| Glucose is an important monosaccharide in nature and the main energy source of cells.It is widely used in the fields of food,medicine and industry.Although glucose is closely related to human life,excessive intake may induce diabetes.Therefore,monitoring the glucose concentration is very important for social production and human health.However,current glucose detection methods have problems such as time-consuming,labor-intensive,low specificity,low sensitivity,and poor stability.In this thesis,a carrier-free enzyme-polymer conjugate nanoparticle with high sensitivity and high stability was prepared based on PEGylation technology,and its application in glucose detection was carried out.For the preparation and characterization of enzyme-polymer conjugate nanoparticles,the mechanism of cross-linking and self-assembly of m PEG-ALD with GOx and HRP to form enzyme-polymer conjugate was studied.And by the method of deposition drying,the conjugate nanoparticle array coating with high specific surface area was prepared on the surface of the glass slide.The results of the study have shown that changing the content(concentration and ratio)of the two enzymes and the Schiff base reaction conditions(p H and t _S)can adjust the activity and particle size of the conjugate respectively;the activity of the conjugate increases with the increase of t _Swithin 24 h,Up to 2.4 times the activity of the free enzyme,which is attributed to the increased utilization efficiency of intermediates due to the shortening of the distance between the two enzymes;adjusting the preparation conditions can make the copolymer form a surface pattern of nanoparticle arrays on the glass slide.We hope that this enzyme-polymer conjugate nanoparticle array coating will be applied in the fields of biosensing,clinical medicine and biocatalysis.For the part of the application of conjugate nanoparticles array to glucose detection,the author designed a glass microchip loaded with conjugate nanoparticles array to conduct research on enhanced cascade enzyme reaction and glucose detection.Studies have shown that,due to the surface pattern of nano-concave-convex-like increasing the specific surface area,the catalytic activity of the array coating is 3 times higher than that of the free enzyme coating;the acid,heat and storage stability of the array coating are significantly higher than that of the free enzyme;owing to the low mass transfer resistanc,this method has high sensitivity(0.0046 Abs/μM)and low detection limit(LOD is 1.6μM)when detecting glucose.It can detect 0.1～0.75 m M glucose colorimetrically whith a short detection time(30 s)and has the ability to detect the glucose concentration of body fluids(saliva,serum and urine).This research provides a new way to improve the performance of glucose detection.For the research part of paper chip coated with conjugate nanoparticles for glucose visual detection,the author designed a circular cellulose paper chip loaded with conjugate nanoparticles,and proved that the nanoparticles can be loaded on the fiber surface of the paper;the detection conditions are optimized to achieve rapid(about 30 s)colorimetric detection of 0.1～3 m M glucose;this paper chip shows more stable detection performance than the paper chip loaded with free enzyme when face with high temperature and long-term storage,and has potential practical application value. |
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