| Fluorescent probes have become an important tool for biological detection due to their simple technology,flexible structure,good sensitivity and non-invasive detection.Then,the cell environment is more complicated,and the fluorescent probe faces many problems whenit acts as a detector.Ideal fluorescent probes should have high permeability,no background interference,low photobleaching and phototoxicity to aid in real-time detection and long-term observation in biological systems without damaging the cells.In order to solve these problems,two-photon fluorescent probes were born.In addition,the perfect combination of two-photon microscopy imaging and two-photon probes facilitates probe advancement.Two-photon microscopy imaging technology uses near-infrared photons as excitation light source and long-wave low-energy excitation light,which improves the stability and space-time resolution of the probe during detection,reduces photobleaching and phototoxicity,and helps the probe in cells,tissue and in-vivo applications.In recent years,two-photon fluorescent probes have become an extremely important research tool in life medicineSubcellular organelles mitochondria not only provide continuous energy to cells,but also related to cell differentiation,proliferation,apoptosis and transmission of information.Intracellular viscosity is closely related to signal transduction,biomolecular interactions,and metabolite diffusion,as well as at subcellular levels.Abnormal changes in mitochondrial matrix viscosity induce changes in mitochondrial network,which in turn affects the spread of metabolism,which is associated with many diseases and dysfunctions(diabetes,Alzheimer's disease,atherosclerosis).Intracellular SO2 is mainly produced by oxidation of sulfur-containing amino acids or oxidative decomposition of sulfinylacetone.The cells are present in the form of HSO3-/SO32-and its salts in addition to pure SO2 gaseous molecules.SO2 can act as a gaseous signal molecule to mediate physiological processes,and can also act as an antioxidant and an anti-reducing agent to regulate redox balance.Mitochondrial SO2 is thought to significantly reduce myocardial damage and isoproterenol-induced mitochondrial swelling and deformation.Lysosomal is a common subcellular organelle in most animal/plant cells and it contains a variety of hydrolases.The lysosomal polarity is an important parameter in the biological microenvironment,which not only affects its shape,structure and membrane permeability,but also determines the interaction of hydrolase.The lysosomal polarity is related to the decomposition of macromolecules,the activity of hydrolase,the digestion of cell debris,autophagy,and the removal of damaged structures.In addition,the polarity of each region of lysosomes is significantly different.In the process of apoptosis,lysosomal polarity can be used as a reaction factor to observe changes in cell microenvironment,which is of great significance for studying cell biology and tumor pathology.Through a large amount of literature reading and the accumulation of work of this group,this paper designed and synthesized two kinds of microenvironment two-photon fluorescent probes with carbazole as the matrix and targeting subcellular organelles.The structural verification of the intermediate product and the target compound was carried out by means of precise chemical structure characterization.The optical properties and biological properties were tested and a series of meaningful results were obtained.1.A two-photon fluorescent probe MCB with cross-talk-free dual-detection mitochondrial viscosity and SO2 derivative without crosstalk emission was designed and synthesized.The strong electron-accept 3-methylbenzothiazole iodonium salt and the weakly electron-donor p-methoxyphenylacetylene are introduced into the N-ethyl-carbazole precursor to form a strong ICT system.When the MCB is monolithic,the 3-methylbenzothiazole iodonium salt can be used as a molecular rotor to detect the viscosity with the N-ethyl-carbazole parent double bond.The red fluorescence emission peak is at 566 nm.When the SO2 derivative is partially reacted with the ?,?-unsaturated vinyl-3-methylbenzothiazolium of MCB by a Michael addition reaction,the conjugated system is destroyed,and the remaining weak ICT system causes short-wave emission,The blue fluorescence emission peak is at 392 nm.As the SO2 derivative is added dropwise,the solution gradually changes from yellow to colorless.The detection of SO2 derivatives by MCB has a low detection limit(2 nM)and a fast reaction time(?60 s),which can efficiently and sensitively detect endogenous SO2 derivatives.In addition,when the MCB detects viscosity and SO2 derivatives,the fluorescence emissions do not interfere with each other.MCB has excellent two-photon performance,low cytotoxicity,and the depth of tissue penetration of mouse liver slices reaches 120 ?m.Cell imaging shows specific localization of mitochondria.MCB can quantitatively measure the viscosity in solution and cells,and quantitatively analyze the viscosity distribution of cells by fluorescence lifetime imaging.MCB can detect endogenous/exogenous SO2 derivatives in solution and cell mitochondria and visually detect viscosity and HSO3-of zebrafish.2.A two-photon polar fluorescent probe PCT with lysosomal localization was designed and synthesized.The two-photon fluorescent probe PCT was designed and synthesized by introducing a lysosomal targeting group and a polar responsive group to the carbazole matrix.In the probe molecule of PCT,the basic tetravinylpyridinyl group is a lysosomal localization group,the nitrogen ethyl carbazole is a two-photon fluorophore,and the triphenylaminoketene group is a polar responsive group and forms a good D-?-A structure,the typical ICT mechanism molecular configuration,is a necessary condition for the design of polar molecules,and each structural unit is connected to form a large conjugated system.It was found by optical test that PCT has a good fluorescence response to the polarity change of the solvent,and the polarity of the solution can be quantitatively detected.PCT has excellent two-photon performance and is unaffected by temperature and pH.Cytotoxicity and colocalization assays in HeLa cells showed low PCT cytotoxicity and better lysosomal localization.In addition,changes in lysosomal polarity during apoptosis were observed by two-photon fluorescence confocal cell imaging. |
PDF Full Text Request