Phosphate-triggered ratiometric multicolor immunosensor based on nanobody-alkaline phosphatase fusion protein for sensitive detection of fenitrothion.

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]