Label-free fluorescent sensor based on aptamer and thiazole orange for the detection of tetracycline

Abstract

A label-free fluorescence aptasensor based on tetracycline-binding aptamers and thiazole orange (TO) was established for the selective and sensitive detection of tetracycline. TO is essentially nonfluorescent in aqueous solution, while TO can intercalate into the tetracycline-binding aptamers with G-quadruplex structures causing a high fluorescence emission intensity. In the presence of the target tetracycline, the specific recognition and binding of aptamers with the targets induce the conformational changes of aptamers from G-quadruplex structures to hairpin structures. Such target-induced conformational changes lead to the quenching of the fluorescence emission from TO, which thus constitutes the mechanism of the aptamer-based label-free fluorescence biosensor for specific target analysis. Under the optimum conditions, the proposed fluorescence assay shows a linear range from 0.05 to 100 μg mL−1 with a detection limit of 0.029 μg mL−1. The cross-reactivity of this approach against other common antibiotics was negligible compared with the response to tetracycline, proving the excellent selectivity of this label-free fluorescent aptasensor. When used in samples of tetracycline-spiked raw milk, this method demonstrated the satisfactory recoveries between 90.0% and 108.0%, and the results were in full agreement with those from HPLC, confirming the high reliability and feasibility of this bioassay for rapid screening of tetracycline in various practical applications. Using the structure-switching aptamers and the fluorescent intercalator of nucleic acids, the proposed label-free strategy avoids all complicated covalent modifications or chemical labeling, and thus offers obvious advantages of simplicity, convenience, specificity and cost efficiency.