Abstract：In this study，the watersoluble CdTe quantum dots （QDs） had been successfully synthesized.The temperature at 105 ℃ had been determined to be the optimum condition by considering the refluxing time，the fluorescence intensity and the diameter distribution.A modified Stober Method for the facile synthesis of CdTe@SiO2 composite particles was also presented in this article.The diameter of composite particles could be easily tuned between 160 nm and 260 nm by changing the rate of Tetraethylorthosilicate （TEOS） and water.We also conjectured the growth pattern of SiO2 on the surface of CdTe QDs according to TEM images.Considering the further applications in biological detection，3aminopropyltriethoxysilanes （APTS） had been introduced to enhance abundant amine groups on the surface of silica shell and the perfect time had been also confirmed to add it in the view of crystal growth theory.Based on the above，the content of amino on particles surface is the highest.These composite microspheres have better water solubility，stability，and reactivity compared with original QDs.It sets the stage for the application of QDs as fluorescent probes.
　　Key words： CdTe quantum dots； silica coating； Stober Method
　　CLC number： TB 383 Document code： A Article ID： 10005137（2015）06062408
　　Received date： 20151001
　　Foundation item： We gratefully acknowledge the financial support by Development Center of Plant Germplasm Resources （B601011001），Agricultural Achievement Transformation Fund （143919N0500），the State Key Laboratory of Dairy Biotechnology Industry （Bright DairyLimited by Share Ltd） （SKLDB201304），Local colleges and universities capacity building Program （13430502900），Shanghai Biomedicine Key Program （10391901700），and the Program of Food Safety and Nutrition Team of Shanghai Normal University （DXL123）.
　　Corresponding author： WEI Xinlin，College of Life and Environmental Sciences，Shanghai Normal University，No. 100，Guilin Rd.，Shanghai 200234，China，Email：wxl@shnu.edu.cn；WANG Yuanfeng，Email：yfwang@shnu.edu.cn
　　1 Introduction
　　Semiconductor quantum dots （QDs） have attracted significant attention for their fascinating optical properties compared with organic fluorescent dyes，such as sizecontrolled tunable maximum wavelength of emission，superior signal brightness，less susceptibility to photobleaching，and narrow emission spectra，which make the QDs a new type of labeling materials useful for versatile biological and biomedical applications[1-6]. 　　However，there are characteristics that limit their effectiveness for QDs using as bioprobes，i.e.，cytotoxicity because of the release of heavy metal ions upon photooxidation[7]，colloidal and chemical instabilities of the QDs in harsh environments，and difficulty to separate QDs bioprobes from the reaction system by centrifugation.In order to solve these problems，coating QDs with silica getting composite particles has been widely investigated.Because of silica showing excellent biocompatibility，stability against degradation，leakage prevention of heavy metal ions into environments，and the welldeveloped silica surface functionalization.Therefore，the silica coating is becoming an ideal means for constructing versatile fluorescent bioprobes based on QDs.
　　So far，coating QDs with silica can generally be classified into two groups based on reverse microemulsion synthesis[8-9]and the Stober Method.Advantages of the microemulsion method are that it is very ′robust′ against many reaction conditions，the resulting silica nanoparticles not only have ″smooth″ surfaces，display good monodisperses，but also control particle size distribution，besides that，some nanoparticles with nonpolar ligands can be directly coated[9].However，this process is not environmentally friendly because of 4 costing massive organic solvent and the yield is lower than Stober Method.In addition，the diameter of composite particles is smaller than 100 nm.From this perspective，nanoparticles with diameters bigger than 100 nm prepared by the Stober Process are more preferable to use as biological labeling.Additionally，only single or a few nanocrystals are encapsulated in silica spheres in most of those preparations，which limits the fluorescence intensity of composite nanoparticles resulting a bottleneck of improving sensitivity of fluorescence biological labeling.
　　Alternatively，the Stober Process can be used to form either aqueous QDs or organic soluble QDs.Ligand exchange is usually required for transferring them from less polar solvents to polar media[10]，either QDs @silica coreshell structures or ′raisin bun′type composite particles[11].In addition，the Stober process allows easily control of the thickness of the silica shell over a range from nanometers to micrometers level by adjusting the content of water，ammonia，alcohol，and silica precursor[12-13].
