Tailoring a Dual-Response Site Ratiometric Luminescent Probe Based on a Ruthenium(II) Complex–Benzothiazole Scaffold for Monitoring Intracellular pH Changes

Organelle-specific pH represents a crucial parameter associated with cellular homeostasis and pathological conditions. Dysregulated intracellular pH has been manifested in a myriad of diseases. Ratiometric luminescence imaging enables precise spatiotemporal mapping of pH dynamics in living systems, offering better accuracy compared with single-wavelength approaches. However, ratiometric probes still face several intractable challenges: (1) significant spectral overlap under a single-wavelength excitation; and (2) single recognition site design, resulting in minimal luminescence ratio variations within the physiological pH range of organelles (4.0-8.0). Herein, a dual-site ratiometric luminescent probe, Ru-HBT, was rationally designed by coupling a Ru(II)-polypyridyl complex with a pH-sensitive benzothiazole derivative (one site) via an amino group as a linker (another site). This molecularly engineered probe exhibited a large emission shift (∼130 nm) under single-wavelength excitation (λex = 405 nm) and a luminescence lifetime change (Δτ = 226 ns) across the pH range of 4.0-9.0. By integrating dual-recognition mechanisms, including the photoinduced electron transfer (PET) and excited-state intramolecular proton transfer (ESIPT), Ru-HBT achieved a remarkable 354-fold emission intensity ratio change within the organelle-relevant pH range, significantly enhancing its detection sensitivity. Ru-HBT was validated as an organelle pH sensor in dexamethasone-induced apoptosis and ammonia-mediated cell death models, effectively monitoring lysosomal membrane permeability increases linked to pathological pH dysregulation.