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

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 (alpha-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 center dot mL(-1) range, with a detection limit of 2 pg center dot 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.