A Turn-On Fluorescent and Ratiometric Electrochemical Dual-Mode Probe for Hydrogen Peroxide Detection in Brain Microdialysates of Alzheimer’s Disease Mice

Hydrogen peroxide (H 2 O 2 ), a pivotal reactive oxygen species (ROS), is closely linked to oxidative stress in the pathogenesis of Alzheimer’s disease (AD). Herein, we report a dual-mode probe (Re-PS) integrating turn-on fluorescence and ratiometric electrochemistry for the selective detection of H 2 O 2 in brain microdialysates of AD model mice. The probe is constructed using resorufin (Re) as a dual-signal reporter and a pentafluorobenzenesulfonyl (PS) group as the H 2 O 2 -responsive unit. Upon reaction with H 2 O 2, the PS group undergoes nucleophilic substitution, leading to the release of Re; this process triggers a fluorescence “turn-on” response and generates a ratiometric electrochemical signal. Compared with ester-based probes, Re-PS shows superior stability due to the strong electron-withdrawing effect of fluorine atoms in the PS group. The fluorescence mode achieves a detection limit (LOD) of 50 nM, while the electrochemical detection mode (using a carbon fiber microelectrode modified with carbon nanotubes (CFME/CNT)) has a detection range of 1.0–50 μM. Both modes exhibit excellent selectivity against other ROS and biomolecules. In vivo microdialysis analysis reveals significantly elevated H 2 O 2 levels in the brains of AD mice (28.6 ± 3.2 μM) compared with wild-type mice (10.3 ± 1.8 μM). This dual-mode strategy enables cross-validation, providing a reliable tool for monitoring oxidative stress in neurodegenerative diseases.