Nanobodies are one-tenth the size of conventional antibodies and are naturally obtained from the atypical heavy-chain-only antibodies present in camelids. Their small size, high solubility, high stability, and strong resilience to organic solvents facilitate their use as novel analytical reagents in immunochemistry. In this study, specific nanobodies against pesticide carbofuran were isolated and characterized from an immunized library via phage display platform. We further established an indirect competitive enzyme-linked immunosorbent assay (ELISA) using nanobody Nb316 to detect carbofuran in vegetable and fruit samples. The results showed a half-maximal inhibitory concentration (IC₅₀) of 7.27 ng/mL and a detection limit of 0.65 ng/mL. A simplified sample pretreatment procedure omitting the evaporation of organic solvent was used. The averaged recovery rate of spiked samples ranged between 82.3% and 103.9%, which correlated with that of standard UPLC–MS/MS method. In conclusion, a nanobody with high specificity for carbofuran was characterized, and a nanobody-based sensitive immunoassay for simple and rapid detection of carbofuran in real samples was validated.

Nanobodies have several advantages, including great stability, sensibility, and ease of production; therefore, they have become important tools in immunoassays for chemical contaminants. In this manuscript, nanobodies for the detection of the toxin Nodularin-r (NOD-R), a secondary metabolite of cyanobacteria that could cause a safety risk for drinks and food for its strong hepatotoxicity, were for the first time selected from an immunized Bactrian camel VHH phage display library. Then, a sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for NOD-R, based on the nanobody N56 with great thermostability and organic solvent tolerance, was established under optimized conditions. The results showed that the limit of detection for NOD-R was 0.67 µg/L, and the average spike recovery rate was between 84.0 and 118.3%. Moreover, the ic-ELISA method was validated with spiked water sample and confirmed by UPLC–MS/MS, which indicated that the ic-ELISA established in this work is a reproducible detection assay for nodularin residues in water samples.