Voltammetric determination of alkaline phosphatase and horseradish peroxidase activity using 3-indoxyl phosphate as substrate Application to enzyme immunoassay

The use of 3-indoxyl phosphate (3-IP) as an electrochemical substrate for ELISAs with voltammetric detection was investigated. Indirect measurements of alkaline phosphatase (AP) and horseradish peroxidase (HRP) activity in solution were carried out. Picomolar levels of both enzymes can be detected, which enables the design of electrochemical immunoassays using this substrate. The enzymatic turnover of the substrate gives indigo blue, insoluble in aqueous solutions. This product is easily converted into its soluble parent compound, indigo carmine (IC), by addition of fuming sulphuric acid to the reaction media. IC shows a reversible voltammetric peak at the formal potential of −0.15 V (versus Ag pseudo-reference electrode) when a screen-printed carbon electrode (SPCE) is used. The peak current of this process constitutes the analytical signal. Using this approach an ELISA assay to quantify pneumolysin (PLY, a toxin related to respiratory infections) was carried out using AP or HRP as enzymatic label. Calibration plots obtained are reported. 3-IP is demonstrated to be the first suitable substrate for the two most common enzyme labels used in immunoassays.