A Coumarin-Based Probe for Sequential ON–OFF–ON Detection of Cu2+ and Biothiols: Naked-Eye Detection, Smartphone RGB Readout and In Vivo Imaging

Copper ions (Cu2+) and intracellular biothiols are tightly coupled in cellular redox regulation, where copper–thiol coordination governs oxidative stress and metal homeostasis. However, analytical platforms capable of sequentially monitoring Cu2+ and biothiols within a single molecular system remain scarce. Herein, we report a coumarin-based fluorescent probe XDP that enables sequential ON–OFF–ON sensing of Cu2+ and biothiols through a coordination–competition mechanism. The imine (C=N) site of XDP selectively coordinates Cu2+, leading to fluorescence quenching arising from coordination-induced electronic perturbation and enhanced nonradiative decay. The probe exhibits a linear response toward Cu2+ over 1–80 μM with a detection limit of 0.108 μM. Subsequent competitive binding of biothiols (GSH, Cys, and Hcy) releases Cu2+ from the complex, thereby restoring fluorescence and enabling detection within 1–30 μM with submicromolar sensitivity. XDP also displays a large Stokes shift (135 nm), which minimizes spectral overlap and improves signal reliability. Notably, Cu2+ binding triggers a distinct color change that supports naked-eye detection and smartphone-based RGB quantification. The probe further enables visualization of Cu2+ and thiol-triggered signal recovery in living cells and zebrafish. This work establishes a versatile analytical platform for probing copper–thiol interactions in environmental and biological systems.