PHB/ErGO electrochemical immunosensor for H5N1 serology: Insights into selectivity and inflammation-mediated matrix effects.

Abstract

Reliable electrochemical immunosensing in biological samples requires not only selective molecular recognition but also preservation of analytical performance in complex matrices. Here, a label-free electrochemical immunosensor based on electrochemically reduced graphene oxide (ErGO) and polyhydroxybutyrate (PHB) nanoparticles was developed for the detection of anti-H5N1 antibodies in chicken serum. PHB nanoparticles were pre-functionalized with H5N1 hemagglutinin and deposited onto disposable ErGO-modified screen-printed electrodes, providing a simplified strategy for biointerface assembly. Structural, morphological, and electrochemical characterization confirmed the formation of the sensing interface and its response to antibody binding. The immunosensor exhibited a linear response from 1 pg mL-1 to 10 ng mL-1, with a detection limit of 1 pg mL-1, and showed selective recognition of anti-H5N1 antibodies in the presence of unrelated viral antibodies and proteins. Systematic comparison of PBS, healthy serum, and inflammatory serum revealed a progressive reduction in analytical sensitivity, with calibration slopes of 5.53, 3.03, and 1.69, respectively, accompanied by increased baseline suppression. Nevertheless, concentration-dependent responses and target discrimination were maintained in serum matrices. These findings support the use of the ErGO/PHB interface for anti-H5N1 detection and quantify the loss of analytical sensitivity observed in the serum conditions investigated.