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.
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.
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