Molecular Self‐Assembly, Chemical Lithography, and Biochemical Tweezers: A Path for the Fabrication of Functional Nanometer‐Scale Protein Arrays

Abstract

Exploration of the immensurable gene products of the ∼25000 human genes and mapping of dynamic protein interaction networks are key challenges in current biological research. [1] Highly parallel protein chip technologies deliver promising tools for these challenges even down to the singlemolecule level. [2] Whereas DNA molecules are robust and easy to immobilize in a functional manner—as seen in the triumph of DNA chip technology [3] —proteins are highly sensitive and chemically heterogeneous entities denaturing rapidly in vitro. Using multivalency as a design principle, a chemical recognition unit with exceptionally high affinity for proteins was designed, while maintaining their functionality. [4] These multivalent chelator heads consist of cumulated N-nitrilotriacetic acid (NTA) moieties, which provide chemical recognition for histidine-tagged proteins by complexation of transition metal ions, for example, Ni(II). These strong as well as specific binding capabilities can easily be switched on and off by addition or removal of Ni(II) ions. Such properties make tris-NTA chelators ideal “biochemical tweezers” for attaching proteins to surfaces and releasing them again. A tris-NTAunit coordinates six imidazole moieties and, thus, perfectly matches the coordination demands of a hexahistidine tag with binding affinities in the nanomolar range. [5] For the generation of structured protein arrays, a lithography step is necessary. Both electron-beam lithography (EBL) or extreme UV interference lithography (EUV-IL) are promising for this task. EBL gives the largest variation of pattern shapes and lateral dimensions in a very controlled manner. [6] The application of focused e-beams provides a chance to even generate structures capable of immobilizing single biological molecules. EUV-IL can create highly parallel patterns over large areas in a single processing step with the resolution approaching the EBL limit, [7] which is of great potential for applications in nanotechnology. In this Communication we present a new path for the fabrication of solid templates for the laterally defined and functional immobilization of proteins. The technology is based on the combination of electron-induced chem