Hairpin DNA assisted dual-ratiometric electrochemical aptasensor with high reliability and anti-interference ability for simultaneous detection of aflatoxin B1 and ochratoxin A

The simultaneous detection of multiple mycotoxins in grains is significant due to the enhanced toxicity induced by their synergistic effects. In this work, a dual-ratiometric electrochemical aptasensing strategy for the simultaneous detection of aflatoxin B1 (AFB1) and ochratoxin A (OTA) was developed. Here, an anthraquinone-2-carboxylic acid (AQ)-labelled complementary DNA (cDNA) was used to provide separate and specific binding sites to assemble the ferrocene-labelled AFB1 aptamer (Fc-Apt1) and methylene blue-labelled OTA aptamer (MB-Apt2). The target-induced current ratios of IFc/IAQ and IMB/IAQ were then used to quantitatively relate to AFB1 and OTA, respectively. Following this principle, two types of aptasensors involving the hairpin DNA (hDNA) and linear single-stranded DNA (ssDNA) as the cDNA were fabricated for performance comparisons. The results revealed that hairpin DNA with a rigid 2D structure can greatly improve the assembly and recognition efficiency of the sensing interface, which makes the hDNA-based aptasensor possess high sensitivity, reliability and anti-interference ability. The hDNA-based aptasensor exhibited a detection range of 10-3000 pg mL−1 for AFB1 and 30-10000 pg mL−1 for OTA, respectively, with no observable cross-reactivity. Furthermore, the aptasensor was applied to analyze corn and wheat samples, and the reliability was validated by HPLC-MS/MS. Our work has presented a novel way for fabricating a high-performance aptasensor for simultaneous detection of multiple mycotoxins.