Synthesis Of Tryptophan Carbon Dots And Application Of Cell And Bacteria Bio-imaging With Carbon Dots

Background:Biological imaging uses bioluminescence as a signal to qualitatively and quantitatively study the tissues,cells and molecules of organisms.Fluorescence imaging is an indispensable tool for observing the cell structure and understanding the life process because of its simple and easy operation and high sensitivity.It has been applied in almost all fields of life sciences.A suitable fluorescent contrast agent is an important factor in obtaining an ideal fluorescence image.From small organic molecules to fluorescent proteins,to quantum dots,to nanoparticles and so on,a wide range of fluorescent contrast agents have been discovered and intensively studied.Organic small molecule dyes are generally smaller in size and have the advantage of extremely subtle interference with biological systems,however,they are easily photobleached and photolyzed and discolored,have short useful lives,and photolysis products often have a killing effect on the organism and more the main problem is that it is difficult to mark multicolor simultaneously,making it difficult to achieve real-time,non-destructive monitoring and research.Quantum dots have the following advantages over fluorescent dyes in that they can withstand multiple excitation and light emission,have a wide excitation spectrum and a narrow emission spectrum,etc.However,the semiconductor quantum dots contain metal ions which cause great toxicity to the living body and the environment.Therefore,how to develop and develop a new type of nanomaterial with low toxicity and multi-color imaging will be of great significance.Carbon dots,also known as carbon nano dots,are a new class of carbon nanomaterials with excellent optical properties,small particle size,strong penetration and low toxicity,which make the wide range of application in the field of biological imaging.Nanoparticles have more photostability and can overcome the disadvantages of the above conventional contrast agents.Recent studies have found that carbon dots have multi-color fluorescence under different excitation states.Since the surface of carbon dots contains hydroxyl groups,it has a high affinity for the cell wall and can adsorb onto the cell membrane,allowing cells and bacteria with different fluorescence characteristics under different excited state.And carbon dots entering into the bacteria can make bacteria have different fluorescence characteristics,which can identify bacteria.Based on the above analysis,through the preparation of tryptophan carbon dots,labeling of cells and bacteria,observe the imaging characteristics,this experiment can lay the foundation for identifying bacteria and the use of the carbon dots observation and analysis of cell morphology and physiological activity.Methods:1.Preparation of tryptophan carbon dotsThe classical hydrothermal method was used to prepare tryptophan carbon dots,then to characterized them.2.Biological safety evaluation of tryptophan carbon dotsThe logarithmic growth phase of RAW264.7,MC3T3-E1,L929 cells cultured in sterile 96-well plates were divided into blank control group,and different concentrations of tryptophan carbon dots solution(10?g.ml^-1?50?g.ml^-1?100?g.ml^-1?200?g.ml^-1?400?g.ml^-1?600?g.ml^-1).After 24 hours culture,tryptophan carbon dots cytotoxicity was detected by MTT assay.3.Biological imaging observation of tryptophan carbon dots labeling cellsIn vitro experiments of cell selection macrophage cell line RAW264.7,pre-mouse osteoblast cell line MC3T3-E1 and mouse fibroblast L929,after with the best concentration of tryptophan carbon dots co-cultured 24h respectively,the laser scanning confocal fluorescence microscope to observe the cell imaging results with different excitation wavelengths.4.Biological imaging research of tryptophan carbon dots labeling bacreriaIn vitro bacterial tests were performed on Escherichia coli?ATCC25922?and Staphylococcus aureus?ATCC25923?.After incubation with different concentrations of tryptophan carbon dots for 24 hours respectively,the best imaging concentration of carbon dots and bacterial were to be determined.Fluorescence spectroscopy was used to determine the fluorescence characteristics of different bacteria.The results of bacterial imaging results under different excitation wavelengths and fluorescence thresholds were observed by laser scanning confocal fluorescence microscopy.Results:1.The preparation of tryptophan carbon dots aqueous solution showed dark blue color and showed colorless after dilution.TEM observation showed that the prepared carbon dots were spherical particles with uniform size and good dispersion,and the diameter was about 3.35 nm.The molecular structure of carbon dots tested by FT-IR,which was only composed of carbon,oxygen,hydrogen and nitrogen,indicating that the surface of the synthesized carbon dots retains the amino groups,carboxyl groups and other groups in the carbon source,and these functional groups can impart good water solubility and dispersibility to the carbon dots.Fluorescence spectra showed that the carbon dots can present different colors under different excitation wavelengths and exhibited excitation dependence.2.MTT resultsThe results of MTT showed that when the concentration of tryptophan carbon dots was 0?g.ml^-1,10?g.ml^-1,50?g.ml^-1,100?g.ml^-1,200?g.ml^-1,cytotoxicity are garde 1 and in the safe range.The concentration of tryptophan carbon dots was 400?g.ml^-1,gradually showing low cytotoxicity with increasesing the concentration of carbon dots.3.Cell imaging resultsRAW264.7,MC3T3-E1,L929 cells were co-cultured with carbon dots respectively,RAW264.7 cells were round or oval,relatively small cells,rare protrusions.MC3T3-E1 cells were fusiform or pyramidal,with larger cells and nuclei.L929 cells were long fusiform,with protrusions.Under confocal laser scanning confocal microscope,the three kinds of cells under different excitation wavelengths showed different colors of fluorescence.The fluorescent regions of RAW264.7 and L929 mainly concentrated in the cell membrane and cytoplasm.The whole cells of MC3T3-E1 had fluorescent and imaging cells clear outline,and the fluorescence intensity was stable,indicating that tryptophan carbon dots have a good effect on the cell bio-imaging.4.Bacterial bio-imaging results4.1 The best concentrations of tryptophan carbon dots and bacteria were 0.4 mg.ml^-1 and 1×10⁸ CFU.ml^-1,in the best binding assay in vitro.4.2 Fluorescence spectrometer test results showed that the fluorescence intensity threshold of tryptophan carbon dots labeled by Staphylococcus aureus was larger than that of tryptophan carbon dots labeled by E.coli under the excitation wavelength of 300nm⁷00nm.4.3 Laser scanning confocal fluorescence microscopy results showed that E.coli could show green and red fluorescence images at 488nm and 548nm excitation wavelength,while S.aureus also showed green and red fluorescence images.However,when the fluorescence intensity threshold was set higher than that of tryptophan carbon dots labeled by E.coli fluorescence intensity threshold,this time only see the fluorescence images of Staphylococcus aureus,by which can identify E.coli and Streptococcus aureus.Conclusions:1.The tryptophan carbon dots were successfully made by hydrothermal method.Processes of preparation were simple and feasible.2.Tryptophan carbon dots were low cytotoxicity and good biocompatibility.3.Tryptophan carbon dots can successfully label cells which morphology iscomplete and the optical properties are stable in vitro,indicating that the tryptophan carbon dots have a great effect on bio-imaging with cells.4.In vitro bacterial experiments,tryptophan carbon dots can successfully label bacteria which have a good morphology.Tryptophan carbon dots can detect and identify bacteria from morphological and fluorescence characteristics,indicating that which have a great effect on bacteria biological imaging.