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

项目摘要 胰腺癌是最致命的实体恶性肿瘤，5年生存率低于7%。晚 分期诊断被认为是导致患者预后不佳的最重要因素。以来 胰腺癌细胞可能需要几十年才能转移，理论上，早期诊断有很大的窗口期。 然而，由于缺乏敏感和特异性的胰腺生物标志物， 对成像方法的空间分辨率的限制。细胞外囊泡（EV）是亚微米级的 几乎所有的细胞释放的囊泡，并正在成为一个有吸引力的非侵入性液体活检目标， 临床癌症诊断与其他液体活检材料相比，EV可能由活细胞释放， 含有比循环肿瘤DNA（ctDNA）长得多的DNA片段，并且比循环肿瘤DNA（ctDNA）更丰富。 循环肿瘤细胞（CTC），以提供更高的灵敏度和更好的临床效用。根据我们初步的 研究使用碳纳米管（CNT）森林作为前所未有的亚微米颗粒分离材料， 我们提出了一种新的CNT集成EV分离和基因组富集微装置（CNT-iEVIGEM）， 与CNT森林集成的连续流动微流体装置。自调节连续流设计 将我们以前的CNT集成设备的样品容量和吞吐量提高了10倍， 因此，该装置可以避免堵塞，同时使装置不受局部CNT森林缺陷的影响并保持高EV捕获效率。 通过在碳纳米管上添加纳米厚度的功能氧化锆，CNT-iEVIGEM可以有效地纯化 EV DNA原位。对于胰腺癌的诊断，我们将开发一种多重数字液滴PCR（ddPCR）检测方法 通过CNT-iIMIGEM检测从血浆中分离的EV的KRAS突变。KRAS突变是 胰腺癌的突变率超过90%。它是一个主要的驱动致癌基因， 胰腺肿瘤发生的最早期事件。使用在健康对照中掺入的癌症衍生EV的模型样品 我们将检测限确定为可检测到的最低突变等位基因分数 毫不含糊该检测方法将使用来自IV期胰腺癌患者的75份血浆样本进行验证 和75名年龄/性别匹配的健康献血者。我们将用此方法对胰腺癌的诊断进行研究 通过招募60名早期（I + II期）和60名III期胰腺癌患者，对不同阶段的癌症进行了研究。 将通过组织中的突变状态来测量不同阶段检测的灵敏度和特异性 样本作为真实值。拟议的研究将为CNT的进一步临床验证铺平道路- iEVIGEM技术用于癌症诊断。EV分离平台也可用于其他DNA突变， 其他EV货物和其他癌症类型作为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.