LASER BASED CELL LOADING & LYSIS FOR SINGLE CELL ELECTROPHORESIS

我们的目标是开发一种技术来检测 单个癌细胞中关键信号转导蛋白的活性。 蛋白激酶是促进生长的关键成分， 信号转导通路。不适当地激活激活酶 导致了不受控制的扩散，这是 致癌状态。我们正在开发一种技术来测量 这些酶在单个细胞中的活性。由于单元格位于 肿瘤在分子水平上是异质性的，特征是 单细胞中的激酶活性将提供关键信息而不是 从当前的方法中可用。具体地说，我们将加载荧光 酶的多肽底物进入细胞，并定量 从该细胞中磷酸化到非磷酸化的多肽底物。 磷酸化的多肽与非磷酸化的多肽的比例为 直接测量父代的激酶。这一衡量标准的准确性 激酶的活性严重依赖于敏感度和 技术的时间分辨率。毛细管的使用-或 基于微芯片的电泳法提供了所需的灵敏度 (10-21至10-19摩尔)用于这些单电池测量。这个 时间分辨率取决于衬底的速度 多肽可以从细胞中移除，涉及到 多肽已终止。蛋白酪氨酸激酶催化磷酸化 因此，在几秒钟内大量的底物， 测量的时间分辨率必须是亚秒级。我们有 使用光声方法在不到30ms内裂解细胞。我们有 将这种裂解技术与一种方法相结合，迅速将 将单个裂解细胞的内容物放入毛细管。我们还有 证明了细胞内荧光标记的鉴定 俄勒冈州绿毛细管区带电泳法利用这一技术。

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.