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, K3[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 K3[Fe(CN)6] into K4[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 MnO2 nanosheet to control the concentration of Na2SO3, 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.