Electrochemical immunoplatform to improve the reliability of breast cancer diagnosis through the simultaneous determination of RANKL and TNF in serum.

Abstract

This paper describes a dual immunosensor using neutravidin-functionalized magnetic microbeads (Neu-MBs) and dual screen-printed carbon electrodes (SPdCEs) for the simultaneous amperometric determination of two emerging biomarkers related to breast cancer (BC) and metastasis: Receptor Activator of Nuclear Factor-κB Ligand (RANKL) and Tumor Necrosis Factor alpha (TNF). In the implemented methodology, sandwich-type immunocomplexes, using biotinylated specific capture, detector antibodies and HRP-labeled secondary anti￾bodies, are formed onto Neu-MBs. Electrochemical detection was performed by amperometry (−0.20 V vs. the Ag pseudo-reference) electrode using the H2O2/hydroquinone (HQ) system upon capturing the Neu-MBs mod￾ified with the sandwich immunocomplexes for each target biomarker on the corresponding working electrode (WE) of SPdCEs. The approach exhibits high sensitivity offering detection limits of 2.6 and 3.0 pg mL−1 for RANKL and TNF, respectively, using simple protocols and taking 90 min as assay time. The usefulness of the dual immunoplatform was tested by determining RANKL and TNF levels in 5 μL of human serum from healthy controls and BC patients diagnosed with different HER2 subtypes. Results showed a higher expression of both biomarkers in BC patients (38 and 17 % higher for RANKL and TNF, respectively) and were in agreement to those obtained using the ELISA methodologies for each target biomarker involving the same immunoreagents. The obtained results show the potential of this immunoplatform to improve the reliability of BC diagnosis using fast and cost-effective procedures.