| Objective:In recent years,since the diseases of gout and other diseases caused by uric acid accumulation in the body are more and more affecting the health and daily life of people.The detection of uric acid in the body in time is necessary for preventing and treating diseases such as gout and hyperuricemia.The common method for detecting uric acid is mainly high-performance liquid chromatography,isotope dilution mass spectrometry,enzyme method and the like.But the prior two detection methods have the disadvantages of complex operation,high instrument,long detection period and the like,and are not suitable for rapid detection of uric acid.The enzyme method was used to detect uric acid to produce allantoin catalyzed by uricase,and the product was proportional to the concentration of substrate.By measuring the absorbance value of allantoin,the concentration of uric acid was calculated.The enzyme method has the advantages of good specificity and high accuracy.However,uricase has high requirements for reaction conditions and can not be recovered and continuously utilized.Therefore,serum uric acid was determined by using mesoporous silicone hollow nanosphere immobilization of uricase by enzyme immobilization technology.Methods:SiO2 nanospheres were synthesized by typical Stober method.Mesoporous organosilicon hollow nanospheres were prepared by one-step growth-induced corrosion method using SiO2 nanospheres as hard templates.Characterization of synthesized SiO2nanospheres by scanning electron microscopy.The synthesized mesoporous organosilicon hollow nanospheres and uricase immobilized with mesoporous organosilicon hollow nanospheres were characterized by scanning electron microscopy,transmission electron microscopy,nitrogen adsorption,infrared spectroscopy and thermogravimetric analysis.The uricase was immobilized with the synthesized mesoporous silicone hollow nanosphere,and the optimum conditions for the immobilization of uricase were selected by orthogonal test.The optimum reaction time and carrier amount of uric acid were selected by single factor test.Under the optimum reaction conditions,the uric acid was detected by uricase immobilized with mesoporous silicone hollow nanosphere.Because the allantoin produced is proportional to uric acid,the concentration of uric acid is calculated by measuring the absorbance of allantoin at 290 nm.Results:SEM and TEM results show that the average size of the synthesized mesoporous organosilicon hollow nanospheres is 380nm and each sphere is hollow,and the hollow cavity is 230 nm,which is the same as that of SiO2 nanospheres.SiO2 nanospheres and mesoporous organosilicon hollow nanospheres are well dispersed.The BET specific surface area and pore volume of mesoporous organosilicon hollow nanospheres are 592.1 m2/g and 0.29 cm3/g,respectively.The BET specific surface area and pore volume of uricase immobilized by mesoporous silicone hollow nanospheres were 47.89 m2/g and 0.059 cm3/g,respectively.The pore sizes of mesoporous organosilicon hollow nanospheres and uricase immobilized with mesoporous organosilicon hollow nanospheres were 2 nm.The results of thermogravimetric analysis showed that the weight loss of mesoporous organosilicon hollow nanospheres was about 3%between 100?and 400?,while that of mesoporous organosilicon hollow nanospheres was 7%after immobilization of uricase,and above 400?,mesoporous organic compounds were immobilized by mesoporous organic nanospheres.Both silica hollow nanospheres and mesoporous organosilicon nanospheres immobilized uricase lose weight rapidly,and the weight loss is about 7%at 400?900?,which is partly due to the bridged organosilane group.The optimal fixation time was 8 hours,and the optimal uricase volume was 2 mL.Under the optimum conditions,the average immobilization rate of uricase was more than 90%.After immobilization,the activity of uricase was more than 90%compared with that of free uricase.The optimum reaction temperature of the immobilized uricase was 40?,and the activity of uricase remained above 40%at70?.The optimum pH was 8.5,and it was stable between the pH=7-11.After storage at 4?for 3 months,the immobilized uricase activity remained above 90%compared with the initial one.The activity of serum uric acid detected by the immobilized uricase was lost after about 40 times of repeated use,and remained more than 70%after 30 times of repeated use.The optimum reaction time and the optimal carrier-uricase quantity were selected by single factor test.The optimum amount of uricase was 3 mg when the optimal reaction time was 20 min.Under the optimum reaction conditions,uricase immobilized by mesoporous organosilicon hollow nanospheres was used to detect uric acid.The concentration of uric acid was calculated by measuring the absorbance value of allantoin at 290 nm.The detection of uric acid in serum was linear in the range of 0.01 mg/mL1.0 mg/mL,the detection limit was 0.0039 mg/mL,the recovery rate is between 93.4%and 96.2%.The intra-day variation rate is between2.52%and 3.05%,and the inter-day variation rate is between 5.20%and 6.87%.The results of determination of serum uric acid by this method and the standard uricase ultraviolet method for the detection of uric acid were tested by independent sample T test.t=0.007,df=16,p=0.995,p>0.05.There was no difference in detection of uric acid between mesoporous organosilicon hollow nanospheres and free uricase.The results of uric acid test showed that urea,bilirubin and sodium cholate did not interfere with the detection of uric acid,but ascorbic acid interfered with the detection of uric acid.Conclusion:1.Monodisperse SiO2 nanoparticles were synthesized by Stober method.Mesoporous silicone hollow nanospheres with hollow cavity,uniform pore size,large specific surface area and good dispersion were synthesized by one-step growth induced corrosion method.2.The uricase was successfully fixed with mesoporous silicone hollow nanosphere,and the enzyme properties after fixation were determined.The stability and thermal stability of uricase pH fixed by mesoporous silicone hollow nanosphere were good,but the enzyme activity and structure remained unchanged.3.A method for the determination of uric acid by mesoporous silicone hollow nanosphere fixation with uric acid enzyme was established.The detection of uric acid can be completed within 20 min.The method has high accuracy and good reproducibility.The fixed uric acid enzyme can still be reused after centrifugation and precipitation. |
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