An indirect perfluorosulfonated ionomer-coated electrochemical immunosensor for the detection of the protein human chorionic gonadotrophin

The development of an amperometric immunosensor for the detection of human chorionic gonadotrophin (hCG) is described. Ln this immunosensor, Nafion wa s used to immobilize an anti-hCG monoclonal antibody onto a glassy carbon e lectrode. A systematic study on the effects of experimental parameters such as the quantity of ethanol present in the Nafion solution, the percentage composition of Nafion, the pH of the immobilization buffer, and the concent ration of antibody used for entrapment experiments on the binding between t he immobilized antibody and I-125-labeled hCG has been carried out. Two imm obilization methods, coimmobilization and adsorption immobilization, have t hen been attempted. A binding of approximately 3% was obtained in the forme r method, while 5.5% binding was achieved in the latter. On the basis of th ese results, adsorption immobilization was employed to entrap antibody on t he electrode surface. A sandwich assay was then developed for hCG in which the enzyme horseradish peroxidase was conjugated to a second anti-hCG monoc lonal antibody. The activity of the enzyme was determined electrochemically by the reduction of benzoquinone to hydroquinone. Binding of hCG to immobi lized antibody determines the quantity of enzyme-conjugated antibody at the electrode surface, permitting the quantification of hCG. By a standard add itions calibration method of hCG performed in blank human serum samples, th e immunosensor exhibits a limit of linearity at 200 mIU mL(-1) and a detect ion limit of 11.2 mIU mL(-1) (based on twice the standard deviation of the blank solution).