Study On The Photo-induced Oxidase-like Activity Of Ru（bpy）₃²⁺ And Its Analytical Application

Natural enzymes,most of them are proteins,can catalyze specific chemical reactions in organisms with high catalytic efficiency and good specificity to the substrate of the reaction.At present,natural enzymes have been widely used in applications such as enzymatic biofuel cells and biosensing.However,the structure of natural enzymes is denatured under high temperature or strong acid and base conditions,high separation cost and low stability limited their practical application.To address the above limitations of natural enzymes,researchers have developed a variety of materials to mimic natural enzymes.Yan’s team first found that ferric oxide nanoparticles have enzyme-like activities,and later researchers found that a variety of nanomaterials have enzyme-like activities.However,nanomaterials also have some shortcomings:first,most nanomaterials with enzyme-like activity have great differences in catalytic activity due to different synthesis batches,leading to poor reproducibility of experimental results;secondly,nanomaterials are easy to agglomerate,which causes the loss of enzyme activity;finally,some nanomaterials with poor water-solubility often need to introduce some hydrophilic groups,the introduction of hydrophilic groups will reduce the enzyme activity dramatically.Therefore,it is necessary to explore new enzyme-like materials.Ru（bpy）₃²⁺is an organo-metallic complex with good photoelectric properties,good photostability,high photoelectric conversion rate and recyclability.At present,Ru（bpy）₃²⁺has been applied in the fields of electrochemistry and organic synthesis as an electrochemiluminescence reagent,photosensitizer and photocatalyst.However,the enzyme-like activities of Ru（bpy）₃²⁺itself are rarely reported.Therefore,this work is devoted to investigating the photoinduced oxidase-like activity of Ru（bpy）₃²⁺,and studying the catalytic mechanism.Based on the oxidase-like activity of Ru（bpy）₃²⁺,a new spectrophotometric method for the detection of biomercaptan,mercuric ion and hyaluronase activities was successfully constructed,and the application of Ru（bpy）₃²⁺in biochemical analysis was expanded.The specific work content is as follows:（1）Study on the photoinduced oxidase-like activity of Ru（bpy）₃²⁺We found for the first time Ru（bpy）₃²⁺possessed the oxidase-like activity under visible light illumination.Under the irradiation of LED lamp,Ru（bpy）₃²⁺can catalyze the oxidation of a typical chromogenic substrate,3,3’,5,5’-tetramethylbenzidine（TMB）to a blue oxidation state TMB.The catalytic reaction follows the typical Michaelis-Menten kinetics equation.The catalytic mechanism of Ru（bpy）₃²⁺was investigated in detail.Through electron spin resonance（ESR）and free radical scavenging experiments,it was confirmed that hydroxyl radical played a major role in the chromogenic reaction catalyzed by Ru（bpy）₃²⁺.Ru（bpy）₃²⁺,as a light-induced oxidase,uses oxygen as an electron acceptor and has high catalytic activity in very little light time.（2）Detection of biothiols and Hg²⁺based on the photoinduced oxidase-like activity of Ru（bpy）₃²⁺In this study,a colorimetric detection method for biothiols and Hg²⁺was established based on the photoinduced oxidase-like activity of Ru（bpy）₃²⁺.Biomercaptans contain sulfhydryl groups that inhibit the chromogenic reaction of TMB catalyzed by Ru（bpy）₃²⁺,and under optimal conditions,the dynamic range of detection Cys is 5.0μmol/L-50μmol/L,the limit of detection is 1.0μmol/L,and the linear range of GSH detection is 5.0μmol/L-60μmol/L,and the detection limit is 1.0μmol/L.Furthermore,there is a strong interaction between Hg²⁺and sulfhydryl groups,which can restore the colorimetric development reaction of Ru（bpy）₃²⁺-TMB system,so as to realize the quantitative detection of Hg²⁺.The linear range of detection Hg²⁺is 0.050μmol/L-20μmol/L and the detection limit is 40 nmol/L.This work broadens the application of Ru（bpy）₃²⁺as a photoinduced oxidase-like enzyme in analytical detection.（3）Detection of hyaluronidase based on the interaction between hyaluronic acid and Ru（bpy）₃²⁺Since hyaluronidase is a potential tumor marker,the establishment of a simple and sensitive hyaluronidase detection method is of great significance for the clinical diagnosis and treatment of cancer.Based on the electrostatic interaction between negatively charged hyaluronic acid（HA）and positively charged Ru（bpy）₃²⁺,a simple and sensitive colorimetric analysis method was designed for the detection of hyaluronidase（HAase）.Ru（bpy）₃²⁺is adsorbed on HA by electrostatic interaction to form the HA@Ru（bpy）₃²⁺complex.Photoinduced oxidase-like activity of Ru（bpy）₃²⁺is inhibited.In the presence of HAase,HA in HA@Ru（bpy）₃²⁺can be broken down into fragments by enzymatic hydrolysis.Therefore,Ru（bpy）₃²⁺attached to HA becomes free,restoring its enzymatic activity so that the chromogenic substrate TMB can be oxidized,which is used to test the concentration of HAase in solution.The dynamic range of HAase is 0.020-1.0 U/m L and the detection limit is 7.0 m U/m L.The system has been used to detect HAase in urine samples with satisfactory results.