Competitive SELEX Strategy for High‐Affinity Aptamer Isolation and Enhanced Biosensing

Aptamers, with their high specificity and affinity, are invaluable for diagnostics and therapeutics. However, conventional SELEX (Systematic Evolution of Ligands by Exponential Enrichment) often favors abundant low‐affinity sequences, limiting the isolation of rare, high‐affinity candidates. To overcome this challenge, we introduce a competitive SELEX strategy that enhances high‐affinity aptamer selection by employing a lower‐affinity, non‐amplifiable aptamer in substantial excess as a competitor. Using Clostridioides difficile ( C. difficile ) RNase H2 (CDH2) as the target and ARH1t6 as the competitor, we isolated CDH2‐1, a high‐affinity aptamer with an apparent K d of 0.050 nM—740‐fold stronger than ARH1t6. The nearly 100‐fold excess of ARH1t6 facilitated the selection of CDH2‐1, which would typically be outcompeted. CDH2‐1 enabled paper‐based C. difficile detection in fecal samples with a limit of 2.9 × 10 3 CFU/mL, a 120‐fold sensitivity improvement over ARH1t6. These findings demonstrate the effectiveness of competitive SELEX in enriching high‐affinity aptamers and enhancing biosensor performance.