Towards Multiplexed Bacteria Detection by Enzyme Responsive Hydrogels

Abstract

In this work, patterned β‐GUS sensing chitosan hydrogels functionalized with three different colorimetric substrates were fabricated for the multiplexed detection of the enzyme β‐glucuronidase (β‐GUS), which is secreted by >98% of all known Escherichia coli ( E. coli ) strains. The immobilization of fluorogenic and chromogenic substrates in specified areas allows a spatially resolved readout of the corresponding colorimetric signal. The apparent initial rate of the β‐GUS induced cleavage of the reporter moieties of chitosan films functionalized with the chromogenic substrate 4‐nitrophenyl‐β‐D‐glucuronide (PNPG), the chromogenic substrate 5‐bromo‐4‐chloro‐3‐indolyl‐β‐D‐glucuronide (X‐Gluc) and the fluorogenic substrate 4‐methylumbelliferyl‐β‐D‐glucuronide (MUG) were analyzed spectroscopically and confirmed a detectable reaction within less than 60 min. Likewise the released dyes were observed in the patterns owing to different colors by naked eye detection under appropriate illumination in less than 80 min. Hence the presence of a characteristic enzyme secreted by E. coli bacteria was successfully detected by three independent sensing moieties, which is important to reduce false positives by introducing redundancy. Patterned enzyme sensing chitosan hydrogels, which are functionalized with different substrates, open the possibility for multiplexed bacteria detection and in the long run also the identification of different bacteria strains.