Electrogenerated chemiluminescence biosensing method for the discrimination of DNA hydroxymethylation and assay of the beta-glucosyltransferase activity
Electrogenerated chemiluminescence biosensing method for the discrimination of DNA hydroxymethylation and assay of the beta-glucosyltransferase activity
复制标题
电化学发光生物传感法鉴别 DNA 羟甲基化和测定 β-葡萄糖基转移酶活性
DOI：
10.1016/j.bios.2015.11.068
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发表时间：
2016
影响因子：
12.6
通讯作者：
Zheng Jianbin
中科院分区：
文献类型：
--
作者：
Sun Huiping;Ma Shangxian;Li Yan;Qi Honglan;Ning Xiaohui;Zheng Jianbin
开发了一种用于高灵敏辨别 DNA 羟甲基化和测定 β-葡萄糖基转移酶 (β-GT) 活性的电生化学发光 (ECL) 生物传感方法。采用金纳米颗粒（AuNPs）/Nafion薄膜制备ECL生物传感电极，然后将三（2, 2′-利吡啶）二氯钌（II）（Ru（bpy）32+）静电吸附到AuNPs/Nafion薄膜上，最后将二茂铁标记的羟甲基化双链DNA（ds-DNA）自组装到AuNPs表面。当引入 β-GT 和尿苷二磷酸葡萄糖 (UDP-Glu) 时，生物传感电极上 ds-DNA 5'-CCGG-3' 内的羟甲基胞嘧啶残基被糖基化。糖基化的生物传感电极经过MspI核酸内切酶处理后，未糖基化的羟甲基胞嘧啶被切割，导致猝灭剂离开电极，导致ECL信号增强。对于 ECL 生物传感方法，β-GT 的检测限极低，为 0.04 U/mL，并且对胞嘧啶、5-甲基胞嘧啶和 5-羟甲基胞嘧啶具有良好的区分度。这项工作表明，酶联反应与高灵敏度 ECL 方法的结合是区分 DNA 羟甲基化、测定 β-GT 活性以及评估 β-GT 抑制剂能力的一种有前途的策略。
An electrogenerated chemiluminescence (ECL) biosensing method for highly sensitive discrimination of DNA hydroxymethylation and assay of the β-glucosyltransferase (β-GT) activity was developed. The ECL biosensing electrode was fabricated by gold nanoparticles (AuNPs)/Nafion film, and then, tris(2, 2′-ripyridine) dichlororuthenium(II) (Ru(bpy)32+) was electrostatically adsorbed into the AuNPs/Nafion film, finally, the hydroxymethylated double-stranded DNA (ds-DNA)-tagged with ferrocene was self-assembled onto the surface of the AuNPs. When β-GT and uridine diphosphoglucose (UDP-Glu) were introduced, the hydroxymethylcytosine residues within 5′-CCGG-3′ of ds-DNA on the biosensing electrode were glucosylated. After the glucosylated biosensing electrode was treated by MspI endonuclease, the unglucosylated hydroxymethylcytosine was cleaved, leading to the quencher leaving the electrode, resulting in an increased ECL signal. For the ECL biosensing method, it showed an extremely low detection limit of 0.04 U/mL for β-GT, and offered a good discrimination toward cytosine, 5-methylcytosine, and 5-hydroxymethylcytosine. This work demonstrates that the combination of the enzyme-linkage reactions with the highly sensitive ECL method is a promising strategy for the discrimination of DNA hydroxymethylation, assay of the activity of β-GT, and evaluation of the capability of inhibitors for the β-GT.