Single-Molecule Imaging of Biological Trauma:Cytokine-Based Intracellular Communication

项目摘要 研究摘要：实时和从单细胞中检测未标记的细胞因子可以提供一种方法， 一个强大的平台，用于理解“生物创伤和疾病的分子语言”。然而，工具 在细胞水平上观察细胞因子，特别是以其分泌形式，是缺乏的。我们已经开发出一种 基于纳米材料的通用近红外荧光传感器和伴随的显微镜平台， 在特定靶分子的存在下产生独特的强度和波长偏移（Zhang*，Landry* 等人Nature Nanotechnology 2013; Landry等人Sensors 2015; Landry等人Nature Nanotechnology 2017）。在 在这个为期5年的计划中，我将（i）开发VEGF、IL-6和IL-8细胞因子的合成传感器，（ii）验证它们的用途 以监测巨噬细胞和上皮细胞的组成性细胞因子分泌，和（iii）直接可视化巨噬细胞和上皮细胞的细胞因子分泌。 细胞间基于精氨酸的协同作用的时空概况。直接细胞测量分泌的 细胞因子将告知来自单个或少数个体细胞的细胞因子分泌谱如何被 趋化因子和细胞因子，其形成目前用于生物标志物的细胞因子分泌谱的基础， 基于诊断。据我们所知，我们在此提出的研究只进行了探索。 理论上（Thurley等人，POLS Comp. Bio. 2015）。 兰德里实验室研究计划：我是一名受过训练的单分子生物药剂学家， 几种能够检测微微牛顿尺度力的仪器（Landry等人，Biophys. J. 2009），以及 纳米级荧光定位（Landry等人，Nucl. Ac. 2012年，我的博士论文。在 在我的博士后职位上，我的目标是利用我在单分子光谱学方面的专业知识 和分子生物物理学来设计纯合成的分子识别工具。我的科学训练， 麻省理工学院化学工程的博士后研究员专注于将这两个以前完全不同的领域合并起来， 科学：光学显微镜和纳米传感器的发展，产生一个平台，光学检测任何 通用分子分析物。我于2016年6月开始在加州大学伯克利分校担任教职， 通过将我的实验室的技术优势转化为显微镜（O 'Donnel等人，Adv. Funct. Mater 2017）， 传感器开发（Beyene et al. ACS Chem Neruo 2017 & Luo et al. ACS Sensors 2017）和分子生物学 识别（Li et al. RSC Chemical Science 2017），以解决开发检测细胞因子的方法的需求 从免疫细胞流出。在我的研究计划的头两年，我的小组将综合和表征 基于纳米材料的细胞因子体外传感器。R35奖励的剩余三年将实施 使用细胞因子传感器测量组成型（第3年）、诱导型（第4年）和细胞内（第5年）细胞因子 从培养的细胞样本中发出信号。我的长期研究目标集中在纳米传感器的应用， 环境中的细胞因子、趋化因子和其他重要生物标志物，如多细胞肿瘤球体 以及活体肿瘤活组织检查，其中生物标志物检测传统上是困难的。 Landry ABSTRACT AB-1

PROJECT ABSTRACT Research Abstract: The detection of unlabeled cytokines in real-time and from single cells could provide a robust platform for understanding the ‘molecular language of biological trauma and disease’. However, tools to visualize cytokines at the cellular level, particularly in their secreted form, are lacking. We have developed a generic nanomaterial-based near-infrared fluorescent sensor and accompanying microscopy platform which produces a unique intensity and wavelength shift in the presence of a specific target molecule (Zhang*, Landry* et al. Nature Nanotechnology 2013; Landry et al. Sensors 2015; Landry et al. Nature Nanotechnology 2017). In this 5-year proposal, I (i) will develop synthetic sensors for VEGF, IL-6, and IL-8 cytokines, (ii) validate their use to monitor constitutive cytokine secretion from macrophage and epithelial cells, and (iii) directly visualize the spatio-temporal profiles of intercellular cytokine-based synergies. Direct cellular measurement of secreted cytokines will inform how cytokine secretion profiles from single or few individual cells are stimulated by chemokines and cytokines, which forms the basis of the cytokine secretion profiles currently used in biomarker- based diagnostics. The research we propose herein has – to the best of our knowledge – only been explored theoretically (Thurley et al. POLS Comp. Bio. 2015). Landry Laboratory Research Program: I am a single-molecule biophysicist by training, having developed several instruments capable of detecting piconewton-scale forces (Landry et al. Biophys. J. 2009), and nanometer-scale fluorescence localization (Landry et al. Nucl. Ac. Res. 2012) for my doctoral work. In transitioning to my postdoctoral position, my goal was to leverage my expertise in single-molecule spectroscopy and molecular biophysics to design purely synthetic molecular recognition tools. My scientific training in as a postdoctoral fellow in Chemical Engineering at MIT focused on merging these two previously disparate areas of science: optical microscopy and nanosensor development, yielding a platform for the optical detection of any generic molecular analyte. I began my faculty appointment at UC Berkeley in June 2016, with a research portfolio motivated by translating the technical strengths of my lab in microscopy (O’Donnel et al. Adv. Funct. Mater 2017), sensor development (Beyene et al. ACS Chem Neruo 2017 & Luo et al. ACS Sensors 2017), and molecular recognition (Li et al. RSC Chemical Science 2017) to addressing the need to develop methods to detect cytokine efflux from immune cells. In the first two years of my research plan, my group will synthesize and characterize nanomaterial-based sensors for cytokines in vitro. The remaining three years of the R35 award will implement the use of cytokine sensors to measure constitutive (year 3), induced (year 4), and intracellular (year 5) cytokine signaling from cultured cell samples. My long-term research goals focus on the application of nanosensors for cytokines, chemokines, and other important biomarkers in environments such as multicellular tumor spheroids and live tumor biopsies, in which biomarker detection has traditionally been difficult. Landry ABSTRACT AB-1