Node-Pore Sensing for Cellular Screening

产品说明：该项目的总体目标是开发节点孔传感（Node-Pore Sensing，NP-Pore Sensing），这是一种创新的无标记微流控技术，因此它可以在标记物数量方面超越流式细胞术，这些标记物可以同时以非破坏性方式进行实际和常规筛选。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于6-10个标记物。当细胞与在已经由节点分段的微流体通道中功能化的抗体相互作用（特异性或非特异性）时，微流控测量细胞的通过时间。细胞表面受体和功能化抗体之间的特异性相互作用阻碍细胞，导致更长的通过时间和随后确定特定表面标记物的存在。 这个高风险、高回报的R21项目有两个具体目标： ·目标1：优化用于高通量筛选和实时分析的设备编码和处理。我们将设计和开发一个独特的基于巴克码的RFID平台，即使在低信噪比（SNR）下也能实现高分辨率检测。巴克码是一种二进制信号，常用于雷达和电信领域，与之类似。 目标2：将分类技术纳入目标1中开发的可持续发展平台。我们打算充分发挥物联网的潜力，并将分拣技术集成到物联网平台。分选技术将基于机械压力驱动，将细胞快速分选为表型亚群，用于下游分析和/或培养。肿瘤学的发展和原理验证将基于对乳腺癌细胞系MCF 10A、MCF-7、MDA-MB-436和MDA-MB-231的筛选和分类，这些细胞系都具有不同的恶性程度和转移状态，用于标记EpCAM、CD 44、CD 24、CD 29、CD 49 f、CD 133、Axl、MUC 1、EGFR和ErbB 2。通过关注这些标志物，我们将对涉及表征转移性乳腺癌患者循环肿瘤细胞亚群的研究产生直接影响。因此，我们对一个完全开发的机器人的原理验证具有很高的影响力。 所提出的集成多标记物测序和分选技术在从基础生命科学研究到即时诊断的许多领域具有变革性影响的潜力。例如，流式细胞术是诊断许多血液恶性肿瘤的基石。通过我们的筛选/分选技术，我们可以检测微小残留病和缓解状态。除了临床诊断，我们的技术还可以用于表征，例如，干细胞分化过程中表面标志物表达的变化，以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.