4.3. PEGylated Gold Nanoparticle Preparation

The thiolated 5K, 10K, and 30K poly(ethylene glycol) monomethyl ether compounds (PEG-SH) were purchased from Laysan Bio, Inc. (Arab, AL, USA). The PEG-SH compounds were dissolved in MilliQ water containing 50 mM TCEP to maintain reducing conditions. A solution of 120 nM AuNPs was incubated with 100 μM of the three different sizes of PEG to fully saturate the AuNP surface. After the overnight incubation at room temperature, the solutions were centrifuged at 9000× g and washed three times with buffer to remove any unbound PEG molecules. Afterward, PEG-coated AuNPs were resuspended in buffer. The PEGylated AuNPs were characterized using UV-Vis spectroscopy and dynamic light scattering to confirm the functionalization. The same extinction coefficient was used for PEGylated AuNPs as for bare AuNPs. Atomic absorption spectroscopy confirmed that this approximation was accurate to within 6% error (Table S2). The PEG density on the AuNP surface was determined as follows. A total of 120 µM thiolated PEG was mixed with 120 nM 15-nm AuNPs. After 1-h incubation, all PEG bound AuNPs were thoroughly spun down (10 min at 21,000× g) and the bound fraction of PEG was determined by the reduction in thiol concentration in the supernatant using Ellman’s reagent (5,5′-dithio-bis (2-nitrobenzoic acid)) [63]. The coordination efficiency (θ) of PEG was calculated using θ = 1 − [SH]_free/[SH]_total, where [SH]_total and [SH]_free correspond to concentration of thiolated PEG in the supernatant before and after binding to AuNP. The PEG grafting density (ρ_PEG) was calculated using formula ρ_PEG = θ ρ_graft, where ρ_graft is the number of initial PEG molecules available per nm² AuNP surface.