Production and Characterization of Biotinylated Anti-fenitrothionNanobodies and Development of Sensitive Fluoroimmunoassay br

A simple and sensitivefluoroimmunoassay (FIA) based on a heavy-chain antibody (VHH) for rapid detection offenitrothion was developed. A VHH library was constructed from an immunized alpaca, and one clone recognizing fenitrothion(namely, VHHjd8) was achieved after careful biopanning. It was biotinylated by fusing with the Avi tag and biotin ligase to obtain afusion protein (VHHjd8-BT), showing both binding capacity to fenitrothion and the streptavidin poly-horseradish peroxidaseconjugate (SA-polyHRP). Based on a competitive assay format, the absorbance spectrum of oxidized 3,3 ',5,5 '-tetramethylbenzidinegenerated by SA-polyHRP overlapped the emission spectrum of carbon dots, which resulted in quenching of signals due to the inner-filter effect. The developed FIA showed an IC50value of 1.4 ng/mL and a limit of detection of 0.03 ng/mL, which exhibited 15-foldimprovement compared with conventional enzyme-linked immunosorbent assay. The recovery test of FIA was validated by standardGC-MS/MS, and the results showed good consistency, indicating that the assay is an ideal tool for rapid screening of fenitrothion inbulk food samples.