Cobalt(III)‐Mediated Permanent and Stable Immobilization of Histidine‐Tagged Proteins on NTA‐Functionalized Surfaces

We present the cobalt(III)‐mediated interaction between polyhistidine (His)‐tagged proteins and nitrilotriacetic acid (NTA)‐modified surfaces as a general approach for a permanent, oriented, and specific protein immobilization. In this approach, we first form the well‐established Co²⁺‐mediated interaction between NTA and His‐tagged proteins and subsequently oxidize the Co²⁺ center in the complex to Co³⁺. Unlike conventionally used Ni²⁺‐ or Co²⁺‐mediated immobilization, the resulting Co³⁺‐mediated immobilization is resistant toward strong ligands, such as imidazole and ethylenediaminetetraacetic acid (EDTA), and washing off over time because of the high thermodynamic and kinetic stability of the Co³⁺ complex. This immobilization method is compatible with a wide variety of surface coatings, including silane self‐assembled monolayers (SAMs) on glass, thiol SAMs on gold surfaces, and supported lipid bilayers. Furthermore, once the cobalt center has been oxidized, it becomes inert toward reducing agents, specific and unspecific interactions, so that it can be used to orthogonally functionalize surfaces with multiple proteins. Overall, the large number of available His‐tagged proteins and materials with NTA groups make the Co³⁺‐mediated interaction an attractive and widely applicable platform for protein immobilization.