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Cite this: DOI: 10.1039/c5cc03046c Received 13th April 2015, Accepted 30th April 2015
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Long-decay near-infrared-emitting doped quantum dots for lifetime-based in vivo pH imaging† Chi Chen,‡ Pengfei Zhang,‡ Li Zhang, Duyang Gao, Guanhui Gao, Yong Yang, Wenjun Li, Ping Gong and Lintao Cai*
DOI: 10.1039/c5cc03046c www.rsc.org/chemcomm
pH-responsive doped quantum dots with an ultrasmall size (B3.5 nm), near-infrared emission (B720 nm) and long lifetime (B1 ls), which exhibit a linear response range from pH 5.5 to 7.0 with the maximum change in the fluorescence lifetime up to B600 nm, were synthesized as lifetime-based pH nanosensors for in vivo imaging.
pH is an essential physiological parameter associated with many important chemical or biological processes, such as cell and enzyme activities.1 It presents a challenge to in vivo sensing and the imaging of pH fluctuations, which is significant for promoting our understanding of pathological processes.2 As a noninvasive technology, optical sensing can provide in situ, fast, quantitative readouts and enable the real-time visualization of parameters which exhibit no intrinsic color or fluorescence.3 More remarkable, near-infrared (NIR)-emitted fluorescence possesses a much higher imaging depth than visible-emitted fluorescence, and can be widely explored for in vivo biosensing and bioimaging.4 Compared to traditional organic dyes, quantum dots (QDs) demonstrate significant advantages not only as biological labels,5 but also as sensors to detect pH values, ions, organic compounds and biomolecules,6 owing to their unique optical properties.7 Traditionally, QDs-based pH-sensors were mainly designed based on the variation in fluorescence intensity.8 However, fluorescence intensity-based methods suffer from many uncertainties because it is difficult to avoid the influence of the excitation light intensity and probe concentration in the medium. As an alternative approach, fluorescence lifetime-based measurement is independent of the fluorescence intensity and fluorophore concentration and offers several advantages via selective reduction of the signal caused by auto-fluorescence.9 More importantly, the appearance Guangdong Key Laboratory of Nanomedicine, CAS Key Laboratory of Health Informatics, Shenzhen Bioactive Materials Engineering Lab for Medicine, Institute of Biomedicine and Biotechnology Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, P. R. China. E-mail: [email protected] † Electronic supplementary information (ESI) available: Experimental procedures, Fig. S1–S7, and Tables S1–S3. See DOI: 10.1039/c5cc03046c ‡ These authors contributed equally.
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of fluorescence lifetime imaging microscopy (FLIM) opened up a new opportunity for visualization of pH fluctuations.10 The combination of FLIM methodologies and QDs has great potential for real-time pH imaging. However, the reported lifetime-based nanosensors have been prepared using visible-emitting QDs with the maximum change in the fluorescence lifetime along the response range of only B10 ns.10,11 Thus, NIR-emitting QDs with much longer decay are very desirable for in vivo pH sensing and imaging with high sensitivity. Band gap engineering12 of QDs has allowed researchers to tune the fluorescence emission spectrum of QDs from ultraviolet (UV) to NIR spectral ranges by designing component (doping,13 cation exchange14) and electron energy band structure (alloying,15 core/shell16). As previously reported in the literature, the photoluminescence mechanism of Cu-doped QDs has been well studied.17 Cu2+ trap states can replace the valence band as ‘‘permanent’’ optically active holes,18 which can strongly influence the optical behavior of QDs. Besides, doping also shows great potential as a strategy to minimize the particle size and tune the fluorescence lifetime of NIR-QDs effectively.19 The ultrasmall-size NIR-emitting QDs offer great opportunities for in vitro and in vivo biosensing and bioimaging applications with high-efficiency and high-sensitivity.20 In addition, the use of alloyed nanostructures also can be regarded as a very common strategy for the tuning of a fluorescence emission spectrum without changing the size of the structure. In general, these alloyed nanostructures can be classified in two categories: homogeneous alloys possessing a uniform internal structure, and gradient alloys composed of a variable elemental distribution.15c It has been reported that the optical properties of gradient-alloyed CdZnS QDs are sensitive to Zn/Cd/S molar ratios.15d However, in the case of homogeneously alloyed CdZnS QDs, the fluorescence emission spectra become blue-shifted systematically with the increase in Zn content.15a In this study, we prepared QDs with NIR-fluorescence emission (650–750 nm) and long-decay (lifetime up to approximately 1 ms) via combining doping with a gradient-alloyed nanostructure. Furthermore, application of the as-prepared long-decay NIR-emitting dopedQDs in lifetime-based in vivo pH imaging has been demonstrated by
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Scheme 1 Scheme for the synthesis of pH-sensitive Cu-doped CdZnS QDs and their use as nanosensors for in vivo pH imaging based on FLIM (the colored spheres represent QDs in different pH standard solutions).
