Interference Reduction Biosensing Strategy for Highly Sensitive microRNA Detection

MicroRNAs are potential biomarkers for human cancers and other diseases due to their roles as post-transcriptional regulators for gene expression. However, the detection of miRNAs by conventional methods such as RT-qPCR, in situ hybridization, northern blot-based platforms, and next-generation sequencing is complicated by short length, low abundance, high sequence homology, and susceptibility to degradation of miRNAs. In this study, we developed a nicking endonuclease-mediated interference reduction rolling circle amplification (NEM-IR-RCA) strategy for the ultrasensitive and highly specific detection of miRNA-21. This method exploits the advantages of the optical properties of long-lived iridium(III) probes, in conjunction with time-resolved emission spectroscopy (TRES) and exponential rolling circle amplification (E-RCA). Under the NEM-IR-RCA-based signal enhancement processes, the limit of detection of miRNA-21 was down to 0.0095 fM with a linear range from 0.05 to 100 fM, which is comparable with the conventional RT-qPCR. Unlike RT-qPCR, the strategy was performed at a lower and constant temperature without heating/cooling cycles and reverse transcription. The strategy could clearly discriminate between matched and mismatched targets, demonstrating high specificity. Moreover, the potential application of this method was demonstrated in cancer cells and mouse serum samples, showing good agreement with RT-qPCR results. Apart from miRNA-21 detection, this platform could be also adapted for detecting other miRNAs, such as let-7a and miRNA-22, indicating its excellent potential for biomedical research and clinical diagnostics.