粟莎莎,张姝,黄健,陈曦,李艳,方立超,邓钧,莫非,郑峻松
SU Shasha,ZHANG Shu,HUANG Jian,CHEN Xi,LI Yan,FANG Lichao,DENG Jun,MO Fei,ZHENG Junsong

• 1:贵州医科大学医学检验学院
• 2:贵州医科大学附院临床检验中心
• 3:陆军军医大学药学与检验医学系

摘要(Abstract)：

目的:构建一种基于5-甲基胞嘧啶(5-mC)抗体的电化学生物传感器,用于DNA多甲基化位点的定量分析。方法:采用纳米金修饰金电极(AuNPs/Au),以自组装方式依次将单链DNA(ssDNA)、巯基己醇(MCH)和靶DNA连接到AuNPs/Au上制备DNA电化学传感器,经过电化学表征检测电极修饰效果;再以5-mC抗体特异性识别靶DNA中的甲基化位点,并以辣根过氧化物酶标记二抗(HRP-IgG)为信号示踪物,优化杂交时间及抗5-mC抗体浓度;采用差分脉冲伏安法(DPV)分别检测非甲基化与不同数量甲基化DNA的电化学信号,同时考察该方法的重复性与稳定性。结果:电化学表征结果表明电极修饰成功,优化实验条件结果显示最优杂交时间为90 min、最佳抗体浓度为10 mg/L,在该条件下DNA电化学传感器能有效区分甲基化与非甲基化DNA,并且DPV峰电流的大小与DNA甲基化位点的数量呈线性关系,具有较好的重复性与稳定性。结论:制备的DNA电化学传感器能对多个位点的DNA甲基化定量检测,有望成为临床DNA甲基化位点定量检测的有效手段。
Objective: To construct an electrochemical biosensor based on 5-methylcytosine(5-mc) antibody for quantitative analysis of DNA polymethylated sites. Methods: The single-stranded DNA(ssDNA), mercaptohexanol(MCH) and target DNA were sequentially attached on the gold nanoparticles(AuNPs) modified electrode through self-assembly. Then, the prepared electrode was incubated with anti 5-mC antibody, and reacted with the horseradish labeled secondary antibody(HRP-IgG). Differential pulse voltammetry(DPV) was used to detect the electrochemical signals of non-methylated DNA and different amounts of methylated DNA, the effects of experimental condition have been investigated. Results: Under the optimal experiment conditions, DNA electrochemical sensor can effectively distinguish methylated DNA from non-methylated DNA, and the DPV peak current is linearly related to the number of DNA methylated sites with good repeatability and stability. Conclusion: This approach can detect multiple methylation sites in the DNA sequence, which can be an effective means for the quantitative detection of DNA methylation site in clinical analysis.

关键词(KeyWords)： DNA甲基化;电化学传感器;5-甲基胞嘧啶;抗体;定量分析
DNA methylation;electrochemistry sensor;5-methylcytosine;antibody;quantitative analysis

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金(81572078)

作者(Author): 粟莎莎,张姝,黄健,陈曦,李艳,方立超,邓钧,莫非,郑峻松
SU Shasha,ZHANG Shu,HUANG Jian,CHEN Xi,LI Yan,FANG Lichao,DENG Jun,MO Fei,ZHENG Junsong

