Biocompatible fluorescent carbon quantum dots

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Biocompatible fluorescent carbon quantum dots prepared from beetroot extract for in vivo live imaging in C. elegans and BALB/c mice† Vikram Singh,‡a Kundan S. Rawat,‡ab Shachi Mishra,a Tanvi Baghel,c Soobiya Fatima,bc Aijaz A. John,d Navodayam Kalleti,c Divya Singh,bd Aamir Nazir,bc Srikanta K. Rathbc and Atul Goel *ab Luminescent carbon quantum dots (CQDs) prepared from aqueous beetroot extract were developed as unique fluorescent nanomaterials for in vivo live animal imaging applications. Blue (B) and green (G) emitting environmentally benign CQDs (particle size of 5 nm and 8 nm, respectively) exhibited bright fluorescence in aqueous medium and were found to be biocompatible, photostable and non-toxic in animal models. The in vivo imaging and toxicity evaluation of both CQDs were performed for the first time in the Caenorhabditis elegans (C. elegans) model, which revealed consistent fluorescence in the gut tissues of the worms without

Received 22nd February 2018, Accepted 19th April 2018

exerting any sign of toxic effects on the nematodes. The in vivo bio-distribution of G-CQDs given by tail vein injection in live BALB/c mice showed optical signals in the lower abdominal regions, mainly in the intestine,

DOI: 10.1039/c8tb00503f

and cleared from the body through faeces. The tremendous potential shown by these eco-friendly CQDs in

rsc.li/materials-b

the C. elegans and mice models advocates new hopes for greener CQD nanomaterials as diagnostic tools in the biomedical field.

Carbon quantum dots (CQDs) have emerged as a new class of environmentally benign fluorescent nanomaterials with particle size below 10 nm. They are potential candidates for numerous modern nanotechnologies and biomedical research.1 Owing to their simple one-pot synthesis, unique excitation-dependent emission, excellent photochemical stability, and high biocompatibility, these water soluble CQDs are now on the verge of becoming an alternative source to conventional organic fluorescent dyes and inorganic semiconductor QDs (mainly composed of toxic heavy metals).2–4 Based on the tuneable optical properties of CQDs with different particle sizes and surface passivating functionalities, they have been judiciously explored for chemical sensing, biosensing, photocatalysis, drug delivery systems, nanomedicine and optoelectronic nanomaterials.1,5–8 A few new lanthanide-doped nanocrystals have been synthesized as potential multifunctional bioprobes

a

Fluorescent Chemistry Lab, Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, Lucknow, 226031, India. E-mail: [email protected] b Academy of Scientific and Innovative Research, New Delhi, India c Division of Toxicology, CSIR-Central Drug Research Institute, Lucknow, 226031, India d Division of Endocrinology, CSIR-Central Drug Research Institute, Lucknow, 226031, India † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c8tb00503f ‡ These authors contributed equally to this work.

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for cell imaging applications.9–11 In the recent past, fluorescent CQDs have been explored in fixed and live cell imaging applications, however, there is a paucity of literature reports on CQDs for live animal imaging. Recently, a few reports on the biomedical applications of CQDs using zebrafish and mice as animal models have generated great enthusiasm in this upcoming field.12–14 Like the zebrafish model, C. elegans, being a small transparent soil worm or nematode, is an extremely useful experimental model for understanding the basic genetic and molecular mechanisms relevant to human development and diseases such as cancer, diabetes and neurodegenerative disorders (Alzheimer’s and Parkinson’s diseases). In spite of the tremendous scope of the C. elegans model in biomedical research, to our surprise, no-one has studied the toxicity evaluation and imaging analysis of CQDs in these systems. In this manuscript, we demonstrate a facile one-pot green synthesis of biocompatible CQDs from aqueous beetroot extract, their characterization, photophysical properties and their potential application in in vivo fluorescence confocal imaging, for the first time in the C. elegans model. Furthermore, toxicity and bio-distribution of these CQDs were successfully demonstrated through live animal imaging in normal BALB/c mice. Numerous methods have been reported for the synthesis of carbon dots from different sources such as microwave irradiation of carbohydrate,15 citric acid,16 and polyethylene glycol (PEG),17 hydrothermal treatment of chitosan,18 nitric

