The Study Of Biosensor Based On The Ni/Co Layered Double Hydrosides Peroxidase Mimic

Natural enzymes have low content in living organisms,and are difficult to purify,high production cost,easy degeneration and inactivation,which severely limit the application of natural enzymes in actual production and life.Therefore,in order to overcome these defects of natural enzyme,the research of nano-enzyme was considerable.Nanomaterials as nanozymes with their unique properties has attracted the attention of many researchers,at present,which has been reported to enzyme properties of nano materials including metal nanomaterials,metal oxide nanomaterials,carbon nanomaterials,metal-organic framework and so on.Our research reported layered double hydroxides?LDHs?nanomaterials as a novel nanozymes and investigate their preparation,characterization,peroxidase-like activity and application in detection small biological molecules,antioxidant effect and antibacterial effect.?1?Synthesized Ni/Co LDHs and studied its minic peroxidase properties.Investigated its peroxidase activity in phosphate buffer solution?pH=7?,Tris-HCl buffer solution?pH=7?,deionized water.Compared the catalytic of Ni/Co LDHs oxidation 2,2-alnitrogen-2?3-ethyl-benzothiazole-6-sulfonic acid?ammonium salt?ABTS?,3,3',5,5'-tetramethyl benzidine?TMB?and o-phenylendiamine?OPD?three kinds of chromogenic substrate and the present of H₂O₂.Electron paramagnetic resonance technique was used to study the catalytic mechanism of Ni/Co LDHs.The results showed that Ni/Co LDHs catalyzed H₂O₂ to produce·OH and ¹O₂.And because of the strong oxidation ability of·OH and ¹O₂,could oxidized the color substrate ABTS.Base on the peroxidase activity of Ni/Co LDHs in water we established the one-step way is to detect glucose and acetylcholine of a colorimetric method.The detection limit of glucose was 0.1?M,the linear range was 0.5?M-100?M.The detection limit of acetylcholine was 1.62?M,linear range was 10?M-150?M.This method is successfully applied in detection glucose in serum samples and the detection of acetylcholine in milk.?2?Ni/Co LDHs peroxide enzyme as the model,we developed three fluorescent peroxidase substrate in alkaline conditions,homovanillic acid?HVA?,Tyramine hydrochloride?Tyramine?,3-?4-hydroxy phenyl?propionic acid?HPPA?.Investigate the influence of pH,temperature,concentration of H₂O₂ and Ni/Co LDHs to the catalytic oxidation of Ni/Co LDHs at the present of H₂O₂.The results showd that Tyramine had stronger resistance to the change of external environment.Steady state dynamics analysis results show that Ni/Co LDHs has a strong affinity to the Tyramine.We applied these three fluorescent substrate to glucose detection,and compared with other fluorescence substrate,the detection limit was significantly reduced and the linear range widened.Finally,we use the Ni/Co LDHs-HVA-H₂O₂ system in the evaluation of ascorbic acid and glutathione antioxidant activity.We concluded that the resistance to oxidation of glutathione was the concentration quenching effect,and the resistance to oxidation of ascorbic acid was irrelevant with the concentration of ascorbic acid.?3?The study showed that the peroxide activity of Ni/Co LDHs was related to the pH of the reaction system.So the peroxides of Ni/Co LDHs could be regulated by changing the pH value of the reaction system.Because of urea can be decomposed by urease to produce ammonia,and then rise the pH value of the system.Penicillin degradation in penicillin enzyme can produce H⁺,thus reduce the pH value of the system.Based on the relationship of fluorescence intensity of Ni/Co LDHs-H₂O₂-HVA system and the p H value,designed the quantitative detection sensor of urea,urease,penicillin,penicillinase.The detection linear range of urea was 1.7×10^-5-1×10^-3 M,and the linear range of the urease was 0.003-2.7 U/mL.The detection linear range of penicillin was 3×10^-6-1.3×10^-3 M,and the linear range of the penicillinase was0.003-0.1 U/mL.We successfully used the established method to determine the concention of urea in human serum and urine sample,and the concentoin of penicillin in milk.?4?Preparation of Ni/Co LDHs with different Ni Co molor rate.Further study the peroxidases activity of Ni/Co LDHs with different molor rate of Ni Co.And concluded that when the molor rate of Ni:Co was4:6 the Ni/Co LDHs had the best catalytic activity and the best antibacterial performance.The antimicrobial properties of Ni/Co LDHs and Ni/Co LDHs+H₂O₂ were investigated for the bacteria of S.aureus and E.coli.