An integrated microfluidic platform for tunable-stringency competitive affinity selection of folate receptor alpha aptamers.

This study developed a new microfluidic device for competitive affinity selection of single-stranded DNA (ssDNA) aptamers targeting folate receptor alpha (FRα), an ovarian cancer (OvCa) biomarker. The device integrated micropumps, microvalves, and micromixers to automate four selection rounds in ∼2.5 h, while washing stringency was tuned using a peristaltic micropump. Increasing the library concentration across rounds yielded a 2.9-fold increase in selection power and a higher binding ratio than the constant-concentration setup. The secondary structures of aptamer candidate sequences were analyzed using mfold, and a weighted structural pattern index (WSPI) combining free energy and structural elements was introduced for ranking; competitive affinity selection increased the WSPI ≥ 12 fraction from 7.4% in conventional systematic evolution of ligands by an exponential enrichment (SELEX) approach to 9.5-11.2% across affinity-selection conditions. FRα-binding aptamers obtained by this approach were characterized by surface plasmon resonance to obtain apparent affinity estimates expressed as the dissociation constant (KD), and a moderate positive correlation was observed between WSPI and -log(KD). This workflow provides a new microfluidic strategy for ranking and selecting FRα-binding aptamer candidates by using PCR-free competitive affinity selection for future biomarker-probe development.