Design And Synthesis Of Organic Molecular Fluorescent Probes And Their Applications In Molecular Recognition And Cell Imaging

Anion-cationic ions,amino acids,and biological enzymes in the organism are involved in the physiological and pathological processes of many life activities,which play an important role in human health.Fluorescent probe is an ideal detection tool for fluorescence analysis.Because of their advantages such as high selectivity,fast response,good biocompatibility,and high space time resolution,which have been widely used used and concerned in bioimaging,medical diagnosis and other fields.Therefore,it is of great significance to design and develop a fluorescent probe with excellent performance for the recognition of specific biomolecules to provide effective information for early clinical disease diagnosis.In this paper,a series of organic molecular fluorescent probes based on coumarin dyes,cyanine dyes,and diphenylpyrrolidine as fluorophore were designed and synthesized for biomolecular recognition and intracellular fluorescence imaging.By studying their spectral properties,detection performance,and response mechanism to achieved specific detection of several bioanalytes and intracellular fluorescence imaging.The main research contents of this paper are summarized as follows:1.Design and synthesis of a simple dual-signal fluorescent probe and application of intracellular biothiol imagingBased on the different reaction activities of biothiols,the 4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole（NBD-Cl）as a sensing group,the coumarin-6-carboxylic acid as the fluorophore to developed a dual-fluorescence signals probe（NC-NBD）for rapid sequence sensing biothiols.When homocysteine（Hcy）and cysteine（Cys）were introduced into the probe,the emission intensity at 550nm gradually increased with 460 nm wavelength excitation,while the fluorescence intensity at 520 nm showed no significant change with 420 nm wavelength excitation.Similarly,when glutathione（GSH）was added,the emission intensity at 520 nm was significantly enhanced under 420 nm wavelength excitation,while the fluorescence intensity at 550 nm was almost unchanged under the excitation wavelength of 460 nm.It can be seen that the Hcy/Cys and GSH can be distinguished by adjusting two different fluorescence signals（green and yellow）.In addition,the probe has a rapid fluorescence response to biothiols,which is suitable for real-time monitoring of biothiols in biological systems.The fluorescence response mechanism of probe to the biothiols is verified and explained by studying the spectral characteristics,the density functional theory,and the HRMS.Moreover,since the probe has high sensitivity and selectivity,it has been successfully used for the determination of the levels of Cys/Hcy and GSH in human serum samples.The results were satisfactory,which indicates that this method is effective and feasible,and it has potential application value in analyzing biological samples.Furthermore,the probe was applied to the cells,showing low toxicity and good biocompatibility,and it successfully used two fluorescence signals to realize fast sequence sensing of intracellular biothiols and fluorescence imaging.It shows that the probe provides an effective way to understand the physiological function of biothiols in cells.2.Synthesis of hydrophilic fluorescent probes and its determination of glutathione metabolites in living cellsAccording to HSO₃^- has good nucleophilicity,a hydrophilic fluorescent BI probe has been synthesized based on the design of cyanine dye as a fluorophore for sensitive detection of intracellular GSH metabolites（SO₂ derivatives）.The BI probe exhibits good fluorescent response and good photostability in PBS media（pH 7.4）.When HSO₃^- is added,the unsaturated bond of BI probe reacts with HSO₃^- by nucleophilic addition.The two corresponding types of excitations(λ_ex:530/400 nm)are generated by two independent emissions(λ_em:575/470 nm),the fluorescence color changed from red to blue to achieve the detection,thus a double-excited BI probe was constructed to realize sensitive detection of SO₂ derivatives in aqueous media.The fluorescence response mechanism was explained by means of ¹HNMR,HRMS,and density functional theory.In addition,BI probe showed pH-sensitive features and is expected to be used as a pH sensor.The application of hydrophilic BI probe in cells,showing good biocompatibility,and it was successfully applied to detect SO₂ derivation and fluorescence imaging in HeLa cells through dual fluorescence signals.Simultaneously,BI probe was used to monitor the level of SO₂ derivatives generated by enzymatic GSH transformation in HepG2 cells using this dual-color pattern,and the results were satisfactory.The probe is expected to provide a reliable tool for monitoring the level of intracellular SO₂ derivatives.3.A sensitive fluorescent bioprobe for detection esterase activity in living cells and its application in intracellular fluorescent imagingTwo sensitive fluorescent probes（VA and VP）were developed by using acetyl and benzoyl as sensing groups as a fluorophore,which were used for biosensing esterase activity in living cells and evaluating cells health status.In different detection systems,the two probes showed strong yellow fluorescence.When probe VA and VP were introduced of esterase into the system,the ester bond of probes was broken due to enzymatic hydrolysis,causing the emission peak to be red shift to 587 nm and 585nm,respectively,which presented red fluorescence.Herein,two different fluorescence signals（yellow-red）were used to visualize detection of esterase activity.The results show that the probe has excellent detection performance,fast response time,and high sensitivity,the detection limit are as low as 7.15×10^-6and 9.10×10^-6 U/mL U/mL.Furthermore,the detection mechanism was explained by HRMS and spectral properties.In living cells,the probe VA showed good biocompatibility and high spatial resolution.The probe VA successfully achieved the intracellular fluorescent imaging and biosensing esterase in living cells as well as the health status of H₂O₂-induced He La cells was evaluated using this dual-colors pattern,suggesting that probe VA could be used to judge and evaluate the health status of cells,which provides an effective means for evaluating intracellular esterase activity.4.Design a fluorescent“Turn-on”biological probe and its imaging study of intracellular hydrogen peroxideIn this chapter,a novel fluorescent“Turn-on”bioprobe 1 was designed and synthesized based on diphenylpyrroline as the fluorophore and benzyl borate ester the recognition group for the sensitive detection of H₂O₂ and glucose.Based on the high oxidation activity of H₂O₂ to benzyl borate,probe 1 has good selectivity and sensitivity,and realizes high efficient fluorescence“Turn on”response to H₂O₂.The detection concentration of H₂O₂ ranges from 6 to 550μM,and its LOD can be as low as 0.11μM.HRMS and HPLC were used to explain the detection mechanism.Probe 1 realized indirect fluorescence“Turn-on”determination of glucose catalyzed by glucose oxidase.The method was used to determine the glucose content in fetal bovine serum simulated human serum,and the results is satisfactory,indicating that probe 1 has a potential application value in diagnosis of diabetes.In addition,probe 1,as a biocompatible molecule,was successfully used to monitor H₂O₂ levels and fluorescence imaging in exogenous living He La and Hep G2cells,indicating that probe 1 has potential biological application prospects.