Fluorometric aptamer assay for ochratoxin A based on the use of single walled carbon nanohorns and exonuclease III-aided amplification.

Abstract

The authors describe an aptamer based assay for the food mycotoxin ochratoxin A (OTA). It is based on the use of exonuclease III (Exo III) which assists in signal amplification, and of single-walled carbon nanohorns (SWCNHs) which act as quenchers of fluorescence. The detection scheme employs a hairpin probe (HP) and a signal probe (SP) labeled with carboxyfluorescein (FAM) at its 5'-end. The fluorescence of intact SPs (best measured at excitation/emission wavelengths of 495/518nm) is quenched by SWCNHs. The HP contains the OTA-specific aptamer sequence and is partially complementary to the SP. After addition of OTA, the aptamer binds OTA and thus exposes a single-stranded sequence that can hybridize with the SP. Exo III digests the SP to liberate the free fluorophore labels. The damaged SPs no longer are adsorbed by the SWCNHs so that fluorescence is no longer quenched. The method has a detection range that is linear from 10nM to 1000nM (with a correlation coefficient of 0.997). The limit of detection (LOD), calculated onthe basis ofa signal to noise ratio of 3, is 4.2nM. The procedure was validated by the quantitation of OTA in spiked real samples and were found to be free of interference by the sample matrix. Recoveries ranged from 93.8 to 113.0% in beer andfrom 92.0 to115.9% in red wine. Graphical abstract After adding ochratoxin A (OTA), the aptamer region in hairpin probe (HP) combined with OTA and thus exposed a single-stranded sequence to hybridize with signal probe (SP). Exonuclease III (Exo III) digested SP to liberate the free fluorophore (FAM).