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. 2011 Oct 25;5(10):7848-57.
doi: 10.1021/nn201904t. Epub 2011 Sep 21.

Single-molecule detection of H₂O₂ mediating angiogenic redox signaling on fluorescent single-walled carbon nanotube array

Jong-Ho Kim  1 , Chitta Ranjan Patra, Jyoti R Arkalgud, Ardemis A Boghossian, Jingqing Zhang, Jae-Hee Han, Nigel F Reuel, Jin-Ho Ahn, Debabrata Mukhopadhyay, Michael S Strano
Affiliations

Single-molecule detection of H₂O₂ mediating angiogenic redox signaling on fluorescent single-walled carbon nanotube array

Jong-Ho Kim (V体育平台登录) et al. ACS Nano. .

Abstract

Reactive oxygen species, specifically hydrogen peroxide (H(2)O(2)), activate signal transduction pathways during angiogenesis and therefore play an important role in physiological development as well as various pathophysiologies. Herein, we utilize a near-infrared fluorescent single-walled carbon nanotube (SWNT) sensor array to measure the single-molecule efflux of H(2)O(2) from human umbilical vein endothelial cells (HUVEC) in response to angiogenic stimulation. Two angiogenic agents were investigated: the pro-angiogenic cytokine, vascular endothelial growth factor A (VEGF-A) and the recently identified inorganic pro-angiogenic factor, europium(III) hydroxide in nanorod form. The nanosensor array consists ofa SWNT embedded within a collagen matrix that exhibits high selectivity and sensitivity to single molecules of H(2)O(2). A calibration from 12. 5 to 400 nM quantifies the production of H(2)O(2) at nanomolar concentration in HUVEC with 1 s temporal and 300 nm spatial resolutions VSports手机版. We find that the production of H(2)O(2) following VEGF stimulation is elevated outside of HUVEC, but not for stimulation via nanorods, while increased generation is observed in the cytoplasm for both cases, suggesting two distinct signaling pathways. .

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Figures

Figure 1
Figure 1
Single molecule detection of H2O2 mediating aniogenic redox signaling. a) Schematic diagram of H2O2 production in angiogenesis and sensing platform. b) nIR fluorescence image of a SWNT/collagen sensor array showing emission from single isolated SWNT sensors. Inset: diffraction-limited spot (2×2 pixels) corresponding to a single SWNT. c) Representative fluorescence time-traces (red) in PBS with and without H2O2 (50 μM), showing clear stepwise fluorescence quenching.
Figure 2
Figure 2
Calibration of SWNT/collagen sensor for H2O2. a) Representative fluorescence time-trace (red) monitored for 20 min showing three transition states, b) four transition states, c) eight transition states, d) nine transition states upon addition of H2O2 (400 nM) in PBS. e) Correlation of the total number of transitions with H2O2 concentration for all 100-SWNT sensors. f) Calibration curve for H2O2 at nanomolar concentration from 12.5 to 400 nM after selecting the traces having sharp real-transitions, showing that the total number of transitions increases with increasing H2O2 concentration.
Figure 3
Figure 3
Single molecule detection of H2O2 produced from living HUVEC for angiogenic redox signaling. a) White-light picture of single cell stimulated by VEGF-A on the top of SWNT/collagen sensor array. b) 100 Diffraction-limited spots of SWNT underneath a cell, which are selected for analysis of fluorescence response to H2O2. c) Representative fluorescence time-traces (red) selectively responding to H2O2 produced under VEGF-A and Eu(OH)3 nanorods stimulation in HUVEC, showing stepwise quenching. d) Spatial distribution plot of the number of transitions after VEGF-A stimulation on HUVEC. e) The total number of transitions calculated from 100-selected SWNTs over the course of 20 min upon treatment of stimuli such as VEGF-A, Eu(OH)3 nanorods and Eu(OH)3/MnTBAP to HUVEC. f) Estimated concentration of H2O2 produced from HUVEC stimulated by VEGF-A, Eu(OH)3 nanorods and Eu(OH)3/MnTBAP using the calibration curve (Fig. 2e).
Figure 4
Figure 4
Intracellular detection of H2O2 in HUVEC after stimulation. a) White-light picture of cells and fluorescence image for unstimulated one, b) for VEGF-A, c) for Eu(OH)3 nanorods, d) for Eu(OH)3/MnTBAP. The results show that H2O2 production increases in the cytoplasm after stimulation. Caboxy-H2DCFDA (25 μM) was used for detection of H2O2 inside of cells.
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References

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