2.5. Gold Nanoparticle Synthesis and Functionalization

Gold NPs with a diameter of 40 nm were prepared by seed-mediated synthesis [36]. The Supplementary Materials contain detailed information on all preparation steps to functionalize the nanoparticles. In brief, to vary the surface density of biotin on the outer surface of poly(ethylene glycol)-brush stabilized gold NPs, they were modified with mixtures of mPEG-SH and HS-PEG-Biotin. HS-PEG-Biotin and mPEG-SH were prepared fresh by dissolving in milli-Q water to a concentration of 3 mM to obtain molar fractions of 0, 5, 10, 20, 40, 50–100% HS-PEG-Biotin/mPEG-SH. Gold NPs were incubated with the respective PEG-thiol mixture under thorough mixing by gentle swirling overnight at 4 °C. The gold particles were purified from excess PEG-thiol and purified by seven repeated sequentl washing steps in Milli-Q water using Amicon Ultra centrifugal ultra-4 filter with 100 kDa cut-off (Merck Millipore, Ltd., Tullagreen, Ireland). The centrifugation was performed at 800× g for 10 min at 4 °C followed by resuspension in Milli-Q water. This yielded colloidally stable particles shielded by a PEG-brush as shown previously [37]. The nanoparticles were diluted in NaCl (10 mM, pH 7.2) solution. Different samples were incubated at room temperature for 5 min and then analyzed; the hydrodynamic diameters and Zeta-potentials for the gold NPs were acquired by dynamic light scattering (DLS) using a Malvern Zeta Sizer, NanoZS (Malvern Instrument Ltd, Malvern, UK) instrument. The size distribution of the gold NPs was determined from transmission electron micrographs (TEM, see below for the description of TEM sample preparation and measurement) using the public domain image processing software ImageJ and its tool to analyze particles. The average gold NP size was calculated by fitting a Gaussian distribution to the size distributions obtained by the TEM analysis. Thermal gravimetric analysis of the nanoparticles was performed on a Mettler Toledo TGA/DSC 1 (Mettler Toledo GmbH, Vienna, Austria), with 80 mL min⁻¹ synthetic air as reactive gas, 20 mL min⁻¹ nitrogen as protective gas and a heating rate of 10 K min⁻¹ from 25 to 650 °C. Using TGA results, the grafting density (σ) was calculated using the following formula:

where (% w/w)_shell is percentage of mass loss in TGA for the organic fraction corresponding to the PEG grafted to the gold core; N_A is the Avogadro constant; ρ_Au is the density of gold; V_core is the volume; A_core is the area of the gold core, calculated from the diameter of the cores measured by TEM; M_PEG is the molecular weight of the PEG; and (% w/w)_core. is the residual mass percentage of the inorganic fraction in TGA.