Preparation And Electrochemical Performance Of Simulated SOD Electrode Modified By Methanobactin

Superoxide dismutase?SOD?is a kind of metalloenzyme which has ability of disproportionation superoxide anion and whose active center has copper ion-bound imidazolyl group.While methanobactin?Mb?also contains an imidazole residue.When combined with copper ions,Mb has SOD mimic enzyme activity which provides a natural active site for simulating natural enzyme.When superoxide anion was produced by DMSO/NaOH system,the stability of Mb and Mb nanoclusters were evaluated by using nitrogen blue tetrazolium?NBT?.On surface of electrodes,Mb nanoclusters were modified by self-assembly method.By using electrochemical method,superoxide anion of DMSO/NaOH system was detected and electrochemical performance of the simulated SOD modified electrode was evaluated.The main research contents are as follows:?1?According to DMSO/NaOH reaction mechanism of producing O₂^-·,reaction system was detected by NBT.Results show that the optimal conditions of producing O₂^-· as followed:the DMSO concentration 5mmol/L,the temperature 250?C and the reaction time 30min.In addition,when the NBT concentration was 510?mol/L,detection of O₂^-· would be with the most sensitive.By comparing the inhibition rate of SOD mimics with SOD enzyme on superoxide anion,the order of simulated SOD activity as described:Mb/Cu???-AuNPs>Mb/Cu???>AuNPs>Mb.By establishing a kinetic model,the chemical reaction rate constant K of SOD mimics and SOD enzyme were 33.8×104 L·mol-1·-s-1 and 37.2× 104 L·mol^-1·s^-1,respectively.These results was indicated that Mb/Cu???-AuNPs had SOD mimic activity?2?Through mixed self-assembly,modified electrodes were prepared by Mb,AuNPs,mercaptocarboxylic acid?2-mercaptoacetic acid,3-mercaptopropionic acid,11-decylundecanoic acid,16-mercaptohexadecanoic acid?.The amount of self-assembly of Mb,AuNPs and self-assembly of mercaptocarboxylic acid on modified electrodes and so on were discussed.Moreover,the variation of the peak current of the CV and the change of the are radius of the AC impedance spectrum in the high frequency region was used to determine the optimal assembly condition.The results showed that the optimal conditions as followed,the amount of Mb solution?5.O×10-4 mol/L?,AuNPs solution?particle size 16 nm?and MUA solution?5 mmol/L?were 300 ?L,200 ?L and 30 ?L,respectively.Since the complex formed by Mb and copper ions has SOD activity,the modified electrodes were coordinated with copper ions.The oxidation-reduction potential of modified electrodes with various mercaptocarboxylic acids were changed.The oxidation-reduction potential of Mb-AuNPs-MUA/Au modified electrode varied from 0.334 V and 0.165 V to 0.304 V and 0.172 V.At the same time,the redox peak current of modified electrodes were enhanced,which indicates that it was easier to expose the active site on Mb after coordiniton of copper ions,which improved the transfer rate of between electrons and electrodes.At the same time,the Mb nanoclusters was modified to the electrodes by the diameter of the circle formed by the high-frequency impedance of the AC impedance and the low-frequency portion Warburg impedance.The entire electrochemical process was controlled from thermodynamic diffusion to kinetic control.The SOD/Au enzyme modified electrode was prepared by using 1 mg/L SOD enzyme to droplet on the surface of the bare gold electrodes.Comparing the SOD modified electrode with the Mb modified electrodes,the peak current and electron transfer resistance were smaller than that of the Mb modified electrodes.The electron transfer resistance of the SOD/Au modified electrode and the Cu/Mb-MUA-AuNPs/Au modified electrode were 4863 ?·cm2 and 4124?·cm2,respectively.Because the spatial structure of the whole enzyme molecule of SOD is large relatively,which make the distance between the electron transfer site and surface of gold electrode be large and the heterogeneous electron transfer rate be reduced.However,due to the small spatial structure of the Mb molecule in the Cu/Mb-AuNPs-MUA/Au modified electrode,and the assembly of AuNPs on the electrode,which could promote the electron transfer rate and increase the electron inversion rate between the Mb modified electrode and the probe solution.?3?The Mb nanoclusters?Mb-AuNPs-MUA?were modified on the surface of gold electrode.And the modified electrode?Cu/Mb-AuNPs-MUA/Au?was prepared by coordination with copper ions,which was used to detect superoxide anion.In this way,the effect of this electron-free medium-modified electrode on the activity of SOD was evaluated.The experimental results showed that the Cu/Mb-MUA-AuNPs/Au modified electrode and SOD/Au modified electrode had obvious catalytic disproportionation on superoxide anion in DMSO/NaOH system.Before and after adding NaOH solution,their peak current went from 1.072×10-7A and 9.145×10-8A to 5.69×10-5A and 7.243×10-5A,respectively.What's more,the change rate of SOD peak current was 78.51%,which indicated that using Cu/Mb-MUA-AuNPs/Au as the simulated SOD modified electrode had the ability to catalyze the superoxide anion produced by disproportionation of DMSO/NaOH system.The heterogeneous electron transfer rate constant?Ks?was 0.041 cm/s and the electron transfer coefficient?a?was 0.435.In the range of 0.06 ?mol/L to 0.2 ?mol/L,the superoxide anion concentration had a better linear relationship with the peak current and the correlation coefficient was 0.9719.Furthermore,the method detection limit?LOD?was 1.129×10-3 ?mol/L?S/N=3?and 3.683×10-3?mol/L?S/N=10?,respectively.And the relative standard deviation?RSD,n=9?was 3.83%,which was measured by the precision experiment.The peak current of detection of superoxide anion had better reproducibility and stability.It was indicated that modified electrodes had good performance with SOD mimic enzyme activity.