Electrochemical immunosensor for antibody recognition against SARS-CoV-2 B-cell epitope: impact of RBD mutations on antigen-antibody binding.

During the SARS-CoV-2 pandemic, the receptor-binding domain (RBD) of the spike protein emerged as a critical target for neutralizing antibodies. While immunoinformatics predicts binding sites, in vitro confirmation of epitope-antibody interactions remains a challenge. Here, we present a modular and highly sensitive square wave voltammetry immunosensor platform based on zinc oxide nanorods (ZnONRs) for detecting antibody responses to SARS-CoV-2 variant epitopes. The device leverages three distinct B-cell peptides (P44; spike415-429) corresponding to the wild-type (WT), gamma, and omicron variants, differing by a single amino acid at the K417 mutation hotspot. This modular design enables rapid adaptation to emerging variants by simply exchanging the peptide recognition element. The immunosensor exhibited detection limits of 0.14 ng mL-1 (WT), 0.19 ng mL-1 (gamma), and 0.35 ng mL-1 (omicron) using the monoclonal neutralizing antibody B38. Clinical validation with human serum samples demonstrated that: (1) WT-infected individuals showed markedly reduced antibody binding to the P44 omicron peptide; (2) BNT162b2-vaccinated individuals displayed strong responses to the WT and gamma peptides but not omicron; and (3) single amino acid mutations at position 417 significantly impacted antibody detection. Importantly, biosensor results showed a strong positive correlation with neutralizing antibody titers measured by pseudovirus assays (r = 0.79). Our results confirm that the modular ZnONRs-peptide biosensor platform is not only sensitive and specific but also versatile, scalable, and rapidly adaptable to future SARS-CoV-2 variants or other emerging pathogens. This approach provides a clinically relevant, point-of-care alternative for serological assessment and monitoring of variant-specific immune responses.