BackgroundThe cellular endoplasmic reticulum (ER) is responsible for various functions, including protein synthesis, folding, distribution, and calcium ion storage. Studies have linked ER stress with acute lung injury (ALI), which can result in oxidative stress and even cell death. Peroxynitrite (ONOO−) is a well-known reactive oxygen species (ROS) that contributes to various physiological and pathological processes in oxidative stress diseases. To understand the role of ER ONOO− in ALI, it is crucial to accurately measure its level in the ER. Unfortunately, there is currently no probe available to detect ER ONOO− in an ALI model. ResultsTo address this, we developed three near-infrared (NIR) fluorescent probes (DCM-F-ONOO, DCM-Cl-ONOO, and DCM-Br-ONOO) for the detection of ONOO− using pentafluorobenzenesulfonate (PFBS) moieties as fluorescence quenchers. Through comprehensive testing, we selected DCM-Br-ONOO as the best NIR fluorescent probe due to its rapid response (within 3 min), high selectivity, good sensitivity (LOD = 2.3 nM), and approximately 66-fold enhanced response to ONOO− in fluorescence intensity. The probe was successfully applied to detect changes in ONOO− levels induced by different drugs in the ER of living cells. Importantly, a significant increase in the level of ONOO− was observed in the ER of an ALI cell model (4.5-fold) and an ALI mouse model (2.5-fold) using the probe, which is essential for understanding the role of ONOO− in ER-associated diseases. SignificanceUsing DCM-Br-ONOO as a probe, present work further validated that the elevated levels of ONOO− secretion were accompanied by the ALI progressed. These findings may provide valuable results for figuring out the biological roles that ONOO− played in ALI.