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.