1D-2D hybrid based electrochemical sensor for nano-architected sensing of ASFV p32 - an early phosphoprotein.

African swine fever (ASF) is a highly contagious and lethal disease with mortality rates approaching 100% in pigs. Early and accurate detection remains critical as no approved vaccine or antiviral treatment exists. We identified ASFV phosphoprotein p32 as a promising early-stage biomarker. Unlike structural proteins such as p54 and p72 detected predominantly during later infection stages, p32 is expressed early during viral replication, highly conserved among ASFV isolates, and strongly immunogenic, making it a reliable target for early and broad-spectrum detection. The ASFV p32 gene was cloned, expressed, and purified using a prokaryotic expression system, with biological specificity validated by western blotting, ELISA and mass spectrometry. Epitope prediction and structural modelling using AlphaFold, PROCHECK and ERRAT identified key antibody-binding regions, and polyclonal antibodies were generated and characterized by SDS-PAGE. A gold nanowire and tungsten disulfide hybrid (AuNW-WS2) was synthesized and characterized by FESEM, HRTEM, SAED, FFT, XRD, XPS and AFM. Antibodies were immobilized onto hybrid-functionalized screen-printed carbon electrodes to fabricate the SPCE/AuNW-WS2/ASFV p32 Ab biosensor. Validated against clinical samples and benchmarked against qPCR, the sensor exhibited a linear detection range of 1 fM-1μM, a limit of detection of ∼1.55 fM, 92.31% sensitivity, 100% specificity and 95% accuracy, underscoring its potential for rapid and reliable ASF diagnostics.