A Triple-Input AND-Logic System Employing an APE1-Propelled Bipedal DNA Walker for Highly Sensitive Detection of Dual miRNAs

The escalating global cancer burden necessitates the development of precise tumor-specific microRNA (miRNA) detection strategies. However, the low-abundance, short-length, and high-sequence homology of miRNAs significantly hinder their accurate detection. Moreover, growing evidence of miRNA codysregulation in tumorigenesis underscores the diagnostic advantage of multiplexed analysis over single-marker approaches. Here, we present a novel bipedal DNA walker-based biosensor (BDWA) assembled on gold nanoparticles for the simultaneous detection of dual-miRNA targets. This system uniquely integrates dual-miRNA responsiveness with tumor-endogenous enzyme-driven signal amplification operating within a triple-input orthogonal AND-logic gate framework to enhance diagnostic specificity. Specifically, the concurrent presence of miR-21 and miR-155 displaces blocking strands to activate the walker, while the tumor-associated endonuclease APE1 serves as an intracellular trigger that propels the walker along engineered hairpin tracks (H1) on AuNPs. APE1-mediated cleavage of abasic (AP) sites results in the release and amplification of fluorescence signals initiated by the dual-targets. The bipedal architecture of the walker ensures a high processivity and efficient signal amplification. Critically, this design produces output signals only when all three inputs─miR-21, miR-155, and APE1─are present, thereby minimizing background interference and false positives. The BDWA system demonstrates high sensitivity and specificity for dual-miRNA detection in tumor cell lysates and intracellular imaging, offering a robust, tumor-activated, and programmable molecular platform for next-generation cancer diagnostics.