Prestructured Lollipop-Like Library for the Discovery of Flagellin Circular Aptamers for Functionalized Hydrogel Biosensing

Circular aptamers, known for their enhanced biological stability and mechanical rigidity, hold significant promise for bioanalytical applications. However, the lack of 3' and 5' termini complicates their modifications and subsequent applications. Additionally, isolating desirable circular aptamers remains challenging due to interference from the constant primer region. Herein, we developed an effective selection strategy by introducing a prestructured lollipop-like circular DNA library with a self-blocking sticklike primer-binding site. This design not only facilitates post-selection modifications but also reduces primer interference during selection. Through this approach, we successfully identified a flagellin-binding circular aptamer, designated as CFla-1, which retained the sticklike structure and bound flagellin with high affinity and selectivity. Furthermore, we developed a CFla-1-functionalized hydrogel biosensor by leveraging the dual liquid-solid characteristics and exceptional immobilization capacity of the DNA hydrogel. The sticklike primer-forming constant region served as a robust framework for click chemistry, enabling sensitive detection of flagellin with an impressive detection limit of 7.4 nM. This study provides an efficient method for developing circular aptamers with enhanced functionality and broader applicability in biosensing.