In Vivo Ratiometric Electrochemical Detection of Hypochlorous Acid in Living Brains Using a Methylene Blue-Benzoyl-Based Microsensor

Hypochlorous acid (HClO), a reactive chlorine species produced by myeloperoxidase (MPO), contributes to neuroinflammation and oxidative stress implicated in neurodegenerative disorders. However, real-time quantification of HClO in the living brain remains challenging due to the lack of selective, miniaturized tools. Here, we report a ratiometric electrochemical microsensor based on a newly synthesized methylene blue-benzoyl (MB-Bz) probe for in vivo detection of HClO in the brain. The MB-Bz probe was immobilized on a three-dimensional (3D) electrochemically reduced graphene oxide-single-walled carbon nanotube (ERGO-SWCNT) nanocomposite-modified carbon fiber microelectrode. Upon reaction with HClO, the probe undergoes oxidative cleavage at the imine linkage adjacent to the benzoyl group, releasing benzoate and regenerating methylene blue, thereby producing two well-separated redox peaks for ratiometric quantification. The optimized microsensor exhibits a linear response toward HClO in the range of 2–40 μM (limit of detection 0.07 μM, S/N = 3) with excellent selectivity, stability, and reproducibility. When implanted into rat and mouse brains, the microsensor enables in vivo, region-specific electrochemical monitoring of HClO. Notably, elevated HClO levels are observed in the cortex, striatum, and hippocampus of PD model mice relative to normal mice, consistent with MPO-mediated oxidative stress. This work provides a molecularly engineered electrochemical platform for in vivo HClO analysis in living brain tissue, offering a valuable approach to investigate reactive chlorine species in neurodegeneration.