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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Imlay, J. A. Cellular defenses against superoxide and hydrogen peroxide. Annu. Rev. Biochem. 77, 755–776 (2008).
Veal, E. A., Day, A. M. & Morgan, B. A. Hydrogen peroxide sensing and signaling. Mol. Cell 26, 1–14 (2007).
Belousov, V. V. et al. Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. Nature Methods 3, 281–286 (2006).
Casanova, D. et al. Single europium-doped nanoparticles measure temporal pattern of reactive oxygen species production inside cells. Nature Nanotech. 4, 581–585 (2009).
Lee, D. et al. In vivo imaging of hydrogen peroxide with chemiluminescent nanoparticles. Nature Mater. 6, 765–769 (2007).
Miller, E. W., Tulyanthan, O., Isacoff, E. Y. & Chang, C. J. Molecular imaging of hydrogen peroxide produced for cell signaling. Nature Chem. Biol. 3, 263–267 (2007).
Zhou, M., Diwu, Z., Panchuk-Voloshina, N. & Haugland, R. P. A stable nonfluorescent derivative of resorufin for the fluorometric determination of trace hydrogen peroxide: applications in detecting the activity of phagocyte NADPH oxidase and other oxidases. Anal. Biochem. 253, 162–168 (1997).
Hong, Y., Blackman, N. M. K., Kopp, N. D., Sen, A. & Velegol, D. Chemotaxis of nonbiological colloidal rods. Phys. Rev. Lett. 99, 178103 (2007).
Cognet, L. et al. Stepwise quenching of exciton fluorescence in carbon nanotubes by single-molecule reactions. Science 316, 1465–1468 (2007).
Jin, H., Heller, D. A., Kim, J.-H. & Strano, M. S. Stochastic analysis of stepwise fluorescence quenching reactions on single-walled carbon nanotubes: single molecule sensors. Nano Lett. 8, 4299–4304 (2008).
Heller, D. A. et al. Multimodal optical sensing and analyte specificity using single-walled carbon nanotubes. Nature Nanotech. 4, 114–120 (2009).
Yarden, Y. & Sliwkowski, M. X. Untangling the ErbB signalling network. Nature Rev. Mol. Cell Biol. 2, 127–137 (2001).
Herbst, R. S. Review of epidermal growth factor receptor biology. Int. J. Radiat. Oncol. Biol. Phys. 59, 21–26 (2004).
Lax, I. et al. Functional-analysis of the ligand-binding site of EGF-receptor utilizing chimeric chicken human receptor molecules. EMBO J. 8, 421–427 (1989).
Masui, H., Castro, L. & Mendelsohn, J. Consumption of EGF by A431 cells—evidence for receptor recycling. J. Cell Biol. 120, 85–93 (1993).
Carpenter, G. & Cohen, S. Epidermal growth-factor. Annu. Rev. Biochem. 48, 193–216 (1979).
Bae, Y. S. et al. Epidermal growth factor (EGF)-induced generation of hydrogen peroxide—role in EGF receptor-mediated tyrosine phosphorylation. J. Biol. Chem. 272, 217–221 (1997).
Foote, C. S. Free Radicals in Biology (Pryor, W. A., ed.) 85–133 (Academic, 1976).
Ziyatdinova, G. K., Gil'metdinova, D. M. & Budnikov, G. K. Reactions of superoxide anion radical with antioxidants and their use in voltammetry. J. Anal. Chem. 60, 49–52 (2005).
Kim, J.-H. et al. The rational design of nitric oxide selectivity in single-walled carbon nanotube near infrared fluorescence sensors for biological detection. Nature Chem. 1, 473–481 (2009).
DeYulia, G. J., Carcamo, J. M., Borquez-Ojeda, O., Shelton, C. C. & Golde, D. W. Hydrogen peroxide generated extracellularly by receptor–ligand interaction facilitates cell signaling. Proc. Natl Acad. Sci. USA 102, 5044–5049 (2005).
Morazzani, M. et al. Monolayer versus aggregate balance in survival process for EGF-induced apoptosis in A431 carcinoma cells: implication of ROS-P38 mapk-integrin A2B1 pathway. Int. J. Cancer 110, 788–799 (2004).
