A FRET-based ratiometric fluorescent chemosensor for mitochondrial ONOO⁻ detection with application to Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disorder characterized by progressive joint destruction, where early diagnosis is crucial for impeding disease progression. Peroxynitrite (ONOO⁻), a key reactive nitrogen species, is critically involved in the pathogenesis of RA and represents a promising diagnostic target for early detection. Herein, we developed MRN, a mitochondria-targeting ratiometric fluorescent probe based on a Förster resonance energy transfer (FRET) mechanism for the dynamic monitoring of ONOO⁻ in RA inflammatory models. The probe integrates a rhodamine-naphthalimide FRET pair and a hydrazide group as the specific recognition unit, which facilitates a rapid, ONOO⁻-triggered ratiometric response within 30   seconds and achieves a detection limit of 7.5   nM. Furthermore, the conjugation of a triphenylphosphonium group ensures precise mitochondrial localization. Cellular assays demonstrated excellent biocompatibility and specific mitochondrial targeting. We successfully applied MRN for real-time ratiometric imaging of both endogenous and exogenous ONOO⁻ fluctuations in live BV-2 cells. Importantly, in a Complete Freund's Adjuvant (CFA)-induced rheumatoid arthritis mouse model, MRN enabled in situ ratiometric fluorescence imaging of ONOO⁻ at articular sites, revealing a strong positive correlation between the fluorescence signal intensity and the degree of inflammatory severity. This work establishes MRN as a novel and robust molecular tool for the early diagnosis of RA.