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acid oxidation of carbon soot prepared from candle,19 laser irradiation on graphite powder,20 a simple colloid-chemical carbonization of vitamin B121 and by microwave and strong alkali treatment of glucose.22,23 However, these methods are not conducive for estabilising a green synthesis method and also increase environmental issues. Recently, there has been a paradigm shift in the synthesis of fluorescent carbon dots using natural products such as vegetable and fruit extracts as green, cost-effective and environmentally benign sources24–27 and some of them have been explored for bioimaging applications by controlled tuning of hydrophilic and hydrophobic surface functionalities to improve cellular uptake.28 We explored a green, environmentally friendly and simple procedure for the synthesis of CQDs from beetroot aqueous extract (Beta vulgaris). The main components of aqueous beetroot extract are vitamins, betalains, sugars, and oxalic and phenolic acids, and the intense red color of beetroot is due to the presence of high concentrations of betalains (about 60% betacyanins and 40% betaxanthins).29 The synthesis of green emitting CQDs was reported via a microwave and strong alkali assisted methodology.30 We developed a new one-pot green synthesis of CQDs from aqueous beetroot extract by hydrothermal and mild acid (ortho-phosphoric acid) treatment, as shown in Scheme 1 (see the detailed synthesis procedure in the ESI†). Briefly, the aqueous beetroot extract (20 mL) was filtered using 11 mm pore size filter paper and the juice was transferred into a 50 mL Teflon-Lined autoclave and heated at 150 1C for 16 h. Subsequently, the resulting CQD mixture was purified by repeated centrifugation at 1500 rpm, 2500 rpm and 4000 rpm for 10 min each. A clear brownish solution of hydrothermally treated extract and a yellowish solution of acid-treated extract were obtained containing the concentrations of 40 mg mL 1 and 328 mg mL 1 CQDs, respectively. The synthesized water soluble beetroot-CQDs from hydrothermal and ortho-phosphoric acid treatments were examined by high resolution transmission electron microscopy (HR-TEM) to determine the morphology and core size. Both (hydrothermal and acid treated) CQDs showed a spherical morphology (Fig. 1a–d). The average core sizes of both CQDs were determined by the particle size distribution plot (inset, Fig. 1a and b), which revealed that they have an average core size of 5 nm and 8 nm, respectively. X-ray photoelectron spectroscopy (XPS) and FT-IR analyses were carried out to determine the surface functionalities. Fig. 2a shows the full XPS spectrum of the beetroot CQDs prepared by

Scheme 1 Schematic representation of the direct one-step synthesis of CQDs from beetroot extract through (a) hydrothermal and (b) H3PO4 treatment.


Journal of Materials Chemistry B

Fig. 1 TEM images of beetroot-CQDs: (a) hydrothermal treatment dispersed in water; the inset shows the particle size distribution, and (b) phosphoric acid treatment dispersed in water; the inset shows the particle size distribution; (c and d) zoomed TEM images of CQDs.

hydrothermal treatment with three peaks at 285, 397 and 534 eV attributed to C1s, N1s and O1s, respectively. The data suggested that the beetroot-CQDs are mainly composed of the elements C, N and O. Binding energy values of these CQDs were obtained from the high resolution spectrum of C1s (Fig. 2c, for O1s, N1s: Fig. S1, ESI†), which showed carbon has three different chemical environments, corresponding to CQC at 284.5 eV, C–C/C–H at 285.6 eV, and CQO at 287.9 eV. Similarly, Fig. 2b shows the full XPS spectrum of the beetroot-CQDs prepared by ortho-phosphoric acid treatment with five peaks at 285, 397, 534, 189, and 132 eV attributed to C1s, N1s, O1s, P2s, and P2p, respectively. Binding energy values of these CQDs were obtained from the high

Fig. 2 Full range XPS analysis of beetroot-CQDs prepared by (a) hydrothermal treatment and (b) phosphoric acid treatment; high resolution XPS spectra of the C1s region of the beetroot-CQDs prepared by (c) hydrothermal treatment and (d) phosphoric acid treatment.

