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Quantitative assay of microRNAs (miRNAs) with mass spectrometric detection currently suffers from two major disadvantages, i.e., being insufficient in sensitivity and requiring an extraction or chromatographic separation prior to MS detection. In this work, we developed a facile and sensitive assay of targeted miRNAs based on the combination of cyclic enzymatic amplification (CEA) with microfluidic voltage-assisted liquid desorption electrospray ionization tandem mass spectrometry (VAL-DESI-MS/MS). The single-stranded DNA (ssDNA) probe was designed to have a sequence complementary to the miRNA target with an extension of a two-base nucleotide fragment (i.e., CpC) at the 3'-position as MS signal reporter, thus being easy to prepare and high in stability. In the proposed CEA-VAL-DESI-MS/MS assay, an ssDNA probe was added to a sample solution, forming a DNA-miRNA hybrid. Duplex-specific nuclease (DSN) was then added to cleave specifically the DNA probe in the heteroduplex strands. As the hybridization-cleavage cycle repeated itself for many rounds, a large quantity of CpC molecules was produced that was quantified by VAL-DESI-MS/MS with accuracy and specificity. miRNA-21 was tested as the model target. The assay had a linear calibration equation in the range from 2.5 pM to 1.0 nM with a limit of detection of 0.25 pM. Determination of miRNA-21 in cellular samples was demonstrated. miRNA-21 was found to be 95.3 ± 13.95 amol ( n = 3) in 100 mouse peritoneal macrophages with a recovery of 94.2 ± 2.6% ( n = 3). Interestingly, analysis of exosomes secreted from these cells revealed that exposure of the cells to chemical stimuli caused a 3-fold increase in exosomal level of miRNA-21. The results suggest that the proposed assay may provide an accurate and cost-effective means for quantification of targeted miRNAs in biomedical samples.

analytical chemistry

Quantification of MicroRNAs by Coupling Cyclic Enzymatic Amplification with Microfluidic Voltage-Assisted Liquid Desorption Electrospray Ionization Mass Spectrometry