Application of Non-G-Quadruplex Hemin Aptamers to Hemin Detection and Heme Oxygenase 1 Activity Evaluation via Spatial Conformational Constraint

The correct spatial folding of the aptamer significantly influences its binding performance with the target. By extending the terminal double-stranded domain of the hemin aptamer, we obtained apt71 with a more stable spatial conformation. The catalytic activity of the resulting apt71/hemin DNAzyme reached 200% of that of the original aptamer. We systematically evaluated the aptamer's performance in terms of apparent dissociation constant, average catalytic efficiency, and substrate affinity. It was successfully applied to the visual detection of hemin, achieving a detection limit of 0.1 nM, with detection performance comparable to previously developed fluorescence and chemiluminescence strategies. High-sensitivity visual detection was demonstrated in both diluted serum and environmental water samples, along with high specificity in distinguishing multiple hemin analogs. Furthermore, the apt71/hemin DNAzyme system was applied to the in vitro activity evaluation of heme oxygenase 1, compared to traditional G-quadruplex aptamers, our optimized non-G-quadruplex hemin aptamer exhibited improved performance, confirming its significant practical value and broad application potential in hemin-related research.