In cancer diagnostics, specific analysis of blood-circulating proteins biomarkers of cancer is often complicated both by their inherently low concentrations and by strong interference from serum/blood proteins. Here, we report a simple and robust electrochemical cellulase-linked sandwich assay on magnetic beads (MBs) for fM-sensitive analysis of the Human Epidermal growth factor Receptor-2 HER-2/neu protein that is over-expressed in most aggressive breast cancers. In the assay, a sandwich is assembled by capturing HER-2/neu on either antibody (Ab) or aptamer-modified MBs accompanied by reaction with the second Ab or aptamer labelled with cellulase. On application of the sandwiches assembled on MBs onto a cost-effective graphite electrode modified with an insulating nitrocellulose film, the cellulase label digests the film. This results in the pronounced changes in the electrical properties of the modified electrodes. The chronocoulometrically-measured extent of the produced changes was proportional to the 10-15-10-10 M HER-2/neu in the analyzed samples, and down to 1 fM of HER-2/neu could be detected in human serum samples in an overall less than 3 h assay. The developed simple and electrochemically label-free methodology is general and can be easily adapted for testing of any other protein.
Keywords: Cellulase; Electrochemical analysis; Electrochemical enzyme-linked sandwich assay; HER-2/neu protein; Magnetic beads.
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