported by a grant from the W.M. Keck Foundation to establish the Keck Institute for Attofluidic Nanotube-based Probes at Drexel University.

    1

    Current address: Argonne National Laboratory, Argonne, Illinois, USA.

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