A targetable fluorescent probe for imaging hydrogen peroxide in the mitochondria of living cells

Abstract

We present the design, synthesis, and biological applications of mitochondria peroxy yellow 1 (MitoPY1), a new type of bifunctional fluorescent probe for imaging hydrogen peroxide levels within the mitochondria of living cells. MitoPY1 combines a chemoselective boronate-based switch and a mitochondrial-targeting phosphonium moiety for detection of hydrogen peroxide localized to cellular mitochondria. Confocal microscopy and flow cytometry experiments in a variety of mammalian cell types show that MitoPY1 can visualize localized changes in mitochondrial hydrogen peroxide concentrations generated by situations of oxidative stress.

Figure 1

Fluorescence responses of 5 µM MitoPY1 to various reactive oxygen species (ROS). Bars represent relative responses at 0, 5, 15, 30, 45, and 60 min after addition of each ROS. Data shown are for 10 mM O₂⁻, 200 µM NO, and 100 µM for all other ROS. Data were acquired at 25 °C in 20 mM HEPES, pH 7, with excitation λ = 503 nm and emission collected between 510 and 750 nm.

Figure 2

Confocal fluorescence images of live HeLa cells with increases in mitochondrial H₂O₂ levels visualized using MitoPY1. Images displayed represent emission intensities collected in optical windows between 527–601 nm upon excitation at 510 nm for MitoPY1. HeLa cells incubated with 5 µM MitoPY1 for 60 min at 37 °C and imaged with MitoPY1 (a), MitoTracker Red and Hoechst (overlay, b), and MitoPY1 with MitoTracker Red (overlay, c). HeLa cells incubated with 5 µM MitoPY1 for 60 min at 37 °C with 100 µM H₂O₂ added for the final 40 min and imaged with MitoPY1 (d), MitoTracker Red and Hoechst (overlay, e), MitoPY1 and MitoTracker Red (overlay, f), and brightfield (g) with 20 µm scale bar. HeLa cells incubated with 5 µM MitoPY1 for 60 min at 37 °C and imaged with MitoPY1 (h), MitoTracker Red and Hoechst (overlay, i), and MitoPY1 with MitoTracker Red (overlay, j). HeLa cells incubated for 24 h with 1 mM paraquat, then washed and incubated with 5 µM MitoPY1 for 60 min at 37 °C and imaged with MitoPY1 (k), MitoTracker Red and Hoechst (overlay, l), MitoPY1 and MitoTracker Red (overlay, m), and brightfield (n) with 20 µm scale bar.

Scheme 1

Synthesis and Activation of MitoPY1