In this manuscript, Chung et al. present an integrated microfluidic platform for competitive affinity selection of aptamers targeting folate receptor alpha. The device combines serial dilution, bead-based affinity selection, and flow-controlled washing in an automated workflow. This platform could help reduce manual handling and minimize PCR amplification bias. However, the current study does not provide sufficient evidence that the selected sequences are sequence-specific FRα-binding aptamers. Several important controls and validation experiments are needed.
1. The authors report SPR-based affinity measurements for selected FRα-binding candidates. However, the current validation is not sufficient to establish accurate Kd values. The authors should perform additional binding assays by immobilizing the aptamer candidates on the sensor surface and testing FRα across a protein concentration series. A random or scrambled DNA control, ideally with the same length as the selected aptamers, should be included and tested under the same SPR conditions and protein concentration range. Without this control, it is difficult to determine whether the observed binding is sequence-specific or reflects nonspecific DNA-protein or DNA-surface interactions.
2. For an affinity-selection platform, the most important parameter is the efficiency of separating binders from non-binders. The authors should demonstrate that non-binding sequences are effectively removed during the washing and bead-retention steps. At present, it is unclear how completely non-binders are removed, how much nonspecific DNA remains after washing, or how much DNA is retained through nonspecific adsorption to beads or the microfluidic device. The authors should quantify the fraction of input DNA recovered in the flow-through, wash fractions, and bead-retained fraction, and compare these results using FRα-coated beads, uncoated beads, and irrelevant protein-coated beads. These data are essential to support the claim that the platform improves partitioning efficiency.
3. The manuscript provides CFD-based estimates of wall shear stress and bead-scale forces, which are useful for describing the device. However, CFD alone does not show that the washing conditions selectively remove weak or nonspecific binders while retaining true binders. The authors should experimentally link pump settings, flow rate, wash duration, DNA removal, bead retention, and final enrichment outcome. For example, known aptamers with different affinities could be used to quantify DNA removal and bead loss under low-, medium-, and high-stringency washing conditions.