Thiol-based non-injection synthesis of near-infrared Ag₂S/ZnS core/shell quantum dots

Abstract

Ag₂S quantum dots (QDs) are a promising candidate for biomedical imaging, labeling and sensing. However, coating a ZnS shell on the Ag₂S QDs to improve the quantum yield (QYs) is still a challenge. Here, we developed a novel non-injection approach for synthesizing Ag₂S and Ag₂S/ZnS core/shell QDs using alkanethiols as both ligand and sulfur sources. The QY was significantly increased after coating a ZnS shell on the Ag₂S core. Moreover, the possible pathway of the sulfur released from the alkanethiol and transferred to the Ag₂S QDs, which is beneficial for understanding the formation mechanism of thiol-based non-injection synthesis of metal sulfide nanocrystal (NCs), was investigated.

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Introduction

The high detection sensitivity, resolution and versatility of fluorescence microscopy have promoted fluorescence imaging for biomedical research, diagnostics, and optically assisted surgery.^1–3 However, the conventional organic dyes cannot meet some requirements of biomedical fluorescence imaging, such as long observation times.³ In the past decade, semiconductor quantum dots (QDs) have attracted great interest owing to their superior optical properties, including high extinction coefficients, size and composition dependent photoluminescence (PL), high quantum yields (QYs) and photostability and large two-photon action cross-sections, which make them promising alternatives for organic dyes.⁴ Currently, QDs have been widely applied for detection and imaging in several areas in the life sciences, such as tumor imaging and virus detection.^5–10 Recently, the successful synthesis of novel near-infrared (NIR) fluorescence I–III–VI and I–VI QDs, including CuInS₂, Ag₂S and Ag₂Se, etc.,^11–16 further promotes the application of QDs for in vivo biological imaging, because the NIR window are more desirable over the visible window (400–750 nm) for the fluorescence imaging owing to the reduced photon scattering, deeper tissue penetration, and lower autofluorescence.^17,18

In consideration of the environmental, health, and safety standpoint,¹⁹ a desired QDs may not contain any toxic element, such as Cd, Pb, and Hg.^20,21 The Ag₂S QDs is promising candidate because of its negligible toxicity,²² simple chemical composition and suitable band gap emitting fluorescence covering the range of visible to NIR. To date, several reports on Ag₂S QDs have been published.^{12–14,23,24} However, the study of new synthetic strategies to improve the QY of Ag₂S QDs are still of great importance. Moreover, coating a ZnS shell on the Ag₂S QDs to improve the QY is still a challenge.

There are two main strategies for synthesizing high-quality metal sulfide QDs, including the hot-injection strategy based on the fast precursor injection and the non-injection strategy using the anion precursor as both ligand and sulfur source (e.g. alkanethiols). In contrast, the non-injection strategy minimizes the number of reagents and simplifies the synthetic steps.¹¹ Thiol-based non-injection method have been successfully applied for synthesizing oil-soluble CuInS₂/ZnS QDs.^11,25,26 In our previous work, we successfully synthesized the water-soluble Ag₂S QDs using carboxylic acid (COOH) groups terminated alkanethiol (3-mercaptopropionic acid) as both sulfur source and ligand.¹³ Moreover, the soft Lewis base alkanethiols are good ligands to suppress the reactivity of the soft Lewis acids Ag⁺ ions,²¹ which is beneficial for synthesizing small size Ag₂S QDs within the quantum confinement regime.

Hence, we developed a novel non-injection approach (Scheme 1) for synthesizing Ag₂S QDs using alkanethiols as both ligand and sulfur source (called as step I). In order to improve the QY of Ag₂S QDs, Zn²⁺ precursor was dropwised into the reaction solution after the formation of the Ag₂S QDs to grow a ZnS shell on the Ag₂S QDs (called as step II). As well known, coating an insulating inorganic shell with a wider band gap on QDs is an effective approach to passivating the surface defect states and preventing the influence of environmental factors (such as oxygen, pH, etc.), thus increasing the PL QY and stability of the QDs.^27–29 The as-prepared oil-soluble Ag₂S/ZnS QDs was transferred to water phase by coating an amphiphilic polymer shell. The cytotoxicity of the obtained water-soluble Ag₂S/ZnS QDs was evaluated through 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-1) assay. Moreover, the pathway of the sulfur released from the 1-octanethiol and transferred to Ag₂S QDs, which is beneficial for understanding the formation mechanism of thiol-based non-injection synthesis of metal sulfide nanocrystal (NCs), was investigated.

