Spectroscopic And Electrochemical Investigations Of Methanobactin Mimic SOD

The determination for mimic superoxide dismutase(SOD)activity of methanobactin(Mb)and copper complexes by spectroscopy and electrochemistry was established and further applied to electrochemical biosensors.Mb is a small molecule fluorescent peptide secreted by methanotrophs to capture and transport Cu2+ extracellularly.It has the ability of coordinating Cu2+ and other metal ions.The copper complex has the ability of scavenging superoxide anion.In the first part of the experiment,Mb was extracted from ethylosinus trichosporium 3011 and combined with Cu2+to form Mb-Cu.The linearization time of pyrogallol autoxidation and the fluorescence excitation wavelength with Mb-Cu added were investigated respectively.The autoxidative linear time of pyrogallol was 90s,and the fluorescence wavelength of pyrogallol system was 446nm.The effects of Tris-HAc concentration,pH,the concentration of EDTA-Na2 and pyrogallol,temperature and the concentration of Mb-Cu on the pyrogallol autoxidation were futher discussed by ultraviolet and fluorescence spectrum.So as to evaluate the SOD activity of Mb-Cu.The results showed that when the concentration of EDTA-Na2 was 2mmol/L,pH value was 8.2,Tris-HAc was 0.05mol/L,the temperature was 20 ?,and the concentration of pyrogallol was 0.363mmol/L,Mb-Cu at a concentration of 0.8 mg/L showed better SOD activity and can inhibit superoxide anion(O2-·)effectively.Under this spectrum analysis conditions,Mb-Cu mimic SOD activity was stable.In the second part of the experiment,AuNps with particle sizes of 11.5,15,35,42 and 50 nm were prepared by sodium citrate reduction method and characterized by TEM.The results showed that the Au nanoparticles(AuNps)prepared have uniform size and smooth surface.Mb and Cu2+ were assembled by AuNps of a particle size of 15 nm successively and characterized by infrared spectrum.It was proved that Mb complexed Cu(?)by the hydroxyl in Mb structure or phenolic hydroxyl group.In turn,15 nm AuNps were bound to crosslinkers of different types of functional groups and characterized by UV,fluorescence,IR and transmission electron microscope.?-mercaptoethylamine can bond closer between AuNps and Mb-Cu,which Forming a dendritic nanocluster structure.The effects of AuNps particle size,molar ratio of AuNps to Mb-Cu,different cross-linking agent and molar ratio of Mb-Cu to cross-linking agent on the pyrogallol autoxidation were investigated respectively.By spectrum analysis,it was considered that when the AuNps particle size was 15nm,molar ratio of AuNps:Mb-Cu was 1:150,the optimal crosslinking agent was P-mercaptoethylamine,molar ratio of Mb-Cu:?-mercaptoethylamine was 1:3,Mb-Cu-Au mimic has the optimum enzyme activity.When the SOD activity measurement system was changed to xanthine oxidase method,it was verified that,the activity of the mimic enzyme activity was basically consistent and the method was stable.The final part of the experiment was performed on blank,Au/?-mercaptoethylamine/AuNps,Au/?-mercaptoethylamine/AuNps/Mb and Au/?-mercaptoethylamine/AuNps/Mb/Cu electrodes to be compared in a probe solution of 0.1m Fe(CN)63-/Fe(CN)64-by cyclic voltammetry(CV)and electrochemical impedance spectroscopy(EIS)scanning.The Au/?-mercaptoethylamine/AuNps/Mb/Cu electrode was assembled as a monolayer on a gold electrode,AuNps can accelerate the transmission of heterogeneous electrons.Otherwise,Cu2+inhibited heterogeneous electron transfer.In the research of SOD mimic enzyme self-assembled membrane,the effects of different electrolytes,electrolyte concentrations,pyrogallol reaction time,pyrogallol dosage and sweeping rate on the electrode response of pyrogallol autotrophic matrix as probe solution.The effects of the current on the electrode current were investigated.When the the concentration of KCl was 0.3 mol/L,the superoxide anion accumulation time was 3 min,the amount of pyrogallol added was 20 ?L and the sweep speed was 300 mV/s,the electrode has the highest degree of induction to the current.The effects of different mimic enzymes,assembly methods and crosslinker on the inhibition of pyrogallol autoxidation by mimic enzymes in the electrochemical method were investigated.It was believed that ?-mercaptoethylamine can be used as a cross-linking agent and can be assembled in layers.The assembled Au/?-mercaptoethylamine/AuNps/Mb/Cu electrode exhibited the highest SOD mimic enzyme activity.The reproducibility of the current measured by self-assembled membrane test was better,and it had good stability and life.In this paper,Mb-Cu was chosen to mimic SOD,and the activity of mimics were determined by spectroscopic and electrochemical methods.The self-assembly system of SOD mimic enzyme has good reproducibility and stability,and has a better application prospect in detecting O2-·.