Optical Detection Of DNA Methylation Based On Metal Nanomaterials

In this paper,copper nanoclusters were synthesized based on long poly T single strands produced by terminal deoxynucleotidyl transferase extension DNA as templates,and gold nanoparticles synthesized by a simple hydrothermal method to construct optical sensors to detect DNA methylation.The thesis mainly consists of the following parts:1.The introduction part mainly introduces the concept of epigenetics and its research content,DNA methylation and its detection methods,metal nanomaterials and their related applications,focusing on the detection methods of methylated DNA and the properties and applications of copper nanoclusters and gold nanoparticles.Finally,the main research contents and significance of this paper are elaborated.2.Based on the long T-templated copper nanoclusters engineered by terminal deoxynucleotidyltransferase,a method for detecting the activity of methyltransferase using a label-free fluorescent probe has been proposed.Based on the specific recognition of MTase and restriction endonucleases Hpall,the extension effect of terminal deoxynucleotidyltransferase and the excellent fluorescence signal of copper nanoclusters,the detection of M.SssI MTase activity can be realized The use of effective polymerization extension reaction catalyzed by terminal deoxynucleotidyl transferase and copper nanoclusters with large Stokes shifts instead of fluorescent dye labeling,has achieved increased sensitivity and expanded detection range.The detection linear range is 1-300 U mL-1,and the detection limit is 0.176 U mL-1.This method is specific to M.SssI,and can be used to screen for methyltransferase inhibitors.3.Based on unmodified gold nanoparticles,a one-step rapid colorimetric detection of DNA methylation without labeling and enzyme was constructed.The method is based on the difference of electrostatic attraction of single-stranded DNA and double-stranded DNA to gold nanoparticles to resist aggregation of gold nanoparticles induced by salting-out effect,so that methylated DNA can be quickly,effectively,economically and easily colorimetric detected by visually observing the color change of unmodified Au NPs solution.The method does not require covalent modification of Au NPs in advance,nor does it require complicated instruments,and expensive biological enzymes or traditional organic dyes are not needed in the reaction process.The detection limit of methylated DNA detected by this method is 8.47 nM,and methylated DNA as low as 20 nM can be clearly detected by naked eyes.In addition,the strategy can detect methylated DNA in the presence of a large amount of unmethylated DNA with a detection limit as low as 0.13%,and can distinguish methylated DNA as low as 1%in heterogeneous samples with naked eyes.The method can also be used to detect methylated DNA in human serum samples,showing great potential in the clinical diagnosis.