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 0.08 mU/mL, with linear range from 0.1 to 50 mU/mL. In addition, the assay was also applied for screening the inhibitors of ALP.