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:探讨甲基化CpG结合蛋白2(MeCP2)和双酶信号放大电化学免疫传感器用于DNA甲基化定量检测的效果。方法:固定在纳米金(AuNPs)修饰电极表面的探针与靶DNA杂交后,将电极于37℃下与MeCP2蛋白溶液孵育,再与葡萄糖氧化酶(GOD)和辣根过氧化物酶(HRP)共同标记的MeCP2-His标签抗体(GOD-HRP/anti-His tag)反应;于含葡萄糖和对苯二酚的检测液中测试其电化学信号,建立电信号大小与DNA甲基化浓度之间的关系,确定该传感器的检测限,考察实验所设计的双酶信号放大策略的放大效果。结果:在1.0×10~(-14)～1.0×10~(-7) mol/L范围内,电信号大小与DNA甲基化浓度的对数呈线性关系,回归方程为I(峰电流值,μA)=1.038 lg C(DNA甲基化的浓度,mol/L)+16.598,相关系数r为0.993,检出限为0.1 fmol/L;双酶标记体系的DPV峰电流最高(9.105μA),单独HRP标记体系的DPV峰电流为1.99μA,单独GOD标记体系的电信号极其微弱、仅为0.969μA;重复性实验得RSD为4.6%;将传感器置于pH 7.4的PBS中,4℃条件下放置28 d后,响应电流大小为最初的93.7%,表明该传感器具有较好的稳定性;与单酶标记体系相比,双酶标记体系产生的电化学信号更强。结论:采用基于MeCP2和双酶信号放大电化学免疫传感器具有较高的灵敏度,能实现痕量DNA甲基化的检测。
Objective: To investigate the effect of bi-enzymatic signal amplification approach on the quantitative detection of DNA methylation of methylated CpG binding protein 2(MeCP2) using electrochemical biosensor. Methods: The probe immobilized on the electrode which was covered with nano-gold(AuNPs) was hybridized with the target DNA, and then the electrode was incubated with the solution containing MeCP2 protein at 37 ℃, followed by His tag antibody conjugated with glucose oxidase and horseradish peroxidase(GOD-HRP) to form MeCP2-His tag~(GOD-HRP). The electrochemical signal was measured in a buffer containing glucose and hydroquinone. Based on electrochemical signal, the correlation between electrical signal size and DNA methylation concentration was established, and the detection limit of the electrochemical biosensor and the amplification effect of the GOD-HRP strategy were determined. Results: In the range of 1.0×10~(-14) mol/L to 1.0×10~(-7) mol/L of DNA methylation concentration, the electrical signal size was linear with the logarithm of DNA methylation concentration. The regression equation was I=1.038 lg C+16.598,(I represents peak current value(μA), C represents the concentration of DNA methylation(mol/L), and correlation coefficient r is 0.993. The detection limit is 0.1 fmol/L. GOD-HRP led to the highest DPV peak current, reaching 9.105 μA, while HRP alone made DPV peak current around 1.99 μA, and GOD alone around 0.969 μA. Using simultaneously prepared five electrochemical biosensors to detect 0.1 μmol/L DNA methylation, RSD was 4.6%. When these five electrochemical immunosensors sensor were placed in PBS of pH 7.4, the response current was very stable, and the current was 93.7% of the initial current after 28 days, suggesting that the prepared electrochemical biosensors had good repeatability and stability. Conclusion: The bi-enzymatic labeling system produces a stronger electrochemical signal than the single enzyme labeling system, and improves the sensitivity of the electrochemical biosensor in detecting DNA methylation.

