Rational Design of Sequentially Activated Dual-Locked Near-Infrared Fluorescent Probe for Simultaneous Imaging of Cerebral Amyloid-β Plaques and ClO ^– in Alzheimer’s Disease

Alzheimer’s disease (AD) is a complex neurodegenerative disorder mediated by multiple pathological factors, including amyloid-β (Aβ) plaque deposition and oxidative stress. Herein, we report a new class of sequentially activated dual-lock-responsive fluorescent probes, D-BDY and T-BDY, which were designed by integrating pyrrole-based electron-donating units and molecular rotors into the BODIPY scaffold. This strategic design enabled photoinduced electron transfer (PET) and twisted intermolecular charge transfer (TICT) modulated fluorescence responses to both Aβ 1–42 aggregates and hypochlorite (ClO – ). The probes exhibited remarkable fluorescence enhancement (50–100-fold) when exposed to a mixed solution containing both ClO – and Aβ 1–42 aggregates. Confocal imaging and staining of Aβ-containing brain slices revealed that the probes preferentially bind to the dense β-sheet-rich cores of plaques, and their colocalization with thioflavin-T (ThT) was further enhanced following ClO – activation. Furthermore, in vivo imaging in an APP/PS1 transgenic AD mouse model demonstrated that D-BDY successfully crossed the blood-brain barrier (BBB), enabling real-time monitoring of Aβ 1–42 plaques and the oxidative microenvironment. The observed fluorescence intensity exhibited a strong correlation with pathological severity and was significantly attenuated upon antioxidant treatment. These results established D-BDY as a promising single-molecule dual-locked fluorescent probe capable of sensitively visualizing Aβ 1–42 aggregation and ClO – -related oxidative stress in vivo, offering a valuable tool for AD diagnosis and pathological progression monitoring.