Precisely Controllable Y‐Shaped DNA Nanomachine‐Immunoassay System: Noninvasive Monitoring of Tumor Progression and Metastasis

ABSTRACT Herein, we developed a DNA scaffold‐mediated Y‐shaped DNA nanomachine immunoassay system (DYN@IA) that enables a homogeneous electrochemical immunoassay for the simultaneous detection of epithelial and mesenchymal circulating tumor cells, applicable to early cancer diagnosis and monitoring. The double Y‐shaped DNA design facilitated the self‐assembly of metal ion‐mediated DNA nanomachines, forming stable and controllable functional nanomachines for the recognition of heterogeneous CTCs. Antigen‐antibody binding formed steric hindrance and exerted local tension on the DNA scaffolds through the biotin‐streptavidin system, triggering conformational changes in the double‐stranded DNA, and releasing Ag + and Hg 2+ . Finally, the practical validation was conducted using primary lung cancer cells (pre‐ and post‐metastasis) and the surface targets epithelial cell adhesion molecule and vimentin. The results indicated that simultaneous detection of different CTCs was achieved within 45 min, with a limit of detection down to the single‐cell level. Additionally, the method was successfully applied to 145 clinical samples for lung cancer early screening and identification of distant metastasis (achieving sensitivity and specificity of 88% and 96%), with results highly consistent with imaging findings and pathology diagnoses. The DYN@IA design outperformed conventional immunoassays by significantly shortening reaction time while improving sensitivity and signal‐to‐noise ratio, providing insights for tumor screening and monitoring.