Electrogenerated Chemiluminescence Biosensing Method For The Determination Of 5-hydroxymethylcytosine Based On Nano-material Signal Amplification

Cancer is one of horrible diseases of threatening human life.Developement of the accurate and effective methods of detecting cancer biomarkers is significant for the early diagnosis and prognosis of cancer.5-hydroxymethylcytosine(5-hmC)is a kind of new epigenetic modification,which plays key roles in nuclear reprogramming,regulates the gene activity,and initiates the DNA demethylation in mammals.In addition,the level of 5-hmC is dramatically reduced in multiple cancers,Lack of 5-hmC may also be a useful indicator in cancer diagnosis.Therefore,the development of reliable,sensitive and accurate technologies for detecting 5-hmC is critical.Electrogenerated chemiluminescence(ECL)represents a marriage between electrochemical and spectroscopic methods.ECL has the advantages of high sensitivity,wide linear range,strong controllability,low background interference,good selectivity and simple instrumentation.It has been widely used in the detection of biomolecules and the diagnosis of diseases.The aim of the thesis is to establish a highly sensitive and highly selective method for the detection of hydroxymethylated DNA by electrochemiluminescence biosensing based on the amplification of nanomaterials and the specificity of biomolecule recognition substances.Research work in this thesis is financially supported by the National Natural Science Fund of China(grant nos.21675124 and 21375102)and the Natural Science Basic Research Plan in Shaanxi Province of China(grant no.2016JM2021).The thesis contains three chapters and are presented as follows:1.Firstly,a review on DNA hydroxymethylation and its research progress are introduced.Secondly,the principle and characteristics of electrochemiluminescence were briefly introduced,and the signal amplification function of nanomaterials in electrogenerated chemiluminescence biosensor are summarized.Finally,the purpose,significance and content of this thesis are described.2.A new and sensitive signal-off ECL biosensing method for the quantification of 5-hydroxymethylcytosine in DNA based on glycosylation modification and quenching function of FcBA on ECL signal was developed.The results showed that the decreased ECL intensity was directly proportional to the concentration of 5.0 × 10-11 to 1.0 × 10-8 M of 5-hmC-DNA.The limit of detection(LOD)was calculated to be 1.63 × 10-11 M(S/N = 3).The method provides the reference for the further development of ECL kit.3.Based on the signal amplification function of nanocomposites and 5-hmC antibody,an ECL biosensing method for quantitative detection of 5-hmC-dsDNA was developed.The results showed that the obtained ECL intensity was directly proportional to the concentration of1×10-1 2× 10-9 M of 5-hmC-dsDNA.The limit of detection(LOD)was calculated to be 3.9× 10-12 M(S/N = 3).The method has the potential value for detecting actual sample.