Three distinct read-out modes for enzyme activity can operate in a semi-wet supramolecular hydrogel

Assays of hydrolytic enzyme activity, such as of glycosidases and phosphatase, as well as several proteases, using a semi-wet supramolecular hydrogel array composed of a glycosylated amino acetate are described. It has been demonstrated that the microcavity formed by gel fibrils is suitable to immobilize native enzymes without denaturation under semi-wet conditions, and thus the nanofiber has been rationally used as a sensing domain to monitor enzymatic reactions. By using a fluorogenic substrate, reducing the size of the hydrogel can significantly improve the problem of suppressed diffusion within the gel matrix thus making the hydrogel a promising semi-wet matrix for evaluating enzyme activity. Confocal laser scanning microscopy observations have shown that an environmentally sensitive fluorescent probe accumulates in the hydrophobic domain of the gel fiber and emits fluorescence more strongly upon hydrolytic cleavage of the substrate peptides. Not only a simple environmentally sensitive probe but also a FRET (fluorescence resonance energy transfer)-type read-out mode can be devised to analyze the enzymatic hydrolysis-triggered redistribution of the probe between the nanospace and the nanofiber to accomplish a more clearly distinguished enzyme assay. Thus, it is clear that three distinct read-out modes, that is, 1) fluorogenic substrates, 2) substrates bearing an environmentally sensitive probe, or 3) a substrate exhibiting FRET, can operate under the semi-wet hydrogel conditions used in these investigations. In addition, owing to the unique properties of the present supramolecular hydrogel in semi-wet conditions, that is, its phase-segregation properties and dynamics, the supramolecular substrate/enzyme array has successfully been used for high-throughput screening of single and multiple enzymes based on their activity, lysate analysis, and quantitative evaluation of inhibitor potency and selectivity.