Protamine Assisted Synthesis And Bioimaging Application Of Multifunction Fluoresccnt Carbon Dots

Fluorescence imaging technology is one of the most extensive and powerful visualization technologies in biological research and clinical diagnosis.It has been widely used in the imaging of molecules,cells,living tissues and other different levels.Fluorescence imaging technology is very suitable for visualizing specific biological processes and transformation processes of specific molecules because it can specifically image some parts or biomolecules in biological systems.In environmental health research,fluorescence imaging technology is often used to detect pollutants accumulated in biological systems,analyze the pathological process caused by metabolism and environmental factors,distinguish and kill harmful pathogens and so on.For fluorescence imaging technology,fluorescent probes play a very important role in labeling and tracing the observed objects.Therefore,the preparation of fluorescent probes with wide emission wavelength range,high fluorescence quantum yield,good biocompatibility and low cytotoxicity has become a research hotspot of chemistry and materials workers.Carbon dots is a kind of new members of carbon-based nanomaterials family.The synthesis methods can be summarized as top-down method and bottom-up method.Among them,the microwave method can provide a lot of energy in a short time,so the synthesis process is the most simple and rapid.Carbon dots have good optical properties,strong light stability,emission wavelength across the visible and near-infrared region;at the same time,they have excellent properties of small size and low toxicity,which have been widely used in various types of biological imaging.Mammalian cell imaging is one of the most widely developed applications of carbon dots,but its application in bacteria and other organisms is relatively blank.In addition,the targeted imaging of specific organelles is relatively less.In this work,a protamine derived carbon dots(CD-PTMs)was constructed for nucleolus imaging and bacterial imaging of living cells.The materials showed potential applications in biomedicine and microbial pollution control.When protamine is selected as a precursor,the following two points should be considered:on the one hand,protamine can naturally bind to nucleic acid and can be used to target nucleolus with RNA as the main component;on the other hand,protamine has some specific biological effects.For example,protamine is a commonly used antimicrobial peptide,which can significantly inhibit the growth of Gram-positive bacteria,yeast and mold,and also has a certain inhibitory effect on some Gram-negative bacteria.The nano probe constructed by modifying protamine on carbon quantum dots is expected to achieve antibacterial effect when detecting bacteria.In this work,CD-PTMs were synthesized by microwave method using ethylene glycol and protamine as precursors,and characterized by means of transmission electron microscopy(TEM),surface zeta potential,1H nuclear magnetic resonance,X-ray photoelectron spectroscopy(XPS),circular dichroism(CD),absolute fluorescence quantum yield,etc,The form of the derivative exists on the surface of the carbon point.In addition,the fluorescence emission spectrum of CD-PTMs was scanned,and the light stability was investigated.The results showed that the fluorescence emission wavelength of the material depended on the excitation wavelength(detection range:?EX=340-500 nm,?EM=350-650 nm),covering the blue,green and red spectral range.After continuous irradiation with excitation light of 340 nm for 15 h,the fluorescence emission intensity remained above 80%.After that,cell titer method was used to prove that CD-PTMs did not produce significant cytotoxicity to HEK-293 cells in the concentration range of less than 40 ?g/ml.The materials were incubated with living cells for 12 h,and the images were collected by confocal fluorescence microscope(excitation wavelength set at 405,488,594 nm),which proved that CD-PTMs could image nucleolus for a long time.Combined with RNase digestion experiment and RNA titration experiment,we believe that this is because protamine derivatives can specifically bind to RNA in nucleolus.Finally,we investigated the imaging and growth inhibition of CD-PTMs for Escherichia coli and Staphylococcus aureus.These two kinds of bacteria are regarded as the representative strains of gram negative and Gram-positive bacteria.The results showed that the bactericidal rate of CD-PTMs to both bacteria was over 99%by plate counting method after the mixture of CD-PTMs and bacteria liquid was cultured for 3 h.In addition,confocal images showed that CD-PTMs could selectively image Gram-positive bacteria.In conclusion,we have successfully synthesized a biocompatible CD-PTMs probe,which can be successfully applied to the biological imaging of human nucleus and bacteria,and inhibit the growth of bacteria.