Recent developments in detection of superoxide radical anion and hydrogen peroxide: Opportunities, challenges, and...

In this review, some of the recent developments in probes and assay techniques specific for superoxide (O₂^-) and hydrogen peroxide (H₂O₂) are discussed. Over the last decade, significant progress has been made in O₂^- and H₂O₂ detection due to syntheses of new redox probes, better understanding of their chemistry, and development of specific and sensitive assays. For superoxide detection, hydroethidine (HE) is the most suitable probe, as the product, 2-hydroxyethidium, is specific for O₂^-. In addition, HE-derived dimeric products are specific for one-electron oxidants. As red-fluorescent ethidium is always formed from HE intracellularly, chromatographic techniques are required for detecting 2-hydroxyethidium. HE analogs, Mito-SOX and hydropropidine, exhibit the same reaction chemistry with O₂^- and one-electron oxidants. Thus, mitochondrial superoxide can be unequivocally detected using HPLC-based methods and not by fluorescence microscopy. Aromatic boronate-based probes react quantitatively with H₂O₂, forming a phenolic product. However, peroxynitrite and hypochlorite react more rapidly with boronates, forming the same product. Using ROS-specific probes and HPLC assays, it is possible to screen chemical libraries to discover specific inhibitors of NADPH oxidases. We hope that rigorous detection of O₂^- and H₂O₂ in different cellular compartments will improve our understanding of their role in redox signaling.