Lipid nanoprobe integrated microdevice for extracellular vesicle isolation and duplex sequencing based mutation detection for non-invasive lung cancer diagnosis

用于细胞外囊泡分离和基于双重测序的突变检测的脂质纳米探针集成微器件用于非侵入性肺癌诊断

基本信息

批准号：
10686287
负责人：
Siyang Zheng
金额：
$ 34.35万
依托单位：
CARNEGIE-MELLON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-08-06 至 2024-07-31
项目状态：
已结题

项目摘要

PROJECT SUMMARY In this proposed research, we will develop a lipid nanoprobe (LNP) integrated microdevice, LiEVchip, for isolation of extracellular vesicles (EVs) from blood plasma of patients with non-small cell lung cancer (NSCLC). The proposed LiEVchip features high-throughput sample processing, one-step enrichment, and low cost. It is expected to isolate EVs from 1 ml of unprocessed plasma in 20 minutes with over 80% isolation efficiency. Furthermore, combination with duplex sequencing based ultrasensitive DNA mutation detection platform, the LiEVchip will significantly prompt EV-based cancer diagnostics in an invasive way. In our previous study, we developed a two-component LNP system for rapid EV isolation and comprehensively EV molecular analyses. Particularly, we successfully identified EV DNA mutations from four patients with advanced NSCLC. The LNP system overcomes low throughput, low purity and other common shortcomings in EV isolation, showing great potential for clinical use. Hence, we proposed to develop a highly integrated one- component LiEVchip for rapid and efficient EV isolation. Firstly, we will design and fabricate the proposed LiEVchip. The LiEVchip integrates the LNP to capture EVs based on their lipid membrane envelope. The EV isolation efficiency is boosted by the design of microchannel curvature to enhance flow-surface interaction by Dean flow, and the empowerment of electrokinetic enhanced EV isolation by the nanoelectrode array. Then, we will optimize its operational parameters to make a balance between EV isolation efficiency and purity. After comprehensive optimization, we will rigorously validate its performance with spike samples by analyzing EV content, including RNA, DNA, and proteins, and compare with those isolated by the prevalent EV isolation method, OptiPrep density gradient ultracentrifugation (odgUC). Subsequently, we will develop duplex sequencing based DNA mutation detection method with 3rd generation high-throughput sequencer (PacBio). The detection sensitivity of point/deletion mutations and complex structural variations (SVs) will be validated with spiked-in samples. Finally, in a cohort clinical study, we will first recruit 40 NSCLC patients at stage IV to validate the overall technology. EV from plasma will be isolated with LiEVchip, and a panel of 20 most common NSCLC mutated genes will be assayed simultaneously by duplex sequencing. We will further perform EV isolation and duplex sequencing in the additional 120 samples covering stage I-III NSCLC to investigate the potential of this technology in early NSCLC diagnosis. Besides, we will use the developed technology to monitor mutation status of 20 NSCLC patients undergoing targeted therapy to explore its clinical utility in treatment monitoring. The cohort clinical studies not only can testify whether LiEVchp combined with duplex sequencing can be routinely applied to detect diverse malignancies in NSCLC, but also can demonstrate the clinical diagnostic values of EV DNA. Altogether, the successful completion of this proposed project will pave the way for clinical translation of EV diagnostics.

项目摘要在这项拟议的研究中，我们将开发脂质纳米探针（LNP）集成的微电位，lievchip，用于从非小细胞肺癌（NSCLC）患者血浆中分离出细胞外囊泡（EV）。拟议的Lievchip具有高通量样品处理，一步富集和低成本。这是预计将在20分钟内从1 mL未加工的血浆中分离出EV，分离效率超过80％。此外，与基于双链测序的超敏DNA突变检测平台的结合， Lievchip将以侵入性的方式显着提示基于EV的癌症诊断。在我们先前的研究中，我们开发了一个两部分LNP系统，用于快速隔离和全面 EV分子分析。特别是，我们成功地发现了四名患者的EV DNA突变高级NSCLC。 LNP系统克服了低吞吐量，低纯度和其他常见的缺点 EV隔离，显示出巨大的临床使用潜力。因此，我们建议开发高度综合的一 lievchip用于快速有效的EV隔离。首先，我们将设计和制造提议的 Lievchip。 Lievchip集成了LNP，以基于其脂质膜包膜捕获电动汽车。 EV 微通道曲率的设计可以提高隔离效率，从而通过院长流量以及纳米电极阵列的电动增强EV隔离的能力。然后，我们将优化其操作参数，以在EV隔离效率和纯度之间达到平衡。后全面优化，我们将通过分析EV来严格地通过尖峰样品验证其性能含量，包括RNA，DNA和蛋白质，并与普遍的EV分离隔离的内容进行比较方法，OptiPREP密度梯度超速离心（ODGUC）。随后，我们将开发双工基于测序的DNA突变检测方法，使用第三代高通量测序仪（PACBIO）。点/缺失突变和复杂结构变化（SV）的检测灵敏度将得到验证带有峰值样品。最后，在一项队列临床研究中，我们将在第四阶段首先招募40名NSCLC患者验证整体技术。来自等离子体的EV将用Lievchip隔离，并且由20个最常见的面板隔离 NSCLC突变基因将通过双链测序同时测定。我们将进一步执行EV 在涵盖I-III阶段NSCLC的另外120个样品中进行的分离和双链测序，以研究该技术在NSCLC早期诊断中的潜力。此外，我们将使用开发的技术来监测20名NSCLC患者接受靶向疗法的监测突变状态，以探索其临床实用性治疗监测。队列临床研究不仅可以证明Lievchp是否与双工合并测序可以通常应用于检测NSCLC中的各种恶性肿瘤，但也可以证明 EV DNA的临床诊断值。总之，这个拟议项目的成功完成将铺路电动汽车诊断临床翻译的方式。

项目成果

期刊论文数量（6）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Enrichment of extracellular vesicles with lipid nanoprobe functionalized nanostructured silica.

DOI：
10.1039/c8lc01359d
发表时间：
2019-07
期刊：
Lab on a chip
影响因子：
6.1
作者：
Y. Wan;Mackenzie Maurer;Hongzhang He;Yiqiu Xia;S. Hao;Wen-Long Zhang;N. Yee;Si-Yang Zheng
通讯作者：
Y. Wan;Mackenzie Maurer;Hongzhang He;Yiqiu Xia;S. Hao;Wen-Long Zhang;N. Yee;Si-Yang Zheng

Small extracellular vesicles in cancer.