Simple Amplifier Coupled with a Lanthanide Labeling Strategy for Multiplexed and Specific Quantification of MicroRNAs,Analytical Chemistry

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Simple Amplifier Coupled with a Lanthanide Labeling Strategy for Multiplexed and Specific Quantification of MicroRNAs
Analytical Chemistry ( IF 6.7 ) Pub Date : 2022-09-07 , DOI: 10.1021/acs.analchem.2c03234
Qi Kang ₁ , Beibei Chen ₁ , Man He ₁ , Bin Hu ₁

Affiliation

Inductively coupled plasma–mass spectrometry (ICP–MS) with elemental labeling is a promising strategy for multiplex microRNA (miRNA) analysis. However, it is still challenging for specific analysis of multiple miRNAs with high homology, and the development of multiplex assays is always limited by the complexity of the sequence design. Herein, a simple and direct ICP–MS-based assay was developed for the simultaneous detection of three miRNAs by combining the lanthanide labeling strategy with entropy-driven catalytic (EDC) amplification. Owing to the specificity of EDC for nucleic acid recognition, it is able to differentiate miRNAs with single-base mutation in each EDC circuit. A universal biotin-labeled DNA strand was designed to hybridize with the DNA substrates for three EDC circuits, targeting miRNA-21, miRNA-155, and miRNA-10b, respectively. All the substrates were loaded on the surface of streptavidin magnetic beads. In the presence of target miRNA, the EDC reaction was initiated, and EDC substrates were dissociated, continuously releasing reporter strands that were labeled with lanthanides (Tb/Ho/Lu). After magnetic separation, the supernatant containing the released reporter strands was introduced into an ICP–MS system for simultaneous detection of ¹⁵⁹Tb/¹⁶⁵Ho/¹⁷⁵Lu and quantification of miRNA-21, miRNA-155, and miRNA-10b, respectively. The limits of detection were 7.4, 7.5, and 11 pmol L^–1 for miRNA-21, miRNA-155, and miRNA-10b, respectively. Overall, this study provides a powerful strategy for simultaneous quantification of multiple miRNAs, with the advantages of flexible probe design, good sensitivity, and excellent specificity.

中文翻译：

简单的放大器与镧系元素标记策略相结合，用于 MicroRNA 的多路复用和特异性定量

带有元素标记的电感耦合等离子体质谱 (ICP-MS) 是一种有前途的多重 microRNA (miRNA) 分析策略。然而，对具有高度同源性的多个miRNA进行特异性分析仍然具有挑战性，并且多重检测的发展总是受到序列设计复杂性的限制。在此，通过将镧系元素标记策略与熵驱动催化 (EDC) 扩增相结合，开发了一种简单而直接的基于 ICP-MS 的检测方法，用于同时检测三种 miRNA。由于 EDC 对核酸识别的特异性，它能够区分每个 EDC 回路中具有单碱基突变的 miRNA。通用生物素标记的 DNA 链被设计用于与三个 EDC 电路的 DNA 底物杂交，分别靶向 miRNA-21、miRNA-155 和 miRNA-10b。所有的底物都装载在链霉亲和素磁珠的表面。在目标 miRNA 存在的情况下，EDC 反应开始，EDC 底物解离，连续释放标记有镧系元素 (Tb/Ho/Lu) 的报告链。磁分离后，将含有释放的报告链的上清液引入 ICP-MS 系统，同时检测^分别对159 Tb/ ¹⁶⁵ Ho/ ¹⁷⁵ Lu 和 miRNA-21、miRNA-155 和 miRNA-10b 进行定量。miRNA-21、miRNA-155 和 miRNA-10b的检测限分别为 7.4、7.5 和 11 pmol L ^–1。总体而言，本研究为多种 miRNA 的同时定量提供了一种强有力的策略，具有探针设计灵活、灵敏度高、特异性好等优点。

更新日期：2022-09-07

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