| Polarity is an important parameter in chemical production,certain inorganic organic reactions are dependent on the participation of polarity.In the biological system,especially at the cellular level,the polarity is an important important component of the micro-environment that determines a series of complex physiological feedback mechanism and physiological maintenance function.Many intracellular physiological processes alter the intracellular polarity,such as the combination of most proteins and enzymes,the production of organelles,the response of the immune system,and the transmembrane transport of molecules.Polarity stability is an important condition for ensuring normal cell proliferation,differentiation,metabolism and functional activity,and abnormal changes in polarity are closely related to the production of some diseases.Therefore,the detection in the polarity of cell or organism is of great significance in the study and treatment of certain diseases.Fluorescence probe is the molecule which fluorescence signal can change with the corresponding changes in a certain system.In recent years,the two-photon fluorescent probe has been widely used in the field of biomedicine by virtue of its high stability,good light bleaching and high bioprecipitation.Therefore,the use of two-photon fluorescent probe to detect the changes in polarity of life has a broad application prospects.Lysosomes contain about 50 hydrolytic enzymes and determines the decomposition of exogenous and endogenous macromolecular substances as an organelle in eukaryotic cell.In autophagy,the material in autophagosomes meets enzyme hydrolytic enzymes in lysosomes when the membrane fusion between autophagosomes and lysosomes occurs.Therefore,in the membrane fusion,the polarity of the lysosomes would be changed,and the corresponding change of fluorescence signal provides us a new method to monitor autophagy.Mitochondria are existed in most cells.Mitochondria are the sites of aerobic respiration,and generally are the major energy production center in eukaryotes.In apoptosis,mitochondrial polarity will changed with the change of membrane potential.Thus,the detection of mitochondrial polarity is of great significance in the study of apoptosis.In addition,in the growth of biological cycle,the mitochondria polarity of cells in different stages of growth and cells in different tissue has some differences,so the detection of mitochondrial polarity plays an important role in study the growth of life.As a solvatochromic group,coumarin is sensitive to the change of polarity in its surrounding solvent with good spectral properties,solubility and low toxicity.Therefore,coumarin is very suitable for a polar probe fluorescence report group.In this paper,coumarin was modified and transformed to lysosomes or mitochondria targeting two-photon fluorescence probes in polarity on the bases of many literatures.These probes were characterized by 1H NMR,13C NMR,and ESI mass spectrometry accurately.The relation of their optical properties and thepolarity were studied furthermore.More importantly,the application of them in cells and organisms has been explored by two-photon fluorescence imaging.(1)we simply introduced structure of fluorescent probes with fluorescence emission mechanism,as well as the advantages of two-photon microscopy imaging technology.(2)A two-photon fluorescent probe Lyso-OC for polarity was designed and synthesized.The probe Lyso-OC molecule mainly consists of a 7-substituted coumarin,a target lysosomal targeting group morpholine and a 1-aceto-4-methoxybenzene and amide structure consisting of "Push-Pull "electronic structure.Among them,1-ethynyl-4-methoxybenzene not only provides a long conjugate structure for the probe which has good two-photon properties,but also larges the Stokes shift of the probe when the solvent polarity changes.More importantly,we observed the changes of polarity at different stages of autophagy with the fluorescence changes of Lyso-OC by two-photon fluorescence microscopy,and successfully monitored the autophagy.(3)A two-photon fluorescent probe Mito-OC for polarity was designed and synthesized.The probe Mito-OC molecule mainly consists of a 7-substituted coumarin,a target lysosomal targeting group morpholine and a 1-aceto-4-methoxybenzene and amide structure consisting of "Push-Pull "electronic structure.Through a series of optical tests,we found that the probe Mito-OC has a good ratiometric response of the solvent polarity,and could be used to detect the changes of the solvent polarity quantitatively.Cytotoxicity and co-localization experiments in MCF-7 cells showed that the probe Mito-OC was low cytotoxic and well targeted in mitochondria.In addition,by two-photon confocal fluorescence imaging and in vivo fluorescence imaging,we found the changes of mitochondrial polarity in apoptosis and the distribution of the polarity in different organs in zebrafish. |
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