Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences , Wuhan University , Wuhan 430072 , China.
ACS Sens. 2018 Jul 27;3(7):1283-1290. doi: 10.1021/acssensors.8b00129. Epub 2018 Jul 9.
In this study, two kinds of sensitive biosensors based on a multipedal DNA walker along a three-dimensional DNA functional magnet particles track for the chemiluminescent detection of streptavidin (SA) are constructed and compared. In the presence of SA, a multipedal DNA walker was constructed by a biotin-modified catalyst as a result of the terminal protection to avoid being digested by exonuclease I. Then, through a toehold-mediated strand exchange, a "leg" of a multipedal DNA walker interacted with a toehold of a catalyzed hairpin assembly (CHA)-H1 coupled with magnetic microparticles (MMPs) and opened its hairpin structure. The newly open stem in CHA-H1 was hybridized with a toehold of biotin-labeled H2. Via the strand displacement process, H2 displaced one "leg" of a multipedal DNA walker, and the other "leg" continued to interact with the neighboring H1 to initiate the next cycle. In order to solve the high background caused by the hybridization between CHA-H1 and H2 without a CHA-catalyst, the other model was designed. The principle of the other model (isothermal strand-displacement polymerase reaction (ISDPR)-DNA walker) was similar to that of the above one. After the terminal protection of SA, a "leg" of a multipedal DNA walker was triggered to open the hairpin of the ISDPR-H1 conjugated with MMPs. Then, the biotin-modified primer hybridized with the newly exposed DNA segment, triggering the polymerization reaction with the assistance of dNTPs/polymerase. As for the extension of the primer, the "leg" of a multipedal DNA walker was displaced so that the other "leg" could trigger the proximal H1 to go onto the next cycle. Due to its lower background and stronger signal, a multipedal DNA walker based on an ISDPR had a lower limit of detection for SA. The limit of detection for SA was 6.5 pM, and for expanding the application of the method, the detections of the folate receptor and thrombin were explored. In addition, these DNA walker methods were applied in complex samples successfully.
在这项研究中，构建并比较了两种基于多足 DNA walker 沿着三维 DNA 功能磁颗粒轨道的敏感生物传感器，用于化学发光检测链霉亲和素（SA）。在 SA 的存在下，通过末端保护构建了多足 DNA walker，以避免被核酸外切酶 I 消化。然后，通过链置换介导的链交换，多足 DNA walker 的“腿”与与磁性微粒（MMP）偶联的催化发夹组装（CHA）-H1 的结合位点相互作用并打开其发夹结构。CHA-H1 中的新打开茎与生物素标记的 H2 的结合位点杂交。通过链置换过程，H2 置换了多足 DNA walker 的一个“腿”，而另一个“腿”继续与相邻的 H1 相互作用以启动下一个循环。为了解决没有 CHA-催化剂时 CHA-H1 和 H2 之间杂交产生的高背景，设计了另一种模型。另一种模型（等温链置换聚合酶反应（ISDPR）-DNA walker）的原理与上述模型类似。在 SA 的末端保护后，多足 DNA walker 的一个“腿”被触发打开与 MMP 偶联的 ISDPR-H1 的发夹。然后，生物素修饰的引物与新暴露的 DNA 片段杂交，在 dNTPs/聚合酶的辅助下引发聚合反应。对于引物的延伸，多足 DNA walker 的“腿”被置换，以便另一个“腿”可以触发近端 H1 进入下一个循环。由于其背景更低且信号更强，基于 ISDPR 的多足 DNA walker 对 SA 的检测下限更低。SA 的检测下限为 6.5 pM，并为了扩展该方法的应用，探索了叶酸受体和凝血酶的检测。此外，这些 DNA walker 方法成功地应用于复杂样本。
