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具有显着的优势，因为它是微创的，并使用生物流体，如血液和尿液，在早期诊断癌症和其他疾病。从癌细胞主动分泌的外泌体携带其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，因此外泌体已成为一种新型的有效生物标志物。然而，常规的外泌体分析方法如免疫印迹或酶联免疫吸附测定是昂贵的，并且需要大约12小时和过量的血清来检测外泌体表面上的跨膜蛋白。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组分。使用微型无标记生物传感器的外泌体的高通量分子谱尚不可用。该项目的目标是开发一种新的能力，根据分子和大小特征快速筛选和分析外来体。这项研究将通过在单个硅芯片上集成两种最先进的技术，导致外泌体分析的变革。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添加新的实验室模块，招募女学生，并为非洲裔美国学生提供暑期实习机会来参与爱荷华州州立大学的项目，并在亚利桑那州州立大学开发与纳米生物技术相关的新的本科生课程，从而与教育相结合。 该项目将导致一个集成的硅基纳米光流体平台，用于快速和连续地分析外泌体的分子和大小特征。将开发级联纳米级确定性横向位移柱阵列以简化外泌体的分离和尺寸分析。外来体将与流体样品中存在的干扰分子有效分离。将开发高性能侧向流通光学生物传感器来量化分离的外泌体。外泌体样品可以流过纳米级生物传感器，并被固定和富集在功能化的传感器表面上。由于分离和检测模块都具有横向流动设计的特征，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带来前所未有的能力，可以连续简化外泌体分离，富集和检测过程，以在一小时内分析多种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定的设备进行，以分选和感应从寄生线虫和人类疾病淋巴丝虫病的病原体释放的外来体。所提出的技术在成本，样品消耗和通量方面优于基于实验室的方法，并且可以扩展到人类或动物生物流体中循环细胞外泌体的分析，以诊断各种疾病，鉴定对药物发现重要的伴随生物标志物，并监测治疗的进展。

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.