Polymetallic Nanozyme With Peroxidase Mimetic Activity For Rapid Evaluation Of Total Antioxidant Capacity In Food

The human body requires natural antioxidants from food to maintain the free radical balance.Different antioxidants present in different foods may have synergistic effects and it is necessary to assess the total antioxidant capacity（TAC）in foods.For the detection of antioxidants TMB（3,3’,5,5’-tetramethylbenzidine）can be used as the peroxidase chromogenic substrate in the colorimetric method.However,the natural peroxidase used in TMB assay has limitations such as low stability and high price.Nanozymes have the unique properties of nanomaterials,such as high catalytic activity,good stability,and low cost.They have the potential to be used as an alternative to natural enzymes.Polymetallic-based nanozymes offer more design options as well as better performing catalysts due to the presence of synergistic effects between different metals.Based on the above aspects,this paper prepared two polymetallic nanozyme by a one-step hydrothermal method,verified their intrinsic peroxide-like enzymatic activities in the presence of TMB and H₂O₂,and realized a rapid detection method for TAC using the colorimetric reaction of nanozyme.The main research contents and results are as follows:（1）Bismuth ferric molybdate trimetallic nanozyme for colorimetric detection of TAC.Different ratios of bismuth ferric molybdate materials were prepared by one-step hydrothermal synthesis,and Bi_0.3Fe_1.7Mo O₆ with optimal simulated enzymatic activity was selected.The morphology,crystal structure,elemental composition and chemical valence of the materials were investigated by means of scanning electron microscopy（SEM）,X-ray diffraction（XRD）and X-ray photoelectron spectroscopy（XPS）characterization,proving the successful synthesis of the materials.After that,the peroxide-like activity of Bi_0.3Fe_1.7Mo O₆catalyst was evaluated and optimized,and the optimal experimental conditions were chosen as a final concentration of 25μg m L^-1 of the material,a reaction time of 10 min,a buffer p H of 3,and an incubation temperature of 25±5°C.Simulated enzymatic reaction kinetic measurements showed that the Bi_0.3Fe_1.7Mo O₆ material exhibited a K_m value of 0.0883 m M and a V_max value of 16.9×10^-8 M s^-1 for the substrate TMB,with a strong affinity for the substrate.The catalytic mechanism suggests that the production of·O₂^-during the catalytic process is the main reason for its excellent peroxidase-like activity.The colorimetric sensing platform has a low detection limit（0.77μM）and a good linear range（0–64μM）in ascorbic acid analysis.Finally,Bi_0.3Fe_1.7Mo O₆ catalyst with high stability and reproducibility was applied to the TAC detection in real food samples.（2）Iron-doped molybdenum oxide bimetallic nanozyme for in situ colorimetric determination of TAC.Fe-Mo O_x nanozymes were prepared by metal-polyphenolic coordination and hydrothermal synthesis strategies.The successful synthesis of the materials was first demonstrated by characterizing Fe-Mo O_x by transmission electron microscopy（TEM）,XRD,and XPS.After that,the simulated peroxidase-like activity of Fe-Mo O_x was optimized and evaluated,and the optimal experimental conditions were selected as a final material concentration of 20μg m L^-1,a reaction time of 6 min,the buffer system and temperature is p H 3 and 20±5°C.Free radical scavenging experiments demonstrated that·OH plays a major role in the catalytic oxidation of TMB.Thus,the colorimetric sensing system using TMB?+?H₂O₂?+Fe-Mo O_x detected ascorbic acid in the range of 10–100μM with the limit of detection is 9.74μM,which has satisfactory sensing performance compared to other Fe-based materials of the same type studied.In addition,it still showed better selectivity for ascorbic acid in the presence of common interferents in foods,such as glucose,sucrose,fructose,and carboxymethyl cellulose（CMC）.Finally,a TAC assay platform with ascorbic acid as the reference standard was constructed and shown to be effective in detecting VC tablets and fruit and vegetable samples.Finally,a TAC assay platform with ascorbic acid as the reference standard was constructed.Further,a hydrogel kit that can be used for TAC colorimetric detection was prepared by combining the test system with hydrogel,and the readings were analyzed with the help of smartphone,showing good potential in food quality studies.