Microdevice for the Measurement of Respiratory Activity of Cells

Abstract

Monitoring of the respiratory activity of animal and microbial cells in solutions of small volumes is required in many situations. For this purpose, devices based on electrochemical principles are advantageous, because miniaturization, batch-fabrication, and integration are easily realized. In our previous work, a device with integrated Clark-type oxygen electrode was developed to measure the bactericidal activity of neutrophils [1][2]. However, problems remain in the preparation of the device by non-experts. In addition, the output current often became unstable by evaporation of water in the electrolyte solution.To solve these problems, we propose a novel structure shown in Figure 1. The structure including a platinum cathode, an Ag/AgCl anode, an electrolyte layer, and a silicone rubber oxygen-permeable membrane were completed in a dry state. The electrolyte and other solution components were impregnated in a thin paper disc. Prior to use, water which is necessary for the reduction of oxygen was introduced into the electrolyte layer through the oxygen-permeable membrane in the form of water vapor simply by immersing the device in water for 8 – 12 hours.In operating the device, −0.8 V was applied to the cathode with respect to the anode using a potentiostat. 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. After the activation, the output current in the air-saturated water was stabilized and the current response to the change in the dissolved oxygen concentration became distinct. The 90% response time of a fully activated device was approximately 30 seconds.To measure the respiratory activity of cells, a chamber made with PMMA (polymethyl methacrylate) was placed on the oxygen permeable membrane of a fully activated device, and a suspension of cells was injected into the chamber. E. coli and MSSA (methicillin-susceptible Staphylococcus aureus) used in this study were purchased from Sigma Aldrich. MRSA (methicillin-resistant Staphylococcus aureus) was isolated from the clinical specimens of a patient in the National Defense Medical College Hospital and grown on the brain heart infusion (BHI) agar supplemented with oxacillin for 48 h at 37 °C under aerobic conditions. Suspensions of bacteria mentioned above were all stored in 20% glycerin at −80 ºC until usage. Prior to the experiments, suspensions of bacteria were thawed and were finally prepared in BHI medium with the desirable concentration. Incubation for the cells was carried out by placing the device on a hotplate maintained at 37 ºC.The respiratory activity of E. coli was first monitored using suspensions of different concentrations. Distinct current changes were observed at E. coli concentrations higher than 106 CFU/mL, and the current decrease was larger with E. coli of higher concentrations. Next, change in the respiratory activity of Staphylococcus aureus suspensions treated with or without antibiotics was monitored for 80 minutes. In this experiment, 1×107 CFU/mL Staphylococcus aureus suspensions of susceptible strain (MSSA) and resistant strain (MRSA) treated with or without 1 µg/mL oxacillin were used as samples. As time elapsed, current decreased distinctly due to the oxygen consumption of bacteria both in the MSSA and MRSA suspensions without oxacillin. Distinct current decrease was also observed in the MRSA suspension treated with oxacillin. On the other hand, in the MSSA suspensions treated with oxacillin, the current decrease was smaller and was almost suppressed following the incubation. These results demonstrated that our device can detect the change in dissolved oxygen concentration caused by the respiration of bacteria.[1] K. Tanabe, M. Kinoshita, M. Nakashima, K. Kariya, M. Yokokawa, Y. Morimoto, H. Suzuki, Med. Devices Sens., 2, 1–7 (2019).[2] A. Yamagishi, K. Tanabe, M. Yokokawa, Y. Morimoto, M. Kinoshita, and H. Suzuki, Anal. Chim. Acta., 985, 1–6 (2017). Figure 1