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Hydrothermal synthesis of highly luminescent blue-emitting ZnSe(S) quantum dots exhibiting low toxicity
Materials Science and Engineering C 64 (2016) 167–172
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Hydrothermal synthesis of highly luminescent blue-emitting ZnSe(S) quantum dots exhibiting low toxicity Fatemeh Mirnajafizadeh a, Deborah Ramsey a, Shelli McAlpine a, Fan Wang b, Peter Reece b, John Arron Stride a,c,⁎ a b c
School of Chemistry, University of New South Wales, Sydney, NSW 2052, Australia School of Physics, University of New South Wales, Sydney, NSW 2052, Australia Bragg Institute, Australian Nuclear Science and Technology Organisation, PMB 1, Menai, NSW 2234, Australia
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Article history: Received 14 April 2015 Received in revised form 14 March 2016 Accepted 21 March 2016 Available online 23 March 2016 Keywords: Water dispersible quantum dots Aqueous synthesis Quantum dot cytotoxicity Cell lines
a b s t r a c t Highly luminescent quantum dots (QDs) that emit in the visible spectrum are of interest to a number of imaging technologies, not least that of biological samples. One issue that hinders the application of luminescent markers in biology is the potential toxicity of the fluorophore. Here we show that hydrothermally synthesized ZnSe(S) QDs have low cytotoxicity to both human colorectal carcinoma cells (HCT-116) and human skin fibroblast cells (WS1). The QDs exhibited a high degree of crystallinity, with a strong blue photoluminescence at up to 29% quantum yield relative to 4′,6-diamidino-2-phenylindole (DAPI) without post-synthetic UV-irradiation. Confocal microscopy images obtained of HCT-116 cells after incubation with the QDs highlighted the stability of the particles in cell media. Cytotoxicity studies showed that both HCT-116 and WS1 cells retain 100% viability after treatment with the QDs at concentrations up to 0.5 g/L, which makes them of potential use in biological imaging applications. Crown Copyright © 2016 Published by Elsevier B.V. All rights reserved.
1. Introduction Semiconductor nanoparticles have unique optical properties such as narrow emission and wide absorption spectra, long decay lifetimes and large absorption cross-sections, promoting their usage in biological and medical imaging applications [1–4]. However, there are considerable restrictions to the effective use of QDs in bio-applications, such as water dispersivity, toxicity and the stability of the QDs in biological environments [5–7]. As toxicity is a serious concern in biological applications, the synthesis of QDs free of heavy metals such as Cd, has attracted much attention over the past decade, with less toxic nanoparticles including ZnSe(S) of particular interest [8–13]. Up until now, ZnSe nanoparticles of various morphologies have been synthesized using both organometallic [15,16,32] and aqueous synthetic pathways [14,17–19,22–31]. By avoiding toxic solvents, aqueous syntheses are more attractive for biological applications than competing organic routes. Moreover, the products of aqueous methods are often water dispersible, which is also an important requirement for bioapplications [20,21]. There have been several reports in the literature using a core experimental method based around the initial formation of an inorganic zinc complex, which is then further reacted to form ZnSe QDs upon addition of selenide under appropriate thermal conditions ⁎ Corresponding author at: Bragg Institute, Australian Nuclear Science and Technology Organisation, PMB 1, Menai, NSW 2234, Australia. E-mail address: [email protected] (J.A. Stride).
http://dx.doi.org/10.1016/j.msec.2016.03.061 0928-4931/Crown Copyright © 2016 Published by Elsevier B.V. All rights reserved.
