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-IgG ^HRP 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-IgG ^HRP as the detection antibody, and the formation of the complete sandwich complex. Upon exposure to the TMB/H₂O₂ substrate, HRP-catalyzed redox reactions generated a measurable electrochemical signal, enabling precise quantification of TBRFV. The developed biosensor exhibited a wide linear detection range from 0 to 10 ⁵ fg/mL, with an ultra-low detection limit of 1.14 fg/mL, corresponding to 65.14 aM. Furthermore, the sensor demonstrated high specificity for TBRFV, effectively distinguishing it from potential interfering agents. The proposed electrochemical immunosensing strategy provides a highly promising platform for the early and accurate detection of TBRFV, offering significant potential for agricultural biosecurity and the prevention of viral outbreaks.