A Double-Locked ESIPT-AIE Fluorescent Probe Detects Esterase with Highly Matched Response Kinetics

作者：Zhuo Ye, Yiting Yang, Yuqi Wang, Ge Wang, Xin Liu, Yafu Wang, Kui Wang, Hua Zhang · 发表于：Analytical Chemistry · 年份：2025 · DOI：10.1021/acs.analchem.4c06390 · 被引用次数：13 · 研究领域：Click Chemistry and Applications、Molecular Sensors and Ion Detection、Biotin and Related Studies

Hydrolyases play an irreplaceable role in complex biological processes, and their dysfunction is a cause of many human diseases. Advanced activatable in situ fluorescence detection methods offer high-resolution spatiotemporal analysis, aiding in the dissection of the complex biological roles of hydrolases. However, current strategies typically focus on only specific stages of enzyme-probe interactions, leading to suboptimal imaging fidelity and sometimes erroneous detection results. Addressing this, we developed a double-locked "Excited State Intramolecular Proton Transfer-Aggregation Induced Emission (ESIPT-AIE)" fluorescent probe (Br-3N-2Et) that matches the entire enzymatic response kinetics for enzyme activity detection. We validated the probe's mechanism by enhancing pre-reaction recognition through double unlockable recognition sites, thereby reducing basal fluorescence (Φ = 0.0183) and increasing resistance to interference signals. Subsequently, the ESIPT fluorophore with multiple hydrogen bonds enhanced the affinity for the hydrolase catalytic site, improving binding kinetics and exhibiting a significant Stokes shift (188 nm). The realization of the ESIPT-AIE dual-emission mechanism facilitated rapid efflux of the fluorophore from the catalytic site and subsequent in situ fluorescence signal enhancement (132.2-fold). This new probe achieved regional differential detection of esterase activity in HepG2 cells and endometrial cancer tissues. Thus, this work paves the way...