Exponential isothermal amplification coupled MALDI-TOF MS for microRNAs detection

MicroRNAs (miRNAs) have attracted significant attention in biomedical research and clinical diagnosis. However, due to their inherent characteristics of low abundance and the high complexity of correspond-ing biological matrices, simultaneous detection of multiple miRNAs at low abundance is still a challenge. In this work, a method coupling exponential amplification reaction (EXPAR) with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is developed for label-free and simultaneous detection of multiple miRNAs. The assay can be performed under isothermal conditions in a single reaction tube, and finished in less than 30 min. It exhibits good quantification ability and with attomolar-level sensitivity for miRNAs detection. It also shows high specificity to distinguish miR-NAs at single-nucleotide resolution. We used the method to detect the miRNA-21, let-7a, miRNA-10 0, and miRNA-125b in samples of spiked human serum and breast cancer cells (i.e., MCF-7, MDA-MB-231 and SK-BR-3). The quantification results were well consistent with the standard real-time fluorescence EXPAR. Consequently, the label-free mass-spectrometric platform could be a potential tool for miRNAs analysis in complex biological samples, and may be used for clinical diagnosis.(c) 2022 Published by Elsevier B.V. on behalf of Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.

Automated summary

In this work, a method combining EXPAR and MALDI-TOF MS is developed for label-free and simultaneous detection of multiple low-abundance miRNAs. The method can be performed in a single reaction tube under isothermal conditions and completed in less than 30 minutes. It demonstrates good quantification ability, attomolar-level sensitivity, and high specificity for miRNAs detection, including single-nucleotide resolution. Therefore, this label-free mass-spectrometric platform has the potential to be a valuable tool for miRNA analysis in complex biological samples and clinical diagnosis.