Competitive Ratiometric Fluorescent Lateral Flow Immunoassay Based on Dual Emission Signal for Sensitive Detection of Chlorothalonil

Abstract

In this study, we develop a competitive ratiometric fluorescent lateral flow immunoassay (CRF-LFIA) based on dual emission fluorescence signal, which has great advantage in visual and quantitative detection of Chlorothalonil (CTN). Red-emitted fluorescent magnetic nanobeads (FMNBs) and green-emitted aggregation-induced emission fluorescent microsphere (AIEFM) are synthesized and conjugated to antibodies and antigens respectively, resulting in competitive binding with the analyte. The ratiometric fluorescence signal which comes from the overlap of these two fluorescence emissions. FMNBs probes also provide immunomagnetic separation (IMS) to enrich the analysts and resist complex matrix effects. This strip generates a visually discernible yellow-to-green fluorescence color change in the presence of CTN (2 ng/mL), which could be incisively observed by naked eye. Moreover, the limit of detection (LOD) reached 0.152 ng/mL by measurement of color (Red-Green-Blue, RGB) signals, and a limit of quantification (LOQ) of 0.2 ng/g. Method validation shows a good correlation between CRF-LFIA and LC-MS/MS.

Keywords: Fluorescent magnetic nanobeads, Aggregation-induced emission, Immunomagnetic separation, Lateral Flow Immunoassay, Ratiometric fluorescence