High Spatiotemporal Profiling of MiRNA and ROS Trafficking at Single Mitochondria with Functionalized θ-Nanopipette

Abstract The dysregulation of mitochondrial microRNA-125b (miR-125b) and overproduction of reactive oxygen species (ROS) are tightly implicated in the pathogenesis and progression of Alzheimer’s disease (AD). However, the dynamic interplay between mitochondrial miR-125b and ROS, as well as their mutual regulatory mechanisms, remains poorly understood. Herein, we develop a functionalized θ-nanopipette integrated with two independent detection channels, which enables simultaneous, crosstalk-free determination of miR-125b and ROS at the subcellular level. Leveraging the high spatiotemporal resolution and sensitivity of nanochannel electrochemistry, the interaction between miR-125b and ROS released from single mitochondria was systematically investigated, and their dynamic level changes in individual mitochondria and the surrounding cytosol were tracked upon stimulation with Okadaic acid (OA), a well-established AD-like pathological inducer. It is demonstrated for the first time that OA stimulation rapidly activates cytosolic miR-125b, which subsequently translocates into mitochondria. Specific enrichment of mitochondrial miR-125b induces a marked mitochondrial ROS burst, followed by ROS diffusion into the cytosol. More importantly, the identified feedback regulatory mechanism between miR-125b and ROS provides critical insights for AD therapy, highlighting that targeting the upstream pathogenic trigger (cytosolic miR-125b translocation into mitochondria) represents a highly effective therapeutic strategy.