12012
J. Phys. Chem. B 2007, 111, 12012-12016
Photochemical Instability of Thiol-Capped CdTe Quantum Dots in Aqueous Solution and Living Cells: Process and Mechanism Jiong Ma,† Ji-Yao Chen,*,†,‡ Yu Zhang,‡ Pei-Nan Wang,*,‡ Jia Guo,§ Wu-Li Yang,§ and Chang-Chun Wang§ Surface Physics Laboratory (National Key Laboratory), Physics Department, State Key Laboratory for AdVanced Photonic Materials and DeVices, and Department of Macromolecular Science and Key Laboratory of Molecular Engineering of Polymers, Fudan UniVersity, Shanghai 200433, China ReceiVed: May 2, 2007; In Final Form: July 8, 2007
The process and mechanism of photochemical instability of thiol-capped CdTe quantum dots (QDs) in aqueous solution were experimentally studied. After laser irradiation, the corresponding Raman bands of the Cd-S bond decreased obviously, indicating bond breaking and thiol detachment from the QD surfaces. Meanwhile, a photoinduced aggregation of QDs occurred with the hydrodynamic diameter 〈Dh〉 increased to hundreds of nanometers from an initial 20 nm, as detected with dynamic light scattering measurements. The bleaching of the photoluminescence of QDs under laser irradiation could be attributed to the enhanced nonradiative transfer in excited QDs caused by increased surface defects due to the losing of thiol ligands. Singlet oxygen (1O2) was involved in the photooxidation of QDs, as revealed by the inhibiting effects of 1O2 quenchers of histidine or sodium azide (NaN3) on the photobleaching of QDs. The linear relationship in Stern-Volmer measurements between the terminal product and the concentration of NaN3 demonstrated that 1O2 was the main pathway of the photobleaching in QD solutions. By comparing the photostability of QDs in C2C12 cells with and without NaN3 treatment, the photooxidation effect of 1O2 on photobleaching of cellular QDs was confirmed.
1. Introduction Colloidal semiconductor quantum dots (QDs), with the radii around several nanometers, have unique photoluminescence (PL) properties due to the quantum confinement effect of the charge carriers.1 Compared with conventional organic fluorescent dyes, QDs possess advantages such as higher PL quantum efficiency, tunable luminescence depending only on their size, wide continuous absorption, narrower PL band, and higher photostability.2 Since hydrophilic QDs were first used as fluorescence probes in cellular labeling in 1998,3,4 QDs have attracted widespread attention from the fields of biology and medicine5-9 and achieved remarkable progress in biomedical applications.10-13 When they are used in biomedical research, water solubility is an elemental requirement for QDs. Whether QDs are prepared in organic solution using the common synthetic way or synthesized directly in water solution with a hydrothermal route,14 the surfaces of QDs should be modified with ligands to become hydrophilic. Among surface-modified QDs, thiolcapped QDs have been used extensively in cellular labeling.15-17 In contrast to organic fluorescent probes, QDs have much better photostability, which is the most attractive characteristic for cellular labeling, especially in long-term imaging such as tracking the transportation processes in cells and probing the path of labeled molecules. However, the photochemical instability of thiol-coated QDs in solution was reported,18 and we also found that thiol-capped CdTe QDs were photobleached in living * Corresponding authors. E-mail:
[email protected] (J.-Y.C.);
[email protected]. (P.-N.W.) † Surface Physics Laboratory (National Key Laboratory), Physics Department. ‡ State Key Laboratory for Advanced Photonic Materials and Devices. § Department of Macromolecular Science and Key Laboratory of Molecular Engineering of Polymers.