Scheme 1 Schematic illustrations of the synthesis of Ag₂S QDs (step I) and Ag₂S/ZnS core/shell QDs (step II) using alkanethiol as both ligand and sulfur source.

Experimental

Materials

1-Octanethiol (≥98.5%), oleylamine (OAm, 70%) and indocyanine green (ICG, polymethine dye) were purchased from Sigma Aldrich. 1-Dodecanethiol (98%) and 1-hexadecanethiol (90%) were purchased from Alfa Aesar. 1-Octadecene (ODE, tech. 90%) was purchased from ACROS. WST-1 was purchased from Beyotime Biotechnology (China). Silver acetate (AgAc, AR) and other reagents were purchased from China National Pharmaceutical Group Corporation.

Synthetic procedures

In a typical synthesis, 0.1 mmol of AgAc, 1.64 mmol of 1-octanethiol and 5 mL of ODE were loaded into a three-neck flask filled with nitrogen. The temperature was increased to 180 °C and kept at this temperature for the growth of Ag₂S nanocrystals. After grown for a certain time, the growth temperature was decreased to 150 °C and the Zn²⁺ precursor (Zn(Ac)₂ dissolved in OAm) was dropwise added to the reaction solution for coating a ZnS shell on the Ag₂S nanocrystals. To monitor the growth of the nanoparticles, aliquots were taken at different reaction times for absorption and FL measurements. The products were mixed with methanol and precipitated through centrifugation at 8000 rpm for 3 min. The precipitate was dispersed in nonpolar solvents for further characterizations.

Phase transfer of Ag₂S/ZnS QDs

The Ag₂S/ZnS QDs were washed with ethanol for several times and redispersed in chloroform before the water solubilization process. Subsequently, excess octylamine-modified polyacrylic acid (OPA) was added to the Ag₂S/ZnS QDs dissolved in chloroform and stirred for several minutes. Then, the solvent was removed by a rotary evaporator and the precipitate was dissolved in borate buffered saline (pH 12.8).

Cell viability assay

MCF-7 cells and Vero cells were incubated in 96 well for 24 h before treatment with Ag₂S/ZnS QDs. The Ag₂S/ZnS QDs was dispersed in 1× PBS and filtered with MILLEX GP Filters (unit 0.22 μm). Then, the MCF-7 cells and Vero cells were treated with the Ag₂S/ZnS QDs of different concentration (0–200 μg mL⁻¹) for 24 h. After the 24 h treatment of Ag₂S/ZnS QDs, the MCF-7 cells and Vero cells were incubated for 4 h with WST-1. Subsequently, the 96 well plates were shook for 1 minute before the absorbance measurement. Finally, the absorbance was recorded at 450 nm and 690 nm (the reference wavelength) using the microplate spectrophotometer system (MULTISKAN MK3).

Characterizations

Transmission electron microscopy (TEM) imaging was performed using Tecnai G2 20 microscope. Copper grids coating with amorphous carbon film was used as substrate for preparing the TEM samples. Powder X-ray diffraction (XRD) data were recorded using Bruker D8 Advanced X-ray diffractometer (Bruker axs) with Cu K-alpha radiation wavelength of 1.5406 Å, the scan rate was set as 0.5 degree min⁻¹. UV-2550 spectrophotometer (SHIMADZU) was used to record absorption spectra. F-4600 fluorescence spectrophotometer (HITACHI) was used to record photoluminescence (PL) spectra. DLS measurements were performed on a Malvern Zetasizer Nano ZS instrument.

