Fluorescent biosensor for sensitive analysis of oxytetracycline based on an indirectly labelled long-chain aptamer

Abstract

A fluorescent assay for oxytetracycline (OTC) detection was presented based on an indirectly fluorescein-labelled aptamer probe, which was fabricated through the partial hybridization of an OTC long-chain aptamer with a FAM-labelled short-chain ssDNA (S1). Upon combination of the target OTC and its aptamer, S1 with quenched fluorescein was released from the probe to the graphene sheet freely. Subsequently, it was hybridized with the complementary ssDNA (C1) and escaped from the quencher graphene to the solution, resulting in the restoration of fluorescence. Benefiting from the labelling of S1 instead of the OTC aptamer directly, the restoration of fluorescence was independent of the long-chain aptamer, perfectly avoiding the negative effects of the intrinsically existing secondary structure. Together with the high affinity of the aptamer for its target, this assay exhibited excellent sensitivity and selectivity. The linear response for OTC was found to be 0.01–0.2 μM with a limit of quantitation of 0.01 μM. Furthermore, the feasibility of the developed assay in a fresh water system and a milk sample was verified through the recovery experiments using spiked samples. This achievement based on such an indirect labelling method is also expected to lay a foundation to realize effective analysis of small molecule pollutants in the environment, for which the specific aptamers are long-chain nucleotide sequences.