Preparation Of Metal Oxide Nanozymes And Its Application In Disease Indicators Detection

Enzymes are catalysts that regulate various biological processes and metabolism in organisms.They have been widely used in medicine and food health,biochemistry and other fields,but they also generally have disadvantages,such as complicated purification,high commercial costs,easy deactivation,etc.Therefore,it is of great research value to discover and obtain substitutes of natural enzymes to make up their defects.Inorganic nanomaterials are generally regarded as inert substances in traditional concepts.Researchers recently have found that some nanomaterials exhibit catalytic properties similar to those of biological enzymes,while retaining the unique physical and chemical properties of materials,which are named“nanozymes”.The advent of nanozymes well reveals the inherent biological effects and new characteristics of nanomaterials.nanozymes not only show considerable catalytic efficiency,but also have the characteristics of heat,acid and alkali resistance,large-scale production,and low cost.At present,various analytical principles and sensors based on nanozymes have been reported,which have realized sensitive detection of various biological macromolecules and small molecules.Herein,based on the development of new types of cobalt-containing nanozymes,the performance of their mimetic enzyme performance,catalytic properties,and mechanism were investigated in detail.Meanwhile,in order to design novel sensors,improve their performance and expand their application,three kinds of sensors for the detection of multiple disease biomarkers are constructed.The three types are colorimetric sensors for determination of neurochemicals,portable hydrogel based colorimetric sensors for qualification of liver-related indicators,and dual-mode sensors for detection of biomarkers of diabetic nephropathy.The main research content of this paper is divided into the following three parts:First,bimetallic oxide Co_1.5Mn_1.5O₄with multiple enzyme-like activities,namely peroxidase-,oxidase-and laccase-mimetics,was prepared by a sol-gel approach.It was found that Co_1.5Mn_1.5O₄ kept high activity in a wide range of temperature and p H,and its catalytical process was in accord with Michaelis kinetic catalysis.Based on the above three different types of mimetic enzymes,the corresponding colorimetric sensors for quantitative analysis of acetylcholine,ascorbic acid and epinephrine were constructed.Specifically,acetylcholine can be hydrolyzed to produce H₂O₂ with the aid of acetylcholinesterase and choline oxidase.Based on peroxidase-like activity of Co_1.5Mn_1.5O₄,the concentration of acetylcholine can be reflected by quantifying H₂O₂.For the determination of ascorbic acid,it can reduce and discolor chromogenic substrate ox TMB that was pre-oxidized via oxidase-like activity of Co_1.5Mn_1.5O₄.Finally,based on laccase mimetic activity,epinephrine can be oxidized in one step to produce red products,thus achieving its direct detection.The detection ranges of the above three targets were0.5～600μmol/L,0.5～100μmol/L and 0.3～100μg/m L,respectively,which covered their normal physiological levels in the human body.The fabricated sensor showed good stability and selectivity,and realized quantitative detection of acetylcholine and ascorbic acid in serum samples,as well as epinephrine in urine samples.Secondly,Au/LDO with peroxidase-like activity was synthesized via combining gold nanoparticles with layered bimetallic oxides.It was found that the synergistic effect of noble metal and metal oxide materials was conductive to improving the activity of nanozyme.We also verified that catalytic performance of Au/LDO was temperature-and p H-dependent,and conformed to Michaelis kinetic curve.The investigation of catalytic mechanism indicated that Au/LDO first catalyzed H₂O₂ to produce O₂^·-,and then oxidized TMB to produce colorimetric signals.In this work,Au/LDO with excellent peroxidase-like performance was used as a colorimetric probe to construct a cascade system by combining with natural enzymes,and relevant strategies for the analysis of three liver-related biomarkers,aspartate aminotransferase,alanine aminotransferase and alkaline phosphate kinase,were designed.Typically,the reagents of different reaction systems and Au/LDO were encapsulated into the hydrogel.By means of the optical transparency,biocompatibility and three-dimensional network structure of hydrogel,the sensitivity of colorimetric signal,the stability of natural enzyme and the diffusion of reactants can be improved.Then,the above functional hydrogels were introduced into a portable device,and the simultaneous detection of three targets was realized with the detection ranges of 10～150 U/L,7～200 U/L and 4～60 U/L,respectively.Compared with traditional assay kits,the fabricated system had the advantages of simple operation procedure,high sensing efficiency,good stability and low cost.Furthermore,by changing the substances immobilized in the hydrogel,it can also be extended to the optical analysis for other biomarkers.Finally,H-LDO with single peroxidase mimetic properties was designed by combining hemin with layered bimetallic oxides.The results showed that its catalytic process was related to temperature and p H,and also in accord with Michaelis kinetic curve.In a typical catalytic process,H-LDO catalyzed H₂O₂ to produce·OH,and then oxidized TMB to ox TMB.The latter was a promising chromogenic product as well as good electrochemical indicator.Based on this property,we established an electrochemical-colorimetric dual-mode sensing system.This unit was composed of nanozyme H-LDO,hydrogel and a portable device,in which H-LDO severed as catalyst and signal amplification probe,hydrogels with porous structure and adhesion can not only be used as carriers to immobilize reaction reagents,but also as outstanding colorimetric signal output terminal and biological interface for electrochemical detection.The integration of hydrogel with portable device simplified the process of sample transformation and detection,thus improving the analysis efficiency and maneuverability.Then,four indicators of diabetes nephropathy（urea,uric acid,glucose and creatinine）were successfully determined using this system.Based on the characteristics of the above four targets,an enzyme inhibition strategy based on p H regulation was designed for urea detection,and a natural enzyme-nanozyme cascade catalysis strategy was proposed for the determination of uric acid,glucose and creatinine.The detection range can well cover their physiological level in human serum.In addition,the current response intensity of the hydrogel modified electrode was about twice that of the bare electrode,and the relative standard deviation of the test results after 15 consecutive tests was 76.8%lower than that of the bare electrode.The above results proved that the system using hydrogel as the sensing interface has improved sensitivity and stability.While the dual-signal mode allowed for mutual verification of results and effectively minimized false negative and positive results.