Electrochemical immunoassay based on aptamer–protein interaction and functionalized polymer for cancer biomarker detection

An electrochemical aptamer-based biosensing assay for MUC1 protein detection by using methylene blue (MB) as electrochemical indicator and modifying the electrode surface using a functionalized conductive polymer was developed. The optimization steps starts with electropolymerization of o-aminobenzoic acid (o-ABA) onto graphite based screen printed electrodes (SPEs). Immobilization of primary antibody as the capturing probe was performed directly on poly o-ABA modified electrodes. Then, a sandwich like structure was fabricated upon MUC1 protein–aptamer complex formation, exploiting aptamer as the detection probe and methylene blue as the electrochemical active marker interacting with the aptamer without previous labeling. The aptamer instead of antibody was successfully used for the electrochemical detection. The recognition of immunoreactions and aptamer binding event was identified via monitoring the interfacial electron transfer resistance with electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). CV and differential pulse voltammetry (DPV) were employed to detect the change of MB oxidization peak current related with the human MUC1 protein concentration. DPV detection showed a reliable and more sensitive quantification of MUC1 with a detection range of 1–12 ppb and a lower detection limit (0.62 ppb).