Circulating tumor DNA (ctDNA)-related liquid biopsy detects DNA fragments released by tumor cells into the bloodstream, providing non-invasive and real-time molecular information for the early diagnosis, efficacy monitoring and individualized treatment of tumors. However, tumor-derived fragments are often scarce among the much larger amount of DNA from noncancerous cells, making reliable detection challenging. Sensitive methods with simpler workflows could therefore complement existing approaches to liquid biopsy. Researchers at West China Hospital, Sichuan University, developed molecular filler-enhanced DNA nanomachines that improve sensing performance and convert the recognition of specific DNA sequences into electrical signals. The platform simultaneously detects two ctDNA mutations without DNA amplification, enzymatic reactions or target labeling.
The researchers built nanoscale structures from self-assembling DNA strands linked by Ag + or Hg 2+ ions. These ions serve as reporters because they produce distinguishable electrical signals. When a matching DNA target binds, the structure disassembles and releases the corresponding ions, allowing the target to be measured. To further improve the sensing performance of the DNA nanomachines, the researchers tested several molecular fillers with different DNA interaction properties and evaluated their binding, loading, and effects on structural stability and reporter retention. By suppressing nonspecific ion leakage and lowering background signals, the incorporation of methylene blue improved analytical sensitivity 100-fold and the signal-to-noise ratio 2.5-fold compared with the corresponding unfilled system. The resulting assays achieved detection limits of 5.2 and 6.0 amol/L for the EGFR L858R and T790M mutations, respectively. The workflow also simplifies sample handling. Plasma required dilution and heating rather than nucleic acid extraction or enrichment. The target reaction involved a 60-minute incubation.
The team evaluated mutation detection in plasma from 66 treatment-naive patients with non-metastatic non-small cell lung cancer, using quantitative qPCR as the reference. Both EGFR assays achieved an area under the receiver operating characteristic curve (AUC) of 0.96 for distinguishing qPCR-positive from qPCR-negative samples. Performance in distinguishing cancer patients from healthy individuals reflected the prevalence and biological roles of the two mutations. L858R occurs in a subset of patients with non-small cell lung cancer, whereas T790M is primarily associated with acquired resistance to EGFR-targeted therapy and is uncommon before treatment. Compared with 30 healthy controls, the L858R-related signal achieved 54.5% sensitivity and 100% specificity. T790M-related signals showed limited discrimination, with an AUC of 0.52, consistent with its low prevalence in this treatment-naive cohort. Larger cohorts of patients receiving EGFR-targeted therapy are needed to assess the platform’s potential for resistance monitoring.
The researchers also adapted the platform to detect BRAF V600E, a mutation associated with papillary thyroid cancer. In 38 patient plasma samples, the assay achieved 94.7% agreement with ARMS-qPCR and an AUC of 0.97 for mutation detection, demonstrating its adaptability to additional cancer-related targets.
Exploratory analyses revealed differences in mutation-associated signals between metastatic and non-metastatic groups, suggesting potential for future disease monitoring. However, larger and longitudinal studies are still needed, and factors such as tumor burden, disease stage, and previous treatment history should be comprehensively considered.
Future development will focus on expanding the range of detectable mutations, optimizing DNA nanostructures and reporter systems for higher multiplexing, and integrating the assay with compact electrochemical devices. Larger, longitudinal clinical studies could further establish its potential for tracking treatment response and emerging resistance, supporting rapid molecular analysis and personalized cancer care.
Science Bulletin
Experimental study