The Direct Electrochemical Research Of Catalase And Artificial Peroxidase

This work was mainly based on the favorable combination of carboxylfunctionalized multi-walled carbon nanotubes and gold nanoparticles,we usedelectrochemical method to achieve a direct electrochemical studies of the protein with heme–centered. then we successfully prepared the third generation enzyme biosensor bycombined catalase and the AP with nanocomposites of AuNPs/L-Cys/MWCNTs-COOHfilms and modified it to the electrode surface of glassy carbon. The electrochemical reactionmechanism of catalase and the AP analysised by cyclic voltammetry was the fixedmechanism, and this biosensor had the higher response to H₂O₂.we measured the simulated enzyme′s structure, the electrochemical characteristics,and enzyme kinetics parameters by the transmission electron microscopy （TEM） and theelectrochemical methods. The main experimental methods were mainly cyclic voltammetry,the time of the current scan and the AC impedance method. The main work were include thefollowings:（1） CVs of different modified electrodes;（2） the study of modified electrodemechanisms in different scanning speed;（3） the effect of different pH to modified electrode;（4） AC impedance of different modified electrodes;（5） The relationship between themodified electrode and the concentration of H₂O₂.First of all, the direct electrochemistry of catalase: By immobilizing catalase on ananocomposite containing functionalized multi-walled carbon nanotubes and L-cysteinemodified gold nanoparticles, a third generation biosensor was developed for determination ofthe hydrogen peroxide. The cyclic voltammograms of catalase on the nanocompositemodified glassy carbon electrode showed a pair of well-defined quasireversible redox peakswith the formal potential of-441±2mV versus Ag/AgCl at a scan rate of0.05V/s.Theenzyme electrode response toward hydrogen peroxide was linear in the concentrations rangingfrom1nmol/l to1μmol/l, with a detection limit of0.5nmol/l. The apparentMichaelis-Menten constant was calculated to be0.34μmol/l. The heterogeneous electron transfer constant was calculated to be8.72s^-1.Secondly, the direct electrochemistry of artificial enzyme constructed by self-assemblyof Gemini+iminidazole+heme+SDS: A nano vesicular was constructed with the complexconsisting of12mmol/l hemin,3mmol/l imidazole,0.8mmol/l Gemini and90mmol/l sodiumdodecyl sulfate （SDS） in50mmol/l phosphate buffer solution （PBS）. The novel nano structureacted as an efficient artificial peroxidase （AP）. The AP was also immobilized on a carboxylfunctionalized multi-walled carbon nano-tubes and gold nano-particlesnano-complexmodified glassy carbon （GC） electrode in50mmol/l PBS. The enzyme biosensor has goodstability and electrochemical activity to build a third-generation biosensors behalf perixidase.the heterogeneous electron transfer rate constant（ks） between the artificial enzyme and themodified electrode was evaluated to be5.4s^-1. under the optimized conditions, The modifiedelectrode can be used to detect hydrogen peroxide in the range from0.03to160μmol/llinearly, with a detection limit of0.03μmol/l. The apparent Michaelis-Menten constant (K_m^app)of AP modified electrode was evaluated to be0.034±0.003mmol/l.Thirdly,the direct electrochemical studie of artificial enzyme which was builtself-assembly based on sodium dodecyl sulfate nano-micelle and cytochrome c. The cyclicvoltammetry analysis was used to observe the electrochemical characteristics of artificialenzyme. The immobilized nano-structured peroxidase not only perform a high stability andactivity and can be immobilized on the electrode. The experimental results suggested thatthe direct electron transport between artificial enzyme and Glassy Carbon electrode wasrealized. The immobilized nano-structured peroxidase modified electrode showed a pair ofwell-defined redox peaks, with a formal potential E′of59±2mV （vs. Ag/AgCl） at a scanrate of0.05V/s. The K_m^appvalue of artificial enzyme based on electrochemical analysis wasevaluated to0.27±0.02μmol/l, the heterogeneous electron transfer constant （ks）is2.1±0.1s^-1.and this modified electrode can be used to detect the hydrogen peroxide,which may be usedas a kind of third generation biosesor.By the experimental data, we have got the following conclusions:1,successfullycombined catalase and catalase and the artificial enzyme with the electrode surface ofglassy carbon;2,catalase and the artificial enzyme has the higher response ability and catalytic efficiency to the lower concentrations substrate;3,successfully build the thirdgeneration biological sensors.