Development and characterization of surface plasmon resonance (SPR)-based immunosensor

Realtime and nonlabelled sensing based on antigen-antibody reaction has been investigated by a surface plasmon resonance (SPR)-based optical biosensor. The sensor detects the change in the refractive index of the metal film surface upon injection of antigen/antibody. in this paper the characteristics of the sensor in immobilized format of antigen (antibody) on gold surface are described; immobilization of antigen/antibody on gold surface was performed either by physical adsorption or by covalent immobilization to an organic film in the monolayer and polyer film. Although the former method was simpler, however, constant response was not obtained in repetitive antigen/antibody bindings. For the detection of antigen/antibody by the SPR sensor with a sensitivity that cannot be easily attained in other methods, it is desired to immobilize antigen/antibody as much as possible in a polymer matrix having low refractive index. Covalent binding of oxidized hydroxyethylstarch polymer was performed onto thin gold film surface via self-assembled monolayer of aminoethanethiol. The immobilized starch layer was reduced by sodium borohydride after antigen/antibody immobilization, because Schiff's base is not necessarily stable through the repeated association-dissociation by an acidic buffer, glycine-HCl (pH 2.5). By the NaBH4 treatment of sensing layer the repeated detection of the determinant was carried out at least 20 times.