Ultraspecific electrochemical DNA biosensor by coupling spontaneous cascade DNA branch migration and dual-signaling sensing strategy

Using spontaneous cascade DNA branch migration and dual-signaling sensing strategy, we developed a novel universal electrochemical biosensor for the highly specific and sensitive detection of nucleic acids. A target strand (Ts) competitively hybridized with a ferrocene (Fc)-labeled signal probe (Fc-S1), which was blocked by a protector strand (Ps), after strand displacement to form the Ts/Fc-S1 duplex. A methylene blue (MB)-modified signal probe (MB-S2) was immobilized on the Au electrode surface by hybridizing with a thiolated capture probe (Cp). Then, the obtained reactants (Ts/Fc-S1 and MB-S2/Cp) suffered spontaneous DNA branch migration and produced two hybridization products (Fc-S1/Cp and MB-S2/Ts). These reactions led to the dissociation of MB molecules and the collection of Fc molecules. The detection mechanism of this DNA biosensor involved distance variation between the redox tags and the Au electrode, which was associated with target-induced cascade DNA branch migration. Moreover, we rationally designed the cascade DNA branch migration to occur spontaneously with ΔG°≈0, at which slight thermodynamic changes caused by base mismatch exerted a disproportionately large effect on the hybridization yield. This “signal-on/off” sensing system exhibited a remarkable analytical performance and an ultrahigh discrimination capability even against a single-base mismatch. The maximum discrimination factor (DF) of base mutations or alterations can reach 17.9. Therefore, our electrochemical biosensor might hold a great potential for further applications in biomedical research and early clinical diagnosis.