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

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(9.105 μA),单独HRP标记体系的DPV峰电流为1.99 μA,单独GOD标记体系的电信号极其微弱、仅为0.969 μA;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.