[14,17–19,22–31]. Most reports have focused on the preparation of ZnSe(S) QDs in which sulfur atoms from thiol groups in the capping agents alloy into the ZnSe lattice at high temperatures [17,18,22–25]. Such particles have been reported to show a higher quantum yield (QY) than native ZnSe QDs [23]. However, a number of reports of alloyed ZnSe(S) QDs prepared under reflux have resulted in relatively poor QYs [18,22–24], which has been overcome in some cases with a post-preparative UV irradiation of the ZnSe(S) QDs [23,24]. The chemical stability of the QDs decreased after post-preparative treatments, making this approach less attractive. An more direct route to alloyed ZnSe(S) QDs prepared them under 24 h of reflux at high pH, yielding QDs with a high QY and chemical stability, without the need for postsynthetic UV irradiation [22]. Metal-doped ZnSe QDs, including Mn, Cd and Cu-doped ZnSe QDs, have also been synthesized [28–30,41]. In order to reduce the reaction time, ZnSe(S) QDs have also been synthesized using microwave assisted methods, but the products had low QYs [17,25]. Hydrothermal reactions have previously been reported, including the synthesis of ZnSe(S)/ZnS nanoparticles with N-acetyl-Lcysteine as the capping agent [31] and ZnSe QDs in a single-step reaction with cetyltrimethyl ammonium bromide [32]. However, the hydrothermal synthesis of ZnSe(S) QDs in the presence of 3Mercaptopropionic acid (MPA) has not been reported to date. Herein, we show that water dispersible ZnSe(S) QDs having high luminescence were synthesized using a hydrothermal method in a short reaction time; the QY is comparable to that obtained by other routes, but without the need for post-synthetic UV irradiation. The cytotoxicity of these
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particles with respect to two cell lines, human colorectal carcinoma cells (HCT-116) and human skin fibroblast cells (WS1) was determined. Images of HCT-116 cells after incubation with the QDs were recorded and the stability of QDs in cell media was investigated using confocal microscopy. 2. Experimental method 2.1. Materials 3-Mercaptopropionic acid (MPA) was obtained from Fluka. ZnCl2, NaBH4, Se (powder), NaOH, bisbenzimide (Hoechst 33342), 17allyamino-17-demethoxy-geldanamycine (Hsp90 inhibitor 17-AAG) and 4′,6-diamidino-2-phenylindole (DAPI) were all obtained from Sigma-Aldrich. Human colorectal carcinoma cells (HCT-116) and human skin fibroblast cells (WS1) were obtained from ATCC (Manassas, Virginia, USA). Dulbecco's modified eagle medium (DMEM), fetal bovine serum (FBS), Penicillin, Streptomycin, L-glutamine and nonessential amino acids were received from Invitrogen. All reagents were used as supplied, without additional purification. Ultra-pure water was used in all syntheses. 2.2. Synthesis of QDs Initially, a fresh sample of aqueous NaHSe (0.5 mmol in 20 mL) was synthesized according to literature methods [33,34]. Then, an aqueous Zn-MPA precursor solution was prepared by dissolving 1.09 g (8 mmol) anhydrous ZnCl2 in 100 mL ultra-pure water and adding 2.5 mL MPA (~29 mmol) to the solution. The pH of the Zn-MPA solution was adjusted to 7.3 using 1 M NaOH. Then, 20 mL of fresh NaHSe (0.5 mmol of Se in 20 mL) was added to the Zn-MPA precursor solution, under a nitrogen atmosphere and a colourless solution was obtained. The Zn:Se:MPA molar ratio in the solution was 16:1:58. This was transferred to a Teflon-lined autoclave and placed in a conventional oven under hydrothermal treatment at T = 150 °C for 165 min to form ZnSe(S) water soluble QDs as a transparent colourless solution. The experimental parameters were based upon a range of experiments found to be optimal for CdSe(S) QDs [35] and were used here without further optimization. 2.3. Characterization of QDs UV–visible absorption spectra were measured with a Varian Cary UV spectrometer. The photoluminescence spectrum of ZnSe(S) QDs was recorded using a J/B SPEX 270 M spectrometer equipped with a power max PMT detector and excitation laser at 350 nm. X-ray powder diffraction spectra (PXRD) were taken on an X'pert PRO Multi-purpose X-ray diffraction System (MPD system). X-ray photoelectron spectroscopy (XPS) was performed using an Escalab 250Xi spectrometer. Transmission electron micrographs (TEM) were recorded by a Philips CM200 instrument. ImageJ software was used to analyse the images.