Results and discussion

Fig. 1 shows the absorption and the corresponding photoluminescence (PL) spectra of Ag₂S core QDs and the Ag₂S/ZnS core/shell QDs. It is obvious that the PL intensity of the Ag₂S core QDs was extremely weak and there is no obvious corresponding first exciton peak appeared in the absorption spectrum. However, it is exciting that the PL intensity was significantly increased after the growth of a ZnS shell on the Ag₂S core. QY measurement shows that the QY was increased from 0.02% to 3.8% (see the QY measurement details and Fig. S1 in the ESI†). Moreover, as a consequence of quantum confinement, an obvious first exciton peak appeared at 803 nm in the absorption spectrum of the Ag₂S/ZnS core/shell QDs. The PL excitation spectrum (Fig. S2 in the ESI†) also displayed a well-defined excitation peak, which agreed well with the absorption spectrum. The temporal evolution of absorption and PL spectra of Ag₂S QDs shows that no obvious corresponding first exciton peak appeared during the whole growth process of 50 min (Fig. 2a) and the PL intensity was not significantly increased with the growth time (Fig. 2b). In contrast, the first exciton peak at 803 nm appeared after grew for only 10 min (after addition of Zn²⁺ precursor) in the ZnS shell growth process (Fig. 2c). Moreover, the PL intensity was significantly increased with the growth time after the addition of Zn²⁺ precursor (Fig. 2d). These results suggest that this method is effective for coating ZnS shell on Ag₂S QDs to improve the fluorescence of Ag₂S QDs.

Fig. 1 PL spectra (a) and the corresponding absorption spectra (b) of the Ag₂S and Ag₂S/ZnS core/shell QDs. A zoom of the Ag₂S PL spectrum (a, inset). The excitation wavelength for PL spectra is 480 nm.

Fig. 2 Temporal evolution of absorption spectra (a) and PL spectra (b) of the Ag₂S QDs grown without Zn²⁺ precursor (step I). Temporal evolution of absorption spectra (c) and PL spectra (d) of the Ag₂S/ZnS core/shell QDs grown after addition of Zn²⁺ precursor (step II). The excitation wavelength for PL spectra is 480 nm.

The Ag₂S core and Ag₂S/ZnS core/shell QDs are monodisperse as shown in the TEM images (Fig. 3a and b). The size distribution histograms (Fig. S3 in the ESI†) show that the size of Ag₂S QDs were increased from 2.1 ± 1.6 nm to 2.4 ± 0.8 nm after grown a ZnS shell and the standard deviations of size distributions were improved. Powder X-ray diffraction (XRD) (Fig. 4) results of the Ag₂S QDs and Ag₂S/ZnS QDs show weak and undistinguishable diffraction peaks owing to their small sizes and amorphous surface ligands.¹⁴ The results of the Ag₂S QDs matches with the reported monoclinic Ag₂S QDs.^14,24 The peak of the as-prepared Ag₂S/ZnS core/shell QDs shows a short shift to that of ZnS crystal (JCPDS Card no. 36-1450), which is similar to other reported core/shell structured QDs such as CdSe/CdS and CdSe/ZnS QDs,^30–32 suggesting the formation of Ag₂S/ZnS core/shell QDs.

Fig. 3 TEM images of the Ag₂S QDs (a) and Ag₂S/ZnS core/shell QDs (b).

Fig. 4 XRD patterns of the Ag₂S QDs (black line) and Ag₂S/ZnS core/shell QDs (red line).

In fact, in addition to the 1-octanethiol, other alkanethiols can also be used as both ligand and sulfur source for synthesizing Ag₂S QDs and Ag₂S/ZnS QDs through our synthetic strategy. In this work, Ag₂S QDs and Ag₂S/ZnS QDs were successfully synthesized using 1-dodecanethiol (CH₃(CH₂)₁₀CH₂SH) or 1-hexadecanethiol (CH₃(CH₂)₁₄CH₂SH) as sulfur precursors. The TEM images, absorption spectra and PL spectra are shown in Fig. S4–S6 in the ESI.†

