Department of Thyroid Surgery, The First Hospital of Jilin University, Changchun 130021, China.
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China.
Molecules. 2021 Mar 15;26(6):1628. doi: 10.3390/molecules26061628.
In this work, a simple enzyme-free flow cytometric assay (termed as TSDR-based flow cytometric assay) has been developed for the detection of papillary thyroid carcinoma (PTC)-related microRNA (miRNA), hsa-miR-146b-5p with high performance through the toehold-mediated strand displacement reaction (TSDR) on magnetic beads (MBs). The complementary single-stranded DNA (ssDNA) probe of hsa-miR-146b-5p was first immobilized on the surface of MB, which can partly hybridize with the carboxy-fluorescein (FAM)-modified ssDNA, resulting in strong fluorescence emission. In the presence of hsa-miR-146b-5p, the TSDR is trigged, and the FAM-modified ssDNA is released form the MB surface due to the formation of DNA/RNA heteroduplexes on the MB surface. The fluorescence emission change of MBs can be easily read by flow cytometry and is strongly dependent on the concentration of hsa-miR-146b-5p. Under optimal conditions, the TSDR-based flow cytometric assay exhibits good specificity, a wide linear range from 5 to 5000 pM and a relatively low detection limit (LOD, 3σ) of 4.21 pM. Moreover, the practicability of the assay was demonstrated by the analysis of hsa-miR-146b-5p amounts in different PTC cells and clinical PTC tissues.
在这项工作中，开发了一种简单的无酶流动细胞检测分析（称为基于 TSDR 的流式细胞检测分析），通过在磁珠（MBs）上的引发链置换反应（TSDR），以高灵敏度检测甲状腺乳头状癌（PTC）相关 microRNA（miRNA）hsa-miR-146b-5p。hsa-miR-146b-5p 的互补单链 DNA（ssDNA）探针首先固定在 MB 表面，与羧基-荧光素（FAM）修饰的 ssDNA部分杂交，导致强荧光发射。在存在 hsa-miR-146b-5p 的情况下，触发 TSDR，由于 MB 表面上形成 DNA/RNA 异源双链体，FAM 修饰的 ssDNA 从 MB 表面释放。MB 的荧光发射变化可以通过流式细胞术轻松读取，并且强烈依赖于 hsa-miR-146b-5p 的浓度。在最佳条件下，基于 TSDR 的流式细胞检测分析具有良好的特异性、从 5 到 5000 pM 的宽线性范围和相对较低的检测限（LOD，3σ）为 4.21 pM。此外，通过分析不同 PTC 细胞和临床 PTC 组织中的 hsa-miR-146b-5p 含量，证明了该测定法的实用性。
