Ratiometric electrochemical immunoassay based on internal reference value for reproducible and sensitive detection of tumor marker

A ratiometric assay in an electrochemical immunosensor for tumor marker, herein carcinoembryonic antigen (CEA) was chosen as a model analyte, was developed to improve simplicity and accuracy. The immunosensor was fabricated via the simple expedient way of using Polythionine-gold (PTh-Au) as electrode modified material to be an internal reference signal and K3[Fe(CN)6] in electrolyte as an indicator signal. When the CEA was fixed on the modified electrode via immunoreaction, only the indicator signal sensitively altered; by contrast, the internal reference signal of PTh-Au remained constant at a suitable pH of the electrolyte. The ratio between the alterations of the indicator signal of K3[Fe(CN)6] and the constant internal reference signal can be used to monitor the concentration of CEA reliably, reproducibly, and sensitively. The prepared ratiometric electrochemical immunosensor could detect CEA with good specificity within a wide linear range from 0.005 ng/ml to 40 ng/ml with a detection limit of 2.2 pg/ml. Additionally, experimental results confirm that our proposed method is practical. Thus, this method can expand to recognize and test other protein markers.