Characterization of Respiratory Burst By 3D CELL Chip with a through-Hole Type Electrochemical Sensing Device
Authors: Kasai Shigenobu , Sato Yusaku , Prasad Ankush , Sato Teruya 
Published: February 2020

1. Introduction
Rapid 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．Experiment
2.1 Fabrication method
A 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 device
THP-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 device
THP-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 Discussion
Figure 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.
Reference
A. 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