Label-free, Ultrasensitive and Electrochemical Immunosensing Platform for microRNA Detection Using Anti-DNA:RNA Hybrid Antibody and Enzymatic Signal Amplification

In this work, we proposed a selective and sensitive biosensor for microRNA detection based on the high specificity and affinity of anti-DNA:RNA hybrids antibody (S9.6 antibody) and alkaline phosphatase catalytic signal amplification. Briefly, after the hybridization of probe DNA and the target microRNA, the S9.6 antibody can be captured on the electrode surface through antigen-antibody immunoreaction. Then, alkaline phosphatase labeled goat anti-mouse IgG (ALP-IgG) was further captured on the electrode surface through the specific recognition effect between the primary antibody and the secondary antibody. Finally, ALP catalyzed the hydrolysis reaction of p-nitrophenyl phosphate to generate p-nitrophenol, resulting in a electrochemical oxidation signal. The simple signal amplification assay performed a successful linear range from 0.5-500 fM with a detection limit of 0.40 fM. Moreover, this biosensor exhibited high selectivity with discriminating only single-base mismatched microRNA sequence. Additionally, the simplicity of this method hold a great promise for further investigation of the role of microRNAs in phytohormone signaling transduction.