AND-logic amplification enables one-pot co-detection of extracellular vesicle miRNA and APE1.

Extracellular vesicles (EVs) are stably present in body fluids and carry diverse molecular cargos, including proteins and nucleic acids, making them an ideal information carrier for liquid biopsy. However, most existing EV assays focus on a single class of markers, and combining multiple markers within the same class often introduces informational redundancy, thereby limiting diagnostic performance. To address this challenge, we developed a one-pot dual-biomarker detection platform driven by an AND logic gate, inspired by the enrichment of the endonuclease APE1 and tumor-associated miRNAs in cancer-related EVs. In our platform, low-abundance target miRNA was first efficiently converted into abundant product probes (Pp) via strand displacement amplification (SDA). The resulting Pp then hybridized with an AP-site containing signal probe to form a cleavage-competent substrate, which was catalytically cleaved by APE1 to restore fluorescence. Meanwhile, the released Pp can repeatedly initiate additional cleavage cycles, enabling cascading signal amplification. The platform achieved a limit of detection of 8 fM for miRNA and 0.008 U/mL for APE1. In plasma EVs from gastric cancer patients and healthy donors, the AND-gated dual-marker readout yields an area under the ROC curve (AUC) of 0.957, significantly outperforming single-marker RT-qPCR miRNA analysis (AUC = 0.823), indicating improved discrimination in clinical samples. Overall, our work provided a simple synergistic logic-gated framework for integrating multi-class EV biomarkers, with potential applications in early cancer screening and dynamic disease monitoring.