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High-Sensitivity and High-Efficiency Detection of DNA Hydroxymethylation in Genomic DNA by Multiplexing Electrochemical Biosensing
Analytical Chemistry ( IF 6.7 ) Pub Date : 2016-03-15 00:00:00 , DOI: 10.1021/acs.analchem.6b00230 Shixing Chen 1 , Yanzhi Dou 1 , Zhihan Zhao 1, 2 , Fuwu Li 1 , Jing Su 1 , Chunhai Fan 1 , Shiping Song 1
Analytical Chemistry ( IF 6.7 ) Pub Date : 2016-03-15 00:00:00 , DOI: 10.1021/acs.analchem.6b00230 Shixing Chen 1 , Yanzhi Dou 1 , Zhihan Zhao 1, 2 , Fuwu Li 1 , Jing Su 1 , Chunhai Fan 1 , Shiping Song 1
Affiliation
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DNA hydroxymethylation (5-hmC) is a kind of new epigenetic modification, which plays key roles in DNA demethylation, genomic reprogramming, and the gene expression in mammals. For further exploring the functions of 5-hmC, it is necessary to develop sensitive and selective methods for detecting 5-hmC. Herein, we developed a novel multiplexing electrochemical (MEC) biosensor for 5-hmC detection based on the glycosylation modification of 5-hmC and enzymatic signal amplification. The 5-hmC was first glycosylated by T4 β-glucosyltransferase and then oxidated by sodium periodate. The resulting glucosyl-modified 5-hmC (5-ghmC) was incubated with ARP-biotin and was bound to avidin-HRP. The 5-hmC can be detected at the subnanogram level. Finally, we performed 5-hmC detection for mouse tissue samples and cancer cell lines. The limit of detection of the MEC biosensor is 20 times lower than that of commercial kits based on optical meaurement. Also, the biosensor presented high detection specificity because the chemical reaction for 5-hmC modification can not happen at any other unhydroxymethylated nucleic acid bases. Importantly, benefited by its multiplexing capacity, the developed MEC biosensor showed excellent high efficiency, which was time-saving and cost less.
中文翻译：
多重电化学生物传感技术高灵敏度，高效率检测基因组DNA中的DNA羟甲基化
DNA羟甲基化（5-hmC）是一种新的表观遗传修饰，在哺乳动物的DNA脱甲基，基因组重编程和基因表达中起着关键作用。为了进一步探索5-hmC的功能，有必要开发灵敏且选择性的检测5-hmC的方法。本文中，我们基于5-hmC的糖基化修饰和酶促信号放大，开发了一种用于5-hmC检测的新型多路电化学（MEC）生物传感器。5-hmC首先被T4β-葡萄糖基转移酶糖基化，然后被高碘酸钠氧化。将所得的经糖基修饰的5-hmC（5-ghmC）与ARP生物素一起孵育，并与抗生物素蛋白-HRP结合。可以在亚毫微克水平检测到5hmC。最后，我们对小鼠组织样本和癌细胞系进行了5-hmC检测。基于光学测量，MEC生物传感器的检出限比市售试剂盒的检出限低20倍。而且，由于5-hmC修饰的化学反应在任何其他未羟甲基化的核酸碱基上均不会发生，因此该生物传感器具有很高的检测特异性。重要的是，得益于其多路复用能力，开发的MEC生物传感器显示出了极高的效率，既节省时间又降低了成本。
更新日期：2016-03-15
中文翻译：
多重电化学生物传感技术高灵敏度，高效率检测基因组DNA中的DNA羟甲基化
DNA羟甲基化（5-hmC）是一种新的表观遗传修饰，在哺乳动物的DNA脱甲基，基因组重编程和基因表达中起着关键作用。为了进一步探索5-hmC的功能，有必要开发灵敏且选择性的检测5-hmC的方法。本文中，我们基于5-hmC的糖基化修饰和酶促信号放大，开发了一种用于5-hmC检测的新型多路电化学（MEC）生物传感器。5-hmC首先被T4β-葡萄糖基转移酶糖基化，然后被高碘酸钠氧化。将所得的经糖基修饰的5-hmC（5-ghmC）与ARP生物素一起孵育，并与抗生物素蛋白-HRP结合。可以在亚毫微克水平检测到5hmC。最后，我们对小鼠组织样本和癌细胞系进行了5-hmC检测。基于光学测量，MEC生物传感器的检出限比市售试剂盒的检出限低20倍。而且，由于5-hmC修饰的化学反应在任何其他未羟甲基化的核酸碱基上均不会发生，因此该生物传感器具有很高的检测特异性。重要的是，得益于其多路复用能力，开发的MEC生物传感器显示出了极高的效率，既节省时间又降低了成本。
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