Diameter-selective dispersion of double-walled carbon nanotubes by lysozyme

Abstract

We have utilized lysozyme to non-covalently functionalize and disperse double-walled carbon nanotubes (DWNTs) in aqueous solution. Lysozyme preferentially binds and disperses DWNTs with larger diameters. This is a facile and effective method to fractionalize and enrich DWNTs with certain diameters.

---

Double-walled carbon nanotubes (DWNTs) have superior structure and characteristics as they bridge the gap between single-walled carbon nanotubes (SWNTs) and multi-walled carbon nanotubes (MWNTs). DWNTs have garnered increasing attention for potential applications including field-emission displays (FEDs), field-effect transistors, atomic force microscope (AFM) tips and solar cells.¹ For example, DWNTs have been successfully used in FEDs,² because DWNTs possess both advantages of SWNTs (low threshold voltage) and MWNTs (high durability), making them the best candidate for emitters. Moreover, DWNTs are also attractive for biological and biomedical applications, such as biosensors.³ However, DWNTs have a strong tendency to aggregate and are practically insoluble in solvents. The dispersion of DWNTs becomes one prerequisite for the advancement of their research. There are two common dispersion strategies for carbon nanotubes (CNTs). One is based on covalent modification.⁴ However, it usually damages the side-wall of CNTs, hence changing their properties. The other is non-covalent modification, which does not disrupt CNTs' electronic structures and thus guarantees the original properties remain intact. Various materials have been employed to disperse CNTs in aqueous system, such as surfactants,⁵DNA,⁶polymers⁷ and peptides/proteins.⁸

Proteins are a common choice to disperse CNTs in aqueous solution. Lysozyme, which consists of a single polypeptide chain of 129 residues, is one of the best water-soluble proteins. The residues containing primary amines make lysozyme a cationic protein with an isoelectronic point (IEP) at 10.7. Because of its inherent hydrophobic domain and good aqueous solubility, lysozyme is utilized to disperse CNTs.^1a

Besides dispersing, sorting CNTs by diameter is also very important for CNT applications, because the diameter is closely related to the properties of CNTs. Some dispersion reagents were utilized to separate CNTs by diameter. For example, Kim et al. used DNA to precipitate large-diameter DWNTs.^6b Hersam et al. used sodium cholate to sort SWNTs by diameter and separate DWNTs from mixture of SWNTs and MWNTs viadensity gradient ultracentrifugation.^5b,c These methods are effective, but there are shortcomings, including high reagent cost, complex separation process, special machinery (ultra centrifuge) and difficulty in mass production.

In this study, we used lysozyme to biofunctionalize and disperse DWNTs. The fact that lysozyme can selectively disperse large-diameter DWNTs provides a facile approach for the enrichment of DWNTs with various diameters in bulk.

The DWNTs used in this work were synthesized by the arc discharge method and then purified.^4,9 The DWNT sample has a purity of ∼97% and outer diameters of 2–6 nm (Fig. 1a and b). Most of the DWNTs exist as bundles due to the van der Waals interactions. The Raman spectrum of DWNTs shows a strong G-band at 1582 cm⁻¹, a weaker peak at 1341 cm⁻¹ (D-band), the G′-band at 2666 cm⁻¹, and a broad peak below 200 cm⁻¹, which are the characteristic Raman signals of DWNTs (Fig. 2a).

Fig. 1 Representative TEM images of DWNTs: DWNTs (a and b) and DWNTs suspension (c and d).

Fig. 2 Wide-range Raman spectra excited at 532 nm (a) for DWNTs (black), suspension (red) and remnant (blue). Corresponding magnified Raman spectra of RBM band (b) and G-band (c) (the intensity of the G-band is set as 1).

To disperse DWNTs, DWNTs powder was mixed with 1.0 mg mL⁻¹ of lysozyme solution to yield a mixture containing 0.2 mg mL⁻¹ of DWNTs. The pH values of the mixture were adjusted by 1 M NaOH or 1 M HCl. After sonication for 30 min by a probe tip sonicator (200 W) in an ice bath, the DWNTs were dispersed in the lysozyme solution (pH 3) forming a dark brown mixture. A brown suspension was obtained after centrifugation at 4000 rpm for 10 min. HRTEM and AFM images show that DWNTs in suspension exist in small bundles or individually, and the DWNTs' surface is coated with plenty of lysozymes (Fig. 1c and d and Fig. S1 in the ESI†).

