Development of a Dual-Readout Multicolor Immunoassay for the Rapid Analysis of Isocarbophos in Vegetable and Fruit Samples

Multicolor immunoassay is a powerful tool for rapid analysis without the use of bulky instruments owing to various color conversions, which is suitable for on-site visual analysis for pesticides. Herein, this study developed a multicolor immunoassay for the rapid detection of isocarbophos. After competitive immunoassay, the secondary antibody (GAM-ALP) catalyzed ascorbyl-2-phosphate (AAP) into ascorbic acid (AA). The AA can reduce K₃[Fe(CN)₆] into K₄[Fe(CN)₆]. The latter can react with Fe³⁺ to form Prussian blue; otherwise, the orange AAP-Fe³⁺ complex was generated. Therefore, the multicolor immunoassay achieved a color conversion of orange-green-blue in response to isocarbophos, allowing for rapid semiquantitative analysis by the naked eye. After parameter optimization, the multicolor immunoassay was developed depending on the ratiometric absorbance between the Prussian blue and AAP-Fe³⁺ complex. Moreover, a smartphone was used to measure the RGB value of the color conversion for the development of portable visual, quantitative analysis. Both the absorbance-based and RGB-based multicolor immunoassays showed good accuracy and practicability in the recovery test. This study provided a common approach for the development of dual-readout multicolor immunoassay, which can be used for on-site rapid screening by quantitative or visual semiquantitative analysis.