Ultrasensitive electrochemical biosensing strategy for microRNA-21 detection based on homogeneous target-initiated transcription amplification

Abstract

In this work, we have developed a simple and rapid biosensing method for highly sensitive detection of microRNA-21 (miRNA-21) by integrating homogeneous target-initiated transcription amplification (HTITA) with interfacial sensing process. MiRNA-21, as a target, recognizes and binds with the corresponding site of the multifunction straight probe, which would trigger extension reaction to form T7 promoters in the presence of Klenow fragment (KF exo−) and dNTPs. The remaining sites of multifunction straight probe serve as templates for transcription amplification based on T7 RNA polymerase (T7 pol.). As a result, a great amount of single-stranded RNA products are obtained, which hybridize with the biotinylated detection probes in the solution, forming transcription products. Finally, the resulted transcription products hybridize with the capture probes immobilized on the surface of electrode, producing electrochemical signal read-out. The developed method achieved a detection limit as low as 0.6fM with a dynamic response range from 1fM to 10nM and could discriminate single-nucleotide differences in microRNA (miRNA) sequences. In addition, the established electrochemical strategy was applied to directly detect miRNA-21 in total RNA extracted from HepG2 cells. Thus, this simple and sensitive electrochemical biosensing method might be a potential alternative tool for the quantitative analysis of miRNAs in physiological fluids, biomedical research and point-of-care testing (POCT) in the future.