Aptamer-Driven PS-THSP Strategy for Rapid and Highly Sensitive Detection of ATP

• A novel ATP detection strategy was developed using an aptamer hairpin probe. • The system detects ATP down to 98 nM within 25 minutes under isothermal conditions. • The modular design allows easy adaptation for detecting diverse biomolecules. We developed an aptamer-mediated system that enables rapid, sensitive, and discriminative quantification of ATP by utilizing phosphorothioate-terminal hairpin formation and self-priming extension (PS-THSP). In this method, the aptamer hairpin probe (AptHP), a key component, binds to ATP through its aptamer moiety, causing disruption of the hairpin structure. This conformational change prevents degradation by the double-strand-specific exonuclease, Exonuclease III, which in turn leads to PS-THSP-based signal amplification. The system exhibited excellent sensitivity with a detection limit of 107 nM and high specificity against structurally similar ATP analogues, completing the entire detection process within 25 minutes. We further demonstrated that this novel platform can reliably quantify ATP in diluted human serum with high accuracy and reproducibility, verifying its potential for practical application in heterogeneous biological and clinical samples. The platform can also be readily adapted to detect various target molecules by simply replacing the aptamer sequence, providing a simple yet robust framework for biosensing.