Park, H. S. et al. Sequential activation of phosphatidylinositol 3-kinase, beta Pix, Rac1, and Nox1 in growth factor-induced production of H2O2 . Mol. Cell. Biol. 24, 4384–4394 (2004).
Ramey, N. A., Park, C. Y., Gehlbach, P. L. & Chuck, R. S. Imaging mitochondria in living corneal endothelial cells using autofluorescence microscopy. Photochem. Photobiol. 83, 1325–1329 (2007).
Welsher, K., Liu, Z., Daranciang, D. & Dai, H. Selective probing and imaging of cells with single walled carbon nanotubes as near-infrared fluorescent molecules. Nano Lett. 8, 586–590 (2008).
Nieva, J. & Wentworth, P. The antibody-catalyzed water oxidation pathway—a new chemical arm to immune defense? Trends Biochem. Sci. 29, 274–278 (2004).
Carpenter, G. The EGF Receptor Family: Biologic Mechanisms and Role in Cancer 33–60 (Academic Press, 2003).
Harbour, J. R. & Issler, S. L. Involvement of the azide radical in the quenching of singlet oxygen by azide anion in water. J. Am. Chem. Soc. 104, 903–905 (1982).
Kuimova, M. K., Yahioglu, G. & Ogilby, P. R. Singlet oxygen in a cell: spatially dependent lifetimes and quenching rate constants. J. Am. Chem. Soc. 131, 332–340 (2009).
Juarez, J. C. et al. Superoxide dismutase 1 (SOD1) is essential for H2O2-mediated oxidation and inactivation of phosphatases in growth factor signaling. Proc. Natl Acad. Sci. USA 105, 7147–7152 (2008).
Yang, J. L., Wang, L. C., Chang, C. Y. & Liu, T. Y. Singlet oxygen is the major species participating in the induction of DNA strand breakage and 8-hydroxydeoxyguanosine adduct by lead acetate. Environ. Mol. Mutagen. 33, 194–201 (1999).
Fridovich, I. Biology of oxygen radicals. Science 201, 875–880 (1978).
Imlay, J. A., Chin, S. M. & Linn, S. Toxic DNA damage by hydrogen peroxide through the fenton reaction in vivo and in vitro. Science 240, 640–642 (1988).
Halliwell, B. & Aruoma, O. I. DNA damage by oxygen-derived species—its mechanism and measurement in mammalian systems. FEBS Lett. 281, 9–19 (1991).
Khan, A. U. & Kasha, M. Singlet molecular-oxygen in the Haber–Weiss reaction. Proc. Natl Acad. Sci. USA 91, 12365–12367 (1994).
Hatz, S., Lambert, J. D. C. & Ogilby, P. R. Measuring the lifetime of singlet oxygen in a single cell: addressing the issue of cell viability. Photochem. Photobiol. Sci. 6, 1106–1116 (2007).
Hardwick, T. J. The rate constant of the reaction between ferrous ions and hydrogen peroxide in acid solution. Canadian J. Chem. 35, 428–436 (1957).
Wentworth, P. et al. Antibody catalysis of the oxidation of water. Science 293, 1806–1811 (2001).
Wentworth, A. D., Jones, L. H., Wentworth, P., Janda, K. D. & Lerner, R. A. Antibodies have the intrinsic capacity to destroy antigens. Proc. Natl Acad. Sci. USA 97, 10930–10935 (2000).
Imlay, J. A. Pathways of oxidative damage. Annu. Rev. Microbiol. 57, 395–418 (2003).
Zanthoff, H. & Baerns, M. Oxidative coupling of methane in the gas phase. Kinetic simulation and experimental verification. Ind. Eng. Chem. Res. 29, 2–10 (2002).
Zhu, J. Y., Dittmeyer, R. & Hofmann, H. Application of sensitivity analysis to the reduction of a complex kinetic model for the homogeneous oxidative coupling of methane. Chem. Eng. Proc. 32, 167–176 (1993).
Mizukawa, H. & Okabe, E. Inhibition by singlet molecular oxygen of the vascular reactivity in rabbit mesenteric artery. Br. J. Pharmacol. 121, 63–70 (1997).
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.
The authors declare no competing financial interests.
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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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