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Gold electrodes have been used in a wide range of electrochemical biosensors because their functionalization process with thiols has been well described and, in general, they offer good chemical stability. However, one of the most commonly used redox-pairs in electrochemical impedance spectroscopy, Hexacyanoferrate (II)/(III), causes corrosion of the gold electrodes and consequently damages the surface modification. This leads to alterations of the sensing signals, and thus, renders the quantitative and sensitive detection of target molecules virtually impossible. To overcome this problem we introduced the in-situ generation of Hexaammineruthenium (II)/(III) as redox-pair during the impedimetric measurement by applying a DC-bias. This DC-bias was chosen in such a way that it supplied Hexaammineruthenium (II) in a suitable concentration at the electrode surface by reducing Hexaammineruthenium (III). We compared the stability of photolithographically fabricated thin-film and screen-printed gold electrodes in Hexacyanoferrate and Hexaammineruthenium solutions. Further, long-time characterization of the electrochemical properties with cyclic voltammetry and electrochemical impedance spectroscopy revealed that Hexaammineruthenium (II)/(III) was an excellent redox-pair for stable impedimetric measurements with gold electrodes. To demonstrate the suitability of Hexaammineruthenium for biosensing we applied it for the impedimetric detection of human-IgG. This biosensor exhibited a linear range from 11.3 ng/mL to 113 μg/mL, which is a suitable range for diagnostic applications.

For most electrochemical impedance spectroscopy measurements Ferro-/Ferricyanide is used as redox-probe, but it has limitations in its application for biosensors based on gold electrodes because of chemical degradation induced by the Ferro-/Ferricyanide. The in-situ reduction of [Ru(NH3)6]3+ to [Ru(NH3)6]2+ by means of a direct current during EIS measurements is introduced to generate a stable redox-probe for biosensors. This method of enhanced EIS measurement has been applied to determine the charge transfer resistance of a human-IgG biosensor with a linear range from 0.9 to 50 mg/L IgG.