The Detection And Mechanism Of H₂O₂ And Glucose Based On Peroxide Mimic Enzymes

Objective:The detection and sensing of human health related factors has been an important research field in preventive medicine.Hydrogen peroxide（H₂O₂）is an industrial product widely used for bleaching,disinfection,sterilization,and oxidation.At the same time,hydrogen peroxide has a certain role in inflammation,oxidative stress,tumor and aging.Glucose is an important component of the human body and a major source of energy for life activities.Serum glucose levels in humans are an indicator of clinical diagnosis of diabetes and therapeutic effects.Therefore,the detection and analysis of H₂O₂ and glucose is of great significance in environmental analysis,biochemical analysis and clinical diagnosis.Nanozymes are a class of artificial mimic enzymes that combine the unique properties of nanomaterials with catalytic functions and can be used in hygienic testing.This research can serve the application research of sanitary detection technology,and lay a foundation for the theoretical research field of nanozymes.Methods:1.VO₂ nanofibers,VO₂ nanosheets and VO₂ nanorods were synthesized by hydrothermal method and characterized by transmission electron microscopy and X-ray powder diffraction.The Michaelis-Menten equation was used to study the catalytic activity of peroxide mimic enzyme.A colorimetric reaction of hydrogen peroxide with the chromogenic substrate 3,3’,5,5’-tetramethylbenzidine（TMB）was used to establish a colorimetric sensing method for the determination of H₂O₂ and glucose.2.The Na₁₀[α-SiW₉O₃₄]was prepared at room temperature,and the Na₁₀[α-SiW₉O₃₄]was characterized by infrared spectroscopy.The peroxide mimic enzyme catalytic activity of Na₁₀[α-SiW₉O₃₄]was studied by using the Michaelis-Menten equation,by reacting hydrogen peroxide with different fluorescent substrates,establish a method for measuring H₂O₂ fluorescence chemical sensing.3.The CdTe quantum dots were synthesized by heating and refluxing.The Na₁₀[α-SiW₉O₃₄]was prepared at room temperature.The Na₁₀[α-SiW₉O₃₄]was characterized by infrared spectroscopy and reacted with CdTe quantum dots by hydrogen peroxide.A method for the determination of H₂O₂ fluorescence chemical sensing was established.The mechanism of fluorescence quenching was determined by capturing the generated hydroxyl radical（·OH）with terephthalic acid（TA）as a fluorescent probe.Results:1.It was found that VO₂ nanofibers,VO₂ nanosheets and VO₂ nanorods have peroxide mimic enzyme catalytic activity and can catalyze color change of chromogenic substrate 3,3’,5,5’-tetramethylbenzidine（TMB）.It was also found that VO₂ nanofibers,VO₂ nanosheets and VO₂ nanorods showed the best catalytic activity in citrate buffer solution（pH=4.0,0.2 M）.VO2 nanofibers,VO2 were measured by steady state kinetics experiments.The K_M values of the nanosheets and VO₂ nanorods relative to the substrate TMB were 0.518,0.111 and 0.801 mM,respectively,and Vmax were 7.95×10^-9,1.44×10^-88 and 4.26×10^-77 M·s^-1,respectively.The K_M values relative to H₂O₂ were 1.043,2.924,and 6.469 mM,respectively,and the Vmax values were 7.64×10^-8,8.31×10^-8,and 1.24×10^-77 M·s^-1,respectively.The linear range of H₂O₂ was determined to be 0.025-10 mM,0.49-60 mM,and 0.49-16 mM,respectively,and the detection limits of H₂O₂ were 0.018,0.266,and 0.41 mM,respectively.The linear range of glucose was determined to be 0.01-10 mM,0.625-15mM,and 0.625-10 mM,respectively,and the detection limits of glucose were 0.009,0.348,and 0.437 mM,respectively.2.Prove that the Na₁₀[α-SiW₉O₃₄]has peroxide mimic enzyme catalytic activity and can be combined with benzoic acid（BA）,thiamine（TH）,and 3-（4-hydroxyphenyl）propionic acid（HPPA）.The fluorogenic substrate reacts.The experimental results show that the Na₁₀[α-SiW₉O₃₄]has the best catalytic activity under alkaline conditions（pH=11.0）.The steady state kinetics experiments show that the Na₁₀[α-SiW₉O₃₄]is relative to the substrate BA.The K_M values of TH and HPPA are 6.03×10^-4,7.95×10^-3and 2.12×10^-22 M,respectively,and the Vmax values are 4.06×10^-7,3.68×10^-8 and5.75×10^-88 M·s^-1,respectively.The K_M values of BA,TH and HPPA relative to H₂O₂were 3.75×10^-7,8.31×10^-5 and 2.51×10^-22 M,respectively,and the Vmax values were5.82×10^-7,3.02×10^-8 and 3.85×10^-88 M·s^-1,respectively.The linear range of H₂O₂ was determined to be 1×10^-8-1.6×10^-66 M,1.6×10^-6-1×10^-44 M,and 1×10^-5-2.5×10^-44 M,and the detection limit of H₂O₂ was determined.They are 6.7×10^-9,2.2×10^-77 and 9.6×10^-6M,respectively.3.The Na₁₀[α-SiW₉O₃₄]reacts with CdTe quantum dots to quench its fluorescence.The experimental results show that the Na₁₀[α-SiW₉O₃₄]has the best catalytic activity under alkaline conditions（pH=8.0）.The linear range of H₂O₂ is7.8×10^-9-2.5×10^-7M.The detection limit of H₂O₂ is 3.8×10^-9M.The H₂O₂ content in the simulated water samples was determined under the optimal conditions.The recoveries were between 93.99%and 110.02%,and the relative standard deviations ranged from 0.88%to 2.78%.Conclusion:1.A method for detecting H₂O₂ and glucose based on the colorimetric VO₂nanoparticles of different morphologies was established.The method was fast,sensitive and low cost.Compared with VO₂ nanorods and VO₂ nanosheets,VO₂nanofibers showed the strongest peroxidase catalytic activity in the detection of H₂O₂and glucose.The detection of H₂O₂ and glucose had the widest linear range and the lowest detection limit.2.Based on Na₁₀[α-SiW₉O₃₄]polyoxometalate,a novel,sensitive,simple and selective fluorescence enhancement method for the determination of H2O2 was developed.This method enables non-fluorescent substrates(benzoic acid（BA）,Thiamine（TH）and 3-（4-hydroxyphenyl）propionic acid（HPPA）react to form a strong fluorescent product.Among them,when BA is used as a substrate,Na₁₀[α-SiW₉O₃₄]catalyzes the detection of H₂O₂ with a wider linear range and lower detection limit.3.A hydrogen peroxide detection method based on polyoxometalate and quantum dot technology was established.The method was simple,rapid,low detection limit,wide detection range,and had good stability and accuracy.4.The CdTe QDs-Na₁₀[α-SiW₉O₃₄]-H₂O₂ system had the lowest detection limit among the three hydrogen peroxide sensing methods.5.Compared to VO₂ nanofibers and nanorods,VO₂ nanosheets have a higher affinity for TMB.Compared with VO₂ nanosheets and nanorods,VO₂ nanofibers have a higher affinity for H2O2.Compared with TH and HPPA,BA has a higher affinity for Na₁₀[α-SiW₉O₃₄]and H₂O₂.The mechanism of fluorescence quenching of CdTe quantum dots is that Na₁₀[α-SiW₉O₃₄]catalyzes the decomposition of H₂O₂ to form hydroxyl radicals（·OH）.