Spectrophotometric determination of the activity of alkaline phosphatase and detection of its inhibitors by exploiting the pyrophosphate-accelerated oxidase-like activity of nanoceria

The oxidase-like activity of nanoceria is low. This limits its practical applications. It is demonstrated here that pyrophosphate ion (PPi) can improve the oxidase-like activity of nanoceria. Specifically, nanoceria catalyzes the oxidation of colorless 3,3′,5,5′-tetramethylbenzidine (TMB) to give a blue product (oxTMB) with an absorption peak at 645 nm in the presence of PPi. If, however, alkaline phosphatase (ALP) is present, it will hydrolyze PPi, and this results in a decreased oxidase-like activity of nanoceria. Hence, less blue oxTMB willl be formed. On the other hand, if the ALP inhibitor Na3VO4 is added to the system, the oxidase-like activity of nanoceria is gradually restored. On the basis of the above results, a spectrophotometric method was developed for determination of the activity of ALP. It works in the 0.5 to 10 mU.mL−1 activity range and has a 0.32 mU.mL−1 detection limit. Na3VO4 causes a 50% ALP inhibition if present in 71 μM concentration. The assay was successfully applied to the determination of ALP in spiked human serum and gave good recoveries.