Node-Pore Sensing for Cellular Screening

描述：该项目的总体目标是开发节点孔传感(NPS)，这是一种创新的无标记微流控技术，使其在标记数量方面可以超越流式细胞术，这些标记可以实际和常规地同时以非破坏性的方式进行筛选。目前，由于同时使用的不同荧光染料的光谱发射重叠，流式细胞术实际上限制在6-10个标记物。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。这个高风险、高回报的R21项目有两个具体目标： ·目标1：优化设备编码和处理，以实现高通量筛选和实时分析。我们将设计和开发一个独特的基于Barker码的NPS平台，即使在低信噪比(SNR)的情况下也能实现高分辨率检测。巴克码是通常用于雷达和电信的二进制信号，与NPS类似。 ·目标2：将分类技术纳入目标1开发的NPS平台。我们打算充分发挥NPS的潜力，并将分类技术整合到NPS平台中。分选技术将基于机械压力驱动，将细胞快速分选到表型亚群，以便进行下游分析和/或培养。NPS的开发和原则证明将基于对乳腺癌细胞系MCF10A、MCF-7、MDA-MB-436和MDA-MB-231进行筛选和分类，这些细胞系都具有不同的恶性肿瘤和转移状态-标记EpCAM、CD44、CD24、CD29、CD49f、CD133、Ax1、MUC1、EGFR和ErbB2。通过专注于这些标志物，我们将在涉及转移性乳腺癌患者循环肿瘤细胞亚群特征的研究中产生立竿见影的影响。因此，我们的原则证明对于一个充分发展的核动力源具有很大的影响。拟议的综合多标记物核动力源和分类技术在从基础生命科学研究到护理点诊断等多个领域具有潜在的变革性影响。例如，流式细胞术是诊断许多血液系统恶性肿瘤的基石。使用我们的筛查/分选技术，我们可以检测到最小的残留病和缓解状态。除了临床诊断，我们的技术可以用来表征，例如，干细胞分化过程中表面标志物表达的变化，以便识别和分离潜在的重要和罕见的亚群。

DESCRIPTION: The overarching goal of this project is to develop Node-Pore Sensing (NPS), an innovative label-free microfluidic technique, such that it could go beyond flow cytometry in terms of number of markers that can be practically and routinely screened simultaneously and in a non-destructive manner. Currently, flow cytometry is practically limited to 6-10 markers due to spectral emission overlap of the different fluorochromes used simultaneously. 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. This high-risk, high-reward R21 project has two Specific Aims: • Aim 1: To optimize device coding and processing for high throughput screening and real-time analysis. We will design and develop a unique NPS platform based on Barker codes that enable high- resolution detection even with low signal-to-noise ratios (SNRs). Barker codes are binary signals that are often used in radar and telecommunications to which NPS is analogous. • Aim 2: To incorporate sorting technology onto the NPS platform developed in Aim 1. We intend to realize the full potential of NPS and integrate a sorting technology to the NPS platform. The sorting technology will be based on mechanical pressure actuation to sort cells rapidly into phenotypic sub- populations for downstream analysis and/or culture. NPS development and proof-of-principle will be based on screening and sorting breast-cancer cell lines, MCF10A, MCF-7, MDA-MB-436, and MDA-MB-231-all of which have different malignancy and metastatic status-for markers EpCAM, CD44, CD24, CD29, CD49f, CD133, Axl, MUC1, EGFR, and ErbB2. By focusing on these markers, we would have an immediate impact in studies involving characterizing sub-populations of circulating tumor cells from patients with metastatic breast cancer. Thus, our proof-of-principle for a fully developed NPS has high impact. The proposed integrated multi-marker NPS and sorting technology proposed has potential for transformative impact in a number of fields ranging from fundamental life sciences research to point-of-care diagnostics. For example, flow cytometry is the cornerstone to diagnosis for many of the hematologic malignancies. With our screening/sorting technique, we could detect minimal residual disease and remission states. Beyond clinical diagnosis, our technology could be employed to characterize, for instance, changes in surface-marker expression during stem-cell differentiation in order to identify and isolate potentially important and rare sub-populations.