Molecular filler-enhanced DNA nanomachines enable one-step multiplex quantification of ctDNA for mutation analysis and metastasis-associated assessment.

Detection of low-abundance biomarkers like circulating tumor DNA (ctDNA) remains challenging for molecular diagnosis and therapeutic management of cancer. Here, we developed a homogeneous electrochemical platform using metal ion-mediated DNA self-assembly enhanced by molecular fillers, enabling simultaneous, amplification-free, label-free, and enzyme-free detection of dual biomarkers. Incorporation of methylene blue into DNA nanostructures can suppress non-specific ion leakage, enhance sensitivity by 100-fold and signal-to-noise ratio by 2.5-fold. Target binding triggered DNA nanomachines disassembly, releasing Ag+ or Hg2+ for simultaneously electrochemical analysis, thereby facilitating precise quantification of the target biomarkers. The system achieved high-performance detection of L858R and T790M with limits of 5.2 and 6.0 amol/L, respectively, when applied to epidermal growth factor receptor mutations in non-small cell lung cancer (NSCLC). Using qPCR as the reference, mutation detection achieved AUCs of 0.96 for both L858R and T790M. In disease discrimination, L858R showed moderate sensitivity, whereas T790M provided limited discrimination in the predominantly treatment-naive cohort. In addition, the platform also demonstrated applicability in detecting BRAF V600E mutations in thyroid cancer. Exploratory analyses further revealed differences in ctDNA-associated signals between non-metastatic and metastatic cases. This filler-enhanced DNA nanomachine strategy offered a high-performance tool for ultrasensitive ctDNA analysis and showed potential for clinical applications.