　　Although these methods have been demonstrated to be successful for the encapsulation of semiconductor nanocrystals in silica spheres，their preparation processes often involve multiple steps and are tedious，and the productions have a badly drawback of dramatic decrease in the fluorescence quantum yield （QY） inevitably occurs during the encapsulation of QDs，which is often interpreted by ligand exchanges of primer[14-15].It is noted that the encapsulation of watersoluble CdTe nanocrystals in silica microspheres without ligand exchange has been reported.Therefore，more simple methods are necessary to synthesis QDssilica composite particles with proper diameter and higher fluorescence QY，which is beneficial to reduce biological toxicity and favorable to improve the sensitivity of bio detections by using QDbased fluorescent probes.In this study，we have improved the traditional Stober Method，under the optimized conditions，using onepot synthesis to prepare the aminomodified CdTe@SiO2NH2 fluorescent microspheres. 　　2 Materials and methods
　　2.1 Reagents and instruments
　　Tellurium （powder，99.99999%），Sodium borohydride （NaBH4） （96%），Cadmium chloridehydrate （CdCl2·2.5H2O）（99%），3Mercaptopropionicacid （MPA，99%），1（3Dimethylaminopropyl）3ethylcarbodiimide hydrochloride （EDC·HCl，99%），3aminopropyltriethoxysilane （APTS，98%），tetraethyl orthosilicate （TEOS） and ammonium hydroxide （25%～28%），were all obtained from Aladdin （Shanghai Aladdin Biochemical Polytron Technologies Inc.）.All chemicals were of analytical grade and used without further purification.Ultrapure water used throughout the experiments was generated by MilliQ purification system （Millipore，Bedford，MA，USA）.
　　2.2 Synthesis of watersoluble CdTe QDs
　　In our first set of experiments，we synthesized the watersoluble CdTe QDs according to a method from literatures[16-17]with some modification.In brief，freshly prepared oxygenfree NaHTe solution was added to nitrogensaturated 1 mmol·L-1 CdCl2 aqueous solution at pH 9.2 in the presence of MPA as a stabilizing agent.NaHTe was produced in an aqueous solution by reaction of NaBH4 with tellurium powder at a molar ratio of 3 ∶1.The molar ratio of MPA ∶Cd2+∶Te- was fixed at 6.25 ∶2.5 ∶1.The CdTe precursor solution was subjected to reflux at 105 ℃ under the protection of nitrogen balloon，and different sizes of CdTe QDs were obtained at different refluxing times.
　　2.3 Synthesis of CdTe@SiO2NH2 composite particles
　　The red CdTe （Em=634 nm） which prepared under 105 ℃ was used to synthesis the follow CdTe@SiO2 composite particles.Influence of the content of water and ammonium hydroxide on the size and morphology of composite nanoparticles were investigated in this part.The asprepared CdTe aqueous solution （9 mL） was introduced into a liquid system containing water （18.2 mL） and ethanol （72 mL），under ultrasonic for 2 min，followed by the addition of ammonium hydroxide （0.8 mL） under stirring for 10 min.TEOS （1 mL） was subsequently added stirring for 30 min，then 40 μL of APTS was introduced for 4 h.Subsequently，the resulting particles were centrifuged at 12000 r/min for 3 min and washed in sequence with ethanol and water respectively to remove any surfactant and unreacted molecules.The particles suspended in liquid media were typically collected by centrifugation and reconstituted with 9 mL water for further characterizations.
　　2.4 Determination of amine groups on CdTe@SiO2 composite particles surface
　　Fluorescamine was used to detect the content of amine groups according to a previous report[18].The procedure was as follows：5 mg of composite particles were dissolved in a mixture of 2.0 mL of NaHCO3 （5 wt.% in water） and 2.0 mL of ethanol under ultraphonic at room temperature.2 mL of suspension above was diluted by 1 mL of NaHCO3 （5 wt.% in water） and 1 mL water.Then 300 μL of fluorescein solution in methanol （5 mmol·L-1） was introduced into the mixture.After oscillating for 3min，the fluorescence intensity at 487 nm of the suspension was determined. 　　2.5 Optical measurements
　　Absorption spectra were measured on a Shimadzu model 3101PCUVVISNIR scanning spectrophotometer over a wavelength range from 300 to 800 nm.The samples were measured against water as reference.Photoluminescence （PL） was measured on a Spex Fluorolog 3111 using a PMT detector for spectra between 200 and 800 nm.All the samples were dispersed in water and loaded into a quartz cell for measurements.
