Synthesis Of ?-NaYF₄:Yb,Er@CaF₂ Upconversion Nanoparticles And Its Application In Single Cell Tracking Imaging

Cell-tracking is a multidisciplinary cross technology which covering biology,physics,materials chemistry,and many other disciplines.It is widely used on the research of cancer mechanism,developmental biology,cell therapy,and so on.However,for early cell-tracking techniques special cells are usually modified with certain markers to track and monitor their group behaviors such as growth,development,division and reproduction.With many important breakthroughs in the biotechnology field people come to realize that the individual information of a single cell is closely related to numerous biological process and genesis of disease,which remain under wraps due to the average effect at cell population level.Therefore,access to individual specific information of cells is getting more and more attention,which has brought forward new requirements on cell-tracking technology.Typical markers for cell-tracking applications,includes fluorescent proteins,quantum dots,magnetic particles,radionuclides,and reporter gene.All of them have some serious defects,e.g.high cost,high biological toxicity,low biological penetration depth,autofluorescence in the tracking process.In recent years,as a kind of new cell-tracking marker,rare-earth-doped upconversion nanoparticles?UCNPs?have attracted extensive attention due to their low biological toxicity,high biological penetration depth and free autofluorescence in the tracking process.In order to further enhance the light transmittance of UCNPs,UCNPs with particular core-shell structure,i.e.?-NaYF₄:Yb,Er@Ca F₂,are prepared and characterized in the present dissertation.The real-time dynamic tracking experiments at single-cell level with UCNPs as markers are successfully demonstrated.The main innovations and research contents are as follows:?1?The ?-NaYF₄:Yb,Er@Ca F₂ UCNPs with uniform size,single crystallization phase,and high brightness are successfully prepared by using improved synthesis process based on traditional solvothermal synthesis method.Compare to traditional preparation technology,the present method has the superiority of low reaction temperature,simple apparatus,succinct operation process.The photoelectric properties of ?-NaYF₄:Yb,Er@Ca F₂ UCNPs have systematically been investigated by means of characterization techniques with Transmission Electron Microscope,X-ray diffraction,fluorescence spectroscopy,and Fourier Transform infrared spectroscopy.The results show that ?-NaYF₄:Yb,Er@Ca F₂ UCNPs have high conversion efficiency,small size,good monodispersity,and biocompatibility.Therefore,the fabricated UCNPs are particularly suitable for tracing applications at single–cell level.The AF₂?A= Ca?Sr?Ba?shell is further coated on the basis of the ?-NaYF₄:Yb,Er UCNPs prepared with solvothermal synthesis.By analyzing the spectral intensities and X-ray diffraction patterns of the nanoparticles with different core shell structures,we show that the Ca F₂ coating layer can be a great option to improve lattice matching and enhance luminescent intensity.The influence of the dopant concentration of Yb3+ ions on the upconversion luminescent intensity of ?-NaYF₄:Yb,Er2%@Ca F₂ UCNPs is systematically analyzed.The results suggest that a 60% dosage concentration can contribute to the optimal luminescent intensity with ⁶50 nm?red light?central wavelength.Comparing with ?-NaYF₄:Yb,Er UCNPs prepared by the same method,the ?-NaYF₄:Yb,Er2%@Ca F₂ UCNPs have higher luminous intensity,smaller size,and better biosecurity.By carrying out experiments towords hydrophilic modification,more appropriate UCNPs for single cell tracking are successfully prepared.?2?Based on ?-NaYF₄:Yb60%,Er2%@Ca F₂ UCNPs,two experimental schemes for single-cell labeling are proposed: microinjection and U-cell methods.The influences of experimental parameters on the effects of single cell labeling and cell activity are systematically studied.The optimal experimental parameters for both shemes are obtained.For the application of single cell tracking,two feasible schemes have been proposed,and the corresponding experimental setups are built as well.Two kinds of single cell markers are realized with the optimal experimental parameters.For the microinjection method,the best injection pressure of single cell hydrophilic UCNPs?0.1 mmol/ml?is optimized as 700 hpa.For U-cell method,the best co-incubation time of UCNPs and cells ranges from 10 to 12 hours.In addition,the optimal ratio of doping between co-cultured cells and unprocessed cells is 1:5.?3?The single-cell tracking experiments with ?-NaYF₄:Yb60%,Er2%@Ca F₂ UCNPs as makers are carried out with the platform of laser scanning confocal optical bioimaging.In this thesis,single cells are successfully labeled by ?-NaYF₄:Yb60%,Er2%@Ca F₂ UCNPs,followed by a series of operations like cell preparation,cell staining,cell immobilization,etc.Based on the laser confocal scanning microscopy platform,single cell tracking experiments are carried out based on He La cell samples.Finally,we real-time and continuously trace single He La cell for over 4 hours.