cells particularly when their intracellular concentration was low.19 Though we have revealed further that photooxidation was the main cause of the QD photobleaching,19 the overall process and related mechanisms of such a photochemical instability of QDs seem complicated and are not well understood yet. Considering the importance of the QD stability in applications especially for cellular labeling, the origination of photobleaching of thiol-capped CdTe QDs in aqueous solutions and living cells was experimentally studied in this work. 2. Experimental Section 2.1. Synthesis of Thiol-Capped CdTe QDs. The details of the synthesis of thiol-capped CdTe QDs can be found in our previous work.20 Briefly, with a molar ratio of 2:1, sodium borohydride was used to react with tellurium in water to prepare sodium hydrogen telluride (NaHTe). Fresh solutions of NaHTe were then diluted by N2-saturated deionized water to 0.0467 M for further use. CdCl2 (1 mmol) and 3-mercaptopropionic acid (MPA) (1.8 mmol) were dissolved in 50 mL of deionized water. Stepwise addition of NaOH solution adjusted the precursor solution to pH 9. Then, 0.096 mL of oxygen-free solution containing fresh NaHTe, cooled to 0 °C, was added to 10 mL of the above prepared precursor solution and stirred vigorously. Finally, the solution with a faint yellow color was put into a Teflon-lined stainless steel autoclave with a volume of 15 mL. The autoclave was maintained at the reaction temperature (200 °C) for a certain time and cooled to room temperature by a hydrocooling process, and then the thiol-capped CdTe QDs, dispersed in water, were obtained. The PL of obtained QDs in aqueous solution peaked at 590 nm, measured by a spectrophotometer (Hitachi, F-2500). 2.2. Experimental Measurements. 2.2.1. Raman Spectral Measurements of QDs. The Raman spectrum is sensitive to some
10.1021/jp073351+ CCC: $37.00 © 2007 American Chemical Society Published on Web 09/21/2007
Photochemical Instability of Thiol-Capped CdTe QDs molecular bonds, and was used to measure the ligand binding on the QD surface here. For thiol-capped CdTe QDs, the thiols were connected to QDs through the Cd-S bond so that the bond breaking of Cd-S could be detected by the intensity change of corresponding Raman peaks. In our case, the surface-bonded thiols and their detachment from the QDs under irradiation were monitored by Raman spectroscopy. The Raman spectrum was measured in a Raman spectrometer (Dilor, Labram-1B) with a CCD detector. A 632.8 nm He-Ne laser with the power of 1 mW was used for the excitation, because this wavelength is much longer than the 590 nm PL band of QDs that can effectively suppress the PL background to obtain satisfactory Raman spectra. The integration time of 100 s was used to acquire the Raman spectra with the spectral resolution of 1 cm-1 in measurements. The QD aqueous solution (1 mg/mL) in a small cuvette was irradiated with a 532 nm laser beam with the power of 250 mW (Coherent, Nd:YVO4) for 2 h. After irradiation, the irradiated QD sample and unirradiated QD sample were put in a small glass cell, respectively, for Raman measurements. 2.2.2. Dynamic Light Scattering (DLS) Measurement. The size distribution of QDs in aqueous solution was measured by a DLS instrument (Malvern, Autosizer 4700) at 25 °C with a detection angle of 90°. A 532 nm laser beam with the power of 250 mW (Coherent, Nd:YVO4) was used to irradiate the QD aqueous solution. The QD samples undergoing different time irradiations were measured by DLS, respectively, to study the effect of laser irradiation on QD aggregation. 2.2.3. Photobleaching Measurement. QD solutions without or with quenchers (histidine or NaN3) were dripped on glass slides and then sealed with coverslips, respectively. Photobleaching was measured by continuously irradiating the QDs with a 488 nm Ar ion laser (Coherent) in a laser scanning confocal microscope (Olympus, FV-300, IX71). The laser beam was focused by an objective to a spot of about 10 µm in diameter. The power density of the focused beam was estimated to be about 20 W/mm2. During laser irradiation, the PL signals were continuously measured in a detection channel with a bandpass filter of 580-640 nm. The PL lifetime of QDs was also measured in this microscope with a filtered (580-640 nm) photomultiplier tube (PMT) by introducing a picosecond laser (28 ps) (PL2143A, EKSPLA) of 400 nm into the microscope to do excitation. 