Abstract
Traditional screening of aptamers for small-molecule targets is often inefficient, typically requiring more than 13 rounds of selection. To address this inefficiency, we optimized the Capture-SELEX process by refining key factors that impact selection efficacy. By extending the length of the capture sequence and reducing the self-dissociation rate to minimize background noise, we significantly enhanced the screening efficiency. Utilizing meperidine (MPD), an addictive substance, as a model, we successfully identified aptamers within just six rounds. We further validated these optimized conditions using a multitarget mixed SELEX approach and successfully obtained high-affinity aptamers. To demonstrate the practical application of these aptamers, we developed two fluorescent biosensors incorporating MPD aptamers. This biosensor achieved a detection limit of 60 nM for MPD in artificial saliva and exhibited excellent specificity even in the presence of various interfering substances. This indicates that our method is effective for both single and mixed targets. By employing a 20-base-long capture sequence to minimize library self-dissociation, we have addressed the long-standing inefficiencies of Capture-SELEX, significantly improving the screening of small-molecule aptamers. This advancement is anticipated to greatly enhance the application of aptamers in small-molecule research and on-site drug detection.
