Research article Received: 05 October 2013,
Revised: 26 November 2013,
Accepted: 03 December 2013
Published online in Wiley Online Library: 16 January 2014
(wileyonlinelibrary.com) DOI 10.1002/bio.2630
Synthesis of highly luminescent and biocompatible CdTe/CdS/ZnS quantum dots using microwave irradiation: a comparative study of different ligands Hua He,* Xing Sun, Xiaojuan Wang and Hai Xu* ABSTRACT: We compared the effects of several ligands frequently used in aqueous synthesis, including L-cysteine, L-cysteine hydrochloride, N-acetyl-L-cysteine (NAC), glutathione and 3-mercaptopropionic acid, for microwave synthesis of CdTe quantum dots (QDs) in a sealed vessel with varied temperatures and times, and then developed a rapid microwave-assisted protocol for preparing highly luminescent, photostable and biocompatible CdTe/CdS/ZnS core–multishell QDs. The effects of molecular structures of these ligands on QD synthesis under high temperatures were explored. Among these ligands, NAC was found to be the optimal ligand in terms of the optical properties of resultant QDs and reaction conditions. The emission wavelength of NAC-capped CdTe QDs could reach 700 nm in 5 min by controlling the reaction temperature, and the resultant CdTe/CdS/ZnS core–multishell QDs could achieve the highest quantum yields up to 74% with robust photostability. In addition, the effects of temperature, growth time and shell–precursor ratio on shell growth were examined. Finally, cell culturing indicated the low cytotoxicity of CdTe/CdS/ZnS core–multishell QDs as compared to CdTe and CdTe/CdS QDs, suggesting their high potential for applications in biomedical imaging and diagnostics. Copyright © 2014 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web-site. Keywords: quantum dots; microwave irradiation; ligands; shell; biocompatibility
Introduction
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* Correspondence to: Hua He, Hai Xu, State Key Laboratory of Heavy Oil Processing and the Centre for Bioengineering and Biotechnology, China University of Petroleum (East China), 66 Changjiang West Road, Qingdao 266580, China. E-mail: [email protected]; [email protected] State Key Laboratory of Heavy Oil Processing and the Centre for Bioengineering and Biotechnology, China University of Petroleum (East China), Qingdao, China
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Owing to their unique electrical and optical properties, colloidal semiconductor nanocrystals or quantum dots (QDs) have attracted considerable attention for their potential applications in optoelectronics and biolabeling (1–4). Currently, most highquality QDs are prepared by the organometallic approach (5,6). Organic-derived QDs show poor dispersion in water and thus require further surface modification with hydrophilic ligands for biological applications, but these modifications are associated with a reduction in quantum yield (QY) and stability (7,8). Aqueous synthesis is an alternative to non-aqueous synthesis, providing QDs with excellent water solubility and stability (9,10). However, the aqueous-based QDs typically require a long reaction time from several hours to several days to prepare and usually possess poor spectral properties and relatively low QYs (such as CdSe and ZnSe QDs) (11,12). Microwave irradiation (MI) as a heating method has been explored as a powerful technique for the preparation of highquality fluorescent QDs in aqueous solution (13–19). Unlike the traditional form of heating, which depends on convection currents and on the thermal conductivity of the material, MI triggers heating by dipolar polarization or ionic conduction mechanisms (20–23). When irradiated at microwave with specific frequencies, the molecular dipoles or charged particles in the reaction mixture oscillate in the alternating field, creating heat through molecular friction (or particle collision) and dielectric loss. Water in the microwave reaction is an excellent solvent and can effectively convert microwave energy into heat because of the
relatively high dielectric loss (tan δ = 0.123) (22). This unique heating mode allows the temperature to be raised uniformly and quickly throughout the whole liquid volume, thereby reducing the thermal gradient effects in favor of uniform nucleation and formation of monodispersed nanoparticles. Importantly, when conducted in a sealed vessel, aqueous solutions can be rapidly heated to above 100 °C under MI. The enhancement of temperature can dramatically accelerate the growth rate of nanocrystals during the Ostwald ripening stage, which is extraordinarily beneficial for reducing the concentration of surface defects of nanocrystals and narrow the particle size distribution (16–19). Microwave methodology is therefore considered a fast and effective strategy to prepare high-quality QDs. For example, Li et al. used MI to synthesize 3-mercaptopropionic acid (MPA)capped CdTe QDs with high QYs (40–60%) within 5 min (13). Qian et al. prepared high-quality CdSe and ZnSe QDs via MI with QYs of up to 10–30% and good crystallinity (14,15), which are usually inaccessible to the conventional aqueous route. Surface ligands play a key role in controlling the nucleation and growth of QDs and strongly affect their optical properties
H. He et al. and stability. In the traditional aqueous synthesis of QDs, the most effective ligands are thioglycolic acid and MPA (10,24,25). To improve the biocompatibility and biofunctionability of the products, some biocompatible ligands, such as L-cysteine (Cys) or its hydrochloride (Cys·HCl), N-acetyl-L-cysteine (NAC), glutathione (GSH) and RGD peptides have recently been explored (26–31). The introduction of these biocompatible molecules has not only produced highly luminescent and biocompatible QDs but also endowed them with more functionality. Nevertheless, for the conventional aqueous synthesis with these ligands, the reaction temperature did not exceed 100 °C. In a microwave synthesis performed in a sealed vessel, however, the elevated temperature and pressure might exert various effects on the ligands and thus affect product quality. The ligands mentioned above have never been used before either in microwave synthesis or lack a reliable examination of the microwave synthesis conditions. An assessment of the effects of these ligands on the microwave synthesis of QDs is therefore required. It is also an accepted wisdom that the epitaxial growth of a suitable inorganic shell on the surface of nanocrystals is a key approach to improve the QYs and stability of QDs as well as to improve the biocompatibility required for their effective use in biomedical applications (32–34). This is because the shell can reduce the nonradiative recombination defects on the nanocrystal surface and the release of toxic metal ions to the biological system. Although it has been reported that MI can be used to synthesize CdTe/CdS core/shell and CdTe/CdS/ZnS core–multishell QDs, they were prepared with MPA as the capping ligand (18,19), and MPA is notorious for its carcinogenic toxicity and its odor. Our aim here is to prepare highly luminescent, photostable, biocompatible core/shell QDs by utilizing the high efficiency and convenience of MI with optimized ligands. We first compared several frequently used ligands in traditional aqueous synthesis of QDs, including Cys, Cys·HCl, NAC, GSH and MPA, in the microwave synthesis of CdTe QDs at a controllable temperature, and then explored the effects of MI temperatures and molecular structures of these ligands on the optical property and stability of QDs. Finally, we employed the optimal ligand, NAC, to prepare highly luminescent and biocompatible CdTe/CdS/ZnS core–multishell QDs.
