Inertial microfluidic circuits for investigating receptor activation dynamics

Abstract

The ErbB family of receptor tyrosine kinases members comprises epidermal growth factor receptor (EGFR), ErbB2, ErbB3 and ErbB4. These receptors recognize external cues (ligands) and subsequently undergo signal transduction that direct the downstream activation of other proteins such as GRB2 and Shc in the signalling cascade and ultimately direct cell fate. Ligand binding induces conformational changes, receptor dimerization and autophosphorylation. Current techniques lack the temporal resolution required to observe receptor activation dynamics that occur over sub-second timescales. Traditionally, quench flow analysis has been used. However, this approach involves turbulent flows that disrupt the cell membrane. Microfluidics involves gentle, laminar flow and has been used for rapid whole cell quench flow analysis to reveal new insights into EGFR autophosphorylation dynamics (Chiang Y. and West J. Lab Chip, 2013, p.1031). In my PhD, I have developed a novel platform based on inertial microfluidics coupled with Dean flow principles. High velocities (m/s) transport within microfluidic channels was used to focus cells to lateral positions within the ligand streams in less than a millisecond (0.5 ms), followed by a curved incubation channel where Dean forces drove cells to a common position, to produce a uniform cell velocity and thus uniform incubation times (CV = <5%). The cells were transported to a second inertial focusing channel and were laterally focused into a quench buffer in order to preserve the receptor intermediates for analysis by multiplexed flow cytometry. The novel microfluidic device successfully monitored the early dynamics of EGFR signalling (25-2000 ms) and unravelled new information on the phosphorylation of two important tyrosine residues, Y1068 and Y1173, found on the EGF receptor. The Y1068 was characterized by high levels of phosphorylation compared to the levels of Y1173 in the sub-second time window. These findings can be used for identifying potential therapeutic targets.

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Identifiers

ORCID for Jonathan Swann: orcid.org/0000-0002-6485-4529

ORCID for Donna Davies: orcid.org/0000-0002-5117-2991

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