A signal-on homogeneous electrochemical biosensor for sequence-specific microRNA based on duplex-specific nuclease-assisted target recycling amplification

Most electrochemical biosensors for microRNA (miRNA) detection need the immobilization of DNA on the electrode; first, the process is tedious and the DNA/RNA hybridization occurs on the electrode/solution interface, resulting in low efficiency. In this study, a homogeneous electrochemical biosensor has been proposed for sequence-specific miRNA in pancreatic cancer cells and lung adenocarcinoma cells, which combines the advantages of the simplicity of immobilization free and high efficiency of duplex-specific nuclease (DSN) assisted target recycling amplification. A DNA probe modified with a methylene blue group at the 3′ terminal (eMB) contains negative charges on its backbone, which cannot diffuse easily to the surface of the negatively charged indium tin oxide (ITO) electrode because of electrostatic repulsion, so a low electrochemical signal was detected. In the presence of miRNA, eMB can recognize and hybridize with miRNA to form a DNA–RNA heteroduplex. The eMB can be hydrolysed by DSN, so miRNA is retained. miRNA can cause a new cycle of hybridization–cleavage–releasing, and this recycling process generates numerous short MB-labelled oligonucleotide fragments (MB-OFs). The MB-OFs with less negative charges can diffuse easily to the surface of the ITO electrode, so an enhanced electrochemical signal was detected. Under the optimal conditions, the differential pulse voltammetric (DPV) response of the system had a linear relationship with the logarithm of the target miRNA concentration in the range of 0.1 pM to 10 nM. The proposed biosensor has been successfully applied to detect miRNA in real samples.