Department of Chemistry, University of Kansas, Lawrence, KS 66045, USA.
Sensors (Basel). 2024 Apr 27;24(9):2805. doi: 10.3390/s24092805.
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.
生物素-链霉亲和素相互作用的高亲和力使得这种非共价偶联成为生物分子亲和试剂固定化和富集的不可或缺的策略。然而，生物素-链霉亲和素键的不可逆性质使得使用这种策略功能化的表面永久修饰，并且不适用于可以提高测定可重复使用性和通量的再生策略。为了增加生物素化靶标的用途，我们在这里介绍了一种使用 6xHis 标记的链霉亲和素作为可再生捕获配体可逆地将生物素化凝血酶结合适体固定到 Ni-硝基三乙酸 (Ni-NTA) 传感器芯片上的方法。这种方法能够在表面等离子体共振测定中重复固定适体并测量适体-蛋白质相互作用。尽管 6xHis-链霉亲和素可逆地附着到 Ni-NTA 表面，但固定化适体表面在两天的五次实验中保持稳定。此外，我们证明了这种固定化方法的重现性以及使用该方法进行的亲和测定的重现性。最后，我们验证了生物素标签-链霉亲和素相互作用的特异性，并评估了一种简单方法的效率，该方法可用于再生和重复使用表面。本文所述的方法将允许研究人员在提高测定效率的同时利用生物素-链霉亲和素相互作用的多功能性和稳定性。
