Electrochemical enzyme immunoassay using immobilized antibody on gold film with monitoring of surface plasmon resonance signal

Sandwich immunoassay was conducted on a thin gold film set in a surface plasmon resonance (SPR) cell. Monochronal antibody (anti-IgG) was immobilized onto the gold film via 4,4′-dithiodibutyric acid (DDA) and avidin-biotin bonding. Next, IgG sample and alkaline phosphatase-conjugated anti-IgG (ALP anti-IgG) were introduced into the cell successively. Finally, p-aminophenyl phosphate (PAPP) was injected as an enzyme substrate, and the produced p-aminophenol (PAP) was electrochemically measured. Flow did not need to be stopped for incubation for the enzyme reaction, because of the thinness of the cell. In these processes, all the antigen-antibody reactions took place on the gold film. Therefore, the immobilization was performed quickly, and each process could be confirmed by SPR signal. This system had the advantage that the middle of the complicated process could be monitored. For example, the amount of antibody immobilized, which affected on the final electrochemical signal, could be confirmed in the course of immobilization. It was also convenient to investigate process conditions, such as removal of used antigens and labeled antibodies. Good correlation was obtained between the electrochemical current and the SPR signals due to the adsorption of IgG and ALP anti-IgG, and the sensitivity of the electrochemical measurement was much higher than the SPR’s.