DOI: 10.19367/j.cnki.1000-2707.2019.11.004

参考文献(References)：

• [1] 王丽娟,刘家熙.表观遗传及表观遗传学概述[J].生物学教学,2017,42(1):2-4.
• [2] FARZEEN K,MEENU G.DNA methylation and application in forensic sciences [J].Forensic Science International,2015,249:255-265.
• [3] DIRK S.Function and information content of DNA methylation [J].Nature,2015,517(7534):321-326.
• [4] 陈笑,李乐赛,陈亦乐.DNA甲基化在肿瘤诊断和治疗中的研究进展[J].现代肿瘤医学,2017,25(9):1476-1480.
• [5] 候海金,张振华,闫慧明.表观遗传在肿瘤中的作用[J].中华临床医师杂志,2016,10(18):2780-2783.
• [6] COPPEDE,FABIO.Epigenetic biomarkers of colorectal cancer:focus on DNA methylation [J].Cancer Letters,2014,342(2):238-247.
• [7] ANDRESEN K,BOBERG K M,VEDELD H M,et al.Four DNA methylation biomarkers in biliary brush samples accurately identify the presence of cholangiocarcinoma [J].Hepatology,2015,61(5):1651-1659.
• [8] XU R H,WEI W,KRAWCZYK M,et al.Circulating tumour DNA methylation markers for diagnosis and prognosis of hepatocellular carcinoma [J].Nature Materials,2017,16(11):1155-1161.
• [9] JONES P A,ISSA J P J,BAYLIN S.Targeting the cancer epigenome for therapy [J].Nature Reviews Genetics,2016,17(10):630-641.
• [10]STIEGLITZ E,MAZOR T,OLSHEN A B,et al.Genome-wide DNA methylation is predictive of outcome in juvenile myelomonocytic leukemia [J].Nature Communications,2017,8(1):2127-2134.
• [11]VON K T,GERBER D,SCHALLER A,et al.Quantitative 1-step DNA methylation analysis with native genomic DNA as template [J].Clinical Chemistry,2010,56(7):1098-1106.
• [12]ASSENOV Y,MULLER,FABIAN,et al.Comprehensive analysis of DNA methylation data with RnBeads [J].Nature Methods,2014,11 (11):1138-1140.
• [13]NAIR S S,COOLEN M W,STIZAKER C,et al.Comparison of methyl-DNA immunoprecipitation (MeDIP) and methyl-CpG binding domain (MBD) protein capture for genome-wide DNA methylation analysis reveal CpG sequence coverage bias [J].Epigenetics,2011,6(1):34-44.
• [14]LAIRD,PETER W.Principles and challenges of genome-wide DNA methylation analysis [J].Nature Reviews Genetics,2010,11(3):191-203.
• [15]BAKSHI A,EKRAM M B,KIM J.Locus-specific DNA methylation analysis of retrotransposons in ES,somatic and cancer cells using High-Throughput Targeted Repeat Element Bisulfite Sequencing [J].Genomics Data,2015,3:87-89.
• [16]BECK D,SADLER-RIGGLEMAN I,SKINNER M K.Generational comparisons (F1 versus F3) of vinclozolin induced epigenetic transgenerational inheritance of sperm differential DNA methylation regions (epimutations) using MeDIP-Seq [J].Environmental Epigenetics,2017,3(3):1-12.
• [17]李博,郑磊,王前,等.电化学DNA生物传感器设计及在医学检验中的应用进展 [J].分子诊断与治疗杂志,2011,3(1):46-50.
• [18]SHEN Q,HAN L,FAN G,et al.Highly sensitive photoelectrochemical assay for DNA methyltransferase activity and inhibitor screening by exciton energy transfer coupled with enzyme cleavage biosensing strategy [J].Biosensors and Bioelectronics,2015,64:449-455.
• [19]PENG X,HU T,BAO T,et al.A label-free electrochemical biosensor for methyltransferase activity detection and inhibitor screening based on graphene quantum dot and enzyme-catalyzed reaction [J].Iournal of Electroanalytical Chemistry,2017,799:327-332.
• [20]YIN H,ZHOU Y,XU Z,et al.An electrochemical assay for DNA methylation,methyltransferase activity and inhibitor screening based on methyl binding domain protein [J].Biosensors and Bioelectronics,2013,41(1):492-497.
• [21]BAO J,GENG X,HOU C,et al.A simple and universal electrochemical assay for sensitive detection of DNA methylation,methyltransferase activity and screening of inhibitors [J].Journal of Electroanalytical Chemistry,2018,814:144-152.
• [22]LI W,LIU X,HOU T,et al.Ultrasensitive homogeneous electrochemical strategy for DNA methyltransferase activity assay based on autonomous exonuclease III-assisted isothermal cycling signal amplification[J].Biosensors and Bioelectronics,2015,70:304-309.
• [23]JING X,CAO X,WANG L,et al.DNA-AuNPs based signal amplification for highly sensitive detection of DNA methylation,methyltransferase activity and inhibitor screening[J].Biosensors and Bioelectronics,2014,58:40-47.
• [24]LU D Q,XU Q D,PANG G C,et al.A novel electrochemical immunosensor based on Au nanoparticles and horseradish peroxidase signal amplification for ultrasensitive detection of α-fetoprotein [J].Biomedical Microdevices,2018,20 (2):46-56.
• [25]GAO F,FAN T,OU S,et al.Highly efficient electrochemical sensing platform for sensitive detection DNA methylation,and methyltransferase activity based on AgNPs decorated carbon nanocubes [J].Biosensors and Bioelectronics,2018,99:201-208.
• [26]HUANG J,ZHANG S,MO F,et al.An electrochemical DNA biosensor analytic technique for identifying DNA methylation specific sites and quantify DNA methylation level [J].Biosensors and Bioelectronics,2019,127:155-160.

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