Single Cell Electroporation

描述(由申请人提供)：广泛的、长期的目标：总体目标是使单细胞电穿孔成为一种定量和可预测的工具，用于输送和采样/检测生物活性分子。对单个活细胞进行分子水平的控制和分析，而不杀死它们，将成为可能。 具体目标：在单个细胞中可靠和可预测地制造瞬时毛孔。对实验参数如何控制孔隙特征有一个完整和定量的了解。开发将已知量的分子可靠地引入细胞或从细胞中移除的能力。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。与健康相关：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。药物在明确的生化过程中的作用将很容易确定，因此与健康有关的不仅仅是疾病和伤害，还包括药物的筛选。设计和方法：首先用脂质体，然后用培养细胞，用微电极和纳米电极进行单细胞电穿孔研究。扫描电化学显微镜、膜片钳和荧光显微镜技术将被用来确定微电极和纳米电极诱导电穿孔的孔的性质。我们将用同样的技术来研究放入和取出分子的效率。用于电传输和输送/取样的充满电解质的毛细管装置将以类似的方式建造和分析。单细胞的氧化还原状态将通过对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.