Dual Signal Enhancement by Magnetic Separation and Split Aptamer for Ultrasensitive T-2 Toxin Detection.

T-2 toxin, a type A trichothecene mycotoxin produced by species, is widely present in cereals and their processed products, posing a significant contaminant in food safety. To address the food safety challenges caused by this toxin, we established a dual signal enhancement by magnetic separation and split aptamer for ultrasensitive T-2 toxin detection. In this method, the introduction of magnetic graphene oxide (MGO) enhanced signal and increased sensitivity by reducing background interference. The shortened split aptamer reduces non-specific binding to MGO via decreased steric hindrance, thereby facilitating rapid target-induced dissociation and signal generation. A FAM fluorophore-labeled split aptamer probe FAM-SpA1-1 was quenched by MGO. While the fluorescence intensity remained nearly unchanged when the unlabeled split aptamer probe SpA1-2 was introduced alone, a significant fluorescence recovery was observed upon simultaneous addition of SpA1-2 and T-2 toxin. This recovery resulted from the cooperative binding of SpA1-1 and SpA1-2 to T-2 toxin, which distanced the FAM-SpA1-1 probe from MGO. Therefore, the proposed biosensor demonstrated excellent stability, reproducibility, and specificity, with a linear response range of 10-500 pM and a limit of detection (LOD) of 0.83 pM. Satisfactory recovery rates were achieved in spiked wheat (86.0-114.2%) and beer (112.0-129.6%) samples, highlighting the biosensor's potential for practical applications in real-sample detection. This study establishes the T-2 toxin split aptamer and demonstrates a novel dual-signal enhancement paradigm that pushes the sensitivity frontier of aptamer-based mycotoxin sensors.