Fluorometric detection of tyrosine and cysteine using graphene quantum dots

Abstract

Herein, a facile fluorescence method has been developed for the detection of tyrosine (Tyr) and cysteine (Cys) based on graphene quantum dots (GQDs) as a sensitive probe. Tyr could be oxidized to dopaquinone by tyrosinase catalyzation, which could efficiently quench the fluorescence of GQDs through an electron transfer process. However, Cys could act as tyrosinase inhibitors and reacted with the generated dopaquinones to give their polyphenol precursors. Therefore, the addition of Cys would make the fluorescence of above system recover. Under optimal conditions, a linear correlation was established between the fluorescence intensity and the concentration of Tyr in the range of 1.0–160 μmol L⁻¹ with a detection limit of 0.5 μmol L⁻¹. The linear range for Cys was from 25 to 2000 μmol L⁻¹ with a detection limit of 5.0 μmol L⁻¹. The fluorescence changes of the as-prepared GQDs as a function of the structure transformation during the interaction process provides a potential fluorescence based tool for monitoring Tyr and Cys.

---

Introduction

Graphene-based materials have attracted tremendous attention in optoelectronics, material science and biology for their extraordinary electronic properties, biocompatibility, and abundance of raw materials in nature.^1,2 Over the past decades, graphene quantum dots (GQDs), as a new member of the graphene family, have become the most famous fluorescent carbon nanomaterials.^3,4 Compared with conventional semiconductor quantum dots, GQDs afford considerably lower toxicity, excellent biocompatibility, stable photoluminescence, high electrical conductivity and thermal conductivity, making them promising for application in the fields of cellular imaging, drug delivery, electrochemical and fluorescent biosensors.^5–8 Over the past few years, most researchers have focused their attentions on the synthesis as well as electrochemical and catalysis properties of GQDs, while investigations on the analytical applications are still very initial.^9,10 It is just the beginning to employ the GQDs as high-efficiency fluorescence probes for the biological application.^11,12 To date, some researches about GQDs based fluorescence probes have been done to explore sensing systems for metal ions,^12–15 glucose,^16,17 melamine,¹⁸ trypsin¹⁹ and others.^20–22

Tyrosinase, an enzyme that playing the central role in fresh-cut browning, is an oligomer metallo-enzyme with an active site containing two copper ions, each coordinated to three histidine residues.^23,24 Tyrosinase catalyzes the oxidation of phenolic substrates by molecular oxygen to give the colored highly reactive o-quinones that can undergo irreversible nonenzyme-catalyzed polymerization to melanin pigments.^24,25 Reducing the undesirable browning process has been widely studied since it lowers the quality and nutritive value of fresh-cut vegetables. Most mechanisms of inhibiting the browning process are either inhibiting the activity of tyrosinase or converting quinones to the colorless adduct. Cysteine (Cys) is well known tyrosinase inhibitors, which could react with quinones to give uncolored catechols.^26–28 Long's group monitored the interaction process of dopamine to form dopamine quinone and the subsequent cysteine residue using dopamine functionalized CdTe/ZnS quantum dots.

In this paper, we proposed a simple, high selective and sensitive fluorescence probes for tyrosine (Tyr) and Cys detection based on the conversion between colorless phenolic compounds and colored o-quinones. The phenolic hydroxyl group of the Tyr could be oxidized to quinone in the presence of tyrosinase and the fluorescence of GQDs could be efficiently quenched via an electron transfer process. Also, the change of the fluorescence intensity of GQDs is proportional to the concentration of tyrosine. Cys which act as tyrosinase inhibitors could react with the resulted dopaquinones to give their polyphenol precursors. The addition of Cys would make the fluorescence recovered and the recovery efficiency is proportional to the concentration of Cys. Therefore, the fluorescence intensity of GQDs is changed as a function of the structure transformation of tyrosine during the interaction process, which provides a potential fluorescence based tool for monitoring Tyr and biothiols.