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resolution spectrum of C1s (Fig. 2d, for O1s, N1s: Fig. S1, ESI†), which showed carbon has three different chemical environments, corresponding to CQC at 284.5 eV, C–C and C–H at 285.6 eV, and CQO at 287.6 eV. In addition, information on the functional groups on the surface of the CQDs obtained from hydrothermal and ortho-phosphoric acid treatments was also obtained from the FTIR spectra. Hydrothermally prepared CQDs showed peaks at 3400 cm 1 for the stretching vibrations of C–OH, 3019 cm 1 for the C–H stretching, 1602 cm 1 for the vibrational absorption band of CQO, 1520 cm 1 for the CQC stretching and 1422 cm 1 due to the C–C vibrations. CQDs synthesized using ortho-phosphoric acid exhibited peaks at 3401 cm 1 for the stretching vibrations of C–OH, 3020 cm 1 for C–H, 1602 cm 1 for the vibrational absorption band of CQO, 1520 cm 1 for CQC, and 1215 cm 1 for the O–C vibrations. Both the XPS and FTIR studies clearly indicated that beetroot-CQDs are functionalized with carbonyl, hydroxyl and carboxylic acid groups. The zeta potential (z) of beetroot-CQDs prepared by hydrothermal treatment and prepared by ortho-phosphoric acid treatment was found to be 1.41 mV and 3.34 mV, respectively. The negative value of zeta potential suggested that these CQDs have acid functionalities on their surface. The presence of these functional groups on both the CQDs suggests excellent solubility in water without further chemical modifications. The blue and green-emitting fluorescent CQDs are denoted as B-CQDs (via hydrothermal treatment) and G-CQDs (via orthophosphoric acid treatment) and their spectroscopic properties are shown in Fig. 3. Since the concentration of these CQDs cannot be calculated accurately, the absorbance of the CQDs was kept in the range of 0.2–0.7 at the excitation wavelength to avoid the inner filter effect. A deep red solution of 0.40 mg mL 1 B-CQDs strongly absorbed at 250–320 nm with the maximum at 282 nm due to the p–p* transition (Fig. 3a, black) and they showed blue fluorescence (inset Fig. 3a) with the emission

Fig. 3 UV-visible absorption and emission spectra of (a) B-CQDs prepared by hydrothermal treatment and (b) G-CQDs prepared by phosphoric acid treatment (lexc = 370 nm); and emission spectra at different excitations of (c) B-CQDs prepared by hydrothermal treatment and (d) G-CQDs prepared by H3PO4 treatment in aqueous medium at pH 7.2. [B-CQDs = 0.40 mg mL 1], [G-CQDs = 11 mg mL 1].

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maximum at 450 nm on excitation at a wavelength of 370 nm (Fig. 3a, blue). A yellow solution of 11 mg mL 1 G-CQDs prepared by ortho-phosphoric acid treatment strongly absorbed at 250–300 nm with the maximum at 285 nm due to the p–p* transition (Fig. 3b, black). G-CQDs emitted with green fluorescence (lmax at 520 nm) on excitation at 370 nm (inset, Fig. 3b). The excitation-dependent emission is an intrinsic property of the carbon quantum dots/nanoparticles. To explore the excitation dependent property of both CQDs, we scanned the samples from 330 to 510 nm excitation wavelength (with 20 nm interval) and collected their emission response from 300 to 650 nm (Fig. 3c and d). A red shift in the case of B-CQDs with the maximum intensity at 330 nm was observed (Fig. S2, ESI†), while in the case of G-CQDs, an increase in the intensity from 350 nm to 430 nm excitation (maximum intensity at 430 nm) with a slight red shift was observed (Fig. S2, ESI†). The relative quantum yield of B-CQDs and G-CQDs in aqueous medium was found to be 6% and 5%, respectively, by taking fluorescein dye as a standard. The photostability of these CQDs was examined by continuous UV light exposure, which revealed that the CQDs showed appreciable fluorescence even after B8 h under direct UV light exposure (Fig. S3, ESI†). To check the pH-dependent stability, these CQDs were dissolved in a solution of different pH values (2–12). B-CQDs exhibited almost stable fluorescence between pH 3 and 10, while G-CQDs showed stability between pH 2 and 12 (Fig. S4, ESI†). To measure the average fluorescence lifetime of the B-CQDs and G-CQDs, time-correlated single photon counting (TCSPC) analysis was performed. The tri-exponential function fitted fluorescence lifetime decay of the B-CQDs and G-CQDs is shown in Fig. 4. The average fluorescence lifetime of B-CQDs (Fig. 4a) and G-CQDs (Fig. 4b) was found to be 5.5 ns and 2.5 ns, respectively, at 370 nm excitation wavelength with good a w2 fitted value. Both B-CQDs and G-CQDs showed bright fluorescence under aqueous conditions with good quantum yields. Therefore, these CQDs were explored for their potential use in bioimaging applications. For this perspective, imaging studies were conducted for the first time in transparent C. elegans nematodes. The model system C. elegans has successfully been employed in various studies towards understanding the effect of xenobiotics and pharmacological agents on physiological endpoints. The model provides a facile test system for studying whether or not a test compound exerts adverse biological effects. C. elegans possesses

