Silver Nanozyme Loaded On Carbon Microspheres For Colorimetric Detection Of Hg(Ⅱ) In Seafood

With the development of economy and the improvement of people’s living standard,food safety has gradually become the focus of global attention.Food safety incidents caused by seafood are global hazards to human health,among which the main hazard factor is heavy metal pollution.After being enriched in the food chain and entering the human body,Hg²⁺will interact with the mercaptan protein system in the human body,resulting in the occurrence of various chronic diseases and even death.Traditional detection methods of Hg²⁺,such as atomic absorption spectrometry,are time-consuming,costly and complicated.Therefore,rapid response,sensitive and convenient analytical methods are urgently needed for trace detection of Hg²⁺,so as to ensure food safety and human health.Colorimetric analysis methods based on nanozymes are not only low cost and easy to operate,but also can realize fast and visual Hg²⁺analysis,which have broad application prospect.Silver nanoparticles（Ag NPs）have been used in the preparation of nanozymes due to their unique optical properties,biocompatibility and structural adjustability.However,pure Ag NPs are easy to aggregate and oxidize,which greatly affects their catalytic performance and limits their practical application.As a typical member of the carbon material family,carbon microspheres（CMs）have good thermal stability,chemical stability and adjustable microstructure.In addition,the surface of CMs can form composites through coordination or electrostatic interactions between surface functional groups and metal ions.Considering the complementary advantages of the Ag NPs and CMs,and the synergistic effect between carbon and metal materials,in this paper,Ag NPs-CMs nanocomposite was obtained by loading Ag NPs on CMs.This nanomaterial presented Hg²⁺-enhanced peroxidase activity.With 3,3,5,5-tetramethylbenzidine（TMB）as the color developing substrate,the colorimetric detection of Hg²⁺in seafood can be realized.The main research results are as follows:1.Ag NPs-CMs nanocomposites were prepared by hydrothermal method and depositional precipitation method,and the morphology and particle size were characterized.SEM and TEM images showed that CMs were dispersed microspheres with a diameter of175.05-440.74 nm.The shape was regular and the outer surface was smooth.Ag nanoparticles with a diameter of～21.51 nm grew on the surface of CMs,which was Ag NPs-CMs.The FTIR spectrum showed that the surface of CMs contained more-OH and-COOH than that of Ag NPs-CMs,indicating that the loading of Ag NPs was formed by the interaction of Ag⁺with-OH and-COOH on the surface of CMs.XRD and XPS characterizations indicated a cubic silver lattice of silver,and that silver existed as Ag⁰ instead of Ag⁺.2.Enzymatic kinetics analysis of Ag NPs-CMs before and after adding Hg²⁺was carried out.Using TMB and H₂O₂ as substrates,the results were consistent with typical Michaelis-Menten model（R²>0.90）,indicating that Ag NPs-CMs had peroxide-like activity.The Michaelis-Menten constant and maximum initial velocity(ν_max)were calculated by the Lineweaver-Burk plot.Compared with natural horseradish peroxidase（HPR）and other silver-based nanozymes,the K_m values obtained were low,indicating that Ag NPs-CMs had a high affinity for TMB and H₂O₂.After adding Hg²⁺,the K_m values were lower,which demonstrated that Hg²⁺enhanced the peroxidase activity of Ag NPs-CMs.Ag NPs-CMs with Hg²⁺were characterized by XPS.The presence of Hg 4f and the valence state of Ag⁺indicated the formation of silver amalgam.Active oxygen scavenging experiment showed that O₂^·-and·OH increased after the addition of Hg²⁺.Therefore,the mechanism of Hg²⁺-enhanced activity of Ag NPs-CMs was that the formation of Ag-Hg amalgam,which leaded to the formation of more O₂^·-and·OH during the catalytic process.3.Under the optimal conditions,that is,the concentration of Ag NPs-CMs was 0.04mg/m L,the incubation time was 5 min,the incubation temperature was 20℃,and the buffer p H was 5.0,the absorbance difference(ΔA₆₅₂)at the maximum absorption wavelength（652nm）before and after adding Hg²⁺was taken as the vertical coordinate and Lg[c(Hg²⁺)]as the horizontal coordinate.Hg²⁺can be detected in the concentration range of 2～833 n M and 2.5～40μM,respectively,with detection limits of 1.10 n M and 1.01μM,respectively.Common metal ions and anions in seafood had no significant effect on the detection of Hg²⁺（P>0.05）,indicating that this method had high selectivity and anti-interference ability for Hg²⁺detection.The recoveries were in the range of 93.39～107.68%,with the relative standard deviations（RSDs,n=3）of 0.96～5.68%.The results showed that this method had high reliability and feasibility for detection of Hg²⁺in seafood.Combining with smart phone APP（Color Picker）,a portable disposable agarose hydrogel detection kit was designed.The detection range was between 0.5 and 5μM,and the field semi-quantitative visual detection of Hg²⁺was realized.In this paper,a colorimetric method with sensitive detection,convenient operation,low cost and strong anti-interference was established for the detection of Hg²⁺in seafood,which provides a new way for the determination of trace contaminants in food by colorimetric method based on nanozymes.