Protein-controlled Synthesis Of Copper Nanoclusters Act As Enzymatic Mimics

Nearly half a century,the research,development and application of metal nanoparticles are increasingly prevalent,metal nanoclusters have been widely concerned as a special kind of metal nanoparticles.Copper nanoclusters are nanoparticles newly appeared after gold and silver nanoclusters,with a synthetic low cost,nature of the stable.Copper nanoclusters,with unique surface activity and biocompatibility,play a pivotal role in sensing,biological imaging.Previous studies mainly focus on the stability and pH responsive of the copper nanoclusters,not too much about its oxidation resistance,or as artificial enzyme mimics.In this article we used the bovine serum albumin?BSA?and egg albumin?CEW?as stabilizer,synthesize three kinds of copper nanoclusters,BSA-Cu NPs 1,BSA-Cu NPs 2 and CEW-Cu NPs simply and greenly,we joined the hydrazine hydrate?N2H4 · 2H2O?as a strong reductant in the synthesis process of BSA-Cu NPs 2.Through the characterization of ultraviolet-visible spectral spectrum,fluorescence spectroscopy,Fourier transform infrared spectroscopy and Malvin particle size instrument,we found that the copper nanoclusters is moderate in size,good in dispersivity,stable in the nature.The beta folding of protein secondary structure is mainly in BSA-Cu NPs 1,BSA-Cu NPs 2,CEW-Cu NPs.Vitro antioxidant experiments of BSA-Cu NPs 1,BSA-Cu NPs 2,CEW-Cu NPs showed that the removal abilities of hydroxyl radicals?· OH?,superoxide anion free radical?O2·-?,DPPH free radicals and ABTS radical were over 30%.The three copper nanoclusters can be good as vitro antioxidants.In further characterization of the peroxidase activity,we found that the three copper nanoclusters has good hydrogen peroxide enzyme activity and horseradish peroxidase activity.To qualify activity of Horseradish peroxidase,it found that,in the presence of H₂O₂,BSA-Cu NPs 1,BSA-Cu NPs 2,CEW-Cu NPs respectively turned four kinds of chromogenic substrate ABTS,DAB,OPD,TMB to green,brown,orange and blue,and the corresponding characteristic absorption peaks in ultravioletvisible are at 410 nm,446 nm to 430 nm,652 nm.After optimizing the dynamics reaction conditions of BSA-Cu NPs 1,BSA-Cu NPs 2,CEW-Cu NPs,we confirmed that the optimum reaction pH value of BSA-Cu NPs 1,BSA-Cu NPs 2 is 5,the optimum reaction temperature is 55 ?,and the optimum reaction pH value of CEW-Cu NPs is 4,the best reaction temperature is 75 ?.Testing kinetic parameters under the standard reaction conditions,experimental results showed that the initial rate of the three nanoclusters in catalytic reaction depended the concentration of substrate TMB,H₂O₂.Enzymatic mimics of BSA-Cu NPs 1,BSA – Cu NPs 2,CEW-Cu NPs followed the rules of mie enzyme reaction.The catalytic reaction mechanism was the ping-pong mechanism,the same as natural HRP.In catalytic process,it firstly combined with the first substrate,released the first product when the second substrate started to react.Activities of the three enzymatic mimics are almost similar,BSA-Cu NPs 2 was slightly higher.By using horseradish peroxidase activity of BSA-Cu NPs 1,BSA-Cu NPs 2,CEW-Cu NPs,we successfully built glucose testing experiment system with high sensitivity and accuracy.The response range of glucose is from 1 to 15 m M at least,and the one of BSA-Cu NPs 2 could reach 40 m M.All in all,we successfully prepared three copper nanoclusters: BSA-Cu NPs 1,BSA-Cu NPs 2,CEW – Cu NPs with uniform particle size,good biocompatibility.The three copper nanoclusters not only can be used as antioxidant,but also can be good peroxide mimic enzymes,especially with strong horseradish peroxidase activity.In addition,the horseradish peroxidase activity can be used for sensitive detection sensor system.The unique nature of BSA-Cu NPs 1,BSA-Cu NPs 2,CEW-Cu NPs is bound to lay a foundation for the development of new nanoscale enzyme,will expand the application field of artificial enzymes,also can be widely used in biology in the future.