Nanoparticle Counting by Microscopic Digital Detection: Selective Quantitative Analysis of Exosomes via Surface-Anchored Nucleic Acid Amplification
Nanoparticle Counting by Microscopic Digital Detection: Selective Quantitative Analysis of Exosomes via Surface-Anchored Nucleic Acid Amplification
复制标题
通过显微数字检测进行纳米颗粒计数：通过表面锚定核酸扩增对外泌体进行选择性定量分析
DOI：
10.1021/acs.analchem.8b00189
复制
发表时间：
2018-06-05
影响因子：
7.4
通讯作者：
Wang, Ben
中科院分区：
文献类型：
--
作者：
Tian, Qingchang;He, Chuanjiang;Wang, Ben
外泌体是细胞分泌的纳米大小的囊泡，具有脂质双层膜以及蛋白质和核酸内容物。在这里，我们提出了第一种通过微芯片中与基于核酸的扩增相结合的数字检测来选择性和定量分析外泌体的方法。与 DNA 寡核苷酸缀合的外部生物相容性锚分子通过表面自组装锚定在外泌体的脂质双层膜上，用于总外泌体分析。然后，应用特异性抗体-DNA缀合物来标记总外泌体中的选择性外泌体。 DNA锚定的外泌体被分配到微芯片室中，每个室有一个或更少的外泌体。通过快速等温核酸检测分析放大了外泌体上 DNA 的信号。具有外泌体的室呈现阳性信号并记录为1，而没有外泌体的室呈现阴性信号并记录为0。10100101数字信号给出了阳性室的数量。根据泊松分布，通过观察到的阳性室分数计算外泌体原液浓度。研究结果表明，可以通过简单的微观设置在微芯片中通过 DNA 介导的信号放大来数字检测纳米级颗粒。该方法可以与多种已建立的核酸检测方法相结合，为定量检测各种纳米体提供了一个通用的平台，从外泌体和包膜病毒等细胞外囊泡到无机和有机纳米颗粒，有望在基础研究领域以及疾病诊断和治疗中具有广泛的应用。
Exosomes are nanosized vesicles secreted by cells, with a lipid bilayer membrane and protein and nucleic acid contents. Here, we present the first method for the selective and quantitative analysis of exosomes by digital detection integrated with nucleic acid-based amplification in a microchip. An external biocompatible anchor molecule conjugated with DNA oligonucleotides was anchored in the lipid bilayer membrane of exosomes via surface self-assembly for total exosome analysis. Then, specific antibody-DNA conjugates were applied to label selective exosomes among the total exosomes. The DNA-anchored exosomes were distributed into microchip chambers with one or fewer exosomes per chamber. The signal from the DNA on the exosomes was amplified by a rapid isothermal nucleic acid detection assay. A chamber with an exosome exhibited a positive signal and was recorded as 1, while a chamber without an exosome presented a negative signal and was recorded as 0. The 10100101 digital signals give the number of positive chambers. According to the Poisson distribution, the exosome stock concentration was calculated by the observed fraction of positive chambers. The findings showed that nanoscale particles can be digitally detected via DNA-mediated signal amplification in a microchip with simple microscopic settings. This approach can be integrated with multiple types of established nucleic acid assays and provides a versatile platform for the quantitative detection of various nanosomes, from extracellular vesicles such as exosomes and enveloped viruses to inorganic and organic nanoparticles, and it is expected to have broad applications in basic research areas as well as disease diagnosis and therapy.