Aptamer-based Fluorescent Assay for Simple and Sensitive Detection of Fipronil in Liquid Eggs

Abstract

In the present study, an aptamer-based fluorescent assay was developed to detect fipronil in liquid eggs, using a carboxyfluorescein (FAM)-labeled aptamer (FAM aptamer) and partially complementary DNA labeled with carboxytetramethylrhodamine (TAMRA cDNA). The fipronil amount in analytical samples was determined by measuring FAM fluorescence at 522 nm. In the absence of fipronil, FAM fluorescence is effectively quenched by fluorescence resonance energy transfer (FRET) phenomenon generated by the hybridized structure of the FAM aptamer and TAMRA cDNA. In contrast, in the presence of fipronil, FAM fluorescence is gradually recovered in a fipronil concentration-dependent manner because the FAM aptamer has a stronger affinity for fipronil than for TAMRA cDNA. The proposed analytical method for fipronil involves a simple one-step procedure of mixing the sample solution without any washing step, and the results can be obtained in approximately 30 min. Under optimized conditions, the developed assay showed a linear response to fipronil in the egg sample in the range of 25-300 ppb with a detection limit of 53.8 ppb (0.068 mg/kg) and recovery rates in the range of 94.7-114.4%. Additionally, the assay selectivity for fipronil was investigated against eight pesticides, and it showed good specificity for fipronil. Compared with conventional liquid chromatography-mass spectrometry (LC-MS), the proposed method provides a simple, easy, and fast method to detect fipronil in egg samples without requiring bulky and expensive instruments and complicated and laborious processes. We believe this detection method could be a promising platform for fipronil detection in real food samples.