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.

抽象的 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，甚至是传染病诊断。了解有序和时机 活化的T细胞分泌的细胞因子（对细胞间信号传导必不可少的水溶性蛋白）可以 另外，提供了通过功能区分分化T细胞的子集的手段。在这里，时间 信息是监测免疫系统行为的整个难题之一。另一个 关键作品是不同细胞类型之间的细胞因子介导的相互作用，其中涉及空间运输 细胞之间的细胞因子。将两个拼图都放在一起，使我们能够捕获 细胞因子释放动力学和细胞因子介导的细胞相互作用，这使我们能够完全理解 免疫力基础的细胞间信号传导过程。但是，由于 缺乏用于实时感知细胞因子介导的信号过程的技术 空间分辨率。这项研究旨在开发一种新颖的无标签成像技术，以充分了解细胞 单细胞水平的细胞因子介导的激活和通信过程中的行为。我们的做法意愿 采用由等离子体纳米annna结构组成的生物传感器，每个结构都针对特定的生物传感器 细胞因子物种。我们将将这些生物传感器集成到包含一系列阵列的微流体系统中 样品/试剂流通通道和单细胞捕获的微孔。微流体传感器集成将提供 在单个芯片上捕获，操纵和激活单个单元格的能力，并 为了实时获得细胞细胞因子分泌过程的时空特征 平行方式。我们还将开发一种理论算法，使我们能够提取 来自测得的图像强度的局部细胞因子浓度分布。 SA 1：我们将高度创造 有序的高密度等离子体纳米antenna生物传感器阵列，每个阵列由高度选择性功能化 针对靶向细胞因子的适体。 SA 2：我们将整合适体偶联的等离子纳米反胶 阵列进入单细胞操纵微流体系统，并在实时单细胞分泌成像 高通量。 SA 3：我们将开发两种模式（荧光和深色场）显微镜成像技术 图像时空细胞因子的秘密分布模式和细胞表面sytokine结合位点。使用此 技术，我们将研究个体人类之间的IL-6介导的动态细胞间通信 肝癌HEP3B细胞和CD 4+ T细胞。