Real Time Monitoring of Layer-by-Layer Polyelectrolyte Deposition and Bacterial Enzyme Detection in Nanoporous Anodized Aluminum Oxide

Porous anodized aluminum oxide (pAAO) is a nanostructured material, which due to its optical properties lends itself to the design of optical biosensors where interactions in the pores of this material are transduced into interferometric reflectance shifts. In this study, a pAAO-based biosensor was developed as a biosensing platform to detect proteinase K, an enzyme which is a readily available model system for the proteinase produced by Pseudomonas aeruginosa. The pAAO pore walls are decorated by means of the layer-by-layer (LbL) deposition technique using poly(sodium-4-styrenesulfonate) and poly-l-lysine as negatively and positively charged polyelectrolytes, respectively. Interferometric reflectance spectroscopy utilized to observe the optical properties of pAAO during LbL deposition shows that the deposition of the polyelectrolyte onto the pore walls increases the net refractive index, thus red-shifting the effective optical thickness (EOT). Upon incubation with proteinase K, a conspicuous blue shift of the EOT is observed, which is attributed to the destabilization of the LbL film upon enzymatic degradation of the poly-l-lysine components. This result is confirmed by scanning electron microscopy results. Finally, as a proof-of-principle, we demonstrate the ability of the label-free pAAO-based biosensing platform to detect the presence of the proteinase K in human wound fluid, highlighting the potential for detection of bacterial infections in chronic wounds.