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

描述（由申请人提供）：提出了用于单细胞代谢表型动态、多参数传感的高通量活细胞微阵列筛选技术。该提案通过扩大向LINCS中心提供签名的范围来解决共同基金的优先事项。一种名为“Cellarium”的三明治微阵列将被开发出来，用于分析单个活细胞。夹层的底层支撑着蚀刻在玻璃上的浅微孔中的细胞。夹层的顶层将细胞密封在150皮升的微孔中，并结合细胞外荧光传感器，用于代谢分析物、氧、pH和葡萄糖的多参数检测。化学隔离是实现当两层被压缩在一起的扁平金属弹簧允许跨膜通量的动态测量没有细胞内探针。单细胞分析，直接揭示异质性代谢反应的扰动内等基因细胞群体是至关重要的生物学推断。这种微阵列是一种可扩展的工具，用于导出标准化的多参数数据集，这些数据集可以以协调的方式集成到LINCS中。该项目的具体目标是：1)开发一种一次性微阵列（“Cellarium”），用于动态，高通量，多参数代谢测量活单细胞的扰动诱导特征；2)修改商用微阵列扫描仪以读出Cellarium；3)通过同时监测02、pH、葡萄糖和ATP的反应，验证该技术在一系列细胞类型中的有效性；4)通过分析单个细胞对扰动响应的代谢特征分布来验证平台；5)制定书面和图形化的标准操作程序，使数据生成可重复；6)与LINCS合作伙伴积极参与仪器开发过程，以确保有效的设备和方法翻译。

DESCRIPTION (provided by applicant): High throughput live-cell microarray screening technology for dynamic, multiparameter sensing of 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 of the sandwich supports cells in shallow microwells etched in glass. The top layer of the sandwich seals the cells in the 150-picoliter microwells, and incorporates extracellular fluorescent sensors for multiparameter detection of the 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 of the 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. PUBLIC HEALTH RELEVANCE: Determinants of human health and disease depend, ultimately, on the biological state of individual cells. This technology will quantify the distributions of key metabolic parameters, on a cell-by-cell basis, among a cell population. The instrument measures previously inaccessible indicators of cell state and cellular responses to perturbations facilitating new insights into underlying molecular pathways and biological mechanisms.