CO2. The thus-prepared samples were used to determine proliferation of the cells using a Cell Counting Kit-8 assay [37]. 2.5. Confocal microscopy studies Live HCT 116 cells were incubated with ZnSe(S) QDs in solution for 24 h at a QD concentration of 100 μg/mL. Images of live cells in the presence of the ZnSe(S) QDs were recorded under a Zias LSM 780 confocal microscope. The cells were stained by adding a Hoechst solution (5 μg/mL) 10 min before recording the images. The emission spectra of the QDs in the cell medium were investigated, initially in the cell media solution, then in both cell media and aqueous solution, as detailed in Supplementary data S2 & S3. 3. Results and discussions 3.1. Synthesis of QDs The hydrothermal synthesis of QDs was found to lead to the formation of highly crystalline ZnSe(S) QDs. PXRD data confirmed that the obtained ZnSe(S) QDs, were cubic in structure, with the particle size calculated using the Scherrer equation of 2.6 ± 0.1 nm, Fig. 1. The diffraction peaks of the QDs were found to be consistent with standard patterns of the cubic phases of ZnSe [38] and ZnS [39], and are characteristic of alloyed ZnSe-ZnS, Table S1, Supplementary data S4. Lattice matching of ZnS and ZnSe facilitates efficient alloying of S into the ZnSe particles [17,40]. This was confirmed using XPS, which revealed the presence of zinc and selenium as the main elements in the QDs, with characteristic peaks of Zn2p at 1021.5 eV and Se3d at 54.5 eV. However, sulfur, originating from the mercaptan group in MPA, was detected at 163.5 eV in addition to three distinct carbons at 285, 286.5 and 288.5 eV, that correspond to the binding energies of C\\S, C_O and C\\H, indicate that ZnSe(S) QDs contain MPA in their structure (Fig. 2, a–d). FT-IR spectroscopy confirmed that MPA is bound to the ZnSe(S) QDs. As shown in Fig. 3, MPA has absorbance bands at 1708, 1251 and 2574 cm− 1, corresponding to the C_O, C\\O and S\\H stretching bands respectively, along with the sharp absorption at 1427 cm− 1 assigned to the OH bending. These peaks all disappeared in the infrared spectrum of ZnSe(S)–MPA capped QDs, confirming the formation of new covalent bonds between sulfur in the structure of MPA and of ZnSe(S) QDs. Meanwhile, two sharp and strong peaks at 1560 and 1406 cm−1, corresponding to the symmetric and asymmetric stretch of COO−, appeared in the FT-IR spectrum of ZnSe-MPA capped QDs, indicating that the carboxylic acid functional group of MPA had ionized
2.4. Cytotoxicity assays A total of five different solutions of QDs were prepared by diluting the aqueous solutions of the ZnSe(S) QDs with ultra-pure water to achieve different concentrations of 25, 50, 100, 250, 500 (μg/mL). Cell cultures of human HCT-116 and WS-1 cell lines were obtained according to the standard protocol [36], as described in the supplementary data S1. The cells were then separately seeded in two 96-well plates (approximately 3000 cells/well) and allowed to adhere to the dish for 24 h in a humidified incubator. As a control, both the cell media alone and cell media in the presence of Hsp90 inhibitor 17-AAG (100 nM = 0.06 μg/mL) were also seeded. Finally, 10 μL of each aqueous solution of QDs (concentrations 25–500 μg/mL) was added to the plates. The plates were incubated in the presence of QDs for 72 h at 37 °C with 5%
Fig. 1. PXRD pattern of ZnSe(S) QDs.
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Fig. 2. XPS spectra of ZnSe(S) QDs binding energy of (a) Zn3p, (b) Se3d, (c) S2pand (d) C1s.
Fig. 3. FT-IR of ZnSe(S)-MPA capped QDs (a), MPA (b) and the structure of MPA (c).
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and MPA was bound to Zn through the thiol group in accord with the FT-IR of the synthesized Mn-ZnSe doped QDs in presence of MPA using reflux, as previously reported [41]. HRTEM images of the as-prepared ZnSe(S) QDs indicated spherical nanoparticles, with a symmetric FFT pattern and clearly visible atomic planes, indicative of a high crystallinity of the nanoparticles. The dspacing was determined to be 3.53 Å, which is approximately equal to the d-spacing of the (111) lattice plane of cubic ZnSe (Fig. 4). Dried samples imaged under the TEM were found to be strongly aggregated, Fig. 4a, largely due to interactions between corresponding groups in the capping agent, MPA. This is in distinct contrast to solubilized samples where the capping agent assists in aqueous dispersion, as evidenced by the PL properties. Optical spectroscopy revealed that the as-synthesized ZnSe(S) QDs have a wide excitation absorption band below 380 nm and a narrow emission band (FWHM = 38 nm) at 415 nm, Fig. 5, in accord with the blue coloration under UV light (Supplementary data S5) and in agreement with the calculated particle size of 2.6 ± 0.1 nm obtained from PXRD pattern. Both the estimated particle size and the observed blue shift (415 nm) were in accord with the quantum confinement effect because as the particle size decreases, the emission peak was blue shifted compared with bulk ZnSe, as previously reported [18,27,31,32,42]. Literature reports on the effects of alloying of sulfur into ZnSe QDs are inconclusive, with other factors such as the capping agent and synthetic parameters also contributing to the optical properties of the final
Fig. 5. Optical spectra of ZnSe (S) QDs.
samples [18,24]. We did not investigate the effect of alloying in our work as our use of MPA resulted exclusively in alloyed ZnSe(S) particles. The QY was determined to be around 29% of that of the fluorescent dye DAPI, or an absolute QY of 1.3%, as detailed in S6. The obtained QY
Fig. 4. TEM image of ZnSe(S) QDs: (a) low resolution, (b, c) high resolution, (d, e) atomic planes in a single particle and its corresponding FFT.
