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

项目总结 在这项拟议的研究中，我们将开发一种脂质纳米探针(LNP)集成的微器件，LiEV芯片，用于 非小细胞肺癌(NSCLC)患者血浆中细胞外小泡的分离 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。它是 预计在20分钟内从1毫升未经处理的血浆中分离出肠道病毒，分离效率超过80%。 此外，结合基于双重测序的超灵敏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。 在我们之前的研究中，我们开发了一种双组分LNP系统，用于EV的快速分离和全面 EV分子分析。特别是，我们成功地从四名患有肠道病毒的患者中鉴定出EV DNA突变。 高级非小细胞肺癌。LNP系统克服了产量低、纯度低等常见的缺点 EV的分离，显示出很大的临床应用潜力。因此，我们提出开发一种高度集成的产品-- 组件LiEV芯片，可实现快速高效的电动汽车隔离。首先，我们将设计和制造拟议的 LiEV芯片。LiEV芯片集成了LNP，根据EV的脂膜膜捕获EV。电动汽车 通过设计微通道曲率来增强流动-表面相互作用，从而提高隔离效率 Dean Flow，以及通过纳米电极阵列增强电动增强型EV隔离的能力。然后, 我们将优化其运行参数，在EV隔离效率和纯度之间取得平衡。之后 全面优化，我们将通过分析电动汽车的SPEKE样本严格验证其性能 内容，包括RNA，DNA和蛋白质，并与流行的EV分离株进行比较 方法采用OptiPrep密度梯度超速离心法。随后，我们将开发双工 基于测序的第三代高通量测序仪DNA突变检测方法 将验证点/缺失突变和复杂结构变异(SVS)的检测灵敏度 以及添加的样本。最后，在一项队列临床研究中，我们将首先招募40名第四至第四阶段的非小细胞肺癌患者 验证整体技术。来自血浆的EV将用LiEV芯片隔离，其中最常见的是20个面板 NSCLC突变基因将通过双序列测定同时进行检测。我们将进一步执行电动汽车 对涵盖I-III期非小细胞肺癌的另外120个样本进行分离和双链测序，以调查 该技术在非小细胞肺癌早期诊断中的潜力。此外，我们将利用开发的技术来 监测20例非小细胞肺癌靶向治疗患者的突变情况，探讨其临床应用价值 治疗监测。队列临床研究不仅可以证明LiEVchp联合双联 测序可以常规应用于检测非小细胞肺癌中的各种恶性肿瘤，但也可以显示 肠道病毒DNA检测的临床诊断价值总之，这一拟议项目的成功完成将为 EV诊断学的临床翻译之道。

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.