2.3. Cell Culture. 2.3.1. Mouse Myoblast (C2C12) Cells. The C2C12 cells procured from the Cell Bank of Shanghai Science Academy were seeded onto a glass coverslip placed in a culture dish containing DMEM-H medium with 10% calf serum, 100 units/mL penicillin, 100 µg/mL streptomycin, and 100 µg/mL neomycin. The cells were then cultured in a fully humidified incubator at 37 °C with 5% CO2. When the cells adhered to the coverslip and reached 80% confluence with normal morphology, QDs were added to the culture dish to achieve a final QD concentration of 100 µg/mL. These cells were then incubated for 4 h. After incubation, the coverslip with adhered living cells was washed with PBS (phosphate-buffered saline) three times to remove the unbound QDs and then sealed on a glass slide for microscopic measurements. 2.3.2. Cellular Images and Photobleaching of QDs. The images of cellular QDs were acquired with the abovementioned confocal laser scanning microscope (CLSM). A water immersion objective (60×) and a matched pinhole were used in measurements. Using the z-scan mode, the images in different layers were recorded to achieve three-dimensional distribution of cellular QDs. With the point-stay mode of CLSM, the photo-
J. Phys. Chem. B, Vol. 111, No. 41, 2007 12013
Figure 1. Raman spectra of MPA-capped CdTe QD solutions before (solid line) and after laser irradiation (dashed line). Excitation: 632 nm. Integration time of CCD: 100 s.
Figure 2. Size distributions of CdTe QD aggregates in solutions (1 mg/mL) after laser irradiation for 0 (1), 2 (9), 4 (b), and 8 h (2). The power density of irradiation at 532 nm was 80 mW/mm2.
bleaching of cellular QDs was measured by focusing the laser beam on the selected spot in the cell and recording the decay of the PL intensity continuously. To confirm the singlet oxygen effect on photobleaching of cellular QDs, QD-loaded cells were incubated with NaN3 (a quencher of singlet oxygen) with a concentration of 10 mM, which is a commonly used concentration for in vitro study.21 3. Results and Discussion To evaluate the effect of laser irradiation on destroying the connection of surface thiols on QDs, samples of QD aqueous solutions before and after laser irradiation were analyzed by Raman spectra. Figure 1 shows the representative Raman spectra of these QD solutions. The highest peak around 184 cm-1 could be assigned as the fundamental longitudinal optical (LO) phonon mode of CdTe, which had a red shift comparable to that of the bulk CdTe,22 demonstrating a similar rule as reported in CdSe QDs.23 This broadened LO phonon band is probably due to the size distribution of QDs and the quantum confinement as well.24 In a recent report, two overlapped broad Raman bands of thioglycollic acid (TGA)-capped CdTe QDs between 240 and 360 cm-1 were considered to be Cd-S bond dependent Raman bands based on the observation in CdTe photoetching experiments.24 There are four bands (268, 287, 334, and 357 cm-1) in the same region as shown in Figure 1. The difference of the Raman bands might result from the ligand difference, because
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Ma et al.
TABLE 1: Size Distributions of CdTe QD Aggregates in Solutions (1 mg/mL) after Laser Irradiationa irradiation time (h) average size (nm) size range (nm)
0 27 33
2 47 38
4 276 395
8 938 1052
a
The power density of laser irradiation at 532 nm was 80 mW/ mm2.
Figure 4. Photobleaching curves of QD solutions (0.05 mg/mL) with and without histidine. The irradiation power density at 488 nm was 20 W/mm2.
Figure 5. Photobleaching curves in aqueous solutions of QDs (0.05 mg/mL) acquired for different NaN3 concentrations: (A) without NaN3 and with (B) 5 mM NaN3, (C) 10 mM NaN3, and (D) 50 mM NaN3. The irradiation power density at 488 nm was 20 W/mm2.