using FLIM. Briefly, as shown in Scheme 1, for the synthesis of Cu-doped gradient-alloyed CdZnS QDs, CuCl2, CdCl2, Zn(OAc)2 and L-glutathione (GSH) were mixed in one pot. The reaction was conducted at 95 1C for 30, 60, 90 and 120 min to obtain QD samples with tunable emission. GSH, a common ligand for the aqueous synthesis of QDs (because it can easily provide S2 for the passivation shell,21 such as CdS and ZnS, which can generally enhance the stability of the nanocrystals in different media and improve the photoluminescence quantum yield, PLQY in abbreviation) was used as the S2 source. In addition, for S2, the reactivity of Cd2+ was higher than that of Zn2+,15a,22 so the composition of the QDs was a Cd-rich core and a Zn-rich shell nanostructure. Then, the QDs-720 (emission at 720 nm) were selected as nanosensors for in vivo pH imaging based on FLIM (further experimental details are given in the ESI†). The optical properties of the QD samples were investigated using an ultraviolet and visible spectrophotometer and fluorophotometer. According to Fig. 1a and b, there was no obvious
Fig. 1 (a) Absorption spectra, (b) photoluminescence spectra, and (c) timeresolved fluorescence decay curves of Cu-doped CdZnS QDs with different heating times (violet: 30 min, blue: 60 min, green: 90 min, and red: 120 min). The excited state lifetimes were calculated to be 807 ns, 835 ns, 854 ns, and 860 ns respectively. (d) TEM image, size distribution (inset) and (e) HRTEM image of Cu-doped CdZnS QDs with a heating time of 90 min.
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change in the absorption spectra of the Cu-doped gradientalloyed CdZnS QDs, but the photoluminescence (PL) spectra can be tuned from 650 nm to 750 nm with increasing the heating reaction time. It was caused possibly by the combined effect of increasing Zn/Cd molar ratios and particle size. The composition of the as-prepared QDs can be further confirmed using inductively coupled plasma optical emission spectroscopy (ICP-OES) (Table S1, ESI†). The nominal Zn/Cd molar ratio of 4 : 1 of the QDs with different reaction times was steadily increased, indicating that the as-prepared QDs have a gradient-alloyed nanostructure. Moreover, no significant change was observed in the photoluminescence excitation (PLE) spectra of the as-prepared QDs at different positions, indicating that the broad fluorescence emission peak was due to the inherent properties of the Cu-doped QDs (Fig. S1, ESI†). According to Fig. 1c, the excited state lifetimes of the as-prepared QDs at different heating times were 807, 835, 854 and 860 ns, respectively (more details in Table S2, ESI†), being consistent with previous reports stating that the excited state lifetime of Cu-doped QDs was on microsecond level.17a To clarify the composition and structure of the as-prepared QDs, powder X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS) and transmission electron microscopy (TEM) were carried out. XRD data (Fig. S2, ESI†) showed that the as-prepared QDs have a zinc-blende dominated structure; it has been reported that this is conducive to improving the efficiency of doping.23 XPS data further demonstrated the gradient-alloyed structure. With increasing heating time, the signal of the characteristic peaks of the Cd 3d level diminished gradually (Fig. S3, ESI†). The mean diameter of the QDs-720 was 3.54 0.43 nm according to the TEM images (Fig. 1d inset), and the morphology was nearly spherical with a narrow size distribution (Fig. 1d). The lattice fringes can be identified in the HRTEM images (Fig. 1e). Furthermore, the colloidal stability of the QDs-720 was evaluated, and up to 80% of the photoluminescence was preserved after a one week incubation. In addition, the photo-stability of the QDs-720 was evaluated, and little photo-bleaching was observed after continuously exciting the QDs in buffers with different pH values using a 400 nm laser for 90 min. The excellent colloidal and photo-stability suggested that the as-prepared QDs are highly stable for long-term imaging (Fig. S4a and b, ESI†). Besides, the cytotoxicity of the QDs-720 was tested using a Cell Counting Kit-8 (CCK-8) assay. There were no significant changes in cell proliferation for the QDs (up to 500 mg mL1), suggesting that the as-prepared QDs were non-cytotoxic (Fig. S4c, ESI†). In order to explore the potential applications of the as-prepared QDs as lifetime-based pH nanosensors, the lifetime of the QDs-720 in buffers with different pH values was measured. As shown in Fig. 2a, there was a significant decline in the PL lifetime as the pH value of the environment decreased (more details in Table S3, ESI†). These changes may be caused by the protonation of the carboxylic acid on the surface of the QDs.8c,d Generally, the lifetime of QDs can be associated with the involvement of surface states and trap states during the carrier recombination process.24 Thus, the results observed here are consistent with protonation/deprotonation of the carboxylic acid affecting the surface of the QDs-720 being
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Fig. 2 (a) Time-resolved fluorescence decay curves of the as-prepared QDs-720 in buffers with different pH values: 5.5 (violet), 5.75 (blue), 6.0 (cyan), 6.25 (green), 6.5 (yellow), 6.75 (orange), and 7.0 (red). (b) pH response of the as-prepared QDs-720 in different pH buffers based on data from the time-resolved fluorescence decay curves. Calibration plot (violet line): tave = 884.97635 205.50544/(1 + exp((pH 6.17267)/0.31999)); R2 = 0.998.
