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

与传统的肿瘤活检相比，液体活检具有显著的优势，因为它是微创的，并且使用血液和尿液等生物液体在癌症和其他疾病的早期阶段进行诊断。癌细胞主动分泌的外体携带其原始细胞的分子成分。由于这些膜外囊泡可以作为细胞替代物，外切体作为一种新型的生物标志物应运而生。然而，传统的外切体分析方法，如免疫印迹或酶联免疫吸附分析，成本较高，需要大约12小时和过多的血清来检测外切体表面的跨膜蛋白。外切体分离需要复杂的步骤来去除碎片或细胞成分，这将扰乱下游分析。使用微型无标记生物传感器的高通量外切体分子图谱是不可用的。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切体。这项研究将通过将两种最先进的技术整合到一块硅芯片上，导致外显体分析的革命性变化。此外，这项研究将与教育相结合，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，招收女学生，为非裔美国学生提供暑期实习机会，让他们参加爱荷华州立大学的项目，并在亚利桑那州立大学开发一门与纳米生物技术相关的新本科水平课程。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切体的分子和大小特征。将开发级联的纳米级确定的横向位移柱阵列，以简化外体的分离和大小轮廓。外体将有效地从流体样本中存在的干扰分子中分离出来。将开发高性能的横向流通式光学生物传感器来定量分离的外切体。外切体样品可以流经纳米生物传感器，并被固定和浓缩在功能化的传感器表面。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。这两项功能的整合将带来前所未有的能力，不断简化外切体分离、浓缩和检测过程，在一小时内为多个蛋白质标记分析多维分子和大小信息。该项目的生物验证计划将使用拟议的设备来分类和检测从一种寄生线虫和人类疾病淋巴丝虫病的病原体释放的外体。该技术在成本、样本消耗和吞吐量方面都优于基于实验室的方法，并可扩展到人或动物生物液中循环细胞外外体的图谱，以诊断各种疾病，识别对药物发现重要的伴随生物标记物，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.