Sensitive colorimetric assay for the determination of alkaline phosphatase activity utilizing nanozyme based on copper nanoparticle-modified Prussian blue

Nanozyme based on Prussian blue nanocubes (PB NCs) loaded with copper nanoparticles (Cu@PB NCs) was synthesized. The peroxidase (POD)-like activity of Cu@PB NCs was studied and utilized for detecting the activity of alkaline phosphatase (ALP). The Cu@PB NCs possess higher POD-like activity compared with PB NCs and natural horseradish peroxidase (HRP) due to the loading of copper nanoparticles. 3,3',5,5'-Tetramethylbenzidine (TMB) can be oxidized to oxTMB in the presence of Cu@PB NCs and H2O2, generating blue-colored compound, while introduction of pyrophosphate (PPi) leads to the POD-like activity of Cu@PB NCs decreased obviously. In the presence of ALP, PPi was hydrolyzed and then the POD-like activity of Cu@PB NCs was restored. So, according to the change of the POD-like activity of Cu@PB NCs, a sensitive colorimetric assay for ALP activity was reported. The limit of detection of the assay is 0.08mU/mL, with linear range from 0.1 to 50mU/mL. In addition, the assay was also applied for screening the inhibitors of ALP.

Automated summary

Cu@PB NCs exhibit enhanced POD-like activity compared to PB NCs and HRP, and can be utilized for colorimetric assay of ALP activity by oxidizing TMB in the presence of H2O2. Introduction of PPi results in decreased POD-like activity of Cu@PB NCs, while ALP hydrolyzes PPi and restores the activity of Cu@PB NCs.