Styryl pyridinium fluorophores with sequential plasma membrane-to-mitochondria targeting and application to sulphite sensing in live cells

The development of fluorescent probes that integrate spatiotemporal organelle targeting with analyte sensing remains a significant challenge. Herein, we report a modular library of four styryl pyridinium fluorophores (Py-Cz-CN, Py-Cz, Py-Am-CN, and Py-Am) based on a donor-π-acceptor (D-π-A) architecture for sequential organelle targeting and sulfite sensing. All four probes exhibited rapid and selective plasma membrane localization, with high colocalization coefficients (PCC = 0.81-0.88). Notably, Py-Cz underwent spontaneous time-dependent translocation from the plasma membrane to mitochondria over 6 h, with the Pearson's correlation coefficient increasing from 0.15 to 0.87, and this mitochondrial accumulation was confirmed to be ΔΨ_m-dependent by CCCP-induced membrane depolarization. In contrast, cyano substitution at the vinyl bridge preserved plasma membrane localization while introducing a Michael acceptor for selective reaction-based fluorescence turn-off sensing of bisulfite/sulfite. Py-Cz-CN exhibited high sensitivity toward bisulfite (LOD = 5.9 μM) and enabled both exogenous and endogenous sulfite imaging in live cells. DFT and TDDFT calculations further supported the excited-state characteristics and sensing mechanism. This work establishes a simple molecular design strategy for combining organelle-specific trafficking with analyte-responsive fluorescence in multifunctional small-molecule probes.