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Combining the Benefits of Biotin–Streptavidin Aptamer Immobilization with the Versatility of Ni-NTA Regeneration Strategies for SPR
Hanson, Eliza K. Whelan, Rebecca J.
Hanson, Eliza K.
Whelan, Rebecca J.
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Abstract
The high affinity of the biotin–streptavidin interaction has made this non-covalent coupling an indispensable strategy for the immobilization and enrichment of biomolecular affinity reagents. However, the irreversible nature of the biotin–streptavidin bond renders surfaces functionalized using this strategy permanently modified and not amenable to regeneration strategies that could increase assay reusability and throughput. To increase the utility of biotinylated targets, we here introduce a method for reversibly immobilizing biotinylated thrombin-binding aptamers onto a Ni-nitrilotriacetic acid (Ni-NTA) sensor chip using 6xHis-tagged streptavidin as a regenerable capture ligand. This approach enabled the reproducible immobilization of aptamers and measurements of aptamer–protein interaction in a surface plasmon resonance assay. The immobilized aptamer surface was stable during five experiments over two days, despite the reversible attachment of 6xHis-streptavidin to the Ni-NTA surface. In addition, we demonstrate the reproducibility of this immobilization method and the affinity assays performed using it. Finally, we verify the specificity of the biotin tag–streptavidin interaction and assess the efficiency of a straightforward method to regenerate and reuse the surface. The method described here will allow researchers to leverage the versatility and stability of the biotin–streptavidin interaction while increasing throughput and improving assay efficiency.
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Date
2024-04-27
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Publisher
MDPI
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Keywords
Surface plasmon resonance (SPR), Ni2+-nitrilotriacetic acid (NTA), Biotin–streptavidin immobilization, Aptamers
Citation
Hanson EK, Whelan RJ. Combining the Benefits of Biotin-Streptavidin Aptamer Immobilization with the Versatility of Ni-NTA Regeneration Strategies for SPR. Sensors (Basel). 2024 Apr 27;24(9):2805. doi: 10.3390/s24092805. PMID: 38732912; PMCID: PMC11086168
DOI
10.3390/s24092805