The Synthesis And Characterization Of CdTe Quantum Dots And Folate Receptor Targeting CdTe/CdS Quantum Dots Fluorescent Probe

Quantum dots?QDs?,semiconductor nanocrystals with the radius of 1 to 20 nm,have been widely applied in optical electrical material science and biomedical sciences because of its unique tunable optical properties.Because of its size is close to or smaller than the exciton Bohr radius,comparing with the traditional organic dyes,QDs possess some unique optical properties including narrow,tunable and symmetric emission spectra,broad excitation spectra,excellent photostability,high fluorescence intensity,tunable fluorescence color varies with particle size,etc.Especially,with the deepening research on quantum dot surface modification in recent years,by coupling of biological macromolecules?e.g.DNA,antibodies or proteins?,QDs have been used in cell labeling,tissue imaging,drug delivery,photosensitization for photodynamic therapy,in vivo tumor detection.CdTe is one of the most important semiconductors in the II-VI group,have received great attention due to their unique tunable optical properties.The synthesis methods of quantum dots are divided into organic phase synthesis and aqueous phase synthesis.Aqueous phase synthesis is simple,low cost,less pollution,and easy modified by some of the amino and carboxyl functional groups which can be directly coupled with biological macromolecularm,for exemple,thioglycollic acid?TGA?,cysteamine and mercaptopropionic acid?MPA?.With the continuous improvement of experimental conditions,Aqueous phase synthesis gradually become the main synthesis methods of quantum dots which are used in biomedicine.There are a lot of reports about the aqueous phase synthesis,but the prepared QDs had low quantum yield and poor stability.How to synthesize high quality of quantum dots has been a big problem in the application of QDS.Cancer has become the first leading cause of death,new cases were still growing every year.Early diagnosis can play a crucial role to reduce cancer mortality.Preparation of a kind of fluorescent probes with high sensitivity and specificity and easy observing for a long time,which will be help for the early diagnosis of diseases.Folate receptor is highly expressed in many tumors including head and neck tumor,and rarely expressed in most normal tissues.Our group previous research identified that folic acid receptors is highly expressed in nasopharyngeal carcinoma,laryngeal carcinoma tissues and theirs cell HNE-1,and Hep-2.Based on the different density of distribution of folate receptor in tumor and nontumor tissues,as well as high specificity,of folate receptor combining with folic acid or folic acid analogues,a folate receptor targeting quantum dots fluorescent probe can be used for tumor cell specific imaging which provided a good basis for tumor of detection,positioning and the evaluation of targeting therapy in the future.This research intends to adopt the method of water heating reflux,and through characterization and the calculation of fluorescence quantum yield to explore the effection of different stabilizers,the mole ratio of Cd²⁺ andTe2-,and pH on CdTe quantum dots synthesis,and to find an optimum synthesis conditions to obtain high quality of CdTe quantum dots.Then,with CdTe quantum dots as the core,Mercaptosuccinic acid?MSA?as the stabilizer,we synthesize and characterize CdTe/CdS core shell QDs.Synthesize folate receptor targeting quantum dots fluorescent probe on the foundation of CdTe/CdS,and make vitro imaging on nasopharyngeal carcinoma cells HNE-1 and human laryngeal carcinoma cells Hep-2 with high expression of folate receptor.This article is divided into the following two parts.The first part:The synthesis and characterization of CdTe quantum dots and CdTe/CdS core shell QDsObjective1.To develop a the best synthetic conditions?stabilizer,n?Te2-?:n?Cd2 +?,PH?to obtain CdTe quantum dots with high quantum yield and stable properties.2.To Synthesize and characterize CdTe/CdS core shell QDs on the foundation of CdTe.MethodsFirst we used water heating reflux method to synthetize CdTe Qds,then Choosing a best synthetic conditions to prepare a kind of CdTe quantum dots with high quantum yield and good stability by comparing the fluorescence quantum yield of different quantum dots which synthetized in different conditions,for example,using different stabilizers?Mercaptosuccinic acid,Mercaptopropionic acid,L-cysteine?,changing the mole ratio?1:2,1:4,1:10?of Cd²⁺ and Te2-,and setting different pH?8.0,9.0,10.0,11.0?.To calculate the fluorescence quantum yield using Rhodamine 6G with 95%quantum yield as reference and measuring the fluorescence emission peak area and the ultraviolet absorption value of Rhodamine 6G and Qds in the light of same wavelength?365nm?.The photoluminescence spectra and ultraviolet absorption spectrum of the Qds were investigated via fluorescence spectrophotometer and UV spectrophotometry.Crystal composition was determined via X-radial Diffractometer.Morphology and surface properties of the prepared QDs was determined by transmission electron microscopy?TEM?.In addition,using the similar water heating reflux method to synthetize core-shell CdTe/CdS quantum dots on the basis of CdTe quantum dots with high quality.Results1.Use the water heating reflux method,CdTe quantum dots which use Mercaptosuccinic acid as stabilizer have higher fluorescence quantum yield than CdTe quantum dots which use mercaptopropionic acid as stabilizer;fluorescence quantum yield in the condition of n?Te2-?:n(Cd²⁺)=1:10 is higher than other two ratios;fluorescence quantum yield in the condition of pH= 10 is higher than other three different pH values.2.In aqueous-phase synthesis,with reaction time prolonging,the diameters of CdTe quantum dots are increasing and the adsorption spectra and emission spectrum is constantly red shifting,they emit visible lights which change from green to yellow,orange and red gradually under ultraviolet light.3.In the condition of pH =10,n(Te²⁺):n?Cd2"?:n?MSA?