Peroxidase Mimetic Activity Of Copper Based Metal-organic Polyhedron And Its Application In Biochemical Analysis

Natural enzymes are a kind of proteins or RNA with high catalytic activity.It has many advantages,such as high catalytic efficiency,good substrate specificity,nontoxicity,etc.,and has been widely used in detection and pharmaceutical industry.However,due to some shortcomings of natural enzymes,such as expensive price,difficult to purify and store,unable to reuse,these have limited their practical applications.To solve these problems,scientists have developed some non protein molecules with catalytic activity,called mimetic enzymes.These artificial enzymes are benefit to apply in many fields due to its advantages of simple synthesis procedure,low price,ability to reuse,comparing to natural enzyme,and can replace natural enzyme to some extent.Although there are many progress in the research of mimetic enzyme,more and more materials have been reported,but there are still some problems in this field.First,most of the best application range of peroxidase is in acid condition,which limits its application in analysis and detection.In addition,the catalytic efficiencies of most mimetic enzymes are lower than natural enzymes,which is difficult to meet the practical needs of high catalytic efficiency.Moreover,the specificity of mimetic enzyme materials is often relatively poor than nature enzyme,so the research on mimetic enzyme needs to be expanded and further explored.Metal-organic polyhedra（Metal-Organic Polyhedra,MOP）is a metal-organic nanocage structure formed by coordination of metal ions or clusters and organic ligands.MOP with multifuctional physical and chemical properties can be obtained through modulating the property of organic ligands and selecting different metal ions.Moreover,the performance of MOP can be further regulated by post modification methods to meet the actual needs.Therefore,MOP has been widely applied in catalysis,gas storage and separation,molecular sensing and other fields.Copper coordination compounds are important catalytic activity centers in many natural enzymes,and they have high catalytic ability.In this paper,the copper based metal-organic polyhedra（Cu-MOP）with different properties is obtained by combining the high catalytic activity of copper ions and the advantages of easy modification and regulation of metal-organic polyhedra,so as to solve some existing problems in the mimetic enzyme field.In this thesis,copper ions with high catalytic performance are selected as the center metal ions to coordinate with isophthalic acid to obtain copper based metal organic polyhedra.By modifying the organic ligands,copper based metal-organic polyhedra with good peroxidase performance were obtained to widen the application fields.It provided a reference for the application of Cu-MOP in analytical chemistry and biomedical fields.The specific contents of this dissertation are as follows:1.In the first chapter,the concept,types and related applications of mimetic enzyme and metal-organic polyhedra are introduced.The research status is summarized,and the research ideas and the significance of this dissertation are put forward.2.In chapter 2,the Br-Cu-MOP nanorods assembly was obtained by using 5-bromo-1,3-isophthalic acid as organic ligand and coordination with copper ions.UV-Vis spectrum showed that the Br-Cu-MOP nanorods had peroxidase activity and showed good catalytic performance in p H 6.0-10.0 range,and can be used to detect H₂O₂ under physiological conditions.Meanwhile,the concentration of glucose can be indirectly determined by combining Br-Cu-MOP nanorods with glucose oxidase.Compared with the previous method of peroxidase materials,Br-Cu-MOP nanorods are used as catalyst to detect glucose without changing buffer solution system,and can be completed in the same p H 7.0 solution.The linear range of H₂O₂and glucose detection by Br-Cu-MOP nanorods is 4.0×10^-6 M-1.0×10^-3 M and 5.0×10^-6 M-3.0×10^-4 M respectively,which can be used for the detection of actual samples.3.In order to further improve the activity of Cu-MOP in acid medium,Hemin was connected to the surface of Cu-MOP by post modification in chapter 3.The results show that Hemin is further coordinated with the copper ions in B-Cu-MOP by free carboxyl group,and then connects to the surface of B-Cu-MOP.The obtained