Engineering diphenyl-coumarin-amide BioAIEgens for selective Cu²⁺ detection and real-time imaging of mitophagy

Coumarins are widely used as natural fluorescent dyes in bioimaging, and integrating them with aggregation-induced emission (AIE) properties enhances their luminescence performance. However, the rational design of coumarin-based AIE probes for Cu²⁺ recognition remains a challenge. Notably, Cu²⁺ concentrations undergo dynamic changes during autophagy, yet the research on tracking this process in biological systems is still limited.

In this study, nine diphenylamine-coumarin derivatives bearing various amide linkages (SL-1 to SL-9) were synthesized and systematically characterized. All compounds exhibited significant intramolecular charge transfer (ICT) characteristics, excellent photothermal stability, and markedly enhanced fluorescence emission in viscous environments. Notably, the dyes incorporating amide or symmetrical dihydrazide bonds displayed higher radiative attenuation efficiency compared to those with carbonyl hydrazine linkages. Furthermore, the compounds that exhibited strong fluorescence in the solid state also demonstrated aggregation-induced emission (AIE) properties. Among them, SL-9 was selected as a representative probe due to its high selectivity, rapid response, excellent sensitivity for detecting Cu²⁺, and outstanding stability. Additional studies revealed that SL-9 possesses good biocompatibility and enables real-time monitoring of dynamic changes in Cu²⁺ during autophagy in living cells (HeLa) and zebrafish.

This study presents a modular strategy based on a diphenyl-coumarin-amide framework. The proposed design principle and mechanism offer a novel approach for the development of high-performance fluorescent probes, with promising applications in bioimaging, metal ion sensing, and environmental monitoring.