| In recent years,near-infrared fluorescence imaging technology,which has the characteristics of high sensitivity,strong tissue penetration,low background fluorescence interference and little tissue damage,has become an important method for detecting biological processes in cells and living bodies.Therefore,the use of near-infrared dyes to develop fluorescent probes is more conducive to cell imaging.As one of the classical fluorophores,xanthene fluorescent dyes are often used to develop small molecular fluorescent probes because of their high molar extinction coefficient,high fluorescence quantum efficiency and good optical stability.However,the maximum absorption wavelength and emission wavelength of traditional xanthene dyes are concentrated in the visible light region,and the Stokes shift is small(<30nm).These deficiencies make it difficult to apply to live cells and in vivo imaging.In recent years,researchers at home and abroad have modified traditional xanthene dyes and reported a variety of fluorescent dyes with long wavelengths.Although the wavelength of these dyes is significantly red-shifted compared with traditional xanthene dyes,their Stokes shift is still not significantly improved(<30 nm).Therefore,it is necessary to develop and design long-wavelength xanthene fluorescent dyes with large Stokes shift.This thesis focuses on modifying traditional xanthene dyes,designing and synthesizing a series of near-infrared xanthene fluorescent dyes with near-infrared emission and large Stokes shift.At the same time,based on the structural characteristics of the dye,the dye is applied to the design of the small molecule fluorescent probe.This paper mainly carried out the following work:(1)On the basis of?-conjugated linear extension xanthene dyes,by introducing electron-withdrawing halogen atoms and fusing terminal nitrogen to increase the rigid plane,a series of long-wavelength near-infrared fluorescent dyes(TJ-Cl R,TJ-FR and TJ-FRN)with large Stokes shift were designed by reacting N-ethyl-2-methoxy phenothiazine with 2,3,4,5-tetrachloro(fluoro)-6-[(2,3,6,7-tetrahydro-8-hydroxy-1,1,7,7-tetramethyl-1H,5H-benzo[ij]quinolizin-9-yl)carbonyl]benzoic acid and 2-[4-(dieth ylamino)-2-hydroxybenzoyl]-3,4,5,6-tetrafluorobenzoic acid respectively.The spectral performance and cell imaging performance of this type of dye are studied in detail.Compared with the classic Rhodamine B dyes,the maximum absorption wavelengths of the three dyes TJ-Cl R,TJ-FR and TJ-FRN in ethanol after the introduction of halogen atoms(Cl/F)in the bottom ring of the spiro ring are respectively 627 nm,622 nm and 632 nm,the maximum emission wavelengths are at753 nm,751 nm and 764 nm;the Stokes shifts of the three dyes are all greater than130 nm,which can effectively reduce the interference of biological background signals.By fusing the terminal nitrogen to increase the rigid structure,the fluorescence quantum efficiency of the new dyes is effectively improved.In addition,the three dyes have the characteristics of a wide p H range,low cytotoxicity and good membrane permeability.From the structural analysis,the dyes not only retain the spiro lactone structure of the classic dyes that can control the“off-on”fluorescence signal,but also the dyes themselves have the S atoms that can be oxidized by active oxides and the 4-position F atom of the bottom ring with high reactivity,which can be used to construct a variety of small molecule fluorescent probes.(2)Based on the above work,the probe TJ-FRP was designed and synthesized by introducing a triphenylphosphine functional group into the F atom at position 4 of the bottom ring of the dye TJ-FR.The photophysical properties and cell imaging properties of TJ-FR and TJ-FRP toward viscosity were explored through spectral testing and cell imaging experiments.The maximum emission wavelengths of probes TJ-FR and TJ-FRP in aqueous solution are 758 nm and 748 nm,respectively.Due to the vibration of the molecular plane,the fluorescence quantum efficiency is low;however,the fluorescence intensity of probes TJ-FR and TJ-FRP in 95%glycerol aqueous solution was significantly enhanced at 735 nm and 725 nm,respectively.Both of them show high selectivity,fast response and wide p H range for environmental viscosity.Based on cell imaging experiments,the probes TJ-FR and TJ-FRP have low cytotoxicity,and can detect the viscosity in the lysosomes and mitochondria of the subcellular organelles.(3)The NIR emission fluorescent dye TJ-FR and the derived probe TJ-FRP contain S atoms reaction sites that can react with active oxides in their structures.The response capability of the two probes to the active oxides were explored through UV absorption spectroscopy and fluorescence emission spectroscopy.The sulfur atoms contained in it are oxidized by hypochlorous acid to sulfoxide,which causes a significant blue shift in the UV absorption spectrum(?abs=524 nm);the fluorescence intensity is significantly enhanced at 608 nm,realizing the specific detection of hypochlorous acid by the probe.In addition,the probes TJ-FR and TJ-FRP not only have lower detection limit(9.5 n M and 17.7 n M,respectively)and fast response time(<10 s),but also can detect of exogenous hypochlorous acid in cells. |
PDF Full Text Request