Comparative Performance of Label-Free HER2 Immunosensors on Bare, Poly(P-Anisidine)-Modified, and Ammonium Alginate-WO3-Modified Glassy Carbon Electrodes

Abstract

The transducer architecture of a label-free electrochemical immunosensor can alter signal transport, calibration shape, and short-term stability, but these effects are not necessarily aligned. This study compared three glassy-carbon platforms for detection of the extracellular domain of human epidermal growth factor receptor 2 (HER2-ECD): activated bare glassy carbon (GCE), poly(p-anisidine)-modified GCE (GCE/PPA), and GCE/PPA coated with an ammonium alginate-tungsten trioxide nanocomposite [GCE/PPA/(AmAlg-WO₃)]. Each platform was combined with trastuzumab (Ab_T), pertuzumab (Ab_P), or an equal-mass trastuzumab-pertuzumab mixture (Ab_TP), producing nine independently calibrated immunosensors. PPA electropolymerization was optimized over 6-15 cyclic-voltammetric cycles; 10 cycles gave the largest charge (72.1 μC). Ferri/ferrocyanide differential-pulse voltammetry was used only to compare electrode architectures. Relative to bare GCE, signal retention decreased to 51.9% after PPA deposition and recovered to 70.7% after AmAlg-WO₃ coating. HER2 measurements were then performed without ferri/ferrocyanide by following the decrease in the approximately 0.9 V peak observed after EDC/NHS treatment. Cumulative HER2 additions covered 1.99-195 ng mL⁻¹, and response was defined as ΔI = I₀ − I_HER2. All configurations followed a baseline-adjusted saturation model, with R² values of 0.9942-0.9998. Model-derived detection and quantification limits ranged from 0.052 to 0.286 ng mL⁻¹ and from 0.158 to 0.911 ng mL⁻¹, respectively. GCE/Ab_TP produced the smallest calibration errors (RMSE 0.0697 μA; MAE 0.0593 μA) and the lowest calculated detection limit (0.052 ng mL⁻¹), whereas GCE/PPA/(AmAlg-WO₃)/Ab_TP showed the smallest 45-min drift (0.92%). Thus, the bare mixed-antibody sensor provided the strongest calibration performance, while the nanocomposite mixed-antibody sensor provided the best short-term operational stability. The results show that increased absolute current or interfacial transport does not alone determine analytical quality and that architecture should be selected against the intended performance criterion.