An “on-off” signal-switchable electrochemiluminescence biosensor for ultrasensitive detection of dual microRNAs based on DNAzyme-powered DNA walker

Herein, an “on-off” signal-switchable electrochemiluminescence (ECL) biosensor for dual miRNAs (miRNA-21 and miRNA-155) detection was constructed based on carbon dots (CDs)-labeled DNA walker, which can effectively avoid problems of the signal overlap or limited available labels that general exist in multiplex assays. In the presence of miRNA-21, the CDs-DNA walker were captured on the electrode via nucleic acid hybridization, producing a strong ECL signal corresponding to miRNA-21 (signal on). For miRNA-155, the biosensor was pre-incubated with CDs-DNA walker and assistant DNA (DNA-21). Target miRNA-155 could trigger metal ion-dependent DNAzyme cleavage. With the assistance of Mn2+, the substrate strands were cleaved and CDs detached from the electrode, resulting in a reduced ECL signal (signal off). The interference of miRNA-21 was eliminated because no more CDs-DNA walker was added. As a result, benefiting from the signal amplification offered by the CDs-DNA walker and the inherent properties of ECL technique, this biosensor achieved low detection limits of 33 fM for miRNA-21 and 33 aM for miRNA-155. This paper also demonstrates the biosensor’s bioanalytical application for multiplex detection of miRNA-21/155 in human serum samples and the total RNA extraction solution from the human cancer cell lines.