基于滑动微流控芯片系统的癌症外泌体即时检测研究

The tumor exosome, which carries the rich biological information of the tumor patients, is an important target for the liquid biopsy of tumor. At present, the methods for exosome separation and analysis still face many problems, and the successful solution of these problems is the key to the realization of the clinical application of exosome. Based on the technology of slipchip, we will design a new chip for separation and purification of exosomes and combine it with visual quantitation chip, to establish a new point-of-care diagnostic platform for exosome. We will use DNA strand displacement reaction to efficiently separate the exosome from cell culture medium and blood in the separation chip, and achieve high sensitivity and visual detection of exosome on chip by means of hybridization chain reaction and multistaged signal amplification strategy. As the final platform will not employ complicated detection and flow control instruments, only needs a small amount of sample and reagent, and also can be used for detection of a variety of exosome surface biomarker at the same time， the platform can be used for point-of-care detection of exosomes. We will develop a set of mature exosome detection system with ovarian cancer as a model to provide a new way for the point-of-care detection of exosome.

肿瘤外泌体携带着肿瘤患者丰富的生物分子信息，是肿瘤液体活检重要的靶标。目前，外泌体分离和分析方法依然面临众多问题，这些问题的成功解决是实现外泌体临床应用的关键。本项目将以滑动微流控技术为基础，设计出全新的外泌体的分离纯化芯片，并且与外泌体可视化定量检测芯片相结合，建立起新的外泌体的即时诊断平台。申报人将利用DNA链置换反应，在分离芯片中实现细胞培养液和血液中的外泌体的高效分离；借助于DNA杂交链反应和级联信号放大策略，在检测芯片上实现外泌体的高灵敏度可视化检测。由于不使用复杂的检测和流体控制装置，并且样品和试剂的使用量较小，可以对多种外泌体表面蛋白标志物同时检测，因此该检测平台能够用于外泌体即时检测中。申请人将以卵巢癌为模型，发展出一套成熟的外泌体的检测系统，为外泌体的即时检测提供一条新的思路。

肿瘤外泌体携带着肿瘤患者丰富的生物分子信息，是肿瘤液体活检重要的靶标。目前，外泌体分离和分析方法依然面临众多问题，这些问题的成功解决是实现外泌体临床应用的关键。在本项目的资助下，我们首次制备了一种具有磁性胶体抗体，用于培养基及血浆中纳米细胞外囊泡的快速识别及分离。相比较于传统的分离方法，该磁性胶体抗体制备简易、易于保存，可以在短时间内实现对外泌体的快速分离和高效地富集，同时可重复使用数次，大大降低的外泌体分离检测的成本。我们其与微流控芯片结合，构建一种基于外泌体的可视化POCT检测方法，分别实时了监测小鼠体内的肿瘤发展和临床卵巢癌血液样本多个特异性标志物准确检测。此外，我们发展了基于金属有机框架的DNA条形码扩增检测技术。通过与CRISPR/Cas12a体系结合，实现了级联信号放大，可用于外泌体中miRNA和肿瘤蛋白标志物的高灵敏检测。这些研究成果为外泌体高效分离和POCT 诊断提供新的思路。