Detection of single-molecule H2O2 signalling from epidermal growth factor receptor using fluorescent single-walled carbon nanotubes

Abstract

An emerging concept in cell signalling is the natural role of reactive oxygen species such as hydrogen peroxide (H2O2) as beneficial messengers in redox signalling pathways. The nature of H2O2 signalling is confounded, however, by difficulties in tracking it in living systems, both spatially and temporally, at low concentrations. Here, we develop an array of fluorescent single-walled carbon nanotubes that can selectively record, in real time, the discrete, stochastic quenching events that occur as H2O2 molecules are emitted from individual human epidermal carcinoma cells stimulated by epidermal growth factor. We show mathematically that such arrays can distinguish between molecules originating locally on the cell membrane from other contributions. We find that epidermal growth factor induces 2 nmol H2O2 locally over a period of 50 min. This platform promises a new approach to understanding the signalling of reactive oxygen species at the cellular level.

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Figure 1: Nanotube sensing platform.
Figure 2: Spatial mapping of quenching transitions over single A431 cells.
Figure 3: SWNT quenching depends of EGFR density.
Figure 4: Quantitative analysis of results from SWNT sensor array.

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Acknowledgements

M.S.S is grateful for a Beckman Young Investigator Award and a National Science Foundation (NSF) Career Award. This work was funded under the NSF Nanoscale Interdisciplinary Research Team on single-molecule detection in living cells using carbon nanotube optical probes. Part of this work was supported by the national grants Ministry of Education of the Czech Republic project no. MSM0021620806 and KAN grant no. 400100701. The authors thank S. Tannenbaum, G.Wogan and L. Trudel and acknowledge a seed grant from the Center for Environmental Health Sciences at MIT. We also thank M. Balastik at Harvard Medical School for assistance with the confocal experiments, K.D. Wittrup, G. Stephanopoulos, J.-H. Ahn, J.-H Han at Chemical Engineering at MIT, S. Sheffield, Mathematics Department, MIT, and Y. Li at University of Illinois Urbana Champaign for helpful discussions.

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H.J. and M.S. conceived the experiments, derived the models and wrote the manuscript. H.J. performed the experiments and analysed the data. D.H., M.K., J.-H.K., J.Z. and A.B. all assisted in the experiments. H.J. and M.S. co-wrote the paper with input from N.M.

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Correspondence to Michael S. Strano.

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Jin, H., Heller, D., Kalbacova, M. et al. Detection of single-molecule H2O2 signalling from epidermal growth factor receptor using fluorescent single-walled carbon nanotubes. Nature Nanotech 5, 302–309 (2010). https://doi.org/10.1038/nnano.2010.24

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