　　2.6 Transmission Electron Microscope （TEM）
　　The size and shape of the products were studied by TEM obtained using a JEOL2100 microscope，operating at 200 kV.The specimens were prepared by drop casting the sample dispersion onto a carbon grid，which was placed on a filter paper to absorb the excess solvent.
　　3 Results
　　3.1 Influences of the refluxing time and temperature on properties of CdTe
　　As shown in Figure 1 along with the passage of the refluxing time，an obvious increasing of the maximumemission wavelength and a significant changing of the color （from green，through yellow to red）occurred，which indicated that the diameter of CdTe grew gradually[19].Meanwhile，the halfpeak width was broadening，and fluorescence intensity enhanced first and then reduced，which were consistent with the ′Ostwald ripening′ during the crystal growth process[20].
　　As illustrated in Table 1 at 100，105，115 ℃，the rates of redshift were 1.98，3.24，4.76 nm/h，respectively.As known that with the raise of thediameter of CdTe QDs，the redshift of maximum emission occurred，hence，it was inferred that a higher temperature can make the rate of growth increase.Otherwise，the fluorescence intensity of the green CdTe QDs which prepared at 105 ℃ was the highest，3.53 and 5.40 times of the particles which prepared at 100 and 115 ℃，respectively； the yellow CdTe QDs was 1.34 and 3.25 times and the red CdTe QDs was 0.87 and 1.68 times.Also，the halfpeak width of the green CdTe QDs which prepared at 105 ℃ was 86% and 82% of the particles prepared at 100 and 115 ℃，the fact indicated that green CdTe QDs （105 ℃） had narrow diameter distribution.Considering the refluxing time，the fluorescence intensity and the diameter distribution，we determined that 105 ℃ is the optimum condition to synthesis CdTe QDs at different PL emission wavelength.
　　3.2 Investigation of the growth pattern of SiO2 on the surface of CdTe QDs
　　Figure 2 presents the general morphology of the fluorescent CdTe@SiO2 composite particles.Products were relatively uniform，CdTe QDs located at the center of each silica particle to form the coreshell structure.Apart from that，the image of 2d shows that the final composite was regular sphere with only one CdTe QD in silica shell；the eventually composite particle which multiple CdTe QDs concentrated in one area of SiO2 was also spherical indicated in Figure 2c； multiple CdTe QDs distributed in two relatively independent areas of the SiO2 particle，and the structure of final composite was pyriform （Figure 2b）； It is also displayed at Figure 2a that three relatively independent areas formed in the SiO2 particle，and the structure of final composite was pyriform as well，but has three distinct parts.Phenomenons above can be explained by the growth process of SiO2：on the initial stage of the reaction，TEOS was hydrolyzed on the surface of CdTe QDs to form the original silicon layer （This is called growth center）； then the hydrolyzing continued to take shape of the final composite particles which CdTe QDs were completely coated by SiO2.During the second phase of hydrolysis，TEOS maybe was condensed with hydroxyl group on a sole growth center，or hydroxyl group on multiple growth centers which depended on the extent of crosslinking between growth centers.So，if there is no crosslinking，TEOS would be hydrolyzed evenly in all directions on a single growth center，eventually forming rules of spherical composite particles （Figure 2d and c）； if there are two growth centers linked together，TEOS would be hydrolyzed to two main directions，eventually forming dimer pyriform composite particles （Figure 2b）； if there are three growth centers linked together，TEOS would be hydrolyzed to three main directions，eventually forming trimer pyriform composite particles （Figure 2a）.Therefore，we can say that the final morphology of the particles was determined by the distribution and the extent of crosslinking of CdTe QDs in the growth center. 　　3.3 Morphology and diameter of CdTe@SiO2NH2 composite particles
　　Figure 3 showed the morphologies of CdTe@SiO2NH2 composite particles which were synthesized in the presence of different amounts of H2O and ammonia.We changed the amount of ethanol to keep the balance of solution system.In combination with Table 2，it could be clearly seen that the nodularity decreased with the increasing of the concentration of ammonia.The reason of the results above is：the hydrolysis rate of TEOS was accelerated caused by the more ammonia which led to the increase of the possible of crosslinking to form more dimer pyriform composite particles or multimer composite particles.In turn，the less ammonia，the less crosslinking.Besides，the excess or lack of H2O would diminish the size of composite particles which was in line with classic Stober Method[21].