We found that the dispersion of DWNTs by lysozyme is pH sensitive. DWNTs are well dispersed, forming a stable brown suspension when the pH value of the lysozyme solution is lower than its IEP (10.7), while DWNTs cannot be suspended in lysozyme solution at pH 10. By testing the various pH values of the systems, we found that the optimal dispersion pH value is 3. At pH 3 the suspension has the highest content of DWNTs (87 mg L⁻¹) (Fig. 3b), whilst the concentration of DWNTs is 67 mg L⁻¹ at pH 5 (Fig. 3c). As the pH is lowered to 1, the content of DWNTs is only 33 mg L⁻¹ (Fig. 3a). Furthermore, the dispersion is reversible. When the pH value of the DWNT suspension changes from 3 to 10 by adding 1 M NaOH, DWNTs precipitate at the bottom after centrifugation. Subsequently, the flocculated DWNTs can be reverted back to the homogeneous suspension by adjusting the pH back to 3 using 1 M HCl with 30 min sonication (Fig. 3e).

Fig. 3 Photographs of the DWNT suspension at pH = 1 (a), pH = 3 (b), pH = 5 (c), pH = 10 (d), and the re-dispersed DWNTs suspension at pH = 3 (e).

The above results suggest that the positive-charge on lysozyme is the major stabilizing factor for the DWNT–lysozyme suspension. As the pH value decreases from 10 to 1, the positive charge of lysozyme increases. However, at pH 1 the stability of lysozyme also decreases, exposing more buried interior hydrophobic residues and increasing the tendency to aggregate.¹⁰ Therefore, a well-folded structure of lysozyme may also be a prerequisite for the efficient suspension of DWNTs.

The suspension and remnants of the DWNT dispersion system were collected for Raman analysis. The Raman spectra of both samples show the characteristic Raman signals of DWNTs (Fig. 2a).

To investigate the Raman changes in detail, we have magnified the RBM band and the G-band (Fig. 2b and c). The frequency of RBM (ω_r) is inversely proportional to the inner diameter (d) of the DWNTs, following the equation ω_r = 6.5 + 223.75/d.⁹ Accordingly, the inner and outer diameters of the DWNTs are in the ranges 0.50–2.80 nm and 1.26–3.56 nm, respectively. Compared to the pristine DWNTs, the RBM band of the DWNTs in suspension obviously shifts to the lower frequencies of 75–155 cm⁻¹ (Fig. 2b), corresponding to the outer diameters of 2.32–4.05 nm. DWNTs in suspension have larger diameters. On the other hand, the apparent red shift of the RBM band, corresponding to the outer diameters of 1.56–2.29 nm, demonstrates that DWNTs in the remnants have smaller diameters (Fig. 2b). This suggests lysozyme has a high binding selectivity towards large-diameter DWNTs. What we should mention is that DWNTs with diameters larger than 4 nm do not show RBM peaks, because their (resonantly enhanced) Raman cross-section is pretty small.¹¹

When looking at the G band in Fig. 2c, the broad and asymmetric Breit–Wigner–Fano (BWF) line^12a from 1525–1575 cm⁻¹ clearly appears in the spectrum of the DWNTs. The BWF line depends on the diameter of the CNTs.¹² The BWF effect increases with decreasing nanotube diameter.^12a Compared to the original DWNTs, the strong depression of the BWF line of the DWNT suspension also proves that DWNTs in suspension have a large diameter (Fig. 2c).

The entire diameter distribution of the DWNTs samples can be obtained by directly measuring the diameter of DWNTs under TEM. Based on 200 nanotubes' statistics, the diameters of the DWNTs are in the range 1–4 nm for the outer (Fig. 4a) and 0.5–3.5 nm for the inner (Fig. 4b). The Gaussian mean outer diameter of the DWNTs is 2.7 ± 0.2 nm. While DWNTs in suspension have outer diameters ranging from 3–5 nm, with a Gaussian mean diameter of 4.0 ± 0.1 nm (Fig. 4a). The DWNTs’ diameter distribution obtained under TEM is consistent with that based on Raman measurement.

Fig. 4 Histograms of the outer diameters (a) and the inner diameters (b) of DWNTs (black) and DWNTs in suspension (gray).

