Direct electrochemical detection of DNA methylation for retinoblastoma and CpG fragments using a nanocarbon film.
Direct electrochemical detection of DNA methylation for retinoblastoma and CpG fragments using a nanocarbon film.
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DOI：
10.1016/j.ab.2010.06.004
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发表时间：
2010-10
影响因子：
2.9
通讯作者：
Keisuke Goto;Dai Kato;Naoyuki Sekioka;A. Ueda;S. Hirono;O. Niwa
中科院分区：
文献类型：
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作者：
Keisuke Goto;Dai Kato;Naoyuki Sekioka;A. Ueda;S. Hirono;O. Niwa
我们描述了通过使用纳米碳膜电极在相对长的序列中直接电化学检测DNA甲基化。该薄膜采用电子回旋共振溅射法制备，具有纳米晶sp 2和sp 3混合键结构。我们的甲基化检测技术可以测量5-甲基胞嘧啶和胞嘧啶的氧化电流之间的差异，而无需亚硫酸氢盐反应或标记。这是可能的，因为该膜电极具有宽的电势窗口，同时保持通过直接氧化定量检测两种碱所需的高电极活性。通过电化学预处理优化电极表面条件，我们使用该膜定量检测单个胞嘧啶甲基化，而不管包括视网膜母细胞瘤基因片段（124聚体）的序列中的甲基化位置如何。这可能是由于该膜电极的高稳定性，我们通过控制表面亲水性来抑制结垢，并通过保持电极对所有碱的活性来实现。寡核苷酸测量的pH优化也可用于分别区分两种碱基。在优化的条件下，该膜电极可通过检测不同甲基化率的5 '-胞嘧啶-磷酸鸟苷（CpG）重复寡核苷酸（60 mer），实现DNA甲基化率的定量检测。
We describe the direct electrochemical detection of DNA methylation in relatively long sequences by using a nanocarbon film electrode. The film was formed by employing the electron cyclotron resonance sputtering method and had a nanocrystalline sp2and sp3mixed bond structure. Our methylation detection technique measures the differences between the oxidation currents of both 5-methylcytosine and cytosine without a bisulfite reaction or labeling. This was possible because this film electrode has a wide potential window while maintaining the high electrode activity needed to quantitatively detect both bases by direct oxidation. By optimizing the electrode surface conditions using electrochemical pretreatment, we used this film to quantitatively detect single cytosine methylation regardless of the methylation position in the sequence including retinoblastoma gene fragments (∼24mers). This was probably due to the high stability of this film electrode, which we achieved by controlling the surface hydrophilicity to suppress the fouling, and by maintaining electrode activity against all the bases. The pH optimization of the oligonucleotide measurements was also useful for distinguishing both bases separately. Under the optimized conditions, this film electrode allowed us to realize the quantitative detection of DNA methylation ratios solely by measuring methylated 5′-cytosine-phosphoguanosine (CpG) repetition oligonucleotides (60mers) with different methylation ratios.