As mentioned above, alkanethiols are good sulfide precursor (as both ligand and sulfur source) for non-injection method and have been successfully applied for synthesizing high-quality metal sulfide QDs. However, to our knowledge, the formation mechanism in the thiol-based non-injection synthesis of metal sulfide NCs has not been reported. In this work, the Ag₂S QDs has been successfully synthesized using the 1-octanethiol as both ligand and sulfur source. Thus, we chose this synthetic strategy as the model system to study how does the sulfur released from the alkanethiol and transferred to metal sulfide NCs. In the reported typical hot-injection method using elemental sulfur solution as sulfur source for the synthesis of metal sulfide NCs, the formation mechanism is that H₂S is produced from elemental sulfur under heating, and then react with metal salts to form metal sulfide NCs.^33,34 We infer that the formation mechanism in this work is also based on the reaction between H₂S and silver salt. In order to confirm the inference, we investigated whether the 1-octanethiol could release H₂S gas at the synthetic temperature. The detection of H₂S gas was proceeded by exposing Pb(Ac)₂ testing paper to the reaction atmosphere above the mixture of 1-octanethiol and ODE under different temperatures. The photographs of Pb(Ac)₂ testing papers are shown in Fig. S7a (ESI†) after exposed for 30 min. The photographs show that the testing paper turned from white to yellow under low temperature (below 100 °C) and no black spots appeared, indicating that H₂S gas could not be produced under low temperature. The yellow is attributed to the formation of lead thiolate salt (Pb(CH₃(CH₂)₇S)₂) from the reaction between the volatilized 1-octanethiol and Pb²⁺ in the testing paper. When the solution temperature was increased above 120 °C, the testing paper would turn to black owing to the formation of black PbS salt, indicating the generation of H₂S gas. The control experiment shows that Pb(Ac)₂ testing paper did not turn black when heating ODE without 1-octanethiol (Fig. S7b and S8 in the ESI†). The grayscale plot of the testing paper shows that the grayscale decreased with the increased temperature, indicating faster generation of H₂S gas at higher temperature (Fig. 5). Thus, the possible pathway of the sulfur released from the 1-octanethiol and transferred to Ag₂S QDs is that H₂S was in situ generated through the pyrolysis of 1-octanethiol under the synthetic conditions and the H₂S can then react with silver salts to form Ag₂S QDs.

Fig. 5 Photographs and grayscale plot of Pb(Ac)₂ testing papers for the H₂S detection after exposed to the reaction atmosphere for 30 min under different temperatures (no silver precursor in the reaction flask).

The as-prepared Ag₂S/ZnS QDs were synthesized in organic phase and have a hydrophobic surface, thus could not be directly used for the application in biological system. Therefore, Ag₂S/ZnS QDs must be transferred to water phase. The water solubilization of Ag₂S/ZnS QDs in this work was realized by coating an amphiphilic polymer shell using octylamine-modified polyacrylic acid (OPA). The PL spectra of the Ag₂S/ZnS QDs before and after transferred from chloroform to water are shown in Fig. 6a. After the phase transfer, the PL emission peak was well maintained and the PL intensity was not significantly decreased. The photographs of the oil-soluble Ag₂S/ZnS QDs and the corresponding water-soluble Ag₂S/ZnS QDs show that the Ag₂S/ZnS QDs are well dispersed in water after the phase transfer. Dynamic light scattering (DLS) measurement shows that the hydrodynamic size of the water-soluble Ag₂S/ZnS QDs is approximately 24 nm (Fig. S9 in ESI†). The photostability of the water-soluble Ag₂S/ZnS QDs was investigated. As shown in Fig. 6b, the PL intensities of the water-soluble Ag₂S/ZnS QDs were not significantly decreased under continuous illumination with mercury lamp, which satisfied the requirement of long observation times for biomedical imaging.

Fig. 6 PL spectra (a) and photographs (a, inset) of the Ag₂S/ZnS QDs before and after transferred from chloroform to water. PL intensities of the water-soluble Ag₂S/ZnS QDs after continuous illumination with mercury lamp (b).

As good potential materials for biomedical optical imaging, the toxicity of the QDs is one of the most concerned problems. The Ag₂S QDs is regarded as low-toxic QDs. In order to confirm this, the cytotoxicity of the water-soluble Ag₂S/ZnS QDs was investigated using WST-1 assay, a colorimetric assay for assessing cell viability.³⁵ The dehydrogenase in the mitochondria of living cells is capable of reducing the water soluble tetrazolium dye WST-1 to its orange-yellow formazan, while dead cells cannot realize it. The influence of Ag₂S/ZnS QDs on cell viability were tested by exposing Vero cells (normal kidney cells of an African green monkey) and MCF-7 cells (human breast adenocarcinoma cell) to Ag₂S/ZnS QDs at different concentrations from 0 to 200 μg mL⁻¹. As shown in Fig. 7, approximately 85% of Vero cells and 81% of MCF-7 cells retained viability after being exposed to the Ag₂S/ZnS QDs with concentration of 200 μg mL⁻¹ for 24 h, implying that the Ag₂S/ZnS QDs are low-cytotoxic and environmentally friendly NIR QDs.

