Preparation Of Fluorescein-Like Carbon Dots And Application In Subcellular Imaging

As a new type of zero-dimensional carbon nanomaterial,carbon dots(CDs)are considered one of the most promising fluorescent probes in the field of biomarkers and biological imaging due to excellent fluorescence performance,good photostability,negligible cytotoxicity,easy surface modification,wide sources,et al.The design and construction of fluorescent CDs with different functions to achieve biological imaging and monitoring is a hot spot in the field of CDs.Fluorescein is a typical xanthene dye with high quantum yield and active reaction characteristics.It as carbon source is expected to synthesize CDs with various reaction characteristics.Therefore,fluorescein is used as the main carbon source,through the coupling and polymerization reaction with citric acid and triethylenetetramine to prepare CDs with excellent fluorescence luminescence properties.The CDs has sensitive pH recognition performance and specific GABAA receptor recognition performance,which realizes the sensitive monitoring of pH in the lysosome and the specific recognition and imaging of liver cancer cells.The results obtained in the study provide new ideas for the design and construction of functionalized CDs,and new ways and new tools for the visual non-destructive detection of lysosomes and liver cancer in living organisms.The specific content is as follows:1.Preparation and performance of functionalized carbon dots mediated by fluoresceinBased on the special chemical reactivity of fluorescein and the coupling and polymerization reaction with citric acid and triethylenetetramine,fluorescent CDs(FL-CDs)were prepared by the microwave method with bright green light emission.Structure and optical properties of FL-CDs are characterized by analysis methods such as Transmission electron microscope(TEM),Fourier transform infrared spectroscopy(FT-IR),X-ray photoelectron spectroscopy(XPS),Ultraviolet-visible spectrophotometer,and Fluorescence spectrophotometer.FL-CDs is a kind of particle with size of 2 nm,and its surface has a lactam spiro-ring structure of fluorescein.The fluorescence intensity of ?em=530 nm increases with the opening of the spiro-ring caused by the rise of pH.The fluorescence quantum yield of FL-CDs is as high as 85%.The fluorescence intensity change of FL-CDs is less than 1%under the continuous excitation at 480 nm wavelength for 1 hour and the common metal ions and amino acids with 100 ?M in cells do not affect fluorescence intensity,which shows their excellent resistance to photobleaching and light stability.In the high concentration of FL-CDs with 150 ?g/mL,the activity of HeLa cells still maintained more than 90%,indicating that FL-CDs has good biocompatibility and low toxicity,which lay the foundation for subsequent bioimaging research.2.Fluorescein-mediated functionalized carbon dots for lysosomal targeted imaging and monitoring cell viabilityThe performance of FL-CDs cell imaging is explored with their excellent fluorescence luminescence performance and biocompatibility.The obtained FL-CDs can be enriched and targeted to the lysosome after entering the HeLa cell(human cervical cancer cells),and fluorescence intensity increases as the chloroquine stimulates the pH of the lysosome to rise due to the performance of pH-sensitive response,indicating that FL-CDs can be used for monitoring pH of lysosome.The FL-CDs escapes from the lysosome and accumulate into the nucleolus of the cell after the cell dies.The mechanism of FL-CDs escaping from lysosome and enriching in the nucleolus is explored,and it is shown that FL-CDs has heterocyclic structures such as pyridine and pyrrole for binding to RNA with high selectivity,through RNase,DNase degradation experiments,which realizes the monitoring of cell viability3.Specific liver cancer imaging and mechanism study of fluorescein-mediated functionalized carbon dotsThe confocal imaging of the FL-CDs in HepG-2 cells(human liver cancer cells),SMMC-7721 cells(human liver cancer cells),7401 cells(human liver cancer cells),MCF-7 cells(human breast cancer cells),A549 cells(Human lung cancer cell),HeLa cells(human cervical cancer cells),4T1 cells(mouse breast cancer cells)and HL7702 cells(human liver cells)is explored through fluorescence confocal microscope imaging analysis.FL-CDs are mainly stained on the cytoplasm in normal cells,on the cell membrane in liver cancer cells,and on lysosomes in other cancer cells.The differences in the spatial distribution of FL-CDs in different cells indicate that FLCDs can specifically recognize liver cancer cells.GABAA receptor is a receptor protein of cell membrane and has been proved that it is closely related to the occurrence and development of liver cancer.The characteristic structure of GABA of FL-CDs is confirmed by specific color reaction with bromocresol green,succinic acid and glyoxylic acid.The picrotoxin site where the FL-CDs binds to the GABAA receptor is proved through the substitution competition experiment of specific binding sites of picrotoxin,baclofen,GABA,etomidate,and propofol,which shows GABAA receptor recognition performance and specific recognition mechanism of liver cancer cells of FL-CDs.The results in the paper provide a significant reference and basis for the design and construction of specific GABAA receptor recognition fluorescent FL-CDs and the specific imaging of liver cancer.