Highly specific β‑glucuronidase light-up natural fluorescent probe with aggregation-induced emission and excited-state intramolecular proton transfer for inhibitors screening and in situ imaging in Escherichia coli

Abstract

Expression of β-glucuronidase (GUS) in intestinal bacteria has attracted more and more attentions because of its relevant to drug-inducing enteropathy and enterohepatic circulation. Hence it is essential to develop a facile method for efficient inhibitors screening and in situ activity tracking of GUS. Here, a novel light-up natural fluorescent probe, quercetin-3-O-β-D-glucuronide (obtained from Bupleurum scorzonerifolium), for rapid, sensitive, and specific analysis of GUS activity was found. Hydrophilic quercetin-3-O-β-D-glucuronide was catalyzed by GUS to produce hydrophobic quercetin aggregates in situ. The occurrence of quercetin fluorescence at 540 nm (Stokes shift = 175 nm) with aggregation-induced emission and excited-state intramolecular proton transfer characteristics was light-up, which can be used to evaluate GUS activity. A good linear range (5.0–60.0 μg mL−1, R2 = 0.9904), high sensitivity (limit of detection at 0.7 μg mL−1), satisfying precision (relative standard deviation values at 1.2% and 3.0% for intra-day and inter-day variations) and superior selectivity to GUS were obtained. Then, a facile platform for screening of GUS inhibitors from natural compounds library was constructed successfully. Three natural compounds, resveratrol (IC50 = 0.45 ± 0.04 μM), hesperidin (IC50 = 1.46 ± 0.07 μM), and ursolic acid (IC50 = 7.85 ± 0.26 μM)) were screened out with stronger inhibitory efficiency. Furthermore, on account of the outstanding photostability and excellent biocompatibility, quercetin-3-O-β-D-glucuronide has been employed for in situ imaging of endogenous GUS in Escherichia coli noninvasively with low background noise. Results demonstrated the advantage of natural products for novel fluorescent probes and the great potential of quercetin-3-O-β-D-glucuronide in high-throughput inhibitors screening, GUS-related drug discovery, and disease diagnosis.