Real-Time Monitoring of NADPH Oxidase Inhibitory Effect of ROS Production from THP-1 Cells Using Iron-Porphyrin Modified Carbon Electrode  

Wednesday, 31 May 2017
Grand Ballroom (Hilton New Orleans Riverside)
S. Kasai (Graduate School of Eng.,Tohoku institute of technology), A. Prasad (Department of Biophysics, Palacky University,), R. Matsuoka (Hokuto Denko Corporation), A. Takahashi (Tohoku Institute of technology), A. Nakagawa (Tokyo university of science), Y. Sugiura (Graduate School of Eng.,Tohoku institute of technology), H. Koriyama (Tohoku Institute of Technology), S. Aoyagi (Hokuto Denko Corporation), T. Aikawa (Tokyo University of Science), T. Kondo (Department of Pure and Applied Chemistry), and M. Yuasa (PIRC)
Immune cells such as monocytes and neutrophils responds to external stimuli and during the invasion by foreign body undergoes phagocytosis during which reactive oxygen species (ROS) such as superoxide anion radical (O2•−), hydrogen peroxide (H2O2) and hydroxyl radical (OH) are produced. The iron-porphyrin modified carbon electrode is prepared by the electropolymerization of 1-methylimidazole-coordinated iron meso-tetra (3-thienyl) porphyrin ([Fe(im)2(ttp)]Br) [1].

The detection of O2•− is based on amperometry comprising of counter electrode and working electrodes. The counter electrode is a platinum plate (2.5×2.5mm) while the working electrodes (φ1mm) is an iron-porphyrin modified carbon electrode. Ag/AgCl was used as a reference electrode. The iron-porphyrin modified carbon electrode acts as O2•−detection sensor (Fig. 1).

The O2•− generation was measured in THP-1 cells (cell lines of myeloid leukemia patients) under the effect of differentiation inducer phorbol 12-myristate 13-acetate (PMA) in the absence and presence of exogenous addition 0.1 wt% Triton X at 25°C at a cell density of 3.0 × 106 cells/well. Triton X is known to permeabilize the membranes of living cells. The results obtained showed no considerable changes in reduction currents for O2•− under the effect of inducer (PMA). However, the addition of Triton-X showed a fast increase in oxidation current for O2•− by about approximately 2 nA followed by a gradual drop which continued for a time span of more than 3 hours. We further investigated the effects of inhibitors for the activity of NADPH oxidase that catalytically produce ROS; apocynin were selected as inhibitors of NADPH oxidase [2].

The production of O2•−of real-time monitoring of NADPH oxidase inhibitory effect of PMA induced THP-1 cells using iron-porphyrin modified carbon electrode were detected by amperometory. The results shown in Fig.2 suggests that amperometory is mainly derived from the O2•−,whose expression is induced by NADPH oxidase.

[1] M. Yuasa et al. Polym. Adv. Technol. 16 (2005)287–292.

[2] S.Kasai et al. Analytica Chimica Acta 549 (2005)14-19.