Rational design of a ratiometric fluorescent probe for selective visualizing peroxynitrite in mitochondria and evaluation of organ injury

Abnormal peroxynitrite (ONOO−) expression is closely associated with the development of various pathological processes. In this study, we devised a series of fluorescent probes, namely DHU-DCM-1, DHU-DCM-2, and DHU-DCM-3, tailored specifically for monitoring ONOO− through rationally molecular design. By modulating the electron-donating site within the fluorophore structure, the optimal probe DHU-DCM-3 exhibited excellent ratiometric detecting performance. DHU-DCM-3 displayed high selectivity and sensitivity towards ONOO−, effectively distinguishing it from other reactive oxygen species. Notably, DHU-DCM-3 was capable of labeling mitochondrial ONOO−, producing an enhanced fluorescence ratio (F656/F550), thereby allowing for the visualization of ONOO− fluctuations in both exogenous and endogenous contexts within living cells. The utility of DHU-DCM-3 was further demonstrated in various applications. For instance, through detecting elevated levels of ONOO−, senile C. elegans was successfully identified by utilizing DHU-DCM-3. Moreover, relying on its ratiometric fluorescence, DHU-DCM-3 was proved to be valuable in accurately diagnosing liver injury in mice with varying degrees of severity. Overall, we have constructed a ratiometric fluorescent probe for detecting and imaging ONOO− in both subcellular organelle and in vivo, highlighting its potential significance in various biomedical applications.