XPS, ToF-SIMS, and SPR characterization of protein immobilization onto self-assembled monolayer surfaces

A thorough understanding of surface-bound proteins (their composition, coverage, orientation, conformation, activity, etc.) is essential for understanding protein-surface interactions and designing biorecognition/biocompatible surfaces. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is uniquely qualified to address this problem due to its molecular specificity and surface sensitivity. However, well-defined model systems, both in terms of the immobilization surface and three-dimensional structure of the protein being immobilized, are required to develop the full power of ToF-SIMS. Studies presented in this work focused on developing model systems with well-defined surface structures and immobilization chemistries for ToF-SIMS investigation.;Initially self-assembled monolayers (SAMs) bearing N-hydroxysuccinimide ester oligo(ethylene glycol) (NHS-OEG) and a mixture of nitrilotriacetic acid (NTA) headgroups and oligo(ethylene glycol) (OEG) moieties were developed. Their surface composition, structure and reactivity were characterized using surface analysis methods that included X-ray photoelectron spectroscopy (XPS), ToF-SIMS, and surface plasmon resonance (SPR). By applying principal component analysis (PCA) to and developing multivariate peak ratios from the ToF-SIMS data, spectral changes were correlated to various surface treatments. For NHS-OEG SAMs, aging in ambient air for up to seven days resulted in some hydrolysis of the bound NHS groups, oxidation of some thiol groups, and deposition of adventitious hydrocarbon contaminants onto the monolayers. Proteins immobilized onto the NHS-OEG SAMs were randomly oriented, whereas histagged proteins immobilized onto the Ni2+-loaded NTA/OEG SAMs were oriented in a specific and controlled manner via the NTA headgroups.;Additionally, the conformational changes that occur when a surface-bound protein goes from a hydrated to a dehydrated state were monitored by cold-stage ToF-SIMS. Two protein model protein systems, histagged humanized anti-lysozyme variable fragment (HuLys Fv) on a NTA surface and human serum fibrinogen on a bare gold surface, were used in these studies. PCA results were used to correlate protein structural changes with the surface coverage and degree of dehydration. Multivariate ToF-SIMS peak ratios were further developed for tracking how protein orientation and conformation change with coverage and degree of dehydration. At low coverage significant conformation changes were observed, while at high coverage these dehydration-induced transitions were inhibited and the native protein structure was better preserved.