It is worthwhile to note that about 10% of the pristine DWNTs with outer diameters less than 1.5 nm, and nearly 30% with outer diameters between 1.5 and 2.0 nm, but none of the DWNTs with outer diameters less than 1.5 nm, and only 3% with outer diameters between 1.5 and 2.0 nm are detected in the suspension. This provides an efficient method for enriching and separating the large-diameter DWNTs.

There are two major factors that account for the diameter-selective suspension of DWNTs by lysozyme. Firstly, the small-diameter tubes tend to form stronger bundles than the large-diameter tubes.¹³ Secondly, it has been reported that the interaction of protein and large nanotube is stronger than that of protein and small nanotube.¹⁴ Our previous computational simulation also shows that the interaction between carbon nanoparticles and proteins increases with increasing size of carbon nanoparticles.¹⁵ Therefore, the large-diameter CNTs are expected to be more easily dissociated from the bundles by sonication than the smaller diameter ones, and hence preferentially bound by proteins.

At present, one troublesome problem often encountered is that the synthesized DWNTs always contain some SWNTs impurities. To test whether lysozyme could realize the separation of the SWNTs impurities from DWNTs post-synthesis, we applied the above procedure to an intentionally-made DWNTs–SWNTs mixture (80% pure DWNTs and 20% pure SWNTs).

After sonication and centrifugation of DWNTs–SWNTs in lysozyme solution, only the large-diameter DWNTs were suspended in the supernatant, thus realizing the separation of SWNTs from DWNTs. By HRTEM observation, there are only DWNTs in the suspension and no SWNTs are found (Fig. 5a and Fig. S2 in the ESI†). The samples were further detected by Raman spectroscopy. The spectrum (RBM band at 50–150 cm⁻¹ and a single G band at 1590 cm⁻¹), is different from that of the SWNTs, which has the characteristic peaks of the RBM band at 135–190 cm⁻¹, G bands with two peaks at 1568 and 1590 cm⁻¹, and a D band at 1337 cm⁻¹ (Fig. 5b). This shows that CNTs in the suspension are DWNTs (Fig. 5b). Combining the above results, DWNTs can be collected in the supernatant, while SWNTs impurities are left in the sediment. This can be used to separate the SWNTs impurities from the DWNTs.

Fig. 5 (a) A TEM image of the suspension of DWNTs–SWNTs mixture; (b) Raman spectra excited at 532 nm for DWNTs–SWNTs mixture (black), suspension (red) and SWNTs (blue).

Conclusions

In summary, this work demonstrates that lysozyme is efficient in the non-covalent functionalization and dispersion of DWNTs. The process is pH and DWNTs diameter dependent. There is an optimal pH value at 3 for DWNT dispersion in lysozyme solution. Notably, DWNTs with a larger diameter preferentially bind lysozyme and are suspended in the solution. This method can be used to realize the efficient fractionalization and enrichment of DWNTs with a large diameter on a large scale. In addition, this method can be employed to remove the SWNTs impurities from the DWNTs–SWNTs mixture, which usually exist in some synthetic processes of DWNTs.

Acknowledgements

We thank Prof. Ya-Ping Sun at Clemson University, USA for his helpful discussion. This work is supported by the National Basic Research Program of China (973 Program) (Nos. 2011CB933402 and 2009CB930200), the Chinese Natural Science Foundation (No. 20871010) and Shanghai Leading Academic Disciplines (S30109).

