MitoNeoD: a mitochondria-targeted superoxide probe

Mitochondrial superoxide (O₂^⋅−) underlies much oxidative damage and redox signaling. Fluorescent probes can detect O₂^⋅−, but are of limited applicability in vivo, while in cells their usefulness is constrained by side reactions and DNA intercalation. To overcome these limitations, we developed a dual-purpose mitochondrial O₂^⋅− probe, MitoNeoD, which can assess O₂^⋅− changes in vivo by mass spectrometry and in vitro by fluorescence. MitoNeoD comprises a O₂^⋅−-sensitive reduced phenanthridinium moiety modified to prevent DNA intercalation, as well as a carbon-deuterium bond to enhance its selectivity for O₂^⋅− over non-specific oxidation, and a triphenylphosphonium lipophilic cation moiety leading to the rapid accumulation within mitochondria. We demonstrated that MitoNeoD was a versatile and robust probe to assess changes in mitochondrial O₂^⋅− from isolated mitochondria to animal models, thus offering a way to examine the many roles of mitochondrial O₂^⋅−production in health and disease.