Enzyme-Sensing Chitosan Hydrogels

Abstract

We\nreport on a chitosan hydrogel-based platform for the detection\nof enzymes, which is compatible with the implementation in infection-sensing\nwound dressings. Thin films of the established wound dressing biopolymer\nchitosan were functionalized with a fluorogenic substrate, which is\nreleased upon enzymatic degradation, resulting in a pronounced increase\nin fluorescence emission intensity. In this first model study, the\nfluorogenic substrate alanyl-alanyl-phenylalanine-7-amido-4-methylcoumarin\n(AAP-AMC) was covalently conjugated via amide bond formation to chitosan\nand was shown to facilitate the detection of the serine protease α-chymotrypsin.\nSystematic investigations established the dependence of hydrogel thickness\nand substrate loading on the hydrogel preparation conditions, as well\nas the dependence of the rate of the reaction on the initial enzyme\nconcentration and the loading of AAP-AMC in the hydrogel. The initial\nrelease rate of the fluorophore 7-AMC was found to be linear with\nenzyme concentration and substrate loading and was independent of\nhydrogel thickness. Under optimized conditions the hydrogel reports\nthe presence of α-chymotrypsin in <5 min with a limit of\ndetection of ≤10 nM. This generic approach, which can be adapted\nto detect different kinds of enzymes by using appropriate fluorogenic\nor chromogenic substrates, is highly interesting for targeting the\ndetection of specific pathogenic bacteria, e.g., in wound dressings.