Highly sensitive electrochemical detection of alkaline phosphatase

Alkaline phosphatase (ALP), a labeling enzyme frequently used for enzyme immunoassay, was determined at the attomole level using a mushroom tyrosinase (TN)-embedded carbon paste electrode. The detection scheme consists of two successive amplifications. The first reaction is the ALP-catalyzed hydrolysis of phenyl phosphate to accumulate phenol as a product. The second is the bioelectrocatalytic detection of phenol with the TN-embedded electrode at 0 V versus Ag/AgCl, which allowed sensitive detection of phenol down to 35 nM. The two-enzymatic reactions of ALP and TN were made to proceed successively in one electrochemical cell by adjusting pH to their optimum ones. This method provided a Linear relation between the ALP concentration and the steady-state current and allowed the ALP detection as low as 7 amol/10 mul sample. The ALP detection system was applied to a streptavidin-biotin binding assay. The lowest detection limit of biotin was 4 fmol/10 mul sample with an ALP reaction time of 50 min. (C) 2000 Elsevier Science B.V. All rights reserved.