Experimental Synthesis of CdTe, CdTe/CdS and CdTe/CdS/ZnS quantum dots
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Tellurium powder (99.997%), CdCl2 (99.99%+), NaBH4 (≥ 98%), L-cysteine (≥ 97%), L-cysteine hydrochloride (≥ 98%), NAC (≥ 99%), GSH (≥ 98%), cysteamine (CA; ≥ 98%) and rhodamine 6G (95%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). MPA (≥ 99.0%) was purchased from Fluka (Buchs, Switzerland). All chemicals were used without additional purification. All solutions were prepared with ultrapure water (18.2 MΩ cm) purified on a Millipore System (Millipore, Bedford, MA, USA). A microwave digestion system (WX-4000; Yi-Yao Instruments, Shanghai, China), equipped with controllable temperature units, was used for the preparation of CdTe QDs. The system works at 0–1000 W power and can be operated at a frequency of 2450 MHz. The reaction temperature and time can be programmed by users. CdTe QDs were prepared using the reaction between Cd2+ and NaHTe solution. Briefly, NaHTe solution was freshly prepared by dissolving 0.05 g NaBH4 in 2 mL ultrapure water and then 0.04 g Te powder was added into the NaBH4 solution. This reaction was conducted at
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room temperature overnight in a syringe with a needle to help release the gas generated during the reaction, and the resulting NaHTe solution was then diluted by injecting into 23 mL ultrapure water degassed with a high-purity N2 (99.999%) flow before use. For a typical CdTe QDs synthesis, Cd2+ ligand (Cys, Cys · HCl, NAC, GSH, MPA) precursor solution was prepared by dissolving CdCl2 and a given ligand in ultrapure water, and then adjusted to pH 8.5 with 1 M NaOH. The NaHTe solution was then injected into a N2-saturated precursor solution under vigorous stirring, along with an increase of the solution pH to 9.5. The typical molar ratio of Cd2+, HTe– and ligand was 2: 1: 4.8 in a total volume of 100 mL. Six mL CdTe precursor solution was transferred into a digestion vessel and placed into the microwave digestion furnace. Under MI (300 W), the reaction system reached a certain temperature and pressure (the temperature decides the pressure). Various sizes of CdTe QDs were prepared by controlling MI temperature and time. The QD samples were taken out from the reaction vessel when the temperature was naturally cooled to lower than 60 °C. For the synthesis of CdTe/CdS QDs, NAC-capped CdTe QDs were first precipitated with 2-propanol and collected via centrifugation. The precipitate was then re-dispersed in 6 mL solution containing 1.25 mM CdCl2 and 6.0 mM NAC (pH 8.5), followed by adding Na2S to a final concentration of 0.31 mM. Next, the CdTe/CdS precursor solution was injected into a digestion vessel. CdTe/CdS core/shell QDs with different emission wavelengths were obtained by controlling MI temperature and time. Similarly, the same protocol was also employed for the synthesis of CdTe/CdS/ZnS QDs except for using CdTe/CdS QDs as cores, which were dispersed in the solution containing 1.25 mM ZnCl2 and 6.0 mM NAC (pH 8.5). Characterization The as-prepared QD samples were diluted for optical characterizations. Their ultraviolet (UV)-visible absorption spectra were obtained using a Shimadzu UV-2450 spectrophotometer (Kyoto, Japan) and fluorescence spectra were recorded on a Hitachi F-2500 fluorescence spectrophotometer (Tokyo, Japan). All optical measurements were performed at room temperature under ambient conditions. The QY of CdTe QDs was measured using rhodamine 6G in an ethanol solution (QY = 95%) as a reference standard as described previously (13,24). Briefly, the absorbance for the standard and the CdTe QD samples at the excitation wavelengths and fluorescence spectra of the same solutions were measured, respectively. Six different concentration of rhodamine 6G and CdTe QD solutions (absorbance at excitation wavelength < 0.1) were used in the measurements. The integrated fluorescence intensity vs. absorbance was plotted. The plot obtained should be a straight line with a gradient M, which was used to calculate the QY according to the following equation: ϕx ¼ ϕs
Mx Ms
2 ηx ηs
(1)
Where the subscripts s and x denote standard and test samples, respectively, ϕ is QY and η is the refractive index of the solvent. It should be noted that the excitation wavelength of QDs was set at the excitonic absorption peak of the CdTe QD samples for measurements of QY. Transmission electron microscopy (TEM) samples were prepared by dropping the aqueous nanocrystals on to carbon-coated copper grids with excess
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Synthesis of quantum dots using microwave irradiation solvent evaporated. TEM and high-resolution transmission electron microscopy (HRTEM) images were recorded on a JEM-2100 electron microscope (JEOL Ltd., Tokyo, Japan) operating at 200 kV. XRD spectra were taken on an X’Pert PRO MPD diffractometer (PANalytical, Almelo, The Netherlands). MTT assays
Figure 1. Molecular structures of L-cysteine (Cys), L-cysteine hydrochloride (Cys·HCl), N-acetyl-L-cysteine (NAC), glutathione (GSH), and 3-mercaptopropionic acid (MPA).
MTT cell viability assays were performed to provide an assessment of the toxicity of QDs against human embryonic kidney 293 (HEK 293) cells. Briefly, the cells were pre-seeded on 96-well plates at a density of 2 × 104 cells/well in 100 μL medium containing 10% fetal bovine serum. The plates were then incubated for 24 h at 37 °C containing 5% CO2. Fifty μL of different concentrations of QDs in phosphate-buffered saline solution, estimated according to Yu et al. (35), were added and the cells were incubated in the dark
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Figure 2. The normalized absorption (except for A,C) and normalized PL spectra of CdTe quantum dots synthesized by using L-cysteine (A,B) and L-cysteine hydrochloride (C,D), NAC (E,F), GSH (G,H) and MPA (I,J) as ligands, respectively, at different microwave irradiation temperatures and times. The excitation wavelength for all PL spectra was 370 nm. The microwave irradiation temperatures and times (from line a to f) are 100 °C/5 min, 100 °C/15 min, and 120 °C/5 min, 120 °C/15 min, 120 °C/30 min and 120 °C/ 45 min, respectively. (K,L) Evolution of the emission FWHM and QY, respectively, of NAC-, GSH- and MPA-capped CdTe quantum dots with the emission peak position. FWHM, full width at half maximum; GSH, glutathione; MPA, 3-mercaptopropionic acid; NAC, N-acetyl-L-cysteine; PL, photoluminescence; QY, quantum yield.
H. He et al. for 12 or 24 h. Wells containing cells without QDs served as controls. Subsequently, 20 μL of MTT solution (5 mg/mL) was added to each well and the plates were incubated for 4 h at 37 °C. The precipitated formazan was dissolved in 200 μL of dimethyl sulfoxide. The absorbance of each sample at 490 nm was measured using a microplate autoreader (Spectramax M2e, Molecular Devices, Sunnyvale, CA, USA). Percentage cell survival is expressed as a percentage ratio of A490 of cells treated with QDs over control cells.
Results and discussion Synthesis of CdTe quantum dots with different ligands Figure 1 presents the ligands used in this study, including Cys, Cys·HCl, NAC, GSH and MPA. Figure 2 shows the absorption and photoluminescence (PL) spectra of the CdTe QDs, which were prepared in the presence of these ligands at different
Figure 3. The normalized PL spectra of CdTe quantum dots prepared by using N-acetyl-L-cysteine as a ligand at 140 °C/5 min, 140 °C/15 min, 140 °C/20 min and 160 °C/5 min. The corresponding PL emission wavelengths were 603 nm, 614 nm, 646 nm and 680 nm, with quantum yields of 21.8%, 20.7%, 10.5% and 6.9%, respectively. PL, photoluminescence.
temperatures (100 and 120 °C) and time under MI. The photographs of CdTe sample solutions are shown in the supporting information (see Supplementary Fig. S1). With prolonging the heating time or increasing the reaction temperature, the PL emission peak positions of the resulting CdTe QDs with these ligands shifted to longer wavelengths. Owing to the size dependence of optical absorption and emission of QDs (5,35,36), the growth rate could be evaluated from the shift of absorption or PL emission peaks. We here observed large differences on the growth rate of QDs with different ligands. For example, under the same MI reaction condition, e.g. 120 °C/15 min, the PL emission peaks of CdTe QDs prepared with Cys, Cys·HCl, NAC and MPA shifted to 623 nm, 638 nm, 571 nm and 575 nm, respectively. Besides, these ligands showed different effects on optical quality and stability of QDs. In the cases of Cys and Cys·HCl, QD solutions became turbid suspensions during the growth process due to the formation of aggregates of QDs, which led to the strong non-zero baseline absorbance in their absorption spectra (Fig. 2A,C) and strong frequency scattering peaks at 740 nm (the excitation wavelength was 370 nm) in their PL emission spectra (Fig. 2B,D). Additionally, all these Cys-capped QDs synthesized at different reaction temperatures and times displayed weak PL (QYs less than 5%). In the case of GSH, CdTe QDs could be well prepared at 100 °C, but upon increasing the reaction temperature to 120 °C, the QD solution gave black precipitates after 10 min MI. In comparison to other ligands, MPA and NAC provided QDs with better optical spectra and high QYs up to 45%, as shown in Fig. 2(E,F,I,J). Figure 2(K,L) are respective plots of emission full width at half maximum (FWHM) and QY versus the emission wavelengths for NAC-, MPA- and GSH-capped CdTe QDs. In general, the narrow emission FWHM and high QY reflects the high quality of the QDs. It is evident that in terms of FWHM and QY, NAC-stabilized CdTe QDs are even slightly superior to those prepared under the same conditions with MPA, which is the most typical ligand in current microwave synthesis of QDs. Comparison of the molecular structures of the ligands (Fig. 1) indicates that the amine group might exert an adverse effect on the microwave synthesis of CdTe QDs. Under the present solution conditions (pH typically in the range of 8.5–9.5), the amine
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Figure 4. The typical absorption (A) and PL spectra (B) of CdTe, CdTe/CdS core/shell and CdTe/CdS/ZnS core–multishell quantum dots in the presence of N-acetyl-L-cysteine. The quantum yield of CdTe quantum dots increased from 38.3% to 53.5% and then 60.1% after CdS and ZnS shell growth at 60 °C/5 min and 65 °C/5 min, respectively. Their emission 2+ 2– 2+ 2– wavelengths were 541, 552 and 558 nm respectively. The precursor ratios of Cd /S and Zn /S were fixed at 1/0.25 for shell growth. (C) The PL spectra of the corresponding quantum dot solutions after 24 h irradiation (ultraviolet lamp, 365 nm, 450 W). The quantum yields of CdTe, CdTe/CdS core/shell and CdTe/CdS/ZnS core–multishell quantum dots decreased to 11.9%, 49.9% and 54.1%, respectively. PL, photoluminescence.