Fig. 7 WST-1 assay on Vero cells and MCF-7 cells exposed to Ag₂S/ZnS QDs at different concentrations from 0 to 200 μg mL⁻¹ for 24 h.

Conclusions

In summary, we have developed a novel thiol-based non-injection approach for the synthesis of low-toxic NIR Ag₂S and Ag₂S/ZnS core/shell QDs. The PL QY of the Ag₂S QDs was significantly increased after coating a ZnS shell on the Ag₂S core. The oil-soluble Ag₂S/ZnS QDs could be transferred to water phase by coating an amphiphilic polymer shell. The WST-1 assay confirms that the Ag₂S/ZnS QDs are low-cytotoxic and environmentally friendly NIR QDs. In this work, alkanethiols (e.g. 1-octanethiol) were used as both ligand and sulfur source. We found the possible pathway of the sulfur released from the 1-octanethiol and transferred to Ag₂S QDs is that the H₂S was in situ generated through the pyrolysis of 1-octanethiol under the synthetic conditions and then react with silver salts to form Ag₂S QDs. The study not only provides a novel protocol for Ag₂S QDs synthesis but also promotes the understanding the formation mechanism in thiol-based non-injection synthesis of metal sulfide NCs.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant nos 21405116, 21375101, 91417301), Postdoctoral Science Foundation of China (Grant no. 2014M552074) and the Fundamental Research Fund for the Central Universities (Grant no. 2042014kf0031).