B-Cu-MOP-Hemin has a good catalytic activity in the range of p H 3.0-7.0.The linear range and the Michaelis-Menten constant value（k_m）of H₂O₂ were measured under the conditions of p H 4.0,5.0 and 7.0.It was found that B-Cu-MOP-Hemin had the strongest affinity with substrate and showed the best catalytic activity compared with Hemin and B-Cu-MOP.It can be applied to detect lower concentration of H₂O₂ under multiple p H conditions.4.In order to expand the application scope of Cu-MOP,in this chapter,NO₂-Cu-MOP was obtained after the coordination of 5-nitro-1,3-isophthalic acid and copper ions.After mixing NO₂-Cu-MOP and water with ultrasonic procedure,NO₂-Cu-MOP hydrogel can be obtained.Through the analysis of its structure,it is found that due to-NO₂ can form hydrogen bonds with water molecules,NO₂-Cu-MOP could directly form hydrogel.Based on the high peroxidase activity of NO₂-Cu-MOP,NO₂-Cu-MOP hydrogel showed good antibacterial activity with good biocompatibility,and had a killing rate of 95%and over 97%for E.coli and S.aureus.The antibacterial mechanism is that NO₂-Cu-MOP hydrogel first destroys the cell membrane after contacting with bacteria,and then stimulates the production of ROS substances after entering the bacteria,destroying the protein and lipid of bacteria and killing bacteria.Based on the above results,NO₂-Cu-MOP hydrogel can be modified by polyacrylic acid（PAA）and glycerol to obtain PAA-NO₂-Cu-MOP hydrogel,which has a better moisture retention property and show high antibacterial activity.It can be used to promote wound healing process and the effect is better than the recombinant human epidermal growth factor.5.Due to the good mimetic properties of NO₂-Cu-MOP,the NO₂-Cu-MOP hydrogel is applied in cell protection field in the fifth chapter.The results showed that yeast coated with NO₂-Cu-MOP hydrogel remain the highest viability among other protective materials like Mn O₂,Si O₂,Ca P under the external stimulation conditions.This is attributed to the unique structure of hydrogel and the NO₂-Cu-MOP hydrogel will form a protective barrier between the stimulants and cells.In the mean time,the NO₂-Cu-MOP hydrogel has a high SOD and CAT activity which can quickly remove ROS;hence,it shows the best cell protection effect.6.In order to study the effect of surface charge on peroxidase enzyme catalytic property,two kinds of isophthalic acid ligands with positive and negative charge were designed and synthesized in chapter 6.After the coordination of copper ions with isophthalic acid,N-IPA and S-BDA,three Cu-MOP were obtained.The results showed that the positive N⁺-Cu-MOP had the best catalytic activity due to the electrostatic interaction with the negative ABTS.The linear range of H₂O₂ detection was 1.0×10^-6 M-1.0×10^-4 M.At the same time,the surface of bacteria is usually negative,the combination of N⁺-Cu-MOP destroys the charge balance of the bacteria surface,which leads to the cell membrane rupture and bacterial death.When N⁺-Cu-MOP enters the cell,it promotes the production of ROS to kill bacteria further.Therefore,N⁺-Cu-MOP also shows the best antibacterial performance.In addition,although H-Cu-MOP has a small size,but it is easy to aggregate and disperse poorly in solvent.However,SO₃^--Cu-MOP has a better dispersion property,which is conducive to expose more catalytic sites.Therefore,compared with H-Cu-MOP,SO₃^--Cu-MOP has a better catalytic activity and antibacterial performance.The results show that the design of organic ligands can control the surface properties of MOP,this could provide a basis of controlling the performance of metal organic polyhedra.7.In order to enhance the catalytic activity of Cu-MOP,three amino acids modified Cu-MOP were obtained in chapter 7.After the structural analysis,it was found that the three amino acids were further coordinated with copper ions in H-Cu-MOP by carboxyl group.The modified H-Cu-MOP has a better catalytic efficiency and lower detection limit because of the increase of the binding ability with H₂O₂.Based on the above results,H-Cu-MOP and H-Cu-MOP-Isoleucine were used to detect glutathione.The results showed that the linear range of H-Cu-MOP and H-Cu-MOP-Isoleucine was 1.0×10^-5 M-1.0×10^-4M,2.5×10^-6 M-1.0×10^-4 M.H-Cu-MOP-Isoleucine had a wider linear range,and it has a better application prospect in the field of analysis and detection.