Experimental

Reagents and chemicals

L-Tyrosine (Tyr), L-cysteine (Cys), L-alanine (Ala), L-serine (Ser), L-threonine (Thr), L-aspartic acid (Asp), L-glycine (Gly), L-lysine (Lys), L-arginine (Arg) and L-histidine (His) were obtained from Sigma-Aldrich Chemical Co. Graphite powder was attained from Sinopharm Chemical Reagent Co. Ltd. (China). Tyrosinase (500 U mg⁻¹), Na₂HPO₄ and NaH₂PO₄ were purchased from Beijing Dingguo Changsheng Biotechnology Co. Ltd. All chemicals used were of analytical reagent grade without further purification. The water used in all experiments had a resistivity higher than 18 MΩ cm⁻¹.

Instruments

Atomic Force Microscopy (AFM) measurements were performed on a NanoScope Multimode AFM (Veeco, USA) using the tapping mode AFM. UV-vis spectrum was obtained with a Shimadzu 3100 UV-VIS-NIR Recording Spectrophotometer. Dynamic Light Scattering (DLS) experiments were carried out with Malvern Instrument Zetasizer Nano ZS equipped with a He–Ne laser (633 nm, 4 mW) and an avalanche photodiode detector. The FT-IR spectra were recorded on a Nicolet 400 Fourier transform infrared spectrometer. Fluorescence measurements were performed on a Shimadzu RF-5301 PC spectrofluorophotometer with a 1 cm path-length quartz cuvette.

Synthesis of graphene oxide

GO was prepared by the improved Hummer's method and characterized in our previously reported work.²⁹ Briefly, graphite powder (2.0 g) was added to 98% H₂SO₄ (12 mL) consisting of K₂S₂O₈ (2.5 g) and P₂O₅ (2.5 g). The mixture was reacted at 80 °C for 4.5 h and then diluted with 0.5 L water. After being filtered and washed with water to remove the residual acid, the product was dried under ambient conditions overnight. This pre-oxidized graphite was stirred in 98% H₂SO₄ (120 mL), KMnO₄ (15 g) was gradually added while keeping the temperature above 20 °C. This mixture was stirred at 35 °C for 30 min and 90 °C for another 90 min. Afterwards, the mixture was diluted with water (250 mL) and kept at 105 °C for 25 min. After the resulting mixture was stirred for 2 h, the reaction was terminated by addition of 0.7 L water and 20 mL of 30% H₂O₂. For purification, the mixture was filtered and washed with 1 : 10 HCl aqueous solution and water many times. Finally, the product was further purified by dialysis for 1 week to remove the remaining metal species. The obtained dispersion was centrifuged at 10 000 rpm to remove the unexfoliated GO. The supernatant solution was collected and freezed dry. The GO powder was redispersed in water to obtain 0.5 mg mL⁻¹ GO stock solution.

Synthesis of graphene quantum dots

Graphene quantum dots were synthesized according to the previous works.^30,31 Briefly, 0.3 g graphene oxide were added into the mixture of 60 mL H₂SO₄ (98%) and 20 mL HNO₃. The solution was sonicated for 2 hours and stirred for 24 hours at 80 °C. The mixture was cooled and diluted with 600 mL water and the pH value was adjusted to 7.0 with NaOH solution. The final product solution was further dialyzed in a dialysis bag (retained molecular weight: 3500 Da) for 7 days.

Fluorescence experiments

For Tyr detection, 100 μL aqueous solution of the as-prepared GQDs (0.5 mg mL⁻¹) and 80 μL tyrosinase (2.0 mg mL⁻¹) were firstly mixed. Then Tyr with different concentrations were added into the above solution and allowed to react for 1 h before measurements. Similarly, for Cys detection, 100 μL GQDs, 20 μL Tyr (10 mmol L⁻¹) and 80 μL tyrosinase (2.0 mg mL⁻¹) were incubated for 1 h, then different concentrations of Cys were added respectively. The final volume of every sample was 2 mL. The fluorescence spectra were recorded in the 500–740 nm emission wavelength range with the excitation of 480 nm, and the emission and excitation slits were both set at 10 nm.