responsible for the changes in the PL lifetime along with pH variation. In comparison, the fluorescence emission peak of the QDs-720 was a little blue-shifted (B20 nm) with the increase in pH values from 4.0 to 9.0 (Fig. S5, ESI†). This pH-dependent shift of the emission peak was caused by increased band gap energy, and it further confirms the protonation/deprotonation of the carboxylic acid of the surface ligand, corresponding with the report by the group of Hall.9 The average PL lifetime of the doped-QDs was calculated by eqn (1), to compare the average PL lifetime at different pH values: P ai ti 2 taverage ¼ P (1) ai ti where ti are the decay times, and ai, the pre-exponential weights. The calculated results were shown in Fig. 2b. The tave of the QDs-720 exhibited a dependency on the pH value, decreasing from 900 ns in an alkaline environment (pH > 9.0) to 200 ns when the pH was below 4.0. The S-shaped curve has a linear response range from pH 5.5 to 7.0 with the maximum change in the fluorescence lifetime up to B600 ns. This range suggested a potential application in the detection of the microenvironment of some tumor cells, because of their lower extracellular pH (pH = 6.4–6.9) compared to normal tissues (pH = 7.2–7.4).25 However, one of the most problematic aspects of in vivo pH sensing is the extremely crowded media. Therefore, the selectivity of the as-prepared QD-based pH nanosensors was evaluated, as illustrated in Fig. S6 (ESI†). The tave from QDs-720 solutions at pH 7.0 showed a negligible response in the absence and the presence of different concentrations of potential interfering ions (K+, Mn2+, Mg2+, and HCO3) and biomolecules (glucose and BSA). It should be noted that the concentration of all tested ions and biomolecules was several orders of magnitude higher than the actual in vivo levels.9 Thus, the as-prepared QDs can be quite useful as lifetimebased pH nanosensors. To demonstrate the use of the as-prepared QD nanosensors for lifetime-based pH imaging, the QDs were incorporated into microbeads and dispersed in buffers with different pH values.
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Fig. 3 (a) FLIM images of microbeads equipped with QDs-720, dispersed in buffers with different pH values (left: pH = 6.0, right: pH = 7.0, scale bar: 100 mm) and (b) PL lifetime histograms collected from the images. (c) In vivo FLIM experiments of the background of the nude mouse (left) and the QDs-720 injected into adjacent locations with different pH values (green: pH = 6.0, red: pH = 7.0) on the back of the nude mouse (right), respectively (scale bar: 10 mm).
