The Preparation of Fluorescent Carbon Dots Based on Element,Functional Group and Spatial Conformation Delivery Strategy and Their Cell Imaging Application

Author:Li Rong Sheng

Supervisor:wang jian huang cheng zhi


Degree Year:2019





The emergence of each new structure of carbon materials has attracted widespread attention in the scientific community as well as in the industry,such as the fullerene,carbon nanotubes and graphene oxide.Carbon dots,a member of the carbon material family,were discovered in the by-products of the preparation of single-walled carbon nanotubes in 2004,and generally refer to photoluminescent or electroluminescent materials that are smaller than 10 nm in size.According to the difference of structure,carbon dots are also called carbon quantum dots,carbon nanodots and polymer dots.Due to the excellent physical and chemical properties of carbon dots,especially the photochemical stability and biocompatibility are good.Carbon dots are widely used in the fields of analytical detection and real-time imaging of biomolecules.Currently,the application of carbon dots in detection and imaging lies in the following three aspects:First,we construct the relationship of the concentration of target molecules and optical signals through the interactionn of target molecules and the surface of the carbon dots.Second,we modify the corresponding target molecule on the surface of carbon dots and then use it for detection or imaging.Third,we use carbon dots as an energy donor or acceptor in fluorescence resonance energy transfer(FRET)system,and other fluorophores as acceptors or quenchers.All of these applications rely on specific elements,functional groups,or spatial structures on the surface of the carbon dots.However,carbon dots prepared directly by a simple method generally have fewer effective functional groups,and subsequent modification of the carbon dots is not only difficult but also causes a large waste of time and material.Therefore,it is very urgent to develop a simple and effective method for preparing functionalized carbon dots.Combining the previous laboratory synthetic practices,we developed a structural delivery strategy for synthesizing functionalized carbon dots.The strategy is as follows:First,due to element conservation,we can control the specific ratio and the carbon doping of special elements by adding two or more precursors with special elements and adjusting the proportion of elements in the precursor.Second,due to the specific conditions required for the breaking of chemical bonds of different functional groups,we can keep special functional groups of carbon dots during the synthesis by adjusting the conditions of temperature,pH,oxygen content and pressure.Third,the chirality of the molecule usually manifests itself as the asymmetry center of the carbon element.The asymmetry not only affects the spatial arrangement of newly formed chemical bonds during the synthesis of carbon dots,but also connects on the surface of carbon dots.Moreover,the formation of a chiral center also induces new chirality in adjacent carbon structures.Therefore,it is theoretically very feasible to prepare chiral carbon dots by chiral precursors.Based on the interaction of DNA and nanomaterials,a series of molecular switches and biosensors with stimuli response have been produced in recent years.It is clear that the high affinity of DNA to nanomaterials facilitates the specificity and selectivity of biosensors.A key step is to increase the affinity of DNA with nanomaterials while maintaining DNA hybridization activity.Therefore,it is very important to adjust the affinity of carbon nanomaterials to DNA because the excessive affinity of DNA and carbon nanomaterials will be detrimental to the hybridization recognition between molecules,and the low affinity of DNA and carbon nanomaterials will be detrimental to the specificity and selectivity when sensing of molecules.Therefore,based on the element transfer strategy,we synthesized a carbon dots co-doped with nitrogen and boron to solve this problem.As the third most abundant transition metal element in the human body,copper ion is an essential cofactor for various enzymes such as tyrosinase,copper/zinc superoxide dismutase and cytochrome c oxidase.On the other hand,since copper ions are capable of generating reactive oxygen species(ROS)involved in cellular metabolism.When copper is excessive in the human body,its redox properties can seriously damage the biological system.The complex regulatory mechanism of copper ions is to maintain a balance between maintaining toxicity and necessity at the cellular level.Optical imaging with fluorescent probes has been an effective way to answer questions about how copper ions work in biological systems.However,fluorescent dyes are easily photobleached,whereas conventional quantum dots have well-known cytotoxicity problems due to their composition of heavy metals.Therefore,based on the functional group transfer strategy,amino functionalized carbon dots were synthesized for sensing and detoxification of intracellular copper ions.The development of subcellular targeting molecules is of great importance in the research of life sciences and the development and synthesis of drugs.As a eukaryotic organelle,the Golgi apparatus is essential for the synthesis,secretion and intracellular distribution of most biological macromolecules.According to reports,the morphological changes of the Golgi are related to external stimuli.Therefore,the state of the Golgi apparatus can effectively reflect the physiological state of the cells.The most widely used Golgi dyes at this stage are ceramide analogs such as NBD C6-ceramide.However,the ceramide analog-based Golgi labeling method has its inherent drawbacks because its Golgi staining is the membrane fusion of ceramide and lipid interactions.After the Golgi staining,it needs to be observed as soon as possible because the ceramide molecules will reach other parts of the cell,which makes the use of nerves ceramide analogs are difficult to observe dynamic changes of the same Golgi over a long period.At the same time,problems such as photo-bleaching of fluorescent molecules coupled to