Localized Surface Plasmon Resonance (LSPR) Biosensing Microarray for Multiplex, Real-time Single-Cell Immunophenotyping

1263889 - kurabayashi免疫系统在保护生物体免受病毒、细菌和寄生虫的入侵以及区分患病组织和健康组织方面起着至关重要的作用。白细胞是支持免疫系统的重要血液成分，有几个细胞亚群，由它们的形态和对抗原暴露的反应决定。免疫细胞功能的全面表征对于诊断、分层和监测感染、恶性肿瘤、自身免疫性疾病和损伤或研究新药的疗效至关重要。特别是，需要对单个细胞的细胞因子分泌谱进行定量分析，以精确确定细胞功能和免疫状态在其亚群中的细微变化。然而，为了实现对白细胞因子分泌谱的快速、高效、动态分析，现有方法存在一些不足，需要克服这些不足。本研究旨在建立一个生物传感平台，使从全血中分离的目标免疫细胞分泌的细胞因子在单细胞水平上实现高通量、多路复用、动态测量。这种单细胞功能分析有效地允许芯片上分离和刺激单细胞和检测细胞分泌的蛋白质。我们的方法将在微室阵列中使用局部表面等离子体共振（LSPR）成像，每个微室阵列捕获单个细胞。由于整个系统的小型化，样品体积和总分析时间都可以减少。微流控平台的微阵列形式与LSPR生物传感相结合，将允许通过简单的光学设置对单个细胞分泌的蛋白质进行大规模并行无标签、实时定量和制图。对大量单细胞进行的分析将允许对白细胞亚群进行免疫表型筛选，同时检测其免疫功能的细微差异和时间变化。如果分离过程只产生相同细胞子集的提取，则每个样本的大量测量将导致高统计准确性（即减少假阳性）。这个项目将开发新的方法来分析血液中存在的免疫细胞。具体来说，该提案将开发捕获单个免疫细胞的设备，然后监测捕获细胞中某些蛋白质的释放。免疫细胞产生的蛋白质的水平和类型反映了一个人的健康状况或疾病状况。因此，拟议的设备将具有重要的诊断意义。

1263889 - KurabayashiThe immune system plays a critical role in protecting a living organism against invasions of viruses,bacteria, and parasitic worms and in distinguishing diseased tissue from healthy tissue. Leukocytes -white blood cells - are important blood constituents that support the immune system and haveseveral cellular subsets determined by their morphology and response to an antigen exposure. Comprehensive characterization of the immune cell functions is critical to diagnose, stratify, and monitor infections, malignancies, autoimmune disorders, and injuries or to study the efficacy of new drugs. In particular, quantitative analysis of the cytokine secretion profiles of individual cells is required for precise determination of the subtle variations in the cellular function and immune status across their sub-groups. However, there are some shortcomings in existing methods that need to be overcome for realizing rapid, efficient, dynamic analysis of leukocyte cytokine-secretion profiles. This research aims to establish a biosensing platform that enables high-throughput, multiplexed, dynamic measurement of cytokines secreted by target immune cells isolated from whole blood at thesingle-cell level. This single-cell functional assay efficiently permits on-chip isolation and stimulation of single cells and detection of cell-secreted proteins. Our approach will employ localized surface plasmon resonance (LSPR) imaging within microchamber arrays, each capturing a single cell. As a result of miniaturization of the whole system, both the sample volume and the total assay time canbe reduced. The microarray form of the microfluidic platform coupled with LSPR biosensing will allow for massively parallel label-free, real-time quantification and mapping of proteins secreted individual cells with a simple optics setup. The analysis performed for a large number of single cells will allow for immunophenotypical screening of leukocyte subsets while detecting subtle differences and time variations of their immune functions. If the isolation process only yields extraction of identical cell subsets, the large number of measurements per sample will lead to high statistical accuracy (i.e., reduced false positives). NontechnicalThis project will develop novel means to analyze immune cells present in blood. Specifically, the proposal will develop devices to capture individual immune cells and then monitor release of certain proteins from the captured cells. The levels and the types of proteins produced by the immune cells reflect on the state of health or disease of an individual. Therefore proposed devices will have significant diagnostic implications.