Unleashing the Power of a Double-Nanobody Sandwich Electrochemical Assay for Rapid Detection of Grapevine Fanleaf Virus

Grapevine fanleaf virus (GFLV) is one of the most severe and widespread viruses affecting grapevines, causing significant economic losses by reducing crop quality and vineyard longevity. As no curative treatment exists, disease prevention relies essentially on prophylactic measures. The standard detection method, double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), is reliable and cost-effective but unsuitable for rapid, on-site testing. Additionally, electrochemical biosensors, which are widely used in medical diagnostics for rapid and user-friendly analysis, are still largely underexplored in agriculture. Here, we present a Double-Nanobody Sandwich Electrochemical (DNS-EC) assay for the rapid amperometric on-field monitoring of GFLV. High-affinity nanobodies, Nb75 and Nb59, targeting distinct viral epitopes, were integrated onto screen-printed gold electrodes for virus capturing, and the detection was achieved through enzymatic signal amplification using Nb75 and Nb59 fused to alkaline phosphatase (Nb75AP and Nb59AP), allowing subnanogram sensitivity within 30 min of total assay time. Validation with 15 field samples demonstrated 93% sensitivity (1 false negative) and 100% specificity (no false positives) compared to DAS-ELISA. This rapid, sensitive, and portable biosensor (with smartphone-based potentiostat compatibility) represents a promising technology for on-site GFLV diagnosis, advancing disease management in vineyards.