Development of DNA methylation measurement device using nano-carbon film electrode

利用电子回旋共振（ECR）溅射形成的纳米碳薄膜，建立了DNA甲基化的直接电化学检测方法。所开发的方法是基于胞嘧啶和甲基化胞嘧啶之间的氧化电位差，而不需要任何标记。通过电化学预处理优化电极表面，我们使用该膜定量检测单个胞嘧啶甲基化，而不考虑甲基化在序列中的位置，包括视网膜母细胞瘤基因片段（24 mer）。纳米碳膜电极还允许我们通过测量不同甲基化率下甲基化的5'-胞嘧啶-磷酸鸟苷（CpG）重复寡核苷酸（60 mer）来实现DNA甲基化率的检测。为了提高检测CpG寡核苷酸碱基的灵敏度，我们成功地利用酶切酶P1将目标CpG寡核苷酸消化为相同的单核苷酸5'-NMP。为了开发DNA甲基化检测装置，采用抑制生物分子吸附的纳米碳薄膜制备了灵敏度进一步提高的微磁盘阵列电极。

We developed direct electrochemical detection of DNA methylation using nanocarbon film formed by electron cyclotron resonance(ECR) sputtering. The developed method is based on oxidation potential difference between cytosine and methylated cytosine without any labeling. By optimizing electrode surface using electro- chemical pretreatment, we used this film to quantitatively detect single cytosine methylation regardless of the methylation position in the sequence including retino- blastoma gene fragment (24 mer). The nanocarbon film electrode also allowed us to realize the detection of DNA methylation ratios by measuring methylated 5'-cytosine- phosphoguanosine (CpG) repetition oligonucleotides (60 mer) with different methylation ratio. To improve the sensitivity for detecting the bases in the CpG oligonucleotides, we successfully used an exonuclease P1 to digest the target CpG oligonucleotide to an identical mononucleotide 5'-NMP. To develop the DNA methylation detection device, the microdisk array electrode which further improves sensitivity was fabricated using nanocarbon film which suppress biomolecules adsorption.