Center for Affordable Nanoengineering of Polymeric Biomedical Devices, The Ohio State University , 174 West 18th Avenue, Room 1012, Columbus, Ohio 43212, United States.
Anal Chem. 2013 Dec 3;85(23):11265-74. doi: 10.1021/ac401983w. Epub 2013 Nov 13.
Noninvasive early detection methods have the potential to reduce mortality rates of both cancer and infectious diseases. Here, we present a novel assay by which tethered cationic lipoplex nanoparticles containing molecular beacons (MBs) can capture cancer cell-derived exosomes or viruses and identify encapsulated RNAs in a single step. A series of ultracentrifugation and Exoquick isolation kit were first used to isolate exosomes from the cell culture medium and human serum, respectively. Cationic lipoplex nanoparticles linked onto the surface of a thin glass plate capture negatively charged viruses or cell-secreted exosomes by electrostatic interactions to form larger nanoscale complexes. Lipoplex/virus or lipoplex/exosome fusion leads to the mixing of viral/exosomal RNAs and MBs within the lipoplexes. After the target RNAs specially bind to the MBs, exosomes enriched in target RNAs are readily identified by the fluorescence signals of MBs. The in situ detection of target extracellular RNAs without diluting the samples leads to high detection sensitivity not achievable by existing methods, e.g., quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Here we demonstrate this concept using lentivirus and serum from lung cancer patients.
非侵入性的早期检测方法有可能降低癌症和传染病的死亡率。在这里，我们提出了一种新的测定方法，通过该方法，含有分子信标的连接阳离子脂质体纳米颗粒可以在一步中捕获癌细胞衍生的外泌体或病毒，并识别包裹的 RNA。首先使用一系列超速离心和 Exoquick 分离试剂盒分别从细胞培养液和人血清中分离出外泌体。阳离子脂质体纳米颗粒链接到薄玻璃片的表面上，通过静电相互作用捕获带负电荷的病毒或细胞分泌的外泌体，形成更大的纳米级复合物。脂质体/病毒或脂质体/外泌体融合导致病毒/外泌体 RNA 与脂质体中的 MB 混合。目标 RNA 与 MB 特异性结合后，富含目标 RNA 的外泌体很容易通过 MB 的荧光信号识别。无需稀释样品即可原位检测靶细胞外 RNA，从而实现了现有方法无法实现的高检测灵敏度，例如定量逆转录聚合酶链反应 (qRT-PCR)。在这里，我们使用慢病毒和肺癌患者的血清证明了这一概念。
