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

描述(申请人提供)：高通量活细胞微阵列筛选技术，用于单细胞代谢表型的动态、多参数检测。该提案通过扩大Lincs中心可获得的签名范围，解决了共同基金的优先事项。一种名为“细胞”的三明治微阵列将被开发出来，并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电池。三明治的顶层密封了150皮升微孔中的细胞，并结合了细胞外荧光传感器，用于代谢分析物、氧、pH和葡萄糖的多参数检测。当两层膜用扁平的金属弹簧压缩在一起时，就可以实现化学隔离，从而无需细胞内探针即可动态测量跨膜通量。单细胞分析直接揭示了同源细胞群体对扰动的代谢反应的异质性，对生物学推断至关重要。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，该数据集可以协调地整合到LINCS中。该项目的具体目标是：1)开发一种一次性微阵列(“细胞”)，用于动态、高通量、多参数地测量活的单细胞的扰动诱导特征的代谢；2)修改商业微阵列扫描仪以读出细胞；3)通过同时监测氧气、pH、葡萄糖和三磷酸腺苷的反应，验证这项技术在一系列细胞类型中的有效性；4)通过分析单细胞对扰动的代谢特征的分布来验证平台；5)开发书面和图形化的标准操作程序，以便能够产生可重复的数据；6)与Lincs合作伙伴积极参与仪器开发过程，以确保高效的设备和方法转换。 公共卫生相关性：人类健康和疾病的决定因素最终取决于单个细胞的生物状态。这项技术将在逐个细胞的基础上量化关键代谢参数在细胞群体中的分布。该仪器测量以前无法获得的细胞状态和细胞对扰动的反应的指示器，有助于对潜在的分子途径和生物机制进行新的洞察。

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.