Research For Electrochemical Biosensor Of DNA Methylation Levels Based On The MeCP2 And Bienzymatic Signal Amplification

Objective: In this study,a simple and sensitive electrochemical biosensor was fabricated for the quantitative detection of DNA methylation detection using methyl-CpG-binding protein 2(MeCP2)as methylation recognization unit,coupled with the gold nanoparticles(AuNPs)modified electrode and bienzyme-labeled antibody as the signal amplification system.Methods: 1.The fabrication of DNA electrochemical biosensor.The AuNPs was first deposited on the electrode surface,and the probe DNA,mercaptohexanol and target DNA were sequentially attached on the AuNPs modified electrode through self-assembly.The morphology of electrodeposited AuNPs and probe DNA were characterized by scanning electron microscope(SEM)and atomic force microscopy(AFM),and investigated the effect of each step by the cyclic voltammetry(CV)and electrochemical impedance spectroscopy(EIS).2.The preparation of bienzyme modified antibody.The glucose oxidase(GOD)and the horseradish peroxidase(HRP)were labeled on the anti-His tag antibody through glutaraldehyde cross-link method and sodium periodate method,and the ultraviolet-visible(UV-vis)was used to characterized the bienzyme-labeled antibody of GOD-HRP/IgG.3.The analysis of feasibility.Designed unmethyalted and one methylation site target DNA with same base sequence to fabricated DNA electrochemical biosensor.After reacting with MeCP2 and GOD-HRP/IgG,the electrode was detected by differential pulse voltammetry in the buffer solution containing glucose and hydroquinone.The feasibility of this method was carried out by comparing the detection results of methylated and unmethylated target DNA and investigated the catalyzed effect of our designed amplification strategy.4.The optimization of experiment parameters.We optimized the experimental conditions including hybridization time,MeCP2 concentration,antibodies and glucose concentration.5.The quantitative analysis of DNA methylation sites: Designed the same sequence target DNA with various numbers of methylation sites and analyzed the current signal by the prepared electrochemical biosensor,and established the relationship between the numbers of methylation site and the electrochemical signal.6.Evaluate the electrochemical biosensor performance,including sensitivity,repeatability and stability.Results: 1.The SEM showed the AuNPs were spherical and well-distributed;The AFM image showed that the size of AuNPs is consistent with the SEM image and a regular distribution of the DNA could be observed on the AuNPs/Au surface;CV and EIS together indicated that each component was successfully modified on the electrode surface.2.The shifts of the GOD-HRP/IgG absorption peak to the maximum peak of GOD and HRP in the UV-vis image,indicating that the successful modification of bienzyme.3.The fesibility test results showed that this method could effectively distinguish between methylated and unmethylated sequence.At the same time,the electrochemical response of the bienzyme strategy is considerably higher than the mono-enzyme labeling system,suggesting that the good amplification effect of the proposed bienzyme system.4.On the basis of experimental results,the opimial hybridization time was 90 min,the MeCP2 concentration was 200 mg/L,the anti-His tag antibody concentration was 10 mg/L and the glucose concentration was 0.5 mmol/L.5.The electrochemical signal of different amounts of methylation sites showed a good linear relationship to the numbers of methylation sites from one methylation site to five methylation sites,and the linear regression equation was I(?A)=7.550 N + 1.328 with the correlation coefficient r was 0.984.6.This method could detect the methylated target DNA as low as 0.1 fmol/L with a broad linear range,and exhibited high sensitivity,good repeatability and stability.Conclusion: We successfully constructed the electrochemical biosensor for the use of DNA methylation detection.It could distinguish multiple methylation sites in the DNA sequence,which could be an effective means for the quantitative detection of DNA methylation in clinical analysis.