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 Na 3 VO 4 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. Na 3 VO 4 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. Schematic presentation of pyrophosphate (PPi)-induced acceleration of the oxidase-like activity of nanoceria (CeO 2 ) for determination of alkaline phosphatase enzyme (ALP) activity and its inhibitor NaVO 3 .