Single Cell Electroporation

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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.