Collaborative Research: Silicon Nano-Opto-Fluidics Enabled Multi-Dimensional, High-Throughput Molecular and Size Profiling of Exosomes

液体活检比传统的肿瘤活检有显著的优势，因为它是微创的，并且使用生物液体，如血液和尿液，在早期阶段诊断癌症和其他疾病。外泌体是由癌细胞积极分泌的，它携带着癌细胞起源细胞的分子成分。由于这些膜状细胞外囊泡可以作为细胞替代物，外泌体已成为一种新型的有效生物标志物。然而，传统的外泌体分析方法，如免疫印迹法或酶联免疫吸附法，是昂贵的，并且需要大约12小时和大量的血清来检测外泌体表面的跨膜蛋白。外泌体分离需要复杂的步骤来去除会混淆下游分析的碎片或细胞成分。使用微型无标签生物传感器的外泌体的高通量分子分析是不可用的。该项目的目标是开发一种基于分子和大小特征快速筛选和分析外泌体的新能力。该研究通过将两种最先进的技术集成到单个硅芯片上，将导致外泌体分析的革命性变化。此外，这项研究将通过在现有的本科生物医学工程辅修课程中增加新的实验模块，招募女学生，并为爱荷华州立大学的非裔美国学生提供暑期实习机会，以及在亚利桑那州立大学开发一门新的与纳米生物技术相关的本科课程，与教育相结合。该项目将导致一个集成的硅基纳米光流体平台，用于快速连续地分析外泌体的分子和大小特征。级联纳米级确定性横向位移柱阵列将被开发，以简化外泌体的分离和大小分析。外泌体将与存在于液体样品中的干扰分子有效分离。将开发高性能的横向流动光学生物传感器来定量分离的外泌体。外泌体样品可以通过纳米级生物传感器流动，并在功能化传感器表面进行固定化和富集。由于分离和检测模块都具有横向流动设计的特点，因此可以使用纳米压印光刻工艺将它们集成在单个硅芯片上。这两种功能的整合将导致前所未有的能力，不断简化外泌体分离，富集和检测过程，以在一小时内分析多个蛋白质标记物的多维分子和大小信息。该项目的生物学验证计划将使用拟议的设备进行分类和检测从寄生虫和人类疾病淋巴丝虫病的病原释放的外泌体。所提出的技术在成本、样品消耗和吞吐量方面优于基于实验室的方法，并且可以扩展到对人类或动物生物体液中循环的细胞外泌体进行分析，以诊断各种疾病，识别对药物发现很重要的伴随生物标志物，并监测治疗进展。

Liquid biopsy has significant advantages over traditional tumor biopsies, because it is minimally invasive and uses biofluids, such as blood and urine, to diagnose cancer and other diseases in their early stages. Exosomes, which are actively secreted from cancer cells, carry molecular constituents of their originating cells. Because these membranous extracellular vesicles can serve as cellular surrogates, exosomes have emerged as a new type of potent biomarkers. However, conventional exosome analysis methods such as immunoblotting or enzyme-linked immunosorbent assays are costly and require approximately twelve hours and excessive volumes of serum to detect transmembrane proteins on the surface of exosomes. Exosome separation requires complex steps to remove debris or cellular components that will confound downstream analysis. High-throughput molecular profiling of exosomes using miniature label-free biosensors is not available. The goal of this project is to develop a new capability to rapidly screen and profile exosomes based on both molecular and size characteristics. This research will lead to a transformative change in exosome analysis by integrating two state-of-the-art technologies on a single silicon chip. In addition, this research will be integrated with education through adding new lab modules to existing undergraduate biomedical engineering minor program curriculum, recruiting female students, and providing summer internship opportunities to African-American students to participate in the project at Iowa State University, and developing a new undergraduate-level course related to nanobiotechnology at Arizona State University. The project will lead to an integrated silicon-based nano-opto-fluidic platform for rapidly and continuously profiling of both molecular and size features of exosomes. Cascaded nanoscale deterministic lateral displacement pillar arrays will be developed to simplify the isolation and size profiling of exosomes. The exosomes will be effectively separated from interference molecules present in the fluid sample. High-performance lateral flow-through optical biosensors will be developed to quantify the separated exosomes. The exosome samples can flow through the nanoscale biosensor and be immobilized and enriched on the functionalized sensor surface. Because both the separation and detection modules have the features of lateral flow designs, they can be integrated on a single silicon chip using the nanoimprint lithography process. The integration of these two functions will lead to an unprecedented ability to continuously streamline exosome separation, enrichment and detection processes to profile multi-dimensional molecular and size information for multiple protein markers within one hour. The biological validation plan of the project will be carried out using the proposed device to sort and sense exosomes released from a parasitic nematode and etiological agent of the human disease, Lymphatic Filariasis. The proposed technology is advantageous over the lab-based methods in terms of cost, sample consumption, and throughput, and could be extended to the profiling of circulating exocellular exosomes in human or animal biofluids to diagnose a variety of diseases, identify companion biomarkers that are important for drug discovery, and monitor the progress of a therapy.