Label-free SELEX of aptamers for ultra-sensitive electrochemical aptasensor detection of amanitin in wild mushrooms

Background: Mushroom poisoning poses a significant global health concern, with high morbidity and mortality rates. The primary lethal toxins responsible for this condition are alpha-amanitin (a-AMA) and beta-amanitin ((1-AMA). As a promising bio-recognition molecules in biosensors, aptamers, have been broadly used in the field of food detection. However, the current SELEX-based methods for screening aptamers for structurally similar small molecules were limited by the labelling or salt ion induction. In this study, we aimed to develop a novel label-free SELEX strategy for the screening of aptamers with high affinity and constructed new aptasensors for the detection of a-AMA and (1-AMA. Results: A novel label-free SELEX strategy based on the positively charged gold nanoparticles (AuNPs) was proposed to simultaneous screening of aptamers for a-AMA and (1-AMA. Only 18 rounds of SELEX were required to obtain new aptamers. The candidate aptamers were analyzed by colloidal gold assay, and the sequences of a30 and (1-37 displayed great affinity with Kd values of 22.26 nM and 23.32 nM, respectively, without interference from botanical toxins. Notably, the truncated aptamers a-30-2 (50 bp) and (1-37-2 (57 bp) exhibited higher affinity than their original counterpart (79 bp). Subsequently, the selected aptamers were utilized to construct recognition probes for electrochemical aptasensors based on hairpin cyclic cleavage of substrates by Cu2+ 2+ dependent DNAzyme and Exo I-triggered recycling cascades. The detection platform showed excellent analytical performance with limits of detection as low as 4.57 pg/mL (a-AMA) and 8.49 pg/mL (/3-AMA). Moreover, the aptasensors exhibited superior performance in mushroom and urine samples. Significance: This work developed a simple and efficient label-free SELEX method for screening new aptamers for a-AMA and /3-AMA, which employed the positively charged AuNPs as the screening medium, without the need for chemical labelling of libraries or induction of salt ions. Furthermore, two novel electrochemical aptasensors were developed based on our newly obtained aptamers, which offer the new biosensing tool for ultrasensitive detection of the AMA poisoning, showing great potential in practical applications.