Retention-Based, Dissociation-Sensitive Pre-Screening of Aptamers Using a Centrifugal Microspin Filter.

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-koff 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 koff. A preliminary analysis of streptavidin aptamers likewise showed a W0-NRT-based ranking consistent with the literature-reported koff 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 koff 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-koff assay therefore provides a simple post-SELEX approach for prioritizing candidates with higher retention under dissociation-sensitive washing before detailed kinetic characterization.