A Palette of Fluorescent Probes with Varying Emission Colors for Imaging Hydrogen Peroxide Signaling in Living Cells

Abstract

We present a new family of fluorescent probes with varying emission colors for selectively imaging hydrogen peroxide (H 2 O 2 ) generated at physiological cell signaling levels. This structurally homologous series of fluorescein-and rhodol-based reporters relies on a chemospecific boronate-tophenol switch to respond to H 2 O 2 over a panel of biologically relevant reactive oxygen species (ROS) with tunable excitation and emission maxima and sensitivity to endogenously produced H 2 O 2 signals, as shown by studies in RAW 264.7 macrophages during the phagocytic respiratory burst and A431 cells in response to EGF stimulation. We further demonstrate the utility of these reagents in multicolor imaging experiments by using one of the new H 2 O 2 -specific probes, Peroxy Orange 1 (PO1), in conjunction with the green-fluorescent highly reactive oxygen species (hROS) probe, APF. This dual-probe approach allows for selective discrimination between changes in H 2 O 2 and hypochlorous acid (HOCl) levels in live RAW 264.7 macrophages. Moreover, when macrophages labeled with both PO1 and APF were stimulated to induce an immune response, we discovered three distinct types of phagosomes: those that generated mainly hROS, those that produced mainly H 2 O 2 , and those that possessed both types of ROS. The ability to monitor multiple ROS fluxes simultaneously using a palette of different colored fluorescent probes opens new opporunities to disentangle the complex contributions of oxidation biology to living systems by molecular imaging.

Epidermal growth factor-induced hydrogen peroxide production is mediated by dual oxidase 1

“…Our data provide the missing link between agonist-evoked calcium signals and ROS production, which were previously described to be connected in epidermal cell lines (9). EGF-induced H 2 O 2 production by A431 cells was originally described by Bae et al (8) and was subsequently confirmed by several other groups (22), (23). In a recent work, the phagocytic NADPH oxidase, Nox2 was detected in A431 cells, however no attempt was made to establish a correlation between ROS production and Nox2 expression (24).…”

Aquaporin-3 mediates hydrogen peroxide uptake to regulate downstream intracellular signaling

“…Accordingly, we developed a unique H 2 O 2 -specific fluorescent indicator, PY1-ME (Fig. S1A), which combines a boronate-masked phenol for the selective detection of H 2 O 2 (10,(33)(34)(35)(36)(37)(38)(39) and an appended methyl-ester group designed to increase cellular uptake and retention upon cleavage by intracellular esterases; this latter feature makes the dye suitable for both fluorescence imaging and flow cytometry experiments. Because this probe relies on a specific molecular reaction between H 2 O 2 and the boronate to generate a fluorescence signal, rather than thiol-based chemistry that can occur by non-specific ROS oxidation or disulfide exchange, our chemical method affords a direct measure of H 2 O 2 in cellular specimens.…”

The invalidity of the photo-induced electron transfer mechanism for fluorescein derivatives

“…3 Thus, for a long period of time, the carboxylic group was considered to be indispensable for strong fluorescence and was included in almost all fluorescein-based fluorescence probes. [4][5][6][7][8][9][10][11] Recently, T. Nagano et al [12][13][14][15][16][17] proposed that the fluorescein molecule can be regarded as a directly linked donoracceptor system, where the benzene moiety and the xanthene moiety were the electron donor and acceptor, respectively. The fluorescence quantum yield (F fl ) of fluorescein is dependent on photoinduced electron transfer (PeT) from donor to acceptor.…”