We report on a
chitosan hydrogel-based platform for the detection of
enzymes, which is compatible with the implementation in
infection-sensing
wound dressings. Thin films of the established
wound dressing
biopolymer chitosan were functionalized with a
fluorogenic substrate, which is released upon enzymatic degradation, resulting in a pronounced increase in fluorescence emission intensity. In this first model study, the
fluorogenic substrate alanyl-alanyl-phenylalanine-7-amido-4-methylcoumarin (
AAP-AMC) was covalently conjugated via
amide bond formation to
chitosan and was shown to facilitate the detection of the
serine protease α-
chymotrypsin. Systematic investigations established the dependence of
hydrogel thickness and substrate loading on the
hydrogel preparation conditions, as well as the dependence of the rate of the reaction on the initial
enzyme concentration and the loading of
AAP-AMC in the
hydrogel. The initial release rate of the fluorophore 7-AMC was found to be linear with
enzyme concentration and substrate loading and was independent of
hydrogel thickness. Under optimized conditions the
hydrogel reports the presence of α-
chymotrypsin in <5 min with a limit of detection of ≤10 nM. This generic approach, which can be adapted to detect different kinds of
enzymes by using appropriate fluorogenic or
chromogenic substrates, is highly interesting for targeting the detection of specific pathogenic bacteria, e.g., in
wound dressings.