Nanoplasmonic Spatiotemporal Imaging of Single-Cell Protein Secretion and Intercellular Communication

单细胞蛋白质分泌和细胞间通讯的纳米等离子体时空成像

• 批准号: 10723157
• 负责人: Katsuo Kurabayashi
• 金额: $ 25.17万
• 依托单位: NEW YORK UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-30 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10723157
• 关键词: Algorithms; Area; Autocrine Communication; Behavior; Behavior monitoring; Binding Sites; Biological; Biological Assay; Biomedical Research; Biosensor; Cell Communication; Cell Culture Techniques; Cell physiology; Cell secretion; Cell surface; Cells; Cellular biology; Communicable Diseases; Communication; Coupled; Coupling; Darkness; Detection; Development; Disease; Enzyme-Linked Immunosorbent Assay; Expression Profiling; Fluorescence; Future; Gene Expression Profiling; Hep3B; Human; Image; Imaging Device; Imaging Techniques; Imaging technology; Immune; Immune response; Immune system; Immunity; Immunology; Immunophenotyping; Individual; Inflammation; Inflammatory; Interleukin-6; Knowledge; Label; Malignant Neoplasms; Marketing; Measures; Mediating; Microfluidics; Microscopy; Molecular; Nanostructures; Pattern; Performance; Population; Primary carcinoma of the liver cells; Process; Protein Secretion; Proteins; Public Health; Reagent; Research; Research Personnel; Research Project Grants; Resolution; Sampling; Scientist; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Spatial Distribution; Structure; System; T cell differentiation; T-Cell Activation; T-Lymphocyte; Techniques; Technology; Theoretical model; Time; Visualization; Water; aptamer; cell behavior; cell type; cellular imaging; commercialization; communicable disease diagnosis; cost; cytokine; density; extracellular; fabrication; human leukocyte antigen testing; insight; intercellular communication; microscopic imaging; nano; nanobiosensor; nanoplasmonic; novel; paracrine; plasmonics; protein expression; real-time images; receptor; secretion process; sensor; single cell proteins; spatiotemporal; tool

ABSTRACT The ability to probe the temporal profile of the protein secretion behavior of individual immune cells will impact future immunology, cell biology, and even infectious disease diagnosis. Knowledge of the ordering and timing of cytokines (water-soluble proteins essential for intercellular signaling) secreted by activated T cells can additionally provide the means to discriminate subsets of differentiated T cells by function. Here, the temporal information is one of the pieces of the whole puzzle in monitoring the behavior of the immune system. The other critical piece is the cytokine-mediated interplay between different cell types, which involves spatial transport of cytokines between cells. Putting both pieces of the puzzle together allows us to capture the full picture of the cytokine release dynamics and cytokine-mediated interactions of cells, which allows us to fully understand the intercellular signaling processes underlying immunity. However, no study has yet obtained such a picture due to the lack of a technology for real-time sensing of intercellular cytokine-mediated signaling processes at high spatial resolution. This research aims to develop a novel label-free imaging technique to fully understand cellular behaviors during cytokine-mediated activation and communication at a single-cell level. Our approach will employ biosensors consisting of plasmonic nanoantenna structures, each specifically targeting a particular cytokine species. We will integrate these biosensors in a microfluidic system incorporating an array of sample/reagent-flow channels and single-cell trapping microwells. The microfluidic sensor integration will provide the ability to capture, manipulate, and activate single cells for cell-to-cell communications on a single chip and to obtain the spatiotemporal profile of cellular cytokine secretion processes in real time, both in a massively, parallel manner. We will also develop a theoretical algorithm that allows us to extract the quantitative values of the local cytokine concentration distributions from measured image intensities. SA 1: We will create highly ordered, high-density plasmonic nanoantenna biosensor arrays, each functionalized by highly selective aptamers against targeted cytokines. SA 2: We will integrate the aptamer-conjugated plasmonic nanoantenna arrays into a single-cell manipulation microfluidic system and achieve real-time single-cell secretion imaging at high throughput. SA 3: We will develop a two-mode (fluorescence and dark-field) microscopy imaging technique to image spatiotemporal cytokine secretomic profile patterns and cell surface sytokine binding sites. Using this technique, we will study the IL-6-mediated dynamic intercellular communication between individual human hepatoma Hep3b cells and CD 4+ T cells.

摘要 探测单个免疫细胞蛋白质分泌行为的时间分布的能力将影响 未来的免疫学、细胞生物学，甚至传染病诊断。了解以下项目的排序和时间安排 由激活的T细胞分泌的细胞因子(细胞间信号转导所必需的水溶蛋白)可以 此外，还提供了根据功能区分分化的T细胞亚群的方法。在这里，世俗 信息是监测免疫系统行为的整个谜题之一。另一个 关键环节是细胞因子介导的不同细胞类型之间的相互作用，它涉及到细胞的空间运输。 细胞间的细胞因子。把这两块拼图放在一起，我们就能捕捉到 细胞因子的释放动力学和细胞因子介导的相互作用，使我们能够充分了解 细胞间信号传递处理潜在的免疫。然而，目前还没有研究得出这样的图景，因为 缺乏一种实时检测细胞间细胞因子介导的信号传递过程的技术 空间分辨率。这项研究旨在开发一种新的无标记成像技术，以充分了解细胞 单细胞水平上细胞因子介导的激活和交流过程中的行为。我们的方法将 使用由等离子体纳米天线结构组成的生物传感器，每个结构专门针对特定的 细胞因子种类。我们将把这些生物传感器集成到一个微流控系统中，该系统集成了一系列 样品/试剂流动通道和单电池捕集微孔。微流控传感器集成将提供 在单个芯片上捕获、操作和激活单个细胞以进行细胞间通信的能力 为了实时获得细胞细胞因子分泌过程的时空分布，既大规模地， 并行方式。我们还将开发一种理论算法，使我们能够提取 根据测量的图像强度，局部细胞因子浓度分布。SA 1：我们将创造高度的 有序的高密度等离子体纳米天线生物传感器阵列，每个都通过高度选择性实现功能化 针对靶向细胞因子的适配子。SA 2：我们将集成适体共轭等离子体纳米天线 阵列集成到单细胞操纵微流控系统中，实现实时单细胞分泌成像 高吞吐量。SA 3：我们将开发一种双模(荧光和暗场)显微成像技术 成像时空细胞因子分泌模式和细胞表面系统因子结合位点。使用这个 ，我们将研究IL-6介导的人与人之间的动态细胞间通讯 肝癌细胞系Hep3b细胞和CD+T细胞。