Figure 3. (A) Photobleaching curve of QD aqueous solution (0.05 mg/mL) under continuous laser irradiation and (B) PL time decay kinetics of QD aqueous solutions after different times of continuous irradiation measured with picosecond laser excitation, respectively.
MPA has a longer chain than TGA that may cause a more complicated Raman spectrum. After laser irradiation, these Raman bands all decreased dramatically, implying the breaking of surface Cd-S bond and the consequent loss of surface connected thiols. Losing the hydrophilic ligands, QDs tended to become aggregates. Figure 2 shows the size distributions of QD aggregates measured by means of dynamic light scattering (DLS) after laser irradiation for different times. DLS is usually used to measure the average hydrodynamic diameter 〈Dh〉 of the micelles, which is normally bigger than the “dry” size.25 The real size of single CdTe QDs in this work was about 4 nm,20 but their 〈Dh〉 was about 27 nm before laser irradiation. Under the irradiation of a 532 nm laser (80 mW/mm2), QDs gradually aggregated and the 〈Dh〉 of QD aggregates became larger, from 27 to 938 nm, and the size distribution became broad as well. The details are summarized in Table 1. DLS measurements directly confirm the photoinduced aggregation of thiol-capped QDs. When photooxidation occurred during the laser irradiation, the hydrophilic surfaces of QDs became
imperfect by losing thiol ligands, which led to the aggregation of QDs. With longer irradiation time, more thiol ligands were detached, resulting in the formation of larger QD aggregates. On the other hand, the loss of thiol ligands would produce more defects on the QD surfaces, resulting in the increase of nonradiative decay and the decrease of the PL of QDs as photobleaching. Figure 3A shows that the photobleaching of QDs really occurred under laser irradiation. Since the enhanced nonradiative decay results in the shortening of PL lifetime, the PL lifetime can be used as a probe to detect the effect of nonradiative decay. The PL lifetimes of QDs after different irradiation times were measured, respectively, as shown in Figure 3B. The consistent results between the photobleaching and PL lifetime can be found by comparing Figure 3A and 3B. Accompanying the photobleaching, the PL lifetimes of QDs were gradually shortened. In our previous study, we found that the photobleaching of thiol-capped QDs was mainly due to oxygen-dependent photooxidation. Therefore, the above observations, such as ligand detachment, increased QD aggregation, as well as the shortened PL lifetime and photobleaching should all result from the QD photooxidation. Then the next question is what the mechanism of such a photooxidation is. By absorbing photons, the excited QDs could react with the surrounding oxygen molecules to produce reactive oxygen species (ROS), which are well-known
Photochemical Instability of Thiol-Capped CdTe QDs
Figure 6. Stern-Volmer relationship of QD aqueous solutions. The QD concentration was 0.05 mg/mL.
agents to initiate the oxidation of the targets in situ. There are several different ROS, such as singlet oxygen (1O2), hydroxyl radical, and superoxide anion. Since the 1O2 intermediates were reported to be produced by QDs during irradiation,26 1O2 is most probably the main reactant for the photooxidation of thiolcapped QDs. Due to the high reaction rate with singlet oxygen,27,28 histidine was chosen as a quencher to test the involvement of 1O2 in photooxidation of QDs. When histidine was added to QD solution with a concentration of 3.2 mM, photobleaching was effectively reduced compared with that without histidine addition (Figure 4). The improved photostability implied that 1O2 was a possible intermediate in the process. However, as a weak acid, histidine may influence the PL intensity of QDs.29,30 It is necessary to confirm the 1O2 mechanism with other 1O2 quenchers.
