We propose a ratiometric electrochemical assay for detecting microRNA (miRNA) on the basis of dual-amplification mechanism by using distinguishable electrochemical signals from thionine (Thi) and ferrocene (Fc). The thiol-modified and ferrocene-labeled hairpin capture probes (CP) are first immobilized on an Au electrode via Au-S reaction. The target miRNA hybridizes with CP and unfolding the hairpin structure of CP to form miRNA-DNA duplexes. Then, kamchatka crab duplex specific nuclease (DSN) specifically cleaves the DNA in miRNA-DNA duplexes, leading to the release of miRNA and another cleaves cycle, meanwhile, numerous Fc leaves away from the electrode surface and leads to the signal-off of Fc. The residual fragment on electrode surface acts as a HCR primer to form dsDNA polymers through in situ HCR with the presence of the primer and two probes (HDNA and HDNA'), resulting in the capture of numerous DNA/Au NPs/Thi and the signal-on of Thi. The dual-amplification mechanism significantly amplifies the decrease of Fc signal and the increase of Thi signal for ratiometric readout (I/I), thus providing a sensitive method for the selective detection of miR-141 with a detection limit down to 11aM. The dual-signal ratiometric outputs have an intrinsic self-calibration to the effects from system, which is promising to be applied in biosensing and clinical diagnosis.
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