Digital Microfluidic Platform Based on Printed Circuit Board for Affinity Evaluation of Mertansine Aptamers

Aptamers make up a class of nucleic acid strands that can specifically recognize target molecules. However, the selection of aptamers typically involves multiple optimizations and an iterative process, including library design, high-affinity library enrichment, sequencing, and affinity assessment of candidate sequences. Among these steps, the affinity evaluation of large numbers of candidate sequences is particularly resource-intensive in terms of labor, materials, and time. To address this, we developed a low-cost digital microfluidic platform based on printed circuit boards using the aptamer of small-molecule tumor drug mertansine as an example. We achieved miniaturization and automation of the affinity determination process. First, we optimized the droplet actuation parameters on the digital microfluidic platform and verified its feasibility for aptamer affinity determination. Then, using this platform, we investigated the affinity of seven candidate sequences for mertansine and successfully found two new aptamers (Seq15-X and Seq19-X) with good affinity for mertansine. The dissociation constants (K_d) of the two aptamers are 70.9 ± 8.5 nM and 43.6 ± 21.6 nM, respectively. Compared with manual methods, the digital microfluidic platform not only reduced the time for a single determination from 105 to 45 min but also significantly decreased the reagent consumption from 1280 to 30.72 μL. Therefore, the digital microfluidic platform offers several advantages, including high automation, low reagent consumption, and strong versatility, not only significantly improving the efficiency of aptamer screening and performance evaluation but also providing a promising tool for the study of interactions between other biomolecules.