| 5-Methylcytosine(5-mC,the fifth base of gene)can be oxidized by the ten-eleven translocation(TET)proteins to form 5-hydroxymethylcytosine(5-hmC,the sixth base of gene),resulting in DNA hydroxymethylation.It is reported that 5-hmC is a vital intermediate in the process of DNA active demethylation and plays a key role in physiological processes,such as differentiation of cells and regulation of gene expression.Furthermore,the changes in 5-hmC content are associated with a variety of human diseases,including neurological diseases(depression),genetic diseases,and cancer.Compared with healthy cells,5-hmC content in cancer cells is reduced significantly.Therefore,5-hmC can be a biomarker for early diagnosis of cancer.The methods for accurate,rapid,and sensitive detection of DNA hydroxymethylation and related transferase activity are required.The methods may clarify the important mechanism of healthy and pathological gene expression,provide an important basis for diagnosis and prognosis evaluation of cancer and hereditary diseases,and are one of the frontier fields of analytical chemistry and molecular biology.Electrochemiluminescence(ECL)method is a rare highly sensitive analytical method in analytical chemistry,which has been widely used in clinical diagnosis,food analysis,and environmental analysis.In the thesis,taking advantages of ECL technique and the specificity of biological molecular recognition including transferase and 5-hmC antibody,four ECL biosensing methods for determination of the activity of?-glucosyltransferase(?-GT)and DNA hydroxymethylation have been developed incorporating nanomaterial amplification and nucleic acid signal amplification.Besides,while studying for Ph.D.,I had been awarded a scholarship under the State Scholarship fund to pursue my study at the Zhou Group in Texas A&M University.I conducted research in the study of the metal-organic frameworks derivated to carbon metal oxides and applied in supercapacitors.The thesis includes six chapters.The main research is as follows:1.An ECL biosensing method was developed for discrimination of DNA hydroxymethylation and assay of the?-GT activity.AuNPs/Nafion/GCE modified electrode was prepared by using gold nanoparticles(AuNPs)/Nafion film modified glassy carbon electrode(GCE),and then,tris(2,2?-ripyridine)dichlororuthenium(II)(Ru(bpy)32+)was adsorbed on the surface of AuNPs/Nafion/GCE by electrostatic adsorption,finally,the hydroxymethylated double-stranded DNA-tagged with ferrocene was self-assembled onto the surface of the AuNPs.In the presence of?-GT and uridine diphosphoglucose(UDP-Glu),the hydroxymethylcytosine within 5?-CCGG-3?of ds-DNA on the electrode was glucosylated.After glucosylation and Msp I endonuclease treating,the unglucosylated hydroxylated sequence was cleaved,resulting in the ferrocene away from the electrode and a recovered ECL intensity.This method showed a good linear relationship for the concentration of?-GT from 0.1-180 U·m L-1,and a low detection limit of 0.04 U·m L-1.This study provides a reference for related methods and technologies for the discrimination of 5-hmC from 5-C and the determination of?-GT.2.A sensitive ECL biosensing method based on Methyltransferase specific recognition and nanomaterial amplification was developed for detection of hydroxylated DNA(5-hmC DNA).The method in chapter 2 was developed for?-GT activity detection,which also could discriminate the 5-hmC from 5-mC.For the problem of low content of 5-hmC,a highly sensitive method was established for detection of DNA hydroxymethylation.First,GCE was modified by electrochemically reduced molybdenum disulfide-poly(acrylic acid)nanosheets.Then,a capture probe with amino group hybridized with the target 5-hmC DNA was assembled to the electrode surface.In the presence of cysteamine and M.Hha I methyltransferase,the hydroxyl group of 5-hmC within DNA would be replaced by thiol and generated the amine-derivated DNA.Finally,surface chemically activated Ru(bpy)32+-doped silica(Ru@Si O2)nanoparticles covalently bonded to the amine-derivated DNA,leading to the ECL signal material close to the electrode.Under the optimized