| AVS 63rd International Symposium & Exhibition | |
| Applied Surface Science | Tuesday Sessions |
| Session AS+AC-TuM |
| Session: | Practical Surface Analysis II: Microanalysis, Nanoanalysis, Atom Probe, and All Things ‘Small’ |
| Presenter: | David Castner, University of Washington |
| Authors: | Y.-C. Wang, University of Washington D.G. Castner, University of Washington |
| Correspondent: | Click to Email |
Nanoparticles (NPs) have been widely used in many fields of science due to their unique physical properties. While many applications of NPs such as imaging probes or drug carriers often require the conjugation of proteins or biomolecules, the surface interactions between NPs and biomolecules remains underexplored. For example, the immobilization of immunoglobulin G (IgG) onto nanoparticle surfaces is critical for the development of many immunosensors and drug delivery nanocarriers. Notably, the orientation of the immobilized IgG can have significant impact on the clinical outcomes of these carriers by impacting its biostability and efficacy.
In this work, Protein G B1, a protein that will selectively bind to the Fc tail of IgG, was immobilized onto gold NPs (AuNPs) functionalized with maleimide and oligo-(ethylene glycol)(OEG) self-assembled monolayers (SAMs). Protein G B1 was immobilized onto AuNPs using either carbonyldiimidazole (CDI) chemistry or through a maleimide-cysteine bond. We used the surface sensitive analysis techniques of x-ray photoelectron spectroscopy (XPS) and time of flight-secondary ion mass spectrometry (ToF-SIMS) to characterize the protein G B1 immobilization. Unlike conventional NP characterization techniques such as dynamic light scattering (DLS) and UV/Vis, XPS and ToF-SIMS can provide additional information on the surface elemental composition, protein coverage and orientation.
XPS analysis confirmed the AuNP functionalization with both the maleimide and OEG-SAMs. After incubation with protein, the immobilization of the protein was demonstrated by the increased nitrogen signal on the surface of both SAMs. Loosely bound protein on the AuNPs was effectively removed through conventional centrifugation-resuspension washes and dialysis cleaning.
ToF-SIMS analysis also confirmed the successful functionalization, CDI activation, and protein immobilization by identifying signature secondary ions from each step of the protein immobilization process. Further, by utilizing high surface sensitivity and small sampling depth (2nm) of ToF-SIMS, the orientation of immobilized protein G B1 was determined by comparing the ratio of secondary ion intensity originating from the opposite end of the protein. As expected, the non-site specific CDI chemistry did not lead to a well-defined orientation on the AuNPs. In contrast, we were able to control the orientation of the immobilized protein using maleimide functionalized AuNPs and cysteine mutants of Protein G B1. The systematic characterization of this study provided detailed information about protein-nanoparticle interactions that advances our understanding of the complex protein-NP interface.