Electrochemical Sandwich Immunoassay for the Ultrasensitive Detection of Human MUC1 Cancer Biomarker

A new electrochemical sandwich immunoassay for the ultrasensitive detection of human MUC1 cancer biomarker using protein G-functionalized magnetic beads (MBs) and graphite-based screen-printed electrodes (SPEs) was developed. Magnetic beads were employed as the platforms for the immobilization and immunoreaction process. A pair of primary and secondary antibodies was used to capture the MUC1 protein. After labeling with a third antibody conjugated with horseradish peroxidase (HRP), the resulting conjugate was trapped at the surface of the graphite-based SPEs and MUC1 determination was carried out by differential pulse voltammetry (DPV) at 0.4？V upon H2O2 addition using acetaminophen (APAP) as the redox mediator. A linear relationship was obtained for the detection of human MUC1 over a range of 0–25？ppb with the lowest detection limit of 1.34？ppb when HRP was applied as a label. Preliminary experiments were performed using disposable electrochemical sensors in order to optimize some parameters (i.e., incubation times, concentrations, and blocking agent). 1. Introduction MUCl mucin is an integral membrane glycoprotein expressed by most if not all “wet” epithelia, such as on bladder, breast, gastric, pancreas, ovary, and respiratory tract epithelium, with a molecular weight varying between 250 and 1000？kD [1]. In normal secretory epithelial cells, MUC1 is expressed at the apical plasma membrane. However, following malignant transformation, MUC1 may be expressed at high levels on the entire membrane surface as well as in the cytoplasm. In addition to the altered cellular localization, changes in glycosylation may occur during malignant transformation [2, 3]. Although the numbers of glycosidic links are increased, the carbohydrate chains are generally shorter [4]. The reduced glycosylation reveals protein epitopes, making MUC1 more accessible to specific antibodies. Thus, overexpression and altered glycosylation allow MUC1 to be used as a cancer marker. Widespread use of tumor markers in healthcare will ultimately depend upon the detection of many tumor markers with high selectivity and sensitivity. This goal has not been obtained with conventional methods which have poor precision or are experiencing difficulties in realizing automatization [5]. Recently, electrochemical immunosensors have received considerable attention for the detection of tumor markers due to their high sensitivity and easy miniaturization [6, 7]. Most immunoassay techniques are based on the separation of free and bound immunospecies. In the electrochemical immunosensors, one of the