Results and discussion

Characterization of GO and GQDs

The graphene oxide (GO) and graphene quantum dots (GQDs) were synthesized according to the previously described method.³¹ To explore the properties of the GO and GQDs, we performed the atom force microscope (AFM) to investigate GO and GQDs morphology and the results were shown in Fig. 1a and b. The height of the GO sheet was measured to be ∼1.2 nm, assuming the single atomic layer motif. More attractively, all GO sheets were cut into GQDs (1–3 graphene layers) and the sizes of GQDs were distributed in the range of 20–40 nm. As illustrated in Fig. 2a, the dynamic light scattering (DLS) showed the diameters of GQDs were about 38 nm, which is in accordance with the results of AFM image. Furthermore, the UV-vis maximum absorption of GO was around 230 nm while the GQDs showed the UV-vis absorption peak around 300 nm (shown in Fig. 2b). As shown in Fig. 2c, the fluorescence emission of GQDs was about 540 nm with the excitation wavelengths at 480 nm. The FT-IR spectrum of GO (Fig. 2d) showed a peak corresponding to C–O stretching of epoxy groups at 1112 cm⁻¹. The emerging peak at 1400 cm⁻¹ was attributed to C–H stretching vibrations. The peaks at 1620 and 1720 cm⁻¹ were associated with the vibrations of C=C and C=O bonds, respectively. The broad adsorption band between 3000 and 3700 cm⁻¹ can be assigned to O–H stretching vibration of hydroxyl groups of GO. Compared with the FT-IR spectra of GO, the peaks of GQDs such as C=C and C–O increased in intensity, resulting from the formation of more hydrophilic functional groups such as epoxyl, carboxyl and hydroxyl groups on the surfaces of GQDs. Also the presence of these groups made them soluble in water.

Fig. 1 The atomic force microscopy image of GO (a) and GQDs (b).

Fig. 2 The DLS (a), UV-vis (b), fluorescence spectra (c) and FT-IR (d) of GO and GQDs.

Design of the florescence sensing system

In this work, a novel optical sensing method based on the conversion between colorless phenolic compounds and colored o-quinones for detection of tyrosine (Tyr) and cysteine (Cys) was developed via the fluorescent signal change of GQDs. Scheme 1 illustrated the principle of our fluorescence sensing system for the Tyr and biothiols determination. Tyrosine could be oxidized to dopaquinone by tyrosinase catalyzation, which could efficiently quench the fluorescence of GQDs. Cys, which act as tyrosinase inhibitors, could also react with the resulted dopaquinones to give their polyphenol precursors. Therefore, the addition of Cys would make the fluorescence recovered and the recovery efficiency is proportional to the concentration of Cys.

Scheme 1 Schematic illustration of the GQDs sensing system for the detection of Tyr and Cys.

The fluorescence spectra of GQDs sensing system in the presence or absence of target analytes were shown in Fig. 3a. When the solution of GQDs just mixed with Tyr or tyrosinase respectively, no obvious GQDs fluorescence intensity changes were observed, which indicated that neither Tyr nor tyrosinase could quench the fluorescence intensity of GQDs. However, the fluorescence of GQDs was effectively quenched in the presence of tyrosinase and tyrosine. These results indicated that the fluorescence quenching was resulted from the product of tyrosinase catalyzed reaction. Meanwhile, the fluorescence recovery could be observed remarkably after the addition of Cys. The UV-vis spectra were used to investigate the change of oxidation and inhibition process. From Fig. 3b, it can be seen that the UV-vis maximum absorption of Tyr was around 275 nm while the absorption peaks of dopaquinone were about 305 and 476 nm. After addition of the Cys, the absorption peaks changed from 305 nm to 288 nm. At the same time, the absorption peak at 476 nm was obviously decreased. The results indicated that the change of fluorescence intensity was resulted from the conversion between colorless phenolic compounds and colored o-quinones.

