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的动态、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LINCS中心可用的签名范围来解决共同基金的优先事项。将开发一种称为“Cellarium”的夹心微阵列，用于分析单个活细胞。夹层的底层支持在玻璃中蚀刻的浅微孔中的细胞。夹层的顶层将细胞密封在150皮升微孔中，并结合了细胞外荧光传感器，用于代谢分析物、氧气、pH值和葡萄糖的多参数检测。当两层用扁平金属弹簧压缩在一起时，实现化学隔离，从而允许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了同基因细胞群内对扰动的代谢反应的异质性，这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，这些数据集可以以协调的方式集成到LINCS中。本计画的具体目标为：1）发展一种可抛弃式微阵列（“Cellarium”），用于活单细胞的扰动诱导特征的动态、高通量、多参数代谢测量; 2）修改商业微阵列扫描仪以读出Cellarium; 3）通过同时监测O2、pH、葡萄糖和ATP响应来验证该技术在一系列细胞类型中的有效性; 4）通过分析单细胞响应扰动的代谢特征的分布来验证平台; 5）开发书面和图形标准操作程序，以实现可重现的数据生成; 6）与LINCS合作伙伴一起积极参与仪器开发过程，以确保有效的设备和方法翻译。 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学状态。这项技术将量化关键代谢参数的分布，在细胞的基础上，在细胞群中。该仪器测量以前无法获得的细胞状态指标和细胞对扰动的反应，促进对潜在分子途径和生物机制的新见解。

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.