AND‐Gated Multipedal 3D DNA Walker Enables Rapid Dual MicroRNA Imaging and Accurate Cancer Discrimination in Cells and Tissues

Real-time monitoring of intracellular microRNA expression is crucial for disease diagnosis, yet existing DNA walker-based detection methods suffer from insufficient specificity and limited reaction efficiency for reliable cancer identification. Herein, we present an AND-gated multipedal 3D DNA walker (AMTW) for simultaneous imaging of miR-21 and miR-155 expression in live cells and clinical tissues. The presence of target miRNAs activates the AND-gated operation, triggering the walking process and generating amplified fluorescence signals. AMTW achieves a detection limit of 2.79 pM and exhibits higher initial reaction rates than bipedal and monopedal walkers (4.38-fold and 8.73-fold increases, respectively). Moreover, the system effectively discriminates between tumor and normal samples at both cellular and tissue levels. Collectively, AMTW advances intracellular miRNA detection by integrating logic-gated recognition with enhanced collision efficiency, providing a robust platform for precise cancer diagnostics and early disease identification in clinical applications.