An impedimetric determination of alkaline phosphatase activity based on the oxidation reaction mediated by Cu²⁺ bound to poly-thymine DNA

Abstract

We herein describe a novel impedimetric method to determine alkaline phosphatase (ALP) activity based on the Cu²⁺-mediated oxidation of ascorbic acid on a specific DNA probe-modified electrode. In this method, pyrophosphate (PPi) capable of complexing with Cu²⁺ is employed as a substrate of the ALP enzyme. In the presence of ALP, PPi is hydrolyzed to phosphate (Pi), which is not able to entrap Cu²⁺. The free Cu²⁺ are specifically bound to a poly-thymine DNA probe immobilized on the electrode surface and reduced to form copper nanoparticles by a concomitant oxidation of ascorbic acid. As a result, the oxidation products of ascorbic acid are accumulated on the electrode surface, which consequently increase electron transfer resistance (<i>R</i> _et) by interrupting the electron transfer on the electrode. On the other hand, in the absence of ALP, PPi remains intact to effectively capture Cu²⁺, consequently preventing the oxidation of ascorbic acid and the subsequent increase of <i>R</i> _et. Based on this design principle, the change in <i>R</i> _et, which is proportional to ALP activity, was measured by electrochemical impedance spectroscopy (EIS) and ALP activities were successfully determined down to 6.5 pM (7.2 U L^-1) with excellent selectivity.