The performance of the reference electrode is sometimes overlooked in the development of new electrochemical biosensors, which may result in a significant reduction in function even under standard operating conditions. This paper investigates the worst-case analytical errors, mainly addressed through finite element modelling and further supported by experimental data. The specific case of a two-electrode mediated amperometric biosensor utilizing Ag/AgCl as a combined counter and

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... electrode was modelled numerically. The reference electrode was found to shift 5 mV for every 20 mM change in analyte concentration. In addition, a gradual increase with time was observed. This deviation in reference potential resulted in a lowering of the current magnitude with increasing analyte concentration due to the operating potential of the working electrode being shifted closer to the reversible potential of the electrode reaction. The analytical deviation was measured to 14% away from the ideal value following a titration curve from 5 mM to 25 mM over 150 s. The combined counter/pseudo-reference electrode was further studied experimentally during current load in a ferro/ferricyanide mediator system and compared to that of using separate counter and pseudo-reference electrodes. A linear 7 mV μA −1 cm −2 potential shift-to-current relation was deduced for the former case, whereas the potential remained stable when separate reference and counter electrodes were used.