Construction Of Sensor For Nanoporphyrin Mimetic Peroxidase And Accurate Detection Of Antioxidants And Pesticides

Peroxidase（PO）is an important endogenous active oxygen scavenger in cells,and plays a significant role in the process of peroxide oxidation.Natural peroxidases,such as horseradish peroxidase（HRP）,lignin peroxidase（LIP）and lactoperoxidase（LP）,their difficulties remain in practical applications due to their thermal instability,high purification costs and scarce sources.Recently,artificial enzymes have been developed to solve this problem.Porphyrin nanocomposites are considered as an ideal peroxidase mimetic enzyme due to their high catalytic activity,excellent biocompatibility and low cost.However,most porphyrin nanocomposites are products derived from single metal nanomaterials or metal oxide nanomaterials.They have some disadvantages such as complex synthetic processes,severe pollution of raw materials,and low bioavailability,which failure to meet requirements of detection in modern biochemical analysis and food analysis.Consequently,development of a highly active,easy to prepare and stable peroxidase mimic catalyst is of great significance for solving the peroxidase application.This paper has been studied the catalytic properties of various nanoporphyrins with inherent peroxidases.According to catalytic oxidation reaction between the peroxidase substrates 3,3’,5,5’-tetramethylbenzidine（TMB）and hydrogen peroxide（H₂O₂）to select 5,10,15,20-tetrakis（4-pyridyl）porphyrin-Zn（II）（ZnTPyP）as the best catalytic enzyme,and propose a variety of sensors for detection of antioxidants and pesticides based on nano-ZnTPyP peroxidase.The main content of this work is as follows:1)Construction and activity detection of nanoporphyrin peroxidaseIn previous study,a novel analytical peroxidase fabricated with dodecyltrimethylammonium bromide（DTAB）nanosized Zn/Cu/Fe/Mn/Sn/Co TPyP has been confirmed as a promising peroxidase mimetic for catalyzing the oxidation of peroxidase substrate TMB in the presence of H₂O₂produces a blue reaction.The optimized nano ZnTPyP showed higher stability and simulated peroxidase activity,and its catalytic activity was much higher than horseradish peroxidase（HRP）.The catalytic mechanism of nano ZnTPyP peroxidase as following:When the nano ZnTPyP were irradiated under the visible light,TPyP molecules from the nano ZnTPyP absorbed specific wavelength of visible light,and the electrons could be transfer from TPyP to DTAB conduction band.Then,the electrons of excition are further transferred to H₂O₂and generated·OH in acidic solution（p H=3.6）.Thus,a simple,inexpensive and highly sensitive assay to detect H₂O₂with the detection limit of 0.5μmol L^-1was developed.Moreover,the proposed nano ZnTPyP-based sensor was applied successfully for the analysis of H₂O₂extraction from human saliva samples.In addition,the nanoporphyrin-based peroxidase sensor can also be used for constructing a corresponding sensor through the elimination of·OH,the generation of H₂O₂and nanoporphyrin.This study will promote the use of nanoporphyrin peroxidase in medical diagnosis and drug testing.2)Accurate detection of catechin and gallic acid in green tea by nanoporphyrin peroxidaseA colorimetric sensor based nano ZnTPyP as a peroxidase mimetic was developed and used for rapid detection of the antioxidant catechins and gallic acid.The typically mechanism is that nano ZnTPyP oxidizes H₂O₂to produce·OH,which catalyzes the oxidation of the chromogenic substrate TMB to produce a color reaction.Then,the catechins and gallic acid scavenge the·OH to inhibit the color development of nano ZnTPyP.The catalytic oxidation of the substrate TMB was inhibited.As a result,the concentration of catechins and gallic acid is inversely proportional to the color development intensity of TMB.In addition,a sensitive colorimetric assay to detect three representative antioxidants,including epigallocatechin gallate（EGCG）,gallocatechin（GC）,and gallic acid（GA）with a range of concentration from0.8 to 8.0,0.05 to 1.00,and from 1 to15μmol L^-1,respectively.The recovery experiments of spiked catechin and gallic acid in green tea clarified that nano ZnTPyP-based peroxidase mimetic sensor for stable and reproducible detection of pesticides in pure solutions and complex matrices.Therefore,the nanoporphyrins peroxidase-like activity of nanomaterials and scavenging effect of catechins on·OH makes them be a sensitively and accurately tool for the hydroxyl radical scavenging ability of antioxidants and compounds in food and drug analysis.3)Accurate detection of glucose by nanoporphyrin peroxidaseA novel sensor for glucose detection in nano ZnTPyP/glucose oxidase（GOx）/3,3’,5,5’-tetramethylbenzidine（TMB）was developed based on the excellent peroxidase-like activity of nano ZnTPyP and the generation of H₂O₂for the detection of.GOx catalyze the oxidation of glucose to produce H₂O₂in the presence of TMB and nano ZnTPyP,which produces blue products of TMB oxidation and visible light absorption at 652 nm.This simple approach towards colorimetric detection of glucose with a linear detection range from 3 to 70μmol L^-1and a detection limit of 0.15μmol L^-1in human serum samples.It proved that such a nano ZnTPyP peroxidase exhibits the excellent stability of and its high biocompatibility in aqueous solution.It can be used as an excellent potential substitute for horseradish peroxidase for low-cost monitoring of glucose in vivo and vitro.In addition,this method is promising to be applied to the monitoring of cholesterol and sarcosine in clinical medicine because cholesterol and sarcosine can also produce H₂O₂under the catalysis of corresponding oxidases.4)Accurate detection of pesticides by nanoporphyrin peroxidaseA new method for the rapid detection of multiple pesticide residues was constructed based on the specific interaction between nanoporphyrins in peroxidase-like mimic and pesticides.Nanoporphyrin can catalyze the production of blue ox TMB（652 nm）by the peroxidase substrate TMB in the presence of hydrogen peroxide.Due to the different interactions between different pesticides and various nanoporphyrins,if pesticides are added to the reaction system,the activity of peroxidase is inhibitted,reduceded the formation of ox TMB and absorbance at 652 nm.Therefore,the amount of pesticide is inversely proportional to the intensity of color development of TMB.Moreover,this experiment tested three pesticides,trichlorfen,paraben,and dichlorvos,the linear range for trichlorfen,thimet and dichlorvo determination is 0.247,0.660,and 1.020 ng m L^-1,respectively.This sensor was successfully applied to the detection of trichlorfen,thimet and dichlorvo with satisfactory recovery results from 97.8%to 105.3%in fresh apple juice,cabbage and human serum respectively.The present study both expands the application of nanoporphyrin as peroxidase mimetic,and provides theoretical basis for further development and research with new rapid and accurate detection of pesticide residues and active compounds.