An impedimetric determination of alkaline phosphatase activity based on the oxidation reaction mediated by Cu(2+) bound to poly-thymine DNA

We herein describe a novel impedimetric method to determine alkaline phosphatase (ALP) activity based on the Cu(2+)-mediated oxidation of ascorbic acid on a specific DNA probe-modified electrode. In this method, pyrophosphate (PPi) capable of complexing with Cu(2+) 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 Cu(2+). The free Cu(2+) 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 (R(et)) by interrupting the electron transfer on the electrode. On the other hand, in the absence of ALP, PPi remains intact to effectively capture Cu(2+), consequently preventing the oxidation of ascorbic acid and the subsequent increase of R(et). Based on this design principle, the change in R(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.