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was larger than that of ZnSe QDs previously obtained from aqueous phase reactions in previous reports [14,43], confirming that the alloyed ZnSe(S) QDs exhibit improved optical properties without the need for post-preparative UV treatment [23]. In addition, the QY of the QDs was found to be significantly larger than that of ZnSe(S) QDs obtained from microwave assisted methods [25]. This outcome indicates that hydrothermal synthesis is a promising alternative approach to other synthetic methods currently in use. Moreover, these ZnSe(S) QDs have a relatively high QY and were obtained over shorter reaction times compared with heating under reflux, [22]. 3.2. Cytotoxicity of QDs Heavy metal-free nanoparticles including ZnSe(S) QDs are expected to have lower toxicity than heavy-metal QDs [44]. However, the cytotoxicity of these nanoparticles is worthy of investigation due to their nanoscale properties and the potential toxicity of their components and capping agents. For example, both zinc and selenium are less toxic than other elements commonly used in QDs, but the presence of MPA acid in the structure of the ZnSe(S) QDs may result in some level of toxicity [45]. Fig. 7. Confocal image of HCT-116 live cells after incubation with ZnSe(S).
3.2.1. Cytotoxicity of QDs in presence of cancer cells HCT-116 cells are cancer cells that have a DNA mutation with the capacity to form tumours when injected into mice and are also fatal in the human body due to metastasis [46,47]. Cytotoxicity assays of the synthesized QDs toward these cancer cells were performed and toxicology data showed that ZnSe(S) QDs are nontoxic over all concentrations at 25 to 100 μg/mL, with 100% cell viability of the HCT-116 cell line after incubation with the as–synthesized ZnSe(S) nanocrystals (Fig. 6). 3.2.2. Cytotoxicity of QDs toward normal cells Cell proliferation of the WS1 cell line in the presence of ZnSe(S) QDs was determined to be 100% at concentrations 25 to 100 μg/mL, similar to the cell viability of HCT-116 cell line after treatment with ZnSe(S) QDs, as shown in Supplementary data S7. 3.3. Confocal microscopy studies Any cytotoxicity or oxidation of the QDs during incubation the cells, would lead to necrosis of the cell nuclei. Confocal microscopy images of live HCT-116 cells were recorded after incubation with the assynthesized QDs (Fig. 7) and were found to display no signs of necrosis at a concentration of 100 μg/mL, confirming the cytotoxicity data. However, the fact that the ZnSe(S) QDs and Hoechst 33342 both have strong
emissions in the blue region hinders facile identification of the QDs in the images. It does demonstrate however, the stability of cells after incubation with the QDs, Fig. 7, indicating a low toxicity of the QDs toward HCT-116 cells. As any interaction of QDs with cell media can alter the emission spectra of QDs [48-50], the emission profiles of the as-synthesized ZnSe(S) QDs in both cell media and water were recorded using confocal microscopy. The results indicated that there is no significant change or shift in the photoluminescence spectrum of the ZnSe(S) QDs in cell media compared to the emission profile of QDs in water (Supplementary data S8), whilst it has been previously reported that water dispersible CdTe-MPA capped QDs are stable only in water and the cell growth media alters their emission profile [51]. These data confirm the potential capacity of the as-synthesized QDs for bio-applications due to low toxicity, water dispersity and stability in cell media.
4. Conclusions This work highlights that a hydrothermal synthetic method is an effective approach to the synthesis of blue emitting ZnSe(S) QDs. The QDs obtained are highly crystalline and stable, displaying high luminescence and dispersity in water. Hydrothermal reaction conditions were used to promote the formation of QDs over short reaction times, without the need for post-preparative UV treatments to obtain a high QY. Moreover, it was clearly shown that the obtained QDs exhibited no toxicity across a range of concentrations, from 25 to 500 μg/mL of QDs, toward both HCT116 and WS1 cell lines. Acknowledgments We would like to thank the School of Chemistry and the Mark Wainwright Analytical Centre of University of New South Wales for facilities, Ms. Kokila Egodage for her help about using photoluminescence instrument, Dr. Renee Wan for her assistance about confocal images and Dr. Alex Macmillan for his good advice about QY. Appendix A. Supplementary data
Fig. 6. The results of cytotoxicity data of ZnSe (S) QDs toward HCT-116 cell line. Both media and 17-AAG were used as control and error bars indicate standard error of the mean.
Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2016.03.061.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Hydrothermal synthesis of highly luminescent blue-emitting ZnSe(S) quantum dots exhibiting low toxicity Fatemeh Mirnajafizadeh a, Deborah Ramsey a, Shelli McAlpine a, Fan Wang b, Peter Reece b, John Arron Stride a,c,⁎ a b c
School of Chemistry, University of New South Wales, Sydney, NSW 2052, Australia School of Physics, University of New South Wales, Sydney, NSW 2052, Australia Bragg Institute, Australian Nuclear Science and Technology Organisation, PMB 1, Menai, NSW 2234, Australia
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Article history: Received 14 April 2015 Received in revised form 14 March 2016 Accepted 21 March 2016 Available online 23 March 2016 Keywords: Water dispersible quantum dots Aqueous synthesis Quantum dot cytotoxicity Cell lines
a b s t r a c t Highly luminescent quantum dots (QDs) that emit in the visible spectrum are of interest to a number of imaging technologies, not least that of biological samples. One issue that hinders the application of luminescent markers in biology is the potential toxicity of the fluorophore. Here we show that hydrothermally synthesized ZnSe(S) QDs have low cytotoxicity to both human colorectal carcinoma cells (HCT-116) and human skin fibroblast cells (WS1). The QDs exhibited a high degree of crystallinity, with a strong blue photoluminescence at up to 29% quantum yield relative to 4′,6-diamidino-2-phenylindole (DAPI) without post-synthetic UV-irradiation. Confocal microscopy images obtained of HCT-116 cells after incubation with the QDs highlighted the stability of the particles in cell media. Cytotoxicity studies showed that both HCT-116 and WS1 cells retain 100% viability after treatment with the QDs at concentrations up to 0.5 g/L, which makes them of potential use in biological imaging applications. Crown Copyright © 2016 Published by Elsevier B.V. All rights reserved.
1. Introduction Semiconductor nanoparticles have unique optical properties such as narrow emission and wide absorption spectra, long decay lifetimes and large absorption cross-sections, promoting their usage in biological and medical imaging applications [1–4]. However, there are considerable restrictions to the effective use of QDs in bio-applications, such as water dispersivity, toxicity and the stability of the QDs in biological environments [5–7]. As toxicity is a serious concern in biological applications, the synthesis of QDs free of heavy metals such as Cd, has attracted much attention over the past decade, with less toxic nanoparticles including ZnSe(S) of particular interest [8–13]. Up until now, ZnSe nanoparticles of various morphologies have been synthesized using both organometallic [15,16,32] and aqueous synthetic pathways [14,17–19,22–31]. By avoiding toxic solvents, aqueous syntheses are more attractive for biological applications than competing organic routes. Moreover, the products of aqueous methods are often water dispersible, which is also an important requirement for bioapplications [20,21]. There have been several reports in the literature using a core experimental method based around the initial formation of an inorganic zinc complex, which is then further reacted to form ZnSe QDs upon addition of selenide under appropriate thermal conditions ⁎ Corresponding author at: Bragg Institute, Australian Nuclear Science and Technology Organisation, PMB 1, Menai, NSW 2234, Australia. E-mail address: [email protected] (J.A. Stride).
http://dx.doi.org/10.1016/j.msec.2016.03.061 0928-4931/Crown Copyright © 2016 Published by Elsevier B.V. All rights reserved.
