Single Cell Electroporation

单细胞电穿孔

• 批准号: 7088726
• 负责人: STEPHEN G. WEBER
• 金额: $ 20.25万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7088726
• 关键词: cell membrane; electrodes; electrolytes; electrophysiology; electroporation; fluorescence microscopy; glutathione; liposomes; oxidation reduction reaction; oxidative stress; reperfusion; single cell analysis; technology /technique development; tissue /cell culture; voltage /patch clamp

DESCRIPTION (provided by applicant): Broad, long-term objectives: The overall aim is to make single-cell electroporation a quantitative and predictable tool for delivery and sampling/sensing of bioactive molecules. Molecular level control and analysis of single, living ceils, without killing them, will become possible. Specific aims: Make transient pores reliably and predictably in single cells. Have a complete and quantitative understanding of how experimental parameters control pore characteristics. Develop the capability to introduce or remove known quantities of molecules to/from a cell reliably. Develop techniques leading to higher spatial resolution, and for determining a cell's redox status. Health relatedness: This is very broad. The determination of the redox state and its effect on transcription are related to oxidative stress and reperfusion injury, and cancer. Our methods will accelerate single-cell proteomic studies. The action of drugs on well-defined biochemical processes will be determined easily, thus the health relatedness is not only in disease and injury, but also in screening for pharmaceuticals. Design and methods: Single cell electroporation with micro- and nano-electrodes will be studied, first with liposomes and then with cultured cells. The techniques of scanning electrochemical microscopy, patch clamp and fluorescence microscopy will be used to determine the properties of pores from micro- and nano-electrode induced electroporation. The efficiency of putting in and taking out molecules will be studied with the same techniques. Electrolyte-filled capillary devices for eiectroporation and delivery/sampling will be constructed and analyzed in a similar way. The redox state of single cells will be determined through analysis of the glutathione system.

描述（由申请人提供）：广泛的长期目标：总体目的是使单细胞电穿孔成为生物活性分子传递和采样/感测的定量且可预测的工具。分子水平的控制和分析单个活着的天花板而无需杀死它们，将成为可能。 具体目的：在单个细胞中可靠和可预测的瞬态孔。对实验参数如何控制孔特征有完整的定量理解。发展能够可靠地从细胞引入或去除已知量分子的能力。开发技术导致较高的空间分辨率，并确定细胞的氧化还原状态。 健康相关性：这非常广泛。氧化还原状态的确定及其对转录的影响与氧化应激和再灌注损伤以及癌症有关。我们的方法将加速单细胞蛋白质组学研究。药物对明确定义的生化过程的作用将很容易确定，因此，健康相关性不仅在疾病和损伤中，而且还在筛查药品。 设计和方法：将研究使用微电极和纳米电极的单细胞电穿孔，首先是脂质体，然后再用培养细胞进行研究。扫描电化学显微镜，斑块夹和荧光显微镜的技术将用于确定微观和纳米电极诱导的电穿孔的孔的性质。将分子和取出分子的效率将通过相同的技术进行研究。将以类似的方式构建和分析用于涂层和采样的电解质毛细管设备。单细胞的氧化还原状态将通过分析谷胱甘肽系统来确定。