Dual-Mode Turn-On Fluorescent and Ratiometric ElectrochemicalDetection of Cu2 + in Alzheimer’s DiseaseMouse Brains Based on a Single Resorufin–Picolinate Probe

Copper ions (Cu2+) are closely implicated in the pathogenesis of Alzheimer's disease (AD) by mediating oxidative stress and promoting β-amyloid (Aβ) aggregation. Accurate quantification of labile Cu2+ in the brain extracellular microenvironment is critical for elucidating its pathological roles and developing AD diagnostic tools. Herein, we report a dual-mode sensing platform integrating "turn-on" fluorescence and ratiometric electrochemistry for sensitive and selective Cu2+ quantification, based on a resorufin-picolinate conjugate (RP) probe and a carbon nanotube-modified carbon fiber microelectrode (CFME/CNT). Resorufin (Re) acts as both the fluorescent reporter and electrochemical redox center, while picolinate serves as the Cu2+-specific recognition moiety. Cu2+ coordinates with picolinate to catalyze the ester hydrolysis of RP, releasing free Re that triggers a fluorescence enhancement at 594 nm and a ratiometric electrochemical response, corresponding to the oxidation currents of free Re and intact RP, respectively. The RP probe exhibits high selectivity for Cu2+ over other metal ions, with limits of detection of 19 nM (fluorescence) and 7.2 nM (electrochemistry). The dual-mode platform was successfully applied to the accurate quantification of endogenous labile Cu2+ in cerebral cortex dialysate from AD mice and age-matched wild-type (WT) mice, and noninvasive in vivo fluorescence imaging further confirmed the elevated Cu2+ level in living AD mouse brains. Quantitative results revealed that the labile Cu2+ concentration in AD mouse cerebral cortex dialysate was significantly higher than that in WT mice. The two detection modes yielded highly consistent results, validating the reliability of the dual-mode platform in complex biological matrices. This dual-mode strategy synergizes the merits of fluorescence and ratiometric electrochemistry, providing a robust and selective tool for investigating AD pathogenesis related to Cu2+ dyshomeostasis and holds great potential for clinical translational applications in AD diagnosis.