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

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Abstract

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.

Schematic presentation of pyrophosphate (PPi)-induced acceleration of the oxidase-like activity of nanoceria (CeO2) for determination of alkaline phosphatase enzyme (ALP) activity and its inhibitor NaVO3.

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Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (21705056), the Young Taishan Scholars Program of Shandong Province (tsqn201812080), the program for Taishan Scholars of Shandong province (ts201712048), the Natural Science Foundation of Shandong Province (ZR2017MB022, ZR2018BB057 and ZR2018PB009) and the start-up funding from University of Jinan (511-1009408, 511-1009424).

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Authors and Affiliations

  1. School of Materials Science and Engineering, University of Jinan, Jinan, 250022, China

    Pengjuan Ni, Chuanxia Chen, Yuanyuan Jiang, Zhenlu Zhao & Yizhong Lu

  2. College of Chemistry, Chemical Engineering and Materials Science, Key Laboratory of Molecular and Nano Probes (Ministry of Eduction), Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in University of Shandong, Institute of Molecular and Nano Science, Shandong Normal University, Jinan, 250014, China

    Junfeng Xie

  3. School of Chemistry and Chemical Engineering, University of Jinan, Jinan, 250022, China

    Yan Zhang & Jinghua Yu

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  1. Pengjuan Ni
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Correspondence to Pengjuan Ni, Yizhong Lu or Jinghua Yu.

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Ni, P., Xie, J., Chen, C. et al. Spectrophotometric determination of the activity of alkaline phosphatase and detection of its inhibitors by exploiting the pyrophosphate-accelerated oxidase-like activity of nanoceria. Microchim Acta 186, 320 (2019). https://doi.org/10.1007/s00604-019-3423-8

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