Design Of Fluorescent Probe Based On ICT Mechanism And Its Application In Imaging Of Organelle Microenvironment

Organic small molecule fluorescent probe has the unique performance of real-time in-situ non-invasive monitoring of biomolecules in living cells,and is a powerful tool in the field of life medicine.Among them,intramolecular charge transfer（ICT）is a common mechanism for constructing fluorescent probe molecules,and D-π-A structure is an important component of typical ICT mechanism.A probe based on this principle usually includes an Acceptor and a Donor,which are connected by a conjugated linking group.Through the process of intramolecular electron transfer,the molecules are in a charge transfer state,and the detection of analytes,such as SO₂,CO and other gas signal molecules,viscosity and polarity in the microenvironment can be realized through the change of fluorescence signals.Viscosity is one of the important factors to measure the microenvironment of cells,which affects the diffusion rate of substances between cells.The change of cell viscosity level can induce various diseases or physiological disorders,such as hypertension,diabetes,atherosclerosis and malignant tumor,so it is of great significance to develop a tool to detect viscosity for biological application.Like viscosity,polarity is also an important parameter to detect the changes of cell microenvironment.Usually,the polarity of tumor cells is different from that of normal cells.Therefore,we can realize the diagnosis of diseases by detecting the parameters of cell polarity,that is,developing a tool that can detect cell polarity is of great significance to biological applications.SO₂ is a gas signal molecule and a double-edged sword.Normal concentration of SO₂ can regulate cardiovascular function,control inflammatory reaction and maintain intracellular redox balance.Excessive intake of exogenous SO₂ will produce uncomfortable physiological reactions such as respiratory insufficiency and allergy,and will also destroy the normal metabolism of the human body,which will lead to lung cancer,cardiovascular diseases and many nervous system diseases.Similar to SO₂,CO is also a gas signal molecule.Appropriate amount of CO can relax blood vessels,regulate blood pressure,prevent infection and inhibit inflammatory reaction.Excessive CO can cause human poisoning,shock and organ failure.At present,the interaction mechanism between CO and SO₂ is still not completely clear,so it is of great significance to develop a tool that can simultaneously monitor the contents of CO and SO₂ for biomedical applications.In this paper,lipid-targeted fluorescent probes A-XB,which can simultaneously detect SO₂ and viscosity,were designed and synthesized based on ICT mechanism.A fluorescent probe KM-D for simultaneously detecting SO₂ and CO and a fluorescent probe WA-C for detecting SO₂;Fluorescent probe XY-CN and fluorescent probe XY-CNN for detecting polarity.（1）Based on ICT mechanism,a fluorescent probe A-XB with push-pull electron effect is designed.Naphthalimide is used as a fluorophore,which is connected with indole salt through C=C to form a conjugated system.The probe is sensitive to viscosity,and the red fluorescence increases at 647 nm with the increase of viscosity.With the addition of SO₂,the fluorescence at 647 nm is weakened,while the fluorescence at 540 nm is enhanced.SO₂ can also be detected by the intramolecular charge transfer（ICT）process of donor 1,8-naphthalimide and acceptor indole salt under the condition of viscosity.A-XB has good ratio characteristics after reacting with SO₂,long emission（647 nm）,high selectivity,low detection limit and rapid identification（<10 s）.In addition,the probe can also be used for the imaging study of SO₂and viscosity in He La and Hep G2 cells.The probe is sensitive to viscosity,and the red fluorescence increases at 647 nm with the increase of viscosity.With the addition of SO₂,the fluorescence at 647 nm is weakened,while the fluorescence at 540 nm is enhanced.SO₂ can also be detected by the intramolecular charge transfer（ICT）process of donor 1,8-naphthalimide and acceptor indole salt under the condition of viscosity.A-XB has good ratio characteristics after reacting with SO₂,long emission（647 nm）,high selectivity,low detection limit and rapid identification（<10 s）.In addition,the probe can also be used for the imaging study of SO₂ and viscosity in He La and Hep G2 cells.（2）Based on the ICT mechanism,using coumarin as fluorophore,the fluorescent probe KM-D which can detect SO₂ and CO at the same time and the ratio fluorescent probe WA-C which can identify SO₂ inside and outside living cells were designed and synthesized respectively.In the absence of SO₂,the probe WA-C emits intense red fluorescence at 672 nm.In the presence of SO₂,the ICT mechanism is destroyed due to the break of the double bond,resulting in green fluorescence at 507 nm and blue shift of the probe fluorescence.The probe achieves excellent specificity for SO₂ and short recognition time.In cells,probe WA-C can detect SO₂ from both exogenous and endogenous aspects.（3）Based on the ICT mechanism,two fluorescent probes XY-CN and XY-CNN were designed and synthesized with dimethylaminonaphthalene formaldehyde as the fluorophore.With the increase of solvent polarity,the emission peak of probe XY-CNN gradually shifted from 555 nm to 675 nm.At the same time,due to ICT effect,the fluorescence quantum yield decreases with the increase of solvent polarity.These results show that the probe XY-CNN is sensitive to environmental polarity.The probe XY-CNN can specifically locate the lipid droplets,which has the specificity of lipid droplet staining and good light stability,and can be used for three-dimensional confocal imaging and visualization of the spatial distribution of lipid droplets.In addition,spectral scanning imaging shows that the polarity sensitive fluorescent probe can also quantitatively determine the polarity of lipid droplets.