As shown in Fig. 3a, the microbeads present different colors indicating the different PL lifetime of the QDs-720 as a result of pH response, which can be clearly distinguished from the FLIM images. According to the PL lifetime histograms of the FLIM images, the average lifetime of the QDs-720 was B500 ns (pH = 6.0) and B800 ns (pH = 7.0), respectively (Fig. 3b). This is consistent with the S-shaped curve result. Robust maintenance of the pH response under a variety of in vitro environmental conditions gives us confidence that the nanosensors can be applied to lifetime-based in vivo pH imaging. The as-prepared QDs were injected subcutaneously at two adjacent locations with different pH values in a nude mouse. According to the FLIM images, the high-lifetime signals of the QDs at different spots could be easily visualized above the background (Fig. 3c). These results demonstrated the advantages of long-decay and NIR-emission of QDs for in vivo imaging. Furthermore, the diverse spots with different pH values presented distinctive colors, which was consistent with the results of the in vitro experiments. In contrast, the same spots could not be distinguished by using NIR-emitting fluorescence imaging (Fig. S7, ESI†). These results suggested that the as-prepared QDs have great potential as nanosensors for lifetime-based in vivo pH imaging. In summary, we prepared Cu-doped CdZnS QDs with ultrasmall size (B3.5 nm), NIR-emission (B720 nm), and long-decay (lifetime up to B1 ms) by combining the use of doping and a gradientalloyed nanostructure. Moreover, the as-prepared QDs showed excellent pH sensitivity and exhibited a linear response range from pH 5.5 to 7.0 with the maximum change in the fluorescence lifetime up to B600 ns. Furthermore, the QD-based in vivo pH imaging has been demonstrated using FLIM. This work will provide a new candidate for NIR-emitting lifetime-based sensors for in vivo microenvironment imaging. The presented research was financially supported by the National Basic Research Program of China (973 Program No. 2011CB933600), the National Natural Science Foundation of China (Grant No. 81401509, 21305152 and 21375141), the Research Foundation of Chinese Academy of Sciences (yz201439), the Guangdong Science and Technology Program (Grant No. 2012A061400013),
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the Shenzhen Science and Technology Program (Grant No. JCYJ20130402103240486, JCYJ20140417113430607, and KQCX20140521115045447), SIAT Innovation Program for Excellent Young Researchers (201412, 201306), Shenzhen Key Laboratory for Molecular Biology of Neural Development (ZDSY20120617112838879) and Guangdong Innovation Research Team of Lowcost Healthcare.
Notes and references ¨ckner, E. B. Pasquale and R. Klein, Science, 1997, 275, 1640; 1 (a) K. Bru (b) J. R. Casey, S. Grinstein and J. Orlowski, Nat. Rev. Mol. Cell Biol., 2010, 11, 50. 2 (a) G. Helmlinger, F. Yuan, M. Dellian and R. K. Jain, Nat. Med., 1997, 3, 177; (b) F. A. Gallagher, M. I. Kettunen, S. E. Day, D.-E. Hu, J. H. Ardenkjaer-Larsen, R. I. T. Zandt, P. R. Jensen, M. Karlsson, K. Golman, M. H. Lerche and K. M. Brindle, Nature, 2008, 453, 940. 3 (a) Y. Zhao, Z. Xie, H. Gu, C. Zhu and Z. Gu, Chem. Soc. Rev., 2012, 41, 3297; (b) Y. Zhao, Z. Xie, H. Gu, C. Zhu and Z. Gu, Angew. Chem., Int. Ed., 2012, 51, 3532. 4 S. Kim, Y. T. Lim, E. G. Soltesz, A. M. De Grand, J. Lee, A. Nakayama, J. A. Parker, T. Mihaljevic, R. G. Laurence, D. M. Dor, L. H. Cohn, M. G. Bawendi and J. V. Frangioni, Nat. Biotechnol., 2004, 22, 93. 5 G. Chen, J. Zhu, Z. Zhang, W. Zhang, J. Ren, M. Wu, Z. Hong, C. Lv, D. Pang and Y. Zhao, Angew. Chem., Int. Ed., 2015, 54, 1036. 6 M. Frasco and N. Chaniotakis, Sensors, 2009, 9, 7266. 7 (a) W. C. W. Chan and S. Nie, Science, 1998, 281, 2016; (b) M. Bruchez, M. Moronne, P. Gin, S. Weiss and A. P. Alivisatos, Science, 1998, 281, 2013. 8 (a) P. T. Snee, R. C. Somers, G. Nair, J. P. Zimmer, M. G. Bawendi and D. G. Nocera, J. Am. Chem. Soc., 2006, 128, 13320; (b) R. C. Somers, R. M. Lanning, P. T. Snee, A. B. Greytak, R. K. Jain, M. G. Bawendi and D. G. Nocera, Chem. Sci., 2012, 3, 2980; (c) Y.-S. Liu, Y. Sun, P. T. Vernier, C.-H. Liang, S. Y. C. Chong and M. A. Gundersen, J. Phys. Chem. C, 2007, 111, 2872; (d) A. S. Susha, A. M. Javier, W. J. Parak and A. L. Rogach, Colloids Surf., A, 2006, 281, 40. 9 M. J. Ruedas-Rama, A. Orte, E. A. H. Hall, J. M. Alvarez-Pez and E. M. Talavera, Chem. Commun., 2011, 47, 2898.
