Isothermal and enzyme-free MicroRNA assay based on catalytic hairpin assembly and rare earth element labeled probes

Abstract

MicroRNAs (miRNAs) are a category of short non-coding RNA molecules which play essential roles in regulating gene expression. Correspondingly, the aberrant expression of miRNAs is commonly related to the development of various diseases including cancers and cardiovascular diseases. Hence, miRNA is considered as an ideal biomarker for disease diagnosis and its quantification is highly demanded. Herein, an isothermal and enzyme-free miRNA assay based on catalytic hairpin assembly (CHA) amplification coupled with inductively coupled plasma mass spectrometry (ICP-MS) detection is proposed. Two hairpin probes are employed in CHA reaction with rare earth element tag on H1 for ICP-MS detection and biotin on H2 for immobilization on magnetic beads. In the presence of target miRNA, the two independent probes open up their stem-loop structure and hybridize. The target miRNA will be released after the hybridization to trigger another CHA reaction. Ultimately, one target miRNA molecule promotes a number of CHA cycles and produces several copies of hybridized complex leading to the enhanced ICP-MS signal. Based on this approach, miRNA-141 was successfully analyzed with high sensitivity and specificity. The limit of detection (LOD) was 88 fmol with a linear range 0.1–2 pmol. Compared with fluorescence mediated CHA method and ICP-MS mediated basic sandwich method, the current strategy displayed the lowest LOD. The developed method was also applied to analyze miRNA-141 and miRNA-21 simultaneously to demonstrate its multiple detecting capability and practical applicability. Furthermore, this miRNA assay has the potential to become a high-throughput method by employing more distinct rare earth elements.