Key Laboratory of Electroanalytical Chemistry of Shaanxi Province, Institute of Analytical Science, Northwest University , Xi'an, 710069, People's Republic of China.
Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University , Xi'an, 710062, People's Republic of China.
Anal Chem. 2016 Oct 18;88(20):9934-9940. doi: 10.1021/acs.analchem.6b01265. Epub 2016 Sep 26.
DNA methylation is used to dynamically reprogram cells in the course of early embryonic development in mammals. 5-Hydroxymethylcytosine in DNA (5-hmC-DNA) plays essential roles in the demethylation processes. 5-Methylcytosine in DNA (5-mC-DNA) is oxidized to 5-hmC-DNA by 10-11 translocation proteins, which are relatively high abundance in embryonic stem cells and neurons. A new method was developed herein to quantify 5-hmC-DNA based on selective electrogenerated chemiluminescence (ECL) labeling with the specific oxidation of 5-hmC to 5-fC by KRuO. A thiolated capture probe (ssDNA, 35-mer) for the target DNA containing 5-hmC was self-assembled on a gold surface. The 5-hmC in the target DNA was selectively transformed to 5-fC via oxidation by KRuO and then subsequently labeled with N-(4-aminobutyl)-N-ethylisoluminol (ABEI). The ABEI-labeled target DNA was hybridized with the capture probe on the electrode, resulting in a strong ECL emission. An extremely low detection limit of 1.4 × 10 M was achieved for the detection of 5-hmC-DNA. In addition, this ECL method was useful for the quantification of 5-hmC in serum samples. This work demonstrates that selective 5-hmC oxidation in combination with an inherently sensitive ECL method is a promising tactic for 5-hmC biosensing.
DNA 甲基化被用来在哺乳动物早期胚胎发育过程中动态地重新编程细胞。DNA 中的 5-羟甲基胞嘧啶（5-hmC-DNA）在去甲基化过程中发挥着重要作用。10-11 易位蛋白将 DNA 中的 5-甲基胞嘧啶（5-mC-DNA）氧化为 5-hmC-DNA，这些蛋白在胚胎干细胞和神经元中含量较高。本文开发了一种新的方法，基于选择性电生成化学发光（ECL）标记，通过 KRuO 将 5-hmC 特异性氧化为 5-fC，从而定量检测 5-hmC-DNA。含有 5-hmC 的目标 DNA 的巯基化捕获探针（ssDNA，35 个碱基）自组装在金表面上。目标 DNA 中的 5-hmC 通过 KRuO 的氧化被选择性转化为 5-fC，然后用 N-(4-氨基丁基)-N-乙基异鲁米诺（ABEI）进行标记。ABEI 标记的目标 DNA 与电极上的捕获探针杂交，产生强烈的 ECL 发射。该方法对 5-hmC-DNA 的检测限低至 1.4×10 M。此外，该 ECL 方法可用于血清样本中 5-hmC 的定量检测。这项工作表明，选择性 5-hmC 氧化与固有灵敏的 ECL 方法相结合，是一种很有前途的 5-hmC 生物传感策略。