Fig. 4 Time-resolved luminescence decay of (a) B-CQDs (lex = 370 nm and lem = 450 nm) and (b) G-CQDs (lex = 370 nm and lem = 520 nm) in aqueous medium with good fit.


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an intricate physiology that exhibits altered functioning in response to a change in the micro-environment of the worms. Its nervous system functions such that even a subtle physical or chemical stress affects the neurotransmission in a way that the worm posture, its sinusoidal wave pattern, locomotion and its body bending get altered as a result of an imbalance between excitatory and inhibitory neurotransmissions. To the best of our knowledge, toxicity and imaging analyses of carbon quantum dots have never been reported in C. elegans prior to this study. In our studies, the C. elegans nematodes exposed to none (as control) or various CQDs (B-CQDs and G-CQDs) were assayed for effects on the overall behaviour and morphology (see the ESI,† for details). At the tested concentration, both the CQDs did not exert any deleterious effects on the nematodes as the behaviour, development and locomotion did not exhibit any changes under treatment conditions when compared to that of the worms from the control group. A careful microscopic examination of the worms also revealed that the pattern of body bends of the worms that is typically affected even under subtle toxic effects did not reveal any change, hence leading us to conclude that the tested CQDs did not exert any toxic effects on the worms. After feeding the nematodes with both CQDs, we went on to carry out confocal fluorescence microscopy towards analysis of their uptake by the worms and the systemic absorption across the gut tissues. The worms, mounted onto glass slides under the influence of sodium azide, were observed for fluorescence using specific excitation/emission filters. We observed that the control worms did not exhibit fluorescence (Fig. S5, ESI†), the worms treated with the B-CQD (Fig. 5a and b) group exhibited a consistent fluorescence in the gut tissue right from the region behind the posterior pharyngeal bulb to the anal orifice. The nematodes treated with G-CQDs (Fig. 5c and d) exhibited a strong fluorescence within the gut and also across it, as it was observed that tissue around phasmid processes around the tail region and possibly neural cells attached to the anal sphincter also exhibited fluorescence, revealing that the compound was systemically absorbed into the worm body and could be localized to non-gut cells as well (see the movie clip, ESI†).


After obtaining convincing results that these CQDs are nontoxic to soil C. elegans nematodes and remain fluorescent in the body of worms, we further explored the application of these CQDs in live animal fluorescence imaging in BALB/c mice. The fluorescent, water soluble G-CQDs were examined for in vivo bioimaging applications, employing the non-invasive live-animal fluorescence imaging technique. A CQD solution (50 mL) was intravenously injected into BALB/c mice through the tail vein for whole-body circulation. Three mice were used at each time point after injection. The mice were imaged with excitation at 430 nm using an in vivo fluorescence-imaging system (an IVIS spectrum). Initially, the mice were imaged for 15 min at 5 min intervals post injection. The B-CQDs did not show fluorescence in mice at 430 nm excitation, while the G-CQDs showed the timedependent in vivo bio-distribution profile of carbon quantum dots in mice. The difference in bioimaging efficiency of both CQDs can be attributed to different surface functionalities present on the B-CQDs and G-CQDs as indicated by different zeta potential values of 1.41 mV and 3.34 mV, respectively. In the case of G-CQDs, no significant difference was observed between 5 and 15 min post injection. After 5 min, an optical signal could be detected in different parts of the body, mainly the stomach and intestinal regions (Fig. 6a). At B24 h post injection, appreciable fluorescence was observed in the intestinal region only, indicating that the CQDs underwent in vivo uptake through circulation, followed by facile excretion from the body through the biliary pathway. This was further confirmed by the clear fluorescence signals observed in the collected faeces. The biodistribution of CQDs was also demonstrated using ex vivo fluorescence imaging, which was performed on the excised major organs, when the mice were sacrificed. Only the dissected stomach and intestine exhibited meaningful fluorescence from the carbon dots (Fig. 6b), consistent with the biliary excretion pathway. No noticeable sign of toxicity from these CQDs to the treated animals was observed, even after two weeks. These results implied that the CQDs prepared from beetroot extract are non-toxic and useful nanomaterials as diagnostic tools and/or contrast agents for biomedical applications.