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ChemComm 10 A. Orte, J. M. Alvarez-Pez and M. J. Ruedas-Rama, ACS Nano, 2013, 7, 6387. 11 (a) M. Tantama, Y. P. Hung and G. Yellen, J. Am. Chem. Soc., 2011, 133, 10034; (b) A. Esposito, M. Gralle, M. A. C. Dani, D. Lange and F. S. Wouters, Biochemistry, 2008, 47, 13115. 12 (a) A. M. Smith and S. Nie, Acc. Chem. Res., 2009, 43, 190; (b) X. G. Peng, Acc. Chem. Res., 2010, 43, 1387. 13 (a) D. J. Norris, A. L. Efros and S. C. Erwin, Science, 2008, 319, 1776; ¨ller, Angew. (b) S. K. Panda, S. G. Hickey, H. V. Demir and A. Eychmu Chem., Int. Ed., 2011, 50, 4432. 14 (a) D. H. Son, S. M. Hughes, Y. Yin and A. Paul Alivisatos, Science, 2004, 306, 1009; (b) C. Chen, X. He, L. Gao and N. Ma, ACS Appl. Mater. Interfaces, 2013, 5, 1149. 15 (a) X. H. Zhong, Y. Y. Feng, W. Knoll and M. Y. Han, J. Am. Chem. Soc., 2003, 125, 13559; (b) S. M. Nie and R. E. Bailey, J. Am. Chem. Soc., 2003, 125, 7100; (c) M. D. Regulacio and M. Y. Han, Acc. Chem. Res., 2010, 43, 621; (d) J. Ouyang, C. I. Ratcliffe, D. Kingston, B. Wilkinson, J. Kuijper, X. Wu, J. A. Ripmeester and K. Yu, J. Phys. Chem. C, 2008, 112, 4908. 16 X. G. Peng, M. C. Schlamp, A. V. Kadavanich and A. P. Alivisatos, J. Am. Chem. Soc., 1997, 119, 7019. 17 (a) B. B. Srivastava, S. Jana and N. Pradhan, J. Am. Chem. Soc., 2011, 133, 1007; (b) G. K. Grandhi, R. Tomar and R. Viswanatha, ACS Nano, 2012, 6, 9751. 18 R. Viswanatha, S. Brovelli, A. Pandey, S. A. Crooker and V. I. Klimov, Nano Lett., 2011, 11, 4753. 19 C. Chen, P. Zhang, G. Gao, D. Gao, Y. Yang, H. Liu, Y. Wang, P. Gong and L. Cai, Adv. Mater., 2014, 26, 6313. 20 (a) Y. He, Y. Zhong, Y. Su, Y. Lu, Z. Jiang, F. Peng, T. Xu, S. Su, Q. Huang, C. Fan and S.-T. Lee, Angew. Chem., Int. Ed., 2011, 50, 5695; (b) J. Wang, Y. Lu, F. Peng, Y. Zhong, Y. Zhou, X. Jiang, Y. Su and Y. He, Biomaterials, 2013, 34, 9509. 21 Y. Zheng, Z. Yang and J. Y. Ying, Adv. Mater., 2007, 19, 1475. 22 W. J. Zhang, H. Zhang, Y. Y. Feng and X. H. Zhong, ACS Nano, 2012, 6, 11066. 23 S. C. Erwin, L. J. Zu, M. I. Haftel, A. L. Efros, T. A. Kennedy and D. J. Norris, Nature, 2005, 436, 91. 24 M. G. Bawendi, P. J. Carroll, W. L. Wilson and L. E. Brus, J. Chem. Phys., 1992, 96, 946. 25 X. Wan, D. Wang and S. Liu, Langmuir, 2010, 26, 15574.
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Cite this: DOI: 10.1039/c5cc03046c Received 13th April 2015, Accepted 30th April 2015
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Long-decay near-infrared-emitting doped quantum dots for lifetime-based in vivo pH imaging† Chi Chen,‡ Pengfei Zhang,‡ Li Zhang, Duyang Gao, Guanhui Gao, Yong Yang, Wenjun Li, Ping Gong and Lintao Cai*
DOI: 10.1039/c5cc03046c www.rsc.org/chemcomm
pH-responsive doped quantum dots with an ultrasmall size (B3.5 nm), near-infrared emission (B720 nm) and long lifetime (B1 ls), which exhibit a linear response range from pH 5.5 to 7.0 with the maximum change in the fluorescence lifetime up to B600 nm, were synthesized as lifetime-based pH nanosensors for in vivo imaging.
