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

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 (H₂O₂) 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 H₂O₂ levels and 5-HT homeostasis. Additionally, the efficacy of HOP in detecting H₂O₂ distribution is validated in vivo and ex vivo using epileptic mouse models. This study presents a robust tool for precise imaging of H₂O₂ 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.