Construction Of Dual-channel Ratio Fluorescence Probes And Their Applications In The Detection Of Sulfur Dioxide

BackgroundSulfur dioxide（SO₂）,as a well-known major environmental pollutant,widely exists in water,food and organic organisms as SO₃^2-/HSO₃^-.And SO₂,as a gas transmitter,is generated endogenously through thiosulfate thiotransferase（TST）pathway or sulfur-containing amino acid metabolism,and participate in the regulation of redox process.Abnormal concentration of SO₂in organisms could lead to many disease such as cardiovascular diseases,endocrine abnormalities and even cancer.Therefore,the development of effective monitoring methods of SO₂and its derivatives is helpful to further study the physiological and pathological effects of SO₂,and has broad application prospects in crop safety,food safety,individual health,judicial identification and other aspects.Small molecule fluorescent probe is considered as a powerful tool because of its excellent characteristics such as instantaneous simplicity,sensitive and non-destructive,and high spatial and temporal resolution.Compared with the reported single-channel fluorescent probes,the ratio fluorescent probes have the ability to resist background interference,self-calibrate,and not be affected by concentration and equipment,which has attracted wide attention.Now the fluorescent probe for SO₂detection suffer from some drawbacks such as low energy transfer efficiency,small distance between two emission wavelength,easily disturbed by other thiols.Therefore,it is valuable to develop novel ratiometric fluorescent probes for SO₂detection with high selectivity.ObjectiveA ratio fluorescent probe with fast response,high selection sensitivity,low cytotoxicity,good living cell biological imaging,and excellent resistance to environmental background interference was designed and synthesized for the detection of exogenous SO₂in cells and food,so as to promote the application of dual-channel ratio fluorescent probe in food safety and explore the metabolic distribution of SO₂in organisms.Methods1.Three fluorescent probes YN-PQ-D,LLZ-IPY,and GXY-IPY for SO₂detection were designed and synthesized based on the Knoevenagel condensation reaction.The structure and purity of the probes were confirmed by HR-MS,¹HNMR,and other analytical equipment.2.UV-vis spectrophotometer and fluorescence spectrophotometer were used to detect the response of the constructed fluorescence probe to Na₂SO₃molecules.3.Cervical cancer cells He La and hepatoma cell Hep G2 were cultured to verify the toxicity of the probe.The feasibility and application value of the probe in living cells was explored by cell fluorescence imaging.4.Fluorescence spectroscopy was used to detect the content of SO₂in yuba and rock sugar,and to explore the application prospect of fluorescence probe in food safety.Results1.Fluorescent probe YN-PQ-D:the probe is constructed by connecting the benzopyran group as the response site of SO₂and the dansyl chloride fluorophore through a piperazine bond.The probe YN-PQ-D is regulated by FRET.When excited by light,the fluorescent group of benzopyran is the energy acceptor and the dansyl chloride unit is the energy donor,showing red fluorescence.In the presence of Na₂SO₃,due to the nucleophilic addition of SO₃^2-to benzopyran groups,FRET was blocked,YN-PQ-D showed red fluorescence weakened,green fluorescence enhanced,and emitted ratio fluorescence.The fluorescence intensity ratio(I₅₅₅/I₆₄₅)of YN-PQ-D has an excellent linear relationship with SO₃^2-in the range of 0-200μM,and the detection limit is 0.327μM.The specific detection of exogenous sulfur dioxide in He La cells was realized.2.Fluorescence probe LLZ-IPY:by combining ESIPT unit 2-（1-phenylimidazolo[1-phenylimidazolo]pyridine-3-yl）phenol group and cyanine dye,a near-infrared fluorescence probe LLZ-IPY with response to Na₂SO₃ was constructed.In the presence of SO₂,the double bond C in the attack probe destroys theπbridge structure between the two groups,which results in the inhibition of the ICT mechanism,the disappearance of red fluorescence,the opening of intramolecular proton transfer effect in ESIPT unit,the emission of blue fluorescence,and the migration of emission peak of 226 nm in the fluorescence spectrum.The existence of SO₃^2-leads to the proportional change of fluorescence intensity of probe LLZ-IPY(I₄₈₀/I₇₀₆).In addition to the selectivity of SO₃^2-being better than that of other ions,LLZ-IPY also has high sensitivity（LOD=0.03μM）.In addition,cell experiments showed that the probe LLZ-IPY had good cell membrane permeability and mitochondrial targeting,and could be used to observe the exogenous SO₃^2-in Hep G2 cells.3.Fluorescence probe GXY-IPY:by combining benzopyran salt unit and IPY aldehyde unit,the fluorescence probe GXY-IPY,IPY aldehyde fluorophore for a specific response to SO₂has ESIPT characteristics.When combined with SO₂,the ESIPT process is turned on,the original red fluorescence(λ_em=745 nm)is quenched,and the spectrum has a large blue shift and emits blue fluorescence(λ_em=480 nm).The probe GXY-IPY has a near-infrared emission wavelength（745 nm）,high sensitivity（0.21μM）,large spectral migration（265 nm）,and proved the reversibility of HCHO and GXY-IPY-SO₂in vitro.It has been successfully applied to the detection of SO₂in food yuba and rock sugar.ConclusionFluorescent probes YN-PQ-D,LLZ-IPY,and GXY-IPY were designed and synthesized based on FRET,ESIPT,and other mechanisms,and were successfully applied to fluorescence imaging experiments of cells and detection of SO₂ in food.