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.