Simple Amplifier Coupled with a Lanthanide Labeling Strategy for Multiplexed and Specific Quantification of MicroRNAs

Inductively coupled plasma-mass spectrometry (ICP-MS) with elemental labeling is a promising strategy for multiplex microRNA (miRNA) analysis. However, it is still challenging for specific analysis of multiple miRNAs with high homology, and the development of multiplex assays is always limited by the complexity of the sequence design. Herein, a simple and direct ICP-MS-based assay was developed for the simultaneous detection of three miRNAs by combining the lanthanide labeling strategy with entropy-driven catalytic (EDC) amplification. Owing to the specificity of EDC for nucleic acid recognition, it is able to differentiate miRNAs with single-base mutation in each EDC circuit. A universal biotin-labeled DNA strand was designed to hybridize with the DNA substrates for three EDC circuits, targeting miRNA-21, miRNA-155, and miRNA-10b, respectively. All the substrates were loaded on the surface of streptavidin magnetic beads. In the presence of target miRNA, the EDC reaction was initiated, and EDC substrates were dissociated, continuously releasing reporter strands that were labeled with lanthanides (Tb/Ho/Lu). After magnetic separation, the supernatant containing the released reporter strands was introduced into an ICP-MS system for simultaneous detection of Tb-159/Ho-165/Lu-175 and quantification of miRNA-21, miRNA-155, and miRNA-10b, respectively. The limits of detection were 7.4, 7.5, and 11 pmol L-1 for miRNA-21, miRNA-155, and miRNA-10b, respectively. Overall, this study provides a powerful strategy for simultaneous quantification of multiple miRNAs, with the advantages of flexible probe design, good sensitivity, and excellent specificity.

Automated summary

A simple and direct ICP-MS-based assay was developed for the simultaneous detection of three miRNAs by combining the lanthanide labeling strategy with entropy-driven catalytic amplification. This method allows for flexible probe design, good sensitivity, and excellent specificity.