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Synthesis of quantum dots using microwave irradiation group is protonated, making the ligands Cys and Cys·HCl electrically neutral. As a result, the interparticle electrostatic repulsion is significantly reduced, thus promoting the initial kineticfavored aggregation growth of QDs and agglomeration of the resulting QDs with increasing incubation time (37). In microwave synthesis of QDs, such processes would be dramatically strengthened because of the rapid heating via MI and the higher temperature in the sealed vessel. Hence, the use of Cys and Cys·HCl as the ligands for microwave synthesis is unfavorable for obtaining dispersible QDs with high quality. In addition, in the case of Cys·HCl, the introduction of HCl is equivalent to the addition of NaCl into Cys solution under alkaline conditions. The resultant increase in ionic strength must further lower the interparticle electrostatic repulsion, thus further accelerating the aggregation and agglomeration of particles (38). As shown in Fig. 2(C,D), larger non-zero baseline absorption and stronger frequency scattering peaks at 740 nm than those shown in Fig. 2 (A,B) suggest that Cys·HCl causes more aggregation of QDs. In the case of GSH, although each GSH molecule contains a terminal amine group, the two carboxyl groups still maintain relatively strong interparticle repulsion, thus favoring the surface protection and stability of the QDs. However, higher temperature (≥ 120 °C) and longer reaction time in the sealed vessel caused the QD solution to produce black precipitates, suggesting the loss of the stabilizing effect of GSH probably due to its decomposition (see Supplementary Fig. S2 in the Supporting Information). For NAC, the N-terminal capping eradicated the adverse effect of the amine group and the interparticle electrostatic repulsion arising from the carboxyl group ensures that the NAC-capped QDs are well dispersed in
aqueous solution. The high temperature under MI shortened the preparation time by increasing the growth rate of QDs. As can be seen in Fig. 2(E,F), the growth rate at 100 °C was relatively slow, but it was markedly accelerated at 120 °C, and QDs with longer emission wavelengths were obtained after the same reaction time. Furthermore, QDs could be successfully prepared in the presence of NAC at higher temperatures, e.g. 140 and 160 °C, and still possessed relatively good optical properties (Fig. 3), suggesting the excellent stabilizing effect of NAC for QDs. For example, at 160 °C, CdTe QDs emitting at 680 nm could be prepared in 5 min with a QY of 6.9%. Therefore, the relatively strong electrostatic repulsion and molecular stability at high temperature gave NAC the exceptional capability of acting as a ligand for a high-temperature reaction, which could accelerate greatly the growth rate of nanocrystals during the Ostwald ripening stage in favor of preparing high-quality QDs with a low concentration of surface defects and a narrow size distribution (16–19,29). In addition, we also examined microwave synthesis of CdTe QDs using CA as ligand, which contains an amine group in place of a carboxyl group in comparison with NAC. In this case, the CdTe precursor solution instantly gave a precipitate upon heating to above 100 °C (see Supplementary Fig. S3 in the Supporting Information), suggesting the poor stabilizing effect of CA for CdTe QDs in the pH range 8.5–9.5. For CA, a slightly acidic environment (pH 5.6–6.9) may be more suitable for stabilizing CdTe QDs (9). However, such conditions might weaken the function of the thiol group, which plays the dominant role in determining the stability and optical properties of QDs in our system (31).
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Figure 5. (A) The normalized PL spectra of NAC-capped CdTe core and CdTe/CdS core/shell QDs prepared by microwave irradiation for 5 min at different temperatures. 2+ 2– The precursor ratio of Cd /S was fixed at 1/0.25. (B) The normalized PL spectra of NAC-capped CdTe/CdS core and CdTe/CdS/ZnS core–multishell QDs prepared by 2+ 2– microwave irradiation at 65 °C for different growth times. The precursor ratio of Zn /S was fixed at 1/0.25. (C) The normalized PL spectra of NAC-capped CdTe core 2+ 2– and CdTe/CdS core/shell QDs prepared at different Cd /S ratios by microwave irradiation for 5 min at 60 °C. (D) The normalized PL spectra of NAC-capped CdTe/CdS core 2+ 2– and CdTe/CdS/ZnS core–multishell QDs prepared at different Zn /S ratios by microwave irradiation for 5 min at 65 °C. NAC, N-acetyl-L-cysteine; PL, photoluminescence; QD, quantum dot.
H. He et al. Synthesis of CdTe/CdS core/shell and CdTe/CdS/ZnS core– multishell quantum dots To improve the optical properties, stability and biocompatibility of nanocrystals, a core/shell structure is usually an ideal choice. The shell not only acts as a physical barrier to protect the optically active core from its surrounding medium against environmental changes, surface chemistry and photo-oxidation, but also provides an efficient passivation of the surface, such as reducing the number of surface dangling bonds, which enhances greatly the PL QYs of QDs. In the choice of shell materials, the most crucial factor is the lattice mismatch between core and shell, which can affect the quality of core/shell QDs by creating excessive crystal strain at core/shell interfaces. For a CdTe core, a CdS shell is more suitable than ZnS because of better overlap between the lattice parameters (19,34). Taking into account the inherent cytotoxicity of Cd-containing QDs in live cell and in vivo biology, however, ZnS is more suitable as a shell material. As a result, the CdTe/CdS/ZnS core–multishell system was considered, where CdS acted as a buffer layer and allows stepwise change of lattice spacing between the CdTe core and ZnS shell, thus reducing the strain within the nanocrystals. Here, the
optimized ligand NAC was used for the microwave synthesis of CdTe/CdS core/shell and CdTe/CdS/ZnS core–multishell QDs with controllable temperature. Figure 4 gives representative absorption and PL spectra of CdTe core, CdTe/CdS core/shell, and corresponding CdTe/CdS/ZnS core–multishell QDs synthesized under MI. The MI temperatures/times were 60 °C/5 min and 65 °C/5 min for CdS and ZnS shell growth, respectively. Note that the same CdTe or CdTe/CdS solution was used for the subsequent preparation of CdTe/CdS or CdTe/CdS/ZnS QDs. A significant red-shift was observed in the absorption and PL emission spectra, which gives an indication of the formation of the intended CdTe/CdS/ZnS structure instead of the CdZnTeSalloyed QDs that have been extensively discussed in the literature (19,39). Owing to the encapsulation of CdS and ZnS shells, the PL intensity of QDs was noticeably enhanced and they displayed better optical stability. Figure 4(C) presents the PL spectra of the corresponding QD solutions exposed to UV irradiation for 24 h (365 nm, 450 W). After continuous strong irradiation, the emission peaks of CdTe /CdS and CdTe/CdS/ZnS QDs were nearly unchanged with QYs remaining above 50%, while the PL spectrum of CdTe QDs became asymmetric with a decrease of QY from 38.3% to 11.9%.
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Figure 6. Transmission electron microscopy (top) and high-resolution transmission electron microscopy (middle) images and size distribution histograms (bottom) of (A,D,G) NAC-capped CdTe core QDs emitting at 541 nm, (B,E,H) NAC-capped CdTe/CdS core/shell QDs emitting at 552 nm and (C,F,I) NAC-capped CdTe/CdS/ZnS core–multishell QDs emitting at 558 nm. NAC, N-acetyl-L-cysteine; QD, quantum dot.
