3D carbon-nanotubes integrated microdevice for extracellular vesicle isolation and in situ sample preparation towards noninvasive pancreatic cancer diagnosis

项目总结 胰腺癌是最致命的实体恶性肿瘤，总体5年生存率不到7%。晚些 分期诊断被认为是导致患者预后黯淡的最重要因素。自.以来 胰腺肿瘤细胞可能需要几十年的时间才能转移，理论上，早期诊断有很大的窗口。 然而，目前的早期诊断往往由于缺乏敏感和特异的胰腺生物标志物和 成像方法的空间分辨率限制。细胞外小泡是亚微米级的。 囊泡由几乎所有的细胞释放，并成为有吸引力的无创液体活检靶点。 临床癌症诊断。与其他液体活检材料相比，EV很可能是由活细胞和 含有比循环肿瘤DNA(CtDNA)长得多的DNA片段，并且比 循环肿瘤细胞(CTCs)提供更高的敏感性和更好的临床应用。根据我们的初步调查 碳纳米管林作为一种前所未有的亚微米颗粒分离材料的研究， 我们提出了一种新的集成CNT的EV分离和基因组浓缩微设备(CNT-iEVIGEM)，它是 与碳纳米管森林集成的连续流动微流控装置。自动调节连续流的设计 将我们以前的CNT集成设备的样本容量和吞吐量提高了10倍，并消除了 堵塞，同时使设备对局部碳纳米管森林缺陷免疫，并保持高电动汽车捕获效率。 通过在碳纳米管上添加纳米厚度的功能氧化锆，碳纳米管-iEVIGEM可以有效地提纯 EV DNA原位检测。对于胰腺癌的诊断，我们将建立一种多重数字液滴聚合酶链式反应(Ddpcr)检测方法。 用CNT-iEVIGEM检测从血浆中分离的EV的KRAS突变。KRAS突变最多的是 常见的胰腺癌突变，90%以上发生。它是癌基因的主要驱动因素，而且通常是 胰腺肿瘤发生的最早事件。使用健康对照组中添加了癌症来源的电动汽车的模型样本 血浆中，我们将检测下限确定为可以检测到的最低突变等位基因比例 毫不含糊。该检测方法将通过75份来自IV期胰腺癌患者的血浆样本进行验证。 75名年龄/性别匹配的健康捐赠者。然后我们将利用这一检测方法来研究胰腺癌的诊断 癌症不同阶段，招募60例早期(I+II期)和60例III期胰腺癌患者。 不同阶段检测的敏感度和特异度将根据组织中的突变状态来衡量 作为真实值的样本。这项拟议的研究将为CNT的进一步临床验证铺平道路。 IEVIGEM癌症诊断技术。EV分离平台还可用于其他DNA突变， 其他EV货物和其他癌症类型作为一种非侵入性液体活检技术。

PROJECT SUMMARY Pancreatic cancer is the deadliest solid malignancy with an abysmal overall 5-year survival less than 7%. Late stage diagnosis is regarded as the most important factor contributing to the bleak patient outcome. Since pancreatic neoplastic cells can take decades to metastasis, in theory, there is a large window for early diagnosis. However, current early diagnosis often fails due to lack of sensitive and specific pancreatic biomarkers and limitations on the spatial resolution of the imaging methods. Extracellular vesicles (EVs) are sub-micrometer vesicles released by almost all the cells and are emerging as an attractive non-invasive liquid biopsy target for clinical cancer diagnosis. Compared with other liquid biopsy materials, EVs are likely released by live cells and contains much longer DNA fragments than circulating tumor DNA (ctDNA), and are more abundant than circulating tumor cells (CTCs) to provide higher sensitivity and better clinical utility. Based on our preliminary study on using carbon nanotube (CNT) forest as an unprecedented material for sub-micrometer particle isolation, we propose a new CNT-integrated EV isolation and genome enrichment microdevice (CNT-iEVIGEM), which is a continuous flow microfluidic device integrated with CNT forest. The self-regulating continuous-flow design boosts the sample capacity and throughput of our previous CNT integrated device by 10 times and eliminates clogging, while making the device immune to local CNT forest defects and maintaining high EV capture efficiency. With additive fabrication of nanometer-thick functional zirconia on CNTs, the CNT-iEVIGEM can efficiently purify EV DNA in situ. For pancreatic cancer diagnosis, we will develop a multiplex digital droplet PCR (ddPCR) assay to detect KRAS mutations from EVs isolated from plasma by CNT-iEVIGEM. KRAS mutations are the most prevalent pancreatic cancer mutations with over 90% occurrence. It is a major driver oncogene and often the earliest event in pancreatic tumorigenesis. Using model samples of cancer-derived EVs spiked in healthy control plasma, we will determine the detection limit as the lowest mutant allele fraction that can be detected unambiguously. The assay will be validated with 75 plasma samples from stage IV pancreatic cancer patients and 75 age-/sex- matched healthy donors. Then we will use this assay to investigate diagnosis of pancreatic cancer at different stages, by recruiting 60 early stage (stage I + II) and 60 stage III pancreatic cancer patients. The sensitivity and specificity of the assay at different stages will be measured with the mutation status in tissue samples as the true values. The proposed study will pave the way for further clinical validation of the CNT- iEVIGEM technology for cancer diagnosis. The EV isolation platform can also be used for other DNA mutations, other EV cargos, and other cancer types as a non-invasive liquid biopsy technology.