A Synergistically Enhanced Near-Infrared ESIPT Fluorescent Probe for High-Performance In Situ Imaging of Cellular Apoptosis.

Monitoring apoptotic progression is critical for tracking disease development and evaluating therapeutic interventions. Fluorescent probes that integrate aggregation-induced emission (AIE) and excited-state intramolecular proton transfer (ESIPT) offer some advantages, including large Stokes shifts, high quantum yields in the aggregated state, and strong photostability. In this study, we developed an ESIPT-based fluorescent probe, ABTT-DEVD, with AIE properties for the highly sensitive detection of caspase-3 and real-time imaging of apoptosis. The probe design incorporates a hydrophilic DEVD peptide substrate linked to a hydrophobic ESIPT fluorophore. Caspase-3-mediated cleavage induces a significant change in water solubility, leading to strong fluorescence enhancement with an ultralow detection limit of 1.2 pM. Molecular docking studies corroborate the specific interaction of the DEVD peptide with the caspase-3 active site. Furthermore, ABTT-DEVD enabled the visualization of endogenous caspase-3 activity in living cells, facilitating the evaluation of antitumor drug efficacy. The probe's effectiveness in tracking apoptosis progression is further supported by fluorescence data and flow cytometry analysis, underscoring its potential as a valuable tool for apoptosis monitoring in biomedical research.