A Pyridinium fluorophore for the detection of zinc ions under autophagy conditions

Molecular probes for sensing and imaging of various analytes and biological specimens are of great importance in clinical diagnostics, therapy, and disease management. Since the cellular concentration of free Zn²⁺ varies from nanomolar to micromolar range during cellular processes and the high affinity Zn²⁺ imaging probes tend to saturate at lower concentrations of free Zn²⁺, fluorescence based probes with moderate binding affinity are desirable in distinguishing the occurrence of higher zinc concentrations in the cells. Herein, we report a new, pentacyclic pyridinium based probe, PYD-PA, having a pendant N,N-di(pyridin-2-ylmethyl)amine (DPA) for Zn²⁺ detection in the cellular environment. The designed probe is soluble in water and serves as a mitochondria targeting unit, whereas the pendent DPA acts as the coordination site for Zn²⁺. PYD-PA displayed a threefold enhancement in fluorescence intensity upon Zn²⁺ binding with a 1:1 binding stoichiometry. Further, the probe showed a selective response to Zn²⁺ over other biologically relevant metal ions with a moderate binding affinity (K_a = 6.29 × 10⁴ M⁻¹), good photostability, pH insensitivity, and low cytotoxicity. The demonstration of bioimaging in SK-BR-3 breast cancer cell lines confirmed the intracellular Zn ion sensing ability of the probe. The probe was successfully applied for real time monitoring of the fluctuation of intracellular free zinc ions during autophagy conditions, demonstrating its potential for cellular imaging of Zn²⁺ at higher intracellular concentrations.