In order to discover the fundamental mechanisms that govern how and why cells mature and differentiate, we need a laboratory technique that can monitor cells’ internal biomarkers and measure changes over time without destroying or disrupting their growth. No current tool has been able to address this intractable technological challenge with versatility and throughput relevant for systems biology analysis. This proposed exploratory project would establish a novel method for non-destructive cytosol sampling of cells adhered to a substrate, one of the key barriers to overcome toward the goal of temporal and longitudinal biomolecular analysis of live single cells. The focus is on development of a microfluidic device for intra-cellular delivery and non-destructive cytosol sampling of cells cultured on a porous membrane. In particular, this project would address the task of cytosol sampling by augmenting the design of a microfluidic device we have previously used, to establish the feasibility for intra-cellular delivery, and to discover key design features that influence the efficiency of sampling and cell viability. To accomplish non-destructive cell sampling of internal biomolecules, “localized” electroporation will be used to sample the cytosol through transient and reversible nanopores induced in the cell membrane. By tuning electroporation parameters such as the applied electrical input, i.e., polarity, voltage, frequency, and duration of input signal, precise and reproducible sampling of cells should be possible based on our preliminary results on luciferase sampling. These preliminary results also suggest high cell viability due to the localized nature of electroporation. For this project, we will sample both housekeeping (GAPDH, β-actin) and biologically relevant mRNA (BRCA, VEGF), in a breast cancer cell line (MDA-MB 231), followed by reverse transcription-polymerase chain reaction (RT-PCR) assay in a commercially available micro-well plate reader. The ability to measure biomarkers temporally as cells are maturing would be a transformative advance for cell biology, bioengineering, and synthetic biology.
|Effective start/end date||9/15/19 → 8/31/23|
- National Institute of General Medical Sciences (5R21GM132709-02)
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