Single Cell Electroporation

描述（由申请人提供）：广泛的长期目标：总体目标是使单细胞电穿孔成为生物活性分子递送和取样/传感的定量和可预测工具。在分子水平上控制和分析单个活细胞而不杀死它们将成为可能。 具体目标：在单细胞中可靠和可预测地制造瞬时孔。对实验参数如何控制孔隙特性有完整和定量的了解。开发可靠地将已知量的分子引入细胞或从细胞中去除已知量的分子的能力。开发技术，导致更高的空间分辨率，并确定细胞的氧化还原状态。 健康相关性：这是非常广泛的。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，而且在药物筛选中。 设计和方法：将研究微电极和纳米电极的单细胞电穿孔，首先使用脂质体，然后使用培养的细胞。采用扫描电化学显微镜、膜片钳和荧光显微镜等技术对微纳米电极诱导的电穿孔孔的性质进行了研究。将用同样的技术研究放入和取出分子的效率。将以类似的方式构建和分析用于电穿孔和递送/取样的电解质填充的毛细管装置。单细胞的氧化还原状态将通过谷胱甘肽系统的分析来确定。

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.