Labeling of ferrocenecarboaldehyde (Fc-CHO) to immunoglobulin G (IgG) via formation of Schiff-base and its reduction was investigated for construction of an electrochemical probe for miniaturized amperometric flow immunoassay. Approximately eight molecules of Fc-CHO were labeled to IgG and the reversible redox property of ferrocene was observed. Labeling efficiency improved by over three times as compared to the conventional method using ferrocenemonocarboxylic acid (Fc-COOH). Also, binding affinity of IgG labeled with Fc-CHO to its antigen, IgE, was investigated by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance assay. IgG labeled with Fc-CHO that retained eight ferrocene moiety showed sufficient binding affinity to its antigen and the current response obtained in the flow electrochemical detection system increased by 14-fold as compared with IgG labeled with Fc-COOH when applying the potential of 390 mV vs. Ag/AgCl. The minimum detectable concentration of IgG labeled with Fc-CHO was 0.06 microM. IgG labeled with Fc-CHO demonstrate biochemical and electrochemical properties that are useful for electrochemical immunosensors.
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