School of Mechanical Engineering, Nanjing University of Science and Technology, Nanjing 210094, China.
Biosens Bioelectron. 2013 Mar 15;41:526-31. doi: 10.1016/j.bios.2012.09.017. Epub 2012 Sep 23.
In this work, we report a novel strategy of electrochemical DNA (E-DNA) sensor based on surface initiated enzymatic polymerization (SIEP). This DNA sensor employs a capture DNA probe labeled with thiol at its 3' terminal to be immobilized at gold electrode via gold-thiol chemistry. Oligo (ethylene glycol) -terminated thiols (SH-OEGs) are then used to prepare an oligonucleotide-incorporated nonfouling surface (ONS). After the sequence-specific recognition of target DNA, terminal deoxynucleotidyl transferase (TdT) is employed to catalyze the sequential addition of deoxynucleotides (dNTPs) at the 3'-OH group of target DNA without template. During the TdT-mediated extension reaction, by using biotinlated 2'-deoxyadenosine 5'-triphosphate (biotin-dATP), biotin labels are incorporated into the SIEP-generated long single-stranded DNA (ssDNA). Specific binding of avidin-horseradish peroxidase (Av-HRP) to the biotin label leads to enzyme turnover-based signal transduction. By using this new strategy, we demonstrated the high picomolar sensitivity and a broad detection range of six orders of magnitude.
在这项工作中，我们报告了一种基于表面引发酶聚合（SIEP）的新型电化学 DNA（E-DNA）传感器策略。该 DNA 传感器采用在 3'末端标记巯基的捕获 DNA 探针，通过金-硫醇化学固定在金电极上。然后使用末端带有乙氧基（OEG）的硫醇（SH-OEGs）来制备带有寡核苷酸的非固着表面（ONS）。在目标 DNA 的序列特异性识别之后，末端脱氧核苷酸转移酶（TdT）被用于在没有模板的情况下催化目标 DNA 的 3'-OH 基团上的脱氧核苷酸（dNTPs）的顺序添加。在 TdT 介导的延伸反应中，通过使用生物素化的 2'-脱氧腺苷 5'-三磷酸（biotin-dATP），生物素标记物被掺入到 SIEP 生成的长单链 DNA（ssDNA）中。生物素标记物与亲和素辣根过氧化物酶（Av-HRP）的特异性结合导致基于酶转化的信号转导。通过使用这种新策略，我们证明了其具有皮摩尔级别的高灵敏度和六个数量级的宽检测范围。
