Figure 3 A–C) Extinction spectra of GNPs incubated with BSA
without removal of unbound proteins; D–F) Extinction spectra of GNPs
samples incubated with BSA after removal of unbound proteins (by
centrifugation and resuspension).
After the “washing” cycle to remove unbound proteins, all three types
of GNPs demonstrated similar behaviour patterns – at least some
aggregation, when the GNPs were preincubated with BSA at concentrations
≤2 mg/mL and stabilization, when preincubated with BSA at concentrations
≥9 mg/mL. It could be due to only very tightly bound BSA molecules
remaining on the GNPS during the “washing”, while weakly bound
proteins were lost.
These observations are supported by zeta potential measurements of the
“washed” samples (Figure 4 ). Aqueous-media exposed –COOH
groups of the capping agents are ionized, creating negative surface
charges and an acidic pH of the colloid GNPs solutions (pH
~ 3 for all three solutions after
synthesis).[35] At a pH lower than its isoelectric
point, BSA molecules have a net positive charge. Thus, after
preincubation with a low concentration of BSA and removal of any unbound
protein, the absolute surface charge of the GNPs@BSA nanoparticles is
much lower than the as-synthesized samples – almost neutral causing
aggregation due to the reduction of interparticle electrostatic
repulsion. We speculate that with the increase of protein bound to the
nanoparticles, the solution pH increases, and GNPs@BSA charge stabilizes
at around –15 mV. It is generally accepted that absolute values of
surface charge lower than 30 mV are not high enough to prevent NPs from
aggregation due to a lack of electrostatic
repulsion.[36,37] However, the stability of the
GNPs@BSA particles is enhanced by the BSA layer, presumably by its
steric repulsion.