Simultaneous determination of zearalenone and ochratoxin A based on microscale thermophoresis assay with a bifunctional aptamer

Highlights: • A bifunctional aptamer for simultaneous determination of zearalenone (ZEN) and ochratoxin A (OTA) was firstly engineered. • MST proved the bifunctional aptamer had higher affinity and specificity for ZEN and OTA. • A bifunctional aptamer-based MST-aptasensor was developed. • The aptasensor achieved highly sensitive and specific for ZEN and OTA in real samples. • The proof-of concept can be referred for other studies of development novel aptamers and aptasensors. Nowadays, contamination of various mycotoxins in crops and their products exposes increasing risks to human health. Efficient determination methods are urgently needed. Herein, a bifunctional aptamer and a simple aptasensor based on microscale thermophoresis assay (MST) were constructed for the first time for simultaneous determination of two mycotoxins, i.e. zearalenone (ZEN) and ochratoxin A (OTA). The bifunctional aptamer was engineered by splicing a ZEN aptamer and an OTA aptamer with a linker according to the structure analysis of aptamers. The binding mechanism of the bifunctional aptamer to ZEN and OTA were revealed basing on the molecular docking studies. The MST assay proved that the bifunctional aptamer showed high affinity and specificity towards ZEN and OTA. Furthermore, a bifunctional aptamer-based MST-aptasensor was developed for simultaneous detection of ZEN and OTA in corn oil sample. The MST-aptasensor provided a limit of detection (LOD) of 0.12 nM, with satisfactory recoveries of 93.31–104.19% and excellent selectivity, indicating that the bifunctional aptamer and MST-aptasensor had great potential in practical applications.