Label-free and real-time synchronized monitoring of extracellular matrix proteolysis using quartz crystal microbalance and nanoplasmonic sensing with morphological validation by atomic force microscopy

While the QCM-D/LSPR signals both detect mass uptake during protein adsorption and mass loss during proteolysis, synchronized measurements, complemented with AFM imaging, reveal more complex responses arising from the differing surface sensitivities of the techniques. Our integrated analysis show substantial differences in adlayer morphology and proteolytic degradation. Collagen forms a vertically heterogeneous adlayer with a dense near-surface layer and a highly viscoelastic outer layer of protruding fibrils (Δf ≈ -100 to -240 Hz, ΔD ≈ 40-70 ppm, and ∆λ ≈ 0.7 nm), whereas elastin adsorbs as a thinner, more rigid film (Δf ≈ -36 to -40 Hz, ΔD ≈ 2-3 ppm, and ∆λ ≈ 0.4 nm). Real-time monitoring reveals that collagenase primarily degrades the protruding collagen fibrils, significantly affecting all QCM-D and LSPR signals-particularly showing a clear overtone dependence in ΔD and Δf shifts-while elastase digestion of elastin occurs without overtone dependence and results in more pronounced changes in Δf and ∆λ, with comparatively low effect on ΔD. Cross-reactivity experiments confirm substrate specificity; however, both proteases show non-specific activity. Inhibition studies demonstrate that QCM-D can detect both true enzymatic inactivity and substrate-inhibitor interactions that mimic inhibition in conventional assays, for example by physically adsorbing to the substrate and thereby shielding it from proteolysis.