## 3 Results and Discussion
The surface wettability of nZnO-F and nZnO samples were investigated by measuring the WCA values before and after each functionalization step. The results are reported in Figure 3. Due to the hydrothermal synthesis process, samples were mostly hydrophobic; the WCA values of \((117\pm 7)^{\circ}\) and \((110\pm 10)^{\circ}\) were measured for nZnO and nZnO-F, respectively. After the plasma oxygen process, the hydroxyl groups activated on the sample surfaces induced a strong decreasing of the WCA values down to \((45\pm 5)^{\circ}\) for the nZnO-F and to \((61\pm 9)^{\circ}\) for the nZnO. As expected, after the amino-silanization process, increased values of WCA up to \((106\pm 12)^{\circ}\) and \((98\pm 2)^{\circ}\) were measured for the nZnO-F and the nZnO, respectively, due to the replacement of -OH groups by the hydro-carbons and -NH\({}_{2}\) functional groups of the APTES molecules. The high increase of the WCA values was attributed to an efficient capping of hydroxyl groups by the amino-silane, probably oriented in a way that mainly exposed the hydrophobic hydro-carbon groups [22]. The BS3 crosslink introduced the hydrophilic N-hydrosulfosuccinimide groups (NHS) on the surfaces, resulting in a WCA value decrease down to \((68\pm 9)^{\circ}\), in the case of the nZnO-F, and to \((54\pm 8)^{\circ}\) for the nZnO. After the incubation with the PrA*, the WCA values of both samples remained unchanged within the errors: This result was compatible with the presence of both the hydrophilic and hydrophobic domains, and characteristic of the protein structure [21]. The fluorescence microscopy imaging was used to support the presence of the PrA* on the sample surfaces after the last functionalization step. The images, reported in Figure 4, showed a fluorescence homogeneously clustered on the whole surface of PrA*-modified nZnO-F (mean fluorescence intensity = 20 \(\pm\) 1 counts), while only few luminescent spots, corresponding to a mean fluorescence intensity of 13 \(\pm\) 1 counts, occurred on the surface of nZnO after PrA*. Analyzing the percentage difference between the values of the mean fluorescence intensities in both samples, we estimated that about 35% as much protein was immobilized on the doped one. The control samples, i.e., the nZnO-F and nZnO without the PrA*, appeared completely dark (mean fluorescence intensity = 5 \(\pm\) 1). The fluorescence microscopy imaging was thus in perfect agreement with the results obtained by FTIR spectroscopy. Figure 3: Wettability investigation of nZnO-F (upper line) and nZnO (lower line) samples. WCA measurements were performed after each functionalization step. Figure 2: FTIR spectra of nZnO-F sample before (black line) and after (red line) functionalization with PrA. After the characterization of the surface functionalization by the standard methods of confirming the immobilization of the labeled protein, new samples of nZnO-F and nZnO were grown on silicon and functionalized with unlabeled PrA at different concentrations; in this case, the surface modification was monitored using two label-free techniques, i.e., the spectroscopic reflectometry and the photoluminescence analysis, exploiting the intrinsic optical properties of the semiconductor film. The layers of nanostructured ZnO acted as Fabry-Perot interferometers from the optical point of view. Their reflectance spectra consisted of fringe patterns due to the interference occurring at air/nZnO-F (or nZnO) and nZnO-F (or nZnO)/bulk silicon interfaces. The optical thickness, OT, of a layer could be quantified as \(nL\) where \(n\) is the average refractive index of the material and \(L\) is the physical thickness. The OT could be calculated counting the fringe maxima satisfying the relationship \(m\lambda=2\) nL, with \(m\) integer and \(\lambda\) wavelength of the incident light. A faster method was based on the fast Fourier transform (FFT) of the reflectance spectrum; FFT of the spectrum was characterized by a peak whose position along \(x\)-axis was two times the optical thickness of the layer.