Selectivity and Regeneratability Evaluation of Antigen Analogous-Modified Surface Film for Polychlorinated Dibenzodioxin Immunosensor Using Surface Plasmon Resonance Measurement

Abstract

For the development of a sensitive and accurate immunosensor, the production of a highly specific surface to the corresponding antibody is essential. Moreover, a specific surface having good reusability is worthwhile for constructing a prompt auto-monitoring immunosensing system. In this study, antigen analogous-modified gold substrates having a specific adsorption property to an anti-dioxin antibody, were prepared by the immobilization of 2,4,5-trichlorophenoxy (TCP) derivatives on a gold surface using various methods. An anti-dioxin antibody, which has a good correlation immunoaffinity with the total toxic equivalent quantity (TEQ) of polychlorinated dibenzo-p-dioxins, was employed as a specific antibody, and the specification of the surface was evaluated by a comparison of the surface plasmon resonance (SPR) responses to a specific anti-dioxin antibody and non-specific anti-hemoglobin antibody. In order to obtain a firm and stable immobilization of TCP, a carboxyl group was introduced on gold by a self-assemble monolayer (SAM) technique, and TCP derivatives were attached to the SAM surface with covalent bonding directly and indirectly through the presence of a hexamethylene chain, bovine serum albumin (BSA), and ethylene glycol oligomer (EGO). A TCP derivative-immobilized substrate, prepared using BSA, showed poor reusability. A TCP derivative-immobilized substrate having EGO was highly specific to an anti-dioxin antibody, while that prepared by the direct immobilization of TCP and with introduction of a hexamethylene chain presented nonspecific adsorption. In addition, a TCP derivative-immobilized substrate having EGO showed a good reproducible response from the second repetition, and was reusable more than seven times.