School of Public Health, Chengdu Medical College, Chengdu, Sichuan, 610500, China.
Clinical Medical College & Affiliated Hospital, Chengdu University, Chengdu, Sichuan, 610041, China.
Anal Chim Acta. 2024 Oct 16;1326:343136. doi: 10.1016/j.aca.2024.343136. Epub 2024 Aug 20.
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 (ɑ-AMA) and beta-amanitin (β-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 ɑ-AMA and β-AMA.
A novel label-free SELEX strategy based on the positively charged gold nanoparticles (AuNPs) was proposed to simultaneous screening of aptamers for ɑ-AMA and β-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 ɑ-30 and β-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 ɑ-30-2 (50 bp) and β-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 (ɑ-AMA) and 8.49 pg/mL (β-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 ɑ-AMA and β-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.
蘑菇中毒是一个全球性的健康问题，具有高发病率和死亡率。导致这种情况的主要致死毒素是α-鹅膏蕈碱（ɑ-AMA）和β-鹅膏蕈碱（β-AMA）。作为生物传感器中有前途的生物识别分子，适体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。在这项研究中，我们旨在开发一种新的无标记 SELEX 策略，用于筛选具有高亲和力的适体，并构建用于检测 ɑ-AMA 和 β-AMA 的新型适体传感器。
提出了一种基于带正电荷的金纳米粒子（AuNPs）的新型无标记 SELEX 策略，用于同时筛选 ɑ-AMA 和 β-AMA 的适体。仅需 18 轮 SELEX 即可获得新的适体。通过胶体金测定分析候选适体，ɑ-30 和 β-37 的序列与 Kd 值分别为 22.26 nM 和 23.32 nM 具有很大的亲和力，且不受植物毒素的干扰。值得注意的是，截短的适体 ɑ-30-2（50 bp）和 β-37-2（57 bp）比其原始对应物（79 bp）具有更高的亲和力。随后，利用所选适体构建了基于 Cu 依赖性 DNA 酶和 Exo I 引发的循环级联反应切割底物的发夹环化的电化学适体传感器的识别探针。该检测平台具有出色的分析性能，检测限低至 4.57 pg/mL（ɑ-AMA）和 8.49 pg/mL（β-AMA）。此外，该适体传感器在蘑菇和尿液样本中表现出优异的性能。
这项工作开发了一种简单高效的无标记 SELEX 方法，用于筛选新的 ɑ-AMA 和 β-AMA 适体，该方法使用带正电荷的 AuNPs 作为筛选介质，无需文库的化学标记或盐离子诱导。此外，基于我们新获得的适体开发了两种新型电化学适体传感器，为 AMA 中毒的超灵敏检测提供了新的生物传感工具，在实际应用中具有很大的潜力。
