Nanoporous Anodic Aluminum Oxide/Silicon – Polyester Hybrid Systems for the Detection of Bacterial Enzymes

The development of targeted diagnostics and therapy approaches aiming at treating bacterial infections are in the focus of research worldwide, driven by the increasing impact of antibiotic-resistant bacteria. One of the diagnostic approaches is based on stimuli-responsive materials, so-called "smart materials". In this Thesis, a stimuli-responsive biodegradable polymer was combined with anodic aluminum oxide (AAO) and porous silicon rugate filters (pSiRF) for the optical detection of fungal and bacterial enzymes.
Firstly, AAO nanostructures were produced via refined anodization processes. The anodization parameters were adjusted to tailor the structure of the AAO to the sensing requirements. The fabricated AAO nanomaterials with well-ordered hexagonal pore structures were further treated with phosphoric acid to widen the pores. The pore diameter, length, and porosity were evaluated by field emission scanning electron microscopy (FESEM). The data showed that the porosity increased linearly with widening time. The dependence of the pore length on the anodization time was also studied, showing also a linear tendency. The fabrication of porous silicon (pSi) nanostructures was also studied in detail. The fabrication process was optimized to obtain the desired structures that fulfill the sensing requirements for bare eye detection. The lectrochemical etching of pSi was carried out in HF/ethanol mixtures and afforded nanopores with pore diameters and pore lengths that increased linearly with the current densities and the etching time, respectively. The optimal regime for electrochemical etching to synthesize pSiRF sensors with a high-quality factor was a 7 sec period time for 100 cycles at minimum and maximum current densities of 15 and 50 mA cm-2, respectively, in hydrofluoric acid/ethanol (1:2 v/v). Spin coating and solvent casting methods were then utilized to modify the AAO, planar Si, and pSiRF sensors with the biodegradable polymer poly(lactic acid) (PLA). The modification of those sensors was investigated using FESEM, energy-dispersive X-ray spectroscopy (EDX), thermogravimetric analysis (TGA), ellipsometry, contact angle, and reflectometric interference spectroscopy (RIfS) techniques.
The enzymatic degradation of PLA in these sensors, catalyzed by proteinase K, a fungal enzyme, and proteases secreted by the pathogen Pseudomonas aeruginosa (PAO1) (P. aeruginosa) (PAO1), was studied. The water contact angle showed a change in the wettability after the enzymatic reaction, which is consistent with a reduction in the calculated carbonyl band observed in Fourier transform infrared (FTIR) spectra. Atomic force microscopy (AFM) roughness data also confirmed the PLA film degradation. RIfS emphasized the occurrence of the degradation of PLA-modified pores of AAO and pSiRF. The data showed a decrease in effective optical thickness (EOT) and a blue-shift in wavelength after treatment with proteinase K. Finally, the AAO and pSiRF sensors were tested in the P. aeruginosa (PAO1) cultures and their corresponding sterile-filtered culture supernatants. The results revealed that the enzymatic reaction of PLA was detectable compared to the free enzyme solution (LB medium). Interestingly, there were noticeable color shifts in the pSiRF sensor, transitioning from green to blue, when exposed to pure proteinase K solution or P. aeruginosa (PAO1) cultures. This color change could be detected by the bare eye after treating the sensor with the enzyme, washing, and drying, suggesting that this principle may be improved and potentially utilized to sense bacterial enzymes of clinically relevant bacterial pathogens visually and rapidly.