Electrogenerated Chemiluminescence Biosensing Method For The Investigation Of Genomic DNA Hydroxymethylation

The recently re-discovered 5-hydroxymethylcytosine?5-hmC?has been recognized as the sixth base after 5-methylcytosine?5-mC?,which can be formed under the catalysis effect of ten eleven translocation?TET?proteins to 5-mC.5-hmC was abundant in many mammalian tissues and cells.Although the content of 5-hmC is much lower than 5-mC,5-hm C plays key roles.Moreover,more and more studies have shown that the level of 5-hmC decreases significantly in various cancers,implying that it may become a biomarker for early diagnosis of cancers.Therefore,it is highly desirable to establish reliable,selective and easy-to-use analytical methods for 5-hydroxymethylcytosine double-stranded DNA?5-hmC-dsDNA?detection in genome DNA.Electrogenerated chemiluminescence?ECL?is a powerful analytical technique featured with controllable potential and high sensitivity.The ECL biosensor has been widely used in bioanalysis,immunoassay,food safety and environmental monitoring.As a result,the aim of this work is to detect 5-hmC-dsDNA in the genome.From the perspective of enriching dsDNA immobilization,we established two kinds of highly selective and sensitive ECL biosensing methods for quantitative detection of 5-hmC-dsDNA in genomic DNA.The full paper is divided into three chapters,the main content is as follows:In chapter one,firstly,the basic principle and application of electrochemiluminescence were introduced.Secondly,the composition and structure of DNA and the basic principle of electrochemiluminescence DNA biosensor were reviewed.Finally,DNA hydroxymethylation and its research progress were presented,and the research content and significance of our work were summarized.In chapter two,a novel ECL biosensing method for ultrasensitive detection of 5-hmC-dsDNA was designed by employing an anchor probe and a ruthenium?II?complex-tagged reporter probe as an ECL probe,combined with oxidation of 5-hmC-dsDNA by KRuO₄.The results showed the ECL intensity was linearly proportional to the content of 5-hmC-dsDNA range from 0.0090%to 0.5761%.The limit of detection?LOD?was calculated to be 0.0036%?S/N=3?.The developed 5-hmC-dsDNA detection strategy was applied in various mice tissues.It was demonstrated that the content of 5-hmC-dsDNA vary significantly in different mice tissues:the content of 5-hmC is highest in brain and lowest in spleen.From these results,we can see that this biosensing method can detect the amount of 5-hmC in real samples.In chapter three,Highlysensitiveelectrochemiluminescencedetectionof5-hydroxymethylcytosine double-stranded DNA in Genome based on polyadenine-Au interaction.Firsty,chemical modification of the hydroxy group of 5-hmC in the DNA sequence is accomplished by using DNA methyltransferase?M.HhaI?,where 5-hmC-dsDNA can be converted to amino-derived 5-hmC-dsDNA.A DNA containing poly-adenine?polyA?is used as an anchor probe to complete the immobilization of 5-hmC-dsDNA through preferential adenine–Au interaction.Finally,streptavidin modified Ru?bpy?₂?cbpy?NHS is bound to anchor probe through high affinity of biotin-streptavidin.Therefore,the analysis of5-hmC-dsDNA can be achieved based on the relationship between ECL intensity and the content of 5-hmC-dsDNA.The detection limit of this method for 5-hmC-dsDNA was 0.0003%?S/N=3?.