Wang Anping, Zhao Siqi, Tian Miaomiao, Yang Jinlan, Yang Li
Department of Chemistry, Northeast Normal University, 5268 Renmin Street, Changchun, Jilin Province, 130024, China.
Institute of Chemical and Industrial Bioengineering, Jilin Engineering Normal University, Changchun, Jilin Province, 130052, China.
Talanta. 2026 Jan 1;296:128441. doi: 10.1016/j.talanta.2025.128441. Epub 2025 Jun 6.
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.
毛细管电泳-质谱联用（CE-MS）是一种强大的工具，结合了CE分离和MS检测的优点，可用于分离和表征多种生物分子；然而，由于其在复杂生物基质中的表达水平极低且家族序列同源性高，其在多重微小RNA（miRNA）分析中的潜力仍未得到充分开发。为了解决这些限制，我们开发了一个集成的微反应器辅助CE-MS平台，该平台能够实现信号放大和自动样品处理。具体而言，在毛细管入口处制造了独特设计的固定化RNA毛细管微反应器（RNA-IMR），并通过与电喷雾电离（ESI）源相同的毛细管出口进行连续的CE-MS分析。通过T7 Exo介导的循环扩增，将目标miRNA转化为大量含有聚（腺嘌呤）或聚（鸟嘌呤）片段的单链DNA（ssDNA，以下简称输出DNA）。随后将所得的输出DNA注入毛细管，被固定化RNA特异性识别。捕获的输出DNA然后进行酸催化的脱嘌呤反应，水解聚（腺嘌呤）或聚（鸟嘌呤）片段中的糖苷键，释放游离腺嘌呤（A）或鸟嘌呤（G）作为信号分子。这些小的核碱基由于其低分子量和极性，在MS中表现出高电离效率，无需标记即可直接进行MS检测。所提出的RNA-IMR CE-MS一次进样即可集成纯化/预浓缩、信号转换、CE分离和MS检测，与RT-qPCR相比减少了制备时间，与基于杂交的方法相比提高了多重检测能力。以miRNA-21和miRNA-155为模型靶点，我们表明该方法在低飞摩尔范围内具有检测限，并且对miRNA的多重检测具有特异性。该方法成功应用于同时检测MCF-7、HepG2癌细胞和HL-7022细胞中的miRNA-21和miRNA-155水平，显示了其在临床应用中的潜在价值。通过将酶促信号放大与自动CE-MS分析相结合，我们的研究建立了一个可推广的框架，将CE-MS用作高灵敏度miRNA检测的通用方法。
