Dual-channel Ratiometric Fluorescent Probe For Detection And Bioimaging Of Reactive Sulfur Species

BackgroundReactive sulfur species（RSS）is a kind of endogenous sulfur bioactive substances,mainly containing hydrogen polysulfide（H₂S_n）and sulfur dioxide（SO₂）,cysteine（Cys）,glutathione（GSH）,homocysteine（Hcy）,etc.The abnormal expression of RSS is often closely related to many diseases.Therefore,it plays an important role in disease diagnosis and forensic identification to efficiently,accurately and real-time detect of them.Fluorescence probe has the advantages of good biocompatibility,high sensitivity,high selectivity,fast response,suitable for biological imaging,and has been widely used in the analysis and detection of RSS.However,there are still some problems to be solved,such as poor selectivity,low sensitivity or limitations in vivo application.And most probes exhibit single channel fluorescence and are easily disturbed by the environment and other factors.The ratiometric fluorescent probe based on the signal ratio of dual channels could effectively eliminate background interference and improve the selectivity and sensitivity of the probe,which has broad application prospects.ObjectivesThe dual-channel ratio fluorescent probes with good biocompatibility,high sensitivity,high selectivity,fast response were designed and synthesized for the detection of reactive sulfur species.They were applied by cell imaging of RSS to study the biological application value.Methods1.The fluorescent probe HF-NA-MC for Na₂S₂ detection,fluorescent probe NG-TCF for Na₂SO₃ detection and fluorescent probe ECH-AC for thiols detection were designed and synthesized,respectively.The structure of these probes were characterized by MS and NMR.2.The response ability of the probes to RSS including Na₂S₂,Na₂SO₃,Cys,GSH and Hcy were detected by UV-vis and fluorescence spectroscopy.3.Hep G2 cells and Hela cells were cultured,and MTT experiments were measured to study the cell viability of the probes.The fluorescence cell imaging was recorded by confocal microscope or inverted fluorescence microscope to study the biomedical application of the probes.Results1.Fluorescent probe HF-NA-MC:In the presence of H₂S_n,the ketone configuration of the probe was transformed into enol form.With the open of ESIPT process,the fluorescence emission shifted from 450 nm to 540 nm,with a red shift of 90 nm.The fluorescence intensity ratio of the probe(I₅₄₀/I₄₅₀)showed a good linear relationship with H₂S_n in the range of 0～120μM,and the ratio detection of exogenous hydrogen polysulfide in He La cells was well conducted.2.Fluorescent probe NG-TCF:In the presence of SO₂,the C=C double bond in the probe was destroyed,which inhibited the ICT process between donor and receptor.The emission of the probe shifted from 670 nm to 550 nm,and ratio intensity(I₆₇₀/I₅₅₀)showed a good linear relationship with the concentration of SO₃^2-in the range of 0-50μM.The probe was used to dual channel detection of exogenous and endogenous SO₂ in He La and Hep G2 cells.3.Fluorescent probe ECH-AC:In the presence of biothiols（Cys,GSH,Hcy）,the yellow fluorescence of the probe was quenched and showed good selectivity.The probe showed a good linear relationship with the concentration of biothiols（Cys,GSH,Hcy）in the range of 0～75μM,and further realized the detection of biothiols in Hep G2 cells.Conclusions1.A fluorescent probe HF-NA-MC based on flavone was synthesized and successfully detected polysulfides and exogenous H₂S_n in living cells by dual emission channels.2.A naphthalene imide based fluorescent probe NG-TCF with ICT mechanism was synthesized and successfully used for ratio detection of SO₂ derivatives.The cell imaging of exogenous and endogenous SO₂ in dual emission channels in He La and Hep G2 cells was realized.3.A fluorescent probe ECH-AC based on hydroxyl chalcone and acryyl groups was designed and synthesized,which was successfully used to selectively detect biothiols（Cys,GSH,Hcy）and fluorescence imaging in cells.