Preparation Of Cobalt-based Single-atom Nanozymes And Their Application In Biological Analysis

As an efficient biocatalyst,enzyme has two main research directions in current studies.One is artificial modification on the basis of natural enzymes,and the other is the synthesis of nanozymes with similar structure and properties to natural enzymes.As an artificial enzyme,nanozyme has the advantages of high stability,low cost and large-scale production.However,it also has weak points,such as poor specificity and selectivity,low catalytic activity and unclear catalytic mechanism,which still needs further research.The research topic of this paper is focused on cobalt-based single-atom nanozymes.Combining the advantages of single-atom nanomaterials in defining catalytic active sites and maximum atomic utilization efficiency with the excellent enzyme-like properties of cobalt-based nanozymes,nanozymes with excellent oxidase-like activity are obtained by rationally regulating the coordination environment of single-atom Co.Their catalytic mechanism is revealed and visual colorimetric detection of ascorbic acid and starch is realized.The main research contents are as follows:1.The preparation and application of Co-N₄ nanozymes:The cobalt-based single-atom nanomaterial（SA-Co/NC）was synthesized by hydrothermal method and high-temperature pyrolysis.The oxidase-like activity and catalytic mechanism of SA-Co/NC were studied by combining experimental and theoretical calculations.Free radical scavenging experiments proved that the main reactive oxygen species（ROS）were superoxide radicals and the source was dissolved oxygen in water.At the same time,the inhibition mechanism of different reducing substances on the activity of SA-Co/NC oxidase was studied:the sacrifice mechanism represented by ascorbic acid（AA）,and the competition mechanism represented by glutathione（GSH）.Finally,we designed a scheme based on time-visualization to determine the total antioxidant capacity and applied it to the detection of actual cosmetics.2.Preparation and application of heteroatom（P,S,B）doped Co-N₄ nanozymes:Based on the synthesized SA-Co/NC,the oxidase-like activity was improved by doping heteroatoms.It was proved by characterization that the introduction of a small amount of heteroatoms would not have a significant effect on the morphology and structure.However,according to the experimental results,the introduction of phosphorus elements had a improving influence on the oxidase-like activity.Furthermore,according to kinetic analysis,it was verified that the doping of phosphorus increased the maximum reaction rate of the catalytic reaction and the affinity with the substrate.Using the enhanced oxidase-like activity of phosphorus-doped cobalt-based single-atom nanomaterial（Co-P/NC）,I^-can be oxidized to I₃^-and further complexed with starch.Based on the above principles,we constructed a scheme on colorimetric detection of starch concentration.3.Co-N₄ coordination environment regulation:Due to the multiple valence states of transition metals,Fenton-like reaction may also be the reason that cobalt-based nanomaterials exhibit oxidase-like activity.Considering the definition of catalyst which the quality and activity before and after the reaction will not change,the nanozyme should also remain unchanged before and after the catalytic reaction.Herein,we synthesized a graphene oxide material loaded with cobalt single atoms（Co N/e GO-900）by hydrothermal method,which had a macroscopic columnar morphology with stable structure and a nanostructure with ultra-high porosity and specific surface area.The structure was favorable for recycling experiments to prove that a catalytic reaction occurred.It was proved by control experiments that the loading of cobalt single atoms and 900°C high temperature treatment were the necessary conditions for graphene oxide to have obvious oxidase-like activity.In addition,the introduction of nitrogen into cobalt-loaded graphene oxide can form a cobalt-nitrogen coordination structure,which can improve the oxidase-like activity.