Ultrasensitive detection of microRNA based on a homogeneous label-free electrochemical platform using G-triplex/methylene blue as a signal generator.

The electrochemical methods for microRNA (miRNA) detection have received increasing attention because high portability and affordability of electrochemical biosensors may facilitate point-of-care quantitative detection of miRNAs. Among these biosensors, the homogenous label-free electrochemical biosensors for miRNAs are rarely reported due to the lack of a universal and efficient signal read-out-mode. A newly discovered G-triplex, 5'-CTGGGAGGGAGGGA-3' (denoted as G3), can specifically bind with methylene blue (MB), leading to a significant decrease of the diffusion current of MB. By using miRNAs as a driving force, a two-stage isothermal exponential amplification reaction was proposed to generate G3 through miRNAs. The generated G3 can combine with MB and produce observable current changes, which depend on the concentration of miRNAs. Therefore, a novel homogeneous label-free electrochemical biosensor for miRNA detection was successfully constructed. By choosing let-7a, the down-regulation of which is possibly associated with the over-expression of RAS and HMGA2 oncogenes, as a model, we discovered that this biosensor demonstrated excellent analytical performance in detecting let-7a, with an ultralow limit of detection (0.45 fM) and high specificity (discriminating one nucleotide variation). Moreover, the proposed biosensor was successfully applied in monitoring the expression levels of the low-abundant miRNAs in the human lung adenocarcinoma cell lines. This assay successfully verified the feasibility of G-triplex/MB as an efficient and sensitive probe for immobilization-free and label-free electrochemical detection of nucleic acids, which would greatly promote the rapid development of homogeneous label-free electrochemical biosensors.