Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China; College of Resources and Environments, Southwest University, Chongqing 400715, PR China.
College of Resources and Environments, Southwest University, Chongqing 400715, PR China.
Anal Chim Acta. 2014 Feb 6;811:70-5. doi: 10.1016/j.aca.2013.11.022. Epub 2013 Nov 25.
In this study, we for the first time presented an efficient, accurate, rapid, simple and ultrasensitive detection system for small molecule ochratoxin A (OTA) by using the integration of loop-mediated isothermal amplification (LAMP) technique and subsequently direct readout of LAMP amplicons with a signal-on electrochemiluminescent (ECL) system. Firstly, the dsDNA composed by OTA aptamer and its capture DNA were immobilized on the electrode. After the target recognition, the OTA aptamer bond with target OTA and subsequently left off the electrode, which effectively decreased the immobilization amount of OTA aptamer on electrode. Then, the remaining OTA aptamers on the electrode served as inner primer to initiate the LAMP reaction. Interestingly, the LAMP amplification was detected by monitoring the intercalation of DNA-binding Ru(phen)3(2+) ECL indictors into newly formed amplicons with a set of integrated electrodes. The ECL indictor Ru(phen)3(2+) binding to amplicons caused the reduction of the ECL intensity due to the slow diffusion of Ru(phen)3(2+)-amplicons complex to the electrode surface. Therefore, the presence of more OTA was expected to lead to the release of more OTA aptamer, which meant less OTA aptamer remained on electrode for producing LAMP amplicons, resulting in less Ru(phen)3(2+) interlaced into the formed amplicons within a fixed Ru(phen)3(2+) amount with an obviously increased ECL signal input. As a result, a detection limit as low as 10 fM for OTA was achieved. The aptasensor also has good reproducibility and stability.
在这项研究中，我们首次提出了一种高效、准确、快速、简单和超灵敏的小分子赭曲霉毒素 A (OTA)检测系统，该系统结合了环介导等温扩增 (LAMP) 技术，随后通过信号放大电化学发光 (ECL) 系统直接读取 LAMP 扩增产物。首先，将由 OTA 适体和其捕获 DNA 组成的 dsDNA 固定在电极上。在目标识别后，OTA 适体与目标 OTA 结合，随后从电极上脱落，这有效地减少了电极上 OTA 适体的固定量。然后，电极上剩余的 OTA 适体作为内引物启动 LAMP 反应。有趣的是，通过监测 DNA 结合的 Ru(phen)3(2+) ECL 指示剂与新形成的扩增子的插入，来检测 LAMP 扩增。由于 Ru(phen)3(2+)-扩增子复合物向电极表面的缓慢扩散，ECL 指示剂 Ru(phen)3(2+)与扩增子结合导致 ECL 强度降低。因此，预计存在更多的 OTA 将导致释放更多的 OTA 适体，这意味着在固定量的 Ru(phen)3(2+)中，电极上用于产生 LAMP 扩增子的 OTA 适体减少，导致更少的 Ru(phen)3(2+)插入形成的扩增子中，从而明显增加 ECL 信号输入。结果，实现了对 OTA 的检测限低至 10 fM。该适体传感器还具有良好的重现性和稳定性。
