Cheeyoon Ahn

Efficient evaluation of aptamer candidates after systematic evolution of ligands by exponential enrichment (SELEX) remains limited by the time and cost required for direct kinetic characterization. We developed a centrifugal microspin filtration (MSF)-based MSF–k_off assay for retention-based, dissociation-sensitive pre-screening of aptamer candidates. Repeated washing preferentially removes rapidly dissociating aptamers from target-immobilized magnetic beads, and the retained aptamers are quantified by quantitative polymerase chain reaction and expressed as Wash 0-normalized retention (W0-NRT). In a thrombin aptamer panel, W0-NRT showed a strong inverse relationship with SPR-derived k_off. A preliminary analysis of streptavidin aptamers likewise showed a W0-NRT-based ranking consistent with the literature-reported k_off values, although the two target systems exhibited distinct empirical relationships. In a wash-based thrombin sandwich assay, two affinity-matched sensor aptamers differing 1.76-fold in k_off were ranked in the same order by post-wash retention as by W0-NRT. The agreement was in rank order rather than in the magnitude of retention. The parallel microspin filter format was also estimated to offer advantages in analysis time and assay-related cost. The MSF–k_off assay therefore provides a simple post-SELEX approach for prioritizing candidates with higher retention under dissociation-sensitive washing before detailed kinetic characterization.