Novel amperometric immunosensor for rapid separation-free immunoassay of carcinoembryonic antigen

A novel immunosensor for rapid separation-free determination of carcinoembryonic antigen (CEA) in human serum is proposed. The immunosensor is prepared by co-immobilizing thionine and horseradish peroxidase (HRP)-labeled CEA antibody on a glassy carbon electrode (GCE) through covalently binding them to GCE with a glutaraldehyde (GA) linkage. The electrochemical behavior of the immobilized thionine displays a surface-controlled electrode process with an average electron transfer rate constant of 4.74 +/- 2.99 s(-1). It can be used as an electron transfer mediator for enzymatic activity detection of the HRP-labeled antibody to CEA. After the immunosensor is incubated with CEA solution at 23 degreesC for 40 min, the access of activity center of the HRP to thionine is partly inhibited, which leads to a linear decrease in the catalytic efficiency of the HR-P to the oxidation of immobilized thionine by H2O2 at - 300 mV over two CEA concentration ranges from 0.5 to 3.0 and 3.0 to 167 ng/ml. Under optimal conditions, the detection limit for the CEA immunoassay is 0.1 ng/ml at three times background noise. The immunosensor shows good accuracy and acceptable storage stability, precision and reproducibility with intra-assay CVs of 6.1% and 5.8% at 2.5 and 50 ng/ml CEA, respectively, and an inter-assay CV of 6.3% at 50 ng/ml. This method is economical and shortens the analytical time, making it potentially attractive for clinical immunoassays. (C) 2004 Elsevier B.V. All rights reserved.