Fig. 3 The fluorescence spectra (a) and the UV-vis absorption (b) of GQDs sensing system. A: GQDs; B: GQDs + Tyr; C: GQDs + tyrosinase; D: GQDs + tyrosinase + Tyr; E: GQDs + tyrosinase + Tyr + Cys.

Optimization of the fluorescence sensing system

In order to optimize the conditions for Tyr and Cys detection, we studied the effects of incubation time, the concentration of tyrosinase, pH and temperature on the fluorescence of GQDs. Fig. 4A showed the fluorescence intensity changes of GQDs as a function of the incubating time that tyrosinase catalyzed oxidation of tyrosine. It could be found that the fluorescence intensity of GQDs were linear decreased with the increasing of the incubation time when the concentrations of tyrosinase were 5.0 and 10 U mL⁻¹. After increasing the concentration of tyrosinase to 40 U mL⁻¹, the fluorescence was rapidly decreased with the incubation time increased from 0 to 30 min and then kept slowly changing after 30 min. At the same time, we could obtain a relationship between the concentration of tyrosinase and the fluorescence quenching of GQDs. As shown in Fig. 4B, the quenching effect was enhanced with the increasing quantity of tyrosinase, and kept slowly changing at 30 U mL⁻¹ of tyrosinase. The fluorescence intensity of the mixture reached a plateau after 30 U mL⁻¹ of tyrosinase added. Thus in order to realize the effectively quenching, we chose 1 h, 40 U mL⁻¹ of tyrosinase as the optimum incubating conditions. We further studied the incubation time of the inhibition process. As shown in Fig. 4C, the fluorescence intensity of the sensing system increased with the increasing of the incubation time and reaching a plateau within 4 h at room temperature. In the following experiments, the incubation time of 4 h at room temperature for the inhibition process was adopted.

Fig. 4 The effect of the incubating time with different concentrations of tyrosinase (1–6 represented the concentrations of tyrosinase of 0, 5.0, 10, 20, 30, 40 U mL⁻¹, respectively.) (A) and the concentrations of tyrosinase (B) on the oxidation process; (C) the influence of the incubation time on the inhibition process; (D) the effect of pH on the fluorescence intensity ratio I/I₀ of GQDs sensing system. I₀ and I were the fluorescence intensity of GQDs sensing system in the absence and presence of tyrosine, respectively.

For the catalytic activity of tyrosinase towards a series of tyrosine depended on the pH value, we therefore studied the effect of pH on the tyrosinase-catalyzed oxidation process. From Fig. 4D, it can be found that the fluorescence intensity ratio I/I₀ (I₀ and I were the fluorescence intensity of GQDs sensing system in the absence and presence of tyrosine, respectively) was rapidly decreased in the pH range of 5.5–7.0, then the remarkable increasing of fluorescence intensity ratio was observed in the pH range of 7.0–9.0 and the largest quenching effect appeared at pH 7.0, which is also in accordance with the previous reported work. The result indicates that the quenching effect on the fluorescence intensity was much greater in neutral medium than that in acidic or alkaline medium. So we further selected the pH value of 7.0 in the following experiments.

