The global spread of African swine fever virus (ASFV) - a 100 % lethal pathogen with pandemic potential in swine populations - underscores the critical need for diagnostic technologies that combine laboratory-grade sensitivity with field-deployable speed. Here, we introduce a nanozyme-based chemical signal cascade electrochemical amplification strategy for ultrasensitive ASFV detection, which integrates a Cu2-xS@Cu-MOF heterojunction nanozyme with significantly enhanced peroxidase-like activity compared to its individual components. This heterojunction architecture achieves synergistic catalytic enhancement through charge separation at the Cu2-xS/Cu-MOF interface, which improves charge carrier mobility, while the porous Cu-MOF scaffold prevents nanoparticle aggregation and increases active site accessibility for H2O2 and 3,3′,5,5′-tetramethylbenzidine (TMB) substrates. The platform employs a dual-stage signal amplification system combining catalytic turnover of TMB oxidation with electrochemical redox cycling, where each H2O2 molecule participates in multiple redox cycles to generate amplified current signals. Notably, this design enables a detection limit of 6.45 × 10−8 TCID50/mL (TCID50 refers to 50 % tissue culture infective dose) for ASFV using the ASFV p54 antibody (Ab) as a biomarker molecule onto the nanozyme surface, which represents one of the lowest reported values for electrochemical ASFV sensors. Our heterojunction nanozyme architecture redefines biosensor design paradigms, simultaneously achieving the sensitivity floor of molecular diagnostics and the operational simplicity of point-of-care devices for pandemic-ready viral surveillance.
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