Live-cell Microarray for high-throughput observation of metabolic signatures

用于高通量观察代谢特征的活细胞微阵列

• 批准号: 8333992
• 负责人: Deirdre R. Meldrum
• 金额: $ 72.54万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-16 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8333992
• 关键词: Address; Biological; Cells; Chemicals; Collaborations; Data; Data Set; Databases; Detection; Development; Device or Instrument Development; Devices; Disease; Effectiveness; Ensure; Funding; Generations; Glass; Glucose; Health; Heterogeneity; Human; Human Pathology; Incubated; Individual; Instruction; Label; Laboratories; Lasers; Libraries; Life; Malignant Neoplasms; Measurement; Measures; Metabolic; Metabolism; Metals; Methods; Molecular; Monitor; Network-based; Noise; Oxygen; Oxygen Consumption; Pathway interactions; Phenotype; Polymers; Population; Procedures; Process; Reading; Resolution; Running; Sampling; Scanning; Screening procedure; Signal Transduction; Speed; Supporting Cell; System; Technology; Translations; Writing; base; biosignature; cell type; extracellular; glucose uptake; insight; instrument; programs; response; seal; sensor; single cell analysis; time interval; tool

High throughput live-cell microarray screening technology for dynamic, multiparameter sensing cf single-cell metabolic phenotypes is proposed. The proposal addresses Common Fund priorities by extending the range of signatures available to the LINCS centers. A sandwich microarray, called the "Cellarium" will be developed and used to analyze individual live cells. The bottom layer ofthe sandwich supports cells in shallow microwells etched in glass. The top layer ofthe sandwich seals the cells in the 150-picoliter microwells, and incorporates extracellular fluorescent sensors for multiparameter detection ofthe metabolic analytes, oxygen, pH and glucose. Chemical isolation is achieved when the two layers are compressed together with a flat metal spring allowing dynamic measurement of transmembrane fluxes without Intracellular probes. Single-cell analysis which directly reveals heterogeneity in metabolic response to perturbations within an isogenic cell population is critical to biological inference. This microarray is an extensible tool for deriving a standardized multiparameter set of data that can be integrated in a coordinated way into LINCS. The specific aims ofthe project are: 1) develop a disposable microarray ("Cellarium") for dynamic, high throughput, multiparameter metabolic measurements of perturbation-induced signatures of live single cells; 2) modify a commercial microarray scanner to read out the Cellarium; 3) verify the effectiveness of this technology across a range of cell types by simultaneously monitoring 02, pH, glucose and ATP responses; 4) validate the platform by analyzing the distribution of metabolic signatures of single cells in response to perturbations; 5) develop written and graphical standard operating procedures that enable reproducible data generation; 6) develop active participation with LINCS partners in the instrument development process to ensure efficient device and methods translation.

用于单细胞动态多参数传感的高通量活细胞微阵列筛选技术 提出了代谢表型。该提案通过扩大范围来解决共同基金的优先事项 LINCS 中心可用的签名。称为“Cellarium”的三明治微阵列将 开发并用于分析单个活细胞。三明治的底层支撑着细胞 玻璃上蚀刻的浅微孔。三明治的顶层将细胞密封在 150 皮升中 微孔，并结合细胞外荧光传感器，用于代谢的多参数检测 分析物、氧气、pH 值和葡萄糖。当两层被压缩时实现化学隔离 与扁平金属弹簧一起可以动态测量跨膜通量，而无需 细胞内探针。单细胞分析直接揭示代谢反应的异质性 同基因细胞群内的扰动对于生物学推断至关重要。该微阵列是 可扩展的工具，用于导出标准化的多参数数据集，这些数据可以集成到协调的 进入 LINCS 的方式。该项目的具体目标是：1）开发一次性微阵列（“Cellarium”） 对扰动引起的特征进行动态、高通量、多参数代谢测量 活的单细胞； 2) 修改商用微阵列扫描仪以读取 Cellarium； 3）验证 通过同时监测 02、pH、葡萄糖，该技术在多种细胞类型中的有效性 和 ATP 反应； 4）通过分析单个人的代谢特征分布来验证平台 细胞对扰动的反应； 5) 制定书面和图形化的标准操作程序 实现可重复的数据生成； 6) 与 LINCS 合作伙伴积极参与该工具 开发流程以确保高效的设备和方法翻译。