Paper BI+AS-ThA6
Controlling and Probing the Orientation of Immobilized Protein G B1 on Gold Nanoparticles Using Time of Flight Secondary Ion Mass Spectrometry and X-ray Photoelectron Spectroscopy

Thursday, November 2, 2017, 4:00 pm, Room 12

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 can 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 through specific maleimide-cysteine interaction. As the wild type Protein G B1 does not contain a cysteine, we can strategically introduce cysteine mutants on Protein G B1 to control the location of the maleimide-cysteine bonding. 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 surface elemental composition, coverage, and orientation of the protein G B1 immobilization process.

XPS analysis confirmed the AuNP functionalization with the maleimide SAMs. After incubation with protein containing cysteine mutant, the immobilization of the protein was demonstrated by the increased nitrogen signal on the surface of the AuNP. Wild type Protein G B1 cannot form the maleimid-cysteine bond and was effectively removed through conventional centrifugation-resuspension washes and dialysis cleaning.

ToF-SIMS analysis also confirmed the successful functionalization and protein immobilization on the AuNPs by identifying signature secondary ions of the maleimide functional group and amino acids. Utilizing the small sampling depth (~2nm) of ToF-SIMS relative to the size of Protein G B1 (~3nm), the orientation of immobilized protein G B1 was determined by comparing the ratio of secondary ion intensity originating from the opposite regions of the protein. Overall, site-specific maleimide-cysteine interaction and systematic surface characterizations enabled us to both control and probe the orientation of immobilized proteins on AuNPs. The systematic characterization of this study provided detailed information about protein-NP interactions and a platform for controlled immobilization for IgGs on NPs.