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.

Figure 1

Nanotube sensing platform. a, A431 cell cultured on collagen-SWNT film. Zoom in on red circle: EGFR domains spanning the cell membrane. Domains I and III bind to EGF (red) and generate H₂O₂. b, NIR image of SWNT underneath A431 (left) and phase contrast image of A431 cell (right) cultured on SWNT sensors (658 nm excitation, 1mW, Alpha Plan-Apo 100x/1.46 oil emersion objective). c, Forward and reverse binding rate of SWNT sensor for various analytes show selectivity for H₂O₂. d, Fluorescence trace for control (no cells) show no steps. e, Trace for SWNT in the red circle in (b) show reversible, stepwise quenching (green trace), modeled by HMM (red).

Figure 2

Spatial mapping of quenching transitions over single A431 cells. a-d, Quenching activity (unit of counts) over the 3000s observation window of each sensor was binned into sixteen categories represented by 16 different color bars with red having the highest quenching activity and black the lowest for live (a, b) and fixed (c, d) A431 cells. e, Control experiment where 10μM H₂O₂ was present in the absence of a cell. Left panels show phase contrast images without the overlap of quenching activities. Fluorescence trace of green star (f) and dark blue star (g) from (a) are shown.

Figure 3

SWNT quenching depends of EGFR density. a, b, Real time quenching rate for live 3T3 cells (a) and live/fixed A431 cells (b). The number of sensors under each single cell is 255, 200, 250, 150, 255, 200 respectively for cell 1, 2, 3, 4, unstimulated and control in (a); 160, 110, 126, 174, 140, 180, 180, 200 respectively for cell 1, 2, 3, 4, fixed cell 1, 2, unstimulated and control in (b). Representative confocal images for 3T3 cells (c) and A431 cells (d) with EGFR (red) labeled with rabbit polyclonal antibody against EGFR and Alexa Fluor 568 donkey anti-rabbit IgG. Nuclei (blue) is stained with 4’,6-diamidino-2-phenylindole (DAPI).

Figure 4

Quantitative analysis of results from SWNT sensor array. a-d, Simulation of sensor response (a), rank-ordered sensor response from a (b), sensor response following beta distribution (c) and rank-ordered sensor response from c (d) of 10⁵ H₂O₂ randomly falling onto 300 sensors (blue), with additional response to local generation (red). After far-field component subtraction from the rank-ordered sensor response (black, -EGF; green, +EGF), the local generation before (blue, star) and after (red, star) EGF stimulation for live (e), fixed (f) A431 cell and live 3T3 cell (g). h, Real-time quenching rate for fixed A431, before (green) and after (blue) EGF stimulation. Sodium azide decreases the quenching, with (red) and without (black) EGF. Extending the singlet oxygen lifetime using D₂O increases the quenching (purple). Concentration profiles on log-log scale for different species from solving Reaction 1–8 (i) and from considering the effect of NaN₃ when solving the reaction network (j). k, Scheme of the proposed pathway for H₂O₂ generation.