Target-driven assembly of DNAzyme probes for simultaneous electrochemical detection of multiplex microRNAs

In this work, we employed target-driven assembly of a Mg²⁺-dependent DNAzyme to develop an ultrasensitive electrochemical biosensor for the simultaneous detection of miRNA-21 and miRNA-141. The target miRNAs could hybridize with two partial DNAzymes, facilitating the formation of a stable and active Mg²⁺-dependent DNAzyme. With the help of the Mg²⁺ cofactor, the DNAzyme could circularly cleave the ferrocene (Fc) or methylene blue (MB) labelled hairpin probes and release Fc and MB labels from the electrode surface, which could significantly amplify the current suppression to achieve multiple detection of small amounts of miRNA-21 and miRNA-141. This electrochemical biosensor showed high sensitivity and selectivity for the simultaneous detection of miRNA-21 and miRNA-141. Furthermore, the proposed method was also successfully applied for the determination of miRNA-21 and miRNA-141 from diluted serum samples. Overall, the proposed sensor showed several considerable advantages including simple preparation, high sensitivity, and enzyme-free signal amplification. Therefore, the proposed electrochemical biosensor could be used as a highly efficient amplification strategy for simultaneous detection of various miRNA biomarkers in bioanalysis and clinical diagnostics.