Fig. 5 Confocal microscopy images of wild-type (N2) C. elegans treated with (a and b) B-CQDs and (c and d) G-CQDs (1.5 mg mL 1, 24 h). Blue channel: lex = 405 nm, lem = 460–510 nm. Green channel: lex = 405 nm, lem = 500–550 nm band pass. Scale bar: 25 and 100 mm.


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Fig. 6 (a) In vivo fluorescence images of intravenously injected G-CQDs (50 mL) at 10 min and 24 h post-injection and (b) ex vivo fluorescence images of major organs of mice sacrificed at 24 h post-injection. (lex = 430 nm, lem = 520 20 nm).

In summary, we have successfully synthesized fluorescent CQDs in aqueous medium from beetroot extract through a green, simple and cost-effective methodology. The as-synthesized CQDs with carbonyl, hydroxyl and carboxylic acid functionalities at the surface exhibited a spherical morphology with average particle sizes of 4–8 nm, excitation-dependent emission, good photostablity, and good biocompatibility in aqueous medium. These CQDs were successfully demonstrated for the first time in the soil nematodes model C. elegans, which revealed stable and consistent fluorescence in the body of the worms without exerting any side effects. Furthermore, the G-CQDs have shown great potential as fluorescent nanomaterials for in vivo non-invasive live animal imaging in BALB/c mice. This study thus opens up new avenues for the development of greener and biocompatible CQDs in aqueous medium for their applications as diagnostics and theranostics in the biomedical research field.

Conflicts of interest There are no conflicts to declare.

Acknowledgements AG thanks the Department of Atomic Energy (DAE-SRC) for Outstanding Investigator Award (21/13/2015-BRNS/35029). The authors thank the SERB (PDF/2016/000208), CSIR and UGC, New Delhi for research fellowships. We acknowledge Mrs Rima A. Sarkar for the confocal imaging of C. elegans slides and SAIFCDRI for providing spectral data. We are grateful to Prof. A. K. Mishra, IIT Madras, Chennai, for time-resolved fluorescence measurements. HR-TEM and XPS analyses were carried out at IIT, Kanpur. The CDRI Communication number is 9674.

Notes and references 1 S. Y. Lim, W. Shen and Z. Gao, Chem. Soc. Rev., 2014, 44, 362–381. 2 J. C. G. Esteves da Silva and H. M. R. Gonçalves, TrAC, Trends Anal. Chem., 2011, 30, 1327–1336. 3 S. N. Baker and G. A. Baker, Angew. Chem., Int. Ed., 2010, 49, 6726–6744.