pH is an essential physiological parameter associated with many important chemical or biological processes, such as cell and enzyme activities.1 It presents a challenge to in vivo sensing and the imaging of pH fluctuations, which is significant for promoting our understanding of pathological processes.2 As a noninvasive technology, optical sensing can provide in situ, fast, quantitative readouts and enable the real-time visualization of parameters which exhibit no intrinsic color or fluorescence.3 More remarkable, near-infrared (NIR)-emitted fluorescence possesses a much higher imaging depth than visible-emitted fluorescence, and can be widely explored for in vivo biosensing and bioimaging.4 Compared to traditional organic dyes, quantum dots (QDs) demonstrate significant advantages not only as biological labels,5 but also as sensors to detect pH values, ions, organic compounds and biomolecules,6 owing to their unique optical properties.7 Traditionally, QDs-based pH-sensors were mainly designed based on the variation in fluorescence intensity.8 However, fluorescence intensity-based methods suffer from many uncertainties because it is difficult to avoid the influence of the excitation light intensity and probe concentration in the medium. As an alternative approach, fluorescence lifetime-based measurement is independent of the fluorescence intensity and fluorophore concentration and offers several advantages via selective reduction of the signal caused by auto-fluorescence.9 More importantly, the appearance Guangdong Key Laboratory of Nanomedicine, CAS Key Laboratory of Health Informatics, Shenzhen Bioactive Materials Engineering Lab for Medicine, Institute of Biomedicine and Biotechnology Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518055, P. R. China. E-mail: [email protected] † Electronic supplementary information (ESI) available: Experimental procedures, Fig. S1–S7, and Tables S1–S3. See DOI: 10.1039/c5cc03046c ‡ These authors contributed equally.
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of fluorescence lifetime imaging microscopy (FLIM) opened up a new opportunity for visualization of pH fluctuations.10 The combination of FLIM methodologies and QDs has great potential for real-time pH imaging. However, the reported lifetime-based nanosensors have been prepared using visible-emitting QDs with the maximum change in the fluorescence lifetime along the response range of only B10 ns.10,11 Thus, NIR-emitting QDs with much longer decay are very desirable for in vivo pH sensing and imaging with high sensitivity. Band gap engineering12 of QDs has allowed researchers to tune the fluorescence emission spectrum of QDs from ultraviolet (UV) to NIR spectral ranges by designing component (doping,13 cation exchange14) and electron energy band structure (alloying,15 core/shell16). As previously reported in the literature, the photoluminescence mechanism of Cu-doped QDs has been well studied.17 Cu2+ trap states can replace the valence band as ‘‘permanent’’ optically active holes,18 which can strongly influence the optical behavior of QDs. Besides, doping also shows great potential as a strategy to minimize the particle size and tune the fluorescence lifetime of NIR-QDs effectively.19 The ultrasmall-size NIR-emitting QDs offer great opportunities for in vitro and in vivo biosensing and bioimaging applications with high-efficiency and high-sensitivity.20 In addition, the use of alloyed nanostructures also can be regarded as a very common strategy for the tuning of a fluorescence emission spectrum without changing the size of the structure. In general, these alloyed nanostructures can be classified in two categories: homogeneous alloys possessing a uniform internal structure, and gradient alloys composed of a variable elemental distribution.15c It has been reported that the optical properties of gradient-alloyed CdZnS QDs are sensitive to Zn/Cd/S molar ratios.15d However, in the case of homogeneously alloyed CdZnS QDs, the fluorescence emission spectra become blue-shifted systematically with the increase in Zn content.15a In this study, we prepared QDs with NIR-fluorescence emission (650–750 nm) and long-decay (lifetime up to approximately 1 ms) via combining doping with a gradient-alloyed nanostructure. Furthermore, application of the as-prepared long-decay NIR-emitting dopedQDs in lifetime-based in vivo pH imaging has been demonstrated by
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Scheme 1 Scheme for the synthesis of pH-sensitive Cu-doped CdZnS QDs and their use as nanosensors for in vivo pH imaging based on FLIM (the colored spheres represent QDs in different pH standard solutions).
using FLIM. Briefly, as shown in Scheme 1, for the synthesis of Cu-doped gradient-alloyed CdZnS QDs, CuCl2, CdCl2, Zn(OAc)2 and L-glutathione (GSH) were mixed in one pot. The reaction was conducted at 95 1C for 30, 60, 90 and 120 min to obtain QD samples with tunable emission. GSH, a common ligand for the aqueous synthesis of QDs (because it can easily provide S2 for the passivation shell,21 such as CdS and ZnS, which can generally enhance the stability of the nanocrystals in different media and improve the photoluminescence quantum yield, PLQY in abbreviation) was used as the S2 source. In addition, for S2, the reactivity of Cd2+ was higher than that of Zn2+,15a,22 so the composition of the QDs was a Cd-rich core and a Zn-rich shell nanostructure. Then, the QDs-720 (emission at 720 nm) were selected as nanosensors for in vivo pH imaging based on FLIM (further experimental details are given in the ESI†). The optical properties of the QD samples were investigated using an ultraviolet and visible spectrophotometer and fluorophotometer. According to Fig. 1a and b, there was no obvious
Fig. 1 (a) Absorption spectra, (b) photoluminescence spectra, and (c) timeresolved fluorescence decay curves of Cu-doped CdZnS QDs with different heating times (violet: 30 min, blue: 60 min, green: 90 min, and red: 120 min). The excited state lifetimes were calculated to be 807 ns, 835 ns, 854 ns, and 860 ns respectively. (d) TEM image, size distribution (inset) and (e) HRTEM image of Cu-doped CdZnS QDs with a heating time of 90 min.
