An ultrasensitive aptasensor for detection of Ochratoxin A based on shielding effect-induced inhibition of fluorescence resonance energy transfer

Abstract

A novel and ultrasensitive aptasensor based on shielding effect-induced inhibition of fluorescence resonance energy transfer (FRET) was developed for detection of Ochratoxin A (OTA). The aptasensor is composed of a free OTA aptamer, a gold nanoparticle coated with biotinylated partially complementary DNA (biotin-cDNA-AuNP), and a Cy3-conjugated streptavidin, each of which acts as a recognition molecule, energy acceptor, and energy donor, respectively. In the absence of target, a half of OTA aptamer hybridizes with biotin-cDNA-AuNP, while another half of the aptamer plays a shielding cover to block the specific interaction between biotin and streptavidin, resulting in inhibition of FRET from Cy3-conjugated streptavidin to AuNP. In the presence of OTA, however, strong and specific interaction between aptamer and OTA reduces the shielding effect-based FRET inhibition, thus leading to dose-dependent fluorescence decrease. Under optimal condition, a limit of detection (LOD) of 1.4pgmL−1 and a wide assay range from 2.5pgmL−1 to 1μgmL−1 was achieved for OTA detection in PBS. A negligible interference from other mycotoxins demonstrates a good selectivity of the developed aptasensor. The proposed method was also successfully applied to measure OTA in spiked wheat and green coffee bean extractives, demonstrating a great feasibility for analysis of various real samples. In addition, the unique shielding effect-based aptasensor could be adapted to the detection of other small molecules with poor immunogenicity by simple change of the aptamer.