Novel electrochemical methodology for activity estimation of alkaline phosphatase based on solubility difference

We propose a novel electrochemical detection system for alkaline phosphatase (ALP) activity using the difference in water and oil solubilities between the substrate, ferrocene ethyl phosphate ester (FcEtOPO₃ ^2-), and the enzymatic product, ferroceneethanol (FcEtOH). In this system, water droplets containing ALP and FcEtOPO₃^2- were placed on a Pt disk microelectrode and surrounded by a mineral oil. By the ALP-catalyzed reaction, FcEtOPO₃^2- was converted to FcEtOH, which was then transferred to the mineral oil from the water droplets with FcEtOPO₃^2- remaining in the water droplets. After partitioning FcEtOH from the water droplets, FcEtOPO₃^2- was detected at the Pt disk microelectrode to estimate the ALP activity. Using this novel system, the ALP activity of embryoid bodies was successfully detected. We believe that the present system will be widely applicable to ALP-based bioassays.