Preparation Of DNA Functionalized Nanomaterials And Its Application In 5-mC Biosensing Analysis

DNA methylation is the most important epigenetic modification identified,by adding a methyl（CH₃） to the fifth carbon of the cytosine（C） to form a 5-methylcytosine（5-mC） without altering the DNA sequence.Due to its regulation of gene expression,gene imprinting and gene silencing in cell differentiation,embryonic development,hereditary diseases and tumorigenesis,DNA methylation has become the most valuable third generation genetic markers after restriction fragment polymorphism and DNA point mutation.DNA methylation is an important mechanism of tumor suppressor gene inactivation and oncogene activation in tumorigenesis.Many studies have shown that tumor hypermethylation and oncogene hypomethylation have been identified as early changes in tumor formation;hereditary diseases such as sclerosis and thalassemia,and methylation bias of target genes characteristic of autoimmune diseases including systemic lupus erythematosus and type I diabetes.The imbalance of DNA methylation has become a new target for early diagnosis and early warning of epigenetic disorders such as tumors.Therefore,quantitative analysis of DNA methylation levels of specific sequences has become a new means of early diagnosis and early warning of epigenetic disorders such as epigenetic research,autoimmune diseases and tumors.Quantitative analysis of DNA methylation has been the frontier of cross-sectional research between experimental diagnostics and epigenetics.Electrochemical biosensors are a class of analytical techniques that perform detection by converting the interaction of a recognition molecule and a target molecule into an electrical response signal.Its advantages were operation convenient,direct interpretation of results,easy clinical laboratory promotion,and have been widely used in biological analysis and detection.Recent years,in order to further improve sensor,various functionalized nanomaterials have been developed and used for the construction of electrochemical biosensors.Among them,DNA nanomaterials have important application value in electrochemical biosensing analysis because of its structural stability,strong coding,simple preparation and good biocompatibility.So,DNA functionalized nanomaterials and bio-assisted amplification techniques are used to construct electrochemical biosensors and photoelectrochemical biosensors for quantitative analysis of DNA methylation in this paper.The details are mainly as follows:1.An electrochemical strategy with tetrahedron rolling circle amplification for ultrasensitive detection of DNA methylationIn this experiment,three thiol-modified single-stranded DNAs and one specific DNA sequences were used to synthesize DNA tetrahedrons,and DNA tetrahedrons were immobilized on the surface of gold electrodes by Au-S bonds to achieve efficient immobilization of the sensing interface probes.After bisulfite the nucleic acid sequence containing the 5-mC target sequence and without 5-mC,only the 5-mC target sequence is cyclized with the linear padlock probe.Using the cyclized nucleic acid sequence as a template,a single-stranded DNA probe extending at the apex of the DNA tetrahedron is subjected to RCA amplification of the primer to produce a long DNA chain rich in G bases,which binds to hemin.Thereafter,a G-quadruplex/hemin mimetic DNAzyme is formed which has horseradish peroxidase activity and catalyzes amplifying a current response signal generated by methylene blue（MB） in the presence of H₂O₂.Under the optimal experimental conditions,the electrochemical biosensor DPV current signal exhibits a good linear correlation with the logarithm of the 5-mC target sequence concentration,with a linear range of 10^-15 M to 10^-9 M and a detection limit of 0.1 fM.The electrochemical biosensor has the advantages of high sensitivity,good selectivity and simple preparation,and is successfully applied to DNA methylation recovery detection.2.A new strategy for 5-mC detection based on photoelectrochemical biosensor using CdS@DNA S1 and PTB7In order to achieve quantitative analysis of low-abundance DNA methylation,in this study,we first used CdS QDs in combination with PTB7 as a photoactive material to construct photoelectrochemical（PEC） biosensors for DNA methylation detection.First,the CdS QDs-labeled signal probe CdS@DNA S1 hybridizes with the DNA S0sequences of IgG/AuNPs on gold nanoparticles（AuNPs） and secondary antibody（IgG） complexes to form IgG/AuNPs/CdS@DNA S1 nanocomposites.In the presence of DNA methylation,the anti-5-methylcytosine antibody specifically recognizes with DNA methylation,followed by binding of the anti-5-methylcytosine antibody to IgG.Because CdS QDs increase the steric hindrance of the electrode and inhibit the transfer of electrons from the ascorbic acid electrolyte to PTB7,the photocurrent signal of PTB7on the electrode surface is reduced,and the decreased photocurrent signal is positively correlated with the concentration of DNA methylation.Under the optimal experimental conditions,the photocurrent response signal of the constructed PEC sensor showed a linear correlation with the logarithm of the 5mC target sequence concentration,with a linear range of 50 aM to 500 pM and a detection limit of 50 aM.This experiment provides based on CdS QDs and PTB7 PEC analysis method,which can significantly improve the photoelectric conversion efficiency,and provide a new idea for the establishment of low-background,high-sensitivity low-abundance DNA methylation quantitative analysis.