Nanostructured immunosensing system for label-free impedimetric detection of multiple breast cancer biomarkers (CEA and HER2) using CoMoO4@PANI-PPy Nanocomposite

Breast cancer patients often exhibit positive expression of selective tumor biomarkers including the carcinoembryonic antigen (CEA), and human epidermal growth factor receptor 2 (HER2). These potential protein biomarkers are highly crucial for the selective and early tumor diagnosis. In this study, disposable dual impedimetric immunosensing systems were constructed using the cobalt molybdate (CoMoO₄) and polyaniline/polypyrrole (PANI-PPy) nanocomposite, for the rapid quantification of CEA and HER2 concentration levels. The designed sensing probes were fabricated by chemically conjugating two-targeting antibodies (anti-CEA and anti-HER2) on the nanostructured electrode surfaces modified with (AuNPs/CoMoO₄@PANI-PPy). These successful chemical antibody immobilizations were enabled by the use of 4-aminothiophenol (4-ATP) as a cross-linking agent. The impedimetric immunosensing assay was fully optimized by screening of nanomaterials, determining the optimal ratio of the nanocomposite, adjusting the time of self-assembled monolayer (SAM) formation, and identifying the degree of cross-reactivity with potential non-targeting molecules. Accordingly, the biosensors demonstrated distinguished linear dynamic ranges of 1.0 fg/mL to 100 ng/mL for the CEA and 25 fg/mL to 100 ng/mL for the HER2, with detection limits of 8.2 fg/mL and 4.9 fg/mL for CEA and HER2, respectively. The immunesensor's reproducibility and stability were studied, showing minimal variation in charge transfer resistance (RSD = 1.33%, n = 5) and maintaining 82.5% functionality after four weeks of storage at 4 °C. These findings highlighted the potential use of these biosensor chips for clinical applications in early breast cancer detection and rapid diagnosis.