Ultrasensitive and multiplexed microRNA analysis: Integrated RNA microreactor-capillary electrophoresis-mass spectrometry platform.

Capillary electrophoresis-mass spectrometry (CE-MS) is a powerful tool for the separation and characterization of a wide range of biomolecules with the joint merits of both CE separation and MS detection; yet its potential in multiplexed microRNAs (miRNAs) analysis remains relatively underdeveloped, owing to their extremely low expression level and high family sequence homology in complex biological matrices. To address these limitations, we develop an integrated microreactor-assisted CE-MS platform that enables signal amplification and automated sample processing. Specifically, the uniquely-designed RNA-immobilized capillary microreactor (RNA-IMR) is fabricated at the inlet of a capillary and the successive CE-MS analysis is performed via the outlet of the same capillary as the electrospray ionization (ESI) source. Target miRNAs are converted into large amount of single-stranded DNAs (ssDNAs, hereafter referred to as outputDNAs) containing poly(adenine) or poly(guanine) segments with the T7 Exo mediated recycling amplification. The resultant outputDNAs are subsequently injected into the capillary, specifically recognized by the immobilized RNAs. The captured outputDNAs then undergo acid-catalyzed depurination to hydrolyze glycosidic bonds in the poly(adenine) or poly(guanine) segments, releasing free adenine (A) or guanine (G) as signaling molecules. These small nucleobases exhibit high ionization efficiency in MS due to their low molecular weight and polarity, enabling direct MS detection without labeling. The proposed RNA-IMR CE-MS integrates purification/pre-concentration, signal conversion, CE separation and MS detection with one injection, with reduced preparation time compared to RT-qPCR and improved multiplexing compared to hybridization-based methods. Using miRNA-21 and miRNA-155 as model targets, we show that the method exhibits detection limits in the low femtomolar range and specificity for multiplexing detection of miRNAs. The method is successfully applied to simultaneously detect miRNA-21 and miRNA-155 levels in MCF-7, HepG2 cancer cells and HL-7022 cells, showing its potential value in clinical applications. By bridging enzymatic signal amplification with automated CE-MS analysis, our study establishes a generalizable framework for the utilization of CE-MS as a universal method for highly sensitive miRNA assays.