[14,17–19,22–31]. Most reports have focused on the preparation of ZnSe(S) QDs in which sulfur atoms from thiol groups in the capping agents alloy into the ZnSe lattice at high temperatures [17,18,22–25]. Such particles have been reported to show a higher quantum yield (QY) than native ZnSe QDs [23]. However, a number of reports of alloyed ZnSe(S) QDs prepared under reflux have resulted in relatively poor QYs [18,22–24], which has been overcome in some cases with a post-preparative UV irradiation of the ZnSe(S) QDs [23,24]. The chemical stability of the QDs decreased after post-preparative treatments, making this approach less attractive. An more direct route to alloyed ZnSe(S) QDs prepared them under 24 h of reflux at high pH, yielding QDs with a high QY and chemical stability, without the need for postsynthetic UV irradiation [22]. Metal-doped ZnSe QDs, including Mn, Cd and Cu-doped ZnSe QDs, have also been synthesized [28–30,41]. In order to reduce the reaction time, ZnSe(S) QDs have also been synthesized using microwave assisted methods, but the products had low QYs [17,25]. Hydrothermal reactions have previously been reported, including the synthesis of ZnSe(S)/ZnS nanoparticles with N-acetyl-Lcysteine as the capping agent [31] and ZnSe QDs in a single-step reaction with cetyltrimethyl ammonium bromide [32]. However, the hydrothermal synthesis of ZnSe(S) QDs in the presence of 3Mercaptopropionic acid (MPA) has not been reported to date. Herein, we show that water dispersible ZnSe(S) QDs having high luminescence were synthesized using a hydrothermal method in a short reaction time; the QY is comparable to that obtained by other routes, but without the need for post-synthetic UV irradiation. The cytotoxicity of these
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particles with respect to two cell lines, human colorectal carcinoma cells (HCT-116) and human skin fibroblast cells (WS1) was determined. Images of HCT-116 cells after incubation with the QDs were recorded and the stability of QDs in cell media was investigated using confocal microscopy. 2. Experimental method 2.1. Materials 3-Mercaptopropionic acid (MPA) was obtained from Fluka. ZnCl2, NaBH4, Se (powder), NaOH, bisbenzimide (Hoechst 33342), 17allyamino-17-demethoxy-geldanamycine (Hsp90 inhibitor 17-AAG) and 4′,6-diamidino-2-phenylindole (DAPI) were all obtained from Sigma-Aldrich. Human colorectal carcinoma cells (HCT-116) and human skin fibroblast cells (WS1) were obtained from ATCC (Manassas, Virginia, USA). Dulbecco's modified eagle medium (DMEM), fetal bovine serum (FBS), Penicillin, Streptomycin, L-glutamine and nonessential amino acids were received from Invitrogen. All reagents were used as supplied, without additional purification. Ultra-pure water was used in all syntheses. 2.2. Synthesis of QDs Initially, a fresh sample of aqueous NaHSe (0.5 mmol in 20 mL) was synthesized according to literature methods [33,34]. Then, an aqueous Zn-MPA precursor solution was prepared by dissolving 1.09 g (8 mmol) anhydrous ZnCl2 in 100 mL ultra-pure water and adding 2.5 mL MPA (~29 mmol) to the solution. The pH of the Zn-MPA solution was adjusted to 7.3 using 1 M NaOH. Then, 20 mL of fresh NaHSe (0.5 mmol of Se in 20 mL) was added to the Zn-MPA precursor solution, under a nitrogen atmosphere and a colourless solution was obtained. The Zn:Se:MPA molar ratio in the solution was 16:1:58. This was transferred to a Teflon-lined autoclave and placed in a conventional oven under hydrothermal treatment at T = 150 °C for 165 min to form ZnSe(S) water soluble QDs as a transparent colourless solution. The experimental parameters were based upon a range of experiments found to be optimal for CdSe(S) QDs [35] and were used here without further optimization. 2.3. Characterization of QDs UV–visible absorption spectra were measured with a Varian Cary UV spectrometer. The photoluminescence spectrum of ZnSe(S) QDs was recorded using a J/B SPEX 270 M spectrometer equipped with a power max PMT detector and excitation laser at 350 nm. X-ray powder diffraction spectra (PXRD) were taken on an X'pert PRO Multi-purpose X-ray diffraction System (MPD system). X-ray photoelectron spectroscopy (XPS) was performed using an Escalab 250Xi spectrometer. Transmission electron micrographs (TEM) were recorded by a Philips CM200 instrument. ImageJ software was used to analyse the images.
