(574g) Identifying and Sensing Proteases As Key Drivers of Chronic Wounds

Chronic wounds, often arising from complications like diabetes, poor circulation, or prolonged inflammation, impose substantial burdens on healthcare systems due to prolonged treatments and high infection rates¹. Proteases, including matrix metalloproteinases (MMPs) and neutrophil elastases (HNE), play essential roles in tissue remodeling and inflammation². In chronic wounds, persistently elevated protease activity leads to excessive degradation of extracellular matrix components, hindering wound closure. Consequently, high protease levels serve as biomarkers for non-healing wounds³. Additionally, microbial infection further complicates wound healing, partly through protease secretion. Pathogenic bacteria secrete proteases as virulence factors that degrade host proteins such as growth factors and their receptors, impair immune responses, and contribute to biofilm formation⁴. This combined proteolytic activity from both host and pathogens severely delays wound healing. Despite this known importance, the current methods for protease sensing remain inadequate for wound diagnostics. Wound exudates are chemically and physically diverse, and traditional fluorescence-based sensors often fail to provide reliable, multiplexed information in such a complex matrix. To address these challenges, we are developing an integrated approach combining high-throughput peptide substrate screening with advanced sensing technologies to profile protease activity in wounds.

First, we are developing an M13 bacteriophage peptide display platform coupled with next-generation sequencing (NGS) or nanopore sequencing to identify protease-specific substrates. The phage library is covalently conjugated to magnetic beads via sortase-mediated ligation, enabling robust manipulation within complex biological matrices, such as wound exudates. This system not only accelerates substrate discovery but also provides diagnostic potential, revealing distinct “proteolytic cleavage signatures” that effectively discriminate chronic wound exudates from acute or healthy samples. Secondly, we have engineered a low-cost protease nanosensor capable of bedside proteolytic through inductive detection of particle release with pulsed magnetic fields in a PDMS fluidic chip. Incorporating pulse and gradiometer coils into a printed circuit board⁵, our device achieves a detection limit below 1 µg of iron using pulses ranging up to tens of millitesla (mT), while the duty cycle can be varied to control temperature through Joule heating. As proof of concept, we demonstrated sensitive detection of chymotrypsin at concentrations in the hundreds of nanomolar. These integrated methodologies have the potential to advance towards disposable fluidic chips and low-cost detection devices, paving the way for wide-spread ubiquitous monitoring of protease activity in wound diagnostics.

References

1 Peña, O.A., Martin, P. Cellular and molecular mechanisms of skin wound healing. Nat Rev Mol Cell Biol 25, 599–616 (2024).

2 Kalogeropoulos, K. et al. Chap. 8 Proteolytic signaling in cutaneous wound healing. Proteolytic Signaling in Health and Disease. Elsevier (2021).

3 International consensus. The role of proteases in wound diagnostics. An expert working group review. London: Wounds International (2011).

4 Uberoi, A., McCready-Vangi, A. & Grice, E.A. The wound microbiota: microbial mechanisms of impaired wound healing and infection. Nat Rev Microbiol 22, 507–521 (2024).

5 Li, F., Sieben, L., Büchler, J. et al. A fluidic device for continuous on-line inductive sensing of proteolytic cleavages. Lab Chip, 25, 500 (2025).