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Synthesis of quantum dots using microwave irradiation Effects of microwave irradiation temperature, growth time and shell–precursor ratio on optical properties To achieve optimal enhancement in the optical property of nanocrystals, the reaction conditions for shell growth must be carefully controlled. The key point is to control the shell thickness. If the shell is too thin, it is inefficient for the desirable passivation of the core nanocrystals. Too thick a shell generally causes deterioration in the optical properties of the resulting core/shell QDs (33,40). In addition, due to the epitaxial growth of the shell on the nanocrystal surface, the red-shifts of the absorption and emission spectra are thickness-dependent (33,40). As the shell thickness increases, the absorption and emission peaks shift toward longer wavelengths. In the present system, we found that MI temperature, growth time and the precursor ratio of Cd2+/S2– and Zn2+/S2– affected significantly the optical properties of the resulted core/shell QDs. By regulating these conditions, we observed that limiting the emission red-shifts to 10–15 nm for CdS shell and 5–10 nm for ZnS shell, respectively, was favorable for obtaining high-quality QDs, which may be consistent with the optimum shell thickness. Under optimal conditions, as shown in Fig. 5, the highest QYs of CdTe/CdS/ZnS core–multishell QDs can reach as high as 74%, retaining narrow FWHMs of 40–60 nm. Figure 5(A) presents the PL spectra of NACcapped CdTe core and the corresponding CdTe/CdS core/shell QDs prepared via MI for 5 min at different temperatures. The precursor ratio of Cd2+/S2– was fixed at 1/0.25. When the reaction temperature increased, the PL emission peak showed a bathochromic shift. The optimal enhancement in PL intensity (10–15 nm red-shifts) could be achieved with temperatures between 60 and 70 °C. Figure 5(B) presents the PL spectra of NAC-capped CdTe/CdS core and the corresponding CdTe/CdS/ ZnS core–multishell QDs prepared via MI at 65 °C for different growth times. The precursor ratio of Zn2+/S2– was fixed at 1/0.25. As the growth time was prolonged, the PL emission peak also shifted to longer wavelengths. Here, the optimal growth time 5–10 min, during which the resultant CdTe/CdS/ZnS core–multishell QDs showed significant improvement in the QYs (5–10 nm red-shifts). Additionally, the emission red-shift was strongly influenced by the precursor ratio. Figure 5(C,D) show the PL spectra of CdTe/CdS core/shell and CdTe/CdS/ ZnS core–multishell QDs grown for 5 min at different precursor ratios of Cd2+/S2– and Zn2+/S2– respectively. The Cd2+/S2– or Zn2+/S2– ratios were regulated by adding different amounts of S2– into the precursor solution at the fixed Cd2+ or Zn2+ concentration. With an increase of the amount of S2–, the PL emission peaks of CdTe/CdS core/shell and CdTe/CdS/ZnS core–multishell QDs shifted to longer wavelengths during the same growth time. Here, the optimal precursor ratio Cd2+/S2– or Zn2+/S2– was 1/0.25. When the precursor ratio was decreased to 1/1, the resultant red-shift after shell growth of 5 min reached 20–30 nm. It should be noted that, at the precursor ratio of 1/1, the emission red-shift could also be adjusted to 10–15 nm by further reducing the growth time, but it was difficult to control the experimental protocol to ensure the reproducibility of results in less than 5 min.
Transmission electron microscopy and X-ray diffraction characterization
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Figure 7. The powder X-ray diffraction patterns of N-acetyl-L-cysteine-capped CdTe, CdTe/CdS core/shell and CdTe/CdS/ZnS core–multishell quantum dots. Standard diffraction lines of cubic CdTe (JCPDS no. 15-0770), cubic CdS (JCPDS no. 80-0019) and cubic ZnS (JCPDS no. 80-0020) are shown for comparison.
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Figure 6 shows TEM and HRTEM images and the corresponding size distribution histograms of the as-prepared CdTe, CdTe/CdS
and CdTe/CdS/ZnS QDs. Their emission wavelengths were 541, 552 and 558 nm respectively. These QDs are slightly spherical with narrow size distributions. The average sizes of CdTe, CdTe/CdS and CdTe/CdS/ZnS nanocrystals were about 3.1 nm, 3.9 nm and 4.8 nm respectively. The CdTe/CdS and CdTe/CdS/ ZnS QDs were prepared from the same CdTe and CdTe/CdS solutions. Accordingly, the increment in size indicates the growth of the shells on the cores. The corresponding shell thicknesses of CdS and ZnS were estimated to be about 0.40 and 0.45 nm respectively. In addition, the existence of lattice planes on the HRTEM images confirmed the crystallinity of these QDs. Figure 7 shows XRD patterns of CdTe, CdTe/CdS and CdTe/CdS/ZnS powders. In the diffraction pattern of the CdTe cores, three peaks can be assigned to {111}, {220}, {311} planes, consistent with the cubic (zinc blende) structure of CdTe. By contrast, the diffraction patterns of CdTe/CdS and CdTe/CdS/ZnS QDs shift slightly to a higher angle, and are positioned successively between those of bulk CdTe and bulk CdS and bulk ZnS due to the consecutive formation of CdS and ZnS shell on CdTe core. Compared with the CdTe core, the diffraction peak widths of CdTe/CdS and CdTe/CdS/ZnS QDs become slightly larger, thereby providing additional evidence of epitaxial shell growth rather than an alloyed structure that would show a narrower XRD peak width with increasing size as discussed in the literature (41).
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Figure 8. MTT assays illustrating the percentage cell viability upon exposing human embryonic kidney 293 cells to different concentrations of N-acetyl-L-cysteine-capped CdTe, CdTe/CdS core/shell and CdTe/CdS/ZnS core–multishell QDs for 12 h (A) and 24 h (B). QD, quantum dot.
Cytotoxicity The biocompatibility of QDs is a crucial issue for their future biological applications, especially those in live cell and in in vivo imaging. One of our purposes for the design of CdTe/CdS/ZnS core–multishell structure is to lower the cytotoxicity of nanocrystals using the protection of a ZnS shell, which can block the release of Cd2+ ions from the QD surface to the biological system. To validate this hypothesis, the cytotoxicity of CdTe, CdTe/CdS core/shell and CdTe/CdS/ZnS core–multishell QDs against human embryonic kidney 293 cells was extensively assessed by MTT assays. Figure 8(A,B) illustrate percentage cell viability upon exposing the cells to different concentrations of QDs for 12 h and 24 h respectively. As expected, CdTe/CdS/ZnS core–multishell QDs have lower cytotoxicity than CdTe and CdTe/CdS core/shell QDs under the same conditions. In addition, the cells exposed to CdTe or CdTe/CdS QDs showed a strong concentration-dependent decrease in cell viability while this trend was significantly reduced in the case of CdTe/CdS/ZnS QDs. Increasing of the incubation time to 24 h had a strong effect on the cytotoxicity difference between these QDs. For instance, when incubated for 24 h with 400 nM QDs, the cell viability values of the CdTe and CdTe/CdS QDs were reduced almost to zero while the cell survival of CdTe/CdS/ZnS QDs was still maintained above 50%, which demonstrates significantly improved biocompatibility due to the ZnS shell coating.
Conclusions
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The results give an insight into the microwave synthesis of CdTe QDs. We have studied five kinds of ligands, including Cys, Cys·HCl, NAC, GSH and MPA, which are frequently used in the traditional aqueous synthesis of QDs. The effects of the molecular structure of these ligands on the optical properties of CdTe QDs were investigated within a temperature range between 100 and 160 °C. Among these ligands, NAC was found to be the optimal ligand in terms of the optical properties and QYs of resultant QDs as well as reaction conditions. It allowed the preparation of CdTe QDs ranging in emission wavelength from 500 to 700 nm with good optical properties within only 5 min
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by controlling the MI temperature up to 160 °C. Based on the ideal ligand (NAC) and utilizing the high efficiency and convenience of MI, we have also successfully prepared highly luminescent CdTe/CdS/ZnS core–multishell QDs with QYs up to 74%. Finally, we have demonstrated their excellent photostability and favorable biocompatibility. These NAC-capped CdTe/CdS/ ZnS QDs thus could serve as promising optical probes for applications in biological and medical fields. Acknowledgments The authors gratefully acknowledge the support from the National Natural Science Foundation of China (20905078, 21103230) and the Fundamental Research Funds for the Central Universities (12CX04053A, 11CX05001A). H.X. acknowledges the support by Program for New Century Excellent Talents in University (NCET-11-0735).
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Supporting information Additional supporting information may be found in the online version of this article at the publisher’s web-site.
