Stable immobilisation of His-tagged proteins on BLI biosensor surface using cobalt

Abstract In biosensor experiments, ligand molecules need to be immobilized tightly on sensor surfaces for sensitive and robust detection of the analyte of interest. Stretches of histidines (His-tags) are typically inserted into recombinant proteins for affinity purification purposes, but can also be used in the immobilization of biosensors functionalized with Ni-NTA (nitrilotriacetic acid). The His-tag-Ni-NTA bond is, however, easily disrupted by factors including low pH, reducing agents, or chelating agents such as EDTA. In biosensing applications, a stable ligand immobilization that tolerates harsh conditions is preferable, because the sample matrix can vary from bodily fluids to waste waters. We describe here Co(III)-NTA bonding for tight immobilization of His-tagged proteins on Octet BLI-biosensors. The performance of the Ni(II)-NTA was compared to that of Co(III)-NTA using His-tagged avidins and norovirus proteins. Comparative studies were performed using biotin-iminodiacetic acid (IDA) for streptavidin sensor functionalization instead of the NTA surfaces provided by the manufacturer. It was noted that Co(III)-NTA offers highly stable immobilization of His-tagged protein, tolerating factors such as 0.7 M imidazole, that is typically used for detaching proteins from Ni(II)-NTA-agarose used in protein purification columns. The good performance of Co(III)-NTA functionalized BLI sensor was demonstrated by detecting norovirus antibodies from human serum samples with sensors functionalized with His-tagged virus-like particles. The use of Co-NTA sensors for attachment of His-tagged proteins is ideal in conditions, where the immobilized ligands need to be firmly attached and tolerate harsh chemical conditions.