Two-Dimensional Multi-Stage Isotachophoretic Technology for Multiplex Analysis of Cancer Exosomes and Proteins Marker Panel

摘要 Exosome是小尺寸(30-120 nm)的细胞外小泡。它们由大多数类型的细胞分泌，并发挥作用 在正常和病理过程中的细胞外通讯中发挥重要作用。衍生的外切体 来自癌症的信号分子(例如蛋白质和miRNAs)从亲代癌细胞和组织穿梭到 远端受体细胞对受体细胞进行重新编程，促进肿瘤生长和转移。因此， 携带肿瘤标志性蛋白标记物的循环肿瘤来源的外切体具有巨大的潜力 用于早期癌症非侵入性诊断的无价液体活组织检查工具。尽管他们有潜在的临床价值 意义，将疾病衍生的外切体转化为用于早期诊断的护理点(POC)应用 一个关键的技术障碍阻碍了癌症的发展：缺乏具有成本效益的能够 临床标本中特异性外切体及其含量标记物的同时分析。尽管有一些人 已经开发了用于外切体分离和鉴定的方法，或者它们是单一官能化的， 不特定、费力或耗时，或者它们缺乏作为经济高效的概念验证采用的稳健性 技术。因此，迫切需要一种有效、准确、易于使用、低成本的方法来 多重POC样本分析以检测特定释放的特定外体群体的痕量水平 在癌症细胞的早期阶段，并全面描述外切体携带的癌症标记物。这 应用程序旨在通过采取多学科方法来填补这一空白，开发一种新颖的、一次性的两种- 基于纸的多阶段等速渗透(ITP)技术平台 以经济高效的方式分析特定的目标外切体和外切体蛋白。我们的目标是发展 一种集成的纸基等渗平台，其上：1)阴离子级联ITP用于耗尽高密度的 在捕获和分析之前丰富血浆蛋白和丰富目标外切体；2)第二次ITP 过程同时分析通过裂解捕获的外体释放的多重外体蛋白 第一ITP过程；以及3)基于智能手机的小型化检测模块对目标外切体进行量化，并 由新的分级结合测试品系捕获的蛋白质标记。我们期待着有效的整合 在经济有效的模块中通过对其内容的多重分析来分离/鉴定特定的外切体 Platform将提供强大的POC方法来推进疾病的基础和临床转化研究- 衍生的外切体。来自乳腺癌的外切体将被用作模型靶点，以验证我们的 技术该项目的成功不仅将提供临床上兼容的POC工具，用于跟踪特定的 外显体和标记用于癌症的早期筛查，但也促进了对轮廓分析的研究 用于其他疾病精确诊断的外体标志物。因此，这项技术可以有广泛的应用前景 在管理恶意疾病患者和提高他们的生活质量方面的翻译潜力。

Abstract Exosomes are small-sized (30–120 nm) extracellular vesicles. They are secreted by most cell types and play important roles in extracellular communication in normal and pathological processes. The exosomes derived from cancers shuttle signaling molecules (e.g. proteins and miRNAs) from parental cancer cells and tissue to distal recipient cells to reprogram the recipient cells and promote tumor growth and metastasis. Therefore, circulating tumor-derived exosomes carrying signature protein markers of the tumor hold great potential as invaluable liquid biopsy tools for the noninvasive diagnosis of early-stage cancers. Despite their potential clinical significance, translating disease-derived exosomes into point-of-care (POC) applications for the early diagnosis of cancers is hampered by a critical technical barrier: lack of a cost-effective POC approach capable of the simultaneous analysis of specific exosomes and their content markers in clinical samples. Although a number of methods for exosome isolation and characterization have been developed, either they are singly functionalized, nonspecific, laborious, or time-consuming, or they lack the robustness to be adopted as a cost-effective POC technique. Therefore, there is an urgent need for an effective, precise, easy to use, low-cost approach to multiplex POC sample analysis to detect trace levels of specific populations of exosomes released by specific cancer cells at an early stage and comprehensively profile the cancer markers carried by the exosomes. This application aims to fill the gap by taking a multidisciplinary approach to developing a novel, disposable two- dimensional paper-based multistage isotachophoresis (ITP) technology platform capable of the simultaneous analysis of specific target exosomes and exosomal proteins in a cost-effective way. Our objective is to develop an integrated paper-based isotachophoretic platform on which: 1) anionic cascade ITP is used to deplete high- abundance plasma proteins and enrich target exosomes before their capture and analysis; 2) a second ITP process simultaneously analyzes multiplex exosomal proteins released by lysing the exosomes captured in the first ITP process; and 3) a miniaturized smartphone-based detection module quantifies the target exosomes and protein markers captured by novel graded-binding test lines. We expect that integrating effective isolation/identification of specific exosomes with multiplex analysis of their contents in a cost-effective modular platform will provide a robust POC approach to advancing basic and clinical translational research on disease- derived exosomes. Exosomes derived from breast cancer will be used as model targets to validate our technology. The success of this project will not only provide a clinically compatible POC tool for tracking specific exosomes and markers for early screening for cancers, but also prompt research on the profile analysis of exosomal markers for the precision diagnosis of other diseases. Therefore, the technique may have broad translational potential in managing patients with malicious diseases and improving their quality of life.