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Copyright © 2014 John Wiley & Sons, Ltd.
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Revised: 26 November 2013,
Accepted: 03 December 2013
Published online in Wiley Online Library: 16 January 2014
(wileyonlinelibrary.com) DOI 10.1002/bio.2630
Synthesis of highly luminescent and biocompatible CdTe/CdS/ZnS quantum dots using microwave irradiation: a comparative study of different ligands Hua He,* Xing Sun, Xiaojuan Wang and Hai Xu* ABSTRACT: We compared the effects of several ligands frequently used in aqueous synthesis, including L-cysteine, L-cysteine hydrochloride, N-acetyl-L-cysteine (NAC), glutathione and 3-mercaptopropionic acid, for microwave synthesis of CdTe quantum dots (QDs) in a sealed vessel with varied temperatures and times, and then developed a rapid microwave-assisted protocol for preparing highly luminescent, photostable and biocompatible CdTe/CdS/ZnS core–multishell QDs. The effects of molecular structures of these ligands on QD synthesis under high temperatures were explored. Among these ligands, NAC was found to be the optimal ligand in terms of the optical properties of resultant QDs and reaction conditions. The emission wavelength of NAC-capped CdTe QDs could reach 700 nm in 5 min by controlling the reaction temperature, and the resultant CdTe/CdS/ZnS core–multishell QDs could achieve the highest quantum yields up to 74% with robust photostability. In addition, the effects of temperature, growth time and shell–precursor ratio on shell growth were examined. Finally, cell culturing indicated the low cytotoxicity of CdTe/CdS/ZnS core–multishell QDs as compared to CdTe and CdTe/CdS QDs, suggesting their high potential for applications in biomedical imaging and diagnostics. Copyright © 2014 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web-site. Keywords: quantum dots; microwave irradiation; ligands; shell; biocompatibility
Introduction
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* Correspondence to: Hua He, Hai Xu, State Key Laboratory of Heavy Oil Processing and the Centre for Bioengineering and Biotechnology, China University of Petroleum (East China), 66 Changjiang West Road, Qingdao 266580, China. E-mail: [email protected]; [email protected] State Key Laboratory of Heavy Oil Processing and the Centre for Bioengineering and Biotechnology, China University of Petroleum (East China), Qingdao, China
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Owing to their unique electrical and optical properties, colloidal semiconductor nanocrystals or quantum dots (QDs) have attracted considerable attention for their potential applications in optoelectronics and biolabeling (1–4). Currently, most highquality QDs are prepared by the organometallic approach (5,6). Organic-derived QDs show poor dispersion in water and thus require further surface modification with hydrophilic ligands for biological applications, but these modifications are associated with a reduction in quantum yield (QY) and stability (7,8). Aqueous synthesis is an alternative to non-aqueous synthesis, providing QDs with excellent water solubility and stability (9,10). However, the aqueous-based QDs typically require a long reaction time from several hours to several days to prepare and usually possess poor spectral properties and relatively low QYs (such as CdSe and ZnSe QDs) (11,12). Microwave irradiation (MI) as a heating method has been explored as a powerful technique for the preparation of highquality fluorescent QDs in aqueous solution (13–19). Unlike the traditional form of heating, which depends on convection currents and on the thermal conductivity of the material, MI triggers heating by dipolar polarization or ionic conduction mechanisms (20–23). When irradiated at microwave with specific frequencies, the molecular dipoles or charged particles in the reaction mixture oscillate in the alternating field, creating heat through molecular friction (or particle collision) and dielectric loss. Water in the microwave reaction is an excellent solvent and can effectively convert microwave energy into heat because of the
relatively high dielectric loss (tan δ = 0.123) (22). This unique heating mode allows the temperature to be raised uniformly and quickly throughout the whole liquid volume, thereby reducing the thermal gradient effects in favor of uniform nucleation and formation of monodispersed nanoparticles. Importantly, when conducted in a sealed vessel, aqueous solutions can be rapidly heated to above 100 °C under MI. The enhancement of temperature can dramatically accelerate the growth rate of nanocrystals during the Ostwald ripening stage, which is extraordinarily beneficial for reducing the concentration of surface defects of nanocrystals and narrow the particle size distribution (16–19). Microwave methodology is therefore considered a fast and effective strategy to prepare high-quality QDs. For example, Li et al. used MI to synthesize 3-mercaptopropionic acid (MPA)capped CdTe QDs with high QYs (40–60%) within 5 min (13). Qian et al. prepared high-quality CdSe and ZnSe QDs via MI with QYs of up to 10–30% and good crystallinity (14,15), which are usually inaccessible to the conventional aqueous route. Surface ligands play a key role in controlling the nucleation and growth of QDs and strongly affect their optical properties
H. He et al. and stability. In the traditional aqueous synthesis of QDs, the most effective ligands are thioglycolic acid and MPA (10,24,25). To improve the biocompatibility and biofunctionability of the products, some biocompatible ligands, such as L-cysteine (Cys) or its hydrochloride (Cys·HCl), N-acetyl-L-cysteine (NAC), glutathione (GSH) and RGD peptides have recently been explored (26–31). The introduction of these biocompatible molecules has not only produced highly luminescent and biocompatible QDs but also endowed them with more functionality. Nevertheless, for the conventional aqueous synthesis with these ligands, the reaction temperature did not exceed 100 °C. In a microwave synthesis performed in a sealed vessel, however, the elevated temperature and pressure might exert various effects on the ligands and thus affect product quality. The ligands mentioned above have never been used before either in microwave synthesis or lack a reliable examination of the microwave synthesis conditions. An assessment of the effects of these ligands on the microwave synthesis of QDs is therefore required. It is also an accepted wisdom that the epitaxial growth of a suitable inorganic shell on the surface of nanocrystals is a key approach to improve the QYs and stability of QDs as well as to improve the biocompatibility required for their effective use in biomedical applications (32–34). This is because the shell can reduce the nonradiative recombination defects on the nanocrystal surface and the release of toxic metal ions to the biological system. Although it has been reported that MI can be used to synthesize CdTe/CdS core/shell and CdTe/CdS/ZnS core–multishell QDs, they were prepared with MPA as the capping ligand (18,19), and MPA is notorious for its carcinogenic toxicity and its odor. Our aim here is to prepare highly luminescent, photostable, biocompatible core/shell QDs by utilizing the high efficiency and convenience of MI with optimized ligands. We first compared several frequently used ligands in traditional aqueous synthesis of QDs, including Cys, Cys·HCl, NAC, GSH and MPA, in the microwave synthesis of CdTe QDs at a controllable temperature, and then explored the effects of MI temperatures and molecular structures of these ligands on the optical property and stability of QDs. Finally, we employed the optimal ligand, NAC, to prepare highly luminescent and biocompatible CdTe/CdS/ZnS core–multishell QDs.
