Synthesis Of Fluorescent Carbon Quantum Dots For Imaging Nucleic Acid Structures In Live Organisms

Nucleic acids,both deoxyribonucleic acid(DNA)and ribonucleic acid(RNA),are one of the most basic life materials.They play an important role in the storage,copy and transfer of genetic information,and the synthesis of protein.Direct monitoring the structure changes of DNA and RNA in real-time,such as the segregation,aggregation,and cleavage,is of great significance for understanding the behavior of cell transcription,division,protein expression and apoptosis.However,due to the lack of probe photostable enough to distinguish DNA and RNA,and more importantly,to cross multiple membrane barriers ranging from cell-organelle to tissue-organ levels,it is still a great challenge to real-time observe the dynamic structure changes of DNA and RNA in living cells,especially in vivo.Compared to traditional semiconductor quantum dots and organic dyes,carbon quantum dots possess superior properties in terms of non-toxic,high resistance to photobleaching,easy modification and so on,making them proficient for real-time in situ imaging analysis in living cells and organisms.Here,we report the discovery of a cationic carbon quantum dot(cQD)probe with the high efficiency of membrane-penetrating by chemical modification to improve the positive charge of the cQD.The cQD probe emits spectrally distinguishable fluorescence upon binding with double-stranded DNA and single-stranded RNA,achieving synchronously real-time in situ fluorescent imaging of the dynamic structure changes of DNA and RNA in living cells and Caenorhabditis elegans(C.elegans).The main research contents are as follows:1 We report the discovery of a cationic cQD probe with the high efficiency of membrane-penetrating.First,The cQD with uniform size was made by oxidizing the conductive carbon nanoparticles in refluxing HNO3 to add a large number of carboxyl groups to the edges of cQDs.Then,further modification with p-phenylendiamine(pda)through acylation produces cQD with modest green fluorescence.Finally,4-carboxybutyl triphenylphosphonium(PPh3+)bromide was chemically linked to cQD through the pda sites to obtain the cationic cQD probe.The cQD interacts with double-stranded DNA(dsDNA)and single-stranded RNA(ssRNA)differently,producing spectrally distinguishable fluorescence.2.We achieved the synchronous in-situ imaging of DNA and RNA and their dynamic changes of structure in live cells by using the cQD probe.The cQD probe can effectively cross the cell membrane and nuclear membrane to bind with the intracellular DNA and RNA.Under excitation of 488 nm,the nucleus emits strong green fluorescence,while under excitation of 543nm,the cytoplasm and nucleoli emit red fluorescence,demonstrating the specific recognition ability of the cQD probe to DNA and RNA in living cells.Furthermore,due to the strong photostability and low toxicity of the probe,we achieved imaging the dynamic structure changes of DNA and RNA during cell division and also the 3D structure reconstruction of the chromosome and nucleolus using stimulated emission depletion microscopy(STED).3.Finally,we explored the possibility of in-situ imaging of DNA and RNA in live organisms.The C.elegans were fed with cQD in culture media.As expected,cQD:initially entered the intestinal pathway,but surprisingly,it can go through interstitial tissues,gonad vesicle with the continuous incubation,and finally the cQD probe permeate trough the germ cell membrane and nuclear membrane into germ cell nuclear.Similar to the results in living cells,the cQD probe labelling DNA and RNA in germ cells also emits green and red fluorescences,respectively.All the results showed the cQD probe possess the ability of selectivity towards dsDNA and ssRNA,and in particular,the unique ability to penetrate through various biological barriers in cells and live organisms.