3-indoxyl phosphate as an electrochemical substrate for horseradish peroxidase

In this work 3-indoxyl phosphate (3-IP), an alkaline phosphatase substrate, is demonstrated to be a suitable substrate for horseradish peroxidase (HRP). HRP catalyzes the oxidation of 3-IP in presence of hydrogen peroxide (H2O2) generating the product indigo blue, which is an aromatic heterocycle compound insoluble in aqueous solutions. This product was easily converted into its soluble parent compound indigo carmine (IC) (by addition of fuming sulfuric acid to the reaction media) which has a reversible voltammetric peak at the formal potential of - 0.15 V (vs. Ag pseudo-reference electrode) when a screen-printed carbon electrode (SPCE) is used. Parameters that influence the enzymatic reaction, such as pH, temperature, substrate concentration and reaction time have been optimized. Moreover, the enzyme apparent kinetic constants (V-max, K-M) for both substrates (3-IP and H2O2) have been calculated. Indirect measurements of HRP activity in solution were carried out not only by cyclic voltammetry but also using amperometric detection in a flow system. The detection limits were 6.86 x 10(-12) and 5.68 x 10(-12) M, respectively. Thus, 3-IP is the first substrate that could be used for alkaline phosphatase (AP) and HRP, the most common enzymatic labels in affinity assays.