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Multimodal Ochratoxin A-Aptasensor Using 3′-FAM-Enhanced Exonuclease I Tool and Magnetic Microbead Carrier
Analytical Chemistry ( IF 7.4 ) Pub Date : 2022-07-29 , DOI: 10.1021/acs.analchem.1c05576
Jin-Yuan Chen 1 , Qing-Xia Wei 2 , Liang-Yong Yang 2 , Jia-Yi Li 2 , Tai-Cheng Lu 1 , Zhou-Jie Liu 2 , Guang-Xian Zhong 3 , Xiu-Hua Weng 2 , Xiong-Wei Xu 2
Affiliation  

Thanks to its preparatory ease, close affinity, and low cost, the aptasensor can serve as a promising substitute for antibody-dependent biosensors. However, the available aptasensors are mostly subject to a single-mode readout and the interference of unbound aptamers in solution and non-target-induced transition events. Herein, we proposed a multimodal aptasensor for multimode detection of ochratoxin A (OTA) with cross-validation using the 3′-6-carboxyfluorescein (FAM)-enhanced exonuclease I (Exo I) tool and magnetic microbead carrier. Specifically, the 3′-FAM-labeled aptamer/biotinylated-cDNA hybrids were immobilized onto streptavidin-magnetic microbeads via streptavidin–biotin interaction. With the presence of OTA, an antiparallel G-quadruplex conformation was formed, protecting the 3′-FAM labels from Exo I digestion, and then anti-FAM-horseradish peroxidase (HRP) was bound via specific antigen-antibody affinity; for the aptamers without the protection of OTA, the distal ssDNA was hydrolyzed from 3′ → 5′, releasing 3′-FAM labels to the solution. Therefore, the OTA was detected by analyzing the “signal-off” fluorescence of the supernatant and two “signal-on” signals in electrochemistry and colorimetry through the detection of the coating magnetic microbeads in HRP’s substrate. The results showed that the 3′-FAM labels increased the activity of Exo I, producing a low background due to a more thorough digestion of unbound aptamers. The proposed multimodal aptasensor successfully detected the OTA in actual samples. This work first provides a novel strategy for the development of aptasensors with Exo I and 3′-FAM labels, broadening the application of aptamer in the multimode detection of small molecules.

中文翻译:

使用 3'-FAM 增强型核酸外切酶 I 工具和磁性微珠载体的多模式赭曲霉毒素 A-适体传感器

由于其制备简便、亲和性强和成本低,适体传感器可以作为抗体依赖性生物传感器的有希望的替代品。然而,可用的适体传感器大多受到单模式读出和溶液中未结合适体的干扰和非目标诱导的转换事件的影响。在此,我们提出了一种多模式适体传感器,用于多模式检测赭曲霉毒素 A (OTA),并使用 3'-6-羧基荧光素 (FAM) 增强型核酸外切酶 I (Exo I) 工具和磁性微珠载体进行交叉验证。具体来说,3'-FAM 标记的适体/生物素化-cDNA 杂合体通过链霉亲和素-生物素相互作用固定在链霉亲和素-磁性微珠上。在 OTA 的存在下,形成了反平行 G-四链体构象,保护 3'-FAM 标记免受 Exo I 消化,然后通过特异性抗原抗体亲和力结合抗FAM辣根过氧化物酶(HRP);对于没有OTA保护的适配体,远端ssDNA从3'→5'水解,将3'-FAM标记释放到溶液中。因此,通过检测HRP底物上的涂层磁性微珠,分析上清液的“信号关闭”荧光和电化学和比色法中的两个“信号开启”信号来检测OTA。结果表明,3'-FAM 标记增加了 Exo I 的活性,由于对未结合的适体进行了更彻底的消化,产生了低背景。所提出的多模式适体传感器成功地检测到实际样本中的 OTA。这项工作首先为开发具有 Exo I 和 3'-FAM 标签的适体传感器提供了一种新策略,
更新日期:2022-07-29
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