Electrogenerated Chemiluminescence Biosensing Method For The Determination Of DNA Methyltransferase Activity Based On Graphene Oxide

DNA methylation is an important epigenetic modification, is achieved by DNA methyltransferase (MTase) catalyzing covalent addition of a methyl group to cytosine or adenine in the presence of S-adenosylmethionine (SAM). Aberrant DNA methylation induced by microbe remodels the structure of DNA, influences the interaction between DNA and protein, and alters gene expression, which may result in tumor occurrence and tumor growth. In addition, the inhibition of MTases may provide a broad spectrum of antimicrobial applications. Therefore, detection of DNA methylation and assay of MTase activities have received more and more research interests. Traditional methods for DNA MTase activity assay rely on radioisotope labeled substrates, various polymerase chain reaction (PCR)-based techniques, gel electrophoresis and high-performance liquid chromatography (HPLC). Such methods are time-intensive, DNA-consuming, requiring laborious treatment and especially isotope labeling.Electrogenerated chemiluminescence is an analytical technique that combined with chemiluminescence method and electrochemical means. The technology integrated with luminescence analysis the advantages of high sensitivity and electrochemical potential controllable, has become one of workers is very interested in research field. The aim of this thesis is to develop two kinds ECL DNA methylation biosensor method based on graphene oxide. Research work in this thesis is financially supported by the National Natural Science Foundation of China(Grant No.21005061). The main contents are as follows:In Chapter1, we generated review from the principles, brief history of electrogenerated chemiluminescence. Summarizing the biological significance, the analysis method of the DNA methylation in disease diagnosis and drug development, and introduces the application of graphene in DNA biosensor. Finally, this paper expounds the research purpose, content and significance of this paper.In Chapter2, A novel electrogenerated chemiluminescence biosensing method for methyltransferase activity based on graphene oxide. The ECL biosensing method for highly sensitive detection of DNA methylation and assay of the methyltransferase (MTase) activity was developed using Dpn I endonuclease and methylation-responsive hairpin-capture DNA probe to improve selectivity and employing signal amplification of graphene oxide (GO) to enhance the assay sensitivity. The ECL biosensing electrode was fabricated by self-assembling a design of5’-thiol modified methylation-responsive hairpin-capture DNA probe on the surface of a gold electrode. When Dam MTase and S-adenosylmethionine were introduced, all adenines in the symmetric tetranucleotide5’-G-A-T-C-3’of hairpin-capture DNA probe on the biosensing electrode were methylated. After the methylated biosensing electrode was treated by Dpn I endonuclease, the methylated adenines were cleaved, methylation-induced scission of hairpin-capture DNA probe would displace the hairpin section and remain the "capture DNA probe" section on the gold electrode. Subsequently, the remained "capture DNA probe" on the gold electrode can hybridize with the5’-amino modified DNA probe. Finally, tris(1,10-phenanthroline) ruthenium (Ru(phen)32+)-assembled GO composites were conjugated to the electrode surface via EDC-NHS coupling, a strong ECL response was electrochemically generated. The increased ECL intensity was proportion to Dam MTase activity in the range from0.05U/mL to40U/mL with a detection limit of0.02U/mL. The present work demonstrates that the combination of the enzyme-linkage and GO as a platform for signal probe is a great promising approach for methyltransferase activity and evaluation of the capability of inhibitors for the methyltransferase.In Chapter3, Label-free electrogenerated chemiluminescence biosensing method for the determination of DNA methyltransferase activity incorporating amplification of signal reagent-assembled graphene oxide. The study using graphene oxide strong noncovalent binding of single-stranded DNA to construct biosensing for methyltransferase activity. An exponential curve is obtained between the ECLintensity and concentration of Dam from0to50U/mL, notably, the ECL intensity exhibits a log-linear relationship with the concentration of Dam from0.02to10U/mL. The detection limit is calculated to be0.01U/mL, R=0.9974. The method can screen associated with DNA methylation enzyme inhibitors.