ELECTRICALLY MEDIATED GENE DELIVERY AND THEIR DIFFUSION MECHANISM ON LOCALIZED SINGLE CELL USING ITO MICROELECTRODE BASED TRANSPARENT CHIP Tuhin Subhra Santra 1 , Sheng-Chiech Chen 1 , Chia-Jung Chang 2 , Tsung-Ju Chen 1 , Pen-Cheng Wan 2 Fan-Gang Tseng 1,2,3 1 Institute of NanoEngineering and MicroSystems, National Tsing Hua University, Taiwan 2 Department of Engineering and System Science, National Tsing Hua University, Taiwan 3 Division of Mechanics, Research Center for Applied Sciences, Academia Sinica, Taiwan ABSTRACT In recent years, more studies focused on single cell electroporation. However, not only the position cannot be confined in the specific region of the cells, but also the employed electrodes are not transparent, causing difficulty on optical observation. Here, we demonstrated localized single cell membrane electroporation (LSCMEP), which an efficient to deliver drugs into single cell by selective and localized way from millions of cells. The ITO microelectrodes fabricated by wet etching as well as FIB technique, which successfully deployed 1μm gap between two microelectrodes. Due to submicron level gap, electric field can more intense in a narrow region. The diffusion mechanism with PI dye intensity variation has been analyzed by image pro plus software. In our approach, we have successfully achieved 0.99 μm electroporation regions on the cell membrane to inject PI dye into the cell with high cell viability by trypan blue test. We demonstrated the cell self-recover process from the injected PI dye intensity variation. Our localized single cell membrane electroporation technique, not only generates well-controlled nano-pore allowing rapid recovery of cell membrane, but also provides a clear optical path potentially monitoring drug/DNA delivered into single cell level even in single molecule level. KEYWORDS Localized single cell electroporation, ITO micro-electrodes, FIB technique, transparent chip INTRODUCTION When a certain strong electric field pulses applied across a cell and tissue, then it’s have ability to rearrangement of their structure causes the permeabilization of the cell membrane named in early 1980’s “electroporation” [1-2]. In the past decades, high electric field pulses were applied to the whole cells between two large electrodes which resulted in permeabilizing the membrane of millions of cells simultaneously without reversibility [3]. In recent years, more studies focused on single cell electroporation on chip. For single cell electroporation the electric field parameter can be controlled better to avoid the cell death. For this electroporation, the electric field applied locally surrounding the single cell whereas, in bulk electroporation a homogeneous electric field applied to suspension of millions of cells together. The success rate like surviving cells for single cell electroporation is far better compare with bulk electroporation. This technique is faster and easy to perform with less toxicity and technical difficulty for application of wider tissue and cell types. However to allow selective manipulation of single organelles with in a cell, the electrode size must be reduce to nano-scale level. Thus the localized single cell membrane electroporation (LSCMEP) concept has come in last few years [4]. By this technique selective manipulation organelles and biochemical effect can be analyzed of the individual cell and this techniques more advance compare to single cell electroporation. The cell rapture and cell death can be minimized because of electric field concentrated in more localized region of the cell membrane surface compare to single cell electroporation. EXPERIMENT Fig. 1(a) demonstrate our experimental setup for localized single cell membrane electroporation (LSCMEP) technique, in where ITO electrodes fabricated on top of the glass (130 μm) substrate. The distance between two electrodes is 1μm. Fig. 1(b) shows an intense electric field in between two microelectrodes (Results simulated by Comsol Multiphysics). 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences October 28 - November 1, 2012, Okinawa, Japan 978-0-9798064-5-2/μTAS 2012/$20©12CBMS-0001 962