Exploration The Enzyme-like Activity Of Copper Prussian Blue Analogues And Its Application In The Analysis Of Dairy Product Quality And Safety

Dairy products are a common part of everyday life and there are many different types of dairy products that are rich in nutrients.But some of the dairy scandals that have broken in the news have made us realize that the issue of food safety in dairy products should not be underestimated.The construction of sensitive,efficient and rapid analytical methods is therefore essential for the quality and safety analysis of dairy products.Existing testing methods often involve large analytical instruments and expensive reagents and drugs,as well as cumbersome experimental procedures.Nanozymes were nanomaterials with similar catalytic activity to natural enzymes which have been widely used in analytical sensing assays due to their low cost of synthesis,easy modulation of activity and high stability.Copper Prussian blue analogues（Cu-PBA）are a class of nanomaterials with a metal-organic framework structure formed by replacing the iron ion attached to the nitrogen end of the cyanide group in Prussian blue（PB）with a copper ion,which has abundant defect sites and excellent electrical conductivity and is often used in energy storage and conversion and electrochemistry.The investigation of the enzyme-like activity of copper Prussian blue analogues and their application to dairy product quality analysis and testing can both extend the application of copper Prussian blue analogues nanomaterials and enhance the assurance of dairy product quality and safety.In this paper,a copper Prussian blue analogue nanozyme（copper hexacyanoferrate nanozyme,Cu-HCF nanozyme）was synthesized and investigated its two mimic enzymatic activities,and furthermore,the structure of Cu-HCF nanozyme was modulated by chemical etch to get the Etch-Cu-HCF nanozyme and enhanced its polyphenol oxidase-like catalytical activity,and then the two nanozymes were applied to the analysis of dairy product quality and safety.The main research contents are as follows:（1）Preparation,characterization and activity studies of Cu-HCF nanozyme and Etch-Cu-HCF nanozyme.The Cu-HCF nanozyme was successfully prepared and characterized.The enzyme-like catalytic activity was investigated and the Cu-HCF nanozyme was found to have peroxidase-like catalytic activity and,for the first time,polyphenol oxidase-like catalytic activity.A kinetic study of the enzyme activity revealed that the affinity of the Cu-HCF nanozyme for the substrate was nearly 7 times greater than that of the natural polyphenol oxidase（PPO）;the stability of the Cu-HCF nanozyme was compared with that of the natural polyphenol oxidase,and the Cu-HCF nanozyme showed superior stability under extreme alkaline,high temperature and high salt concentration conditions,and could be stored and reused for long periods of time.The Etch-Cu-HCF nanozyme was obtained by ammonia etch on the Cu-HCF nanozyme and characterized.The activity study revealed that the ammonia etches increased the polyphenol oxidase-like catalytical activity by approximately twofold,and the kinetic study revealed that the maximum initial reaction rate of the Etch-Cu-HCF nanozyme reached the level of the natural polyphenol oxidase and still maintained a high affinity for the substrate.（2）Quantification of hydrogen peroxide（H₂O₂）in cow and goat milk using the peroxidase-like activity of Cu-HCF nanozyme.It was found that the peroxidase-like catalytic activity of Cu-HCF nanozyme could catalyze the oxidation of the chromogenic substrate in the presence of hydrogen peroxide,generating a colorimetric signal,and by optimizing the concentration of the chromogenic substrate and other reaction conditions,a highly sensitive quantitative detection of hydrogen peroxide was achieved and successfully applied to milk and goat milk.（3）Quantitative colorimetric detection of alkaline phosphatase（ALP）in pasteurized milk was achieved using the polyphenol oxidase-like activity of the Cu-HCF nanozyme.The polyphenol oxidase-like catalytic activity of Cu-HCF nanozyme was found to be inhibited by pyrophosphate（PPi）,alkaline phosphatase can hydrolyze pyrophosphate to restore nanozyme activity.The quantitative colorimetric detection of alkaline phosphatase was achieved by using pyrophosphate as the sensing unit and optimizing its concentration in the detection system and other conditions such as alkaline phosphatase incubation with pyrophosphate,and successfully applied to pasteurized milk.（4）Quantification of bisulfite in poor quality white sugar illegally added to dairy processed products using the polyphenol oxidase-like activity of Etch-Cu-HCF nanozyme.The investigation revealed that the intermediate product of the chromogenic reaction of the chromogenic agent 2,4-dichlorophenol with 4-aminoantipyrine catalyzed by the polyphenol oxidase-like activity of Etch-Cu-HCF nanozyme can be consumed by an addition reaction with bisulfite,causing a change in the colorimetric signal.By optimizing the conditions of the stepwise reaction,a new method for the colorimetric detection of bisulfite by the polyphenol oxidase-like activity of Etch-Cu-HCF nanozyme was developed and applied to a milk system that can detect the use of poor quality white granulated sugar in dairy products.