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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.

The excessive use of carbaryl has resulted in the risk of its exposure. In this study, we isolated six nanobodies (Nbs) from a camelid phage display library against the biomarker of carbaryl, 1-naphthol (1-NAP). Owing to its characteristics of easy genetic modifications, we produced a nanobody-alkaline phosphatase (Nb-CC4-ALP) fusion protein with good stability. A dual-emission system based ratiometric fluoroimmunoassay (RFIA) for quick and highly sensitive determination of 1-NAP was developed. Silicon nanoparticles (SiNPs) was used as an internal reference and for aggregation-induced emission enhancement (AIEE) of gold nanoclusters (AuNCs), while AuNCs could be quenched by MnO2 via oxidation. In the presence of ALP, ascorbic acid phosphate (AAP) can be transformed into ascorbic acid (AA), the later can etch MnO2 to recover the fluorescence of the AuNCs. Based on optimal conditions, the proposed assay showed 220-fold sensitivity improvement in comparison with conventional monoclonal antibody-based ELISA. The recovery test of urine samples and the validation by standard HPLC-FLD demonstrated the proposed assay was an ideal tool for screening 1-NAP and provided technical support for the monitoring of carbaryl exposure.

Aristolochic acid (AA) toxicity has been shown in humans regarding carcinogenesis, nephrotoxicity, and mutagenicity. Monitoring the AA content in drug homologous and healthy foods is necessary for the health of humans. In this study, a monoclonal antibody (mAb) with high sensitivity for aristolochic acid I (AA-I) was prepared. Based on the obtained mAb, a chemiluminescent immunoassay (CLEIA) against AA-I was developed, which showed the 50% decrease in the RLUmax (IC50) value of 1.8 ng/mL and the limit of detection (LOD) of 0.4 ng/mL. Carbon dots with red emission at 620 nm, namely rCDs, were synthesized and employed in conventional indirect competitive enzyme-linked immunosorbent assay (icELISA) to improve the assay sensitivity of a fluoroimmunoassay (FIA). Oxidized 3,3′′,5,5′′-tetramethylbenzidine dihydrochloride (oxTMB) can quench the emission of the rCDs through the inner-filter effect; therefore, the fluorescence intensity of rCDs can be regulated by the concentration of mAb. As a result, the assay sensitivity of FIA was improved by five-fold compared to CLEIA. A good relationship between the results of the proposed assays and the standard ultra-high performance liquid chromatography-triple quadrupole mass spectrometer (UPLC-QQQ-MS/MS) of real samples indicated good accuracy and practicability of CLEIA and FIA. [ABSTRACT FROM AUTHOR]

Multicolor immunosensor is a promising tool for on-site rapid analysis by naked eye without bulky instruments. Herein, a multicolor immunosensor employed a specific nanobody-alkaline phosphatase fusion protein (VHHjd8-ALP) for biosensing of fenitrothion was developed. After one-step indirect competitive immunoassay, the binding VHHjd8-ALP in microplate catalyzed ascorbyl-2-phosphate (AAP) into ascorbic acid, which propose dual readout including multicolor ratiometric visible immunoassay (MRVIA) and multicolor ratiometric fluoroimmunoassay (MRFIA). For MRVIA, K 3 [Fe(CN) 6 ] and Fe3+ was employed for multicolor generation. The substrate AAP can chelate with Fe3+ to form orange AAP-Fe3+ complex. When ALP catalyzed AAP into ascorbic acid, the latter can reduced K 3 [Fe(CN) 6 ] into K 4 [Fe(CN) 6 ], which reacted with Fe3+ to produced Prussian blue via self-assembly. A visible color transition from orange to blue against fenitrothion was obtained. For MRFIA, the generated ascorbic acid etched MnO 2 nanosheet to control the concentration of Na 2 SO 3 , which can convert 9-anthraldehyde into sulfuretted anthracene accompanied by a fluorescent color transition from green to blue. These two methods showed visible multicolor transition against fenitrothion for naked-eye analysis. The recovery and blind samples test verified good accuracy and practicability, which can be used for rapid screening of fenitrothion by a naked-eye semiquantitative analysis mode. [Display omitted] • Multicolor immunoassays were achieved without precious-metal materials. • Color regulation relies on ratiometric changing of dual signals. • The VHHjd8-ALP-based strategy can develop a dual multicolor immunosensor. • They can be used for semiquantitative detection without complex apparatus. [ABSTRACT FROM AUTHOR]