Monitoring Of Lysozyme Amyloid Fibrosis Process And Screening Of Inhibitors Based On Gold Nanocluster Fluorescence Assay

Alzheimer's,huntington's disease and other diseases related to the old have received extensive attention,while one of the most important reason for the emergence of these diseases is that the related proteins denatured and deposited in the human body,i.e,protein amyloide fibrillation.With the development of science and technology,scientists have established many methods for the detection of protein amyloid fibrils.However,it is still necessary to establish a facile method for the detection of protein amyloid fibers because these methods have some shortcomings.There are many proteins and peptides in the human body,and under certain conditions,they will deform and lose their functions,and even aggregate in the human body and cause diseases.The amyloid fibrosis process of protein is a process from water-soluble monomer to insoluble fiber,and the hydrophobic group is exposed continuously,its hydrophobicity is continuously enhanced.Lysozyme is a typical amyloid fibrosis protein,which has a similar structure to human lysozyme protein.Therefore,lysozyme was used as a model protein to study the amyloid fibers of proteins in this work.Gold nanocluster is a novel fluorescent probe with many excellent properties,such as low toxicity,high stability,large Stokes shift,infrared emission,long fluorescence life,and good biocompatibility.Therefore,it has been applied in sensors,biological imaging and diagnosis and treatment,especially in sensing of metal ions and small molecules.Based on the hydrophobicity enhancement after lysozyme amyloidosis fibrosis and the fluorescence emission change of gold nanoclusters in different solvents,herein,gold nanoclusters were used to detect the process of lysozyme amyloidosis by using the aggregation induction effect of gold nanoclusters in hydrophobic solvent.In addition,a metal organic framework material with fluorescence properties,was synthesized by one-pot method and applied to the detection of 2,4,6-trinitrophenols(TNP).Finally,platinum nanoparticles(Pt NPs)were synthesized and used for the detection of iodide by using its peroxidase properties.The thesis is mainly composed of the following four parts:Chapter one,introduction.Firstly,the different synthesis methods,properties and applications of gold nanoclusters were summarized.Then,the process of protein amyloidosis and some related human diseases are introduced.Next,the properties of the model protein lysozyme and the process of amyloidosis are summarized.Finally,the detection methods of protein amyloidosis are summarized.In chapter two,the gold nanoclusters were used in the detection of lysozyme amyloidosis fibrosis and the screening of amloide fibrillation inhibitors.Firstly,gold nanoclusters with aggregation induction effect were synthesized and characterized based on the methods reported in literature.Then,gold nanoclusters were applied to detect lysozyme amyloid fibers.When native lysozyme was added to the gold nanoclusters,the fluorescence intensity did not increase,while when lysozyme fiber was added to the gold nanoclusters,the fluorescence of gold nanoclusters significantly increased.Under optimized conditions,when the concentration of lysozyme fiber was less than 0.05 mg/mL,there was a good linear relationship between the fluorescence intensity of gold nanoclusters and lysozyme fiber concentration.When the gold nanoclusters were added to lysozyme incubated for different time,the fluorescence of the gold nanoclusters gradually increased and finally did not change,which reflected the complete growth process:nucleation stage,growth stage and maturity stage.In addition,we selected ascorbic acid as the representative of inhibitors,and used this method to screen inhibitors.If inhibitors were added when incubating lysozyme,lysozyme fibrosis process would be crimped,the amount of lasozyme fibers would decrease.Then,this method is used to inspect,and we got to the same conclusion:the addition of ascorbic acid inhibited amyloid fibrosis process of lysozyme,and inhibiting levels associated with the concentration of ascorbic acid,with the increase of ascorbic acid concentration,the degree of inhibition became stronger.This method has potential application value in the detection of protein amyloid fibrosis.In chapter three,composite material TPE@ZIF-8 was synthesized by one pot method and used for the detection of explosive TNP.ZIF-8 is a kind of metal-organic frame material with good stability and water solubility.We added the aggregation induced emission molecule tetrstyrene(TPE)in the synthesis process to synthesize the metal-organic frame material with fluorescence properties.The study has showed that the explosive TNP has a strong fluorescence quenching ability,so we used TPE@ZIF-8 with fluorescence property for the detection of TNP.The linear range of this method is 10-300 ?M,and the detection limit is 3.16 ?M.This method is expected to be applied to the detection of explosives.In chapter four,based on the properties of Pt NPs for catalytic oxidation of 3,3,5,5-tetramethylbenzidine(TMB),a colorimetric method for detecting iodide ion was established.Platinum nanoparticles can catalyze the oxidation of TMB,and the presence of iodide ion will inhibit this process.The oxidation degree of TMB will be reduced,and the ultraviolet absorbance at 652 nm will be reduced,so the purpose of detecting iodide ion could be acheived.The linear range of iodide ion was 0.05-1.0 ?M,and the detection limit was 0.03 ?M.Moreover,this method has good anti-interference ability,and the common anion and anion do not have obvious influence on the detection of iodide by this method.The results of standard addition method also showed that the method has certain reliability.