As we know, tyrosinase is also sensitive to temperature, so we investigated the effects of temperature on the oxidation and inhibition process, respectively. Fig. 5A showed the effect of temperature on the tyrosinase catalytic process, we found that the fluorescence intensity ratio I/I₀ decreased rapidly with the increasing of time when the temperature was lower than 37 °C and could also reach the balance within 60 min. The change of temperature just altered the reaction rate but not the quenching efficiency. However, the fluorescence intensity ratio I/I₀ decreased more slowly at 45 °C due to the inactivation of tyrosinase at higher temperature. Fig. 5B shows the change of the quenching efficiency after incubating 60 min, it could be more intuitive to see that the quenching efficiency was lower at 45 °C on the oxidation process. The effect of temperature on the inhibition process is shown in Fig. 5C, the change of fluorescence intensity ratio I/I₀ has been enhanced after addition of Cys when the temperature below 25 °C. But there are two turning points at 37 (10 and 180 min) and 45 °C (10 and 90 min). The decrease in recovery efficiency at the first turning point was due to the oxidation of product at high temperature. The reason for the increase of the recovery efficiency at second turning points was the formation of melanin. And we found that the turning point appeared earlier at higher temperature. Fig. 5D was the changes of fluorescence intensity ratio I/I₀ after incubating 4 h and the maximum fluorescence recovery efficiency was obtained at 25 °C. Therefore, we choose 25 °C as the optimal reaction temperature for the oxidation and inhibition process.

Fig. 5 The change of fluorescence intensity ratio I/I₀. The oxidation process under different incubating time at 4, 18, 25, 37 and 45 °C (A) and at different temperature after 60 min incubation (B). The inhibition process under different incubating time at 4, 18, 25, 37 and 45 °C (C) and at different temperature after 4 h incubation (D). I₀ and I were the fluorescence intensity of GQDs sensing system in the absence and presence of target analytes, respectively.

Tyr or Cys detection

To further study the performance of the sensing system for Tyr or Cys detection, we investigated the fluorescence emission spectra of this system with different concentrations of Tyr or Cys under the optimized conditions. As shown in Fig. 6, the fluorescence intensity of sensing system increased upon the increasing concentration of Tyr from 0 to 300 μmol L⁻¹. The inset of Fig. 6 showed a good linear relationship between the fluorescence intensity ratio I₀/I (I₀ and I were the fluorescence intensity of the sensing system in the absence and presence of Tyr, respectively) and the concentrations of Tyr in the range of 1.0–160 μmol L⁻¹ with a correlation coefficient R² = 0.997. The linear regression equation was I₀/I = 1.019 + 0.009 × C_Tyr (μmol L⁻¹), and the detection limit for Tyr was 0.5 μmol L⁻¹ based on 3σ rule, the relative standard deviation (RSD) for six replicate measurements of 5.0 μmol L⁻¹ Tyr was 3.2%. The fluorescence spectra of the sensing system with or without Cys were shown in Fig. 7, and the inset of Fig. 7 revealed a good linear correlation between the fluorescence intensity ratio I/I₀ (I₀ and I were the fluorescence intensity of the sensing system in the absence and presence of Cys, respectively) and the concentration of Cys in the range of 25–2000 μmol L⁻¹. The linear regression equation for Cys was as follows: I/I₀ = 1.075 + 3.904 × 10⁻⁴ C_Cys (μmol L⁻¹) (R² = 0.996), and Cys could be detected as low as 5.0 μmol L⁻¹. The RSD for six replicate measurements of 100 μmol L⁻¹ Cys were 4.8%, indicating the excellent repeatability and precision of the proposed detection method.

Fig. 6 Fluorescence emission spectra of GQDs sensing system with different concentrations of Tyr (0, 1.0, 3.0, 10, 20, 30, 55, 80, 100, 130, 160, 230 and 300 μmol L⁻¹), the inset showed the calibration curve between the fluorescence intensity ratio I₀/I and the concentration of Tyr in the range of 1.0 to 160 μmol L⁻¹. I₀ and I were the fluorescence intensity of the sensing system in the absence and presence of Tyr, respectively.

Fig. 7 Fluorescence emission spectra of GQDs sensing system with different concentrations of Cys (0, 25, 250, 500, 750, 1000, 1250, 1500, 1750, 2000, 3000, 4000 and 5000 μmol L⁻¹), the inset showed the calibration curve between the fluorescence intensity ratio I/I₀ and the concentration of Cys in the range of 25 to 2000 μmol L⁻¹. I₀ and I were the fluorescence intensity of the sensing system in the absence and presence of Cys, respectively.