Experimental Synthesis of CdTe, CdTe/CdS and CdTe/CdS/ZnS quantum dots
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Tellurium powder (99.997%), CdCl2 (99.99%+), NaBH4 (≥ 98%), L-cysteine (≥ 97%), L-cysteine hydrochloride (≥ 98%), NAC (≥ 99%), GSH (≥ 98%), cysteamine (CA; ≥ 98%) and rhodamine 6G (95%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). MPA (≥ 99.0%) was purchased from Fluka (Buchs, Switzerland). All chemicals were used without additional purification. All solutions were prepared with ultrapure water (18.2 MΩ cm) purified on a Millipore System (Millipore, Bedford, MA, USA). A microwave digestion system (WX-4000; Yi-Yao Instruments, Shanghai, China), equipped with controllable temperature units, was used for the preparation of CdTe QDs. The system works at 0–1000 W power and can be operated at a frequency of 2450 MHz. The reaction temperature and time can be programmed by users. CdTe QDs were prepared using the reaction between Cd2+ and NaHTe solution. Briefly, NaHTe solution was freshly prepared by dissolving 0.05 g NaBH4 in 2 mL ultrapure water and then 0.04 g Te powder was added into the NaBH4 solution. This reaction was conducted at
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room temperature overnight in a syringe with a needle to help release the gas generated during the reaction, and the resulting NaHTe solution was then diluted by injecting into 23 mL ultrapure water degassed with a high-purity N2 (99.999%) flow before use. For a typical CdTe QDs synthesis, Cd2+ ligand (Cys, Cys · HCl, NAC, GSH, MPA) precursor solution was prepared by dissolving CdCl2 and a given ligand in ultrapure water, and then adjusted to pH 8.5 with 1 M NaOH. The NaHTe solution was then injected into a N2-saturated precursor solution under vigorous stirring, along with an increase of the solution pH to 9.5. The typical molar ratio of Cd2+, HTe– and ligand was 2: 1: 4.8 in a total volume of 100 mL. Six mL CdTe precursor solution was transferred into a digestion vessel and placed into the microwave digestion furnace. Under MI (300 W), the reaction system reached a certain temperature and pressure (the temperature decides the pressure). Various sizes of CdTe QDs were prepared by controlling MI temperature and time. The QD samples were taken out from the reaction vessel when the temperature was naturally cooled to lower than 60 °C. For the synthesis of CdTe/CdS QDs, NAC-capped CdTe QDs were first precipitated with 2-propanol and collected via centrifugation. The precipitate was then re-dispersed in 6 mL solution containing 1.25 mM CdCl2 and 6.0 mM NAC (pH 8.5), followed by adding Na2S to a final concentration of 0.31 mM. Next, the CdTe/CdS precursor solution was injected into a digestion vessel. CdTe/CdS core/shell QDs with different emission wavelengths were obtained by controlling MI temperature and time. Similarly, the same protocol was also employed for the synthesis of CdTe/CdS/ZnS QDs except for using CdTe/CdS QDs as cores, which were dispersed in the solution containing 1.25 mM ZnCl2 and 6.0 mM NAC (pH 8.5). Characterization The as-prepared QD samples were diluted for optical characterizations. Their ultraviolet (UV)-visible absorption spectra were obtained using a Shimadzu UV-2450 spectrophotometer (Kyoto, Japan) and fluorescence spectra were recorded on a Hitachi F-2500 fluorescence spectrophotometer (Tokyo, Japan). All optical measurements were performed at room temperature under ambient conditions. The QY of CdTe QDs was measured using rhodamine 6G in an ethanol solution (QY = 95%) as a reference standard as described previously (13,24). Briefly, the absorbance for the standard and the CdTe QD samples at the excitation wavelengths and fluorescence spectra of the same solutions were measured, respectively. Six different concentration of rhodamine 6G and CdTe QD solutions (absorbance at excitation wavelength < 0.1) were used in the measurements. The integrated fluorescence intensity vs. absorbance was plotted. The plot obtained should be a straight line with a gradient M, which was used to calculate the QY according to the following equation: ϕx ¼ ϕs
Mx Ms
2 ηx ηs
(1)
Where the subscripts s and x denote standard and test samples, respectively, ϕ is QY and η is the refractive index of the solvent. It should be noted that the excitation wavelength of QDs was set at the excitonic absorption peak of the CdTe QD samples for measurements of QY. Transmission electron microscopy (TEM) samples were prepared by dropping the aqueous nanocrystals on to carbon-coated copper grids with excess
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Synthesis of quantum dots using microwave irradiation solvent evaporated. TEM and high-resolution transmission electron microscopy (HRTEM) images were recorded on a JEM-2100 electron microscope (JEOL Ltd., Tokyo, Japan) operating at 200 kV. XRD spectra were taken on an X’Pert PRO MPD diffractometer (PANalytical, Almelo, The Netherlands). MTT assays
Figure 1. Molecular structures of L-cysteine (Cys), L-cysteine hydrochloride (Cys·HCl), N-acetyl-L-cysteine (NAC), glutathione (GSH), and 3-mercaptopropionic acid (MPA).
MTT cell viability assays were performed to provide an assessment of the toxicity of QDs against human embryonic kidney 293 (HEK 293) cells. Briefly, the cells were pre-seeded on 96-well plates at a density of 2 × 104 cells/well in 100 μL medium containing 10% fetal bovine serum. The plates were then incubated for 24 h at 37 °C containing 5% CO2. Fifty μL of different concentrations of QDs in phosphate-buffered saline solution, estimated according to Yu et al. (35), were added and the cells were incubated in the dark
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Figure 2. The normalized absorption (except for A,C) and normalized PL spectra of CdTe quantum dots synthesized by using L-cysteine (A,B) and L-cysteine hydrochloride (C,D), NAC (E,F), GSH (G,H) and MPA (I,J) as ligands, respectively, at different microwave irradiation temperatures and times. The excitation wavelength for all PL spectra was 370 nm. The microwave irradiation temperatures and times (from line a to f) are 100 °C/5 min, 100 °C/15 min, and 120 °C/5 min, 120 °C/15 min, 120 °C/30 min and 120 °C/ 45 min, respectively. (K,L) Evolution of the emission FWHM and QY, respectively, of NAC-, GSH- and MPA-capped CdTe quantum dots with the emission peak position. FWHM, full width at half maximum; GSH, glutathione; MPA, 3-mercaptopropionic acid; NAC, N-acetyl-L-cysteine; PL, photoluminescence; QY, quantum yield.
H. He et al. for 12 or 24 h. Wells containing cells without QDs served as controls. Subsequently, 20 μL of MTT solution (5 mg/mL) was added to each well and the plates were incubated for 4 h at 37 °C. The precipitated formazan was dissolved in 200 μL of dimethyl sulfoxide. The absorbance of each sample at 490 nm was measured using a microplate autoreader (Spectramax M2e, Molecular Devices, Sunnyvale, CA, USA). Percentage cell survival is expressed as a percentage ratio of A490 of cells treated with QDs over control cells.
Results and discussion Synthesis of CdTe quantum dots with different ligands Figure 1 presents the ligands used in this study, including Cys, Cys·HCl, NAC, GSH and MPA. Figure 2 shows the absorption and photoluminescence (PL) spectra of the CdTe QDs, which were prepared in the presence of these ligands at different
Figure 3. The normalized PL spectra of CdTe quantum dots prepared by using N-acetyl-L-cysteine as a ligand at 140 °C/5 min, 140 °C/15 min, 140 °C/20 min and 160 °C/5 min. The corresponding PL emission wavelengths were 603 nm, 614 nm, 646 nm and 680 nm, with quantum yields of 21.8%, 20.7%, 10.5% and 6.9%, respectively. PL, photoluminescence.
temperatures (100 and 120 °C) and time under MI. The photographs of CdTe sample solutions are shown in the supporting information (see Supplementary Fig. S1). With prolonging the heating time or increasing the reaction temperature, the PL emission peak positions of the resulting CdTe QDs with these ligands shifted to longer wavelengths. Owing to the size dependence of optical absorption and emission of QDs (5,35,36), the growth rate could be evaluated from the shift of absorption or PL emission peaks. We here observed large differences on the growth rate of QDs with different ligands. For example, under the same MI reaction condition, e.g. 120 °C/15 min, the PL emission peaks of CdTe QDs prepared with Cys, Cys·HCl, NAC and MPA shifted to 623 nm, 638 nm, 571 nm and 575 nm, respectively. Besides, these ligands showed different effects on optical quality and stability of QDs. In the cases of Cys and Cys·HCl, QD solutions became turbid suspensions during the growth process due to the formation of aggregates of QDs, which led to the strong non-zero baseline absorbance in their absorption spectra (Fig. 2A,C) and strong frequency scattering peaks at 740 nm (the excitation wavelength was 370 nm) in their PL emission spectra (Fig. 2B,D). Additionally, all these Cys-capped QDs synthesized at different reaction temperatures and times displayed weak PL (QYs less than 5%). In the case of GSH, CdTe QDs could be well prepared at 100 °C, but upon increasing the reaction temperature to 120 °C, the QD solution gave black precipitates after 10 min MI. In comparison to other ligands, MPA and NAC provided QDs with better optical spectra and high QYs up to 45%, as shown in Fig. 2(E,F,I,J). Figure 2(K,L) are respective plots of emission full width at half maximum (FWHM) and QY versus the emission wavelengths for NAC-, MPA- and GSH-capped CdTe QDs. In general, the narrow emission FWHM and high QY reflects the high quality of the QDs. It is evident that in terms of FWHM and QY, NAC-stabilized CdTe QDs are even slightly superior to those prepared under the same conditions with MPA, which is the most typical ligand in current microwave synthesis of QDs. Comparison of the molecular structures of the ligands (Fig. 1) indicates that the amine group might exert an adverse effect on the microwave synthesis of CdTe QDs. Under the present solution conditions (pH typically in the range of 8.5–9.5), the amine
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Figure 4. The typical absorption (A) and PL spectra (B) of CdTe, CdTe/CdS core/shell and CdTe/CdS/ZnS core–multishell quantum dots in the presence of N-acetyl-L-cysteine. The quantum yield of CdTe quantum dots increased from 38.3% to 53.5% and then 60.1% after CdS and ZnS shell growth at 60 °C/5 min and 65 °C/5 min, respectively. Their emission 2+ 2– 2+ 2– wavelengths were 541, 552 and 558 nm respectively. The precursor ratios of Cd /S and Zn /S were fixed at 1/0.25 for shell growth. (C) The PL spectra of the corresponding quantum dot solutions after 24 h irradiation (ultraviolet lamp, 365 nm, 450 W). The quantum yields of CdTe, CdTe/CdS core/shell and CdTe/CdS/ZnS core–multishell quantum dots decreased to 11.9%, 49.9% and 54.1%, respectively. PL, photoluminescence.
