MOE Key Laboratory for Analytical Science of Food Safety and Biology, Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, College of Chemistry, Fuzhou University, Fuzhou, Fujian 350116, China; Fujian Provincial Key Laboratory of Modern Analytical Science and Separation Technology, College of Chemistry and Environment, Minnan Normal University, Zhangzhou 363000, China.
MOE Key Laboratory for Analytical Science of Food Safety and Biology, Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, College of Chemistry, Fuzhou University, Fuzhou, Fujian 350116, China.
Biosens Bioelectron. 2018 May 15;105:182-187. doi: 10.1016/j.bios.2018.01.043. Epub 2018 Jan 31.
The difference of electrostatic interaction between free Ru(phen) and Ru(phen) embedded in double strand DNA (dsDNA) to the negatively charged indium tin oxide (ITO) electrode has been applied to develop a homogeneous and label-free electrochemiluminescence (ECL) aptasensor for the first time. Ochratoxin A (OTA) has been chosen as the model target. The OTA aptamer is first hybridized with its complementary single strand DNA (ssDNA) to form dsDNA and then interacted with Ru(phen) via the grooves binding mode to form dsDNA-Ru(phen) complex, which remains negatively charged feature as well as low diffusion capacity to the negatively charged ITO electrode surface owing to the electrostatic repulsion. Meanwhile, the intercalated Ru(phen) in the grooves of dsDNA works as an ECL signal reporter instead of the labor-intensive labeling steps and can generate much more ECL signal than that from the labeling probe. In the presence of target, the aptamer prefers to form an aptamer-target complex in lieu of dsDNA, which induces the releasing of Ru(phen) from the dsDNA-Ru(phen) complex into the solution. With the assistance of RecJ exonuclease (a ssDNA specific exonuclease), the released ssDNA and the aptamer in the target-complex were digested into mononucleotides. In the meantime, the target can be also liberated from OTA-aptamer complex and induce target cycling and large amount of free Ru(phen) present in the solution. Since Ru(phen) contains positive charges, which can diffuses easily to the ITO electrode surface because of electrostatic attraction, causing an obviously enhanced ECL signal detected. Under the optimal conditions, the enhanced ECL of the system has a linear relationship with the OTA concentration in the range of 0.01-1.0 ng/mL with a detection limit of 2 pg/mL. This innovative system not only expands the immobilization-free sensors in the electrochemiluminescent fields, but also can be developed for the detection of different targets easily with the same strategy by changing the aptamer used.
已将游离 Ru(phen) 和嵌入双链 DNA(dsDNA)中的 Ru(phen) 与带负电荷的铟锡氧化物(ITO)电极之间的静电相互作用差异应用于首次开发均质且无标记的电化学发光(ECL)适体传感器。选择赭曲霉毒素 A(OTA)作为模型靶标。OTA 适体首先与其互补单链 DNA(ssDNA)杂交形成 dsDNA，然后通过沟结合模式与 Ru(phen)相互作用形成 dsDNA-Ru(phen)复合物，由于静电排斥，复合物仍保持带负电荷的特征以及低扩散能力到带负电荷的 ITO 电极表面。同时，嵌入 dsDNA 沟中的 Ru(phen) 充当 ECL 信号报告器，而不是繁琐的标记步骤，可以产生比标记探针更多的 ECL 信号。在存在靶标的情况下，适体更喜欢形成适体-靶复合物而不是 dsDNA，这会导致 Ru(phen)从 dsDNA-Ru(phen)复合物中释放到溶液中。在 RecJ 核酸外切酶（一种 ssDNA 特异性核酸外切酶）的协助下，释放的 ssDNA 和目标复合物中的适体被消化成单核苷酸。同时，目标也可以从 OTA-适体复合物中释放出来，并诱导目标循环和溶液中存在大量游离的 Ru(phen)。由于 Ru(phen) 带正电荷，由于静电吸引，很容易扩散到 ITO 电极表面，从而导致检测到的 ECL 信号明显增强。在最佳条件下，该系统的增强 ECL 与 0.01-1.0ng/mL 范围内的 OTA 浓度呈线性关系，检测限为 2pg/mL。该创新系统不仅在电化学发光领域扩展了无固定化传感器，而且还可以通过改变使用的适体轻松用于检测不同的靶标。
