Biosensing based on electrochemical impedance spectroscopy: Influence of the often-ignored molecular charge

When measurement of electrochemical impedance is used as an analytical method for affinity biosensors, its increase is in the most cases considered as a proof of specific binding. The increase of the mass in the protein layer is expected to be the cause. However, we encountered severe inconsistencies for these assumptions. In our experiments during the development of an immunosensor, proteins with diverse charges were immobilised and captured on the electrode surface. The measured values of impedance were predominantly affected by the charge of these proteins - negatively charged proteins caused (an expected) increase of the impedance values. Contrary, immobilization/capture of positively charged proteins and other compounds caused (a surprising) relative decrease of the impedance. The obvious and logical origin of this behaviour - a repulsive or attractive ionic interactions between protein layers and negatively charged redox probe [Fe(CN)(6)](4-/3-) (commonly used in electrochemistry) - is usually neglected throughout the literature although some fragmental information could be traced. Besides, we want to emphasize here the importance of the additional characterization of the studied system using for example gel electrophoresis, since many unexpected and overlooked factors can influence the electrochemical response upon analyte binding.