Preparation And Application Of Carbon-based Quantum Dots In Zebrafish Bio-imaging And Study Of The Relevant Toxicity

Fluorescent carbon-based quantum dots(CDs),as a new organic carbon material,had been praised as the bellwether of materials because of the specific fluorescence properties,stable physical and chemical properties,high biocompatibility and other excellent properities.Nowadays,it has become increasingly popular to research the interdisciplinary study in materials,chemistry and biology.Recently,extensive researches on carbon-based quantum dots had become largely relevant to biological and environmental fields,which gained tremendous attention on the biocompatibility and toxicological risk in the wide range of applications.In our work,two kinds of biomass(hair and pig skin)and a low toxicity and harmless substance(citric acid)were used as raw materials to prepare three kinds of low toxicity and high biocompatible carbon quantum dots,which were subsequently applied in fluorescence imaging in zebrafish.On the other hand,the reduced graphene oxide quantum dots with excellent fluorescence properties were prepared and applied in zebrafish fluorescence imaging and relavent toxicology study.Firstly,hair derived CDs(HCDs)and skin derived CDs(SCDs)were obtained by using human hair and pig skin as precursors,the microwave-assisted carbonization method was implemented to synthesize the fluorescent HCDs and SCDs from the two protein-rich biomass without using any dopant or toxic reagents.At the same time,the citric acid carbon dots(CCDs)were synthesized by chemical hydrothermal synthesis using the citric acid and urea as raw materials,which were chosen as control to investigate the effects of precursors on photoluminescence properties.All three carbon quantum dots were nanoparticles and sized in 10 nm,an the chemical composition and fluorescence properties were changed fron the raw materials,which caused the different fluorescence colour of the three CDs and the quantum yield(QY)were 86.06%(HCDs),51.35%(SCDs)and 19.73%(CCDs),respectively.The three CDs have a broad excitation-dependent photoluminescence that can be used as a fluorescent bioprobes for applications.Secondly,the modified Hummer's method was performed to synthesize the graphene oxide(GO)with high proportion of oxidant(KMnO4)and the DMF hydrothermal treatment approach was carried out to reduce the GO and enhance the fluorescence intensity of the as-prepared reduced graphene oxide quantum dots(rGOQDs).The GO were used as precursors and DMF acted as a nitrogen source and reducing agent to enhance the PL properties of rGOQDs.After that,the rGOQDs were modified by epoxy groups,carbonyl,hydroxyl and C-N functional groups,which largely improved their fluorescence properties.The QY of the as-prepared rGOQDs was increased from 6.53% to 24.62%,and the resulting rGOQDs had an excitationdependent photoluminescence performance.Afterwards,the three CDs of HCDs,SCDs and CCDs were successfully applied in fluorescence imaging in zebrafish.To demonstrate the feasibility of CDs as fluorescent probes,the effects of a series concentrations of CDs,the absorption,distribution,metabolism and excretion(ADME)of CDs were tested in zebrafish.The results displayed that the fluorescent imaging of the embryo was excellent in the first 48 hours after the immersed in HCDs and SCDs solution.As time went on,the fluorescence became increasingly weaker until the fluorescence completely diminished.Moreover,the quenching assays were performed to detect the decaying mechanism of CDs in zebrafish by adding ATP,NADH and Fe3+ ions to CDs solution,the results showed that all three substances could quench the CDs in different levels.Eventually,for rGOQDs,it mainly reserched the fluorescence imaging in zebrafish,as well as their corresponding developmental toxicology,genetic toxicology studies.The hatch rate,mortality,heart rate,body length and malformation were obtained statistically,and all indexes had a certain weakened,indicating that rGOQDs induced developmental toxicity in zebrafish.For deeply explore the perturbation of rGOQDs in gene expression of zebrafish,the genes of cyp1 a,cyp1c and cyp7a1 which related to aromatic hydrocarbon receptor(AhR),and another hsp70 which was sensitive to the external stress were researched by real-time fluorescence quantitative polymerase chain reaction(RT-PCR).The results showed that cyp1 a,cyp1c,cyp7a1 and hsp70 were up-regulated by 8.49,4.65,5.93 and 3.63 folds,respectively,which confirmed the perturbation of rGOQDs in the AhR signal transduction pathway of zebrafish.Meanwhile,the Tg(cyp1a: gfp)zebrafish was used to evaluate the rGOQDs induced capture cyp1a:gfp expression.Moreover,the western blot analysis result also verified that the rGOQDs induced an increase of green fluorescent protein expression in Tg(cyp1a: gfp)zebrafish in a dose-dependent manner,which was in line with the RT-PCR results.These studies will precisely guide us to refer their potential hazards of graphene-based materials before applied in biomedical and biological fields,which was of great reference value.