A hydrogel film microarray stabilizes very short structure switching aptamer duplexes to achieve enhanced sensing of small molecules

• Developed hydrogel film microarray is able to physically entrap and thermally stabilize FRET-based structure switching aptamer constructs ( i.e. , duplexed aptamers) assembled with short quencher stems for improved binding affinity of small molecules. • Using an adenosine aptamer reporter, complexed with rationally located 7-bp quencher stems under decreasing aptamer surface density, hydrogel sensors can achieve detection limit between 1.2–3 µ m and binding affinity between 35.5–14.7 µ m , comparable to the native aptamer. The reduction of affinity associated with the modifications required to create FRET- aptamer constructs represents a key challenge hindering the practical adaptation of such systems for small molecule detection. While the use of shorter and rationally positioned quencher stems is desirable to enhance target-induced fluorescence recovery, the weak hybridization potential of such stems results in high levels of background fluorescence and poor sensitivity. Herein, we introduce a hydrogel microarray sensor able to thermally stabilize very short aptamer duplexes ( i.e. , quencher stems ≤10-bp), resulting in improved FRET efficiency and reduced background levels across a broad range of conditions. The optimal hydrogel microarray can physically entrap >75 % of loaded aptamer reporters while maintaining high accessibility to the target molecules, enabling accurate quantification of dose-responsive affinity interactions. Using an ATP aptamer reporter, the hydrogel sensor can achieve a binding affinity as low as 14.7 µ m − comparable to the native aptamer – and very low limits of detection (LOD) of 1.2 µ m in a pure buffer and 4.6 µ m in 50 % serum. Moreover, the developed hydrogel microarray is reusable, amenable to printing fabrication, and provides protection against nuclease-based degradation. This simple approach thus holds promise for advancing microarray aptamer technology in critical biosensing applications.