Off-on fluorogenic substrate harnessing ESIPT and AIE features for in situ and long-term tracking of β-glucuronidase in Escherichia coli

Detection and isolation of both O157:H7 and non-O157:H7 Escherichia coli are essential for the evaluation and surveillance of the associated foodborne disease risk. However, the development of a chromogenic/fluorogenic culture medium for detection and isolation is challenging yet urgent. beta-Glucuronidase (GUS) serves as an important biomarker in E. coli. A novel and simple off-on fluorogenic substrate (BTBP-Gluc) was developed, based on a solid-state fluorophore (BTBP) with excited-state intramolecular proton transfer (ESIPT) and aggregation-induced emission (AIE) effects. BTBP-Gluc could be well-suited for visually detecting GUS activity under acidic conditions (3.0 <= pH<7.0). It was successfully applied for detection of endogenous intracellular GUS activity in living E. coli and could produce the in situ and long-term localization of the target colonies and cell imaging of living E. coli. Additionally, it showed high selectivity, little toxicity, excellent cell permeability and sensitivity. BTBP-Gluc was combined with two tightly localizing chromogenic substrates, Magenta-Gal and X-Glu. These combinations were applied to the development of a new chromogenic-fluorogenic culture medium (C-F E. coli agar). On this new agar simultaneous visual detection and isolation of O157:H7 and non-O157:H7 E. coli with relatively high accuracy was achieved. C-F E. coli agar shows potential in serving as a simple sensitive tool for detecting and isolating O157:H7 and non-O157:H7 E. coli in food samples such as milk.