Indirect determination of alkaline phosphatase based on the amperometric detection of indigo carmine at a screen-printed electrode in a flow system

Amperometric analysis of indigo carmine at a bare screen-printed electrode placed in an FIA system is reported. This compound is easily detected at a potential of -0.3 V (vs. Ag pseudo-reference electrode) without observing any fouling of the electrode surface, thus allowing the repetitive use of the same electrode in a reproducible manner (coefficients of variation down to 7% for more than 20 consecutive determinations). A linear range of three orders of magnitude and a limit of detection in the sub-micromolar range were attained for this molecule. Based on these studies, indirect amperometric measurements of alkaline phosphatase (ALP) activity in solution were easily carried out using 3-indoxyl phosphate substrate. Its hydrolysis catalyzed by ALP gave rise to indigo product. This product is insoluble in aqueous solutions but it was easily converted into its soluble parent compound, indigo carmine, by addition of fuming sulfuric acid to the reaction media. Using this approach. we achieved a linear range of more than one order of magnitude and a limit of detection of 1 U/l ALP. for an enzymatic reaction time of 60 min.