A trifluoromethyl-triggered J-aggregation switch for Cu+-Responsive fluorescence imaging and in vivo application in mice.

Herein, we elucidate the structural basis of J-aggregation in our previously reported dual-locked fluorescent probe HBimmCue, a trifluoromethyl-substituted sensor for neuronal mitochondrial Cu+. By designing a methyl-substituted control probe HBimmCH3, we demonstrate that the trifluoromethyl group is the critical moiety triggering J-aggregation in PBS. This aggregation enables HBimmCue to function through a photoinduced electron transfer (PET) + J-aggregation-caused quenching (J-ACQ) dual-quenching mechanism, affording an exceptional 59-fold fluorescence turn-on response. In contrast, HBimmCH3 lacks J-aggregation entirely and exhibits only a 13.2-fold enhancement via single PET quenching. DFT/TD-DFT calculations and electrostatic potential (ESP) analysis further corroborate the PET mechanism, energy-level shifts, and substituent-dependent assembly behaviors. Given its excellent photophysical performance, we further explored the in vivo imaging capability of HBimmCue. This work demonstrates a facile substituent-engineering approach to modulate J-aggregation, which provides valuable insights for the rational design of high-performance activatable fluorescent probes.