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

Sanna Auer, Latifeh Azizi, Felix Faschinger, Vesna Blazevic, Timo Vesikari, Hermann J. Gruber, Vesa P. Hytönen
Sens. Actuators, B, 2017, 243, 104 - 113
Analyte Matrix Technique


blood serum


Subject category(s): General analytical chemistry; Sensors; Clinical and biochemical analysis; Immune response

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.

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