Label-free and isothermal multiplex detection of miRNAs using self-priming hairpin probes and light-up aptamer...

MicroRNAs (miRNAs) are key post-transcriptional regulators of gene expression and serve as valuable biomarkers for the early diagnosis and prognosis of various diseases, including cancer. However, conventional miRNA detection methods often involve complex probe designs, multi-step reactions, and limited multiplexing capabilities.

In this study, we developed a label-free, isothermal multiplex miRNA detection system that integrates a self-priming hairpin probe and transcription of fluorescent light-up aptamers (FLAPs). Each hairpin probe encodes a target-specific FLAP sequence, enabling fluorescence signal generation via T7 RNA polymerase-mediated transcription following target miRNA recognition and polymerase-driven extension. The method enabled sensitive and selective detection of three target miRNAs (miR-21, miR-141, and let-7d), with detection limits in the low picomolar range. Efficient multiplex analysis was achieved through FLAP-fluorogen pairs with minimal spectral cross-talk.

The developed method reproduced the expected differential miR-21 patterns across cancer and normal cell lines using cell-derived RNA extracts, consistent with stem-loop RT-qPCR. Notably, the method requires only a single hairpin probe per target miRNA and operates in a label-free format, enabling streamlined multiplex readout with minimal mutual interference. This platform offers a practical route toward efficient multiplex miRNA profiling in bioanalytical and diagnostic settings.