Enzyme-Modified\nMicroelectrode for Simultaneous Local\nMeasurements of O<sub>2</sub> and pH

The use of miniaturized probes opens a new dimension\nin the analysis\nof (bio)chemical processes, enabling the possibility to perform measurements\nwith local resolution. In addition, multiparametric measurements are\nhighly valuable for a holistic understanding of the investigated process.\nTherefore, different strategies have been suggested for simultaneous\nlocal measurements of various parameters. Electroanalytical methods\nare a powerful strategy in this direction. However, they have been\nmainly restricted to coupling concurrent independent measurements\nwith different miniaturized probes. Here, we present an enzymatic\nmicrobiosensor for the simultaneous detection of O<sub>2</sub> and\npH. The sensing strategy is based on the pH-dependent bioelectrocatalytic\nprocess associated with O<sub>2</sub> reduction at a gold microelectrode\nmodified with a multicopper oxidase. After initial investigations\nof the bioelectrocatalytic reaction over gold macroelectrodes, the\nfabrication and characterization of micrometer-sized probes are presented.\nThe microbioelectrode exhibits a linear current increase with O<sub>2</sub> concentration extending to 17.2 mg L<sup>–1</sup>,\nwith a sensitivity of (5.56 ± 0.13) nA L mg<sup>–1</sup> and a limit of detection of (0.5 ± 0.3) mg L<sup>–1</sup>. Moreover, a linear response allowing pH detection is obtained between\npH 5.2 and 7.5 with a slope of −(47 ± 8) mV per pH unit.\nIn addition, two proof-of-concept analytical examples are shown, demonstrating\nthe capability of the developed sensing system for simultaneous local\nmeasurements of O<sub>2</sub> and pH. Compared with other miniaturized\nprobes reported before for simultaneous detection, our strategy stands\nout as the two investigated parameters are acquired from the very\nsame measurement. This strategy greatly simplifies the analytical\nsetup and for the first time provides truly simultaneous local detection\nin the micrometer scale.