A Microfluidic Platform for Detecting Circulating Endothelial Cells at the Point-of-Care

项目目标：开发一个检测循环内皮细胞的护理点平台，用于诊断或监测血管疾病或损伤。非技术摘要：循环内皮细胞(CECs)是血管损伤和/或疾病的指示物。临床研究表明，患有血管疾病，如缺血、血管损伤、急性心肌梗死、镰状细胞性贫血、血管炎、肺动脉高压和深静脉血栓形成的人，其CECs水平高于健康对照组(CECs不少或没有)。重要的是，CEC的数量与损伤或疾病的严重程度密切相关。因此，CEC具有诊断和预后的重要性。目前检测CEC的方法总体上是不够的，因为它们缺乏敏感性，需要训练有素的内科科学家来解释结果，并且不能在医生的办公室进行。该项目将开发一个微流体平台，该平台可以在护理点检测患者血液中的CECs，使医生能够诊断和监测患者的血管疾病或损伤，并在急性病例中快速做出反应。除了明显的医学/临床影响外，该项目还将产生强大的社会影响，因为将举办研讨会，向学生展示工程和科学领域的不同学科如何能够结合起来解决实际和重要的问题，就像这个医学项目所做的那样，以及学生如何能够同时进行工程和科学思考。技术摘要：该项目的总体目标是开发一种新型的无标记微流控平台，用于筛选循环内皮细胞(CECs)。内皮祖细胞是血管损伤和/或疾病的指示物，可以从血管壁脱落(成熟的内皮细胞)，也可以从骨髓中招募(内皮祖细胞或内皮祖细胞)。CECs占外周血中单个核细胞总数的0.01%至0.0001%，检测具有挑战性。即将开发的平台将利用收缩-扩张阵列(CEA)中的惯性流体动力学，根据大小从全血中分离候选CEC。然后，该平台将使用节点孔传感(NPS)来筛选分离的细胞，不仅可以识别CEC和白细胞，还可以根据特定的表型特征区分成熟的CEC和EPC。当细胞与微流体通道中的抗体相互作用(特异性或非特异性)时，NPS测量细胞的传输时间，微流体通道已被节点分割。细胞表面受体和功能化抗体之间的特定相互作用会延缓细胞，导致更长的转运时间，并随后确定特定表面标志物的存在。总体而言，拥有在护理点识别和区分患者血液中成熟的CEC和EPCS的能力将使医生能够诊断和监测患者的血管疾病或损伤，并在急性心肌梗死等情况下快速做出反应。这项为期三年的项目有三个具体目标：-目标1：优化收缩-扩张阵列(CEA)设备，使其从全血中丰富CEC。CEA设备将依靠惯性力来根据大小对血液和CEC进行分级。该设备将进行优化，以100%恢复癌细胞。-目标2：扩大NPS的能力，以筛选成熟的CECs和EPCs。将设计和开发具有优化的编码和处理(最佳巴克码、匹配滤波、稀疏反卷积、线性分类器，所有这些都受到雷达和电信理论的启发)的独特NPS平台，以实现对CEC的高分辨率检测和分类。-目标3：将优化的NPS与CEA设备集成，并在下游包括一个分类器。带有NPS的CEA设备将集成到单一平台上，能够从外周血中分离、分析和分选CEC。

Proposal Title: A Microfluidic Platform for Detecting Circulating Endothelial Cells at the Point-of-CareProject Goals: This project involves developing a point-of-care platform to detect circulating endothelial cells for diagnosing or monitoring vascular disease or injury.Nontechnical Abstract: Circulating endothelial cells (CECs) are indicators of vascular injury and/or disease. Clinical studies have shown that individuals with vascular disorders, such as ischemia, vascular trauma, acute myocardial infarction, sickle-cell anemia, vasculitis, pulmonary hypertension, and deep-vein thrombosis, have higher levels of CECs than healthy controls (who had no little to no CECs). Importantly, the number of CECs strongly correlated with the severity of injury or disease. Thus, CECs have diagnostic and prognostic importance. Current methods to detect CECs are overall inadequate, as they lack sensitivity, require a highly trained physician-scientist to interpret results, and cannot be performed in a physician's office. This project will develop a microfluidic platform that can detect CECs in patient blood at the point-of-care, enabling a physician to diagnose and monitor a patient's vascular disease or injury and also to respond quickly in acute cases. Beyond the obvious high medical/clinical impact, this project will have strong societal impact, as workshops will be held to demonstrate to students how different disciplines in engineering and science can come together to address real and important problems, just as had done for this medical project, and how students are capable of both engineering and scientific thinking.Technical Abstract: The overarching goal of this project is to develop a novel label-free microfluidics platform that screens for circulating endothelial cells (CECs). CECs are indicators of vascular injury and/or disease and are either shed from the vascular wall (mature CECs) or recruited from the bone marrow (endothelial progenitor cells or EPCs). Representing 0.01% to 0.0001% of the total mononuclear cells in peripheral blood, CECs are challenging to detect. The platform to be developed will utilize inertial fluid dynamics within a contraction-expansion array (CEA) to isolate candidate CECs from whole blood based on size. The platform will then screen the isolated cells using Node-Pore Sensing (NPS) to not only identify CECs from white blood cells but also to differentiate mature CECs from EPCs based on specific phenotypic profiles. NPS measures the transit time of a cell as it interacts (specifically or non-specifically) with antibodies functionalized in a microfluidic channel that has been segmented by nodes. Specific interactions between cell-surface receptors and the functionalized antibody retard the cell, leading to longer transit times and subsequent determination of a particular surface-marker presence. Overall, having the ability to identify and distinguish between mature CECs and EPCS in patient blood at the point-of-care would enable a physician to diagnose and monitor a patient's vascular disease or injury and also respond quickly in cases such as an acute myocardial infarction.This three-year project has three specific aims:-Aim 1: To optimize a contraction-expansion array (CEA) device such that it enriches CECs from whole blood. The CEA device will rely upon inertial forces to fractionate blood and CECs based on size. The device will be optimized for 100% recovery of cancer cells.-Aim 2: To expand the capabilities of NPS to screen for mature CECs and EPCS. A unique NPS platform with optimized coding and processing (optimum Barker codes, matched filtering, sparse deconvolution, linear classifiers, all of which are inspired by radar and telecommunications theory) will be designed and developed to enable high-resolution detection and classification of CECs.-Aim 3: To integrate the optimized NPS with the CEA device and also include a sorter downstream. The CEA device with NPS will be integrated onto a single platform, enabling isolation, analysis, and sorting of CECs from peripheral blood.