Thesis (Ph.D.)--Chulalongkorn University, 2007

This thesis studied the electrochemical properties of the products of seven substrates for the enzyme label, alkaline phosphatase, which is commonly used in electrochemical immunosensors. Cyclic voltammetry and amperometry of these products were carried out at glassy carbon disk, gold disk, and screen-printed carbon electrodes. Among the products, L-ascorbic acid showed the most sensitive and well-defined amperometric response, obtaining from 2-phospho-L-ascorbic acid substrate for the electrochemical detection with an alkaline phosphatase label. Using MIgG as a model analyte, the alkaline phosphatase-amplified sandwich-type amperometric immunosensor was constructed. The immunosensor was fabricated by electropolymerization of o-aminobenzoic acid (o-ABA) on the electrode surfaces. Covalently immobilized onto the poly-o-ABA modified electrodes, the anti-MIgG was bound with the target MIgG that was caught with the alkaline phosphatase conjugated anti-MIgG to form a sandwich-type immunosensor. The alkaline phosphatase is capable of changing 2-phospho-L-ascorbic acid to electroactive L-ascorbic acid, which can be determined by the amperometric detection with the signal directly proportional to the quantity of MIgG. The best performance for the poly-o-ABA modified Au disk immunosensor was 297-to-1 current density response ratio for 1000 and 0 ng mL¯¹ MIgG and detected MIgG down to the concentration of 1 ng mL¯¹ with the linear range of 3-200 ng mL¯¹. Boron-doped diamond, the new electrode material was developed for the MIgG immunosensor. The poly-o-ABA modified boron-doped diamond was characterized by scanning electron microscopy and X-ray photoelectron spectroscopy. X-ray photoelectron spectroscopic results revealed the presence of carboxyl groups at the modified electrode surface. These carboxyl groups were used to covalently attach to the anti-MIgG. The amperometric sensing of MIgG from poly-o-ABA modified glassy carbon plate immunosensor was compared. The LODs of 0.30 and 3.50 ng mL¯¹for MIgG at boron-doped diamond and glassy carbon plate electrodes were obtained. Three orders of magnitude of the wide linear range (1-1000 ng mL¯¹) was obtained from the boron-doped diamond whereas the narrower linear range (10-500 ng mL¯¹) was found at the glassy carbon plate. In addition, the proposed method was successfully applied to a real mouse serum sample containing MIgG.