Study On The Detection Of Human Methyltransferase (DNMT1) Activity By Electrochemiluminescence

Three different kinds of DNA MTases have been identified in mammals. They were named as DNMT1, DNMT3A and DNMT3B. Among them, DNMT1 is the most abundant DNA MTase in mammals, and considered as the key methyltransferase.Studies show that abnormal of the activity of DNA methyltransferase associates with many types of genetic defects and the effect of malignant tumors. And alterations of the DNMT activity may lead to aberrant gene-specific methylation. Both hypomethylation and hypermethylation have been identified in multifarious types of cancer, such as breast cancer, ovarian cancer, lung cancer, colon cancer, urological cancer and leukemia. The 5-carbon position of the pyrimidine ring which occured DNA methylation of cytosine in the CpG site in mammal genome. And DNA MTases catalyzes the methylation by transferring a methyl group from S-adenosyl-L-methionine to cytosine at the CpG site. Recently, some researches also suggest that the DNA methyltransferase is one kind of potential biomarker and drug targets of different cancers in the process of diagnosis, because the aberrant methylation progress often occurs earlier than other symptoms of malignant tumors.Therefore, it is significant to develop a simple and sensitive detection strategy used for detecting the activity of DNA MTase in real sample. Electrochemiluminescence which combines electrochemistry and chemiluminiscence is a promising method in analytical applications due to its wide dynamic range, low background and highly sensitivity. The aim of thesis is to develop ECL approaches to detect methylation level in early diagnosis, and to provide rapid sensitive method for the early diagnosis and treatment of diseases. We built electrochemiluminescence sensors to detecting the activity of DNA methyltransferase (DNMT1) in cancer cells. The main results are follows:1. We reports a sensitive electrochemiluminescence assay based on hybridization chain reaction coupled with G-quadruplex/hemin DNAzyme for detecting the DNMT1 methyltransferase activity from cancer cells. In this assay, the MPA modified Eu3+-doped CdS nanocrystals (MPA-CdS:Eu NCs) were selected as the ECL emitter,then linked MPA-CdS:Eu NCs modified GCE with dsDNA. When DNMT1 and SAM were introduced in the system, cytosine in the sequence fragments of hemimethylated 5’-CGCGCG-3’ were methylated, after been digested by BssHII, the residual helices possess ablunt 3’ end of the (hemimethylated DNA), which could be cleared by ExoIII.The sequence of the DNA fragments could hybrid with prime DNA, which could trigger a chain reaction of hybridization events between alternating H1 DNA and H2 DNA to form nicked double-helix. Since the H1 DNA and H2 DNA could form G-quadruplex/hemin DNAzymes in the presence of hemin, a lot of DNAzymes were yielded on the dsDNA super-structure. On the basis of these pricinples, this assay showed a high sensitivity in the detection of DNMT1 from cancer cells, with a detection limited of 0.09 U/mL. In addition, it also applied in detecting the activity of DNMT1 in cancer cells, and the detection number of A549 cells can be up to 2. The proposed method held great potential for biomedical research.2. We designed a novel dual-potential ECL detection approach to detect DNMT1 activity from cancer cells. In this work the ration of ECL increments of both CdS:Eu NCs and luminol showed a constant value and could be used to judge the reliability of the detection results, providing a great promise in clinical diagnosis.CdS:Eu NCs were used as one of the ECL emitters was modified on the electrode,after hybridized with the dsDNA-Au NPs. It not only decreases the CdS:Eu NCs ECL intensity but also enhances the ECL signal of luminol, due to the SPR and the superior catalytic effect of Au NPs. Results demonstrated that the ECL ratio was judge the accuracy of DNMT1. With the use of this dual-potential system, the limit of detection can down to 0.07 U/mL, and it was lower than the use of single signal changes. This method is promising for screening the inhibitor of MTase, which may be a powerful tool for the discovery of anticancer drugs, and applied in the clinical diagnostics and biomedical researches.