Paper BI+AS-MoA1
Characterization of Protein G B1 Immobilized Gold Nanoparticles using Time of Flight Secondary Ion Mass Spectrometry and X-ray Photoelectron Spectroscopy

Monday, October 19, 2015, 2:20 pm, Room 211D

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 NP surfaces is critical for the development of many immunosensors and drug delivery nanocarriers. Notably, the orientation of the immobilized IgG can have a significant impact on clinical outcomes of nanocarriers by impacting its biostability and efficacy. One approach to control the proper orientation of IgG is by utilizing the IgG Fc tail binding proteins.

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 on AuNPs using either carbonyldiimidazole (CDI) chemistry or maleimide-cysteine interaction. We use the surface sensitive analysis techniques of x-ray photoelectron spectroscopy (XPS) and time of flight-secondary ion mass spectrometry (ToF-SIMS) to characterize the immobilization of protein G B1. 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 CDI activation of the OEG-SAMs AuNPs by detecting the nitrogen containing active intermediate and the attenuation of gold signal. After incubation with protein, the immobilization of the protein was demonstrated by the increased nitrogen signal on the surface. 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.

By comparing the ratio of secondary ion intensity originating from opposite ends of the protein, it was possible to determine the orientation of immobilized protein G B1. As expected, the non-site specific CDI chemistry did not lead to a specific protein orientation on the AuNPs. In contrast to CDI chemistry, we expect to control the orientation of the immobilized protein using maleimide functionalized AuNPs and cysteine mutants of Protein G B1 through site-specific carbon-sulfur interaction.

Overall, the systematic characterizations in this study will provide detail information of protein-NP interactions and serve as a platform for controlling the orientation of IgG on AuNPs.