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

摘要 探测单个免疫细胞的蛋白质分泌行为的时间曲线的能力将影响免疫细胞的免疫功能。 未来的免疫学，细胞生物学，甚至传染病诊断。知识的顺序和时间， 由活化的T细胞分泌的细胞因子（细胞间信号传导所必需的水溶性蛋白质）可以 另外提供了通过功能区分分化的T细胞亚群的手段。在这里，时间 信息是监测免疫系统行为的整个难题中的一部分。另 关键的部分是不同细胞类型之间的精氨酸介导的相互作用，这涉及到空间运输， 细胞之间的细胞因子。把这两块拼图放在一起，我们就可以捕捉到整个画面， 细胞因子的释放动力学和细胞的甘氨酸介导的相互作用，这使我们能够充分了解 免疫系统的细胞间信号传导过程。然而，目前还没有研究获得这样的图片， 缺乏一种技术，用于实时感测细胞间精氨酸介导的信号传导过程在高 空间分辨率本研究旨在开发一种新的无标记成像技术，以充分了解细胞 在单细胞水平上，在马槟榔碱介导的激活和通信期间的行为。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，每个纳米天线结构特异性地针对特定的 细胞因子种类。我们将把这些生物传感器集成在一个微流体系统中， 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， 为了获得真实的时间内细胞因子分泌过程的时空分布，无论是大规模的， 平行的方式。我们还将开发一种理论算法，使我们能够提取的定量值 局部细胞因子浓度分布来自测量的图像强度。第1章：我们要创造 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的功能化， 针对靶向细胞因子的适体。SA 2：我们将整合适体共轭等离子纳米天线 阵列到一个单细胞操纵微流控系统，并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式（荧光和暗场）显微成像技术 以成像时空细胞因子分泌谱模式和细胞表面细胞因子结合位点。使用此 技术，我们将研究IL-6介导的动态细胞间通讯个体之间的人 肝癌Hep 3b细胞和CD 4+ T细胞。

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.