Cu-induced Assembly Of Methanobactin-Functionalized Gold Nanoparticles And Its Mimic Enzyme Activity

Peroxidase(POD)can catalyze the oxidation of organic compounds by hydrogen peroxide.Natural peroxidase can be used in industrial wastewater treatment,medical clinical analysis,analysis of trace phenols in food,phenolic polymer synthesis and many other fields.At present,horseradish peroxidase have been widely used and most of peroxidase were isolated and extracted from plants.Natural horseradish peroxidase was expensive and easily inactivated.Due to its large molecular weight,it was difficult for antigens-antibodies bind during enzyme-linked immunosorbent analysis and it was difficult to label.Looking for a mimic enzyme and catalytic system to replace natural horseradish peroxidase,and exploring an oxygen donor substrate to enhance the sensitivity of mimic enzyme catalytic reaction was the hot spots of the enzyme catalysis field.In this thesis,with the characteristics of peroxidase activity of methanobactin and copper complexes(Mb/Cu),the large surface area and high electronic turnover rate of gold nanoparticles,Mb-functionalized gold nanoparticles assembled by copper ion coordination was used as mimic peroxidase.An efficient POD mimic enzyme was designed and synthesized.The main research contents were as follows:Using the metal ions chelation character of methanobactin(Mb),UV-visible absorption spectra and fluorescence spectra of Mb following addition of Cu(?)showed that Cu(?) and Mb had three different coordination forms of Mb-Cu,Mb2-Cu and Mb4-Cu.The peroxidase activity of three kinds of coordination modes was studied.The results showed that three kinds of Mb/Cu complexes were good catalysts for the catalytic oxidation of hydroquinone with Kobs of 6.11×10-4 s-1,8.28×10-4 s-1 and 4.16×10-4 s-1,respectively.And different coordination reaction rate constant increased 400?800 times.Among them,the activity of dimmer peroxidase was the highest,which indicated that the peroxidase activity of Mb/Cu complexes was affected by the coordination mode.The structure of Mb2-Cu complex may be more conducive to the formation of transition state,so it showed the highest activity.From this,it was confirmed that the optimal coordination mode of Mb/Cu was Mb2-Cu.The Mb and Cu coordination was used to mediate the assembly of Mb-functionalized gold nanoparticles to construct the nano-enzyme complex system.The addition of Cu(?)or Mb-Cu to Mb-modified gold nanoparticles showed that both Mb(?)and Mb-Cu formed Mb2-Cu coordination on the surface of AuNPs.Peroxidase activity was tested on both assembly formats,As a result,it was found that their Kobs were 3 and 1 orders of magnitude faster than those of Cu(?)or Mb-Cu.On this basis,On this basis,we studied the kinetics of Mb2-Cu stable nanocluster structure and determined the optimal reaction conditions:the optimum pH was 7.0,the optimum concentration of hydrogen peroxide was 6×10-3mol/L,the optimum concentration of hydroquinone was 4×10-4mol/L,the optimum reaction temperature was 70 ?.The experimental results showed that the constructed nano-enzyme conformed to the general catalytic rules of the biocatalyst and had higher stability than the native peroxidase,which made it possible to find a new replacement of peroxidase.The following experiments mainly focused on the direct electrochemical study on Mb-functionalized gold nanoparticles.The effects of scanning speed,pH and modification time on the modified electrode were discussed to determine the best electrochemical reaction condition.The results showed that for the Mb2-Cu mediated nanoclusters modified electrode,the optimal reaction conditions were as follows:the scanning speed was 100 mV/s,the pH was 7.0 and the modification time was 16h for the mimic enzyme-modified electrode.The chronoamperometry of Mb2-Cu stabilized gold nanoparticles and Mb2-Cu mediated nanoclusters were studied.The results showed that there was a good linear relationship between the current of two modified electrodes and H2O2 concentration in the range of 5.0×10-5?5.0 × 10-4 mol/L.The apparent Michaelis-Menten constant were 0.805mmol/L and 0.793mmol/L,respectively.This value was small,indicating that both self-assembled electrodes had high affinity for the reduction of H2O2.The detection limits of H2O2 were 1.1 ×10-5 mol/L and 0.9×10-5 mol/L,respectively.In summary,Mb2-Cu mediated nanoclusters was considered to have a higher peroxidase catalytic activity than Mb2-Cu stabilized gold nanoparticles,it was consistent with the conclusions obtained in solution.