Monitoring of the Respiratory Activity of Cells Using a Microdevice with Microfabricated Clark-Type Oxygen Electrode

Abstract

Introduction With the progress of cell engineering and the increase in the necessity to understand status of human body, measurement of the respiratory activity of animal and microbial cells in solutions of small volumes is required in many situations. In this trend, we have also developed a device with integrated Clark-type oxygen electrode to measure the antibacterial activity of neutrophils [1][2]. Although obtained results demonstrated the utility of the device, preparation and handling of the device needed expertise to obtain stable current output by avoiding unintended introduction of small bubbles into the electrolyte solution that influence the output current of the oxygen electrode. In addition, evaporation of water limited the lifespan of the oxygen electrode.To solve the problems, we propose a novel oxygen electrode structure. The structure including the cathode and anode, an electrolyte layer, and an oxygen-permeable membrane were completed in a dry state. Prior to use, water necessary for the reduction of oxygen was introduced into the electrolyte layer in the form of water vapor through the oxygen permeable membrane by simply immersing the chip in water, which remarkably simplified handling by end-users. In addition, we used sorbitol in the electrolyte solution to suppress the evaporation of water. We employed the device to monitor the respiratory activity of E.coli. Furthermore, the device was used to measure the antibiotic resistance of bacteria. Method Figure 1 shows the structure of the device. The oxygen electrode consisted of a platinum cathode (diameter: 1 mm) and Ag/AgCl anode formed on a glass substrate, a reservoir to accommodate an electrolyte solution, a circular paper to impregnate the electrolyte solution, and a silicone rubber oxygen-permeable membrane. Electrode patterns were formed by sputtering and lift-off. Platinum patterns were formed for the cathode and the base layer of the anode with a chromium intermediate layer. Silver patterns was additionally formed only on the anode area. A polyimide insulating layer was formed to delineate the active areas of the electrodes. The reservoir for an electrolyte solution was formed with SU-8 (Kayaku Advanced Materials). A circular paper (diameter: 3 mm) immersed in pure water was moved onto the cathode and anode in the SU-8 reservoir. Following this, 2 μL of 2% PVP solution was poured onto the paper. The solution was prepared with 1.0 M Tris-HCl buffer solution (pH 8.3) containing 1.0 M KCl, 4.0wt% sorbitol. After drying the electrolyte layer at ~30%RH for an hour, a silicone adhesive (KE-3475-T, Shin-Etsu Chemical) was spin-coated to form the oxygen-permeable membrane (thickness: ~100 μm). The oxygen electrode was activated by introducing water into the electrolyte layer through the oxygen permeable membrane in the form of vapor by immersing the entire chip in pure water. To measure the respiratory activity of cells, a PMMA chamber was formed and was tightly fixed on the oxygen electrode.In operating the device, -0.8 V was applied to the cathode with respect to the anode using an Autolab PGSTAT 128N potentiostat (Metrohm Autolab, Netherlands). The response of the oxygen electrode was checked by immersing the active area into air-saturated pure water and removing dissolved oxygen by adding Na2SO3. To measure the respiratory activity of E. coli, freeze-dried E.coli was activated by resuspending it in PBS containing 100 mM glucose. Solutions of different cell densities were prepared. The solutions were injected into the sample chamber, and the respiratory activity of E. coli was measured. Evaluation of the antibiotics resistance was performed using 1×107 CFU/mL E. coli suspension including 5 mg/mL piperacillin sodium salt. Results and Conclusions The output current was stabilized after 8 - 12 h of activation (Figure 2). Figure 3 shows the response curve of the oxygen electrode. The output current in air saturated water was stable, and the current decreased clearly when the dissolved oxygen was removed by adding Na2SO3. The 90% response time was 30 s. Figure 5 shows that glucose enhances the respiratory activity of E. coli at glucose concentrations higher than 100 mM. Figure 6 shows the current decrease depended on the density of E. coli and was significant in the range of E. coli density higher than 106 CFU/mL. The current decrease is directly correlated with oxygen consumption by E. coli., which also reflects the number of living E. coli. Figure 7 shows the response curves obtained with E. coli suspension containing piperacillin. In the presence of the antibiotics, current decrease was suppressed compared with the case without the antibiotics, demonstrating that the device can also be used for the determination of the minimum inhibitory concentration of antibiotics.