ceramides have also hampered their long-term imaging applications.Therefore,it is very urgent to design and develop new optical probes with long-term Golgi imaging capabilities.Inspired by the importance of L-cysteine of N-acetylglucosaminyltransferase for golgi targeting,we have developed chiral and thiol-rich carbon dots based on chiral and functional group transfer strategies.The main points of our work are summarized as follows:1.Boron and nitrogen co-doped carbon dots:a high-affinity platform for visualizing the dynamic invasions of HIV DNA into living cells through fluorescence resonance energy transfer.High-affinity binding of carbon nanomaterials with necleobases,which is still a challenge,is the basis for DNA directed assembly and sensing.In this work,boron and nitrogen co-doped single-layered graphene quantum dots(BN-SGQDs)are designed as a high-affinity platform for nucleic acid detection and imaging in living cells,which has been confirmed by density functional theory(DFT)simulation and experiment.Owing to their excellent absorption and photoluminescent ability,the high quantum yield(QY 36.5%)yellow fluorescence BN-SGQDs could act as an energy donor in the fluorescence resonance energy transfer(FRET)process for the nucleic acid detection.Furthermore,this BN-SGQDs based sensing platform has been successfully adopted to visualize the dynamic invasions of Human immunodeficiency virus(HIV)DNA into HeLa cells.The high-affinity platform has shown its potential for biosensing in complicated biological samples.2.Highly fluorescent amino carbon dots as selective and visual probes for sensing copper ions in living cells via an electron transfer process.An As an integral part of many important enzymes,Cu2+is involved in a number of vital biological processes,which is linked to the oxidative damage and environmental contamination when Cu2+is excessive.In this work,Cu2+can be captured by the amino groups of carbon dots(CDs)to form complexes,resulting in a strong fluorescence quenching of CDs via a nonradiative electron transfer process,which offered a rapid,visual,and selective methodology for Cu2+detection.The probe exhibited a wide response concentration range(0.01-2μM)to Cu2+with a detection limit of 6.7 nM.Significantly,the CDs presented excellent biocompatibility and high photostability,which were applicable to the visualization of Cu2+dynamic invasion into living cells.Furthermore,the toxicity of Cu2+ions to living cells could be inhibited with CDs by forming the complexes.3.Chiral carbon quantum dots for targeting and long-term imaging of the Golgi apparatus.The Golgi apparatus is an essential subcellular organelle.Targeting and monitoring the Golgi change at single-cell level over a long time scale are critical but challenges that have not yet been tackled.Inspired by the precise Golgi positioning ability of galactosyltransferase and protein kinase D owing to their cysteine residues,we developed a method for long-term Golgi imaging.Carbon quantum dots(CQDs)could target the Golgi when they are modified with L-cysteine.L-cysteine-rich chiral carbon quantum dots(LC-CQDs),which hold the merits of high Golgi specificity from L-cysteine and the excellent photostability and biocompatibility from CQDs,are proved to be highly suitable for long-term in situ imaging of the Golgi.Mechanism investigations showed that free thiol groups and L-type stereo configuration of LC-CQDs are indispensable for specific targeting of the Golgi.With the aids of the as-prepared LC-CQDs,the dynamic changes of the Golgi in the early stage of virus infection were visualized.The Golgi targeting and imaging strategy used in this work is beneficial for Golgi-targeted drug delivery,early diagnosis and therapy of Golgi diseases.In addition,based on the targeting abiligy of the Golgi-targeted chiral carbon dots,we have explored the method of Golgi targeting based on cysteine.By precisely connecting different numbers of cysteines to fluorescent molecules,the multivalent effect of polycysteine on the Golgi was confirmed by analyzing the relationship between the number of cysteine and the Golgi targeting ability of cysteine labelled molecules.Based on the importance of polycysteine,a Golgi targeting peptide with a cysteine tandem structure was developed,and the targeting peptide was applied to the Golgi function regulation.First,we linked the cysteine targeting peptide to the horseradish peroxidase system to achieve Golgi-targeted horseradish peroxidase.Based on the specific catalytic polymerization of DAB molecules and hydrogen peroxide by horseradish peroxidase,the polymer on the Golgi produced by the polymerization of DAB molecules could lock the cancer cells in the middle of the cell mitosis.Second,we coupled the Golgi targeting peptide with energy transfer system,furin recognizing site,and nanofiber polypeptide precursor to form a new functional tag.The work mechanismis as follows:the Golgi targeting peptide has high affinity of to transferrin receptor of cancer cells;the Golgi targeting peptide is oxidatively coupled in the higher pH environment of the Golgi of cancer cells;furin is highly expressed in the tans-Golgi of cancer cells;furin can cutting the carbon end of the recognizing site,inducing the disconnecton of the FRET system and the self-assembly of nanofiber polypeptide precursor in the Golgi of cancer cells.Hence,high-selective imaging and killing of the cancer cell Golgi were realized by the new functional tag.In summary,in this study,carbon dots with special element doping(nitrogen and boron),special functional grouping(amino),and specific spatial structure(chiral)were prepared by simple operation based on the structural genetic strategy in the process of carbon synthesis.These functional carbon dots are applied to intracellular metal ion detection,nucleic acid detection,and organelle targeted imaging,respectively.Inspired by the ability of cell specific imaging ability of carbon dots,we have successfully developed Golgi targeting vectors.This study provides the method of preparing biofunctional carbon dots and also provides new ideas for the application of carbon dots in biochemical analysis and targeted imaging.