Spectrophotometric Determination of Alkaline Phosphatase in Serum by A Copper Prussian Blue Analog as A Novel Polyphenol Oxidase-Like Nanozyme

Nanomaterials with natural enzyme-like activities have attracted widespread interest and hence their development is significant. Here, a copper Prussian blue analogue named Cu-HCF was prepared and its polyphenol oxidase (PPO)-like activity was investigated for the first time. Cu-HCF nanozyme showed nearly 7-fold higher substrate affinity than natural PPO and was more stable under alkaline conditions, high temperature, high salt concentrations, and long-time storage. The catalytic activity of Cu-HCF nanozyme was reduced by pyrophosphate (PPi). Alkaline phosphatase (ALP) hydrolyzed PPi to orthophosphate (Pi) and hence the PPO-like activity of Cu-HCF nanozyme was restored. A method was therefore developed for selective determination of the ALP activity. The linear range was from 1 to 50 mU/mL with a limit of detection (LOD) of 0.35 mU/mL. This approach was validated in human serum samples, showing its potential in diagnostic applications.