Stochastic Sensing of His-Tagged Proteins with a NTA-Functionalized Solid-State Nanopore

Abstract

Artificial nanopores in solid-state membranes have emerged as a versatile tool to study single biomolecules. Employing nanopores as stochastic sensors offers the attractive prospect of detecting and quantifying molecular interactions and bindings on a single-molecule level. Individual interactions between analyte molecules and receptor sites within the pore can be observed as transient modulations of the trans-pore current.Here we demonstrate the stochastic sensing of single proteins in a solid-state nanopore functionalized with nitrilotriacetic acid (NTA) chelator groups as specific binding sites for histidine-tagged proteins. The non-covalent yet selective His-tag/NTA interaction is well suited as a model system, since the binding strength depends on NTA valency and presence of competitive binders.NTA affinity groups are tethered within the nanopores by first coating the walls of pores in SiN membranes with gold, and subsequently adsorbing NTA-functionalized alkane-thiol self-assembled monolayers.Individual binding events of His-tagged proteins were successfully detected in real-time. Analysis of the binding times provide kinetic information and corroborate the occurrence of bimolecular interactions. The binding is shown to be reversible and specific, as the kinetics can be significantly altered by the addition of imidazole as a competitive binding agent and is completely inhibited by removal of the chelated metal ions. In concentration dependent studies we elucidate the influence of the competitive binder on the single-molecule binding times.We systematically studied the influence of the NTA valency on the binding strength, comparing mono-, bis-, and tris-NTA receptors and present a ranking of single-molecule binding times. Our results indicate that multivalent interactions are strong enough to stably immobilize His-tagged proteins within the pore, so their interactions with third-party proteins can be studied. This paves the way for the use of NTA functionalized metal nanopores as generic tools to study protein-protein interactions one-on-one.