Design Of Novel Fluorescent Probes For Imaging Analysis Of Environmental Related Bioactive Molecules

Fluorescence imaging analysis method has the advantages for real-time,in situ,dynamic detection with high sensitivity and high selectivity.It has been widely used in the fields of environmental analysis and biological analysis.In this paper,based on environmental imaging analysis technology,we focus on the development of new fluorescent probe tools with excellent fluorescence properties for fluorescence imaging analysis of small bio-reactive molecules under environmental stress.By analyzing the distribution,transport,variation and interaction rule of all reactive species in cells,tissues and in vivo,we evaluate the relationship between environment and health.The details of this article are as follow:1.A ratiometric near-infrared fluorescent probe for the analysis of cysteine hydropersulfide in living cells and in vivo.Cysteine hydropersulfide?Cys-SSH?plays primary roles in synthesis of sulfur containing cofactors,regulation of cellular signaling,activation or inactivation of enzyme activities,and modulation of cellular redox milieu.The concentration fluctuation of Cys-SSH is closely related to the occurrence and development of diseases.Herein,we design and synthesize a novel ratiometric near-infrared fluorescent probe Cy-Dise for the fluorescence imaging of Cys-SSH in living cells and in vivo.Cy-Dise is composed of three moieties: bis?2-hydroxyethyl?diselenide,heptamethine cyanine,and D-galactose.Cy-Dise exhibits satisfactory linear ratio response to Cys-SSH in the range of 0-12 ?M Cys-SSH?r = 0.9913?.The experimental detection limit is determined to be 0.12 ?M.Based on a detection mechanism of selenium–sulfur exchange reaction,the concentrations of Cys-SSH in Hep G2 cells,HL-7702 cells and primary mouse hepatocytes have been determined as 1.85 ± 0.2 ?M,0.80 ± 0.1 ?M and 2.51 ± 0.4 ?M,which are further verified by HPLC-MS.The probe is also suitable for deep tissue ratio imaging.Organ targeting tests with Cy-Dise in normal rats and hepatic carcinoma rats verify its predominant localization in liver and quantification ability.The probe has been applied for fluorescent imaging of Cys-SSH in living zebrafish.The probe is promising for revealing the roles of Cys-SSH in physiological and pathological processes.2.A near-infrared fluorescent probe for detection of sulfane sulfur in different kinds of cells and organs.Sulfane sulfur refers to ionized sulfur that is reversibly attached to other sulfur atoms in the form of 6-valence electrons?S0?.Some sulfane sulfur possesses stronger nucleophilicity and reducibility than hydrogen sulfide in a series of physiological reactions,which makes sulfide sulfur may be the actual signal molecule in cells.Herein,we design and synthesize a near-infrared fluorescent probe BD-di SH for sensitive detection and imaging of sulfane sulfur in living cells and in vivo.The probe BD-di SH is composed of two moieties: the fluorophore BODIPY and the sulfane sulfur recognition unit 2-mercapto benzoate.The mercapto group of 2-mercapto benzoic acid can nucleophilicly capture the sulfur atom of thiosulfoxide tautomers in sulfane sulfur to form –SSH.The group –SSH will soon induce intramolecular cyclization reaction and release the BODIPY fluorophore to emit fluorescence.BD-di SH displays high sensitivity and selectivity towards sulfane sulfur.The experimental detection limit is determined to be 10 n M.The probe BD-di SH targets well in cytoplasm of the living cells.The results illustrate that the endogenous and exogenous sulfane sulfur level changes depending on different cell lines.BD-di SH is also capable of imaging the level changes of sulfane sulfur in mice.The probe has been applied for fluorescent imaging of the level changes of sulfane sulfur in living zebrafish.The above applications make our new probe a potential chemical tool for the study of physiological and pathological functions of sulfur sulfide.3.A ratiometric near-infrared fluorescent probe for analysis the relationship between GSH concentration and apoptosis under temperature stress.Environmental temperature changes can abnormally decrease intracellular glutathione?GSH?concentrations and horribly induce apoptosis.As the most abundant intracellular nonprotein biothiol,GSH can protect cells from apoptosis.Considering the important roles of GSH in the process of anti-apoptotic in cells and in vivo,we design and synthesize a near-infrared ratiometric fluorescent probe?Cy O-Dise?for the qualitative and quantitative detection of GSH concentration fluctuations in cells and in vivo upon temperature stress.Cy O-Dise is composed of three moieties: bis?2-hydroxyethyl?diselenide,keton cyanine,and D-galactose.There existed a linear relationship between logarithm of the ratio and the concentrations of GSH,in the range of 0-10 m M GSH?r = 0.9947?.The experimental detection limit was determined to be 50 ?M.The probe has been used to assess the changes of GSH levels in Hep G2 and HL-7702 cells by the stimulations of temperature and find that human normal liver HL-7702 cells have stronger abilities to fight against temperature stress than human liver carcinoma Hep G2 cells.Temperature stress can also improve drug resistance of cis-Dichlorodiamineplatinum?II??DDP?