Dual-Amplification\nSingle-Particle ICP-MS Strategy\nBased on Strand Displacement Amplification–CRISPR/Cas12a Amplification\nfor Homogeneous Detection of miRNA

MicroRNAs (miRNAs) regulate a myriad of biological processes\nand\nthus have been regarded as useful biomarkers in biomedical research\nand clinical diagnosis. The specific and highly sensitive detection\nof miRNAs is of significant importance. Herein, a sensitive and rapid\ndual-amplification elemental labeling single-particle inductively\ncoupled plasma-mass spectrometry (spICP-MS) analytical method based\non strand displacement amplification (SDA) and CRISPR/Cas12a was developed\nfor miRNA-21 detection. Taking gold nanoparticles (AuNPs) as the elemental\nlabels, the Au NP probe initially hybridized with linker DNA, forming\nlarge aggregates. In the absence of target miRNA-21, large aggregates\nof AuNPs will produce high pulse signals in spICP-MS detection. In\nthe presence of the target miRNA-21, it triggered the SDA reaction,\nand the SDA products activated CRISPR/Cas12a’s trans-cleavage\nactivity to cleave the linker DNA, resulting in disassembly of the\nAuNP aggregates. The AuNP aggregates with smaller size displayed lower\npulse signals in spICP-MS detection. Under the optimal conditions,\na good relationship between the average pulse signal intensity of\nAuNP aggregates and the concentration of miRNA-21 was obtained in\nthe range of 0.5 fmol L<sup>–1</sup>–100 pmol L<sup>–1</sup> with a quantification limit as low as 0.5 fmol L<sup>–1</sup>. The developed method was successfully used for determination\nof miRNA-21 in human breast cancer cell lines (SK-BR-3 and MCF-7)\nand real blood samples from breast cancer patients. It is versatile,\ncan be adapted to detect other targets by modifying the specific sequence\nof the SDA template chain that is complementary to the analytes, and\noffers a promising strategy for detecting various biomarkers with\nhigh sensitivity and specificity.