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change in the absorption spectra of the Cu-doped gradientalloyed CdZnS QDs, but the photoluminescence (PL) spectra can be tuned from 650 nm to 750 nm with increasing the heating reaction time. It was caused possibly by the combined effect of increasing Zn/Cd molar ratios and particle size. The composition of the as-prepared QDs can be further confirmed using inductively coupled plasma optical emission spectroscopy (ICP-OES) (Table S1, ESI†). The nominal Zn/Cd molar ratio of 4 : 1 of the QDs with different reaction times was steadily increased, indicating that the as-prepared QDs have a gradient-alloyed nanostructure. Moreover, no significant change was observed in the photoluminescence excitation (PLE) spectra of the as-prepared QDs at different positions, indicating that the broad fluorescence emission peak was due to the inherent properties of the Cu-doped QDs (Fig. S1, ESI†). According to Fig. 1c, the excited state lifetimes of the as-prepared QDs at different heating times were 807, 835, 854 and 860 ns, respectively (more details in Table S2, ESI†), being consistent with previous reports stating that the excited state lifetime of Cu-doped QDs was on microsecond level.17a To clarify the composition and structure of the as-prepared QDs, powder X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS) and transmission electron microscopy (TEM) were carried out. XRD data (Fig. S2, ESI†) showed that the as-prepared QDs have a zinc-blende dominated structure; it has been reported that this is conducive to improving the efficiency of doping.23 XPS data further demonstrated the gradient-alloyed structure. With increasing heating time, the signal of the characteristic peaks of the Cd 3d level diminished gradually (Fig. S3, ESI†). The mean diameter of the QDs-720 was 3.54 0.43 nm according to the TEM images (Fig. 1d inset), and the morphology was nearly spherical with a narrow size distribution (Fig. 1d). The lattice fringes can be identified in the HRTEM images (Fig. 1e). Furthermore, the colloidal stability of the QDs-720 was evaluated, and up to 80% of the photoluminescence was preserved after a one week incubation. In addition, the photo-stability of the QDs-720 was evaluated, and little photo-bleaching was observed after continuously exciting the QDs in buffers with different pH values using a 400 nm laser for 90 min. The excellent colloidal and photo-stability suggested that the as-prepared QDs are highly stable for long-term imaging (Fig. S4a and b, ESI†). Besides, the cytotoxicity of the QDs-720 was tested using a Cell Counting Kit-8 (CCK-8) assay. There were no significant changes in cell proliferation for the QDs (up to 500 mg mL1), suggesting that the as-prepared QDs were non-cytotoxic (Fig. S4c, ESI†). In order to explore the potential applications of the as-prepared QDs as lifetime-based pH nanosensors, the lifetime of the QDs-720 in buffers with different pH values was measured. As shown in Fig. 2a, there was a significant decline in the PL lifetime as the pH value of the environment decreased (more details in Table S3, ESI†). These changes may be caused by the protonation of the carboxylic acid on the surface of the QDs.8c,d Generally, the lifetime of QDs can be associated with the involvement of surface states and trap states during the carrier recombination process.24 Thus, the results observed here are consistent with protonation/deprotonation of the carboxylic acid affecting the surface of the QDs-720 being
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Fig. 2 (a) Time-resolved fluorescence decay curves of the as-prepared QDs-720 in buffers with different pH values: 5.5 (violet), 5.75 (blue), 6.0 (cyan), 6.25 (green), 6.5 (yellow), 6.75 (orange), and 7.0 (red). (b) pH response of the as-prepared QDs-720 in different pH buffers based on data from the time-resolved fluorescence decay curves. Calibration plot (violet line): tave = 884.97635 205.50544/(1 + exp((pH 6.17267)/0.31999)); R2 = 0.998.
