LASER BASED CELL LOADING & LYSIS FOR SINGLE CELL ELECTROPHORESIS

基于激光的细胞装载

• 批准号: 6308188
• 负责人: Nancy L. Allbritton
• 金额: $ 2.46万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6308188
• 关键词: Mammalia; biological products; biomedical resource; enzymes; lasers; model design /development; neoplasm /cancer; technology /technique

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.

我们的目标是开发一种技术， 单个癌细胞中关键信号转导蛋白的活性。 蛋白激酶是促生长的关键组分， 信号转导途径 激酶激活不当 导致了不受控制的扩散， 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。 由于细胞内的 肿瘤在分子水平上是异质的， 单细胞中激酶活性将提供关键信息， 从目前的方法。 具体来说，我们将加载荧光 激酶的肽底物进入细胞，并定量 从该细胞磷酸化为非磷酸化的肽底物。 磷酸化肽与非磷酸化肽的比率为 直接测量亲本激酶。 这种测量的准确性 激酶活性严重依赖于敏感性， 技术的时间分辨率。 使用毛细管-或 基于微芯片的电泳提供了所需的灵敏度 （10-21至10-19摩尔）用于这些单细胞测量。 的 时间分辨率取决于衬底 可以从细胞中除去肽， 肽终止。 激酶催化磷酸化 因此， 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30 ms的时间内裂解细胞。 将这种裂解技术与一种方法相结合， 单个裂解细胞的内容物进入毛细管。 我们还 证明了细胞内荧光标记物的鉴定 俄勒冈州绿色采用毛细管区带电泳技术。