The Design,Synthesis And Performance Research Of Fluorescent Probes For Biothiols And Hypochlorite

Small molecules biothiols and HClO/ClO^-play important physiologicals role in biological systems.Once their concentration in organism out of balance,it will cause a lot of diseases.Development of simple,sensitive and reliable method to detect such molecules has very important significance.Fluorescent probe molecule design flexible,easy to modify.This technology has a lot of advantages such as simple operation,fast response,high sensitivity,good selectivity,real-time quantitative detection of analytes and effective detection of biological molecules in biological systems.Therefore,fluorescence probe plays a significant role in chemical analysis,environmental science,biotechnology and so on.Fluorescence analysis method has a lot of advantages such as simple operation,fast response,high sensitivity,good selectivity,and can achieve real-time visualization tracer and nondestructive analysis in vivo and single cell,in areas such as chemical analysis,environmental science,biotechnology has important applications.Based on this,we designed and synthesized three organic small molecular fluorescent probe,which could recognize glutathione?GSH?,hypochlorous acid?HClO/ClO^-?and cysteine?Cys?in vivo and cells,respectively.And we studied the spectral characteristics and the recognition mechanism in detail.The concrete research content of the paper is as follows:In Chapter 2,the N-ethyl carbazole-3-formaldehyde and nitromethane as raw materials,the CZ-Nm probe for GSH was further prepared according to the Knoevenagel condensation.The CZ-Nm probe showed a weak fluorescence,upon treatment with GSH,owing to the Michael addition reaction,blocking the PET process and leading to the fluorescence enhance.The probe CZ-Nm could high selectively detect the GSH and barely affected by other amino acids and thiol compounds.CZ-Nm is a“turn-on”fluorescent probe,when increasing concentration of GSH was added,the fluorescence intensity of CZ-Nm dispersed in solution increased gradually.The CZ-Nm with GSH has a wide linear range?0-10 mM?and low detection limit?6.4?M?.The CZ-Nm has applied to image of intracellular GSH in PC12 cells successfully.Furthermore,the reaction between probe CZ-Nm and GSH accompanied by a marked change color of the solution,can recognize GSH by naked eye,to further expand the application range of CZ-Nm probe.In Chapter 3,the commercially available 7-hydroxy-4-methylcoumarin as the probe for the selective detection for hypochlorite.Under the test conditions,the probe showed a weak fluorescence.Upon the addition of the hypochlorite,it could promote the probe undertake intermolecular dehydration reaction,the fluorescence intensity increased obviously.And the fluorescence intensity increased 86eq,the fluorescent quantum yield enhances from 0.005 to 0.44.The probe with hypochlorite has a wide linear range?0-150 mM?and the detection limit as low as 67 nM.The probe was successfully applied for the quantitative detection of hypochlorite in tap water.And,the fluorescence imaging of hypochlorite in A549cells was also carried out successfully.In Chapter 4,we use the natural pigment curcumin as the mother molecule synthesized a new type fluorescent probe CAC for Cys.The probe CAC contains two acrylate groups as the thiol reaction site.And the selectivity of CAC to Cys was hardly affected in the presence of GSH,Hcy,H₂S and some common amino acids.The detection limit of CAC to Cys as low as 0.19?M.Upon the addition of the Cys,the color of the CAC solution from colorless to yellow,can recognize Cys by naked eye.In the presence of the Cys,the probe CAC underwent conjugate addition and subsequently followed by cyclization to release the curcumin molecule and generate two seven membered-ring cyclic amide,so as to realize the detection of the Cys.In order to further explore the application prospect in molecular biology and fluorescence imaging of curcumin-based fluorescent probes,we carried out the fluorescence imaging experiment in PC12 cells and zebrafish.We successfully achieved the detection of Cys concentration in vivo,for this kind of probe laid a foundation in the application of molecular biology.