Glucose Oxidase Immobilization With Amino Dendritic Mesoporous Silica Nanoparticles And Its Application In Glucose Detection

Objective:Glucose is the main component of animal and plant carbohydrates,and its quantitative determination plays an important role in life sciences.The existing glucose detection methods mainly include:high performance liquid chromatography,optical rotation,spectrophotometry,optical rotation,biosensor method,etc.However,these methods generally have the disadvantage of being expensive and complicated to operate.Therefore,it is of great application value to construct a new detection method with high sensitivity,high selectivity,rapid and simple.In recent years,glucose oxidase has been widely used in biochemistry,clinical chemistry and food analysis because of its high accuracy and selectivity.However,free enzymes are sensitive to pH and temperature and are easily deactivated,which cannot be reused.The immobilization of enzymes can largely solve such problems.In this study,amino-dendritic mesoporous silica nanomaterials were synthesized by template method,which was used to immobilize glucose oxidase,and a new method for immobilized glucose oxidase to detect glucose was established,which expanded the new development in the field of health detection technology.Methods:1.Dendritic mesoporous silica nanoparticles?DMSNs?were synthesized by template method using triethylamine as the base source,and modified with3-aminopropyl triethoxysilane?APTES?to modify the amino group.Finally,aminodendritic mesoporous silica nanoparticles?DMSNs-NH₂?were synthesized.This material was used as a carrier to fix glucose oxidase.The optimum reaction conditions for immobilized glucose oxidase were investigated.The carrier and immobilized enzyme were characterized by scanning electron microscope,transmission electron microscope,infrared spectroscopy scanner,nitrogen adsorption apparatus and thermogravimetric analyzer.2.Glucose was detected by immobilized glucose oxidase,and the optimum reaction conditions were discussed.Methodological evaluation includes determination of detection limits,controlled experiments,accuracy,specificity,repeatability,etc.Finally,the established methods were used to detect glucose in real samples such as clinical blood samples and drinks..SPSS 24.0 was used to analyze the experimental data,and the results were expressed as ???±s.Results:1.Scanning electron microscopy and transmission electron microscopy showed that the aminoized dendritic mesoporous silica nanoparticles synthesized in this experiment were dispersed spherical particles,evenly dispersed,with particle size of about 200nm,with dendritic pores directly through the interior.The overall openness was good,which could provide more space for the immobilized enzyme.The infrared spectrogram showed that there was a-NH₂ vibration peak at 1636 cm^-1,indicating that the amino group had been successfully modified to dendritic mesoporous silica,and that the 1646 cm^-1 and 1526 cm^-1 were respectively the absorption peaks of the amide I bond and the amide II bond,indicating that the glucose oxidase had been successfully fixed to the amino dendritic mesoporous silica.Nitrogen adsorption desorption curve showed that the specific surface area and pore volume of amino dendrimer mesoporous silica were 215.19 m²/g and 0.88 cm³/g,respectively,and the specific surface area and pore volume of immobilized enzyme were 165.06 m²/g and0.64 cm³/g,respectively,indicating once again that glucose oxidase had been fixed on the carrier.Thermogravimetry showed a total weight loss of 15%of the immobilized enzyme,while a weight loss of 27%of the immobilized enzyme.The difference between the two was the fixed amount of glucose oxidase,further indicating that glucose oxidase was successfully immobilized on the carrier.In addition,when the immobilization condition was pH 5.0,the time was 6.0 hours,and the enzyme concentration was 0.3 mg/mL,the immobilization effect was the best,the protein load was 225.0 mg/mL,and the enzyme activity was 215.0 U/mg.2.The results of enzymatic property analysis of immobilized enzyme and free enzyme showed that the optimal pH of free enzyme and immobilized enzyme was 5.0,the optimal temperature of free enzyme was 30?,and the optimal temperature of immobilized enzyme was 40?.The relative enzyme activity of immobilized enzyme was remaining 60.0%and the free enzyme was only 40%under the condition of partial acid and alkaline.After incubation at a temperature of 60?for 2.5 hours,the immobilized enzyme activity remained 75.0%,and the relative enzyme activity was relatively high,while the free enzyme activity was only 57.0%.After storage for 2months,the activity of the immobilized enzyme was 93.0%and that of the free enzyme was 76.0%.3.The immobilized enzyme glucose detection experiment was optimized to determine the optimal detection conditions as follows:The optimal detection time was20 min,and the optimal immobilized enzyme content was 3.0 mg.In the methodological evaluation,the detection limit was 0.0014 mg/mL,and the glucose concentration was within the range of 0.001 mg/mL⁰.500 mg/mL,showing a good linear relationship.The linear equation was y=4.9883x+0.0025,R²=0.9987,After repeated use for 45 times,the activity of the fixed enzyme remained above 80%.The results of the control experiment show that there is no significant difference between this method and the national standard.To verify the accuracy of the experiment,Relative Standard Deviation?RSD?was calculated between 1.60%and 7.40%,and the Standard recovery was between 90%and 120%,indicating that the accuracy of the method was good.In the repetitive experiments,the intraday variation rate was2.56%⁷.14%,and the intraday variation rate was 4.20%⁶.07%.It indicates that this method has good specificity in the detection of glucose and is free from the interference of fructose,lactose,maltose,sodium cholate,ascorbic acid,uric acid and other substances.The results of blood samples and beverage samples were consistent with the actual values,indicating that the method was reliable.Conclusion:1.Dendritic mesoporous silica was prepared by template method using triethylamine as base source,and then modified with amino group.The material is uniformly dispersed and has dendritic pores,which provide a large space for the immobilization of enzymes.In this study,the immobilization of glucose oxidase was realized by using this material as the carrier,and the immobilized enzyme was used to determine the actual sample.2.Compared with free enzyme,the thermostability,pH stability and storage stability of immobilized enzyme are significantly higher than that of free enzyme,which has high reusability and greatly improves the performance of glucose oxidase.3.This method has a good linear relationship,and the results of serum samples and beverage samples are accurate and fast,free from interference from other substances,stable and reliable.