Study On The Electrogenerated Chemiluminescence Of Hydroxymethyl DNA Sensor

As a mark for transcriptional silencing,DNA methylation is central to many important cellular processes.The methylation state of DNA is found to be dynamically changed during mammalian early development,partially because of active demethylation.DNA demethylation is a reverse of DNA methylation that methyl group is removed from the C-5 position of cytosine in the dinucleotide sequence CpG by DNA demethylase.Additionally,the discovery of the ten-eleven translocation(Tet)oxidation products 5-hydroxymethyl cytosine(5-hmC),5-formylcytosine(5-fC),and 5-carboxylcytosine(5-caC)suggests a possible demethylation pathway.In recent years,many studies have indicated that 5-hmC plays an important role in the epigenetic,and the content and distribution of 5-hmC have become one of the important markers for the diagnosis of cancer.Therefore,it is important to develop a simple,sensitive and selective method for analyzing the activity of DNA demethylase and assaying 5-hmC-DNA.However,most of present methods suffer from expensive and large-volume instruments,multiple separation and wash steps,which might limit their applicability for routine assays.The electrogenerated chemiluminescence techniques have the advantages of instrument miniaturization,operation simplification,low consumption of reagent and high sensitivity,which make ECL technique as a suitable platform for DNA demethylase activity assay and hypermethylation state of DNA sequence.The aim of the present work is to develop hinghly sensitive and selective electrogenerated cheniluminescence(ECL)biosensors for the detection of 5-hmC-DNA and DNA demethylase.The main contents are as follows:In Chapter 1,we generate review from the principles,brief history of electrogenerated chemiluminescence.Summarizing the biological significance,the analysis method of the DNA methylation and DNA hydroxymethylation.Finally,this paper expounded the research purpose,content and significance of this paper.In Chapter 2;we introduce a new method for 5-hmC quantification based on electrogenerated chemiluminescence(ECL)assay combined with KRuO4 oxidation.5-hmC can be selectively transformed into 5-fC by the oxidation of KRuO4 and then labeled with N-(4-Aminobutyl)-N-ethylisoluminol(ABEI).Thus,significant ECL from ABEI-labeled DNA can be observed if the DNA contains the 5-hmC modification.However,no ECL occurs if the DNA does not contain the 5-hmC modification because the normal DNA and 5-mC-DNA can not be labeled with ABEI.In Chapter 3.we describe a rapid,sensitive,and selective electrogenerated chemiluminescence method for DNA demethylase activity assay by coupling the dual signal amplification of MoS2-thionin composite and supersandwich approach with the site-specific cleavage of Hpall to improve the selectivity.MoS2-thionin composite and super sandwich approach can provide an amplification platform for assembling a large number of signal probe,and thus resulting in a high sensitive ECL detection of demethylase(MBD2).This assay showed a low detection limit of 0.16 pg/mL for MBD2 and recognize MBD2 from other possibly coexisting proteins such as MBD1,MBD4,and MeCP2.This assay might provide a useful electrochemical platform for assay of relative-enzyme activity and screening of relative drug for inhibition or promotion DNA demethylase activity.