Ferrocenylethyl Phosphate: An Improved Substrate for the Detection of Alkaline Phosphatase by Cathodic Stripping Ion-Exchange Voltammetry. Application to the Electrochemical Enzyme Affinity Assay of Avidin

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Abstract
The ferrocenylethyl phosphate disodium salt was synthesized and used as a new substrate for alkaline phosphatase (AP). The enzyme-generated ferroceneethanol was selectively and sensitively detected at a Nafion film-coated electrode by anodic preconcentration of the ferricinium salt, followed by cyclic voltammetry. The accumulated ferricinium units could be expelled from the polymer film in their neutral form by cathodic stripping, and so the Nafion-modified electrode could be reused for more than 10 measurements with a standard deviation less than 3%. Values of 0.75 mM for the Michaelis constant and 1.42 μmol s-1 (mg of protein)-1 for the maximal velocity were found. The regenerable Nafion-coated electrode was employed for the indirect detection of AP down to 2 × 10-12 M and for the noncompetitive heterogeneous enzyme assay of avidin, whose detection limit was 5 × 10-12 M.