Notes and references

1. (a) S. Kuwahara, S. Akita, M. Shirakihara, T. Sugai, Y. Nakayama and H. Shinohara, Chem. Phys. Lett., 2006, 429, 581; (b) S. Wang, X. L. Liang, Q. Chen, Z. Y. Zhang and L. M. Peng, J. Phys. Chem. B, 2005, 109, 17361; (c) J. Q. Wei, Y. Jia, Q. K. Shu, Z. Y. Gu, K. L. Wang, D. M. Zhuang, G. Zhang, Z. C. Wang, J. B. Luo, A. Y. Cao and D. H. Wu, Nano Lett., 2007, 7, 2317; (d) X. C. Dong, D. L. Fu, Y. P. Xu, J. Q. Wei, Y. M. Shi, P. Chen and L.-J. Li, J. Phys. Chem. C, 2008, 112, 9891.
2. H. Kurachi, S. Uemura, J. Yotani, T. Nagasako, H. Yamada, T. Ezaki, T. Maesoba, R. Loutfy, A. Moravsky, T. Nakazawa, S. Katagiri and Y. Saito, IDW Proc., 2001, 1237.
3. (a) Y. F. Li, T. Kaneko and R. Hatakeyama, Small, 2010, 6, 729; (b) C. Lamprecht, J. Danzberger, P. Lukanov, C.-M. Tîlmaciu, A.-M. Galibert, B. Soula, E. Flahaut, H. J. Gruber, P. Hinterdorfer, A. Ebner and F. Kienberger, Ultramicroscopy, 2009, 109, 899.
4. H. Nie, W. Guo, Y. Yuan, Z. Dou, Z. Shi, Z. Liu, H. Wang and Y. Liu, Nano Res., 2010, 3, 103.
5. (a) S. D. Bergin, V. Nicolosi, H. Cathcart, M. Lotya, D. Rickard, Z. Y. Sun, W. J. Blan and J. N. Coleman, J. Phys. Chem. C, 2008, 112, 972; (b) M. S. Arnold, S. I. Stupp and M. C. Hersam, Nano Lett., 2005, 5, 713; (c) A. A. Green and M. C. Hersam, Nat. Nanotechnol., 2009, 4, 64.
6. (a) M. Zheng, A. Jagota, E. D. Semke, B. A. Diner, R. S. Mclean, S. R. Lustig, R. E. Richardson and N. G. Tassi, Nat. Mater., 2003, 2, 338; (b) J. H. Kim, M. Kataoka, Y. A. Kim, D. Shimamoto, H. Muramatsu, T. Hayashi, M. Endo, M. Terrones and M. S. Dresselhaus, Appl. Phys. Lett., 2008, 93, 223107.
7. L. Vaisman, G. Marom and H. D. Wagner, Adv. Funct. Mater., 2006, 16, 357.
8. (a) A. Oritiz-Acevedo, H. Xie, V. Zorbas, W. M. Sampson, A. B. Dalton, R. H. Baughman, R. K. Draper, I. H. Musselman and G. R. Dieckmann, J. Am. Chem. Soc., 2005, 127, 9512; (b) J. Zhong, L. Song, J. Meng, B. Gao, W. S. Chu, H. Y. Xu, Y. Luo, J. H. Guo, A. Marcelli, S. S. Xie and Z. Y. Wu, Carbon, 2009, 47, 967; (c) D. Nepal and K. E. Geckeler, Small, 2007, 3, 1259.
9. H. X. Qiu, Z. J. Shi, L. H. Guan, L. P. You, M. Gao, S. L. Zhang, J. S. Qiu and Z. N. Gu, Carbon, 2006, 44, 516.
10. A. N. Cao, G. Wang, Y. Q. Tang and L. H. Lai, Biochem. Biophys. Res. Commun., 2002, 291, 795.
11. W. C. Ren, F. Li, J. Chen, S. Bai and H. M. Cheng, Chem. Phys. Lett., 2002, 359, 196.
12. (a) S. D. M. Brown, A. Jorio, P. Corio, M. S. Dresselhaus, G. Dresselhaus, R. Saito and K. Kneipp, Phys. Rev. B: Condens. Matter, 2001, 63, 155414; (b) A. Jorio, A. G. S. Filho, G. Dresselhaus, M. S. Dresselhaus, A. K. Swan, M. S. Ünlü, B. B. Goldberg, M. A. Pimenta, J. H. Hafner, C. M. Lieber and R. Saito, Phys. Rev. B: Condens. Matter, 2002, 65, 155412.
13. (a) H. C. Chae, T. V. Sreekumar, T. Uchida and S. Kumar, Polymer, 2005, 46, 10925; (b) T. Dumitrica, C. M. Landis and B. I. Yakobson, Chem. Phys. Lett., 2002, 360, 182.
14. Q. X. Mu, W. Liu, Y. H. Xing, H. Y. Zhou, Z. W. Li, Y. Zhang, L. H. Ji, F. Wang, Z. K. Si, B. Zhang and Y. Bing, J. Phys. Chem. C, 2008, 112, 3300.
15. X. Wu, S. T. Yang, H. F. Wang, L. Y. Wang, W. X. Hu, A. N. Cao and Y. F. Liu, J. Nanosci. Nanotechnol., 2010, 10, 6298.

---

Footnote

† Electronic supplementary information (ESI) available: AFM images of DWNTs suspension and TEM images of the suspension of DWNTs–SWNTs mixture. See DOI: 10.1039/c0nr00831a

---