Attomolar electrochemical direct and sandwich immunoassays for the ultrasensitive detection of tomato brown rugose fruit virus.

Tomato brown rugose fruit virus (TBRFV; Tobamovirus fructirugosum) is a highly virulent tobamovirus that has emerged as a major global threat to tomato and pepper crops over the past decade. Early and ultra-sensitive detection of TBRFV is critical for effective disease management and the mitigation of agricultural losses. In this study, a highly sensitive electrochemical immunosensor was developed based on both direct and sandwich immunoassays for the detection of TBRFV. The assay employs TBRFV-CP-IgG and TBRFV-CP-IgGHRP antibodies, with the latter conjugated to horseradish peroxidase (HRP). The immunoassays were assembled on a nanoporous gold electrode, providing an enhanced electroactive surface for efficient antigen capture and signal amplification. Electrochemical characterization confirmed the successful immobilization of TBRFV-CP-IgG, its specific interaction with the recombinant coat protein of TBRFV (rp-CP-TBRFV), the subsequent binding of TBRFV-CP-IgGHRP as the detection antibody, and the formation of the complete sandwich complex. The assays achieved linear detection ranges of 10–10⁵ fg/mL. The direct assay yielded a limit of detection (LoD) of 1.14 fg/mL (65.14 aM), while the sandwich assay, enhanced by enzymatic amplification, achieved 1.06 fg/mL (60.57 aM). The direct assay’s simplicity suits rapid diagnostics, whereas the sandwich assay’s superior sensitivity is ideal for low-concentration samples. Electrochemical characterization confirmed specific antigen capture and signal amplification. The biosensor demonstrated high specificity, distinguishing TBRFV from other viruses, and detected TBRFV in leaf and seed extracts, offering a promising platform for agricultural biosecurity.