## 3 Results  
### Synthesis and Physicochemical Characterization  
The synthesis of gold nanoparticles was carried out by the chemical reduction method with citrate acting both as reducing and stabiliser agent and resulted, at the used experimental conditions, in a monodisperse colloidal solution of spherical nanoparticles having diameter of 12 nm [37] and a plasmonic band centered at 518 nm (Figure 1). The strong absorption in the visible region spectra of AuNPs is the characteristic feature of gold plasmonic resonance. A red shift and broadening of this band upon the addition of a ligand can be related to the surface coverage and/or aggregation of nanoparticles [38]. Indeed, Figure 1 displays noticeable red shifts of the plasmonic band upon the addition of the peptide in the 25 nM-125 nM concentration range, with total shifts measured of 10 nm for VEGFGlyFAM and 6 nm for VEGFCysFAM, respectively. As the concentration of the added peptide increases, in the range between 125 nM and 175 nM, the width of the plasmonic bands increase up to FWHM of 130 nm compared to 81 nm for the bare AuNPs. Such findings are explained in terms of two main contributions, i.e., a predominant particle coverage by the peptide molecules in the lower concentration range, and a predominant colloidal aggregation in the higher concentration range, respectively. To confirm this hypothesis, the flocculation assay by addition of an electrolyte solution (56 nM NaCl) has been performed for the gold nanoparticle-peptide assemblies formed in the 25-100 nM range of peptide concentration. Figure 1c, d show that the addition of 75 nM VEGFGlyFAM and 25 nM VEGFCysFAM were enough to stabilise the gold colloids, which exhibit a decrease in the intensity of the plasmonic intensity respectively of 30%, 26%, and 9% for bare, VEGFGlyFAM- and VEGFCysFAM-coated nanoparticles. Itwas, therefore, inferred that the cysteine residue was more effective as anchoring site to the metal nanoparticle surface than the glycine residue, thus owing the chemisorption forces that complement the energy balance of the peptide adsorption at the nanoparticle surface. To remove the loosely bound and/or free peptide molecules from the peptide-coated NPs, a centrifugation step was performed. The supernatant and the pellet obtained were collected. Figure 2 shows the UV-vis spectra of recovered supernatants and re-suspended pellets and demonstrate a good stability of the prepared systems. Figure 3 shows the TEM images of the re-suspended samples. The nanoparticles appear well-dispersed with an average dimension of 30 nm, as dynamic light scattering analysis confirmed. Instead, the average size of hydrodynamic di  
Figure 1: Changes in the AuNP (1.3 nM) plasmonic band by addition of: VEGFGlyFAM (**a**) and VEGFCysFAM (**b**) at increasing concentrations (from 2.5 \(\times\) 10\({}^{-8}\) M to 17.5 \(\times\) 10\({}^{-8}\) M). In (**c**,**d**) are shown the UV-visible spectra of bare and peptide-coated AuNPs upon the addition of 56 \(\upmu\)M NaCl. Figure 2: UV-visible spectra of AuNP/VEGFGlyFAM (**a**) and AuNP/VEGFCysFAM (**b**) dispersions before (solid line) and after (dot line: pellet; dash-dot line: supernatant) centrifugation. The spectra for bare AuNP are shown for comparison. AuNPs/VEGFCys FAM reached \(\sim\)159.3 \(\pm\) 11.9 nm in comparison to AuNPs/VEGFGly FAM (\(\sim\)67.4 \(\pm\) 0.8 nm). It was also possible to note a shell around the grouped nanoparticles suggesting the effectively coating of the peptide at the nanoparticles surface. On the other case, AuNPs/VEGFGly FAM image did not clearly evidence the same coating, but only a slight increasing of the average dimensions. The change of \(\zeta\)-potential values further confirms the effective functionalization of AuNP (Figure 4). Indeed, for bare NPs we found an average ZP of \(\sim\)\(-\)60.6 mV, while the surface charge was almost neutralized after the peptide immobilization (ZP\(\sim\)\(-\) 7.7 mV and 4.4 mV for AuNPs/VEGFGly FAM and AuNPs/VEGFCys FAM, respectively).