Synthesis Of Fluorinated Graphene Quantum Dots And Its Application In Cell Imaging And Tumor Photodynamic Therapy

GQDs are mainly composed of sp2 hybridized carbon atoms with the same lattice spacing as graphite and less than 10 atomic layers.As a new type of fluorescent carbon material,GQDs can be produced by "Top-down" method and "Bottom-up" method.The preparation methods of ’Top-down" method mainly include concentrated acid oxidation method,hydrothermal method,electrochemical cutting method and solvothermal method.The preparation methods of "Bottom-up" method mainly include microwave heating method and pyrolysis method and hydrothermal method.Because GQDs have special quantum confinement effects and quantum size effects,have good water dispersibility,good biocompatibility and easy surface modification,they can be used in biomedical fields(such as bioimaging,biosensing and photosensitizers,etc.).Among the many excellent properties of GQDs,the study of fluorescence characteristics is of most importance.At the same time,it has been reported that the combination of GQDs and photosensitizers(PS)for tumor photodynamic therapy(PDT),but the therapeutic efficiency of using GQDs as PS in PDT is still low.Therefore,the synthesis of GQDs and heteroatom-doped GQDs with high fluorescence intensity and high singlet oxygen(1O2)yield(QY)by various methods has broader application prospects.At present,it has been reported that nitrogen doped GQDs(N-GQDs)have good hydrophilicity and biocompatibility,but can be applied to PDT only after combining with PS.Therefore,GQDs doped with other atoms have been extensively studied.In addition,there have been related reports on the synthesis of fluorinated graphene quantum dots using fluorinated graphite as a raw material,but the methods used are solvothermal method and microwave-assisted hydrothermal method,and the synthesis process is relatively complicated.How to develop a simple and convenient method to synthesize GQDs with high yield of 1O2 is still a challenge.In view of the reasons for the low QY of the above GQDs,we synthesized fluorine-doped graphene quantum dots(F-Doped GQDs)and fluorinated graphene quantum dots(FGQDs)by oxidative cutting method.The morphology,structure were characterized utilizing modern characterization techniques.The cytotoxicity of the GQDs,two-dimensional(2D)cell imaging,three-dimensional multi-cell cluster(3D MCs)imaging,and the ability to produce 102 under LED light were further studied.The significant research outcomes and results obtained are as follows:1:Graphite oxide(GO-1)was synthesized by the Hummers method using graphite as a precursor,and then reacted with ammonium fluoride to perform oxidative cutting with hydrogen peroxide to obtain F-Doped GQDs.The control group was synthesized without the addition of ammonium fluoride,oxidative cleavage was performed to obtain graphene quantum dots(GQDs-1).The morphology,structure,and surface groups of the F-Doped GQDs and GQDs-1 were characterized by using transmission electron microscope(TEM),X-ray photoelectron spectroscopy(XPS),ultraviolet-visible absorption spectrum(UV-Vis),and fluorescence emission spectrum(PL).The results showed that the average sizes of F-Doped GQDs and GQDs-1 were around 2.9 nm and 2.5 nm,respectively,and obvious graphitized crystalline carbon lattices could be observed inside diffraction fringes.The F-Doped GQDs have a fluorine doping amount of 1.21%.In terms of optical properties,F-Doped GQDs and GQDs-1 show excitation-independent fluorescence.Using quinoline sulfate as a reference,the fluorescence QY of F-Doped GQDs and GQDs-1 in aqueous solutions were determined to be 12.3%and 10.4%,respectively.In addition,when F-Doped GQDs and GQDs-1 were mixed with various solutions of different metal ions(K+,Gd2+,Ba2+,Sn4+,Mn2+,Cu2+,Fe3+)at the same molar concentration,the observed change in fluorescence intensity of F-Doped GQDs show some ion selectivity.F-Doped GQDs and GQDs-1 were studied as a bioimaging probe.First,human liver cancer cells(HepG2)and human cervical cancer cells(Hela)were used as a 2D cell model to perform cytotoxicity studies by the tetramethylazozolium colorimetric method(MTT).When the concentration of F-Doped GQDs or GQDs-1 reaches 300 μg mL-1,cell viability is still above 80%,which proves that F-Doped GQDs and GQDs-1 have excellent biocompatibility.Then,HepG2 cells and Hela cells were used as a 2D cell model for cell imaging tests,the results showed that when co-cultured with 200 μg mL-1 for 6 h,both F-Doped GQDs and GQDs-1 could emit bright light inside the cells.F-Doped GQDs demonstrated much brighter fluorescence than GQDs-1.In addition,HepG2 3D MCs was used as a 3D MCs model to study the imaging effect of the material in the cell mass,similarly,when F-Doped GQDs and GQDs-1 were co-cultured with HepG2 3D MCs for 6 h,F-Doped GQDs could show bright fluorescence,indicating that F-Doped GQDs have promising cell imaging effect.2:Fluorinated graphite is used as a raw material to synthesize fluorinated graphene oxide(FGO)as a precursor by Hummers method,and then stripped in a mixture of sodium hydroxide and potassium hydroxide,and finally oxidized by hydrogen peroxide to obtain FGQDs.Comparative sample graphene quantum dots(GQDs-2)were synthesized by using graphite as a raw material.The morphology,structure and surface groups of the obtained FGQDs and GQDs-2 were characterized by means of TEM,XPS,UV-Vis,PL,and electron spin resonance spectroscopy(ESR).As a result,the average size of FGQDs and GQDs-2 is about 2.1 nm and 3.6 nm,respectively.In addition,the FGQDs aqueous solution can emit bright green fluorescence at an excitation wavelength of 365 nm,while using quinoline sulfate as a reference,the QY of FGQDs and GQDs-2 in the aqueous solution were measured to be 13.7%and 5.8%,respectively.FGQDs display excitation-independent fluorescence.First,the cytotoxicity study was performed using HepG2 cells as a 2D cell model by the MTT method.When the concentration of FGQDs or GQDs-2 reaches 300 μg mL-1,survival rate of the cells could still be above 85%indicating remarkable biocompatibility.Then,HepG2 cells were used as a 2D cell model to carry out cell imaging studies.The results showed that when co-cultured with FGQDs for 6 h,the cell imaging effect was excellent.At the same time,HepG2 3D MCs was used as the 3D MCs to study the imaging of materials in cell clusters.When FGQDs and HepG2 3D MCs were co-cultured for 6 h,the cell clusters could show bright fluorescence and exhibit good cell imaging effects.The ability of FGQDs and GQDs-2 aqueous solution to produce 1O2 under the induction of LED light was tested by ESR.As the light irradiation time increases,FGQDs produce a gradually enhancement in the 1O2 signal under the light of LED light,while the signal of GQDs-2 increased relatively slowly.Using rose bangle(RB)as a reference and 9,10-anthranilide bis(methylene)bis-aloic acid(ABDA)as a quenching agent,a working curve of absorbance and light time was evaluated by UV-Vis test to evaluate the value of 102.The 1O2 QY of FGQDs and GQDs-2 are 0.49 and 0.24 respectively.Then,using HepG2 cells as a model,the PDT effect of FGQDs and GQDs-2 induced by LED light was detected by the MTT method.The results showed that under the same light conditions,as the material concentration increases,FGQDs are more cytotoxic than GQDs-2.Subsequently,HepG2 3D MCs were used to simulate the tumor microenvironment,and it was found that when co-cultured with FGQDs,the tumor size gradually decreased with the increase of light time.Therefore,FGQDs have a better inhibitory effect on tumor growth than GQDs-2 and are expected to be used in tumor photodynamic therapy.