A New Method For Fluorescence Sensing Of DNA Methyltransferase And Glucose

Methyltransferase-catalyzed DNA methylation is a very important process of epigenetic modification and plays a key role in the modification of DNA conformation,chromatin structure,gene expression and other biological processes.Aberrant DNA methyltransferase activity and methylation levels can lead to multiple diseases,and DNA methyltransferases have become important targets for medical diagnosis and treatment of some complex diseases.Therefore,the development of a simple,sensitive method for the detection of DNA methyltransferase activity is of great significance not only in the study of epigenetics and other basic biological processes but also in the diagnosis of related diseases and drug development.In addition,glucose is an important biochemical indicator for the diagnosis of diabetes.It is necessary to study and establish a glucose quantitative detection method with simple operation,high sensitivity and low price for the treatment and control of diabetes.In this paper,we developed new fluorescence sensing methods for methyltransferases and glucoses,based on simple and highly efficient nucleic acid amplification techniques and superior performance of CeO2 nanoparticles.The main contents are as follows:1.In the second chapter of the thesis,we established a novel method for detecting DNA methyltransferase activity based on nucleic acid amplification technology mediated by terminal deoxynucleotidyl transferase?TdT?.In this method,it is only necessary to design a simple stem-loop structural DNA molecule probe whose stem contains the Dam methyltransferase specifically recognizing the 5'-G-A-T-C-3' sequence and the 3'end is blocked by phosphoric acid.When Dam methyltransferase acts on the stem-loop DNA probe,the DNA probe is methylated to a structure containing the 5'-G-mA-T-C-3' sequence.This methylation site can be specifically recognized by the Dpn I enzyme and cleaved,releasing the 3 '-OH end.In the presence of the TdT enzyme and dTTP,DNA template is not required and a polyT of several kilobases in length can be extended.OligoGreen was then added to stain it.Sensitive measurement of DNA methyltransferase can be achieved by detecting changes in the resulting fluorescence intensity.The novel DNA methyltransferase fluorescence method has a simple design,high sensitivity,no sequence dependence,can be detected in the homogeneous phase,and this method can also be applied to the detection of other proteases through rational probe design.2.In the third chapter of the thesis,we established an efficient fluorescence detection method by using CeO₂ as an excellent fluorescence quencher.In this method,we find that both free Ce3+ ions in solution and Ce3+ ou CeO2 surface can efficiently quench the fluorescence of calcein while the Ce4+ will not.However,when the system contains H2O2,H2O2 may rapidly and efficiently regulate the Ce3+/Ce4+ redox state on the surface of CeO2 nanoparticle by oxidizing Ce3+ to Ce4+,leading to gradually restored fluorescence of calcein.Based on this method,we can detect the content of H2O2.The detection limit of this method for H2O2 is 8 nM,which is 3 orders of magnitude lower than that of the pure Ce3+/calcein-based sensing system..As a product of the glucose-glucose oxidase?GOx?reaction,H2O2 also serves as the indirect target molecule.And with the help of glucose oxidase,we can detect the glucose activity of fluorescence by using CeO2 as a fluorescence quencher method.The method is simple,inexpensive,fast and sensitive,and can accurately detect glucose down to 110 nM.