## 3 Results
### Synthesis and Physicochemical Characterization
Interestingly, although the free peptides exhibit different peak shapes, the peak fitting analysis resulted in the same components of the S 2p peak, i.e., S1, S2 and S3, at different relative intensity ratio. Specifically, the S1/(S2 + S3) ratio is 1.2 for VEGFGlyFAM and 0.3 for VEGFGlyFAM. This finding can be explained by the tendency of cysteine groups to oxidise in a non-reducing environment [44]. As to the peptides bound to the AuNPs, for both AuNP/VEGFGlyFAM and AuNP/VEGFCysFAM the component S0 of chemisorbed sulphur is found. In the case of the peptide containing the cysteine such a component is expected owing to the high binding affinity of thiol groups towards metal surfaces [37; 39]. On the other hand, in the methionine residue, the presence of electron pairs of the sulphur 2p orbitals is likely to prompt, e.g., by coordinative binding, the formation of S-Au bonds corresponding to S0 component. It must be noted that the relative intensity ratio between the components assigned respectively to chemisorbed sulphur (S-Au) and S-C bonds, correspond to the S0/S1 values of 3.1 for AuNP/VEGFCysFAM and 1.7 for AuNP/VEGFGlyFAM, thus confirming the higher chemisorption process occurred in the presence of the cysteine residue. As to the overall oxidation state of sulphur in the AuNP/peptide assemblies, the values of the intensity ratio S1/(S2 + S3 + S4) of 0.7 for AuNP/VEGFCysFAM and 0.3 for AuNP/VEGFGlyFAM point to a less extent of oxidation of the sulphur moieties for the nanoparticle-bound peptide in comparison to the free peptide in the case of VEGFCysFAM. The spectra of Au 4f region exhibit symmetric peaks with comparable full widths at half maximum (FWHM) of about 0.9 eV for all samples (Figure 7). For the bare AuNP, the distinctive doublet peaks can be observed at the BE of 86.9 \(\pm\) 0.2 eV(Au 4f5/2) and 83.2 \(\pm\) 0.2 eV(Au 4f7/2), respectively. The peak separation of 3.7 eV and an area ratio of 3:4 are in good agreement with literature XPS data of gold nanoparticles [45]. For both AuNPs/VEGFCysFAM and AuNPs/VEGFGlyFAM, the Au 4f signals display a shift of 0.4 eV and 0.5 eV respectively towards lower binding energies with respect to the bare  
Figure 6: Representative high-resolution spectra and peak fitting curves of S 2p in AuNPs/VEGFGlyFAM (**a**) and AuNPs/VEGFCysFAM (**c**) hybrids, as well as in VEGFGlyFAM (**b**) and VEGFCysFAM (**d**) peptides, respectively. AuNP sample located at 83.2 eV. Such a shift can be attributed to charge-transfer effects from core electrons of gold to the peptide molecules [37; 39; 46].The spectra of Au 4f region exhibit symmetric peaks with comparable full widths at half maximum (FWHM) of about 0.9 eV for all samples (Figure 7). For the bare AuNP, the distinctive doublet peaks can be observed at the BE of 86.9 \(\pm\) 0.2 eV (Au 4f\({}_{5/2}\)) and 83.2 \(\pm\) 0.2 eV (Au 4f\({}_{7/2}\)), respectively. The ToF-SIMS technique has been extensively used to study the interaction of biomolecules with surfaces including nanofilms and nanoparticles, often as complementary tool of XPS studies [47; 48]. The principal component analysis (PCA) method [49] used to thoughtfully analyse the ToF-SIMS spectra, and the corresponding results are shown in Figure 8. In this plot, each individual point represents the ToF-SIMS spectrum acquired at a specific location of the sample. PC1, represented on the vertical axis, from the PCA study of positive ToF-SIMS spectra for AuNP, AuNP/VEGF peptide and VEGF peptide samples reveals that the bare AuNP and the peptide-coated AuNP samples are clearly discriminated, i.e., located in distinct regions (i.e., PC1 \(<\) 0) with respect to the free peptide one (PC1 \(>\) 0). This result is found for both VEGFGlyFAM (left hand side panel) and VEGFGlyFAM (right hand side panel), respectively.