关键词(KeyWords)： DNA甲基化;电化学;生物传感技术;甲基化CpG结合蛋白2;双酶催化;免疫分析
DNA methylation;electrochemistry;biosensing technology;methylated CpG binding protein 2;bi-enzyme catalysis;immunoassay

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.1000-2707.2019.09.003

参考文献(References)：

• [1] SUZUKI M M,BIRD A.DNA methylation landscapes:provocative insights from epigenomics [J].Nature Reviews Genetics,2008,9(6):465- 476.
• [2] SMITH Z D,MEISSNER A.DNA methylation:roles in mammalian development [J].Nature Reviews Genetics,2013,14(3):204-220.
• [3] 冯敏,曾景棠,谭益清,等.表观遗传学与肿瘤及其临床应用[J] .国际遗传学杂志,2016,39(5):264-268.
• [4] LAI H C,WANG Y C,YU M H,et al.DNA methylation as a biomarker for the detection of hidden carcinoma in endometrial atypical hyperplasia [J].Gynecologic Oncology,2014,135(3):552 -559.
• [5] BAEK S J,YANG S,KANG T W,et al.MENT:methylation and expression database of normal and tumor tissues [J].Gene,2013,518(1):194 - 200.
• [6] WAN Y,WANG Y,LUO J,et al.Bisulfite modification of immobilized DNAs for methylation detection [J].Biosensors and Bioelectronics,2007,22(11):2415-2421.
• [7] 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.
• [8] LOPEZ T A,YANEZ B E,WROBEL K,et al.Selective derivatization of cytosine and methylcytosine moieties with 2-Bromoacetophenone for submicrogram DNA methylation analysis by reversed phase HPLC with spectrofluorimetric detection [J].Analytical Chemistry,2011,83(20):7999-8005.
• [9] 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.
• [10]HEAD J A,MITTAL K,BASU N.Application of the luminometric methylation assay to ecological species:tissue quality requirements and a survey of DNA methylation levels in animals [J].Molecular Ecology Resources,2014,14(5):843-952.
• [11]HUANG W,QI C B,LV S W,et al.Correction to determination of DNA and RNA methylationin circulating tumor cells by mass spectrometry [J].Analytical Chemistry,2016,88(8):4581.
• [12]SAGE A T,BESANT J D,LAM B,et al.Ultrasensitive electrochemical biomolecular detection using nanostructured microelectrodes [J].Accounts of Chemical Research,2014,47(8):2417-2425.
• [13]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.
• [14]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.
• [15]WU W,WANG L,LIU Y,et al.Site-specific DNA methylation detection based on enzyme-linked immunosorbent assay using recombinant methy-CpG binding protein [J].Clinical Chemistry & Laboratory Medicine,2018,56(7):164-166.
• [16]AVRAMEAS S J.Coupling of enzymes to proteins with glutaraldehyde.Use of the conjugates for the detection of antigens and antibodies [J].Immunochemistry,1969,6(1):43-52.
• [17]SAIKANT R,SHILPA R,VIJAYAN A L,et al.Effect of lyophilization on HRP-antibody conjugation:an enhanced antibody labeling technology [J].BMC Research Notes,2018,11(1):596-601.
• [18]POVEDANO E,VARGAS E,MONTIEL,et al.Electrochemical affinity biosensors for fast detection of gene-specific methylations with no need for bisulfite and amplification treatments [J].Scientific Reports,2018,8(1):6418-6423.
• [19]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.
• [20]DU Q,LUU P L,STIRZAKER C,et al.Methyl-CpG-binding domain proteins:readers of the epigenome [J].Epigenomics,2015,7(6):1051-1073.
• [21]YIN H,ZHOU Y,XU Z,et al.Ultrasensitive electrochemical immunoassay for DNA methyltransferase activity and inhibitor screening based on methyl binding domain protein of MeCP2 and enzymatic signal amplification [J].Biosensors and Bioelectronics,2013,49(35):39-45.
• [22]LIU P,LIU M,YIN H,et al.Electrochemical biosensor for DNA methyltransferase detection based on DpnI digestion triggering the formation of G-quadruplex DNAzymes [J].Sensors and Actuators B:Chemical,2015,220:101-106.
• [23]KOO K M,WEE E J H,RAUF S,et al.Microdevices for detecting locus-specific DNA methylation at CpG resolution [J].Biosensors and Bioelectronics,2014,56:278-285.

文章评论(Comment)：