Differential pulse voltammetric ochratoxin A assay based on the use of an aptamer and hybridization chain reaction

Abstract

A differential pulse voltammetric aptasensor based on hybridization chain reaction was developed for determination of the mycotoxin ochratoxin A (OTA) with very low detection limits. In this assay, a capture probe is immobilized on an electrode and then hybridized with an aptamer to form an aptamer/DNA duplex. In the presence of OTA, the formation of an aptamer-OTA complex results in the dissociation of the aptamer from the electrode. Next, the detection probe and two hairpin-helper DNAs are added, which leads to the formation of extended dsDNA polymers through HCR on the electrode surface. Finally, a streptavidin-alkaline phosphatase conjugate that binds to the remaining biotinylated hairpin DNAs is added. The ALP hydrolyzes a synthetic enzyme substrate (α-naphthyl phosphate), which is electroactive and produces a DPV current that increases with increasing OTA concentration. Under optimal conditions, the signal is linearly related to the logarithm of the OTA concentration in the 0.005 to 100 ng·mL−1 range, with a detection limit of 2 pg·mL−1. The assay was successfully applied to the determination of OTA in cereal samples. The results showed satisfactory recovery and good agreement with those of HPLC.