Quantitative Determination Of VEGF165 In Cell Culture Medium By Aptamer Sandwich Based Chemiluminescence Assay

Vascular endothelial growth factor(VEGF)is secreted by endothelial cells and tumor cells and is a signaling protein.It is considered to be an important biomarker for the diagnosis of cancer,the over expression or down-regulation of VEGF are closely related with some human diseases in clinical,so to establish a simple and rapid high sensitivity detection method of VEGF is very important significance.This study main elements are bellowed as the following:The first chapter,a briefly introduction of the relationship between VEGF protein expression and tumor,VEGF protein structure and subtype specific recognition of VEGF protein aptamer selection,chemiluminescence system.And we put forward the research ideas and methods of this study of VEGF assay.The second chapter,we established a chemiluminescence method that used magnetic particles as carriers,aptamer captured VEGF protein,then formed the sandwich structure which the second aptamers connected alkaline phosphatase(ALP),using chemiluminescence intensity to achieve the purpose of quantitative detection of VEGF.The chemiluminescence conditions of the system were optimized,such as the washing solution,the adding order of reagents,the pH of substrate and the best detection time,the best concentration of aptamers,the volume of magnetic particles and the concentration of substrate.After optimize the above aspects,the CL could attach the maximum luminous intensity.Then the standard curve could be established to detect the 1ng/mL of VEGF,and the selectivity of established method was good,which the quantitative determination of VEGF was achieved.Compared with the traditional method ELISA,the results of VEGF detection is almostly the same.The third chapter,the established chemiluminescence detection method has been applicated in real samples of cell medium.We successfully detected the secreted VEGF 165 of tumor HepG2 cells and normal human endothelial cells(HUVEC)under normoxic and hypoxic condition,and the VEGF 165 secretion of HepG2 and HUVEC cultured alone or co-culture condition.The culture of HepG2 cells and HUVEC cells are cultured in a microfluidic chip.The results were basically consistent with ELISA.The establishment of this method can be successfully applied to the detection of cell samples which the cell culture medium does not need to be pretreatment.It is possible to specifically detect other proteins by replacing the aptamer with a suitable ligand for the corresponding protein.It is expected to be applied to clinical medicine and has a practical application prospect.