To discover the fundamental mechanisms that govern how cells mature and differentiate, researchers need to monitor a cell's gene expression profile and measure changes over time without destroying or disrupting its growth. No current tool can meet this technological challenge with a throughput high enough to enable systems biology analyses. This project proposes to establish the feasibility of using localized electroporation for temporal sampling in individual living cells. Specifically, the nanofountain probe electroporation system will be used to identify localized molecular-transport mechanisms, employed in cell sampling, by monitoring the loss of fluorescence intensity in a tdTomato-expressing cell line. These experimental data will be used to validate model parameters in a multi-physics framework based on pore-evolution models. Single-cell RNA sequencing will be used to investigate any off-target effects due to repeated sampling in localized electroporated, bulk electroporated, and unelectroporated (control) cells. The ability to measure gene expression over time in single cells as they are maturing would be a transformative advance for cell biology, bioengineering, and biopharmaceutics.
No existing high-throughput laboratory technique can measure changes over time in cells' internal biochemistry without destroying the cells or disrupting their growth. This project proposes to demonstrate feasibility of using localized electroporation for temporal sampling, in individual living cells, leading to the design of a microfluidic device for non-destructive cytosol sampling of cells adhered to a substrate, overcoming the key barrier to temporal and longitudinal biomolecular analysis of live single cells. Such device would enable cell biologists and bioengineers to correlate gene expression and biochemistry with individual cell phenotypes, providing a transformative opportunity to discover disease mechanisms and potential therapies.
