Simultaneous Detection of Hydrogen Peroxide Production and Oxygen Consumption By Electrochemical Method in THP-1 Cells during Respiratory Burst

1. Introduction

Rapid consumption of oxygen and generation of various reactive oxygen species (ROS), collectively referred to as respiratory burst, occur in immune cells while they are in the processes of bacterial killing and phagocytosis as biological defense mechanisms. It is of significant importance in clinical fields to know the ROS generation mechanism and quantity therein. We have been studying electrochemical means to measure the respiratory burst in THP-1, a cell line from myeloid leukemia patients^{1) 2)}.

In this study, we aimed to develop an electrochemical sensor for simultaneous and real-time monitoring of the rapid oxygen consumption and hydrogen peroxide generation and to examine the time and amount of hydrogen peroxide generation and oxygen consumption using the sensor.

2.Experimental

2-1 Composition of 2ch-type electrochemical chip biosensor

A chip-type electrochemical sensor was used that comprised working electrode 1 (Pt electrode; f 20 µm) for oxygen detection, working electrode 2 (Os-HRP-modified Au electrode; f 1 mm) for hydrogen peroxide detection, a platinum electrode (1 × 3 mm) as the counter electrode, and a measurement well (internal diameter 5 mm, height 10 mm).

2-2 Measurement conditions

THP-1 cell suspension (3.0 × 10⁵ cells/well) was introduced into a well of this sensor, and phosphate buffered saline (PBS; Sigma chemicals, USA) and 11.4 mM glucose solution were used as measurement solutions. Ag/AgCl electrode was used as a control electrode and -0.5 V vs. Ag/AgCl and 0.0 V vs. Ag/AgCl were applied to working electrodes 1 and 2, respectively; the sample was measured for 90 minutes with 1-second sampling time. Twenty minutes after starting the measurement, PMA was added in drops to reach a final concentration of 20 nM or 200 nM, and each reduction current was monitored.

3. Results and Discussion

Simultaneous measurements of oxygen consumption and hydrogen peroxide generation in THP-1 using a 2ch-type electrochemical chip biosensor

Fig. 1 shows the result of the simultaneous measurements of the oxygen reduction current and hydrogen peroxide reduction current during the respiratory burst in THP-1 cells using a chip-type device. The value of oxygen reduction current dropped immediately after the drop-wise addition of PMA, and 10-20 minutes later, it came back to the same level as before the addition of PMA. This is likely due to the respiratory burst that occurred in THP-1 cells as a result of the rapid consumption of oxygen. Meanwhile, the hydrogen peroxide reduction current increased 15 minutes after starting PMA addition, and continuous generation was observed for approximately 60 minutes. This result suggests that hydrogen peroxide was generated for a long period of time.

When it contained no cells, the oxygen reduction current was about -5.5 nA. With this value as a standard (dissolved oxygen concentration 209 µM ³⁾), the dissolved oxygen concentration during normal respiration and respiratory burst in THP-1 cells was calculated and the generated hydrogen peroxide concentration was further determined from a calibration curve. As a result, the dissolved oxygen concentration during the normal respiration in THP-1 cells was determined to be around 125 µM. On the other hand, the dissolved oxygen concentration during the respiratory burst was determined to be 119 µM and 115 µM when the PMA concentration was 20 nM and 200 nM, respectively. Furthermore, the generated hydrogen peroxide was calculated to be 4.5 nM and 8.3 nM when the PMA concentration was 20 nM and 200 nM.

Based on these results, the oxygen consumption and hydrogen peroxide generation during the respiratory burst in THP-1 cells was suggested to be dependent on PMA concentration. Moreover, we confirmed that during the respiratory burst, rapid consumption of oxygen occurs first, and then the generation of hydrogen peroxide starts in a process returning to normal respiration and continues for about one hour. In the future, in addition to the bactericidal effect triggered by PMA stimulation, we plan to examine the respiratory burst and generation of hydrogen peroxide during phagocytosis and conduct more quantitative analysis.

References

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2. S. Kasai, T. Numata, H. Shiku, H. Abe, T. Matsue, Y. Niizeki, 215 th ECS meeting (2009).

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