Hydrogen Peroxide Production In Cells Research Articles

Characterization of Respiratory Burst By 3D CELL Chip with a through-Hole Type Electrochemical Sensing Device

1. IntroductionRapid consumption of oxygen and generation of various reactive oxygen species (ROS), collectively referred as respiratory burst, occur in immune cells while they are in the processes of bacterial killing and phagocytosis as biological defence mechanisms. The production of ROS is in trace amount compared to the oxygen consumption of immune cells. Therefore, it is important to simultaneously measure oxygen consumption and reactive oxygen species and to measure the conversion efficiency of respiratory burst phenomenon. In order to capture this phenomenon, we have developed a through-hole 2ch device for 3-dimensional cultured cells.2．Experiment2.1 Fabrication methodA through-hole type electrochemical sensing device fabrication was carried out in the following step [1-8].1) Sputtering film formation of Cr (20 nm) and Au (500 nm) on Silicon or glass substrate.2) Spin coating of positive photoresist at 3000 rpm, pre-baking at 100°C for 10 min, exposure, development, post-baking at 120°C for 10 min to form the electrode shape. 3) Using photoresist as a mask, wet etching is performed in the order of Au and Cr to form an electrode and the photoresist is removed by immersing in an acetone solution.4) Spin coating the photoresist at 3000 rpm, prebake at 100°C for 2 minutes, exposure, post-exposure bake at 100°C for 2 minutes and developed to form the electrode shape.5) Sputtering film formation in the order of Cr (20 nm) and Pt (300 nm).6) Ultrasonic cleaning while dipping in acetone, forming the electrode lift-off.7) The pore portion of the device was formed by Deep RIE.8) Spin coating the negative type photoresist at 3000 rpm, prebake at 100 °C for 10 minutes, exposure and development, post-baking at 120°C for 10 minutes to form the insulating film shape. 2.2 Preparation of THP-1 cell chip of through-hole type electrochemical sensing deviceTHP-1 cells (7.3 × 105 cells / mL) on the fourth day after passage were suspended in a mixed solution of Cellmatrix Type IA, 5×RPMI-1640 culture solution and buffer for reconstitution, 1uL of which was inoculated into a through hole type device. Thereafter, the cells were incubated at 37 °C and 5% CO2 for 15 minutes to jellify the collagen, thereby forming a cell chip. 2.3 Real-time monitoring of respiratory burst of THP-1 cells with a through-hole electrochemical sensing deviceTHP-1 cells were measured in the sensor well at a constant temperature of 30 ± 0.5° C. Cells were suspended in PBS in presence of glucose at the concentration of 11.4 mM. PMA was added dropwise after 5 min of starting of measurement with a concentration of 200 nM and simultaneous reduction currents were measured. Oxygen reduction current was measured at −0.5 V vs. Ag/AgCl and oxidation current for H2O2 was measured at 0.7 V vs. Ag/AgCl at room temperature. 3. Results and DiscussionFigure 2 shows the result of the simultaneous measurement of the oxygen reduction current and reduction current for H2O2 during the respiratory burst in THP-1 cells with a chip type biosensor device. Oxygen reduction current value was found to decrease immediately after dropwise addition of 200nM PMA. It can be concluded that oxygen consumption and H2O2 production during respiratory burst are a phenomenon which are linked together. The continuous production of H2O2 during the respiratory burst and its longer half-life due to low reactivity can be correlated to the involvement of H2O2 in signalling. ReferenceA. Prasad, H. Kikuchi, K. Inoue, M. Suzuki, Y. Sugiura, T. Sugai, A. Tomonori, M. Tada, M. Kobayashi, T. Matsue, S. Kasai: Simultaneous Real-Time Monitoring of Oxygen Consumption and Hydrogen Peroxide Production in Cells Using Our Newly Developed Chip-Type Biosensor Device.Frontiers in Physiology 7, 109, 1-10 (2016) Figure 1

Simultaneous Real-Time Monitoring of Oxygen Consumption and Hydrogen Peroxide Production in Cells Using Our Newly Developed Chip-Type Biosensor Device

All living organisms bear its defense mechanism. Immune cells during invasion by foreign body undergoes phagocytosis during which monocyte and neutrophil produces reactive oxygen species (ROS). The ROS generated in animal cells are known to be involved in several diseases and ailments, when generated in excess. Therefore, if the ROS generated in cells can be measured and analyzed precisely, it can be employed in immune function evaluation and disease detection. The aim of the current study is to introduce our newly developed chip-type biosensor device with high specificity and sensitivity. It comprises of counter electrode and working electrodes I and II. The counter electrode is a platinum plate while the working electrodes I and II are platinum microelectrode and osmium-horseradish peroxidase modified gold electrode, respectively which acts as oxygen and hydrogen peroxide (H2O2) detection sensors. Simultaneous measurement of oxygen consumption and H2O2 generation were measured in animal cells under the effect of exogenous addition of differentiation inducer, phorbol 12-myristate 13-acetate. The results obtained showed considerable changes in reduction currents in the absence and presence of inducer. Our newly developed chip-type biosensor device is claimed to be a useful tool for real-time monitoring of the respiratory activity and precise detection of H2O2 in cells. It can thus be widely applied in biomedical research and in clinical trials being an advancement over other H2O2 detection techniques.

Hydrogen peroxide and lignification

The production of hydrogen peroxide in plant tissue is demonstrated quickly with a simple histochemical test. The test solution, 50 mM potassium iodide in a 4% (w/v) potato starch suspension, is applied to the cut surface of the tissue to be tested. Hydrogen peroxide oxidizes iodide ions to iodine; the iodine is complexed by the starch to form a blue‐purple color. This test detects hydrogen peroxide production in cells undergoing lignification, i.e. tracheary elements and phloem fibers, and in some epidermal cells. In addition there is a rapid production of hydrogen peroxide in crushed cells. The test is negative under (i) anaerobic conditions and (ii) in the presence of catalase.