Construction And Imaging Application Of Fluorescence Probe Based On ICT Mechanism

Fluorescent probes can track small biologically active molecules in real-time in situ and non-invasively,so they are widely used in the field of life sciences.ICT mechanism is often used to construct fluorescent probes,which have typical“D-π-A”structure.Intracellular viscosity is an important microenvironment factor,which determines the diffusion and control process of species,and plays a crucial role in signaling,enzyme catalysis and other life activities.Abnormal viscosity is often associated with a variety of diseases,including hypercholesterolemia and diabetes.Therefore,simple and rapid detection of viscosity changes in vivo is of great significance for disease prevention and early diagnosis.Nitroreductase（NTRs）is an important member of the flavin protease family and is often highly expressed in tumor cells.NTRs are important markers of tumors and have been used as targets for diagnosis and treatment of cancer.Therefore,it is of great significance to monitor the viscosity and NTRs levels in organisms in related physiological and pathological processes.Esterase is a kind of lipid hydrolase,which is involved in many physiological activities such as gene expression and metabolism,and is also an important index to evaluate cell morphology and vitality.Therefore,abnormal esterase activity is associated with many diseases,such as obesity and cancer.As a common gas messenger molecule,SO₂is a double-edged sword.The duality of SO₂is closely related to its concentration.In general,low concentrations of SO₂play a positive role in regulating cardiovascular function and lowering blood pressure,among other physiological processes.However,high concentrations of SO₂may cause some damage to physiological functions,resulting in some diseases,such as chronic poisoning,nervous system diseases and cancer.However,the exact concentration threshold is still unclear.Therefore,it is of great physiological significance to detect SO₂and esterase and their interactions separately and simultaneously.Based on ICT mechanism,fluorescence probes TC-P,NVP and ES were designed and synthesized,and their optical properties and biological applications were studied.The specific research content of this paper is as follows:（1）Based on the ICT fluorescence mechanism,an electroneutral LDs viscosity fluorescence probe TC-P was constructed by introducing electroneutral electron-pulling group（-CN）,which was used to selectively track the viscosity changes of LDs.The molecular skeleton of probe TC-P has a typical"D-π-A"structure,consisting of7-diethylamine-coumarin and benzothiazol-2-acetonitrile coupled by the C=C bond.The spectral results showed that the probe TC-P improved the sensitivity to viscosity response and had higher fluorescence contrast（the relative difference of fluorescence intensity at different viscosities）by introducing cyanogen group（-CN）with stronger electroneutral electron-pulling effect.In addition,the probe TC-P has the advantages of good sensitivity,selectivity and optical stability to viscosity.Imaging results demonstrated that TC-P can be rapidly and selectively anchored to LDs in living cells and can be used to track in situ changes in LDs viscosity during monensin and nystatin induction and ferroptosis.（2）Based on the ICT fluorescence mechanism,we constructed a double-recognition fluorescence probe NVP,which can achieve specific responses to the viscosity and NTRs in cellular lysosomes and zebrafish through the change of fluorescence signals with different colors.The probe NVP connects the electron donor（N,N-diethyl coumarin）to the electron acceptor（p-nitrobenzene）byπ-bridge conjugation.In addition,the viscosity response site of NVP was C-N in coumarin,and the nitro group was the response site of NTRs.The spectral test results show that the probe NVP has good response ability to viscosity and NTRs.In addition,NVP has high selectivity,good photostability and low biological toxicity.At the same time,the results of cell imaging show that NVP can be accurately aggregated in lysosomes,and the changes of lysosomal viscosity under chloroquine stimulation and the changes of lysosomal NTRs activity under hypoxia are detected.NVP further realized the visual detection of viscosity changes in living zebrafish and NTRs activity changes in zebrafish embryos.（3）Based on ICT fluorescence mechanism and logic gate strategy,a novel dual-site fluorescence probe ES was designed and synthesized to detect SO₂and esterase in mitochondria of living cells,and to explore the role of SO₂in cells.ES selected 1,8-naphthalimide with high fluorescence quantum yield,large Stokes shift and easy modification as fluorophore,and olefin bond and acetyl phenol hydroxyl group as response sites of SO₂and esterase,respectively.The spectral test results show that the probe ES has excellent response to SO₂and esterase,and has a low detection limit.In addition,ES has high selectivity,low biotoxicity and precise mitochondrial targeting.Studies have found that lower concentrations of SO₂have more positive effects on organisms,while higher concentrations have more negative effects.So,SO₂is a double-edged sword,but the specific concentration threshold is still unclear.The results of biological imaging show that probe ES can not only detect SO₂and esterase in mitochondria of living cells separately and simultaneously,but also successfully achieve the purpose of further measuring cell activity through esterase activity after responding to different concentrations of SO₂.