| Capillary electrophoresis（CE）is an ideal analytical method for extremely volume limited biological microenvironments owing to its high separation efficiency,high selectivity and simple operation.Chemiluminescence detection（CL）is characterized by providing low background with excellent sensitivity because of requiring no light source.The coupling of CL with CE has become a powerful analytical method because that both high separation efficiency,high selectivity of CE and excellent sensitivity of CL can be achieved at the same time.So far,this method has been widely applied to chemical analysis,food analysis,drug analysis,environment analysis and other aspects.This thesis is mainly about the separation and detection of glycated hemoglobin in human blood sample by CE-CL.We applied the luminol-hydrogen peroxide CL reaction and the lab-built apparatus to perform our detection.As a result,we successfully achieved the separation and quantitative detection of glycated hemoglobin in blood sample of diabetic patients.Firstly,we optimized the experimental conditions such as the separation and reaction buffer as well as the CL reagents.Secondly,we investigated the linearity range and detection limit of hemoglobin standard substance and hemoglobin in blood sample.Wide linearity ranges(1×10-88 M to1×10-5 M,with the R2 of 0.933 and0.943)and low detection limits(1.28×10-10 M and 1.77×10-10 M)were achieved.Then we performed the standard sample recovery experiment.The recoveries of added standard substance were 103.1%to 108.8%,which were all within the reasonable range.Finally,we performed the reproductivity experiment and obtained the result that all relative standard deviation（RSD）of different capillaries in the same day and during three days were less than 10%.To realize the separation and detection of glycated hemoglobin in blood sample,the resolution and sensitivity of the system needed to be increased.We added3,5-dicarboxybenzeneboronic acid to the system in order to increase the difference of the electric charge between normal hemoglobin and glycated hemoglobin.We introduced the orthogonal experimental design method to optimize the experimental conditions such as the concentration of luminol and 3,5-dicarboxybenzeneboronic acid.Then we investigated the linearity range and detection limit of hemoglobin and glycated hemoglobin standard substance.The result showed wide linearity ranges(1×10-7 M to 7.5×10-6 M for hemoglobin with the R2 of 0.963,2.5×10-6 M to 1×10-5M for glycated hemoglobin in high concentration range with the R2 of 0.996 and 1×10-7 M to 2.5×10-6 M in low concentration range with the R2 of 0.994)and low detection limit(1.86×10-8 M and 1.07×10-8 M)as well.After that we performed the standard sample recovery experiment and the reproductivity experiment.The recoveries of added standard substance were 101.1%to 105.2%and the RSD of different capillaries in the same day and during three days were less than 10%except two results.Finally,we detected the content of glycated hemoglobin in blood sample of diabetic patients.We achieved the content was 9.6%in blood sample of diabetic patients and 5.2%in blood sample of healthy adults after adjusting the different catalytic ability of normal hemoglobin and glycated hemoglobin.This analysis system has the advantages of simple,sensitive,fast as well as low detection limit and low sample consumption.The results show that it’s very suitable for the separation and detection of glycated hemoglobin in blood sample and can provide useful information to the dignose and monitoring of diabetes. |
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