Attachment of Streptavidin to β‐Cyclodextrin Molecular Printboards via Orthogonal Host–Guest and Protein–Ligand Interactions

Abstract

Streptavidin (SAv) is attached to beta-cyclodextrin (beta-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 beta-CD SAMs. SAv complexed to excess monovalent linker in solution and then attached to a beta-CD SAM could be removed by rinsing with a 10 mM beta-CD solution. When SAv was attached to the beta-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 beta-CD SAM resulting in (labile) divalent and (stable) tetravalent beta-CD-adamantyl interactions for the mono- and divalent linkers, respectively. This was confirmed by experiments at varying beta-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 beta-CD SAM allows the stepwise assembly of the complex at the beta-CD SAM by first adsorbing the linker, followed by SAv. This stepwise assembly allows the controlled heterofunctionalization of surface-immobilized SAv.

Orthogonal switching of a single supramolecular complex

“…Recent efforts have been dedicated to gaining orthogonal control over supramolecular systems, whereby different responses can be triggered on demand with a specific order of input stimuli, representing versatility and complexity beyond unifunctional systems 6 . One attractive way to achieve this is to delicately combine host-guest molecular recognition with other non-covalent interactions, for example, metal-ligand or protein-ligand interactions [7][8][9][10] . A major limitation in the field, however, has been the incorporation of multiple responsive components into one single supramolecular entity directly.…”

Multivalent Binding of Small Guest Molecules and Proteins to Molecular Printboards inside Microchannels

“…Compounds 1, 2, and b-CD-heptamine were synthesized as described previously. [35,39,43] Freshly distilled toluene was used for microchannel functionalization. SAv-and FITC-labeled IgG from human serum were obtained from Sigma Aldrich.…”

“…[35] This method also allows control over the orientation of the proteins to the surface because the linker can be designed in such a way that it will bind to a specific region of the protein. Protein adsorption onto the host-guest binding platform can be conducted with complete suppression of nonspecific interactions, by employing a monovalent oligo-(ethylene glycol) masking molecule during the adsorption.…”

“…[28][29][30][31][32][33] We have previously shown that it is possible, by means of host-guest chemistry, to control the binding properties of proteins to b-cyclodextrin (b-CD) monolayers [34] by using orthogonal linkers. [35] Such linkers can be designed with different numbers of guest groups, which results in protein complexes with a varying number of interactions, and thereby, a tunable binding strength to the b-CD monolayer. Proteinlinker complexes with a low number of interactions (e.g., 2) to the surface can be removed from the surface in a competition experiment with b-CD in solution (b-CD l ), whereas a Abstract: b-Cyclodextrin (b-CD) monolayers have been immobilized in microchannels.…”

“…[35] Linker 2, which consists of two adamantyl functionalities to ensure binding to b-CD monolayers and a biotin functionality to ensure binding to SAv, was washed over the surface followed by a wash with SAv. This assembly process leaves two biotin binding pockets available for further functionalization with, for example, fluorescently labeled biotin moieties (3 and 4; Scheme 2a), or biotinylated protein A (bt-PA) or protein G (bt-PG), which in turn can bind to fluorescently labeled human-IgG (5) and goat-IgG (6), respectively (Scheme 2b and c).…”