Transient Incomplete Separation of Species with Close Diffusivity to Study the Stability of Affinity Complexes

作者：Tong Ye Wang, Jean‐Luc Rukundo, An T. H. Le, Nikita A. Ivanov, J. Leblanc, Boris I. Gorin, Sergey N. Krylov · 发表于：Analytical Chemistry · 年份：2022 · DOI：10.1021/acs.analchem.2c03313 · 被引用次数：13 · 研究领域：Monoclonal and Polyclonal Antibodies Research、Advanced biosensing and bioanalysis techniques、Molecular Junctions and Nanostructures

Large molecules can be generically separated from small ones, though partially and temporarily, in a pressure-driven flow inside a capillary. This transient incomplete separation has been only applied to species with diffusion coefficients different by at least an order of magnitude. Here, we demonstrate, for the first time, the analytical utility of transient incomplete separation for species with close diffusion coefficients. First, we prove in silico that even a small difference in diffusivity can lead to detectable transient incomplete separation of species. Second, we use computer simulation to prove that such a separation can be used for the reliable determination of equilibrium dissociation constant ( K d ) of complexes composed of similar-sized molecules. Finally, we demonstrate experimentally the use of this separation for the accurate determination of K d value for a protein–aptamer complex. We conclude that “accurate constant via transient incomplete separation” (ACTIS) can serve as a reference method for affinity characterization of protein–aptamer binding in solution.