DNA Hairpins and Dumbbell-Wheel Transitions Amplified Walking Nanomachine for Ultrasensitive Nucleic Acid Detection

Nucleic acids, including circulating tumor DNA (ctDNA), microRNA, and virus DNA/RNA, have been widely applied as potential disease biomarkers for early clinical diagnosis. In this study, we present a concept of DNA nanostructures transitions for the construction of DNA bipedal walking nanomachine, which integrates dual signal amplification for direct nucleic acid assay. DNA hairpins transition is developed to facilitate the generation of multiple target sequences; meanwhile, the subsequent DNA dumbbell-wheel transition is controlled to achieve the bipedal walker, which cleaves multiple tracks around electrode surface. Through combination of strand displacement reaction and digestion cycles, DNA monolayer at the electrode interface could be engineered and target-induced signal variation is realized. In addition, pH-assisted detachable intermolecular DNA triplex design is utilized for the regeneration of electrochemical biosensor. The high consistency between this work and standard quantitative polymerase chain reaction is validated. Moreover, the feasibilities of this biosensor to detect ctDNA and SARS-CoV-2 RNA in clinical samples are demonstrated with satisfactory accuracy and reliability. Therefore, the proposed approach has great potential applications for nucleic acid based clinical diagnostics.