An Integrated Microfluidic Platform for Tunable-Stringency Competitive Affinity Selection of Folate Receptor Alpha Aptamers

Abstract

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 by 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 by 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 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 estimate their 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.