Structure Activity Study Of Nickel Based Oxidase And Its Application In Fluorescence Detection

Biological enzymes are biocatalysts with high catalytic activity,excellent substrate specificity and biocompatibility.It can be widely used in the fields of biosensor,disease diagnosis and treatment,environmental monitoring and food processing.However,biological enzymes have some disadvantages,such as variability,high cost,low stability and difficult storage,which hamper their further applications.In order to break through these limitations,researchers have made great efforts,such as enzyme immobilization,enzyme modification,artificial enzymes and so on.Recently,nanomaterials with enzyme-like activity,which combine nanotechnology and biology,are defined as nanozymes and have attracted the attention of researchers.Nanozymes possess the specific physical and chemical properties of nanomaterials,show excellent catalytic performance,high stability in harsh environment,and have the advantages of low preparation cost,easy purification and easy regulation of activity.Therefore,nanozymes show the trend of replacing biological enzymes in many fields.In particular,different nanomaterials,such as noble metals,transition metal oxides,carbon materials and composites,can exhibit oxidase-or peroxidase-like activity and catalyze the color development or luminescence of substrate dyes.Based on this,combined with their own physicochemical properties,they can be designed into different biosensors,showing high application potential in the field of sensing.Owing to its unique redox characteristics,nickel oxide（NiO）is widely used in various applications,such as gas sensors,supercapacitors,adsorption and degradation of pollutants,etc.At present,studies have confirmed that NiO can catalyze the oxidation of enzyme substrate dyes and is one of the substitutes of natural oxidase.However,there is lack of research on the structure-activity relationship between NiO and its oxidase-like activity,which is not conducive to our purposeful improvement of NiO catalytic activity.In this paper,NiO with different morphologies were prepared,and their physicochemical properties,such as morphology,crystal surface,oxygen vacancy and specific surface area,were tested and characterized.The effects of these factors on the oxidase-like activity of NiO were analyzed,and the catalytic mechanism of NiO in the catalytic process was discussed.Finally,the oxidase-like activity of NiO was regulated,and NiO/Amplex Red（AR）system was designed as a biosensor to detect L-cysteine（L-Cys）.The research contents and conclusions are as follows:（1）Here,NiO nanoparticles with different morphologies were synthesized,including cube,flake and rod.TEM,HRTEM and XRD were used to explore the morphology and structure of different materials.XPS was used to explore the valence state of NiO and the existence of surface oxygen vacancies.EPR,Raman,free radical capture and quenching experiments showed that oxygen vacancies were defect sites,and the content of oxygen vacancies was intrinsically related to Ni³⁺/Ni²⁺,which played a key role in promoting the oxidation of AR.The catalytic activities of various NiO nanoparticles in NiO/AR reaction system were studied.Based on this,the effect of specific surface area,oxygen vacancies and different valence states of nickel on the performance of simulated oxidase were further studied.The result showed that oxygen vacancies were the active centers of NiO nanozymes and the key to oxidase-like activity of NiO.The samples with larger surface could provide more adsorption sites,which was also the reason why the samples with larger surface have stronger activity.（2）Two different nickel oxide nanosheets were successfully synthesized in different gas atmospheres.XRD showed that the two materials had the same structure,but the grain size of NiO-O_V was smaller.TEM showed that the two different nanosheets were hexagonal sheets with porous structure,but the size of NiO nanosheets calcined in anoxic environment was smaller.XPS and EPR results showed that there was a significant difference in oxygen vacancy content between the two samples.The K_m value obtained from the steady-state dynamic analysis of NiO-O_V was as low as 2.83μM.Compared with other reported materials,the K_mvalue was smaller,indicating that it had stronger affinity for substrate dye AR.At the same time,the reaction conditions of NiO/AR system were optimized.The system showed excellent properties at pH6.5-8.5 and 25-75℃.Under the optimal conditions,based on the fluorescence quenching effect of L-Cys on NiO/AR system,the highly sensitive detection of L-Cys was realized by monitoring the change of fluorescence intensity of the reaction product resorufin,and the detection limit was as low as 37.8nM.