Notes and references

1. R. Weissleder, Nat. Biotechnol., 2001, 19, 316–317.
2. W. B. Edwards, B. Xu, W. Akers, P. P. Cheney, K. Liang, B. E. Rogers, C. J. Anderson and S. Achilefu, Bioconjugate Chem., 2008, 19, 192–200.
3. X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G. Sundaresan, A. M. Wu, S. S. Gambhir and S. Weiss, Science, 2005, 307, 538–544.
4. I. L. Medintz, H. T. Uyeda, E. R. Goldman and H. Mattoussi, Nat. Mater., 2005, 4, 435–446.
5. C. Zhang, X. Ji, Y. Zhang, G. Zhou, X. Ke, H. Wang, P. Tinnefeld and Z. He, Anal. Chem., 2013, 85, 5843–5849.
6. P. Zhang, S. Liu, D. Gao, D. Hu, P. Gong, Z. Sheng, J. Deng, Y. Ma and L. Cai, J. Am. Chem. Soc., 2012, 134, 8388–8391.
7. X. H. Gao, Y. Y. Cui, R. M. Levenson, L. W. K. Chung and S. M. Nie, Nat. Biotechnol., 2004, 22, 969–976.
8. E. Q. Song, Z. L. Zhang, Q. Y. Luo, W. Lu, Y. B. Shi and D. W. Pang, Clin. Chem., 2009, 55, 955–963.
9. E. Q. Song, J. Hu, C. Y. Wen, Z. Q. Tian, X. Yu, Z. L. Zhang, Y. B. Shi and D. W. Pang, ACS Nano, 2011, 5, 761–770.
10. S. L. Liu, Z. Q. Tian, Z. L. Zhang, Q. M. Wu, H. S. Zhao, B. Ren and D. W. Pang, Biomaterials, 2012, 33, 7828–7833.
11. D. Deng, Y. Chen, J. Cao, J. Tian, Z. Qian, S. Achilefu and Y. Gu, Chem. Mater., 2012, 24, 3029–3037.
12. Y. P. Du, B. Xu, T. Fu, M. Cai, F. Li, Y. Zhang and Q. B. Wang, J. Am. Chem. Soc., 2010, 132, 1470–1471.
13. P. Jiang, C.-N. Zhu, Z.-L. Zhang, Z.-Q. Tian and D.-W. Pang, Biomaterials, 2012, 33, 5130–5135.
14. P. Jiang, Z.-Q. Tian, C.-N. Zhu, Z.-L. Zhang and D.-W. Pang, Chem. Mater., 2012, 24, 3–5.
15. C.-N. Zhu, P. Jiang, Z.-L. Zhang, D.-L. Zhu, Z.-Q. Tian and D.-W. Pang, ACS Appl. Mater. Interfaces, 2013, 5, 1186–1189.
16. Y.-P. Gu, R. Cui, Z.-L. Zhang, Z.-X. Xie and D.-W. Pang, J. Am. Chem. Soc., 2012, 134, 79–82.
17. V. J. Pansare, S. Hejazi, W. J. Faenza and R. K. Prud’homme, Chem. Mater., 2012, 24, 812–827.
18. T. Y. Lim, S. Kim, A. Nakayama, N. E. Stott, M. G. Bawendi and J. V. Frangioni, Mol. Imaging, 2003, 2, 50–64.
19. D. B. Warheit, Nano Lett., 2010, 10, 4777–4782.
20. J. Gao, K. Chen, R. Xie, J. Xie, Y. Yan, Z. Cheng, X. Peng and X. Chen, Bioconjugate Chem., 2010, 21, 604–609.
21. R. G. Xie, M. Rutherford and X. G. Peng, J. Am. Chem. Soc., 2009, 131, 5691–5697.
22. Y. Zhang, Y. Zhang, G. Hong, W. He, K. Zhou, K. Yang, F. Li, G. Chen, Z. Liu, H. Dai and Q. Wang, Biomaterials, 2013, 34, 3639–3646.
23. H. Liu, W. W. Hu, F. Ye, Y. L. Ding and J. Yang, RSC Adv., 2013, 3, 616–622.
24. A. Sahu, L. J. Qi, M. S. Kang, D. N. Deng and D. J. Norris, J. Am. Chem. Soc., 2011, 133, 6509–6512.
25. B. Chen, H. Zhong, W. Zhang, Z. a. Tan, Y. Li, C. Yu, T. Zhai, Y. Bando, S. Yang and B. Zou, Adv. Funct. Mater., 2012, 22, 2081–2088.
26. L. Li, A. Pandey, D. J. Werder, B. P. Khanal, J. M. Pietryga and V. I. Klimov, J. Am. Chem. Soc., 2011, 133, 1176–1179.
27. M. Bruchez, M. Moronne, P. Gin, S. Weiss and A. P. Alivisatos, Science, 1998, 281, 2013–2016.
28. J. McBride, J. Treadway, L. C. Feldman, S. J. Pennycook and S. J. Rosenthal, Nano Lett., 2006, 6, 1496–1501.
29. S. Pokrant and K. B. Whaley, Eur. Phys. J. D, 1999, 6, 255–267.
30. J. J. Li, Y. A. Wang, W. Z. Guo, J. C. Keay, T. D. Mishima, M. B. Johnson and X. G. Peng, J. Am. Chem. Soc., 2003, 125, 12567–12575.
31. X. Peng, M. C. Schlamp, A. V. Kadavanich and A. P. Alivisatos, J. Am. Chem. Soc., 1997, 119, 7019–7029.
32. B. O. Dabbousi, J. Rodriguez-Viejo, F. V. Mikulec, J. R. Heine, H. Mattoussi, R. Ober, K. F. Jensen and M. G. Bawendi, J. Phys. Chem. B, 1997, 101, 9463–9475.
33. Z. Li, Y. J. Ji, R. G. Xie, S. Y. Grisham and X. G. Peng, J. Am. Chem. Soc., 2011, 133, 17248–17256.
34. J. W. Thomson, K. Nagashima, P. M. Macdonald and G. A. Ozin, J. Am. Chem. Soc., 2011, 133, 5036–5041.
35. S. Stolte, J. Arning, U. Bottin-Weber, M. Matzke, F. Stock, K. Thiele, M. Uerdingen, U. Welz-Biermann, B. Jastorff and J. Ranke, Green Chem., 2006, 8, 621–629.

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Footnotes

† Electronic supplementary information (ESI) available: Experimental details and additional figures as described in the text. See DOI: 10.1039/c5ra08008h

‡ These authors contributed equally.

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