THREE DIMENSIONAL ELECTRODE STRUCTURE CONTROLLED BY DIELECTROPHORESIS FOR FLOW-THROUGH MICRO ELECTROPORATION SYSTEM Youn-Suk Choi 1,2 , Young-Ji Kim 1 , Maesoon Im 1 , Byoung-Gyun Kim 1 , Kwang-Seok Yun 3 , and Euisik Yoon 4* 1 Korea Advanced Institute of Science and Technology (KAIST), Daejeon, KOREA 2 Kyungwon Tech, Kyunggido, KOREA 3 University of California, Los Angeles, CA, USA 4 University of Minnesota, Minneapolis, MN, USA *Corresponding author: yoon@umn.edu ABSTRACT A three dimensional electrode structure for flow-through micro electroporation is proposed to achieve well-controlled cell positioning in a confined region. In this structure, cell position is actively performed by using dielectrophoresis in a three dimensional electrode structure. Compared with conventional planar electrode structures, the proposed electrode structure can achieve excellent aligning of cells not only in horizontal dimension but also in vertical dimension for receiving uniform electrical field during electroporation. The effectiveness of the proposed structure is validated by both numerical simulation using a commercial tool, CFD- ACE+, and experiments using the fabricated microfluidic chip. The results are compared with conventional parallel finger electrode structures under the same conditions. 1. INTRODUCTION Successful molecular therapies require that any type of agents (e.g. drug, gene, enzymes, antibodies, and other biochemical reagents for intracellular assays) should effectively reach the target cells [1]. Several methods can facilitate the entry of agents into cells: for example, the use of viruses, liposomes, chemicals (e.g. calcium chloride, calcium phosphate), particle bombardment, fine needle naked DNA injection or any combination of these methods [2]. More recently, electroporation has been found to be an effective technique to overcome the membrane barrier and has been widely used in gene therapy, DNA vaccination, cancer therapy, electroinsertion, electroencapsulation, intraocular therapy, vascular therapy and transdermal gene or drug delivery. Electroporation is a technique that introduces foreign materials into cells by applying short electric pulses (typically 1~1.5 kV/cm for a few s to a few ms where the cell size is in the range of 10 m in diameter) to create multiple transient pores in the cell membrane through a dielectric breakdown of the cell membrane. Commercial electroporators are operated in macro scale cuvettes with a batch process. However, electroporation efficiency is quite low because cells experience a non- uniform electric field inside a cuvette. In recent years, microfabrication technologies have been applied to realize electroporators in micro scale. These micro electroporators have some advantages over macro scale counterparts in two ways: low cost due to low reagent consumption and minimal heat generation due to low power consumption. And moreover if a micro electroporator is designed to be operated in flow-through fashion, it can be implemented in a seamless batch type process and easily integrated with other microfluidic devices. Also, the continuous flow cools down the device in the case that there are any local heatings from the applied electroporation signals. By now several groups have developed flow-through micro electroporation chips [3- 5]. However, in these previous works they could not control cell position accurately. Therefore, it was difficult to apply to all the cells the same number of electric pulses with the same electric field strength. These are the important factors in flow-through electroporation. In this work, we propose a three dimensional electrode structure in order to address the cell positioning problem for effective flow-through micro electroporation. In the proposed structure, cell position is controlled actively by dielectrophoresis (DEP) [6-7] with simple modification from the conventional planar electrode structure. We have fabricated two types of electrode structures for comparison: the proposed three-dimensional structure and the conventional planar structure. We report the analysis of DEP operation by numerical simulations as well as experiments confirming the analysis. 2. DESIGN Figure 1 shows the schematic view and the operation principle of the proposed three dimensional electrode structure. This structure consists of two pairs of parallel bottom electrodes (E1~E4) and one top ITO electrode. The cell positioning is actively controlled by these electrodes in a negative DEP regime. Cell positioning is performed in the 466  