Attachment of streptavidin to β-cyclodextrin molecular printboards via orthogonal host-guest and protein-ligand interactions

Streptavidin (SAv) is attached to β-cyclodextrin (β-CD) self-assembled monolayers (SAMs) via orthogonal host-guest and SAv-biotin interactions. The orthogonal linkers consist of a biotin functionality for binding to SAv and adamantyl functionalities for host-guest interactions at β-CD SAMs. SAv complexed to excess monovalent linker in solution and then attached to a β-CD SAM could be removed by rinsing with a 10 mM β-CD solution. When SAv was attached to the β-CD SAM via the divalent linker, it was impossible to remove SAv from the surface by the same rinsing procedure. This is interpreted by assuming that two SAv binding pockets are oriented towards the β-CD SAM resulting in (labile) divalent and (stable) tetravalent β-CD-adamantyl interactions for the mono- and divalent linkers, respectively. This was confirmed by experiments at varying β-CD concentrations. When the [linker]/[SAv] ratio is reduced, a clear trend in the divalent-linker case is seen: the less linker the more protein could be removed from the surface. It is proven that the orthogonality of the binding motifs and the stability of the divalent linker at the -CD SAM allows the stepwise assembly of the complex at the β-CD SAM by first adsorbing the linker, followed by SAv. This stepwise assembly allows the controlled heterofunctionalization of surface-immobilized SAv.