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首页> 外文期刊>RSC Advances >An indirect competitive fluorescence assay for ochratoxin A based on molecular beacon
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An indirect competitive fluorescence assay for ochratoxin A based on molecular beacon

机译：基于分子标识的Ochratoxin A的间接竞争荧光测定

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摘要

A novel, simple and efficient method based on a molecular beacon (MB) probe was developed to detect ochratoxin A (OTA) in malts, which is a common starting material in the brewery industry. With the critical site for OTA binding in capture aptamer in mind, a MB probe containing 20 bases with a fluorophore–quencher pair at the stem ends was designed and synthesized. In the “off” configuration, the fluorescein (FAM) is internally quenched due to close contact with dabcyl and the fluorescence signal is recovered after hybridization with the capture aptamer at the “on” state. In the presence of OTA, the MB probe competes for binding at the loop region of the aptamer, resulting in a decrease in fluorescence signal. Using this indirect competitive assay, the detection of OTA in malt samples was accomplished for the first time. In addition, the effect of binding affinity of the capture aptamer and OTA on the assay performance was investigated. Under optimal conditions, this method allowed for OTA detection at a linear range of 0.0001–1 μg mL ~(?1) (correlation coefficient, R = 0.9920) with superior sensitivity and a detection limit as low as 0.05 ng mL ~(?1) . The sensing system also displayed an excellent selectivity and perfect anti-interference capacity in the matrix. Moreover, the entire process of detection was accomplished in less than 20 min. The recovery from spiked malt samples ranged from 81.0% to 95.2% with RSDs below 5.4%. The performance of our method was further validated by ultra-fast liquid chromatography coupled with tandem mass spectrometry. Compared with similar fluorescence assays, the proposed method is simple, efficient and does not require complicated conjugation steps. Taken together, this novel detection strategy could be a promising tool for hand-held devices used during on-site monitoring of contaminants.
机译：基于分子信标（MB）探针的新型，简单且有效的方法是为了检测麦芽的Ochratoxina（OTA），这是啤酒厂中的常见原料。对于在捕获Aptamer中的OTA结合的关键位点，设计并合成含有含有荧光团猝灭剂对的MB探针20碱基。在“关闭”配置中，由于与丁酰基与丁酰基接触，荧光素（FAM）在内部淬火，并且在“ON”状态下与捕获适体杂交后回收荧光信号。在OTA存在下，MB探针在适体的环形区域竞争结合，导致荧光信号的降低。使用该间接竞争性测定，首次完成麦芽样品中OTA的检测。此外，研究了捕获适体和OTA对测定性能的结合亲和力的影响。在最佳条件下，该方法允许OTA检测在0.0001-1μgmL〜（α1）（相关系数，r = 0.9920）的线性范围内，具有优异的敏感性，并且检测极限低至0.05 ng ml〜（？1 ）。传感系统还在矩阵中显示出优异的选择性和完美的抗干扰能力。此外，在少于20分钟的情况下完成了整个检测过程。 Spiked麦芽样品中的回收率范围为81.0％至95.2％，RSDS低于5.4％。通过与串联质谱耦合的超快速液相色谱法进一步验证了我们的方法的性能。与类似的荧光测定相比，所提出的方法简单，有效，不需要复杂的共轭步骤。在一起，这种新颖的检测策略可能是用于在现场监测污染物期间使用的手持设备的有希望的工具。

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