Comparative study of 4-aminophenyl phosphate and ascorbic acid 2-phosphate, as substrates for alkaline phosphatase based amperometric immunosensor

The performance of ascorbic acid 2-phosphate (AAP), an alternative substrate for alkaline phosphatase (AP), was compared with a widely used 4-aminophenyl phosphate (pAPP). AP is used here as a label in enzyme immunoassay with electrochemical detection. Linear sweep voltammetry and amperometry were used. Optimal working potentials were investigated at four electrode materials such as Pt, Au, glassy carbon, and carbon screen printed electrode (SPE). Two configurations of SPE were investigated, such as amperometry in a stirred batch and a single-drop analysis at a coplanar three-electrode strip. In both systems, at all four electrodes, pAP required significantly lower detection potentials yielding significantly higher signals. Enzyme kinetics of both substrates with AP was examined. The system has been applied to an enzyme immunoassay of rabbit IgG with AP as a label.