3370 | J. Mater. Chem. B, 2018, 6, 3366--3371

4 F. R. Baptista, S. A. Belhout, S. Giordani and S. J. Quinn, Chem. Soc. Rev., 2015, 44, 4433–4453. 5 Y. Wang and A. Hu, J. Mater. Chem. C, 2014, 2, 6921. 6 S. Qu, D. Zhou, D. Li, W. Ji, P. Jing, D. Han, L. Liu, H. Zeng and D. Shen, Adv. Mater., 2016, 28, 3516–3521. 7 K. Jiang, S. Sun, L. Zhang, Y. Lu, A. Wu, C. Cai and H. Lin, Angew. Chem., Int. Ed., 2015, 54, 5360–5363. 8 Z. Gan, H. Xu and Y. Hao, Nanoscale, 2016, 8, 7794–7807. 9 (a) F. Li, C. Li, X. Liu, Y. Chen, T. Bai, L. Wang, Z. Shi and S. Feng, Chem. – Eur. J., 2012, 18, 11641–11646; (b) F. Li, C. Li, J. Liu, X. Liu, L. Zhao, T. Bai, Q. Yuan, X. Kong, Y. Han, Z. Shi and S. Feng, Nanoscale, 2013, 5, 6950; (c) F. Li, C. Li, X. Liu, T. Bai, W. Dong, X. Zhang, Z. Shi and S. Feng, Dalton Trans., 2013, 42, 2015–2022. 10 H. Wang, D. Tu, J. Xu, X. Shang, P. Hu, R. Li, W. Zheng, Z. Chen and X. Chen, J. Mater. Chem. B, 2017, 5, 4827–4834. 11 G. Wang, Q. Peng and Y. Li, Acc. Chem. Res., 2011, 44, 322–332. 12 Y.-F. Kang, Y.-H. Li, Y.-W. Fang, Y. Xu, X.-M. Wei and X.-B. Yin, Sci. Rep., 2015, 5, 11835. 13 S.-T. Yang, L. Cao, P. G. Luo, F. Lu, X. Wang, H. Wang, M. J. Meziani, Y. Liu, G. Qi and Y.-P. Sun, J. Am. Chem. Soc., 2009, 131, 11308–11309. 14 L. Cao, S. T. Yang, X. Wang, P. G. Luo, J. H. Liu, S. Sahu, Y. Liu and Y. P. Sun, Theranostics, 2012, 2, 295–301. 15 X. Wang, K. Qu, B. Xu, J. Ren and X. Qu, J. Mater. Chem., 2011, 21, 2445. 16 X. Zhai, P. Zhang, C. Liu, T. Bai, W. Li, L. Dai and W. Liu, Chem. Commun., 2012, 48, 7955–7957. 17 A. Jaiswal, S. S. Ghosh and A. Chattopadhyay, Chem. Commun., 2012, 48, 407. 18 X. Liu, J. Pang, F. Xu and X. Zhang, Sci. Rep., 2016, 6, 31100. 19 S. C. Ray, A. Saha, N. R. Jana and R. Sarkar, J. Phys. Chem. C, 2009, 113, 18546–18551. 20 S.-L. Hu, K.-Y. Niu, J. Sun, J. Yang, N.-Q. Zhao and X.-W. Du, J. Mater. Chem., 2009, 19, 484. 21 S. K. Bhunia, N. Pradhan and N. R. Jana, ACS Appl. Mater. Interfaces, 2014, 6, 7672–7679. 22 H. Li, X. He, Y. Liu, H. Huang, S. Lian, S. T. Lee and Z. Kang, Carbon, 2011, 49, 605–609. 23 V. Singh and A. K. Mishra, Sens. Actuators, B, 2016, 227, 467–474. 24 S. Y. Lim, W. Shen and Z. Gao, Chem. Soc. Rev., 2014, 44, 362–381.


View Article Online


Paper

25 V. Sharma, P. Tiwari and S. M. Mobin, J. Mater. Chem. B, 2017, 5, 8904–8924. 26 R. Wang, K.-Q. Lu, Z.-R. Tang and Y.-J. Xu, J. Mater. Chem. A, 2017, 5, 3717–3734. 27 V. Singh and A. K. Mishra, J. Mater. Chem. C, 2016, 4, 3131–3137. 28 (a) Y. Zhang, C. Zhang, J. Chen, L. Liu, M. Hu, J. Li and H. Bi, ACS Appl. Mater. Interfaces, 2017, 9, 25152–25163; (b) J. Chen, X. Zhang, Y. Zhang, W. Wang, S. Li, Y. Wang,



M. Hu, L. Liu and H. Bi, Langmuir, 2017, 33, 10259–10270; (c) X. Teng, C. Ma, C. Ge, M. Yan, J. Yang, Y. Zhang, P. C. Morais and H. Bi, J. Mater. Chem. B, 2014, 2, 4631. 29 J. Wruss, G. Waldenberger, S. Huemer, P. Uygun, P. Lanzerstorfer, ¨glinger and J. Weghuber, J. Food Compos. ¨ller, O. Ho U. Mu Anal., 2015, 42, 46–55. 30 G. R. S. Andrade, S. S. L. Costa, C. C. Nascimento and I. F. Gimenez, MRS Adv., 2016, 1, 1371–1376.

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