CO2. The thus-prepared samples were used to determine proliferation of the cells using a Cell Counting Kit-8 assay [37]. 2.5. Confocal microscopy studies Live HCT 116 cells were incubated with ZnSe(S) QDs in solution for 24 h at a QD concentration of 100 μg/mL. Images of live cells in the presence of the ZnSe(S) QDs were recorded under a Zias LSM 780 confocal microscope. The cells were stained by adding a Hoechst solution (5 μg/mL) 10 min before recording the images. The emission spectra of the QDs in the cell medium were investigated, initially in the cell media solution, then in both cell media and aqueous solution, as detailed in Supplementary data S2 & S3. 3. Results and discussions 3.1. Synthesis of QDs The hydrothermal synthesis of QDs was found to lead to the formation of highly crystalline ZnSe(S) QDs. PXRD data confirmed that the obtained ZnSe(S) QDs, were cubic in structure, with the particle size calculated using the Scherrer equation of 2.6 ± 0.1 nm, Fig. 1. The diffraction peaks of the QDs were found to be consistent with standard patterns of the cubic phases of ZnSe [38] and ZnS [39], and are characteristic of alloyed ZnSe-ZnS, Table S1, Supplementary data S4. Lattice matching of ZnS and ZnSe facilitates efficient alloying of S into the ZnSe particles [17,40]. This was confirmed using XPS, which revealed the presence of zinc and selenium as the main elements in the QDs, with characteristic peaks of Zn2p at 1021.5 eV and Se3d at 54.5 eV. However, sulfur, originating from the mercaptan group in MPA, was detected at 163.5 eV in addition to three distinct carbons at 285, 286.5 and 288.5 eV, that correspond to the binding energies of C\\S, C_O and C\\H, indicate that ZnSe(S) QDs contain MPA in their structure (Fig. 2, a–d). FT-IR spectroscopy confirmed that MPA is bound to the ZnSe(S) QDs. As shown in Fig. 3, MPA has absorbance bands at 1708, 1251 and 2574 cm− 1, corresponding to the C_O, C\\O and S\\H stretching bands respectively, along with the sharp absorption at 1427 cm− 1 assigned to the OH bending. These peaks all disappeared in the infrared spectrum of ZnSe(S)–MPA capped QDs, confirming the formation of new covalent bonds between sulfur in the structure of MPA and of ZnSe(S) QDs. Meanwhile, two sharp and strong peaks at 1560 and 1406 cm−1, corresponding to the symmetric and asymmetric stretch of COO−, appeared in the FT-IR spectrum of ZnSe-MPA capped QDs, indicating that the carboxylic acid functional group of MPA had ionized
2.4. Cytotoxicity assays A total of five different solutions of QDs were prepared by diluting the aqueous solutions of the ZnSe(S) QDs with ultra-pure water to achieve different concentrations of 25, 50, 100, 250, 500 (μg/mL). Cell cultures of human HCT-116 and WS-1 cell lines were obtained according to the standard protocol [36], as described in the supplementary data S1. The cells were then separately seeded in two 96-well plates (approximately 3000 cells/well) and allowed to adhere to the dish for 24 h in a humidified incubator. As a control, both the cell media alone and cell media in the presence of Hsp90 inhibitor 17-AAG (100 nM = 0.06 μg/mL) were also seeded. Finally, 10 μL of each aqueous solution of QDs (concentrations 25–500 μg/mL) was added to the plates. The plates were incubated in the presence of QDs for 72 h at 37 °C with 5%
Fig. 1. PXRD pattern of ZnSe(S) QDs.
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Fig. 2. XPS spectra of ZnSe(S) QDs binding energy of (a) Zn3p, (b) Se3d, (c) S2pand (d) C1s.
Fig. 3. FT-IR of ZnSe(S)-MPA capped QDs (a), MPA (b) and the structure of MPA (c).
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and MPA was bound to Zn through the thiol group in accord with the FT-IR of the synthesized Mn-ZnSe doped QDs in presence of MPA using reflux, as previously reported [41]. HRTEM images of the as-prepared ZnSe(S) QDs indicated spherical nanoparticles, with a symmetric FFT pattern and clearly visible atomic planes, indicative of a high crystallinity of the nanoparticles. The dspacing was determined to be 3.53 Å, which is approximately equal to the d-spacing of the (111) lattice plane of cubic ZnSe (Fig. 4). Dried samples imaged under the TEM were found to be strongly aggregated, Fig. 4a, largely due to interactions between corresponding groups in the capping agent, MPA. This is in distinct contrast to solubilized samples where the capping agent assists in aqueous dispersion, as evidenced by the PL properties. Optical spectroscopy revealed that the as-synthesized ZnSe(S) QDs have a wide excitation absorption band below 380 nm and a narrow emission band (FWHM = 38 nm) at 415 nm, Fig. 5, in accord with the blue coloration under UV light (Supplementary data S5) and in agreement with the calculated particle size of 2.6 ± 0.1 nm obtained from PXRD pattern. Both the estimated particle size and the observed blue shift (415 nm) were in accord with the quantum confinement effect because as the particle size decreases, the emission peak was blue shifted compared with bulk ZnSe, as previously reported [18,27,31,32,42]. Literature reports on the effects of alloying of sulfur into ZnSe QDs are inconclusive, with other factors such as the capping agent and synthetic parameters also contributing to the optical properties of the final
Fig. 5. Optical spectra of ZnSe (S) QDs.
samples [18,24]. We did not investigate the effect of alloying in our work as our use of MPA resulted exclusively in alloyed ZnSe(S) particles. The QY was determined to be around 29% of that of the fluorescent dye DAPI, or an absolute QY of 1.3%, as detailed in S6. The obtained QY
Fig. 4. TEM image of ZnSe(S) QDs: (a) low resolution, (b, c) high resolution, (d, e) atomic planes in a single particle and its corresponding FFT.