-resistant Hep G2/DDP cells.The probe Cy O-Dise has been employed to evaluate the relationship between the concentration of GSH and the treatment of Hep G2 and Hep G2/DDP xenografts nude mice,respectively.The probe can help evaluate the relationship between environmental tempreture and organism health.4.A ratiometric near-infrared fluorescent probe for bioimaging and evaluating endogenous superoxide anion changes during ischemia/reperfusion injury.Ischemia/reperfusion?I/R?process is a typical process of hypoxia,which will cause the outburst of superoxide anion?O2·-?in mitochondrial and then lead a series of oxidative damages including polarity loss of mitochondrial membrane potential,overload of secondary cellular calcium,and cascade apoptosis.To monitor the O2·-level fluctuations as well as to evaluate the relationship between O2·-concentration and the degree of cell apoptosis during I/R process,we propose a ratiometric near-infrared mitochondrial targeting fluorescent probe Mito-Cy-Tfs for the detection of level changes of O2·-in cells and in vivo.The probe Mito-Cy-Tfs is composed of three moieties: near-infrared heptamethine cyanine as fluorescence signal transducer,trifluoromethanesulfonamide as fluorescence modulator,and lipophilic triphenylphosphonium cation as mitochondrial guider.There existed a linear relationship between logarithm of the ratio and the concentrations of O2·-?r = 0.9950?.The experimental detection limit was determined to be 30 ?M.The probe can well locate in mitochondria and respond the concentration changes of endogenous O2·-selectively and sensitively.The probe has been successfully utilized to image the endogenous O2·-fluctuations in four kinds of cell I/R models?glucose deprivation/reperfusion,serum deprivation/reperfusion,oxygen deprivation/reperfusion and glucose-serum-oxygen deprivation/reperfusion?.The probe also exhibits deep tissue penetration for real-time imaging of O2·-concentration in liver of I/R mice model.The probe can be employed to accurately indicate and evaluate the mutual relationship between the levels of O2·-and the degrees of organ damage during I/R process.The above applications make our new probe a potential candidate for research the relationship between hypoxia and health.5.A ratiometric near-infrared fluorescent probe for analysis selenol level in CCl4-induced acute inflammation.Selenocysteine?Sec?is a primary kind of reactive selenium species?RSe S?in cells.It often incorporates into selenoproteins and functions as the active sites of enzyme.Sec has been featured its antioxidant roles in a series of liver diseases,such as oxidative damages and inflammatory processes.In this work,we report a ratiometric near-infrared fluorescent probe?Cy-SS?for qualitative and quantitative determination of Sec in living cells and in vivo.The probe is composed of heptamethine cyanine fluorophore,the response unit bis?2-hydroxyethyl?disulfide,and the liver-targeting moiety D-galactose.The concentrations of Sec in Hep G2 cells,HL-7702 cells and primary mouse hepatocytes have been determined as 3.08 ± 0.11 ?M,4.03 ± 0.16 ?M and 4.34 ± 0.30 ?M,which are further verified by HPLC-MS.There was an good linearity between logarithm of the ratio and the Sec concentrations?r = 0.9912?.The experimental detection limit was estimated to be 90 n M.The probe can selectively accumulate at liver.The ratio fluorescence signal of the probe can be employed to quantitatively analyze the fluctuation of Sec concentrations in CCl4-induced mice models of acute hepatitis.The experimental results demonstrate that Sec plays important antioxidant and anti-inflammatory roles during inflammatory process.And the levels of intracellular Sec have a close relationship with the degree of liver inflammation.The probe provides a new tool for evaluating redox level in organs under environmental stress.6.A ratiometric near-infrared fluorescent probe for analysis selenol concentration changes in CS₂-induced acute/chronic inflammation.Carbon disulfide?CS₂?is a common solvent in industry,which can inhibit the activities of oxidoreductase and induce oxidative stress.As important active sites of oxidoreductase in mitochondria,selenocysteine?Sec?takes the responsibility for cytoprotective effect and intracellular redox homeostasis.It is necessary to investigate the cytoprotective effect of Sec against CS₂ exposure.After integrated the response moiety 2,4-dinitrobenzenesulfonamide and mitochondrial targeting moiety into the near-infrared heptamethine cyanine fluorophore,a mitochondrial-targeting ratiometric near-infrared fluorescent probe Mito-di NO2 for the selective and sensitive detection of Sec level changes in cells and in mice models under the stimulation of CS₂ was developed.There was an good linearity between logarithm of the ratio and the Sec concentrations?r = 0.9921?.The experimental detection limit was estimated to be 40 n M.The probe can well accumulate in mitochondria and selectively detect the concentrations of endogenous Sec in different cell lines.The probe can detect the relationship between Sec level and acute inflammation in CS₂-induced mitochondria related inflammation.In the CS₂-induced acute and chronic hepatitis mice models,we examine the relationship between the concentration of Sec and the degree of CS₂-induced liver damage.The above applications make our probe a potential candidate for fluorescence analysis the organism health under CS₂ poisoning.