贵州医科大学学报

2019, v.44;No.228(09) 1005-1010+1015

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基于MeCP2蛋白和双酶信号放大的DNA甲基化电化学免疫分析
Electrochemical Immunoassay for DNA Methylation Analysis Based on MeCP2 and Bienzymatic Signal Amplification

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;School of Clinical Laboratory Science, Guizhou Medical University;Department of Clinical Laboratory, the Affiliated Hospital of Guizhou Medical University;College of Laboratory Science, Army Medical University;

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

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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;School of Clinical Laboratory Science, Guizhou Medical University;Department of Clinical Laboratory, the Affiliated Hospital of Guizhou Medical University;College of Laboratory Science, Army Medical University;

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DOI: 10.19367/j.cnki.1000-2707.2019.09.003

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