Electrogenerated Chemiluminescence Biosensing Method For The Detection Of Hydroxymethylated DNA And Thymine DNA Glycosylase

In mammalian genome DNA,the newly discovered 5-hydroxymethylcytosine(5-hmC)is considered to be the "sixth base" following 5-methylcytosine(5-mC,the "fifth base").5-hmC has been found in human and mouse embryonic stem cells and brain tissues,as an intermediate of DNA demethylation,5-hmC is generated by TET(ten-eleven translocation)oxidation that participates in the process of active demethylation to regulate the degree of DNA methylation in cells.Several research shows that the content of 5-hmC in a variety of cancer cells is lower than normal cells,its expression level may be used as a marker of tumor detection.In addition,the recent study indicated that thymine DNA glycosylase(TDG)also plays an important role in DNA active demethylation.Therefore,it is necessary to develop a highly sensitive method for the determination of hydroxymethylated DNA(5-hmC-DNA)and TDG.Compared with the reported methods,electrogenerated chemiluminescence(ECL)has dual advantages of electrochemical and chemiluminescence method,such as high sensitivity,good controllability,low cost,easy miniaturization,which makes ECL technique become the powerful tools for detection of tumor markers.The aim of this thesis is to design two kinds of highly sensitive and excellently selective ECL biosensing method for the determination of 5-hmC-DNA and TDG.Research work in this thesis is financially supported by the National Natural Science Foundation of China(Grant No.21675124,No.21375102,No.21005061).This thesis includes three chapters.In Chapter 1,general introduction to the definition and characteristics of the ECL.Additional,the strategy and application of signal amplification of nucleic acids and nanomaterials was summarized.Finally,the content and significance of this thesis was presented.In Chapter 2,a novel electrogenerated chemiluminescence(ECL)biosensing method was developed for quantitative detection of DNA hydroxymethylation(5-hydroxymethylcytosine,5-hmC)using T4 ?-glucosyltransferase(?-GT)and graphene oxide(GO)for enzymatic and chemical modification of the 5-hmC.T4 ?-GT selectively glucosylated the hydroxymethyl group of 5-hmC to improve selectivity,whereas GO,which loads substantial amounts of the ECL reagent,was used as the signal amplification element to enhance assay sensitivity.The ECL bioassay involved the capture of hydroxymethylated DNA(5-hmC-DNA),which was chemically modified with a glucose moiety,through hybridization with complimentary 5'-thiol-modified DNA on a glassy carbon electrode(GCE)under the catalysis of ?-GT.The dichlorotris(1,10-phenanthroline)ruthenium-assembled GO(Ru(phen)32+/GO)composite was coupled to the electrode surface via a bridging agent,and provided excellent ECL signal.The bridging agent used was 3-aminophenylboronic acid(APBA),boronic acid and its derivatives can interact with 1,2-or 1,3-diols(such as glucose)to generate 5-or 6-membered cyclic boronate esters.The obtained ECL intensity was positively correlated with the concentration of 5-hmC-DNA,the linear range was from 0.01 pM to 1.00 nM,with a low limit of detection of 3.84 fM 5-hmC-DNA was achieved.In Chapter 3,a highly sensitive and an excellently selective electrogenerated chemiluminescence biosensing method for detection of thymine DNA glycosylase(TDG)activity was developed based on dual signal amplification strategy of bio-barcode and hybridization chain reaction.The thiol modified hairpin probe with 5,overhangs and one G:T mismatch in the stem part was self-assembled on a gold electrode.In the presence of TDG,T base of the G:T mismatch could be specifically identified and removed to produce abasic sites(AP sites)through the N-glycosidic bond hydrolysis.Then,endonuclease IV(En?)was introduced to cleave intact AP sites,producing dsDNA containing a free 3' end in the long sequence,which serves as a complementary sequence to hybrid with the specific sequence of bio-barcode.The carried initiator in bio-barcode could trigger the HCR to form nicked double helices that caused the intercalation of numerous ECL indicators(Ru(phen)32+)into the dsDNA grooves,resulting in significantly amplified ECL signal output.Combining the amplification power of bio-barcode and HCR with the inherent high sensitivity of the ECL technique,a highly sensitivity and excellently selectivity ECL biosensing method was developed,and the detection limit of as low as 1.1×10-5 U/?L TDG was achieved.