Freeze-synthesized bio-barcode immunoprobe based multiplex fluorescence immunosensor for simultaneous determination of four nitrofuran metabolites

Here we have reported a simple and sensitive bio-barcode immunosensor for simultaneous detection of 3-amino-2-oxazolidinone (AOZ), 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), 1-aminohydantoin (AHD), and semicarbazide (SEM) in aquatic products. According to freeze-thaw strategy, four fluorophores (FAM, HEX, ROX, Cy5) labeled single-stranded DNA (ssDNA) were conjugated onto the surface of gold nanoparticles (AuNPs) with corresponding four nitrofuran metabolites monoclonal antibodies (mAbs) for forming four bio-barcode fluorescence immunoprobes. The fluorescence of immunoprobes was quenched by AuNPs. In test progress, the ssDNA with fluorophores were released by adding the dithiothreitol (DTT) and the fluorescence recovered. The immunosensor exhibited sensitive and specific detection of nitrofuran metabolites from 0.05 to 28 μg/L. The limit of detection (LOD) was 0.01, 0.02, 0.02, and 0.05 μg/L for AOZ, AMOZ, AHD, and SEM, respectively. The recoveries of four nitrofuran metabolites in spiked aquatic products have been confirmed by UPLC-MS/MS. The bio-barcode based multiplex immunosensor provides a promising strategy for simultaneous detection of multiple targets.