J. Phys. Chem. B, Vol. 111, No. 41, 2007 12015 Sodium azide (NaN3), a physical quencher for 1O2, was used to further explore the 1O2 involvement in QD photooxidation. Figure 5 shows that NaN3 could also inhibit QD photooxidation. The photostability of QDs was enhanced with increased concentration of NaN3. With 50 mM NaN3, photobleaching of QDs was almost suppressed totally, indicating that 1O2 was really an intermediate in the photooxidation of QDs. In photooxidation studies, the Stern-Volmer relationship is commonly used to determine the main oxidizing agent.31 Using the bleached percentage of QDs as the terminal product R and measuring the R values with different concentrations of NaN3 in QD solutions, the dependence of R on the NaN3 concentration was obtained as shown in Figure 6. The linear relationship in Figure 6 indicates that 1O2 was the dominant intermediate in the reaction and was responsible for the photobleaching of QDs. Summarizing the above results, the mechanism of photochemical instability of thiol-capped QD solutions becomes clear. The QDs absorbed the photoenergy from laser irradiation to produce 1O2 intermediate in the solution, which oxidized the thiol ligands to detach from the QD surface, resulting in photobleaching and aggregation of QDs. Though 1O2 was the main reason for the photoinstability, the 1O2 quantum yield was as low as 5%.26 Therefore, QDs still showed better photostability than the commonly used organic fluorophores.18,19 However, used as fluorescent probes especially for long-term cellular imaging, improving the photostability of QDs is necessary. 1O2 is the product of energy transfer from excited QDs to ground state oxygen via a Type II process. The design of a multilayer shell structure of QDs may decrease the energy transfer and lower the 1O2 quantum yield, thus increasing the photostability, which is a consideration for our future studies. The 1O2 effect was evident in photooxidation of QDs in aqueous solutions. For cellular QDs, such a mechanism should
Figure 7. Micrographs of QD-labeled C2C12 cells. (A) PL image of QD-loaded cells (without NaN3 treatment) and (B) differential interference contrast (DIC) image. (C) PL image of QD-loaded and NaN3-treated cells and (D) DIC image of the cells.
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Ma et al. Acknowledgment. Financial support from the Shanghai Municipal Science and Technology Commission (06ZR14005, 05QMX1404) and the National Natural Science Foundation of China (10774027, 50525310, and 50103011) is gratefully acknowledged. References and Notes
Figure 8. Photobleaching curves of QDs in C2C12 cells with (2) and without (9) NaN3 incubated. The incubation concentration of NaN3 was 10 mM.
be confirmed since photostability is particularly important for cellular detection. When C2C12 cells had been incubated with thiol-capped CdTe QDs (100 µg/mL) for 4 h, cellular QDs could clearly be seen in the confocal fluorescence images (Figure 7A), where the main image exhibits the QD distribution in an X-Y plane inside the cell and the images displayed below and at the right side of the main one are the X-Z and Y-Z profiles of cellular QDs obtained by z-scanning along the marked lines in the main image. The three-dimensional image indicates that QDs localize inside cells. Selecting some certain spots in cells with known initial PL intensity from the image, the laser irradiation on the spot was carried out with the point-stay mode of CLSM and the photobleaching of cellular QDs was measured (Figure 8). After QD-loaded cells were additionally incubated with 10 mM NaN3, the image of cellular QDs was measured first as seen in Figure 7C and no interference to QDs could be found; then the corresponding spots in these NaN3-treated cells that have the same level of PL intensity as that for the previous measurement (without NaN3 treatment) were chosen to perform the photobleaching. As shown in Figure 8, the photostability of cellular QDs was obviously improved after NaN3 treatment. This means that 1O2 was still a pathway of photooxidation of cellular QDs. 4. Summary Under photoexcitation, the thiol-capped CdTe QDs in aqueous solution transferred the absorbed photoenergy to the surrounding oxygen molecules to produce 1O2 via a Type II process. The thiol ligands of QDs were probably oxidized to detach from the core surfaces. The increased surface defects resulted in the photobleaching of QDs due to the enhanced nonradiative decay. Meanwhile, the detachment of hydrophilic ligands led to the aggregation of QDs. The effect of 1O2 on the photobleaching of QDs in C2C12 cells was confirmed by the NaN3 treatment.
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