Discovery of high-specificity DNA aptamers for progesterone using a high-throughput array platform

Aptamer-based biosensors offer several advantages for detecting small molecules, including chemical stability and compatibility with diverse sensing formats. However, developing highly specific DNA aptamers that can distinguish between structurally similar small-molecule analytes remains a major challenge. Steroid hormones share a common four-ring scaffold, and even small modifications in functional groups can lead to distinct biological activities. Although DNA aptamers targeting the female reproductive hormone progesterone have been previously reported, many exhibit substantial cross-reactivity with other steroid hormones. In this work, we report the discovery of DNA aptamers with high affinity and specificity for progesterone using our aptamer array platform, which can characterize millions of aptamer candidates in a single, automated experiment. This platform allows us to profile aptamer libraries from earlier rounds of systematic evolution of ligands by exponential enrichment (SELEX), while preserving sequence diversity. Using this strategy, we identified multiple aptamers with nanomolar affinity for progesterone and minimal cross-reactivity to structurally related steroids. These high-specificity aptamers provide a strong foundation for the development of biosensors applicable to both clinical diagnostics and biological research.