　　3.4 Comparison offluorescence intensity and maximum emission wavelength of CdTe and CdTe@SiO2NH2 composite particles
　　Figure 4 shows thatmaximum emission wavelength of CdTe was 10 nm redshift after encapsulated into SiO2，because of the changes of electron cloud density which caused by the reaction between CdTe and SiO2.Moreover，the intensity of composite particles was 80% of CdTe QDs，mainly because of the inevitable loss during the washing process and the changes of surface state.
　　3.5 Determination of the optimum time for introducing APTS to enhance the amine group which on the surface of CdTe@SiO2NH2 composite particlesGiven that the further applications in biology，sufficient amino on the surface ofsilica shell was needed to combine with biomacromolecule via chemical bond.Therefore，the optimum time for introducing alkylating agent APTS to enhance the amino group was investigated in our article.According to the theory，free primary amine binding with fluorescamine would form fluorescent products and the more amine groups on the surface of particles，the stronger fluorescence of the products.Hence，we measured fluorescence intensity at 487 nm after composite particles were reacted with excessive fluorescamine under the same condition The comparison of the fluorescence intensity was showed in Figure 5.It indicated that particles which prepared by added APTS after the reaction of 30 min had the most amine groups on the surface.Crystal growth includes two processes，fast nucleation and slow growth[22].APTS would be encapsulated in inner layer which resulted in a lower amino content on its surface if it was introduced too early.APTS was probably connected with silane through the condensation of trimethoxy and carboxyl[18].With the SiO2 growing longer and more complete，the contents of APTS on its surface were lower，causing the lower amino content on composite particles′ surface finally. 　　4 Discussions
　　Overall，the watersoluble CdTe QDs were synthesized with MPA as a stabilizing agent.In this synthesis，the growth rate of CdTe QDs increased with increasing reflux temperature，and halfpeak width also increased.The method we have reported allows for the straightforward production of CdTe@SiO2 composite particles.Spherical CdTe@SiO2 composite with good dispersion，wonderful crystallization，and excellent fluorescence was first synthesized by the modified Stober Method.The diameters of the CdTe@SiO2 composites were controllable between 160 nm and 260 nm by simply changing the rate of TEOS and water.The growth pattern of SiO2 on the surface of CdTe QDs were explained by two phases of TEOS hydrolysis.And the final product of CdTe@SiO2 composite has abundant amino group which could easily couple with carboxyl group or amino of biological macromolecules.
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　　摘要：本研究成功地合成了水溶性CdTe量子点（QDs）.考察温度对回流时间、荧光强度及粒径大小等因素的影响，实验优化合成条件之后确定最佳反应温度为105 ℃.接着，本研究运用改进后的Stober法更为简易地合成了硅包CdTe量子点复合微球，该微球的粒径可通过改变正硅酸乙酯及水的含量控制在160～260 nm之间，同时根据复合微粒的电镜拍摄图初步探讨了SiO2在CdTe表面的生长模式.为了将来能运用到生物检测中，此实验加入3-氨基丙基三乙氧基硅烷（APTS），引入氨基团，并优化了 APTS 加入时间，得到的粒子表面氨基含量最高.这种复合微粒具有与原来量子点相当的量子产率，同时具有良好的水溶性、胶体稳定性、化学反应性，为量子点作为荧光标记物的应用打下了基础.
　　关键词：CdTe量子点； 硅包； Stober法
　　（责任编辑：顾浩然，郁 慧）