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

基本信息

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
项目类别：
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

项目摘要

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.

抽象的探测个体免疫细胞蛋白质分泌行为的时间特征的能力将影响未来的免疫学、细胞生物学，甚至传染病诊断。了解顺序和时间安排激活的 T 细胞分泌的细胞因子（细胞间信号转导必需的水溶性蛋白质）可以另外还提供了根据功能区分分化 T 细胞亚群的方法。这里，时间信息是监测免疫系统行为的整个难题的一部分。另一个关键部分是不同细胞类型之间细胞因子介导的相互作用，其中涉及细胞因子的空间运输细胞之间的细胞因子。将拼图的两部分放在一起使我们能够捕捉到整个拼图细胞因子释放动力学和细胞因子介导的细胞相互作用，这使我们能够充分了解细胞间信号传导过程是免疫的基础。然而，目前还没有研究获得这样的图片，因为缺乏实时传感细胞间细胞因子介导的高信号传导过程的技术空间分辨率。本研究旨在开发一种新型无标记成像技术，以充分了解细胞单细胞水平上细胞因子介导的激活和通讯过程中的行为。我们的方法将采用由等离子体纳米天线结构组成的生物传感器，每个纳米天线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体系统中，该系统包含一系列样品/试剂流动通道和单细胞捕获微孔。微流体传感器集成将提供在单个芯片上捕获、操纵和激活单个细胞以进行细胞间通信的能力实时获取细胞因子分泌过程的时空概况，无论是在大规模、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得出局部细胞因子浓度分布。 SA 1：我们将创造高度有序、高密度等离激元纳米天线生物传感器阵列，每个阵列均通过高度选择性功能化针对目标细胞因子的适体。 SA 2：我们将集成适配体共轭等离子体纳米天线将阵列集成到单细胞操纵微流控系统中，并在高吞吐量。 SA 3：我们将开发一种双模式（荧光和暗场）显微镜成像技术对时空细胞因子分泌谱模式和细胞表面细胞因子结合位点进行成像。使用这个技术，我们将研究 IL-6 介导的人类个体之间的动态细胞间通讯肝癌 Hep3b 细胞和 CD 4+ T 细胞。