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Synthesis of quantum dots using microwave irradiation group is protonated, making the ligands Cys and Cys·HCl electrically neutral. As a result, the interparticle electrostatic repulsion is significantly reduced, thus promoting the initial kineticfavored aggregation growth of QDs and agglomeration of the resulting QDs with increasing incubation time (37). In microwave synthesis of QDs, such processes would be dramatically strengthened because of the rapid heating via MI and the higher temperature in the sealed vessel. Hence, the use of Cys and Cys·HCl as the ligands for microwave synthesis is unfavorable for obtaining dispersible QDs with high quality. In addition, in the case of Cys·HCl, the introduction of HCl is equivalent to the addition of NaCl into Cys solution under alkaline conditions. The resultant increase in ionic strength must further lower the interparticle electrostatic repulsion, thus further accelerating the aggregation and agglomeration of particles (38). As shown in Fig. 2(C,D), larger non-zero baseline absorption and stronger frequency scattering peaks at 740 nm than those shown in Fig. 2 (A,B) suggest that Cys·HCl causes more aggregation of QDs. In the case of GSH, although each GSH molecule contains a terminal amine group, the two carboxyl groups still maintain relatively strong interparticle repulsion, thus favoring the surface protection and stability of the QDs. However, higher temperature (≥ 120 °C) and longer reaction time in the sealed vessel caused the QD solution to produce black precipitates, suggesting the loss of the stabilizing effect of GSH probably due to its decomposition (see Supplementary Fig. S2 in the Supporting Information). For NAC, the N-terminal capping eradicated the adverse effect of the amine group and the interparticle electrostatic repulsion arising from the carboxyl group ensures that the NAC-capped QDs are well dispersed in
aqueous solution. The high temperature under MI shortened the preparation time by increasing the growth rate of QDs. As can be seen in Fig. 2(E,F), the growth rate at 100 °C was relatively slow, but it was markedly accelerated at 120 °C, and QDs with longer emission wavelengths were obtained after the same reaction time. Furthermore, QDs could be successfully prepared in the presence of NAC at higher temperatures, e.g. 140 and 160 °C, and still possessed relatively good optical properties (Fig. 3), suggesting the excellent stabilizing effect of NAC for QDs. For example, at 160 °C, CdTe QDs emitting at 680 nm could be prepared in 5 min with a QY of 6.9%. Therefore, the relatively strong electrostatic repulsion and molecular stability at high temperature gave NAC the exceptional capability of acting as a ligand for a high-temperature reaction, which could accelerate greatly the growth rate of nanocrystals during the Ostwald ripening stage in favor of preparing high-quality QDs with a low concentration of surface defects and a narrow size distribution (16–19,29). In addition, we also examined microwave synthesis of CdTe QDs using CA as ligand, which contains an amine group in place of a carboxyl group in comparison with NAC. In this case, the CdTe precursor solution instantly gave a precipitate upon heating to above 100 °C (see Supplementary Fig. S3 in the Supporting Information), suggesting the poor stabilizing effect of CA for CdTe QDs in the pH range 8.5–9.5. For CA, a slightly acidic environment (pH 5.6–6.9) may be more suitable for stabilizing CdTe QDs (9). However, such conditions might weaken the function of the thiol group, which plays the dominant role in determining the stability and optical properties of QDs in our system (31).
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Figure 5. (A) The normalized PL spectra of NAC-capped CdTe core and CdTe/CdS core/shell QDs prepared by microwave irradiation for 5 min at different temperatures. 2+ 2– The precursor ratio of Cd /S was fixed at 1/0.25. (B) The normalized PL spectra of NAC-capped CdTe/CdS core and CdTe/CdS/ZnS core–multishell QDs prepared by 2+ 2– microwave irradiation at 65 °C for different growth times. The precursor ratio of Zn /S was fixed at 1/0.25. (C) The normalized PL spectra of NAC-capped CdTe core 2+ 2– and CdTe/CdS core/shell QDs prepared at different Cd /S ratios by microwave irradiation for 5 min at 60 °C. (D) The normalized PL spectra of NAC-capped CdTe/CdS core 2+ 2– and CdTe/CdS/ZnS core–multishell QDs prepared at different Zn /S ratios by microwave irradiation for 5 min at 65 °C. NAC, N-acetyl-L-cysteine; PL, photoluminescence; QD, quantum dot.
H. He et al. Synthesis of CdTe/CdS core/shell and CdTe/CdS/ZnS core– multishell quantum dots To improve the optical properties, stability and biocompatibility of nanocrystals, a core/shell structure is usually an ideal choice. The shell not only acts as a physical barrier to protect the optically active core from its surrounding medium against environmental changes, surface chemistry and photo-oxidation, but also provides an efficient passivation of the surface, such as reducing the number of surface dangling bonds, which enhances greatly the PL QYs of QDs. In the choice of shell materials, the most crucial factor is the lattice mismatch between core and shell, which can affect the quality of core/shell QDs by creating excessive crystal strain at core/shell interfaces. For a CdTe core, a CdS shell is more suitable than ZnS because of better overlap between the lattice parameters (19,34). Taking into account the inherent cytotoxicity of Cd-containing QDs in live cell and in vivo biology, however, ZnS is more suitable as a shell material. As a result, the CdTe/CdS/ZnS core–multishell system was considered, where CdS acted as a buffer layer and allows stepwise change of lattice spacing between the CdTe core and ZnS shell, thus reducing the strain within the nanocrystals. Here, the
optimized ligand NAC was used for the microwave synthesis of CdTe/CdS core/shell and CdTe/CdS/ZnS core–multishell QDs with controllable temperature. Figure 4 gives representative absorption and PL spectra of CdTe core, CdTe/CdS core/shell, and corresponding CdTe/CdS/ZnS core–multishell QDs synthesized under MI. The MI temperatures/times were 60 °C/5 min and 65 °C/5 min for CdS and ZnS shell growth, respectively. Note that the same CdTe or CdTe/CdS solution was used for the subsequent preparation of CdTe/CdS or CdTe/CdS/ZnS QDs. A significant red-shift was observed in the absorption and PL emission spectra, which gives an indication of the formation of the intended CdTe/CdS/ZnS structure instead of the CdZnTeSalloyed QDs that have been extensively discussed in the literature (19,39). Owing to the encapsulation of CdS and ZnS shells, the PL intensity of QDs was noticeably enhanced and they displayed better optical stability. Figure 4(C) presents the PL spectra of the corresponding QD solutions exposed to UV irradiation for 24 h (365 nm, 450 W). After continuous strong irradiation, the emission peaks of CdTe /CdS and CdTe/CdS/ZnS QDs were nearly unchanged with QYs remaining above 50%, while the PL spectrum of CdTe QDs became asymmetric with a decrease of QY from 38.3% to 11.9%.
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Figure 6. Transmission electron microscopy (top) and high-resolution transmission electron microscopy (middle) images and size distribution histograms (bottom) of (A,D,G) NAC-capped CdTe core QDs emitting at 541 nm, (B,E,H) NAC-capped CdTe/CdS core/shell QDs emitting at 552 nm and (C,F,I) NAC-capped CdTe/CdS/ZnS core–multishell QDs emitting at 558 nm. NAC, N-acetyl-L-cysteine; QD, quantum dot.
