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MicroRNAs (miRNAs) are important biomarker candidates for cancer screening and early detection research. Generally, miRNAs undergo synergistic adjustments in tumor cells. Herein, a mass-spectrometric method based on a duplexspecific-nuclease (DSN)-enzyme-assisted signal-amplification technique was proposed for label-free and multiplexed detection of multiple miRNAs, and applied to the quantification of three miRNAs (i.e., miRNA-141, miRNA-21, and let-7a) in samples of HeLa and MDA-MB231 cell extracts. Experimental results showed that the digestion modes of DSN against three different DNAs complementary to miRNA-141, miRNA-21, and let-7a in their DNA−miRNA heteroduplexes were quite different, verifying the multiplexed-detection capability of the proposed method. Moreover, an advanced calibration model was derived for the quantitative analysis of the complex mass-spectral data measured during the label-free and multiplexed detection of miRNA-141, miRNA-21, and let-7a by the proposed massspectrometric method. With the aid of the advanced calibration model, the proposed mass-spectrometric method achieved quite reliable quantitative results for miRNA-141, miRNA-21, and let-7a in samples of HeLa and MDA-MB231 cell extracts, with recovery rates within the range of 89.2 to 111.6%. The limits of detection (LODs) of the proposed mass-spectrometric method for miRNA-141, miRNA-21, and let-7a in standard samples were estimated to be 42, 41, and 95 pM, respectively. Therefore, it is reasonable to expect that the proposed mass-spectrometric method can be a competitive alternative for the label-free and multiplexed detection of multiple miRNAs in clinical diagnosis.

A platform for microRNA detection was developed based on understanding the hydrolysis patterns of duplex-specific nuclease against probe DNAs.