Labeling Digoxin Antibody with Colloidal Gold and Ferrocene for Its Use in a Membrane Immunostrip and Immunosensor

Current trends in the development of on-site diagnostic tests using immunodetection principles are proceeding in two directions: toward the immunochromatography test strip and toward the immunosensor. To develop both applications for the detection of digoxin, we prepared multilabeled antibody conjugates using colloidal gold and ferrocene. Prior to applying the digoxin antibody in immunochromatography, we constructed a dose–response curve with the digoxin antibody by enzyme immunoassay under optimized conditions. From the results, we found it was possible to detect as little as 10−5 μg/ml (1.28 × 10−11 M) digoxin, which is within the therapeutic and toxic ranges of digoxin. First, this antibody was labeled with ferrocene as a sensor signal generator. As the ferrocene moiety increased, electroactivity also increased, but immunoreactivity between digoxin and its antibody was constant as ascertained from the molar ratio of its conjugate which was up to 1:35. And the immunoreactivity of the conjugate with molar ratio 1:125 dropped by 50%. Thus, after determining the optimum molar ratio (1:35) of antibody to ferrocene, this ferrocene-conjugated antibody was labeled with colloidal gold for the visual recognition of color in the strip. This immunostrip could detect a digoxin level of 1 μg/ml (1.28 × 10−6 M) within 2 min.