High-Sensitivity Simultaneous Discrimination and Detection of Structurally Similar Adenosine Phosphates via DNA Aptamer Conformational Changes in α-Hemolysin Nanopore

Adenosine phosphates (ATP, ADP, and AMP) play critical roles in cellular processes, yet their simultaneous detection remains challenging due to high structural similarity. To overcome this limitation, we developed a nanopore-aptamer sensing platform capable of tracking single-molecule binding dynamics in real time. This method leverages the specific interactions between aptamers and each adenosine phosphate, enabling discrimination among ATP, ADP, and AMP at the single-molecule level. We demonstrate the high sensitivity and selectivity of this approach, which supports high-throughput analysis even in complex matrices. The platform was successfully applied to quantify ATP, ADP, and AMP in human serum, with excellent recovery rates (85.33%-97.53%) and precision (RSD < 7.03%), demonstrating its practical utility for multiplexed detection in biologically relevant environments. This work provides a powerful strategy for precise metabolite analysis and facilitates new studies on nucleotide-dependent pathways.