LASER BASED CELL LOADING & LYSIS FOR SINGLE CELL ELECTROPHORESIS

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Our goal is to develop a technology to detect alterations in the activities of key signal transduction proteins in single cancer cells. The protein kinases are critical components of growth-promoting, signal transduction pathways. Inappropriate activation of kinases leads to the uncontrolled proliferation that characterizes the oncogenic state. We are developing a technology to measure the activities of these enzymes in single cells. since cells within a tumor are heterogeneous at the molecular level, characterization of kinase activity in single cells will provide key information not available from current methods. Specifically we will load fluorescent peptide substrates of kinases into a cell and quantitate the phosphorylated to nonphosphorylated peptide substrates from that cell. The ratio of the phosphorylated to nonphosphorylated peptide is a direct measure of the parent kinases. The accuracy of this measure of kinase activity is critically dependent on the sensitivity and temporal resolution of the technique. The use of capillary- or microchip-based electrophoresis provides the sensitivity required (10-21 to 10-19 moles) for these single-cell measurements. The temporal resolution depends on the speed with which the substrate peptides can be removed from the cell and the reactions involving the peptide terminated. Kinases catalyze the phosphorylation of significant amounts of substrate in periods of seconds therefore, the temporal resolution of the measurements must be subsecond. We have used a photoacoustic method to lyse cells in less than 30 ms. We have combined this lysis technique with a method to rapidly introduce the contents of a single lysed cell into a capillary. We have also demonstrated identification of the intracellular, fluorescent marker Oregon Green by capillary zone electrophoresis using this technology.