Reactive fluorescent probe for covalent membrane-anchoring: enabling real-time imaging of protein aggregation dynamics in live cells

a two-stage mechanism: first, rapid electrostatic interaction-mediated localization to the cell membrane, followed by chemoselective macrocyclization with thiol and amine groups on membrane proteins to form a fluorescent conjugate, whose emission is substantially enhanced due to restriction of twisted intramolecular charge transfer (TICT) within the confined microenvironment induced by protein aggregation. Leveraging this mechanism, the probe successfully reports membrane protein aggregation triggered by diverse stressors, such as redox imbalance and chemotherapeutic agents, while also capturing distinct membrane reorganization dynamics. With features of biocompatibility, wash-free performance, and long-term membrane retention, this probe provides an alternative tool for evaluating the complex structural dynamics of membrane proteins and offers potential for developing targeted therapeutic strategies.