Novel and sensitive electrochemical/fluorescent dual-mode biosensing platform based on the cascaded cyclic amplification of enzyme-free DDSA and functional nucleic acids

Herein, we present a novel electrochemical (EC)/fluorescent (FL) dual-mode biosensor for sensitive and accurate detection of target nucleic acids, which was based on the functional nucleic acids-involved enzyme-free dynamic DNA self-assembly of catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR) for cascaded cyclic amplification. Originally, the CHA reaction of three well-designed hairpin probes were initiated by target sequence, forming abundant Mg2+-dependent three-way DNAzyme junctions (MTWDJ) which could recognize and cleave the methylene blue-labeled substrate hairpin (MB-Hs) to generate the MB-labeled fragments s1 (MB-s1) and the HCR initiator s2. Then, s2 triggered the HCR of four hairpins to produce long DNA nanowires which contained numerous G-quadruplex sequences and the same Mg2+-dependent DNAzyme (MNAzyme) sequences as MTWDJ. Therefore, the HCR copolymer could not only emerge the fluorescent signals through combining thioflavin T with G-quadruplex, but also generate MB-s1 and s2 via MNAzyme cleavage of MB-Hs to continue initiating the HCR. Meanwhile, MB-s1, the cleavage product of MTWDJ and MNAzyme, was captured on the DNA tetrahedron nanostructure modified electrode surface to bring electrochemical signals. Benefiting from integrating the efficient cyclic cleavage of MTWDJ and MNAzyme, the concatenated CHA and HCR amplification circuit, and the dual-mode detection, the sensitivity and accuracy of this biosensor were significantly improved. Under the optimal conditions, the proposed EC/FL dual-mode sensing strategy exhibited a superior analytical performance toward target nucleic acids, showing the promising application in bioanalysis and early disease diagnosis.