Study On Preparation Of ZnCo₂O₄ Microsphere-based Peroxidase And Its Colorimetric Detection Of L-cysteine

Natural enzymes have been widely used in industrial,medical,and biological fields because of their high catalytic activity and substrate specificity,but they have inherent disadvantages such as high cost,poor stability in practical application,and difficulty in recovery.Compared with natural enzymes,artificial mimetic enzymes such as those based on bimetallic materials have the advantages of simple preparation,low cost,good stability,high catalytic efficiency,and non-degradability,which have received wide attention from researchers.As a spinel-type material,ZnCo2O4 has good optical,electrical,and magnetic properties and excellent chemical and structural stability,and has been widely used in catalysis,energy storage,gas sensing,and other fields,but little has been reported in biochemical analysis.As a semi-essential amino acid in the human body,L-cysteine(L-Cys)is involved in several important physiological processes,such as protein folding,catalysis by cysteine proteases,regulation of oxidative stress,and nutrient metabolism levels in the body,and participation in physiological signal transduction.Therefore,developing a fast,inexpensive,and simple method to detect L-Cy is necessary.Given the excellent properties of ZnCo2O4 materials,this thesis prepared Pt@ZnCo2O4 microspheres with different ZnCo2O4 microspheres and different Pt doping amounts,investigated their catalytic performance and catalytic mechanism as artificial mimetic enzymes,and established a simple,rapid and visual colorimetric detection of L-Cys based on the peroxidase-like activity of ZnCo2O4 and Pt@ZnCo2O4 The method was successfully applied to the sensitive detection of L-Cys in real samples.The main studies are as follows.1.Exploration of ZnCo2O4 microsphere-like peroxidase activity and its application in L-cysteine assay.A series of ZnCo2O4 microspheres were easily prepared by the solvothermal method,and the morphology,structural composition,and properties of the synthesized ZnCo2O4 microspheres were studied by SEM,EDS,XRD,XPS,and other material characterization methods.The synthesized ZnCo2O4 microspheres possess peroxidase-like(POD)activity and can oxidize colorless 3,3’,5,5’-tetramethylbenzidine(TMB)to a blue product(oxTMB)in the presence of H2O2.The peroxidase-like activities of different ZnCo2O4 microspheres were compared,among which the single cubic phase structure of ZnCo2O4 microspheres had the best peroxidase-like activity,and their peroxidase-like catalytic mechanism was explored utilizing fluorescence and electrochemical methods.Enzyme kinetic experiments showed that the affinity of this ZnCo2O4 microsphere for either TMB or H2O2 was higher than that of horseradish peroxidase(HRP).A colorimetric sensor for the detection of L-Cys was constructed on the peroxidase-like activity of the ZnCo2O4 microspheres.Under the optimized detection conditions,the linear range of the colorimetric sensor was 2-50 μM,and the detection limit was 0.104 μM,with good selectivity,stability,and reusability.In addition,the colorimetric sensor was used to analyze L-Cys in actual pharmaceutical L-Cys capsule samples with a relative deviation of 2.6%and a spiked recovery of 97.2%-102.3%,which is of good practicality,indicating that the ZnCo2O4 material has an important application in the field of biocatalysis.2.Exploration of Pt@ZnCo2O4 microsphere-like peroxidase activity and its application in L-cysteine assay.In this chapter,based on the work in Chapter 1,the peroxidase-like activity of ZnCo2O4 microspheres was further improved by doping with the noble metal Pt.ZnCo2O4 microspheres were selected as precursors and Pt-doped ZnCo2O4 microspheres were successfully synthesized by a one-step method,varying the Pt doping amount,and Pt@ZnCo2O4 microspheres with different Pt doping amounts.The effect of the change of Pt doping on the structure and composition of ZnCo2O4 microspheres was investigated by SEM,EDS,XRD,XPS,and other characterization methods.The results show that the Pt doping has some effects on the microsphere size,zeta potential,and oxygen vacancies,but does not affect the phase purity of ZnCo2O4 microspheres.Comparing the peroxidase-like activity of Pt@ZnCo2O4 microspheres with different Pt doping amounts,it was found that the Pt doping could significantly improve the peroxidase-like activity of ZnCo2O4 microspheres,and the enzyme activity was positively correlated with the Pt doping amount,in which,12.5%Pt@ZnCo2O4 microspheres(12.5%was the doping atomic ratio)had the best peroxidase-like catalytic activity.Fluorescence and electrochemical methods were used to explore its peroxidase-like catalytic mechanism.Enzyme kinetic experiments showed that the affinity of 12.5%Pt@ZnCo2O4 microspheres for both TMB and H2O2 was higher than that of HRP and ZnCo2O4.Based on this,12.5%Pt@ZnCo2O4 microspheres with the best peroxidase-like activity were selected to construct a colorimetric sensing method for the detection of L-Cys.Compared with the colorimetric system constructed in the previous work,this system showed a linear range of 0.1 μM-50 μM(wider linear range)and a detection limit of 0.0163 μM(lower detection limit)for L-Cys under the optimized conditions.Meanwhile,the relative deviation of L-Cys detection in the actual pharmaceutical L-Cys capsule samples was 1.2%,and the spiked recoveries were 96.9%-103.7%,indicating that the method possesses good accuracy and reliability.In conclusion,Pt@ZnCo2O4 microspheres are simple to synthesize and have high enzymatic activity,and their potential applications in the field of biocatalytic sensing.