The Determination And Analysis Of Hepatopathy Patient’s Serum Proteins Based On Capillary Electrophoresis-LED Induced Fluorescene

There are no obvious specific symptoms in the early stage of cirrhosis and primary liver cancer, the patients are in the latter stage when diagnosed. The early diagnosis of cirrhosis and liver cancer is particularly important for the treatment. The difference of serum proteins is one of the characteristic values of cirrhosis and liver cancer from the normal. As its high separation efficiency, rapid analysis, micro-injection for protein separation analysis, capillary electrophoresis was developed in recent years. Compared with UV detection, induced fluorescence capillary electrophoresis with higher sensitivity can reveal the changes of serum proteins more precisely between patients and normal people. To establish serum protein capillary electrophoresis fluorescence spectra, the serum proteins of normal people, cirrhosis and hepatocellular carcinoma patients was detected by Capillary Electrophoresis Light-Emitting-Diode induced Fluorescence (CE-LED-IF). The difference of serum proteins between the normal people and patients was found to support the diagnosis of liver disease.Objective1. The difference of serum proteins is one of the characteristic values of cirrhosis and liver cancer from the normal. To establish serum protein capillary electrophoresis fluorescence spectra, the serum proteins of normal people, cirrhosis and hepatocellular carcinoma patients was detected by CE-LED-IF. The difference of serum proteins between the normal people and patients was found to support the diagnosis of liver disease.2. There are some structural problems in the capillary electrophoresis apparatus we used, such as the location of the waste reservoir, the way which the waste discharge from the waste reservoir, which would reduce the repeatability of results. In order to detect the serum proteins precisely, some changes were made to the capillary electrophoresis apparatus to improve the repeatability.Methods1. The detectioin of serum proteins by Light Emitting Diode-induced fluorescence-capillary electrophoresis(CE-LED-IF) depends on the FITC fluorophore binding to the protein. It is necessary to detect the FITC standard reagent, to estimate the repeatability of the capillary electrophoresis apparatus. The reason why the repeatability was reduced was found during the estimation. To impove the repeatability, a new reasonable structure was designed.2. The influence factors of serum protein CE-LED-IF detection was detected, such as the type of light source, the length of the separation capillary, the buffer pH, the buffer concentration, the separation voltage, the serum dilution ratio, the mass ratio between FITC and serum proteins. The separation of the serum proteins of the normal people, cirrhosis and hepatocellular carcinoma patients was took under the optimal electrophoresis conditions, and the serum protein capillary electrophoresis fluorescence spectra was established.Result1. The adverse effects caused by the height difference between the outlet end and inlet end of separation capillary was eliminated by the change of the waste reservoir’s location. The 2.5×10-3 mg/mL FITC solution was tested 10 times before and after the change. The RSD of the migration time and peak area before the change was 8.74% and 6.57%, while the RSD of the migration time and peak area after the change was 1.14% and 3.23%, which reveal that the change made to the capillary electrophoresis apparatus improved the repeatability.2. The influence factors of serum protein CE-LED-IF detection was estimated, such as the type of light source, the length of the separation capillary, the buffer pH, the buffer concentration, the separation voltage, the serum dilution ratio, the mass ratio between FITC and serum proteins. The optimal conditions are as follows:center wavelength 470nm LED as the excitation light source, separation capillary length is 53cm(effective separation length 50cm), the buffer pH is 9.6, the buffer concentration is lOmmol/L, the separation voltage is 20kV, the serum dilution ratio is 1:40, the mass ratio between FITC and serum proteins is 3:20.3. The separation of 60 cases serum of the normal people, cirrhosis and hepatocellular carcinoma patients was took under the optimal electrophoresis conditions, and the serum protein capillary electrophoresis fluorescence spectra was established. There are characteristic protein peaks among the serum protein capillary electrophoresis fluorescence spectra of normal, cirrhosis and hepatocellular carcinoma.Conclusion1. The changes made to the capillary electrophoresis apparatus were reasonable, and the repeatability of CE-LED-IF was improved.2. The results were affected by the separation influence factors such as the type of light source, the length of the separation capillary, the buffer pH, the buffer concentration, the separation voltage, the serum dilution ratio, the mass ratio between FITC and serum proteins.3. There are characteristic protein peaks among the serum protein capillary electrophoresis fluorescence spectra of normal, cirrhosis and hepatocellular carcinoma to support the clinical diagnosis of liver disease.