The Central Laboratory, Fujian Key Laboratory of Precision Medicine for Cancer, Key Laboratory of Radiation Biology of Fujian Higher Education Institutions, the First Affiliated Hospital, Fujian Medical University, Fuzhou 350005, China.
Department of Pharmacy, First Affiliated Hospital of Fujian Medical University, Fuzhou 350005, China.
Anal Chem. 2022 Aug 9;94(31):10921-10929. doi: 10.1021/acs.analchem.1c05576. Epub 2022 Jul 29.
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.
由于其制备简便、亲和力强、成本低廉，适体传感器可以作为抗体依赖型生物传感器的有前途的替代品。然而，现有的适体传感器大多受到单模式读出以及溶液中未结合的适体和非靶诱导转换事件的干扰。在此，我们提出了一种使用 3'-6-羧基荧光素（FAM）增强的外切酶 I（Exo I）工具和磁性微球载体的多模式适体传感器，用于多模式检测黄曲霉毒素 A（OTA），并进行交叉验证。具体来说，将 3'-FAM 标记的适体/生物素化-cDNA 杂交体通过链霉亲和素-生物素相互作用固定在链霉亲和素磁性微球上。在 OTA 的存在下，形成了反平行 G-四链体构象，保护了 3'-FAM 标记物免受 Exo I 的消化，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 保护的适体，从 3'→5'水解远端 ssDNA，将 3'-FAM 标记物释放到溶液中。因此，通过分析上清液的“信号关闭”荧光和通过 HRP 底物检测包被的磁性微球的电化学和比色法中的两个“信号开启”信号，检测到 OTA。结果表明，3'-FAM 标记物增加了 Exo I 的活性，由于未结合的适体更彻底的消化，产生了较低的背景。所提出的多模式适体传感器成功地检测了实际样品中的 OTA。这项工作首次为开发具有 Exo I 和 3'-FAM 标记物的适体传感器提供了一种新策略，拓宽了适体在小分子多模式检测中的应用。