responsible for the changes in the PL lifetime along with pH variation. In comparison, the fluorescence emission peak of the QDs-720 was a little blue-shifted (B20 nm) with the increase in pH values from 4.0 to 9.0 (Fig. S5, ESI†). This pH-dependent shift of the emission peak was caused by increased band gap energy, and it further confirms the protonation/deprotonation of the carboxylic acid of the surface ligand, corresponding with the report by the group of Hall.9 The average PL lifetime of the doped-QDs was calculated by eqn (1), to compare the average PL lifetime at different pH values: P ai ti 2 taverage ¼ P (1) ai ti where ti are the decay times, and ai, the pre-exponential weights. The calculated results were shown in Fig. 2b. The tave of the QDs-720 exhibited a dependency on the pH value, decreasing from 900 ns in an alkaline environment (pH > 9.0) to 200 ns when the pH was below 4.0. The S-shaped curve has a linear response range from pH 5.5 to 7.0 with the maximum change in the fluorescence lifetime up to B600 ns. This range suggested a potential application in the detection of the microenvironment of some tumor cells, because of their lower extracellular pH (pH = 6.4–6.9) compared to normal tissues (pH = 7.2–7.4).25 However, one of the most problematic aspects of in vivo pH sensing is the extremely crowded media. Therefore, the selectivity of the as-prepared QD-based pH nanosensors was evaluated, as illustrated in Fig. S6 (ESI†). The tave from QDs-720 solutions at pH 7.0 showed a negligible response in the absence and the presence of different concentrations of potential interfering ions (K+, Mn2+, Mg2+, and HCO3) and biomolecules (glucose and BSA). It should be noted that the concentration of all tested ions and biomolecules was several orders of magnitude higher than the actual in vivo levels.9 Thus, the as-prepared QDs can be quite useful as lifetimebased pH nanosensors. To demonstrate the use of the as-prepared QD nanosensors for lifetime-based pH imaging, the QDs were incorporated into microbeads and dispersed in buffers with different pH values.
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Fig. 3 (a) FLIM images of microbeads equipped with QDs-720, dispersed in buffers with different pH values (left: pH = 6.0, right: pH = 7.0, scale bar: 100 mm) and (b) PL lifetime histograms collected from the images. (c) In vivo FLIM experiments of the background of the nude mouse (left) and the QDs-720 injected into adjacent locations with different pH values (green: pH = 6.0, red: pH = 7.0) on the back of the nude mouse (right), respectively (scale bar: 10 mm).
As shown in Fig. 3a, the microbeads present different colors indicating the different PL lifetime of the QDs-720 as a result of pH response, which can be clearly distinguished from the FLIM images. According to the PL lifetime histograms of the FLIM images, the average lifetime of the QDs-720 was B500 ns (pH = 6.0) and B800 ns (pH = 7.0), respectively (Fig. 3b). This is consistent with the S-shaped curve result. Robust maintenance of the pH response under a variety of in vitro environmental conditions gives us confidence that the nanosensors can be applied to lifetime-based in vivo pH imaging. The as-prepared QDs were injected subcutaneously at two adjacent locations with different pH values in a nude mouse. According to the FLIM images, the high-lifetime signals of the QDs at different spots could be easily visualized above the background (Fig. 3c). These results demonstrated the advantages of long-decay and NIR-emission of QDs for in vivo imaging. Furthermore, the diverse spots with different pH values presented distinctive colors, which was consistent with the results of the in vitro experiments. In contrast, the same spots could not be distinguished by using NIR-emitting fluorescence imaging (Fig. S7, ESI†). These results suggested that the as-prepared QDs have great potential as nanosensors for lifetime-based in vivo pH imaging. In summary, we prepared Cu-doped CdZnS QDs with ultrasmall size (B3.5 nm), NIR-emission (B720 nm), and long-decay (lifetime up to B1 ms) by combining the use of doping and a gradientalloyed nanostructure. Moreover, the as-prepared QDs showed excellent pH sensitivity and exhibited a linear response range from pH 5.5 to 7.0 with the maximum change in the fluorescence lifetime up to B600 ns. Furthermore, the QD-based in vivo pH imaging has been demonstrated using FLIM. This work will provide a new candidate for NIR-emitting lifetime-based sensors for in vivo microenvironment imaging. The presented research was financially supported by the National Basic Research Program of China (973 Program No. 2011CB933600), the National Natural Science Foundation of China (Grant No. 81401509, 21305152 and 21375141), the Research Foundation of Chinese Academy of Sciences (yz201439), the Guangdong Science and Technology Program (Grant No. 2012A061400013),
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the Shenzhen Science and Technology Program (Grant No. JCYJ20130402103240486, JCYJ20140417113430607, and KQCX20140521115045447), SIAT Innovation Program for Excellent Young Researchers (201412, 201306), Shenzhen Key Laboratory for Molecular Biology of Neural Development (ZDSY20120617112838879) and Guangdong Innovation Research Team of Lowcost Healthcare.
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