Study On Biomimetic Enzyme Activity Of Two Novel Iron-based Functional Materials And Their Application In Biological Analysis

Natural bio-enzymes are involved in many important life activities and have also been widely studied and applied in a variety of fields such as medicine,environment,and food industry,owing to their excellent properties including high catalytic efficiency and good substrate specificity.However,the application of these bio-enzymes is severely restricted by their inherent drawbacks such as instability,high-cost preparation and purification processes.As a consequence,more and more attention has been paid to discovery and construction of alternative artificial enzymes in recent years.In comparison with natural enzymes,enzyme mimics exhibit high stability against harsh reaction conditions.Moreover,they possess the advantages such as low cost,flexibility in structure and composition,and tunable catalytic activities.In this thesis,two novel iron-based functional materials Fe₃?PO₄?₂?OH?₂ and MIL-53?Fe?have been developed.Their peroxidase/oxidase mimic activities and the catalysis mechanisms have also been investigated.These two iron-based functional materials are further employed for colorimetric analysis of hydrogen peroxide,glucose and biothiols.Firstly,in this part,an iron hydroxyl phosphate Fe₃?PO₄?₂?OH?₂ microoctahedron was prepared with a one-pot hydrothermal synthesis method.The Fe₃?PO₄?₂?OH?₂ is demonstrated for the first time to be an efficient peroxidase mimetic.The method of colorimetric analysis of hydrogen peroxide and glucose has been further developed by using its peroxidase-like activity.The morphology,elemental composition,crystal structure and metal ion valence state of the peroxidase were analyzed by the Field Emission Scanning Electron Microscopy?FE-SEM?,X ray spectrum?EDS?,X-Ray Diffraction?XRD?,X-Ray Photoelectron Spectroscopy?XPS?and Ultraviolet–visible spectroscopy?UV-vis?.It was used as an efficient peroxidase mimetic for sensitive colorimetric detection glucose.In the presence of H₂O₂,Fe₃?PO₄?₂?OH?₂ catalyzes the oxidation of 3,3',5,5'-tetramethylbenzidine?TMB?to produce the blue-colored oxidized TMB,which is quantitatively measured with UV-Vis spectrophotometer.Under the optimal experimental conditions,the detection limit of hydrogen peroxide can be as low as 0.17?M.The catalytic behavior of Fe₃?PO₄?₂?OH?₂ is in accordance with the typical Michaelis-Menten kinetic model,and its catalytic mechanism follows the ping-pong mechanism.Based on the above colorimetric method,a highly sensitive and selective glucose colorimetric method was further constructed.Glucose oxidase catalyzes the oxidation of glucose to produce gluconic acid and hydrogen peroxide and successfully converts glucose detection into hydrogen peroxide detection.A linear range of 5–100?M and a LOD of 1.2?M are achieved under optimal conditions.In the detection of actual serum sample,the error between the detection value and the standard value is small,indicating its good feasibility of in practical application.This work not only reports a new kind of peroxidase mimetic,but also develops a simple,robust and sensitive colorimetric sensing platform for hydrogen peroxide and glucose detection,showing its great potential applications in biomedicine and food industry.Secondly,metal–organic frameworks?MOFs?,a new type of functional materials,have attracted considerable attention in the field of enzyme mimics.In particular,Fe?III?-based MOFs such as MIL-68,MIL-100 and MIL-88 have been reported as peroxidase mimics.Owing to their large surface area,high stability and active metal center,MOF-based enzyme mimics exhibit excellent catalytic activity.MOF MIL-53?Fe?with abundant active sites was synthesized by a facile one-pot hydrothermal synthesis method using DMF as solvent.The intrinsic oxidase-like catalytic ability of the prepared MIL-53?Fe?has been demonstrated for the first time.The MIL-53?Fe?is then applied for a colorimetric detection of biothiols.The morphology,crystal structure and functional group of MIL-53?Fe?were characterized by the SEM,XRD,XPS,Fourier Transform infrared spectroscopy?FTIR?and UV-vis.Experimental parameters,such as pH,temperature,incubation time and MIL-53?Fe?concentration were investigated to find an optimal condition.Oxidase mimic MIL-53?Fe?can efficiently catalyze TMB oxidation to produce a blue-colored oxidized TMB,which can be inhibited by the addition of biothiols.Thus,a novel and simple colorimetric method for detection of biothiols is developed based on oxidase mimic MIL-53.Under optimal condition,the absorbance at 652 nm against Cys concentration shows a linear relationship within a range from 1 to 100?M with detection limit of 0.12?M.MIL-53?Fe?was used successfully for the highly sensitive colorimetric detection of biothiols in serum samples.The new method for simple detection of biothiols is particularly important in developing countries or poor areas where high-tech diagnostic equipment is not widely available.The difference of biothiols level between cancer cells and normal cells is regarded as one of most important physiological biomarkers for cancer diagnosis.Furthermore,assessment of the level of biothiols has been performed with the developed method in normal and cancer cells.The experimental results show that the level biothiols in cancer cells is significantly higher than that in normal cells.This method provides a powerful tool for the identification cancer cells in a simple way and shows its broad application prospect in biomedical field.