School of Mechanical Engineering, Nanjing University of Science and Technology, Nanjing 210094, China.
School of Environmental and Biological and Engineering, Nanjing University of Science and Technology, Nanjing 210094, China.
Biosens Bioelectron. 2014 May 15;55:231-6. doi: 10.1016/j.bios.2013.11.077. Epub 2013 Dec 14.
In this work, two electrochemical DNA sensors was developed based on the target induced structural switching of stem-loop probe (SLP) and surface initiated enzymatic polymerization (SIEP). Both of the electrochemical DNA sensors employed SLPs with the same sequence. However, one had a thiol label at its 3' terminal (the probe was named 3-SLP and the sensor was named 3-SLP-SENS) and the other at its 5' terminal (the probe was named 5-SLP and the sensor was named 5-SLP-SENS). In the initial state of the sensors, both of the probes adopted the stem-loop structure, which shielded the unlabeled terminals of capture probes from being approached. When the loop regions of the capture probes hybridized with the target DNA the conformation of the SLPs was changed to a rigid double-strand, as a result, the 5-SLP released a 3'-OH terminal for SIEP which could be catalyzed by terminal deoxynucleotidyl transferase (TdT). And the 3-SLP released a 5' phosphate terminal which is not suit for SIEP. Thus a signal probe was employed to hybridize with the 5 terminal of 3-SLP and provide a 3'-OH. Both of the sensors were then submitted to the TdT-mediated SIEP. By using biotinylated 2'-deoxyadenosine 5'-triphosphate (biotin-dATP), biotin labels are incorporated into the SIEP-generated long single-stranded DNA. Then avidin-horseradish peroxidases (Av-HRPs) were employed for specific binding to the biotin labels to produce electrochemical signals. The detection performances of two electrochemical DNA sensors were investigated and compared. It was demonstrated that though the 3-SLP-SENS employed extra signal probes, the background current was lower leading to a better detection limit. By taking advantage of SLP and SIEP, this 3-SLP-SENS has been able to detect as low as 0.1pM DNA targets with excellent differentiation ability for even single mismatches.
在这项工作中，基于茎环探针（SLP）的目标诱导结构切换和表面引发酶聚合（SIEP），开发了两种电化学 DNA 传感器。这两种电化学 DNA 传感器都使用了相同序列的 SLP。然而，其中一个在其 3'端带有硫醇标记（探针命名为 3-SLP，传感器命名为 3-SLP-SENS），另一个在其 5'端带有硫醇标记（探针命名为 5-SLP，传感器命名为 5-SLP-SENS）。在传感器的初始状态下，两个探针都采用了茎环结构，这屏蔽了未标记的捕获探针的末端，使其无法接近。当捕获探针的环区与目标 DNA 杂交时，SLP 的构象发生变化，形成刚性双链，从而 5-SLP 释放出 3'-OH 末端，用于可被末端脱氧核苷酸转移酶（TdT）催化的 SIEP。而 3-SLP 释放出 5'磷酸末端，不适合 SIEP。因此，使用信号探针与 3-SLP 的 5 端杂交，并提供 3'-OH。然后，将两个传感器提交给 TdT 介导的 SIEP。通过使用生物素化 2'-脱氧腺苷 5'-三磷酸（biotin-dATP），生物素标记被掺入 SIEP 生成的长单链 DNA 中。然后，使用亲和素辣根过氧化物酶（Av-HRPs）特异性结合生物素标记物，产生电化学信号。研究并比较了两种电化学 DNA 传感器的检测性能。结果表明，虽然 3-SLP-SENS 使用了额外的信号探针，但背景电流较低，因此具有更好的检测限。通过利用 SLP 和 SIEP，这种 3-SLP-SENS 已经能够检测低至 0.1pM 的 DNA 靶标，并且具有出色的区分能力，甚至可以区分单个错配。
