Multiplexed Single-Cell Plasmonic Immunoassay of Intracellular Signaling Proteins Enables Non-Destructive Monitoring of Cell Fate.

It is of significant importance in cancer biology to identify signaling pathways that play key roles in cell fate determination. Dissecting cellular signaling pathways requires the measurement of a large number of signaling proteins. However, tools for simultaneously monitoring multiple signaling pathway components in single living cells remain limited at present. Herein, we describe an approach, termed multiplexed single-cell plasmonic immunosandwich assay (mxscPISA), for simultaneous detection of multiple signaling proteins in individual living cells. This approach enabled simultaneous non-destructive monitoring of multiple (up to five, currently the highest multiplexing capacity in living cells) cytoplasmic and nucleus signaling proteins in individual cells with ultrahigh detection sensitivity. As a proof of principle, the epidermal growth factor receptor (EGFR) pathway, which plays a central role in cell fate determination, was investigated using this approach in this study. We found that there were differential attenuation rate of pro-survival and accumulation rate of pro-death signaling protein of the EGFR pathway in response to EGFR inactivation. These findings implicate that, after EGFR inactivation, a transient imbalance between survival and apoptotic signaling outputs contributed to the final cell fate of death. The mxscPISA approach can be a promising tool to reveal a signaling dynamic pattern at the single-cell level and to identify key components of signaling pathways that contribute to the final cell fate using only a limited number of cells.