Impedance spectroscopy on immobilized streptavidin horseradish peroxidase layer for biosensing

In this work, a mixed self-assembled monolayer (SAM) formed by 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl) (biotinyl-PE) and 16-mercaptohexadecanoic acid was grafted on gold electrode. Horseradish peroxidase conjugated streptavidin was attached on the SAM through biotin–streptavidin interaction. Cyclic voltammetry and impedance spectroscopy were applied to characterize the electric properties of the self-assembled monolayer and to monitor biosensor building-up process. During impedance measurements, the charge transfer resistance decreased significantly after enzymatic reaction with hydrogen peroxide. The response was very sensitive in the range of 10–100 mM and the detection limit attained 10 μM. The constructed biosensor was successfully tested in milk samples and was able to detect as low as 50 μM of hydrogen peroxide.