MNAzyme-Catalyzed Amplification Assay with Lanthanide Tags for the Simultaneous Detection of Multiple microRNAs by Inductively Coupled Plasma-Mass Spectrometry

Quantification of multiple disease-related microRNAs (miRNAs) is of great significance for clinical diagnosis. Based on the simultaneous multiple element detection ability of inductively coupled plasma-mass spectrometry (ICP-MS) and good specificity of multicomponent nucleic acid enzymes (MNAzymes), a novel and simple method based on the MNAzyme amplification strategy and lanthanide labeling coupled with ICPMS detection was proposed for the sensitive and simultaneous detection of three miRNAs (miRNA-21, miRNA-155, and miRNAlob). Specifically, a probe consisting of streptavidin-modified magnetic beads (SA-MBs) and three DNA substrates labeled with lanthanide tags (Tb-159/ Ho-165/Lu-175) was constructed. In the presence of target miRNAs, three pairs of MNAzymes were assembled where each pair was hybridized with the corresponding miRNA, and then the substrates on the SA-MBs were cleaved by the activated MNAzymes, continuously releasing the fragment with lanthanide tags. The released lanthanide tags in the supernatant were collected after magnetic separation and analyzed by ICP-MS, realizing the simultaneous quantification of multiple miRNAs. The correlation of the lanthanide tag signal with the miRNA concentration fitted well in a linear model in the range of 50-1000 pmol L-1 (miRNA-21) and 50-2000 pmol L-1 (miRNA-155 and miRNA-10b). The limits of detection for three miRNAs were 11-20 pmol L-1, with the relative standard deviations of 2.2- 2.7%. The recoveries of target miRNAs in the human serum and HepG-2 cells were in the range of 87.2-111% and 93.3-111%, respectively. Overall, the method is ideal for the simultaneous quantification of multiple miRNAs with advantages of low spectral interference, high sensitivity, good selectivity, and strong resistance to the complex matrix.

Automated summary

A novel method based on MNAzyme amplification strategy and lanthanide labeling coupled with ICPMS detection was proposed for the sensitive and simultaneous detection of three miRNAs. The technique showed good correlation and low detection limits for the quantification of multiple miRNAs.