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Synthesis of quantum dots using microwave irradiation Effects of microwave irradiation temperature, growth time and shell–precursor ratio on optical properties To achieve optimal enhancement in the optical property of nanocrystals, the reaction conditions for shell growth must be carefully controlled. The key point is to control the shell thickness. If the shell is too thin, it is inefficient for the desirable passivation of the core nanocrystals. Too thick a shell generally causes deterioration in the optical properties of the resulting core/shell QDs (33,40). In addition, due to the epitaxial growth of the shell on the nanocrystal surface, the red-shifts of the absorption and emission spectra are thickness-dependent (33,40). As the shell thickness increases, the absorption and emission peaks shift toward longer wavelengths. In the present system, we found that MI temperature, growth time and the precursor ratio of Cd2+/S2– and Zn2+/S2– affected significantly the optical properties of the resulted core/shell QDs. By regulating these conditions, we observed that limiting the emission red-shifts to 10–15 nm for CdS shell and 5–10 nm for ZnS shell, respectively, was favorable for obtaining high-quality QDs, which may be consistent with the optimum shell thickness. Under optimal conditions, as shown in Fig. 5, the highest QYs of CdTe/CdS/ZnS core–multishell QDs can reach as high as 74%, retaining narrow FWHMs of 40–60 nm. Figure 5(A) presents the PL spectra of NACcapped CdTe core and the corresponding CdTe/CdS core/shell QDs prepared via MI for 5 min at different temperatures. The precursor ratio of Cd2+/S2– was fixed at 1/0.25. When the reaction temperature increased, the PL emission peak showed a bathochromic shift. The optimal enhancement in PL intensity (10–15 nm red-shifts) could be achieved with temperatures between 60 and 70 °C. Figure 5(B) presents the PL spectra of NAC-capped CdTe/CdS core and the corresponding CdTe/CdS/ ZnS core–multishell QDs prepared via MI at 65 °C for different growth times. The precursor ratio of Zn2+/S2– was fixed at 1/0.25. As the growth time was prolonged, the PL emission peak also shifted to longer wavelengths. Here, the optimal growth time 5–10 min, during which the resultant CdTe/CdS/ZnS core–multishell QDs showed significant improvement in the QYs (5–10 nm red-shifts). Additionally, the emission red-shift was strongly influenced by the precursor ratio. Figure 5(C,D) show the PL spectra of CdTe/CdS core/shell and CdTe/CdS/ ZnS core–multishell QDs grown for 5 min at different precursor ratios of Cd2+/S2– and Zn2+/S2– respectively. The Cd2+/S2– or Zn2+/S2– ratios were regulated by adding different amounts of S2– into the precursor solution at the fixed Cd2+ or Zn2+ concentration. With an increase of the amount of S2–, the PL emission peaks of CdTe/CdS core/shell and CdTe/CdS/ZnS core–multishell QDs shifted to longer wavelengths during the same growth time. Here, the optimal precursor ratio Cd2+/S2– or Zn2+/S2– was 1/0.25. When the precursor ratio was decreased to 1/1, the resultant red-shift after shell growth of 5 min reached 20–30 nm. It should be noted that, at the precursor ratio of 1/1, the emission red-shift could also be adjusted to 10–15 nm by further reducing the growth time, but it was difficult to control the experimental protocol to ensure the reproducibility of results in less than 5 min.
Transmission electron microscopy and X-ray diffraction characterization
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Figure 7. The powder X-ray diffraction patterns of N-acetyl-L-cysteine-capped CdTe, CdTe/CdS core/shell and CdTe/CdS/ZnS core–multishell quantum dots. Standard diffraction lines of cubic CdTe (JCPDS no. 15-0770), cubic CdS (JCPDS no. 80-0019) and cubic ZnS (JCPDS no. 80-0020) are shown for comparison.
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Figure 6 shows TEM and HRTEM images and the corresponding size distribution histograms of the as-prepared CdTe, CdTe/CdS
and CdTe/CdS/ZnS QDs. Their emission wavelengths were 541, 552 and 558 nm respectively. These QDs are slightly spherical with narrow size distributions. The average sizes of CdTe, CdTe/CdS and CdTe/CdS/ZnS nanocrystals were about 3.1 nm, 3.9 nm and 4.8 nm respectively. The CdTe/CdS and CdTe/CdS/ ZnS QDs were prepared from the same CdTe and CdTe/CdS solutions. Accordingly, the increment in size indicates the growth of the shells on the cores. The corresponding shell thicknesses of CdS and ZnS were estimated to be about 0.40 and 0.45 nm respectively. In addition, the existence of lattice planes on the HRTEM images confirmed the crystallinity of these QDs. Figure 7 shows XRD patterns of CdTe, CdTe/CdS and CdTe/CdS/ZnS powders. In the diffraction pattern of the CdTe cores, three peaks can be assigned to {111}, {220}, {311} planes, consistent with the cubic (zinc blende) structure of CdTe. By contrast, the diffraction patterns of CdTe/CdS and CdTe/CdS/ZnS QDs shift slightly to a higher angle, and are positioned successively between those of bulk CdTe and bulk CdS and bulk ZnS due to the consecutive formation of CdS and ZnS shell on CdTe core. Compared with the CdTe core, the diffraction peak widths of CdTe/CdS and CdTe/CdS/ZnS QDs become slightly larger, thereby providing additional evidence of epitaxial shell growth rather than an alloyed structure that would show a narrower XRD peak width with increasing size as discussed in the literature (41).
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Figure 8. MTT assays illustrating the percentage cell viability upon exposing human embryonic kidney 293 cells to different concentrations of N-acetyl-L-cysteine-capped CdTe, CdTe/CdS core/shell and CdTe/CdS/ZnS core–multishell QDs for 12 h (A) and 24 h (B). QD, quantum dot.
Cytotoxicity The biocompatibility of QDs is a crucial issue for their future biological applications, especially those in live cell and in in vivo imaging. One of our purposes for the design of CdTe/CdS/ZnS core–multishell structure is to lower the cytotoxicity of nanocrystals using the protection of a ZnS shell, which can block the release of Cd2+ ions from the QD surface to the biological system. To validate this hypothesis, the cytotoxicity of CdTe, CdTe/CdS core/shell and CdTe/CdS/ZnS core–multishell QDs against human embryonic kidney 293 cells was extensively assessed by MTT assays. Figure 8(A,B) illustrate percentage cell viability upon exposing the cells to different concentrations of QDs for 12 h and 24 h respectively. As expected, CdTe/CdS/ZnS core–multishell QDs have lower cytotoxicity than CdTe and CdTe/CdS core/shell QDs under the same conditions. In addition, the cells exposed to CdTe or CdTe/CdS QDs showed a strong concentration-dependent decrease in cell viability while this trend was significantly reduced in the case of CdTe/CdS/ZnS QDs. Increasing of the incubation time to 24 h had a strong effect on the cytotoxicity difference between these QDs. For instance, when incubated for 24 h with 400 nM QDs, the cell viability values of the CdTe and CdTe/CdS QDs were reduced almost to zero while the cell survival of CdTe/CdS/ZnS QDs was still maintained above 50%, which demonstrates significantly improved biocompatibility due to the ZnS shell coating.
Conclusions
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The results give an insight into the microwave synthesis of CdTe QDs. We have studied five kinds of ligands, including Cys, Cys·HCl, NAC, GSH and MPA, which are frequently used in the traditional aqueous synthesis of QDs. The effects of the molecular structure of these ligands on the optical properties of CdTe QDs were investigated within a temperature range between 100 and 160 °C. Among these ligands, NAC was found to be the optimal ligand in terms of the optical properties and QYs of resultant QDs as well as reaction conditions. It allowed the preparation of CdTe QDs ranging in emission wavelength from 500 to 700 nm with good optical properties within only 5 min
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by controlling the MI temperature up to 160 °C. Based on the ideal ligand (NAC) and utilizing the high efficiency and convenience of MI, we have also successfully prepared highly luminescent CdTe/CdS/ZnS core–multishell QDs with QYs up to 74%. Finally, we have demonstrated their excellent photostability and favorable biocompatibility. These NAC-capped CdTe/CdS/ ZnS QDs thus could serve as promising optical probes for applications in biological and medical fields. Acknowledgments The authors gratefully acknowledge the support from the National Natural Science Foundation of China (20905078, 21103230) and the Fundamental Research Funds for the Central Universities (12CX04053A, 11CX05001A). H.X. acknowledges the support by Program for New Century Excellent Talents in University (NCET-11-0735).
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Supporting information Additional supporting information may be found in the online version of this article at the publisher’s web-site.
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