Oriented and reversible immobilization of His-tagged proteins on two- and three-dimensional surfaces for study of protein–protein interactions by a QCM biosensor

A two-dimensional (2D) and a three-dimensional (3D) His-tag capture surfaces were fabricated for oriented and reversible immobilization of His-tagged proteins on quartz crystal microbalance (QCM) biosensor surfaces, which can be used for label-free and real-time detection of the interactions between His-tagged protein and its interacting protein (analyte). His-tagged proteins immobilized on the 2D His-tag capture surface maintained a higher binding activity than those immobilized on a 2D carboxyl surface via amine coupling. The 3D His-tag capture surface has about twice the amount of immobilization capacity as the 2D His-tag capture surface, which enables a higher sensitivity for detection. His-tag capture surface can be optionally regenerated to remove the His-tagged protein as well as the analyte for the next cycle of His-tagged protein immobilization, or to only selectively remove the analyte, leaving the His-tagged protein on the surface for the next cycle of analyte binding. Furthermore, the kinetic and affinity studies of the interactions between the His-tagged protein and its interacting protein were performed. This study provides an efficient way to study protein–protein interactions by oriented and reversible immobilization of His-tagged proteins on QCM biosensor surfaces.