Abstract
The detection of single-nucleotide variants (SNVs) in circulating tumor DNA (ctDNA) in liquid biopsies has increasingly been shown to exhibit unique benefits for early detection or minimal residual disease monitoring in cancer. Yet, current clinically validated assays for ctDNA SNV detection are challenged by (i) time-consuming and laborious spin column-based ctDNA purification protocols, (ii) limited detection specificity to discriminate between mutated SNVs from large excess of closely similar wild-type sequences, and (iii) insufficient detection sensitivity required for trace ctDNA target analysis in blood. Herein, a ctDNA assay is demonstrated to tackle these triple key issues by fusing magnetics for quick ctDNA enrichment directly from unprocessed blood, selected bioenzyme activities for rapid discrimination, and molecular amplification of target SNVs, and designed magnetic-assisted bioelectrocatalytic cycling of DNA-intercalating and freely diffusing redox probes for electrochemical signal intensification. The described ctDNA SNV assay enables the detection of clinically relevant ctDNA SNVs in melanoma (BRAFV600E, KITL576P, and NRASQ61K) from unprocessed plasma samples with unprecedented 0.005% detection sensitivity, ultrabroad dynamic range over four orders of magnitude, and excellent single-base specificity.
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