Surface characterization of mixed self-assembled monolayers designed for streptavidin immobilization

The self-assembly of streptavidin onto biotinylated alkylthiolate monolayer s on gold has served as an important model system for protein immobilizatio n at surfaces. Here, we report a detailed study of the surface composition and structure of mixed self-assembled monolayers (SAMs) containing biotinyl ated and diluent alkylthiolates and their use to specifically immobilize st reptavidin. X-ray photoelectron spectroscopy (XPS), angle-resolved XPS (ARX PS), near-edge X-ray absorption fine structure (NEXAFS), and surface plasmo n resonance (SPR) have been used to characterize the films produced on gold from a range of binary mixtures of a biotinylated alkylthiol (BAT) and eit her a Cls methyl-terminated thiol (mercaptohexadecane, MHD) or a C-11-oligo (ethylene glycol)-terminated (OEG) thiol in ethanol. The correlation betwee n the solution mole fraction of BAT and its surface mole fraction (chi (BAT ,sur)) indicates that it adsorbs similar to4-fold faster than OEG but sligh tly slower than MHD. ARXPS analysis demonstrates that the biotin terminus o f the BAT is exposed at the surface of mixed monolayers with chi (BAT,sur) < 0.5 but is randomly distributed through BAT-rich films. Thus, the OEG dil uent not only adds nonfouling properties but induces an improved concentrat ion of biotin at the surface and reduces the exposure of the methylene segm ents of BAT. NEXAFS characterization demonstrates that pure OEG and mixed B AT/OEG SAMs do not show significant anisotropy in C-C bond orientation, in contrast to MHD and mixed BAT/MHD SAMs, whose aliphatic segments exhibit ps eudo-crystalline packing. SPR measurements of streptavidin binding to and c ompetitive dissociation from the different mixed SAMs indicate that strepta vidin binds both specifically and nonspecifically to the BAT/MHD SAMs but p urely specifically to BAT/OEG SAMs with chi (BAT,sur) < 0.5 For BAT/OECT mi xtures with chi (BAT,sur) = 0.1-0.5, specifically bound streptavidin covera ges of similar to 80% of the C(2,2,2) two-dimensional streptavidin crystall ine density (similar to 280 ng/cm(2)) can be reproducibly achieved. These c omposite results clarify the relationship between the specificity of strept avidin recognition and the surface architecture and properties of the mixed SAMs.