Heterogeneous electrochemical DNA biosensors have attracted huge attention due to their enhanced signal sensitivity, compared to homogeneous biosensors. However, the high cost of probe labeling and the reduced recognition efficiency associated with current heterogeneous electrochemical biosensors confine their potential applications. In the present work, a dual-blocker assisted and dual-label-free heterogeneous electrochemical strategy based on multi-branched hybridization chain reaction (mbHCR) and reduced graphene oxide (rGO) was fabricated for ultrasensitive detection of DNA. The target DNA could trigger the mbHCR of two DNA hairpin probes, resulting in the generation of multi-branched long chain of DNA duplexes with bidirectional arms. One direction of the multi-branched arms in the mbHCR products were then bound to the label-free capture probe on the gold electrode through multivalent hybridization with enhanced recognition efficiency. The other direction of multi-branched arms in mbHCR product could adsorb rGO via π-π stacking interactions. Two DNA blockers were ingeniously designed to block the binding of excessive H1-pAT on electrode and to prevent the adsorption of rGO by residual unbound capture probes. As a result, with the electrochemical reporter methylene blue selectively intercalated into the long chain of DNA duplex and absorbed on rGO, a remarkable electrochemical signal rise was observed. Thus, a dual-blocker aided and dual-label-free electrochemical strategy for ultrasensitive DNA detection is readily realized with the merit of cost-effective. The as-developed dual-label-free electrochemical biosensor has great potential to be employed in nucleic acid related medical diagnostics.
Keywords: DNA detection; Dual-label-free; Heterogeneous electrochemical biosensor; Multi-branched HCR; Reduced graphene oxide.
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