## 3 Results and Discussion
### Protein Corona Quantification  
AuNPs are known to exhibit a fluorescence quenching effect due to surface plasmon resonance band which absorbs close to the wavelength emission band of the FITC molecule (Figure 4). For the fluorescence intensity quantification, a complete dissolution of the gold nanoparticles was mandatory. This was accomplished by adding CN\({}^{-}\) in excess, which led to the formation of complex AuCN\({}^{2-}\), a colorless non-fluorescent ion. The addition of 50 mM KCN had no impact on the fluorescence of the FITC-BSA, as Figure 4 shows; however, as a precautionary measure, KCN was also added to the dilutions used for the calibration curve in order to elude any effect of the cyanide ions on the fluorescence. The calibration curve used for protein corona quantification is presented in Figure 5. A linear plot was obtained over the range 0.125-120 ng, with a correlation coefficient of R\({}^{2}\) = 0.9996. An amount of 1, 50 and 100 ng of FITC-labeled protein was used to estimate the BSA-FITC % recovery. The experimental data are presented in Table 2.  
Figure 4: Normalized fluorescence emission spectra of BSA-FITC. Figure 5: The calibration curve obtained after plotting the fluorescent intensities against the amount of BSA-FITC. Figure 6 shows the total amount of proteins adsorbed on each type of nanoparticles. As expected, the amount of protein adsorbed on the 56 nm AuNPs was higher compared to smaller particles due to a higher surface area (9850 nm\({}^{2}\)/NP vs. 1820 nm\({}^{2}\)/NP) for all types of surface chemistries. As described by [PERSON] et al., particle size plays a crucial role into formation of protein corona, with bigger particles forming a thicker layer of proteins due to different forming kinetics compared to the smaller ones [27]. Another main factor that controls protein affinity for nanoparticles' surfaces is represented by surface chemistry. Several studies have clearly established that surface chemistry quantitatively and qualitatively influences the protein corona (by modulating the type of proteins that are adsorbed on the surface of the nanoparticles from a mixture of proteins) [10; 28; 29]. Our results indicate that the highest amount of proteins was adsorbed by the positively charged, PEG-NH\({}_{2}\) coated AuNPs. In contrast, the neutrally charged PEG-OCH\({}_{3}\) AuNPs absorbed the least, while the negative charged PEG-COOH-coated AuNPs adsorbed an intermediate amount of proteins. The results can be explained by considering the intrinsic properties of BSA, which has an isoelectric point of 4.7 [30] and a surface charge of the nanoparticles. At pH = 7.4, the protein was slightly negatively charged, which was translated into a higher affinity for the positively charged NH\({}_{2}\) modified surface, as the zeta-potential measurements indicated. We also examined the influence of the molecular weight of the PEG ligand bearing the same functional group on the protein corona. Our results indicated that the PEG chain length had no influence on the quantity of protein attached to the surface of AuNPs. No significant differences were \begin{table}
\begin{tabular}{c c c c c} \hline \hline \multirow{2}{*}{**Nanoparticle**} & \multirow{2}{*}{**PEG Ligand**} & \multicolumn{3}{c}{**\% Recovery**} \\ \cline{3-5}  & & **1 ng** & **50 ng** & **100 ng** \\ \hline \multirow{4}{*}{AuNP 24 nm} & PEG-COOH 5 kDa & 98.1 & 98.1 & 101.1 \\  & PEG-COOH 20 kDa & 98.2 & 98.2 & 99.2 \\  & PEG-OCH\({}_{3}\) & 99.5 & 99.5 & 98.5 \\  & PEG-NH\({}_{2}\) & 97.1 & 97.1 & 98.4 \\ \hline \multirow{4}{*}{AuNP 56 nm} & PEG-COOH 5 kDa & 97.2 & 99.7 & 98.2 \\  & PEG-COOH 20 kDa & 98.4 & 102.4 & 99.4 \\ \cline{1-1}  & PEG-OCH\({}_{3}\) & 99.7 & 99.8 & 98.2 \\ \cline{1-1}  & PEG-NH\({}_{2}\) & 96.4 & 98.6 & 100.3 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: BSA-FITC recovery data. Figure 6: The amount of fluorescently-labeled BSA adsorbed onto the AuNPs (24 and 56 nm). (concentration of AuNPs: \(1\times 10^{10}\) NP/mL). Results are given as mean +/- standard deviation of three independent replicates.