Paper BI-WeM13
XPS, ToF-SIMS, NEXAFS and SPR Characterization of Nitrilotriacetic Acid-Terminated Self-Assembled Monolayers For Controllable Immobilization of Proteins

Wednesday, October 17, 2007, 12:00 pm, Room 609

For immobilization of proteins onto surfaces in a specific and controlled manner it is important to start with a well-defined surface that contains specific binding sites surrounded by a nonfouling background. For immobilizing histidine-tagged (histagged) proteins, surfaces containing nitrilotriacetic acid (NTA) headgroups and oligo(ethylene glycol) (OEG) moieties are a widely used model system. The surface composition, structure and reactivity of mixed NTA/OEG self-assembled monolayers (SAMs) on Au substrates were characterized in detail using X-ray photoelectron spectroscopy (XPS), near-edge X-ray absorption fine structure spectroscopy (NEXAFS), time-of-flight secondary ion mass spectrometry (ToF-SIMS) and surface plasmon resonance (SPR) biosensing. XPS results for sequentially adsorbed NTA thiols followed by OEG thiols showed that OEG molecules were incorporated into a incompletely formed NTA monolayer until a complete mixed SAM was formed. Surface concentration of NTA headgroups was estimated to be 0.9 molecule / nm² from XPS results. Angle-resolved XPS and polarization-dependent NEXAFS results both indicated NTA headgroups were slightly reoriented toward an upright position upon OEG incorporation. Nitrogen-containing and OEG-related secondary ion fragments from the ToF-SIMS experiments confirmed the presence of NTA headgroups and OEG moieties in the monolayer. These fragments along with secondary ion amino acid fragments are being used to investigate the orientation and conformation of histagged proteins immobilized onto the NTA/OEG SAMs. SPR measurements of a histagged, humanized anti-lysozyme variable fragment (HuLys Fv) immobilized onto Ni(II) -treated mixed NTA/OEG SAMs demonstrated the reversible, site-specific immobilization of histagged HuLys Fv (170 ± 7 ng / cm²) with strong binding affinity (approximately 43 nM). The mixed NTA/OEG SAMs without Ni(II) treatment exhibited good resistance to nonspecific adsorption of histagged HuLys Fv ( < 2 ng / cm²).