Rapid and regenerable surface plasmon resonance determinations of biomarker concentration and biomolecular interaction based on tris-nitrilotriacetic acid chips

The tris-nitrilotriacetic acid (tris-NTA) chip has been used for surface plasmon resonance (SPR) kinetic studies involving histidine (His)-tagged proteins. However, its full potential, especially for analyte quantification in complex biological media, has not been realized due to a lack of systematic studies on the factors governing ligand immobilization, surface regeneration, and data analysis. We demonstrate that the tris-NTA chip not only retains His-tagged proteins more strongly than its mono-NTA counterpart, but also orients them more uniformly than protein molecules coupled to carboxymethylated dextran films. We accurately and rapidly quantified immunoglobulin (IgG) molecules in sera by using the initial association phase of their conjugation with His-tagged protein G densely immobilized onto the tris-NTA chip, and established criteria for selecting the optimal time for constructing the calibration curve. The method is highly reproducible (less than 2% RSD) and three orders of magnitude more sensitive than immunoturbidimetry. In addition, we found that the amount of His-protein immobilized is highly dependent on the protein isoelectric point (pI). Reliable kinetic data in a multi-channel SPR instrument can also be rapidly obtained by using a low density of immobilized His-tagged protein. The experimental parameters and procedures outlined in this study help expand the range of SPR applications involving His-tagged proteins. (c) 2021 Elsevier B.V. All rights reserved.

Automated summary

The tris-NTA chip shows strong retention and uniform orientation of His-tagged proteins, enabling accurate quantification of IgG molecules and establishing optimal calibration curve construction time. The amount of immobilized His-protein is highly dependent on protein pI. The study outlines experimental parameters and procedures to expand SPR applications involving His-tagged proteins.