Development of an scFv-based electrochemical biosensor for robust detection of Tilapia Lake Virus

Tilapia Lake Virus (TiLV) poses a severe threat to global tilapia aquaculture, causing high mortality rates and significant economic devastation. Conventional diagnostic methods are limited by their operational complexity and requirement for centralized laboratory infrastructure. To address these challenges, this study reports the development of a label-free electrochemical biosensor utilizing a newly engineered recombinant single-chain variable fragment (scFv) antibody targeted against TiLV (scFv-TiLV). The scFv-TiLV construct was expressed in Escherichia coli BL21(DE3) using a low-temperature induction strategy assisted by chemical chaperones, yielding a highly purified (>95%) and soluble bioreceptor. The purified scFv-TiLV was immobilized onto gold nanoparticle-modified electrodes via EDC/NHS coupling chemistry. The immunoelectrode monitored concentration-dependent suppression in square-wave voltammetry (SWV) peak currents resulting from surface alterations upon viral capture. The biosensor demonstrated a broad dynamic linear range from 10² to 10⁶ copies/mL in both standard PBS buffer (R² = 0.9918) and a fish serum matrix (R² = 0.9914), achieving high sensitivity and matrix tolerance. The platform achieved a limit of detection (LOD) of 42.85 copies/mL in the complex serum matrix. Furthermore, preliminary cross-reactivity trials against off-target viral pathogens confirmed excellent selectivity and negligible false-positive responses. This scFv-based electrochemical platform offers a robust, simplified, and accessible alternative for decentralized viral diagnostics.