Interference study

To further testify the selectivity of the present fluorescence method, we investigated the fluorescence response of the sensing system to other interfering substances included alanine (Ala), serine (Ser), threonine (Thr), aspartic acid (Asp), glycin (Gly), lysine (Lys), arginine (Arg) and histidine (His). Blank represented the fluorescence intensity of the sensing system for Tyr and Cys detection without target analytes, respectively. The concentrations of coexisting substances were all 2000 μmol L⁻¹. Fig. 8 showed the changes in the fluorescence intensity ratio I/I₀ of the detection system for Tyr (I₀ and I were the fluorescence intensity of the detection system in the absence and presence of Tyr, respectively) and Cys (I₀ and I referred to the fluorescence intensity of the sensing system in the absence and presence of Cys, respectively) with the addition of various substances. The results demonstrated that no obvious influences were found both for Tyr and Cys detection system, suggesting that this proposed strategy exhibited excellent selectivity for Tyr and Cys determination.

Fig. 8 The interference of potentially interfering substances on the determination sensing system for Tyr and Cys determination, including alanine (Ala), serine (Ser), threonine (Thr), aspartic acid (Asp), glycin (Gly), lysine (Lys), arginine (Arg) and histidine (His).

Conclusion

In summary, we have developed a GQDs based fluorescence method for Tyr and Cys detection. Tyr could be oxidized to dopaquinone by tyrosinase catalyzation, which could efficiently quench the fluorescence of GQDs through an electron transfer process. But Cys could act as tyrosinase inhibitors and reacted with the resulted dopaquinones to give their polyphenol precursors. Therefore, the addition of Cys would make the fluorescence recovered. Under the optimized conditions, good linear relationships were obtained between the fluorescence intensity ratio and the concentrations of Tyr or Cys in the range of 1.0–160 or 25–2000 μmol L⁻¹, respectively. Also, the interference study showed the proposed fluorescence method exhibited excellent selectivity for Tyr and Cys. All these results indicated our proposed fluorescence method showed the potential application for the detection of Tyr and Cys in biological samples.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (No. 21075050, No. 21275063), the Science and Technology Development project of Jilin province, China (No. 20150204010GX).

