Dye-integrated photocrosslinkable polymers and networks for the visual chromogenic detection of a bacterial enzyme

In this work, a copolymer of poly[(hydroxy ethyl acrylamide)-co-(4-benzophenone acrylamide)-co-(hexamethylene diamine acrylamide)-co-(ECOSURF EH-3 acrylate)] was synthesized via free radical polymerization, followed by multi-step modification using click chemistry. The copolymer was subsequently functionalized with either 5-bromo-4-chloro-3-indolyl β-D-glucuronide (X-GLUC) or 4-nitrophenyl-β-D-glucuronide (PNPG), two enzyme-labile chromogenic substrates used to visually observe bacterial β-glucuronidase activity. This enzyme is secreted by over 98% of Escherichia coli strains, a common cause of healthcare-associated infections. The use of two substrates demonstrates the system's versatility in detecting β-glucuronidase activity across different bacterial species, wherein enzymatic cleavage of the dye-sugar bond produces a visible chromogenic signal. Both copolymers were found to be non-cytotoxic to human lung fibroblasts and were independently crosslinked under UV light to form distinct polymer network structures. Upon water swelling, each hydrogel enabled qualitative detection of β-glucuronidase-producing bacteria through the release of indigo or yellow dyes, corresponding to the chromogenic response of X-GLUC or PNPG, respectively. This colorimetric response was confirmed both visually and spectroscopically, underscoring the potential of these polymers for development into multiplexed enzyme-based bacterial detection platforms. The technology offers promising applications in microbiological diagnostics, particularly in food safety and medical contexts.