A novel electrochemical impedance spectroscopy (EIS) sensor design, based on a standard interdigitated electrode arrangement in which the working electrode consists of gold and the combined counter and reference electrodes of polypyrrole doped with polystyrene sulfonate (PPy:PSS), is evaluated for biosensing applications. The performance is successfully proved by immobilization of a thiolated biotin as a self‐assembled monolayer (SAM), followed by streptavidin and a biotinylated horseradish peroxidase. It is shown that specific binding of biomolecules takes place only at the gold electrode. The binding activities are not influenced by the addition of small amounts of the nonionic surfactant Pluronic F‐68. The immobilization process is monitored online with EIS showing an excellent repeatability of the EIS signals in comparison with Au–Au electrode configuration even after electrode regeneration.

Monitoring glycosyltransferases on biosensors is of great interest for pathogen and cancer diagnostics. As a proof of concept, we here demonstrate the layer-by-layer immobilization of a multivalent neoglycoprotein (NGP) as a substrate for a bacterial fucosyltransferase (FucT) and the subsequent binding of the fucose-specific Aleuria aurantia lectin (AAL) on an electrochemical impedance spectroscopy (EIS) sensor. We report for the first time the binding kinetics of a glycosyltransferase in realtime. Highly stable EIS measurements are obtained by the modification of counter and reference electrodes with polypyrrole: polystyrene sulfonate (PPy:PSS). In detail, the N-acetyllactosamine (LacNAc)-carrying NGP was covalently immobilized on the gold working electrode and served as a substrate for the FucT-catalyzed reaction. The LacNAc epitopes were converted to Lewis x (Le x ) and detected by AAL. AAL binding to the Le x epitope was further confirmed in a lectin displacement and a competitive lectin binding inhibition experiment. We monitored the individual kinetic processes via EIS. The time constant for covalent immobilization of the NGP was 653 s. The FucT kinetics was the slowest process with a time constant of 1121 s. In contrast, a short time constant of 11.8 s was determined for the interaction of AAL with the modified NGPs. When this process was competed by 400 mM fucose, the binding was significantly slowed down, as indicated by a time constant of 978 s. The kinetics for the displacement of bound AAL by free fucose was observed with a time constant of 424 s. We conclude that this novel EIS biosensor and the applied workflow has the potential to detect FucT and other GT activities in general and further monitor protein−glycan interactions, which may be useful for the detection of pathogenic bacteria and cancer cells in future biomedical applications.