References

1. K. S. Novoselov, A. K. Geim, S. V. Morozov, D. Jiang, Y. Zhang, S. V. Dubonos, I. V. Grigorieva and A. A. Firsov, Science, 2004, 306, 666–669.
2. L. Liao, Y. C. Lin, M. Bao, R. Cheng, J. Bai, Y. Liu, Y. Qu, K. L. Wang, Y. Huang and X. Duan, Nature, 2010, 467, 305–308.
3. Y. Li, Y. Hu, Y. Zhao, G. Q. Shi, L. E. Deng, Y. B. Hou and L. T. Qu, Adv. Mater., 2011, 23, 776–780.
4. L. A. Ponomarenko, F. Schedin, M. I. Katsnelson, R. Yang, E. W. Hill, K. S. Novoselov and A. K. Geim, Science, 2008, 320, 356–358.
5. S. J. Zhu, J. H. Zhang, C. Y. Qiao, S. J. Tang, Y. F. Li, W. J. Yuan, B. Li, L. Tian, F. Liu, R. Hu, H. N. Gao, H. T. Wei, H. Zhang, H. C. Sun and B. Yang, Chem. Commun., 2011, 47, 6858–6860.
6. X. Wang, X. Sun, J. Lao, H. He, T. Cheng, M. Wang, S. Wang and F. Huang, Colloids Surf., B, 2014, 122, 638–644.
7. J. Ju and W. Chen, Anal. Chem., 2015, 87, 1903–1910.
8. Y. Dong, G. Li, N. Zhou, R. Wang, Y. Chi and G. Chen, Anal. Chem., 2012, 84, 8378–8382.
9. L. Lin, X. Song, Y. Chen, M. Rong, T. Zhao, Y. Wang, Y. Jiang and X. Chen, Anal. Chim. Acta, 2015, 869, 89–95.
10. H. Razmi and R. Mohammad-Rezaei, Biosens. Bioelectron., 2013, 41, 498–504.
11. F. Wang, Z. Gu, W. Lei, W. Wang, X. Xia and Q. Hao, Sens. Actuators, B, 2014, 190, 516–522.
12. H. Sun, N. Gao, L. Wu, J. Ren, W. Wei and X. Qu, Chem.–Eur. J., 2013, 19, 13362–13368.
13. Y. Q. Dong, W. R. Tian, S. Y. Ren, R. P. Dai, Y. W. Chi and G. N. Chen, ACS Appl. Mater. Interfaces, 2014, 6, 1646–1651.
14. J. Ju and W. Chen, Biosens. Bioelectron., 2014, 58, 219–225.
15. R. Z. Zhang and W. Chen, Biosens. Bioelectron., 2014, 55, 83–90.
16. Y. Z. He, X. X. Wang, J. Sun, S. F. Jiao, H. Q. Chen, F. Gao and L. Wang, Anal. Chim. Acta, 2014, 810, 71–78.
17. Z. B. Qu, X. Zhou, L. Gu, R. Lan, D. Sun, D. Yu and G. Shi, Chem. Commun., 2013, 49, 9830–9832.
18. L. Li, G. Wu, T. Hong, Z. Yin, D. Sun, E. S. Abdel-Halim and J. J. Zhu, ACS Appl. Mater. Interfaces, 2014, 6, 2858–2864.
19. X. Li, S. Zhu, B. Xu, K. Ma, J. Zhang, B. Yang and W. Tian, Nanoscale, 2013, 5, 7776–7779.
20. L. Fan, Y. Hu, X. Wang, L. Zhang, F. Li, D. Han, Z. Li, Q. Zhang, Z. Wang and L. Niu, Talanta, 2012, 101, 192–197.
21. J. J. Liu, X. L. Zhang, Z. X. Cong, Z. T. Chen, H. H. Yang and G. N. Chen, Nanoscale, 2013, 5, 1810–1815.
22. Z. S. Qian, X. Y. Shan, L. J. Chai, J. J. Ma, J. R. Chen and H. Feng, Nanoscale, 2014, 6, 5671–5674.
23. T. S. Chang, Int. J. Mol. Sci., 2009, 10, 2440–2475.
24. R. N. Gacche, A. M. Shete, N. A. Dhole and V. S. Ghole, Indian J. Chem. Technol., 2006, 13, 459–463.
25. R. Yoruk and M. R. Marshall, J. Food Biochem., 2003, 27, 361–422.
26. H. M. Ali, A. M. El-Gizawy, R. E. I. El-Bassiouny and M. A. Saleh, J. Food Sci. Technol., 2015, 52, 3651–3659.
27. A. Altunkaya and V. Goekmen, Food Chem., 2008, 107, 1173–1179.
28. C. Queiroz, M. L. Mendes Lopes, E. Fialho and V. L. Valente-Mesquita, Food Rev. Int., 2008, 24, 361–375.
29. Y. Gao, Y. Li, L. Zhang, H. Huang, J. J. Hu, S. M. Shah and X. G. Su, J. Colloid Interface Sci., 2012, 368, 540–546.
30. D. Pan, J. Zhang, Z. Li and M. Wu, Adv. Mater., 2010, 22, 734–738.
31. J. Peng, W. Gao, B. K. Gupta, Z. Liu, R. Romero-Aburto, L. Ge, L. Song, L. B. Alemany, X. Zhan, G. Gao, S. A. Vithayathil, B. A. Kaipparettu, A. A. Marti, T. Hayashi, J. J. Zhu and P. M. Ajayan, Nano Lett., 2012, 12, 844–849.

---