Measuring toxicant effects on cellular function in a microarray format

项目摘要/摘要-美国环保局维护一份&gt；650种有毒化学品的有毒物质释放清单，这些化学品 从美国20,000个工业用地处置或释放，而HHS和美国农业部维护着一份 超过60种对人类、动物和植物健康构成严重风险的精选制剂和毒物。这些 化合物只代表已知和未知环境毒物中的一小部分，这些毒物可能对 影响人类健康。新的和未知的毒物以前所未有的速度合成，并且 大量的这种物质正在迅速进入环境。因此，迫切需要一种高含量的分析方法 毒物对细胞功能的影响。这样的平台将拓宽我们对环境的理解 可能代表自身免疫性疾病、免疫缺陷和肿瘤风险的成分。 干细胞是毒物的重要和敏感目标，因为它们是胚胎的关键组成部分。 对成人组织的发育和维持是不可或缺的。目前测量茎干变化的方法 细胞分化和对毒性效应的编程反应为深入了解AFP的发病机制提供了线索 毒物暴露，但这些技术不能达到保持最快速度所需的吞吐量 新的毒物进入环境。此外，执行劳动密集型黄金的费用- 标准测试限制了为细胞数量、有毒物质身份和暴露而收集的数据量 集中精神。尽管如此，这些测试是定期进行的，因为这些测量对我们的 了解这些代理人所代表的风险，以及我们缓解或消除这些风险的能力。 该项目的目标是开发能够检测毒物对干细胞影响的仪器。 在灵敏的高含量分析中的分化和行为。这项化验将基于对 人巨噬细胞表面标志物表达和细胞因子分泌谱的变化 干细胞。这些细胞将暴露在一个或多个同时的毒物浓度梯度中，这 将表示细胞可能遇到的暴露或双因素联合暴露的光谱 环境。西恩西亚提出了一种既能测量光栅耦合表面等离子激元又能测量表面等离子激元的仪器 来自三个不同荧光团的共振以及等离子体增强的荧光发射。这 该系统还将采用变焦镜头，允许全芯片成像和高倍率单点成像 图像。它将支持芯片上孵化和允许细胞暴露在扩散中的样本室 毒物的梯度，使其成为实验室评估毒物对干细胞分化影响的理想工具。 也可以同时评估对其他细胞系的影响。这一建议的工具 将具有高度的通用性，随着项目的进展，模块化组件将提供额外的实用程序 第三阶段。经过充分验证的这种性质的平台可能很容易适用于药物发现努力和领导 化合物验证，并可能最终发现作为临床诊断的价值。

Project Summary/Abstract – The EPA maintains a Toxics Release Inventory of >650 toxic chemicals that are disposed or released from >20,000 industrial sites in the United States, while HHS and USDA maintain a list of over 60 select agents and toxicants that pose a severe risk to human, animal, and plant health. These compounds represent only a fraction of the known and unknown environmental toxicants that may adversely affect human health. New and uncharacterized toxicants are synthesized at an unprecedented rate, and trace quantities of these are rapidly entering the environment. There is thus a critical need for a high-content assay of the effects of toxicants on cell function. Such a platform would broaden our understanding of environmental components that may represent risks for autoimmune disease, immunodeficiencies, and neoplasia. Stem cells are an important and sensitive target for toxicants as they are critical components of embryonic development and are integral to the maintenance of adult tissues. Current assays measuring changes in stem cell differentiation and programming in response to toxic effects provide insight into the pathogenesis of toxicant exposure, but these techniques are not capable of the throughput necessary to stay apace the speed of new toxicants entering the environment. Further, the expense of performing the labor-intensive gold- standard tests limits the amount of data gathered for a cell population, toxicant identity, and exposure concentration. Nonetheless, these tests are regularly performed since such measurements are critical to our understanding of the risks these agents represent, and to our ability to moderate or eliminate those risks. The goal of this project is the development of instrumentation capable of detecting toxicant effects on stem cell differentiation and behavior in a sensitive high-content assay. This assay will be based on the detection of changes in the surface marker expression and cytokine excretion profile of a spatially encoded microarray of stem cells. These cells will be exposed to one or more simultaneous toxicant concentration gradients, which will represent the spectrum of exposures or two-agent co-exposures that might be encountered by cells in the environment. Ciencia is proposing instrumentation that can measure both grating-coupled surface plasmon resonance as well as plasmonically-enhanced fluorescent emission from three distinct fluorophores. This system will also incorporate a zoom lens that permits whole-chip imaging and high-magnification single-spot images. It will support on-chip incubation and a sample chamber that permits exposure of cells to a diffusing gradient of toxicant, making it ideal for the laboratory evaluation of toxicant effects on stem cell differentiation. Concurrent assessments of effects on other cell lineages may also performed. This proposed instrumentation would be highly versatile, with modular components suggesting additional utility as the project progresses to Phase III. A well-validated platform of this nature may be readily applicable to drug discovery efforts and lead compound validation, and may ultimately find value as a clinical diagnostic.