Binding Energy Difference-Directed Sequential Fluorescence Switching with a Built-In Threshold for Multicolor Sensing

Multicolor fluorescent sensors offer self-calibration and visual readout, but their performances across broad concentration ranges are limited by ambiguous switching boundaries in segmented quantification. Alternately, the sequential response design could extend the quantification range but lacked well-defined boundaries; however, the threshold-switching strategy with high-contrast jumps could not span broad ranges. In this work, we proposed a strategy driven by binding energy differences: two recognition units with substantially different binding energies toward the same analyte were integrated into a single probe. The analyte preferentially occupied the high binding energy sites, and their saturation served as an intrinsic threshold that triggered the secondary response, enabling both sequential activation and threshold switching. A Eu-metal−organic framework (MOF)-on-Zn-MOF composite (ZPE) was constructed as a model system with tetracycline antibiotics (TCs) as targets. DFT calculations confirmed that TC (tetracycline) bound to Eu-MOF with substantially higher binding energy (−26.83 kcal/mol) than to Zn-MOF (−6.8 kcal/mol). At low concentrations, the 617 nm emission of Eu-MOF was enhanced via the antenna effect, while the 420 nm emission of Zn-MOF was quenched by the inner filter effect. Upon saturation of Eu-MOF sites, excess TC accumulated in the Zn-MOF pores, triggering aggregation-induced emission at 515 nm. The two distinct intervals with independent ratiometric modes (617/420 and 515/617) gave a detection limit of 26 nM. This work provided a new paradigm for resolving the boundary precision issue in segmented quantification of multicolor fluorescent sensors.