Abstract
It is demonstrated that the adsorption of bovine serum albumin (BSA) to aqueous gold colloids can be quantified with molecular resolution by differential centrifugal sedimentation (DCS). This method separates colloidal particles of comparable density by mass. When proteins adsorb to the nanoparticles, both their mass and their effective density change, which strongly affects the sedimentation time. A straightforward analysis allows quantification of the adsorbed layer. Most importantly, unlike many other methods, DCS can be used to detect chemisorbed proteins (“hard corona”) as well as physisorbed proteins (“soft corona”). The results for BSA on gold colloid nanoparticles can be modeled in terms of Langmuir-type adsorption isotherms (Hill model). The effects of surface modification with small thiol-PEG ligands on protein adsorption are also demonstrated.
Highlights
The phenomenon of protein adsorption to colloidal particles has been studied for over 100 years
Fluorescence correlation spectroscopy (FCS) and plasmon scattering correlation spectroscopy (PSCS) have been used to obtain diffusion coefficients and hydrodynamic radii of nanoparticles within protein containing fluids.[20,22−25] These can yield thermodynamic data on corona formation and have shown that the process can be quantitatively described in terms of the Hill model, i.e., Langmuir-type adsorption
The data shown here were measured with bovine serum albumin (BSA) in the differential centrifugal sedimentation (DCS)-gradient fluid at the same concentration as used during incubation, distributions measured without BSA in the gradient fluid are shown in Figure S2 in the Supporting Information
Summary
Article thickness of thiolate stabilized gold nanoparticles.[21]. We demonstrate here that this technique is suitable to monitor protein adsorption to gold nanoparticles and to determine the thermodynamic constants that govern this process. In the absence of BSA in the gradient fluid, on the other hand, a slightly thinner protein layer is found at higher BSA concentrations, indicating the loss of some of the adsorbed proteins upon injection This loss of part of the corona within the time scale of the DCS experiment, i.e., within a few minutes, is in agreement with previous reports of such rapid removal of the physisorbed corona.[3,6] even the complete removal of excess BSA by repeated centrifugation prior to the DCS measurement (open circle in Figure 2A) or incubation in BSA-free citrate solution for 24 h (open triangle) do not significantly affect the measured corona thickness. Covalent binding between BSA and the NP makes it likely that the protein would not exchange with other “hard corona” proteins in solution and may be the reason for the memory effect that has been described in the literature, i.e., the fact that the corona composition after incubation in complex biological samples depends on details of the incubation history.[11,55] In this context, it is interesting to note that the chemisorbed BSA corona on citrate-stabilized gold NPs is formed within seconds of incubation in BSA solution, which is much faster than the usual hour time scales for hard corona formation in serum.[3,8,15] This is due to the competition between serum components with different binding modalities, including proteins with weaker but faster adsorption which rapidly cover the NP surface during the initial phase of corona formation, preventing fast formation of the hard corona which eventually develops
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