High-performance strategy for the construction of electrochemical biosensor for simultaneous detection of miRNA-141 and miRNA-21 as lung cancer biomarkers.

In this study, the dual signal-labeled hairpin-structured DNA (dhDNA)-based probes have been developed to construct a novel nano-biosensor. This one hairpin-structured probe consists of a thiolated methylene blue-labeled hairpin capture probe (MB-HCP) as an inner reference probe and a ferrocene-modified anti-miRNA-21 DNA probe (Fc-AP-21). This novel integrated structure of MB-HCP and Fc-AP-21 was designed on one sensing interface for sensitive and simultaneous detection of the miRNA-141 and miRNA-21 in one single assay. The proposed strategy has a good ability to reduce the interference of environmental factors and it was designed to control the initial responses of Fc-AP to MB-HCP ((I/I)) at a 1:1 ratio, which is desirable for further increase in the sensitivity and signal-to-noise ratio of the biosensor operation. Besides, the biosensor was first prepared by immobilizing the dhDNA (Fc-AP-21/MB-HCP) onto the modified glassy carbon electrode. After hybridization with the anti-miRNA-141 complementary sequence (ACP-141), the dhDNA structure was compelled to open and form the final structure of the biosensor. Also, the miRNA-141 and miRNA-21 dissociate duplex structures due to the highly matched sequences between the miRNA-141 and ACP-141 and the miRNA-21 and Fc-AP-21. A linear relationship was found between the logarithm of miRNA-141 and miRNA-21 concentrations and the signal changes. This feature was used to detect the two miRNAs. This sensitive biosensor provided low detection limits of 0.89 and 1.24 fM for the miRNA-141 and miRNA-21, respectively. Also, it has wide linear ranges of 2.0 to 10 fM, with highly selective and accurate results for its application in plasma samples. Therefore, this strategy can be promising as a suitable platform for simultaneous and early detection of various cancer biomarkers.