A combined approach of surface passivation and specific immobilization to study biomolecules by ATR-FTIR spectroscopy

Abstract

Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy is a surface-sensitive and label-free technique, which is applied to obtain dynamic structural information of biomolecules. The study of proteins by ATR-FTIR spectroscopy can be impeded by their tendency to adsorb to solid surfaces. Furthermore, the adsorption process of proteins is often accompanied with conformational changes, which can interfere with the intended structural analysis. We efficiently modified a silicon ATR crystal surface with polyethylene glycol and thereby create a protein-repellent surface. To achieve a high sensitivity, which enables the study of small conformational changes of biomolecules, we combine surface passivation with specific immobilization. This is accomplished via the biotin-streptavidin interaction, which is one of the strongest known non-covalent protein-ligand interactions. As a proof of concept we present the specific immobilization of DNA. The modified surface is stable against elevated temperatures and 8 M urea and can therefore be used to study a wide range of biochemical systems and reactions. The surface chemistry is simple and performed under mild conditions, which leads to a high applicability of the presented approach.