Construction Of Dual-fluorophore And Dual-site Multifunctional Fluorescent Sensors And Study On Their Sensing Performance For Glutathione

In recent years,fluorescence sensors have been recognized as powerful tools for sensing and imaging in vivo due to their advantages of high sensitivity and selectivity,high spatiotemporal resolution and easy manipulation.In particular,multifunctional fluorescent sensors that can analyze multiple substances simultaneously have been gradually developed to monitor the delicate life’s events,providing an efficient analysis platform for revealing the interaction mechanisms and metabolic pathways of various biomolecules.As the most abundant non-protein thiol,major antioxidant and antidote in mammalian cells,glutathione（GSH）participates in many physiological processes including biocatalysis,protein synthesis,cell metabolism,signal transduction and maintenance of redox homeostasis,and its abnormalities in concentration and metabolism are closely related to human chronic diseases involving neurological disease,liver damage,cardiovascular,cerebrovascular disease and alzheimer’s disease.However,to date,multifunctional fluorescent sensors for studying the properties of GSH have been rarely reported.Since mitochondria is the main source site of reactive sulfur（RRS）and reactive oxygen species（ROS）,this paper aims to investigate the regulation of GSH on oxidative stress induced by excess peroxynitrite（ONOO^-）and the metabolic process of its conversion to sulfur dioxide（SO₂）,as well as its complex relationship with the toxicological mechanism of high-does SO₂ in mitochondria.For the purpose of research,two mitochondria targeted multifunctional fluorescence sensors were constructed by bridging two fluorophores with rigid piperazine and integrating two recognition groups.Moreover,their sensing performance for GSH and ONOO^-/SO₂ were also explored.The details are as follows:Chapter 1.This part briefly described the construction and recognition mechanism of fluorescence sensors and the research progress of multifunctional fluorescent sensors.Based on the research status of multifunctional fluorescent sensors for exploring the properties of GSH and the main difficulties at present,the research purpose and details of this paper were put forward.Chapter 2.Coumarin derivative and benzopyrylium derivative were selected as the donor and acceptor fluorophores,respectively,to develop a mitochondria targeted multifunctional fluorescent sensor Mito-CM-CD,in which the oxonium cation or carbon-carbon double bond（-C=C）as was applied for identifying ONOO^-or GSH.In the physiological p H range,the sensor exhibited high sensitivity,fast response and excellent selectivity for both GSH and ONOO^-.In the presence of GSH（or ONOO^-）,with the addition of ONOO^-（or GSH）,the sensor generated a blue-shifted signal in which red fluorescence was quenched but blue fluorescence enhanced（or blue fluorescence-enhanced signal）,so that continuous detection of GSH and ONOO^-could be achieved.Furthermore,Mito-CM-CD has been successfully applied to the visualization of mitochondrial GSH and ONOO^-in living cells and the dynamic monitoring of drug-induced intracellular GSH and ONOO^-levels by three-channel fluorescence imaging.Chapter 3.By choosing two-photon（TP）absorbing fluorophores?naphthalimide derivative and benzopyrylium derivative as the donor and acceptor fluorophores respectively,a TP multifunctional fluorescence sensor Mito-Na-BP was constructed.The sulfoxide functional group（-S=O）was used as the quenching group and the binding site of GSH,and the-C=C was employed for recognizing SO₂.Under single-wavelength excitation,the sensor responded to GSH and SO₂ with signal modes of enhanced both red and green fluorescence and quenched red fluorescence,respectively.In the presence of GSH,Mito-Na-BP produced an obvious blue-shifted fluorescence signal for SO₂.After binding SO₂,the sensor could continue to respond to GSH with a significant change in green fluorescence intensity.Given its excellent spectral response properties（high sensitivity,good selectivity and fast response rate）,Mito-Na-BP has successfully revealed the close correlation of GSH with the toxicological mechanisms of excess SO₂ and visualized in real time the metabolism process of GSH to SO₂ by TP imaging.Chapter 4.The summary and prospect,including the conclusion of the experimental results of this paper,a generalized analysis of the advantages and disadvantages of the two multifunctional fluorescent sensors reported in this paper,and the prospect for the development of multifunctional fluorescent sensors.