Ultrasensitive electrochemical immunoassay for DNA methyltransferase activity and inhibitor screening based on methyl binding domain protein of MeCP2 and enzymatic signal amplification.
Biosens Bioelectron
; 49: 39-45, 2013 Nov 15.
Article
in En
| MEDLINE
| ID: mdl-23708816
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.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Biosensing Techniques
/
DNA-Cytosine Methylases
/
DNA Methylation
Type of study:
Diagnostic_studies
/
Evaluation_studies
/
Screening_studies
Limits:
Humans
Language:
En
Journal:
Biosens Bioelectron
Journal subject:
BIOTECNOLOGIA
Year:
2013
Document type:
Article
Country of publication:
United kingdom