Visualization of peroxynitrite/GSH cross-talk in the oxidant-antioxidant balance by a dual-fluorophore and dual-site based mito-specific fluorescent probe

Peroxynitrite (ONOO-) and glutathione (GSH) play mutually regulating roles in the oxidant-antioxidant balance of organisms, which has a profound relationship with people's health and disease. In this study, we designed a two-photon fluorescent probe CD-NA that could simultaneously detect ONOO- and GSH via dual-fluorophore and dual-site properties. CD-NA shows different fluorescence responses to ONOO- (annihilated red fluorescence) and GSH (enhanced green emission) with high specificity and sensitivity. Notably, the response of CD-NA to ONOO- was unaffected by GSH, and the reverse is also true. It allows the ONOO-/GSH cross-talk to be successfully imaged. Given these excellent properties, CD-NA has been favorably employed in detecting ONOO- and GSH in living cells with the ability to target mitochondria. Therefore, CD-NA offers an efficient method for understanding the oxidant-antioxidant balance and interrelated physiological functions of ONOO- and GSH in living systems, and provides a new strategy to sort out the complex relationships and roles of various analytes in complex physiological processes. By employing the dual-fluorophore and dual-site strategy, a two-photon mito-specific fluorescence probe was designed for detecting both ONOO- and GSH. It provides a method to sort out the relationships of various analytes in physiological processes.