Institute of Basic and Translational Medicine & Shaanxi Key Laboratory of Brain Disorders, Xi'an Medical University, Xi'an 710021, Shaanxi Province, P. R. China.
Engineering Research Center of Brain Diseases Drug Development, Universities of Shaanxi Province, Xi'an Medical University, Xi'an 710021, Shaanxi Province, P. R. China.
Anal Chem. 2024 Aug 20;96(33):13734-13741. doi: 10.1021/acs.analchem.4c03092. Epub 2024 Aug 8.
A one-step, enzyme-free, and highly sensitive digital microRNA (miRNA) assay is rationally devised based on flow cytometric counting of target miRNA-clicked nanobead dimers via a facile mix-and-read manner. In this strategy, highly efficient miRNA-sandwiched click chemical ligation of two DNA probes may remarkably stabilize and boost the dimer formation between two kinds of fluorescence-coded nanobeads, and the number of as-produced bead dimers will be target dose-responsive, particularly when the trace number of miRNA is much less than that of employed nanobeads. Finally, each fluorescence-coded bead dimer can be easily identified and digitally counted by a powerful flow cytometer (FCM) and accordingly, the amount of target miRNA can be accurately quantified in a digital way. This new digital miRNA assay can be accomplished with a facile mix-and-read operation just by simply mixing the target miRNA with two kinds of preprepared DNA probe-functionalized nanobeads, which do not require any nucleic acid amplification, purification, and complex operation procedures. In spite of the extremely simple one-step operation, benefiting from the low-background but high target-mediated click ligation efficiency, and the powerfully digital statistical capability of FCM, this strategy achieves high sensitivity with a quite low detection limit of 5.2 fM target miRNA as well as high specificity and good generality for miRNA analysis, pioneering a new direction for fabricating digital bioassays.
一种一步法、无酶且高灵敏度的数字 microRNA（miRNA）分析方法是基于通过简便的混合读取方式对目标 miRNA-click 纳米珠二聚体进行流式细胞术计数而合理设计的。在该策略中，两种 DNA 探针的高效 miRNA 夹心点击化学连接可能会显著稳定并促进两种荧光编码纳米珠之间的二聚体形成，并且产生的珠二聚体的数量将与靶剂量呈响应关系，特别是当痕量 miRNA 远少于所使用的纳米珠时。最后，每个荧光编码珠二聚体可以通过强大的流式细胞仪（FCM）轻松识别和数字计数，因此可以以数字方式准确定量目标 miRNA 的量。这种新的数字 miRNA 分析可以通过简单地将目标 miRNA 与两种预先制备的 DNA 探针功能化纳米珠混合来完成，而无需任何核酸扩增、纯化和复杂的操作步骤。尽管操作非常简单，但由于低背景但高靶介导的点击连接效率以及 FCM 的强大数字统计能力，该策略实现了高灵敏度，其检测限低至 5.2 fM 目标 miRNA，并且具有高特异性和良好的 miRNA 分析通用性，为数字生物测定的制备开辟了新的方向。
