Concentration and composition of the protein corona as a function of incubation time and serum concentration: an automated approach to the protein corona.

Nanoparticles in contact with proteins form a "corona" of proteins adsorbed on the nanoparticle surface. Subsequent biological responses are then mediated by the adsorbed proteins rather than the bare nanoparticles. The use of nanoparticles as nanomedicines and biosensors would be greatly improved if researchers were able to predict which specific proteins will adsorb on a nanoparticle surface. We use a recently developed automated workflow with a liquid handling robot and low-cost proteomics to determine the concentration and composition of the protein corona formed on carboxylate-modified iron oxide nanoparticles (200 nm) as a function of incubation time and serum concentration. We measure the concentration of the resulting protein corona with a colorimetric assay and the composition of the corona with proteomics, reporting both abundance and enrichment relative to the fetal bovine serum (FBS) proteins used to form the corona. Incubation time was found to be an important parameter for corona concentration and composition at high (100% FBS) incubation concentrations, with only a slight effect at low (10%) FBS concentrations. In addition to these findings, we describe two methodological advances to help reduce the cost associated with protein corona experiments. We have automated the digest step necessary for proteomics and measured the variability between triplicate samples at each stage of the proteomics experiments. Overall, these results demonstrate the importance of understanding the multiple parameters that influence corona formation, provide new tools for corona characterization, and advance bioanalytical research in nanomaterials.