Scalable Multiparametric Characterization of Aptamer–Target Interactions

Structure-switching aptamers transduce target-induced conformational changes into detectable signals, enabling the specific detection of small molecules with limited surface area and charge. Understanding these structural transitions is critical for the rational design of aptamers in downstream biosensing. However, current methods lack the scalability and high spatiotemporal resolution to characterize and resolve these structural dynamics within a single unified platform. Here, we report a scalable droplet microfluidic platform that fills this technological gap by integrating Forster resonance energy transfer with automated imaging for the multiparametric profiling of aptamer−target interactions. This integrated system enables the detailed analysis of aptamer−target interactions in picoliter volumes under physiologically relevant conditions across the millisecond-to-hour time scales. Investigating serotonin aptamers with varying stem lengths, we systematically explore structure−function relationships and translate molecular-level insights into the application-driven selection of optimal candidates. By bridging low-throughput structural characterization with a rapid, low-volume, and multiparametric readout, our platform overcomes a key barrier in translational biosensor development and lays the foundation for data-driven engineering of structure-switching aptamers tailored for diagnostics and beyond.