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 (-AMA) and beta-amanitin (-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 -AMA and -AMA. A novel label-free SELEX strategy based on the positively charged gold nanoparticles (AuNPs) was proposed to simultaneous screening of aptamers for -AMA and -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 -30 and -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 -30-2 (50 bp) and -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 Cu 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 (-AMA) and 8.49 pg/mL (-AMA). Moreover, the aptasensors exhibited superior performance in mushroom and urine samples. This work developed a simple and efficient label-free SELEX method for screening new aptamers for -AMA and -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.

中文翻译：

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无标记适体 SELEX 用于超灵敏电化学适体传感器检测野生蘑菇中鹅膏蕈碱


蘑菇中毒造成了严重的全球健康问题，发病率和死亡率很高。造成这种情况的主要致命毒素是α-鹅膏蕈碱（-AMA）和β-鹅膏蕈碱（-AMA）。适配体作为生物传感器中一种很有前景的生物识别分子，已广泛应用于食品检测领域。然而，目前基于 SELEX 的筛选结构相似小分子适体的方法受到标记或盐离子诱导的限制。在本研究中，我们旨在开发一种新型的无标记SELEX策略来筛选具有高亲和力的核酸适体，并构建了用于检测-AMA和-AMA的新型核酸适体传感器。提出了一种基于带正电的金纳米粒子（AuNPs）的新型无标记SELEX策略来同时筛选-AMA和-AMA的适配体。只需要 18 轮 SELEX 即可获得新的适配体。通过胶体金分析，-30和-37序列显示出良好的亲和力，Kd值分别为22.26 nM和23.32 nM，且不受植物毒素的干扰。值得注意的是，截短的适体-30-2（50 bp）和-37-2（57 bp）比其原始对应物（79 bp）表现出更高的亲和力。随后，基于 Cu 依赖的 DNAzyme 和 Exo I 触发的循环级联对底物的发夹循环切割，利用选定的适体构建电化学适体传感器的识别探针。该检测平台表现出优异的分析性能，检测限低至 4.57 pg/mL (-AMA) 和 8.49 pg/mL (-AMA)。此外，适体传感器在蘑菇和尿液样本中表现出优异的性能。 本工作开发了一种简单高效的免标记SELEX方法来筛选新的-AMA和-AMA适配体，该方法采用带正电荷的AuNPs作为筛选介质，无需对文库进行化学标记或诱导盐离子。此外，基于我们新获得的适配体开发了两种新型电化学适配体传感器，为超灵敏检测AMA中毒提供了新的生物传感工具，在实际应用中显示出巨大的潜力。