Jiangsu Key Laboratory of Biomedical Materials, Jiangsu Collaborative Innovation Center of Biomedical Functional Materials, National and Local Joint Engineering Research Center of Biomedical Functional Materials, Jiangsu Key Laboratory of New Power Batteries, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210023, P. R. China.
Analyst. 2018 Apr 30;143(9):2051-2056. doi: 10.1039/c7an02049j.
The levels of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in DNA (5-mC-DNA and 5-hmC-DNA) are strongly correlated with cancer occurrence and development. The ability to distinguish and quantitatively detect them is important for cancer research. We have developed a hybridization chain reaction (HCR)-based electrochemical assay for the signal-amplified detection of the relative contents of 5-mC-DNA and 5-hmC-DNA. The DNA duplexes (containing 5-mC-DNA and 5-hmC-DNA with different percentages) were modified on a gold electrode. Electroactive [Ru(NH3)6]3+ (RuHex) was used as the signal reporter, because it binds to DNA double strands. The duplexes can be cleaved by MspJI endonuclease without HCR, and result in a small peak current. However, the cleavage can be blocked after the 5-hmC-DNA duplex is converted to β-glucosyl-5-hydroxymethylcytosine (β-glu-5-hmC) by T4 β-glucosyltransferase (T4 β-GT), and with the addition of helper DNA, a long double-helix DNA was formed through HCR. A significantly amplified peak current can be achieved due to the adsorption of numerous RuHex. The electrochemical signal of RuHex is correlated to the content of 5-hmC-DNA. Upon fixing the total quantity of 5-mC-DNA and 5-hmC-DNA on the electrode, the signals increase with the increase in the percentage of 5-hmC-DNA for the HCR. With this assay, a detection limit of 0.05% for 5-hmC-DNA was achieved.
DNA（5-甲基胞嘧啶-DNA 和 5-羟甲基胞嘧啶-DNA）中的 5-甲基胞嘧啶（5-mC）和 5-羟甲基胞嘧啶（5-hmC）水平与癌症的发生和发展密切相关。区分和定量检测它们的能力对于癌症研究非常重要。我们开发了一种基于杂交链反应（HCR）的电化学分析方法，用于信号放大检测 5-mC-DNA 和 5-hmC-DNA 的相对含量。将包含不同百分比 5-mC-DNA 和 5-hmC-DNA 的 DNA 双链体修饰在金电极上。电活性[Ru(NH3)6]3+（RuHex）被用作信号报告分子，因为它与 DNA 双链结合。双链体在没有 HCR 的情况下可以被 MspJI 内切酶切割，导致小的峰电流。然而，在 5-hmC-DNA 双链体被 T4 β-葡萄糖基转移酶（T4 β-GT）转化为β-葡萄糖基-5-羟甲基胞嘧啶（β-glu-5-hmC）后，切割可以被阻断，并且在添加辅助 DNA 后，通过 HCR 形成长双链体 DNA。由于大量 RuHex 的吸附，可以实现显著放大的峰电流。RuHex 的电化学信号与 5-hmC-DNA 的含量相关。在将 5-mC-DNA 和 5-hmC-DNA 的总量固定在电极上的情况下，随着 HCR 中 5-hmC-DNA 百分比的增加，信号增加。使用该测定法，5-hmC-DNA 的检测限达到 0.05%。
