Ratiometric fluorescent aptasensor for convenient detection of ochratoxin A in beer and orange juice

Abstract

Based on the principle of fluorescence resonance energy transfer (FRET), a simple ratiometric fluorescent aptasensor for convenient detection of ochratoxin A (OTA), a Group IIB carcinogen secreted by some fungi, was established. Initially, the anti-OTA aptamer with a quadruplex structure was flanked with FAM and BHQ1, and its partially complementary DNA (cDNA) was tagged with Cy3. In the absence of OTA, this aptamer hybridized with the cDNA strand forming a DNA duplex structure, in which BHQ1 was adjacent to Cy3 and distant from FAM. Due to the FRET principle, the fluorescence intensity emitted by Cy3 (F_Cy3) was quenched by BHQ1, and the fluorescence intensity emitted by FAM (F_FAM) recovered. In the presence of OTA, the prepared aptamer preferred to bind with OTA instead of cDNA, forming an aptamer–OTA complex structure in which BHQ1 was adjacent to FAM and distant from Cy3. As a result, F_FAM was quenched and F_Cy3 was restored. OTA can be accurately detected via the determination of the F_Cy3/F_FAM ratio value. Under optimal conditions, this ratiometric fluorescent aptasensor offers excellent OTA detection in the range of 0.6 nmol L⁻¹–5 μmol L⁻¹, with a limit of detection (LOD) of 0.3 nmol L⁻¹. This ratiometric aptasensor showed the advantages of easy operation, accuracy and sensitive analysis. Good specificity of this aptasensor was demonstrated. This ratiometric aptasensor could be used for the detection of OTA in real samples, e.g. beer and orange juice, showing its promising application potential.