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

项目摘要 在这项拟议的研究中，我们将开发一种脂质纳米探针（LNP）集成微器件，LiEV芯片，用于 从非小细胞肺癌（NSCLC）患者的血浆中分离细胞外囊泡（EV）。 所提出的LiEV芯片具有高通量样品处理、一步富集和低成本的特点。是 预期在20分钟内以超过80%的分离效率从1 ml未处理的血浆中分离EV。 此外，结合基于双链测序的超灵敏DNA突变检测平台， LiEV芯片将以侵入性的方式显著促进基于EV的癌症诊断。 在我们以前的研究中，我们开发了一种双组分LNP系统，用于快速EV隔离和全面 EV分子分析。特别是，我们成功地从四名患者中鉴定了EV DNA突变， 晚期NSCLC。LNP系统克服了低通量、低纯度和其他常见缺点， EV分离，显示出巨大的临床应用潜力。因此，我们建议开发一个高度集成的- 组件LiEV芯片，实现快速高效的EV隔离。首先，我们将设计和制作拟议的 Liev芯片。LiEV芯片集成LNP以基于其脂质膜包膜捕获EV。的EV 通过微通道曲率的设计来提高隔离效率， Dean流，以及通过纳米电极阵列增强电动力学的EV隔离。然后， 我们将优化其操作参数，以在EV分离效率和纯度之间取得平衡。后 全面优化，我们将严格验证其性能与尖峰样本，通过分析EV 内容，包括RNA，DNA和蛋白质，并与流行的EV分离物进行比较 方法，OptiPrep密度梯度超离心（odgUC）。随后，我们将开发复式 使用第三代高通量测序仪（PacBio）的基于测序的DNA突变检测方法。 将验证点/缺失突变和复杂结构变异（SV）的检测灵敏度 加了毒的样本最后，在一项队列临床研究中，我们将首先招募40名IV期NSCLC患者， 验证整体技术。来自血浆的EV将用LiEV芯片隔离， NSCLC突变基因将通过双链体测序同时测定。我们将进一步执行EV 在另外120份涵盖I-III期NSCLC的样本中进行分离和双链体测序，以研究 该技术在早期NSCLC诊断中的潜力。此外，我们将利用先进的技术， 监测20例接受靶向治疗的NSCLC患者的突变状态， 治疗监测。队列临床研究不仅可以证明LiEVchp联合双功 测序可常规应用于检测NSCLC中的多种恶性肿瘤，但也可证明 EV DNA的临床诊断价值总而言之，本拟议项目的成功完成将为 EV诊断的临床转化途径。

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.