experimental conditions,the method provides a wider linear range for the 5-hmC DNA(5.0×10-14M to 1.0×10-11 M),and a lower detection limit of 1.2×10-14 M.3.A simple ECL biosensing method was established for detection of DNA hydroxymethylation based on glycosylation and multifunctional ECL signal compound.A multifunctional ECL signal compound(N-(phthalhydrazide-4-yl)-thiophen-2-amino-5-boronic acid,PTAB)was prepared to achieve rapid and sensitive detection of DNA hydroxymethylation.PTAB includes the three main modules:4-aminophthalhydrazide(isoluminol module),borinic acid module,and thiophene module.In the presence of?-GT and UDP-Glu,5-hmC in double-stranded DNA(5-hmC-dsDNA)was glycosylated to 5-ghmC,and then a borate ester covalent bond was formed between the PTAB and the cis diol of 5-ghmC,so PTAB(isoluminol)was added to each 5-hmC site.5-ghmC-dsDNA labeld with PTAB(thiophene module)can form Au-S bond with the gold electrode surface,and then self-assembled to the electrode surface.After the voltage is applied,a strong ECL signal is generated,which can be used for quantitative analysis of 5-hmC-dsDNA,The linear range of 5-hmC-dsDNA content(mass percentage concentration)was 0.0090%-0.5761%,and the detection limit was 0.0058%.Due to the boric acid group of the PTAB,the biosensing method is expected to be used for the detection of substances containing 1,2-or 1,3-diol functional groups.4.An ECL biosensing method which combined poly(diallyldimethyl ammonium chloride)/ferrosoferric oxide/molybdenum disulfide(PDDA/Fe3O4/Mo S2)nanocomposite and DNA walking machine was developed for determining 5-hmC-dsDNA.A new strategy for the detection of 5-hmC-dsDNA was developed by combining nanomaterial signal amplification with nucleic acid signal amplification.This strategy reduced the steric hindrance effect of DNA assembly on the electrode surface and achieve a high sensitivity detection of 5-hmC-dsDNA content.The established method overcomes the drawbacks of the steric hindrance which results from the step-wise DNA assemble and DNA directly immobilized on the surface of the electrode and improves the sensitivity of the DNA hydromethylation detection method.Firstly,gold nanoparticles(Au)with a relatively large surface area were immobilized with the DNA walker probe,Ru(bpy)32+labeled DNA(Ru-S1),and 5-hmC antibody(Ab)for the formation of the DNA nanomachine.At the presence of 5-hmC-dsDNA,an immune complex was acquired through the immunoreaction between Ab immobilized on Au and 5-hmC-dsDNA loaded on the positively charged PDDA/Fe3O4/Mo S2 nanosheets.As the DNA walker probe paired with Ru-S1,a recognition site for Nb.Bv CI nicking endonuclease formed.Therefore,under the shearing of Nb.Bv CI,Ru-S1?would be produced accompany the releasing of DNA walker probe which would match with the next Ru-S1 from near to far on the Au surface.In this way,a certain amount of 5-hmC-dsDNA was converted to a large amount of Ru-S1?.Furthermore,the obtained Ru-S1?was concentrated on the surface of the fabricated electrode by electrostatic adsorption between DNA and polyethylenimine(PEI)which could further introduce massive Ru-S1?on the electrode,resulting in an enhanced ECL signal.And the ECL signal increased with increasing the concentration of 5-hmC-dsDNA in a broader linear range from 0.0090%to 0.5761%with a low detection limit of 0.0024%.5.ZIF-67 was used for cobalt precursors and sacrifice template and together with the introduction of carbon nanotubes and transition metal manganese copper nanoparticles,generated the crosslinking structure of Cu-Mn/ZIF-67/CNTs-600nanocomposite,which prevents the aggregation of the nanoparticles and improves the constraint capacitance,the electrolyte ion transport,the electrical conductivity and the cycle stability of the electrode materials.Also,Cu-Mn/ZIF-67/CNTS-600nanocomposite was used as the electrode material of the supercapacitor to test its electrochemical performance in the three-electrode system.The results show that the specific capacitance of the electrode material is 1040 F·g-1 when the current density is2 A·g-1. |
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