H 2 O 2 ‐Activated Serotonin Precursor Probe for Mapping Neuronal Redox Homeostasis Reveals 5‐HT Interactions with Neighboring Proteins Under Oxidative Stress

Abstract Serotonin (5‐HT) is a critical neurotransmitter that regulates various neurophysiological processes. However, the role of 5‐HT under oxidative stress remains largely unexplored. Here, the development of a novel intramolecular charge transfer (ICT)‐based fluorescent probe is reported, termed HOP , designed using a tandem sensing and labeling strategy. HOP is selectively activated by hydrogen peroxide (H 2 O 2 ) in neuronal cells undergoing oxidative stress. Upon activation, HOP emits fluorescent signals and covalently cross‐links with nearby proteins, which not only anchors it to the local microenvironment to avoid diffusion of the fluorophore, but also simultaneously releases 5‐HT in situ. The locally released 5‐HT further interacts with nearby functional proteins such as myeloperoxidase (MPO) and sirtuin 1 (SIRT1), as confirmed through mass spectrometry analyses. Furthermore, HOP is employed in high‐throughput screening to identify the antioxidant, hesperidin, that is effective in modulating H 2 O 2 levels and 5‐HT homeostasis. Additionally, the efficacy of HOP in detecting H 2 O 2 distribution is validated in vivo and ex vivo using epileptic mouse models. This study presents a robust tool for precise imaging of H 2 O 2 in living neuronal systems and for exploring 5‐HT‐associated protein modifications under oxidative stress, thus providing new avenues for investigating the role of serotonin in neurological disorders, such as epilepsy.