Rapid Detection of Escherichia coli via Enzymatically Triggered Reactions in Self-Reporting Chitosan Hydrogels

In this work, a self-reporting hydrogel for the rapid in situ detection of bacterial enzymes is reported. To implement the reporting function for the bacterium Escherichia coli into a film-based sensing format, chitosan hydrogel films on solid backing supports were equipped with a reporting function for the enzyme β-glucuronidase (β-GUS), which is secreted by >98% of all known E. coli strains. Covalent coupling of the fluorogenic substrate 4-methylumbelliferyl-β-D-glucuronide or the complementary chromogenic substrate 4-nitrophenyl-β-D-glucuronide via amide bond formation afforded an attachment that is stable for >24 h under physiological conditions. By contrast, in the presence of β-GUS, the reporter dyes were very rapidly cleaved and produced a signal for the presence of the enzyme, which was detectable by bare eye under appropriate illumination. Detailed investigations of the enzymatic reaction for both types of substrates in neat enzyme solution as well as in bacterial supernatant revealed the apparent reaction kinetics and allowed us to determine the concentration of β-GUS in the supernatant. Under optimized conditions, the 4-methylumbelliferyl-β-D-glucuronide-functionalized hydrogel reported the presence of β-GUS within 15 min with a limit of detection of <1 nM. Finally, the function of the generally applicable hydrogel-film-based sensing approach, which is compatible with polymer-film-based applications, including wound dressings and packaging materials, and is also amenable to address noncultivatable pathogenic bacteria by using appropriate fluorogenic or chromogenic substrates, was demonstrated by direct application with bacterial medium.