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

Abstract

Quantification of multiple disease-related micro-RNAs (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 ICP− MS detection was proposed for the sensitive and simultaneous detection of three miRNAs . Specifically, a probe consisting of streptavidin-modified magnetic beads (SA-MBs) and three DNA substrates labeled with lanthanide tags ( 159 Tb/ 165 Ho/ 175 Lu) 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.

Atomic spectrometry update: review of advances in the analysis of clinical and biological materials, foods and beverages

“…Disease-related microRNAs (miRNAs) are an emerging class of biomarkers for which various ICP-MS based detection methodologies have been described using different amplication strategies and elemental tags. [186][187][188][189] Sandwich hybridization on streptavidin coated microtitre plates was used to detect three target miRNAs with three NP probes (Ag, Au and Pt), quantied by ICP-MS. 186 Improved selectivity, sensitivity and dynamic range was achieved in a multiple-metal-NP-tagging method with hybridization chain reaction amplication. 187 Here, three metal NP probes (Ag, Au and Pd) interacted with one target miRNA and ICP-MS response patterns were interrogated to enhance selectivity compared with other miRNA detection methods.…”

“…187 Here, three metal NP probes (Ag, Au and Pd) interacted with one target miRNA and ICP-MS response patterns were interrogated to enhance selectivity compared with other miRNA detection methods. Kang et al 188 presented a multiplexed assay using three MNAzymes (multicomponent nucleic acid enzyme) paired with three target miRNAs and three lanthanide tags with MB-DNA probes. In a different MNAzyme-based approach, cleavage of linker DNA by MNAzyme in the presence of target miRNA resulted in smaller AuNP aggregate size detected by spICP-MS. 189 In addition to solution-based immunoassay platforms, elemental tagging with LA-ICP-MS and LIBS has demonstrated potential for quantitative biomarker imaging in different sample types.…”

Multistage Nucleic Acid Amplification Induced Nano-Aggregation for 3D Hotspots-Improved SERS Detection of Circulating miRNAs

“…and MNAzyme-assisted inductively coupled plasma − mass spectrometry detection technology [12], have been used for miRNA detection and intracellular miRNA imaging. However, the expression levels of circulating miRNAs are low, and various types of miRNAs can coexist in circulating blood.…”

A homogeneous nucleic acid assay for simultaneous detection of SARS-CoV-2 and influenza A (H3N2) by single-particle inductively coupled plasma mass spectrometry

“…Wang’s group had proposed a sensitive and multiplex method for viral DNAs detection [ 17 ]. With multicomponent nucleic acid enzymes (MNAzymes) amplification strategy, lanthanide labeling ICP-MS has also been used for simultaneous detection of three miRNAs recently [ 18 ]. Simultaneous detection of circulating tumor cell of HepG2 and MCF-7 cells in human peripheral blood was achieved by ICP-MS using Au NPs and CdSe quantum dots labels combined with magnetic separation [ 14 ].…”