Development of stable and reproducible biosensors based on electrochemical impedance spectroscopy: Three-electrode versus two-electrode setup

•We compared the performance of a two-electrode and a three-electrode setup.•We carried out electrochemical impedance spectroscopy (EIS) measurements of the double layer capacitance upon changes of the molecular layer.•We performed and followed in-situ and in real-time the hybridization of DNA monolayers self-assembled on the gold electrodes’ surface.•We showed that the three-electrode setup is the most stable configuration, reproducible and suitable for real-time measurements.

This work focuses on the development of electrochemical impedance biosensors based on capacitance readout, for the detection of biomolecules in small sample volumes. We performed electrochemical impedance spectroscopy (EIS) measurements of DNA hybridization in electrochemical cells with microfabricated gold electrodes. The time stability of the device was tested in two different configurations: two microelectrodes in a microfluidic channel; two microelectrodes plus a reference electrode in an electrochemical cell. Our results demonstrate that the three-electrode setup is more stable, more reproducible, and suitable for real-time measurements. In the last part of the work we perform a test study of DNA hybridization in real time, and we show that the three-electrode configuration can measure the process in situ and in real time.