Bipolar Clark-Type Oxygen Electrode Arrays for Imaging and Multiplexed Measurements of the Respiratory Activity of Cells.

Various miniature Clark-type oxygen electrodes (COEs), which are typically used to measure dissolved oxygen (DO) concentration in cellular respiration, have been developed since the 1980s. Arrays with individually addressable electrodes that constitute the sensor were used for various applications. However, the large number of leads and contact pads required for connecting the electrodes and the external instrument complicate the electrode layout and make the operation of integrated COE arrays challenging. Here, we fabricated closed bipolar electrochemical systems comprising 6 × 8 and 4 × 4 arrays of COEs for imaging and multiplexed detection. The cathodic compartment was sealed with a hydrophobic oxygen-permeable membrane to separate the internal electrolyte solution from the sample solutions. Using the bipolar Clark-type oxygen electrode (BCOE) arrays and electrochemiluminescence (ECL), we measured the DO concentration at each cathode. The results revealed that the ECL intensity changed linearly with the DO concentration. In addition, we used ECL imaging to investigate the respiratory activity of Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa) in suspensions with different cell densities. The ECL images showed that the ECL intensity changed noticeably with the bacterial density. The bacterial respiratory activity was then qualitatively analyzed based on the ECL images acquired successively over a time duration. Further, we measured the antibiotic efficacy of piperacillin, oxacillin, gentamicin, and cefmetazole against E. coli and P. aeruginosa using the BCOE. We found that the ECL intensity increased with the antibiotic concentration, thus indicating the suppression of the bacterial respiratory activity.

Bipolar Electrode Arrays for Chemical Imaging and Multiplexed Sensing.

Bipolar electrodes (BPEs) with arrays of cathodic and anodic poles were developed for use in closed bipolar systems. To increase the number of BPEs in the array, the anodic and cathodic poles were connected with each other using thin leads. A further increase in the number of BPEs was achieved by forming the cathodic and anodic poles of the BPEs and the leads in different layers. A device with 9 × 10 arrays of cathodes and anodes was thus realized. When using this device to sense hydrogen peroxide (H2O2), the sensitivity and linear range of calibration plots could be adjusted by changing the driving voltage and the area ratio between the cathodic and anodic poles. The devices were used to image H2O2 and obtain time-lapse images for the diffusion and dilution of H2O2. Furthermore, DNA detection was demonstrated using an electroactive intercalator. The sensitivity could be improved by making the anodic poles smaller with respect to the cathodic pole and concentrating the electrochemiluminescence (ECL) in a small area. The ECL intensity changed according to the target DNA concentration in the solution.

Wired Microfabricated Electrochemical Systems.

Metal wires have been used as an alternative to liquid junctions for the connection of solutions in microfabricated electrochemical devices. They exhibit similar performance to liquid junctions, provided that the interfacial potentials at both ends of the wires were appropriately canceled. Cyclic voltammograms of devices with liquid junctions and metal wires were very similar when no current or a low current flowed through the metal wire between the working and reference electrodes. Iridium wires with iridium oxide at both ends facilitated canceling of the interfacial potentials at either end of the junction particularly well, and were used effectively for voltammetry, amperometry, and potentiometry by adjusting the pH of the solutions in the working and reference electrode compartments to be equal. This approach was used to effectively integrate a reliable common reference electrode between multiple working electrodes and to conduct automated electrochemical control of solution transport in microfluidic systems.

Microdevice with an Integrated Clark-Type Oxygen Electrode for the Measurement of the Respiratory Activity of Cells.

A microdevice for the measurement of the respiratory activity of cells was fabricated using a microfabricated Clark-type oxygen electrode. The oxygen electrode was completed in a dry state and was activated by introducing water necessary for the reduction of oxygen in the form of water vapor through an oxygen-permeable membrane, which significantly facilitated handling of the device even by nonspecialists. The use of a thin paper layer stabilized the current response and enabled stable continuous operation of the oxygen electrode without current disturbance caused by the evaporation of water. The microdevice was tested in some model experiments including the measurement of the respiratory activity of Escherichia coli (E. coli), evaluation of the efficacy of antibiotics, and measurement of the antibacterial activity of neutrophils, all of which demonstrated that the consumption of dissolved oxygen by cells can be monitored clearly by following an easy procedure for the preparation of the measurements.