A Reliable Ratiometric Fluorescent Probe for the Quantification of Mitochondrial Peroxynitrite in Cells and Tissues

Abstract Peroxynitrite (ONOO−) is a reactive oxygen species predominantly generated in mitochondria. Its concentration-dependent dynamics exhibit complex bidirectional effects, making quantitative measurement essential for understanding its pathological roles. Despite advances in fluorescent probes for ONOO− imaging, design limitations and interference from complex cellular environments still compromise detection reliability. Herein, we report the first reliable ratiometric fluorescent probe RDC for quantitative detection of ONOO− in mitochondria. Under single-wavelength excitation, the probe exhibits two emission peaks at 563 nm and 690 nm. Upon reaction with ONOO−, the 690 nm signal remains constant as an internal reference, while the 563 nm signal increases significantly, allowing the ratio (F563/F690) to achieve quantitative detection. The probe exhibits excellent selectivity and high sensitivity (LOD = 96 nM). Cell imaging reveals a linear relationship between the ratiometric signal and exogenous ONOO− concentration, validating its quantitative capability in living cells. Moreover, during pyroptosis, the probe reveals a marked increase in mitochondrial ONOO− levels, which is reversed by a pyroptosis inhibitor, indicating ONOO− as a key effector in pyroptosis. The probe also enables ONOO− detection in tissue samples, demonstrating its potential in complex biological systems. This probe serves as a reliable tool for quantifying mitochondrial ONOO− in cells and tissues and for studying its roles in pyroptosis and related diseases.