Ultrasensitive Visual Detection of Chitin Degradation via Surface Pitting on a Nanofibrous N-Acetylchitosan Hydrogel

Microbial enzymes play a central role in biomass valorization, and their discovery and characterization are increasingly driven by data-driven molecular approaches. However, for enzymes acting on water-insoluble polymeric substrates, enzymatic reactions are inherently confined to solid-liquid interfaces, and interfacial properties critically govern reaction efficiency and detectability. Here, we report an interfacial sensing strategy that enables ultrasensitive visual detection of microbial chitinolytic activity by exploiting a nanofibrous N-acetylchitosan hydrogel. The hydrogel consists of an entangled nanofiber network that provides a markedly increased surface area, thereby facilitating interfacial enzymatic reactions. When chitinolytic microorganisms are grown on the hydrogel surface, extracellular chitinases locally disrupt the nanofibrous network, producing nanoscale depressions that evolve into optically visible pits upon incubation. This approach enables visual detection of chitin degradation at the nanogram scale, with profilometric quantification down to ∼1 ng and naked-eye recognition at several tens of nanograms. By explicitly linking interfacial structure, surface morphology evolution, and enzymatic reaction kinetics, this work introduces a materials-based perspective that complements data-driven molecular approaches for the discovery and evaluation of microbial enzymes acting on insoluble polymeric substrates.