This study reports a novel bio-layer interferometry (BLI)-based SELEX for generation of high affinity aptamers against patulin. Unlike conventional SELEX, the present method enabled real-time monitoring of increasing affinity of the oligonucleotides to the toxin. After seven rounds of selection cycles, the enriched pool of aptamers was characterized by cloning and sequencing and clustered into two families based on similarity. Two sequences, PAT C3 and PAT C4, each belonging to different clades, were further evaluated for their binding affinity. SPR studies determined the dissociation constants (K_D) of 8.2 × 10^–8 and 1.9 × 10^–7 M for aptamer PAT C3 and PAT C4, respectively. The highest affinity PAT C3 aptamer was used to develop a patulin BLI aptasensor, which indicated a linear detection range from 0.045 to 100 ng/mL [limit of detection (LOD) = 0.173 ng/mL; limit of quantification (LOQ) = 0.526 ng/mL]. The aptasensor displayed no cross-reactivity with its structural analogue isopatulin or any of the other mycotoxin groups tested. Spiking studies in simulated apple juice samples showed recoveries in the range of 82.11 to 100.23%, indicating good sensor performance. The study is the first report of BLI-based SELEX for a non-protein toxin, which resulted in the generation of high affinity aptamers and development of an aptasensor which can have wide application in the food industry for high throughput screening of samples for patulin contamination within a short span of time.

中文翻译：

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基于生物层干涉的 SELEX 和使用生成的适体对棒曲霉素进行无标记检测

本研究报告了一种新的基于生物层干涉仪 (BLI) 的 SELEX，用于生成针对棒曲霉素的高亲和力适体。与传统的 SELEX 不同，本方法能够实时监测寡核苷酸对毒素的亲和力增加。经过七轮选择循环后，通过克隆和测序对富集的适体库进行了表征，并根据相似性分为两个家族。进一步评估了各自属于不同进化枝的两个序列 PAT C3 和 PAT C4 的结合亲和力。SPR 研究确定解离常数 ( K _D ) 为 8.2 × 10 ^–8和 1.9 × 10 ^–7M 分别代表适体 PAT C3 和 PAT C4。使用亲和力最高的 PAT C3 适体开发棒曲霉素 BLI 适体传感器，其线性检测范围为 0.045 至 100 ng/mL [检测限 (LOD) = 0.173 ng/mL；定量限 (LOQ) = 0.526 ng/mL]。适体传感器与其结构类似物异棒曲霉素或任何其他测试的霉菌毒素基团没有显示出交叉反应性。对模拟苹果汁样品的加标研究表明，回收率在 82.11% 到 100.23% 之间，表明传感器性能良好。该研究是基于 BLI 的 SELEX 用于非蛋白质毒素的第一份报告，