Development of an automated microfluidic device for high-throughput single cell kinase analysis

In this work, development of a microfluidic device to perform high-throughput single cell analysis is described. The objective is to be able to separate and detect the intracellular contents of hundreds of individual mammalian cells in a short time period. The current biological target is to assess kinase enzyme activity within single cells to study the role these enzymes play in intracellular signaling transduction pathways. Microfluidic devices are well-suited to address this type of high-throughput assay because of their ability to precisely manipulate the sub-picoliter volume contained within a mammalian cell and to achieve rapid electrophoretic separation of the cellular contents.;This dissertation describes continued development of a microfluidic network in which a constant stream of cells is pulled through an electric field. Once cells enter the electric field region, electrical cell lysis occurs and the resulting cell lysate is electrokinetically injected into a perpendicular separation channel. Electrophoretic separation of the intracellular contents then occurs. The development of this microfluidic device includes investigations into channel surface coatings to reduce cell adhesion and cellular debris buildup on the glass microfluidic channels, development of electrophoretic separation conditions for kinase substrate and product peptides, control of the cell flow through the lysis intersection to improve the lysate injection efficiency, assessment of the hydrodynamic flow on the separation conditions and modifications to the channel network to increase the sample throughput.;A significant portion of this research also involved development of a new microfluidic network for high-throughput single cell analysis. The new device design utilizes a modified patch-clamp trapping method and is capable of rapidly trapping and lysing individual cells in succession. Development, unique fabrication aspects and implementation of automation to control cell flow on this device is described. Additionally, demonstration of data collection on both the new and older designs is demonstrated.