Simultaneous and ultrasensitive detection of multiple microRNAs by single-molecule fluorescence imaging,Chemical Science

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Simultaneous and ultrasensitive detection of multiple microRNAs by single-molecule fluorescence imaging
Chemical Science ( IF 7.6 ) Pub Date : 2020-03-24 , DOI: 10.1039/d0sc00580k
Hongding Zhang _{1,

2} , Xuedong Huang ₁ , Jianwei Liu ₁ , Baohong Liu ₁

Affiliation

Cell status changes are typically accompanied by the simultaneous changes of multiple microRNA (miRNA) levels. Thus, simultaneous and ultrasensitive detection of multiple miRNA biomarkers shows great promise in early cancer diagnosis. Herein, a facile single-molecule fluorescence imaging assay was proposed for the simultaneous and ultrasensitive detection of multiple miRNAs using only one capture anti-DNA/RNA antibody (S9.6 antibody). Two complementary DNAs (cDNAs) designed to hybridize with miRNA-21 and miRNA-122 were labelled with Cy3 (cDNA1) and Cy5 (cDNA2) dyes at their 5′-ends, respectively. After hybridization, both miRNA-21/cDNA1 and miRNA-122/cDNA2 complexes were captured by S9.6 antibodies pre-modified on a coverslip surface. Subsequently, the Cy3 and Cy5 dyes on the coverslip surface were imaged by the single-molecule fluorescence setup. The amount of miRNA-21 and miRNA-122 was quantified by counting the image spots from the Cy3 and Cy5 dye molecules in the green and red channels, respectively. The proposed assay displayed high specificity and sensitivity for singlet miRNA detection both with a detection limit of 5 fM and for multiple miRNA detection both with a detection limit of 20 fM. Moreover, it was also demonstrated that the assay could be used to detect multiple miRNAs simultaneously in human hepatocellular cancer cells (HepG2 cells). The proposed assay provides a novel biosensing platform for the ultrasensitive and simple detection of multiple miRNA expressions and shows great prospects for early cancer diagnosis.

中文翻译：

通过单分子荧光成像同时超灵敏检测多种 microRNA

细胞状态的变化通常伴随着多种 microRNA (miRNA) 水平的同时变化。因此，同时超灵敏地检测多种 miRNA 生物标志物在早期癌症诊断中显示出巨大的前景。在此，提出了一种简便的单分子荧光成像测定法，仅使用一种捕获抗 DNA/RNA 抗体（S9.6 抗体）即可同时超灵敏地检测多个 miRNA。设计用于与 miRNA-21 和 miRNA-122 杂交的两个互补 DNA (cDNA) 的 5' 末端分别用 Cy3 (cDNA1) 和 Cy5 (cDNA2) 染料进行标记。杂交后，miRNA-21/cDNA1 和 miRNA-122/cDNA2 复合物均被盖玻片表面上预先修饰的 S9.6 抗体捕获。随后，通过单分子荧光装置对盖玻片表面上的 Cy3 和 Cy5 染料进行成像。通过分别计算绿色和红色通道中 Cy3 和 Cy5 染料分子的图像点来定量 miRNA-21 和 miRNA-122 的量。所提出的检测方法对单线态 miRNA 检测（检测限为 5 fM）和多 miRNA 检测（检测限为 20 fM）显示出高特异性和灵敏度。此外，还证明该检测可用于同时检测人肝细胞癌细胞（HepG2细胞）中的多种miRNA。所提出的检测方法为多种miRNA表达的超灵敏和简单检测提供了一种新颖的生物传感平台，并为早期癌症诊断显示了巨大的前景。

更新日期：2020-04-24

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