Practical considerations for using redox probes in electrochemical sensor characterization

Despite advancements in electrochemical sensor fabrication, particularly through electrode manufacturing using 3D printing technology, their electrochemical characterization remains challenging. The main obstacles arise from potential misinterpretation and incorrect conclusions when characterizing electrochemical sensors using redox probes. [Ru(NH3)6]3+/2+ and [Fe(CN)6]3- /4- , the most common redox probes, are often categorized as outer-sphere redox species, however, their electron transfer processes may differ significantly. Additional challenges emerge when using these redox probes to assess the working electrode area, which is frequently mistaken for the electrochemically active surface area (the so-called "real area"). Also, the effect of the working electrode roughness on the analytical performance of the sensor is frequently overestimated, and generalizations such as "nanostructured electrodes always lead to superior analytical performance" is commonly find in literature. In this study, we demonstrated that for outer-sphere electron transfers, the roughness of the working electrode has minimal effect on the voltammetric response. We have also shown that the charge transfer resistance (Rct) determined by Electrochemical Impedance Spectroscopy (EIS) in the presence of [Fe(CN)6]3-/4- is highly dependent on the working electrode area. Thus, increasing the working electrode area always decreases Rct, which should not be misinterpreted as an improved electron transfer rate. Therefore, this study provides insights that could be useful in preventing common mistakes and misinterpretations during the characterization of electrochemical sensors. Furthermore, it constitutes a valuable resource for the training of early-career scientists in the field of electroanalysis.