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was larger than that of ZnSe QDs previously obtained from aqueous phase reactions in previous reports [14,43], confirming that the alloyed ZnSe(S) QDs exhibit improved optical properties without the need for post-preparative UV treatment [23]. In addition, the QY of the QDs was found to be significantly larger than that of ZnSe(S) QDs obtained from microwave assisted methods [25]. This outcome indicates that hydrothermal synthesis is a promising alternative approach to other synthetic methods currently in use. Moreover, these ZnSe(S) QDs have a relatively high QY and were obtained over shorter reaction times compared with heating under reflux, [22]. 3.2. Cytotoxicity of QDs Heavy metal-free nanoparticles including ZnSe(S) QDs are expected to have lower toxicity than heavy-metal QDs [44]. However, the cytotoxicity of these nanoparticles is worthy of investigation due to their nanoscale properties and the potential toxicity of their components and capping agents. For example, both zinc and selenium are less toxic than other elements commonly used in QDs, but the presence of MPA acid in the structure of the ZnSe(S) QDs may result in some level of toxicity [45]. Fig. 7. Confocal image of HCT-116 live cells after incubation with ZnSe(S).
3.2.1. Cytotoxicity of QDs in presence of cancer cells HCT-116 cells are cancer cells that have a DNA mutation with the capacity to form tumours when injected into mice and are also fatal in the human body due to metastasis [46,47]. Cytotoxicity assays of the synthesized QDs toward these cancer cells were performed and toxicology data showed that ZnSe(S) QDs are nontoxic over all concentrations at 25 to 100 μg/mL, with 100% cell viability of the HCT-116 cell line after incubation with the as–synthesized ZnSe(S) nanocrystals (Fig. 6). 3.2.2. Cytotoxicity of QDs toward normal cells Cell proliferation of the WS1 cell line in the presence of ZnSe(S) QDs was determined to be 100% at concentrations 25 to 100 μg/mL, similar to the cell viability of HCT-116 cell line after treatment with ZnSe(S) QDs, as shown in Supplementary data S7. 3.3. Confocal microscopy studies Any cytotoxicity or oxidation of the QDs during incubation the cells, would lead to necrosis of the cell nuclei. Confocal microscopy images of live HCT-116 cells were recorded after incubation with the assynthesized QDs (Fig. 7) and were found to display no signs of necrosis at a concentration of 100 μg/mL, confirming the cytotoxicity data. However, the fact that the ZnSe(S) QDs and Hoechst 33342 both have strong
emissions in the blue region hinders facile identification of the QDs in the images. It does demonstrate however, the stability of cells after incubation with the QDs, Fig. 7, indicating a low toxicity of the QDs toward HCT-116 cells. As any interaction of QDs with cell media can alter the emission spectra of QDs [48-50], the emission profiles of the as-synthesized ZnSe(S) QDs in both cell media and water were recorded using confocal microscopy. The results indicated that there is no significant change or shift in the photoluminescence spectrum of the ZnSe(S) QDs in cell media compared to the emission profile of QDs in water (Supplementary data S8), whilst it has been previously reported that water dispersible CdTe-MPA capped QDs are stable only in water and the cell growth media alters their emission profile [51]. These data confirm the potential capacity of the as-synthesized QDs for bio-applications due to low toxicity, water dispersity and stability in cell media.
4. Conclusions This work highlights that a hydrothermal synthetic method is an effective approach to the synthesis of blue emitting ZnSe(S) QDs. The QDs obtained are highly crystalline and stable, displaying high luminescence and dispersity in water. Hydrothermal reaction conditions were used to promote the formation of QDs over short reaction times, without the need for post-preparative UV treatments to obtain a high QY. Moreover, it was clearly shown that the obtained QDs exhibited no toxicity across a range of concentrations, from 25 to 500 μg/mL of QDs, toward both HCT116 and WS1 cell lines. Acknowledgments We would like to thank the School of Chemistry and the Mark Wainwright Analytical Centre of University of New South Wales for facilities, Ms. Kokila Egodage for her help about using photoluminescence instrument, Dr. Renee Wan for her assistance about confocal images and Dr. Alex Macmillan for his good advice about QY. Appendix A. Supplementary data
Fig. 6. The results of cytotoxicity data of ZnSe (S) QDs toward HCT-116 cell line. Both media and 17-AAG were used as control and error bars indicate standard error of the mean.
Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2016.03.061.
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