## 3 Results and Discussion
The covalent immobilization of PrA on the sample surfaces induced the red-shift of their reflectance spectra due to an increase of the optical thicknesses. Since the physical thickness \(L\) was fixed, the optical thickness increased due to the change of the average refractive index caused by the infiltration of PrA inside the voids of the layers. Figure 5A,C report the reflectance spectra of nZnO-F and nZnO layers after the interaction with different concentrations of PrA (i.e., 2 mg/mL; 4 mg/mL; 6 mg/mL); the increment of the OT was well evident from Figure 5B,D, which show the FFT of the spectra. By calculating the differences between the OT of the samples before and after the protein functionalization, maximum \(\Delta\)OT of 110 nm and 70 nm were determined in the case of nZnO-F and nZnO, respectively. The result suggested that about 37% as much protein was immobilized on the surface of nZnO-F sample with respect to nZnO one; this value, calculated as the percentage difference between the two maxima \(\Delta\)OT, was in agreement with that calculated by fluorescence imaging. Fitting \(\Delta\)OT data with a dose-response  
Figure 4: Fluorescence microscopy imaging of nZnO-F (upper line) and nZnO (lower line) before and after functionalization with PrA*. curve, the PrA concentration useful for surface saturation was estimated to be 4 mg/mL for both samples (Figure 5E). The PL emission of the nanostructured ZnO is sensitive to the chemical modification of the material surface and it can be used for monitoring the biomolecular interactions in the experiments of label-free optical sensing [14]. For this reason, the interaction between the nZnO-F and nZnO samples and the PrA was also investigated measuring their PL spectra. Figure 6A,B report the PL spectra of the nZnO-F and nZnO, respectively, after the surface functionalization with 2 mg/mL, 4 mg/mL and 6 mg/mL of PrA; the spectra of functionalized samples were compared with the spectra acquired before the functionalization. For both the materials, any variation of the peak at 380 nm ascribed to near band edge excitonic transitions, was not observed. On the contrary, the intensity of the broad visible band, included between 500 and 800 nm and mainly related to the surface defects, decreased at the increasing of protein concentration. By calculating the integrated PL (IPL) intensities and plotting them as a function of the PrA concentration (Figure 6B,D), a variation of 20% was obtained for the  
Figure 5: Reflectivity spectra of nZnO-F (**A**) and nZnO (**C**) before and after modification with different concentrations of PrA; corresponding Fourier transforms (**B**,**D**). \(\Delta\)OT as function of PrA concentrations, calculated for nZnO-F and nZnO; experimental data were fitted by using a dose-response curve (**E**). fluorine doped sample and only 5% for the undoped sample. The integrated PL intensity variations were calculated as:  
\[\Delta I_{PL}=\left(\frac{I_{PL}(0)-I_{PL}(6)}{I_{PL}(0)}\right)\star 100 \tag{1}\]  
The results of the optical investigations, i.e., the spectroscopic reflectometry and the photoluminescence spectroscopy, clearly demonstrated that nZnO-F could be functionalized with a higher efficiency with respect to the undoped nZnO. This effect was mainly ascribed to the peculiar flake-like morphology of the fluorine-doped material, made of nanograins much larger than the nZnO nanocolumns and with a more exposed area that could sustain a stronger surface interaction with the PrA biomolecules. The silanization procedure for the functionalization of nanostructured zinc oxide, undoped and fluorine doped, with protein A was already successfully used in the case of porous silicon [23]. Since silanization was well established in the substitution of Si-OH bonds by Si-C ones, which were much more stable from the thermodynamic point of view, we firstly verified that silanization was able to substitute Zn-OH bonds by Zn-Si-C ones by grafting FITC-labelled protein A to both surfaces.