Multiplexed detection and differentiation of bacterial enzymes and bacteria by color-encoded sensor hydrogels

We report on the fabrication and characterization of color-encoded chitosan hydrogels for the rapid, sensitive and specific detection of bacterial enzymes as well as the selective detection of a set of tested bacteria through characteristic enzyme reactions. These patterned sensor hydrogels are functionalized with three different colorimetric enzyme substrates affording the multiplexed detection and differentiation of alpha-glucosidase, beta-galactosidase and beta-glucuronidase. The limits of detection of the hydrogels for an observation time of 60 min using a conventional microplate reader correspond to concentrations of 0.2, 3.4 and 4.5 nM of these enzymes, respectively. Based on their different enzyme expression patterns, Staphylococcus aureus strain RN4220, methicillin-resistant S. aureus (MRSA) strain N315, both producing alpha-glucosidase, but not beta-glucuronidase and beta-galactosidase, Escherichia coli strain DH5 alpha, producing beta-glucuronidase and alpha-glucosidase, but not beta-galactosidase, and the enterohemorrhagic E. coli (EHEC) strain E32511, producing beta-galactosidase, but none of the other two enzymes, can be reliably and rapidly distinguished from each other. These results confirm the applicability of enzyme sensing hydrogels for the detection and discrimination of specific enzymes to facilitate differentiation of bacterial strains. Patterned hydrogels thus possess the potential to be further refined as detection units of a multiplexed format to identify certain bacteria for future application in point-of-care microbiological diagnostics in food safety and medical settings.