The Research On The Assays To Neurodegenerative Diseases’ Biomarkers Based On The Interaction Of Fluorescence-Labeled DNA And Polymer Nanomaterials

The mechanism of neurodegenerative diseases research and the development of effective early detection and treatment are the current research hotspots at home and abroad.Fluorescence-labeled DNA probe has the advantages of simple operation method,rapid signal response,good selectivity,high sensitivity,free from electric field and magnetic field,little influence on biomolecules,accurate and reliable analysis results,and can realize single molecule analysis,real-time and in situ detection.Polymer nanomaterials have also been developed as a new kind of nanomaterials in recent years due to their unique and excellent physicochemical properties.Therefore,the combination of fluorescence-labeled DNA and polymer nanomaterials for the detection of small biological molecules,proteins and heavy metal ions associated with neurodegenerative diseases is one of the forefront tasks in analytical chemistry which is an important research in the fields of medical treatment,agriculture,food safety and environmental science.This paper focus on novel biochemical analysis methods based on the energy resonance transfer between the fluorescence-labeled DNA probe and the polymer nanomaterials.Through the change of the fluorescence signal,the detection of antioxidants,copper ions,cysteine and histidine is achieved.Combined with micro-dialysis sampling technology,we can realize the analysis of the target in cerebrospinal fluid.In this dissertation,a series of new strategies by the target-mediated interaction between polymer nanomaterials and DNA probes for the regulation of fluorescence signal changes have been developed,which has led to the development of new simple,rapid,sensitive and selective sensor assays method for the rapid detection of neurodegenerative diseases related to small molecules,heavy metal ions and other targets.It also provides a new design ideas and methods for biochemical analysis,medical diagnosis and other important areas.The full text is divided into four parts,as follows:Chapter 1.OverviewFirst of all,this chapter introduce several kinds of common fluorescence-labeled DNA,polymer nanomaterials and their current research progress,and then emphatically discusses the mechanism of neurodegenerative diseases and the research progress andanalysis methods of related targets.At last,the significance and main contents of this thesis are illustrated.Chapter 2.Design and application of cerebral antioxidants based on the dye-labeled DNA/polydopamine conjugatesA simple and novel method for evaluating antioxidants in complex biological fluids has been developed based on the interaction of dye-labeled single-strand DNA（ssDNA）and polydopamine（PDA）.Due to the interaction between ssDNA and PDA,the fluorescence of dye-labeled ssDNA（e.g.FITC-ssDNA,as donor）can be quenched by PDA（as acceptor）to the fluorescence“off”state through Fo?rster resonance energy transfer（FRET）.However,in the presence of various antioxidants,such as glutathione（GSH）,cysteine（Cys）,ascorbic acid（AA）and homocysteine（Hcys）,the spontaneous oxidative polymerization reaction from DA to PDA would be blocked,resulting in the freedom of FITC-ssDNA and leading to the fluorescence“on”state.The sensing system shows great sensitivity for the monitoring of antioxidants in a fluorescent“turn on”format.The new strategy also exhibits great selectivity and is free from the interferences of amino acids,metal ions and the biological species commonly existing in brain systems.Moreover,by combining the microdialysis technique,the present method has been successfully applied to monitor the dynamic changes of the striatum antioxidants in rat cerebrospinal microdialysates during the normal/ischemia/reperfusion process.Chapter 3.Design and application of Cu²⁺based on coumarin-labeled DNA coupled with Cu²⁺-induced oxidation of o-phenylenediamineA novel and facile ratiometric fluorescent method for evaluating Cu²⁺in rat brain has been developed based on coumarin-labeled single-strand DNA（C-ssDNA）coupled with Cu²⁺-induced oxidation of o-phenylenediamine（OPD）.The oxidative reaction of OPD with Cu²⁺can form fluorescent ox OPD with emission peak maximum at 576 nm.Meanwhile,the resultant ox OPD can readily interact with the C-ssDNA to effectively quenched the fluorescence of C-ssDNA with emission peak maximum at 452 nm.Thus,this work unveiled a novel and facile ratiometric fluorescent method for accurate,sensitive and selective measuring of Cu²⁺.The limit of detection was estimated to be4.32 nM.What’s more,by combining the microdialysis technique,this method has also been successfully exploited to monitor the Cu²⁺in rat brain.The fluorescence method is simple in design,easy to operate and has great application prospect for biochemical research and brain chemistry.Chapter 4.Design and application of histidine and cysteine based on coumarin-labeledDNAcoupledwithCu²⁺-inducedoxidationof o-phenylenediamine.Because of the coordination of cysteine and histidine and copper ion,the production of oxOPD will be inhibited.The work further designed a new and simple ratiometric fluorescence method for cysteine and histidine detection based on the interaction between single-stranded DNA and dimer.The method can also be used for the design of logic gates and for the detection of histidine and cysteine in real samples with good result in human urine samples.In addition,this shows that the method has a broad application prospect in practical application.