Peptide-Based Dual-Readout Biosensor for the Detection of the Anti-Foot and Mouth Disease Virus Nonstructural Protein Antibodies

Serodiagnosis of antibodies against the foot-and-mouth disease virus (FMDV) nonstructural protein 3ABC is essential for surveillance of infection and vaccination status. Here, we report a single-probe dual-readout peptide-based biosensor that combines electrochemical and fluorescence readouts for rapid and selective detection of anti-3ABC antibodies directly from serum. FMDV-inspired peptides, site-specifically labeled with 5(6)-carboxyfluorescein (FAM) or 7-methoxycoumarin-4-acetic acid, were immobilized on indium tin oxide electrodes via click chemistry. Antibody binding to the peptides produced a signal-off response in both modalities. FAM-probe electrodes exhibited superior performance, achieving detection limits of 0.38-1.14 (electrochemical) and 0.49-1.49 ng mL^-1 (fluorescence) with <3% interference from nontarget antibodies. In validation with 36 bovine, ovine, and caprine sera, the platform distinguished infected from vaccinated animals with 93 ± 2% accuracy (electrochemical) in only 30 min. This low-cost dual-readout sensor demonstrates a broadly adaptable strategy for high-accuracy serodiagnostics, provided that suitable redox-active fluorophores and antibody-recognizable peptide epitopes are employed, with potential for portable bioanalytical devices.