= 1:10:10,the quantum yield of CdTe quantum dots is higher,The quantum yield of CdTe quantum dots had reached 72.5%in the reaction of ten minutes.There are three obvious peak in the XRD picture.the values of 2 0 are 24.449,40.695,48.799 respectively,corresponding to?111?,?220?and?311?lattice faces of cubic crystal of CdTe.The picture of TEM show the CdTe nanoparticles are nearly spherical,their particle sizes distribution is uniform,the average particle diameter is 3nm,and the degree of dispersion is well.4.On the foundermation of CdTe quantum dots synthesis,we successfully prepared CdTe/CdS quantum dots.Keep purified and liquid quantum?CdTe,CdTe/CdS?under seal in the condition of 4 ?,deposition and fluorescent intensity decreased did not appear in half an year,they had a very steady state.The second part:The synthesis and characterization of folate receptor targeting CdTe/CdS quantum dots fluorescent probe purposes:Synthetize folate receptor targeting CdTe/CdS quantum dots fluorescent probe and to detect its targeting.MethodFirst using dicyclohexylcarbodiimide?DCC?and N-Hydroxysuccinimide?NHS?as Coupling agent to prepare diaminopolyethylene glycol,then on the foundermation of CdTe/CdS quantum dots synthesis in the preceding chapter,using folate-conjugated polymer as bridging material to synthetize the folate receptor targeting quantum dots fluorescent probe?FA-PEG-CdTe/CdS?.The Coupling effect was evaluated by agarose gel electrophoresis and spectral analysis.FR-positive human nasopharyngeal carcinoma cells?HNE-1 cells?and laryngeal carcinoma cells?Hep-2 cells?were cultured for 6 hours in medium contained FA-PEG-CdTe/CdS quantum dots fluorescent probe,and compared with FR-negative human nasopharyngeal carcinoma cells?CNE-2?.Then we observed the result of cell imaging through Inverted Flurescence Microscop.Store the purified Qds-PEG-FA in a water solution of 4 ? or cell culture fluid?RPMI-1640?for three months,observing the fluorescent intensity and stability.Store the cell labeled by Our folate receptors targeted quantum dots fluorescent probe under the ordinary temperature and away from light for one week,then observed the fluorescence through Inverted Flurescence Microscop.Results1.After coupling CdTe/CdS quantum dots to biomacromolecule,the emission spectrum was red shifting and the fluorescent intensity was decreased.these proved that CdTe/CdS QDs coupling with PEG-FA is succeeded.2.The results of agarose gel electrophoresis proved that QDS-PEG-FA is stable,residual reactants are less after purification and indicated that QDS-PEG-FA quantum dots fluorescent probe are in high purity also.3.After culturing FR-positive human nasopharyngeal carcinoma cells?HNE-1 cells?and laryngeal carcinoma cells?Hep-2 cells?in cell culture medium containing QDs-PEG-FA for 6 hours,through fluorescence microscope,we can see the strong fluorescent appeared on the surfaces of almost all cells,and the fluorescence intensity of the lignt expressed on the surfaces of the two cells were a little different.The light intensities of HNE-1 cells is lower than Hep-2 cells.But after culturing FR-negative human nasopharyngeal carcinoma cells?CNE-2 cell?respectively in the cell culture medium containing three different levels of QDs-PEG-FA for 6 hours,all of the cells'surface did not observe any fluorescent.4.Store the purified fluorescent probes which dissolved in water solution or cell culture fluid?RPMI-1640?in 4 degrees Celsius for three months,deposition and fluorescent intensity decreased did not appear.The fluorescent on the surface of the cell can still be observed after labled by Qds-PEG-FA quantum dots fluorescent probes and stored under the ordinary temperature and dark storage one week later.ConclusionsIn the process of Quantum dots synthesis,stabilizer,reactant molar ratio,pH and the action time have an effect on the nature of the quantum dots,such as fluorescence quantum yield,particle size and stability.In the condition of pH = 10,n(Te²⁺):n?Cd2-?:n?MSA?= 1:10:10,with Mercaptosuccinic acid?MSA?as the stabilizer,we can obtain QDs with a high quantum yield,the highest quantum yield increase to 72.5%.In the basic of CdTe,we succeed in making CdTe/CdS quantum dots.Keep purified and liquid quantum?CdTe,CdTe/CdS?under seal in the condition of4 ?,deposition and fluorescent intensity decreased did not appear in half an year,they had a very steady state.We had prepared a kind of folate receptor targeting CdTe/CdS quantum dots fluorescent probes with good stability and strong specificity.This fluorescent probes can make vitro tumor imaging on nasopharyngeal carcinoma cells HNE-1 and human laryngeal carcinoma cells Hep-2 with high expression of folate receptor,but is less able to lock onto nasopharyngeal carcinoma cells CNE-2 with low expression of folate receptor.