Preparation Of Nanozyme Based On Immobilized Ionic Liquid And Its Application In Colorimetric Sensor

Diabetes is a chronic disease characterized by elevated glucose levels.The dysfunction of pancreatic beta cells severely affects glucose levels in the blood.Hydrogen peroxide(H₂O₂)is an oxidant and a byproduct of glucose oxidase(GOD)oxidation of glucose.There are many methods for detecting H₂O₂and glucose.As a kind of optical sensor,colorimetric sensor has the characteristics of simple operation,fast and convenient,and can be widely used in the fields of biological analysis,environmental detection,food safety and medical diagnosis.Nanozymes have both enzyme activity and the characteristics of nanomaterials.Compared with natural enzymes,nanozymes have the advantages of high catalytic activity,good stability and low cost.The magnetic nanoparticles(Fe₃O₄MNPs)first discovered in 2007 had peroxidase activity,and successfully constructed a sensitive and rapid colorimetric method for the detection of H₂O₂and glucose.However,Fe₃O₄tends to aggregate,so nano-enzymes that improve Fe₃O₄are also of concern to researchers.This article aims to use Si O₂to improve the dispersion of Fe₃O₄nanoparticles and increase the coating of ionic liquids;it will also explore solid-supported functionalized ionic liquids and ordered mesoporous silicones as nanozymes with catalase activity for the ratio of H₂O₂to glucose Possibilities in color determination.The research work and results of the thesis are as follows:(1)Using N-methylimidazole,?-chloropropyltriethoxysilane,Tetraethyl orthosilicate(TEOS),Fe Cl₃·6H₂O,Fe Cl₂·4H₂O as raw materials,Fe₃O₄@Si O₂@IL nanomaterials were synthesized and characterized by SEM and FTIR.Research has found that the material has peroxidase activity.Fe₃O₄@Si O₂@IL nanomaterials have excellent catalytic activity at a p H of3.0 and an incubation temperature of 40?.The kinetic study found that it is in accordance with the typical Michaelis-Menten model and the ping-pong mechanism.The Michaelis-Menten(K_m)represents the affinity of the enzyme for the substrate.The Michaelis-Menten(Km)represents the affinity of the enzyme for the substrate.The smaller the K_mvalue,the higher the affinity for the substrate.The K_mof the material for the substrates TMB and H₂O₂were 0.107 m M and 0.245 m M,respectively,and the Km for the substrates TMB and H₂O₂lower than horseradish peroxidase(HRP)were 0.434 m M and 3.700 m M,indicating that the two All kinds of substrates have high affinity.Therefore,based on the peroxidase properties of Fe₃O₄@Si O₂@IL,a colorimetric method was developed for the detection of H₂O₂and glucose.The linear range of H₂O₂and glucose is 4×10^-6mol/L?1×10^-4mol/L and 8×10^-6mol/L?2×10^-4mol/L,and the detection limit is 0.698×10^-6mol/L And 2.39×10^-6mol/L.(2)Using N-methylimidazole,?-chloropropyltriethoxysilane,template agents CTAB,TEOS and anhydrous Fe Cl₃as raw materials,Imm-Fe³⁺-IL nanomaterials were prepared,and the prepared materials were subjected to SEM And FTIR characterization.The study found that the nanomaterial has peroxidase activity.It has the best catalytic activity at the optimal p H of 3.0 and incubation temperature of 50?.Steady-state kinetics studies show that,in accordance with the typical Michaelis-Menten model and ping-pong mechanism,K_mrepresents the affinity of the enzyme for the substrate.The K_mof the material for the substrates TMB and H₂O₂were 0.324 m M and 0.467 m M,respectively,and the Km for the substrates TMB and H₂O₂lower than HRP is0.434 m M and 3.700 m M,indicating that the The material has a high affinity for both substrates.Based on the activity of Imm-Fe³⁺-IL with peroxidase,it was developed for colorimetric detection of H₂O₂and glucose.The linear range of H₂O₂and glucose is 1×10^-6mol/L?2×10^-4mol/L and 1×10^-5mol/L?2×10^-4mol/L,and the detection limit is 0.25×10^-6mol/L and 3.31×10^-6mol/L.(3)Cu@PMO-bis-ILnanomaterialswerepreparedusingimidazole,?-chloropropyltriethoxysilane,template agent CTAB,TEOS and Cu Cl₂·2H₂O The prepared material was characterized.It was found that the prepared Cu@PMO-bis-IL nanomaterial has the properties of peroxidase.The catalytic activity of the natural enzyme HRP and other nanomaterial-based peroxidase mimics depends on p H and temperature.For the catalyst Cu@PMO-bis-IL,the optimal p H is 3.5,the optimal incubation temperature is 45?,and the optimal catalyst concentration is 0.6 mg/m L.Kinetic studies show that it is found to conform to the typical Michaelis-Menten model,where K_mrepresents the affinity of the enzyme for the substrate,and the smaller the K_mvalue,the higher the affinity for the substrate.Studies have shown that for the substrates TMB and H₂O₂,the K_mis 0.139 m M and 1.092 m M,respectively,and the K_mfor the substrates TMB and H₂O₂is lower than HRP is 0.434 m M and 3.700 m M,indicating The material has a strong affinity for the substrate H₂O₂and a high affinity for the substrate TMB.The catalytic mechanism shows that the peroxidase activity of the catalyst Cu@PMO-bis-IL cannot be attributed to the production of hydroxyl radicals(·OH),but may be attributed to the process of electron transfer.Based on its Cu@PMO-bis-IL with peroxidase activity,it was developed for colorimetric H₂O₂and glucose detection.The linear range of H₂O₂and glucose is 1×10^-5mol/L?1×10^-4mol/L and 1×10^-5mol/L?1×10^-4mol/L,and their detection limits are 8.6×10^-6mol/L and 3.86×10^-6mol/L.(4)The synthesized three nanomaterials with peroxidase activity Fe₃O₄@Si O₂@IL,Imm-Fe³⁺-IL,Cu@PMO-Dual-IL were used for the determination of H₂O₂in milk samples,and their recovery rates were 92?111.4%,96?108.3%,93?105.6%,RSD values are 4.19?4.8%,3.1?8.6%,4.2?5.6%respectively,indicating that the three materials used in the determination of H2O2 in milk have good accuracy And repeatability.Therefore,this article studies three kinds of nanomaterials,namely Fe₃O₄@Si O₂@IL,Imm-Fe³⁺-IL,Cu@PMO-dual-IL nanomaterials.Compared with other reported nanozymes,these three nanomaterials have the advantages of easy preparation and low cost.By coating the magnetic Fe₃O₄,the defect of the magnetic material being easily aggregated is improved;by adding an ionic liquid,the substrate is increased Solubility,dissolved oxygen and stability.Compared with the Mie constant of HRP,these three nanomaterials have low Km and high affinity for substrates.Their glucose detection limit is lower than that of bare Fe₃O₄MNPs(3×10^-5mol/L).Compared with other reported nanozymes,these three materials have a wide linear range and a low detection limit.Therefore,the properties of these three nanomaterials with peroxidase are expected to be used in biomedical sensing analysis of metal ions,dopamine,hydrogen peroxide(H₂O₂)and glucose.It has potential application value in food safety,ecological protection and biomedical diagnosis.