0018-9294 (c) 2018 IEEE. Personal use is permitted, but republication/redistribution requires IEEE permission. See http://www.ieee.org/publications_standards/publications/rights/index.html for more information. This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. Citation information: DOI 10.1109/TBME.2019.2914689, IEEE Transactions on Biomedical Engineering > REPLACE THIS LINE WITH YOUR PAPER IDENTIFICATION NUMBER (DOUBLE-CLICK HERE TO EDIT) < 1 Abstract— Objective: Irreversible electroporation (IRE) is a less invasive therapy to ablate tumor cells by delivering short intensive electric pulses more than a few kV via needle-like electrodes. For reducing the required voltage for the IRE, a durable comb-shaped miniature electrode was designed to use in contact with the lesion surface for a new method named contact IRE. Methods: A miniature electrode was newly fabricated by a fine inkjet patterning and the subsequent etching of a copper-clad polyimide film. A train of 10-µs or 100-µs long electric pulses were applied 90 times at the interval of 1 s to a tissue phantom, and its cross section was observed to measure the necrotized area. Results: Cell experiments showed that the maximum ablation depth increased as a function of the applied voltage and reached 400 µm at 20 V. Furthermore, insulation of the lateral space between electrode teeth with a resin and administration of adjuvants to reduce the IRE threshold of the cell membrane did increase the ablation depth by 26% and the ablation area by 40%. Conclusion: The miniature electrode developed in this study successfully necrotized cells in a tissue phantom 400 µm deep from the surface with the electric pulses of only 20 V. Significance: The contact IRE for the surface of skin and gastrointestinal tract will ablate cutaneous and subcutaneous tumors by applying only several tens of volts. Index Terms—Irreversible electroporation, Contact electrode, Cell necrosis, Electric field, Adjuvant. I. INTRODUCTION RREVERSIBLE electroporation (IRE) has been studied as a less invasive method for tumor treatment [1-3]. In a clinical application, a pair of needle-like electrodes is inserted into a target tissue to apply electric pulses to expose the tumor to the electric field strength more than a few kV/cm [4]. An overdose of the electric pulses induces irreversible perforation of the cell membrane, and thereby necrotizes the cells around the electrodes. The IRE therapy has a big advantage of a small surgical invasion; it only requires the insertion of the electrodes for delivering the electric pulses to the target lesion. However, a considerably high voltage causes muscle convulsions and contractions during the treatment. Thus, general anesthesia with a heavy burden is needed for the Manuscript submitted February 21, 2019. This study was supported by JSPS KAKENHI Grant Number 26249021. K. Kurata and H. Takamatsu are with Department of Mechanical Engineering, Kyushu University, Fukuoka, Japan. Shuto Yoshimatsu was with Graduate School of Engineering, Kyushu University, and is now with Miura Co. Ltd., Ehime, Japan. (correspondence e-mail: kurata@mech.kyushu -u.ac.jp). therapy to avoid the discomfort of patients [5] although the procedure of the IRE itself is minimally invasive. The fate of cells during IRE is mainly determined by the magnitude of the electric field. According to a number of previous studies, the transmembrane potential required for irreversible breakdown is within the range from 0.5 to 2 V [6-13]. One of the solutions to decrease the applied voltage while maintaining the aforementioned potential difference is to reduce the distance between the electrodes. To realize this idea, we have fabricated a comb-shaped miniature electrode with teeth and gaps of the order of hundred microns by using a photolithography technique [14]. Due to the geometrical restriction of the surface electrode formed on a flat glass substrate, this electrode was designed for a new method named contact IRE, where the electrode is used in contact with the surface of skin and gastrointestinal tract. In our previous study using fibroblasts cultured in an agarose gel, the electric pulses delivered through the miniature surface electrode successfully necrotized the cells underneath the electrode. However, the maximum ablation depth was only ~70 µm at 100 V, which was not adequate for treating cutaneous and subcutaneous tissues such as skin cancers and gastrointestinal tumors. This was attributed to the low durability of thin electrodes and electrical short-circuit through the lateral spaces between the electrode teeth. Therefore, the aim of this study was to maximize the ablation depth achieved by the contact IRE under the constraint of the limited applied voltage. In order to improve the durability of the electrode, a new comb-shaped miniature electrode thicker than that made by photolithography was fabricated using a fine inkjet technique. The lateral spaces between the electrode teeth were filled with a non-conductive resin to prevent short-circuit between the teeth. In the experiments, the efficacy of cell ablation was evaluated by fluorescent staining as a function of the applied voltage and the pulse width. By comparing numerical simulation with experiments, we determined the critical electric field strength to estimate the ablation depth. Furthermore, the effect of adjuvant surfactants was examined to decrease the threshold of IRE by disturbing the repairing process of the perforated membrane. II. MATERIALS AND METHODS A. Fabrication of a Comb-Shaped Miniature Electrode A comb-shaped miniature electrode with 10 pairs of anode and cathode was designed as shown in Fig. 1. The designed Low-Voltage Irreversible Electroporation Using a Comb-Shaped Contact Electrode Kosaku Kurata * , Shuto Yoshimatsu, and Hiroshi Takamatsu I