Preparation And Characterization Of Carbon Dots Derived From Poria Cocos Polysaccharides And Their Application In Ion Detection And Cellular Dynamics

Carbon dots,as a new nano sc ale luminescent material,excellent photoluminescence property and good biocompatibility.At present,the synthesis of carbon dot has many disadvantages,such as complicated operation,high cost and raw material not environmental protection.Therefore,based on the chemical concept of green and environmental protection,it has become a hot research topic to design and synthesize carbon dots derived from traditional Chinese medicine with cheap,easily obtained,renewable and good biocompatibility of traditional Chinese medicine or Chinese medicine ingredients as raw materials to improve the inherent characteristics,such as solubility,particle size distribution,etc.,and to explore and expand the application of carbon dots derived from traditional Chinese medicine.Objective:In this study,alkali-soluble Poria cocoa polysaccharide was used as carbon source and ethylenediamine as nitrogen source.Through single factor and orthogonal experiments,Poria cocoa polysaccharide-derived carbon quantum dots were prepared.Due to its excellent fluorescence characteristics,low cytotoxicity and good imaging properties,this paper tries to explore its application in ion detection and cellular dynamics studies and evaluates the feasibility of its application in the detection of heavy metals in environmental water samples and biological studies.Methods:(1)Nitrogen-doped carbon quantum dots(N-CDs)were prepared by one-step hydrothermal method using alkali-soluble Poria cocoa polysaccharide as carbon source and ethylenediamine as nitrogen source and were characterized by transmission electron microscopy(TEM),UV-vis absorption spectrum(UV-Vis),photoluminescence(PL)spectrum,X-ray photoelectron spectroscopy(XPS)and Fourier infrared spectroscopy(FTIR).(2)N-CDs is specific for metal ions and can be selectively quenched by Cr6+,which can be used as a label-free fluorescent probe for rapid detection of Cr6+.The detection mechanism was discussed by fluorescence lifetime and Stern-Volmer equation,and successfully applied to actual water samples.(3)Two different cell lines,4T1 mouse breast cancer cells and immune-mouse RAW264.7 cell,were used as models.The uptake kinetics,uptake mechanism,cell distribution and excretion process of N-CDs were observed by confocal laser scanning microscope(CLSM)and fluorescence activating cell sorter(FACS).Results:(1)The carbon dots prepared in this experiment have good water solubility,uniform particle distribution,and an average particle size of 4.61 nm.Under the irradiation of 365 nm UV lamp,the solution of N-CDs emitted a strong blue light.UV-Vis spectra and fluorescence spectra show that N-CDs has excellent fluorescence characteristics,fluorescence quantum yield is 4.82%,and good fluorescence stability under strong acidic conditions,which can be considered for imaging application in cancer cells.What's more,XPS and FTIR spectrum showed that the nitrogen element was successfully doped,the surface was rich in hydrophilic groups,and had good hydrophilicity.Moreover,N-CDs prepared in this experiment had good biocompatibility and low cytotoxicity,which could be used in later application such as cell imaging and cell labeling.(2)By means of various metal ions filter found N-CDs for Cr6+,has high sensitivity,within 0-100 ?M showed a good linearity,the linear equation for the F0/F=0.01472C+0.9942(R2=0.9922),the detection limit of 0.25 ?M,ion detection in water samples could be successfully applied in practice,the quenching mechanism was mainly through the filtering effect and static fluorescence quenching process.(3)Cell behavior results showed that N-CDs intake cells had a certain dose and time dependency,for different cell types take it up in different ways,in view of RAW264.7 cells mainly by phagocytosis and caveolae-mediated endocytosis,for 4T1 cells mainly by clathrin-mediated endocytosis and macropinocytosis process of cells to intake of N-CDs distributed mainly in soluble enzymes in the body,after a small amount of distribution in the mitochondria,almost not into the nucleus,and can be accessed by the cellular internalization of N-CDs cell vomiting effect or lysosome degradation to pass.Conclusion:In this study,N-CDs were successfully prepared using alkali-soluble Poria cocos polysaccharide as the raw material,which have the advantages of good water solubility,small particle size,low toxicity,and high biocompatibility.N-CDs is exclusive to metal ion Cr6+and can be used as fluorescent probe to detect Cr6+through internal filtering effect and static quenching mechanism,which can be applied to the detection of environmental water sample.In addition,due to its excellent fluorescence properties and low cytotoxicity,N-CDs can be used for intracellular stable imaging.Through the feasibility analysis of its biological research,it was found that the uptake rate,pathway,and effusion rate of N-CDs were different in different cell lines,which showed certain lysosomal targeting,proving that the N-CDs prepared by us has a certain potential in biological imaging and biomarkers.