title: Ultrasensitive electrochemical immunoassay for DNA methyltransferase activity and inhibitor screening based on methyl binding domain protein of MeCP2 and enzymatic signal amplification.
abstract: In this work, we fabricated a novel electrochemical immunosensor for detection of DNA methylation, analysis of DNA MTase activity and screening of MTase inhibitor. The immunosensor was on the basis of methyl binding domain protein of MeCP2 as DNA CpG methylation recognization unit, anti-His tag antibody as "immuno-bridge" and horseradish peroxidase labeled immuneglobulin G functionalized gold nanoparticles (AuNPs-IgG-HRP) as signal amplification unit. In the presence of M. SssI MTase, the symmetrical sequence of 5'-CCGG-3' was methylated and then recognized by MeCP2 protein. By the immunoreactions, anti-His tag antibody and AuNPs-IgG-HRP was captured on the electrode surface successively. Under the catalysis effect of HRP towards hydroquinone oxidized by H2O2, the electrochemical reduction signal of benzoquinone was used to analyze M. SssI MTase activity. The electrochemical reduction signal demonstrated a wide linear relationship with M. SssI concentration ranging from 0.05 unit/mL to 90 unit/mL, achieving a detection limit of 0.017 unit/mL (S/N=3). The most important advantages of this method were its high sensitivity and good selectivity, which enabled the detection of even one-base mismatched sequence. In addition, we also verified that the developed method could be applied for screening the inhibitors of DNA MTase and for developing new anticancer drugs.
authors.affiliation: College of Chemistry and Material Science, Shandong Agricultural University, 271018, Taian, Shandong, PR China.
authors.forename: Huanshun
authors.initials: H
authors.lastname: Yin
authors.forename: Yunlei
authors.initials: Y
authors.lastname: Zhou
authors.forename: Zhenning
authors.initials: Z
authors.lastname: Xu
authors.forename: Mo
authors.initials: M
authors.lastname: Wang
authors.forename: Shiyun
authors.initials: S
authors.lastname: Ai
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D019175:DNA Methylation; D015257:DNA-Cytosine Methylases / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D055664:Electrochemical Techniques / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006735:Horseradish Peroxidase / Q000737:chemistry; D006801:Humans; D007118:Immunoassay / Q000379:methods; D007074:Immunoglobulin G / Q000737:chemistry; D057230:Limit of Detection; D051783:Methyl-CpG-Binding Protein 2 / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D007074:Immunoglobulin G; D051783:Methyl-CpG-Binding Protein 2; D006735:Horseradish Peroxidase; C081573:DNA modification methylase SssI; D015257:DNA-Cytosine Methylases
keywords: Anti-histidine tag antibody; DNA methylation; Electrochemical detection; Horseradish peroxidase labeled immuneglobulin G; Methyl binding domain protein