| As the field of fluorescence continues to grow steadily in both fundamental aspects and in applications,fluorescent probes and fluorescence imaging have become indispensable tools in molecular biology,biophysics,biochemistry,clinical diagnostic analysis and environmental chemistry.Due to the development of technology in different fields,for example,from high pressure mercury lamp to laser device that can generate steady laser,from multilayer coating technology to diffraction grating,fluorescence microscope has gone through fast development during last century.From wide-field fluorescence microscope to confocal laser scanning microscope and super-resolution fluorescence microscope that breaks the diffraction limit,functions of fluorescence microscope are becoming more and more powerful,so do the ability of imaging and imaging resolution.Fluorescence microscope cannot work without fluorescent probes,development and improvement of fluorescent probe have becoming key of the key to polish fluorescence imaging technology.As early as in last century,people found that different side chains can change the permeability of fluorescent probes and influence cellular imaging of fluorescent probes.However,the effects of side chains on fluorescent probes are more than that.Modulation/modification of side chains can change the lipophicity,cellular delivery and selectivity of fluorescent probes in a controlled manner without changing photophysical property.In addition,side chains can also be second targeting group due to the hydrophobic interaction between side chains and plasma membrane.In combination with successful applications of side chains in our group,this paper takes side chains modulation/modification as methods to design fluorescent probes and mainly carried out the following work:(1)Fluorescent probes that utilized staining localization to visulally discriminate normal situation and near-zero situation of cell membrane potential(CMP)were developed.Although CMP is typically maintained around-60 mV,in some special cases or in some special cells,CMP can be greatly depolarized to near zero,in which situation they are called "near-zero CMP".Up to now,the fluorescent probes that can discriminate the two CMP situations are labor-intensive and time-consuming,and all need tedious calibration and complex data processing.By modulating alkyl chains in widely used carbazole fluorophore,we developed two fluorescent dyes which can visually discriminate near-zero and normal situations of CMP in a point-to-point manner by two fluorescent images.They can enter the cytoplasm of normal cells due to the electrostatic interation with CMP.Meanwhile,when cells were in near-zero situations,they stay on the plasma membrane due to the hydrophobic interactions of the alkyl chain.Although they are cationic dyes,they are not affected by mitochondrial membrane potential.Despite that they cannot measure CMP quantitatively like traditional potentiometric fluorescent probes,they avoid calibration and data processing in the application.(2)Most mitochondrial probes will fall off during depolarization of mitochondrial membrane potential(MMP),which makes them hard to track mitochondria in long term Present mitochondrial tracking probes untilze covalent bonds to anchor on mitochondria through chemistry reactions.Although this methods realized long-term tracking of mitochondria,they can cause irreversible damge to mitochondrial proteins and lead to high cytotoxicity.In combination with previous research,this paper obtained a reaction-free,MMP-independent fluorescent probe for long-term mitochondria tracking,SP-NBD,by introducing NBD group into the side chain of a traditional mitochondrial probe.The introduction of NBD dramatically increase the lipophilicity of SP-NBD.And the planar structure of NBD increase the rigidity of the molecule,which decrease the permeability of the probe dramatically.When MMP decreases,low permeability of SP-NBD makes it hard to diffuse across mitochondrial membrane and fall off mitochondria,which enable SP-NBD to track mitochondria in long term.(3)Design strategies of cell viability probes are numerous nowadays,among them,the simplest and most effective one is to monitor change of cell membrane permeability.Because no matter in apoptosis cells or in necrosis cells,permeability of cell membrane will increase dramatically in the end.The difficulty of the design strategy is modulation of permeability in order to make probes impermeable to live cells and permeable to dead cells.Herein,by further decrease the permeability of CPS,green cell viability fluorescent probe ECPS was developed.This probe cannot diffuse across cell membrane of live cells,but can diffuse across cell membrane of dead cells and stain cytoplasm and nucleolus simultaneously.There are two main point in this design strategy.Firstly,positive charge have electrostatic interaction with CMP,and can drive probes enter into live cells.So,sulfonate that can neutralize positive charge and increase hydrophilicity were introduced.Secondly,long alkyl chains have hydrophobic interaction with cell membrane which make probes possessing alkyl chains insert cell membrane.Considering that cell membrane have exchange of substance with intracellular organisms,long alkyl chains can cause internalization of probes.Shortening long alkyl chains will make probes unable to insert into cell membrane and drift away from cells,avoid internalization.This probe can mark dead cells in live cells communities.After that,red cell viability fluorescent probe SQ was synthesized based on the same design strategy.The subsequent experiment proved that SQ had the same ability of ECPS,which demonstrate the universality of this design strategy.(4)Present lipid droplet probes target lipid core of lipid droplets by increasing lipophilicity of probes.Experiments in this paper found that excessively high lipophilicity of probes can be counterproductive.We reduced two benzene rings and introduced a hydroxyl group on a fluorescent dye BPAPY which has no selectivity.Finally,we obtained an excellent lipid droplet fluorescent probe BPANA.The modification of functional groups decreases clog P of BPANA from 7.13 to 5.73.In combination with previous work in our group,the membrane of lipid droplets is comprised of amphiphilic phospholipid molecule,in which the hydrophilic part is oriented toward outside of lipid droplets.So,excessively high lipophilicity may hinder probes diffusing acorss lipid droplets membrane.In addition,fluorescence of BPANA is very stable,independent of polarity and viscosity,even do not quench in water.This property means that the reason for the probe lightening up lipid droplets only can be high selectivity of the probe to lipid droplets.Apart from high selectivity,BPANA has excellent photostability,low cytotoxicity,is a powerful tool for lipid droplets imaging.In all,we designed and synthesized a series of functional fluorescent probes for different uses based on side chains modulation/modification strategy in this paper.The strategy takes full advantage of side chains' effects on lipophilicity,targeting ability,permeability of fluorescent probes,endowing them with different staining properties from original fluorescent probes. |
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