A simple
and sensitive fluoroimmunoassay (FIA) based on a heavy-chain
antibody (VHH) for rapid detection of fenitrothion 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 a fusion protein (VHHjd8-BT), showing both binding
capacity to fenitrothion and the streptavidin poly-horseradish peroxidase
conjugate (SA-polyHRP). Based on a competitive assay format, the absorbance
spectrum of oxidized 3,3′,5,5′-tetramethylbenzidine
generated 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 IC50 value of 1.4 ng/mL
and a limit of detection of 0.03 ng/mL, which exhibited 15-fold improvement
compared with conventional enzyme-linked immunosorbent assay. The
recovery test of FIA was validated by standard GC–MS/MS, and
the results showed good consistency, indicating that the assay is
an ideal tool for rapid screening of fenitrothion in bulk food samples.