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Protein Dispersant Binding on Nanotubes Studied by NMR Self-Diffusion and Cryo-TEM Techniques ,† and Oren Regev*,‡ Anton E. Frise,† Eran Edri,‡ Istv an Furo ‡
Department of Chemical Engineering and the Ilse Katz Institute for Meso and Nanoscale Science and Technology, Ben-Gurion University of the Negev, 84105 Beer-Sheva, Israel, and †Division of Physical Chemistry, Department of Chemistry, Royal Institute of Technology, SE-10044 Stockholm, Sweden
ABSTRACT Carbon nanotubes can be dispersed by a variety of molecules. We investigate the dynamics of protein-assisted carbon nanotube dispersion in water. We find that in equilibrium, only a small fraction of the dispersants is indeed adsorbed to the nanotube surface, while there is a fast exchange process between the adsorbed and free protein molecules. Self-diffusion NMR spectroscopy in combination with cryo-transmission electron microscopy imaging are employed. SECTION Nanoparticles and Nanostructures
arbon nanotubes (CNTs) possess unique electrical, mechanical, and optical properties and are hence implemented in applications ranging from nanoelectronics1-3 through biosensors4 to drug delivery.5 A major drawback of as-produced CNTs is their bundling due to strong van-der-Waals interactions. Therefore, the advantages with the high aspect ratio (>1000) and other unique properties of individual single-wall carbon nanotubes (SWNTs) are lost. Hence, substantial effort has been devoted to dispersing and debundling CNTs through either covalently grafting dispersing molecules onto the CNT surface6 or noncovalently adsorbing it on the CNT surface.7 To preserve the original CNT structure together with the associated electrical and mechanical properties, the hybridization of the carbon atoms should be minimally perturbed during the debundling process, which calls for noncovalent dispersion. Moreover, the noncovalent approach, investigated here, does not require chemical reactions. While the noncovalent dispersion and exfoliation of CNTs with surfactants,8-10 polysaccharides,7,11 and polymers12,13 is well-established, biomaterials such as proteins are rather new candidates for dispersing NTs. Nonetheless, proteins gain a rapidly growing interest as an alternative route for the debundling and dispersion of CNTs, especially for biomedical applications. This stems from the hope that they also enhance the biocompatibility of SWNTs.14-16 Lately, an efficient uptake of protein (drug)-dispersed SWNT by cells, probably via an endocytosis17 pathway, has been reported. Here, the SWNTs are employed as molecular transporters for various cargos.18 Indeed, natural water-soluble proteins are reported to successfully disperse NTs through noncovalent interactions.19-22 Short synthetic peptides were also shown to successfully exfoliate, debundle, and disperse nanotubes via specifically designed adsorption23,24 or helical-like wrapping.25 Indeed, the use of peptides and proteins has opened a door to many potential applications.26 However, the dispersant (e.g., surfactant, polymer, or protein) configuration
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on the nanotubes surface remains, among other issues, ambiguous.27,28 As concerns dispersion mechanisms, for polymer-type dispersants, it was suggested that a weak, long-ranged entropic repulsion among polymer-decorated tubes acts as a steric barrier preventing the tubes from approaching the attractive part of the intertube potential.7,29 Others suggested a wrapping-type mechanisms.12,30 However, while there is plenty of data on the conformation of the dispersing agent in dry samples,31,32 there is lack of experimental information on the dispersant configuration or the density of dispersant molecules on the nanotube in situ. For surfactants, various anchoring possibilities on nanotubes were suggested, primarily involving hydrophobic interactions.33 Interactions involved in protein adsorption on NT surfaces can be electrostatic and hydrophobic. While some researchers suggest that hydrophobic interactions20,34 dominantly control protein adsorption, others35 claim that the electrostatic contribution is the major one. Recently, the distribution of the dispersant-dispersant distance on the nanotubes (L) (Figure 1) has been reported for BSA as the dispersant.36 Despite significant research, little is known on the nature of noncovalent dispersion of nanotubes by protein or other dispersants. How much dispersant is adsorbed on a nanotube? What is the fraction of surface area covered? Are the CNT-adsorbed molecules in equilibrium with the freely dispersed ones? These questions are extremely relevant to applications where nanotubes are exploited as transporters of drugs (proteins) to cells.17 In many studies, one assumed a full coverage,28,33 which is, however, difficult to prove experimentally.
Received Date: March 16, 2010 Accepted Date: April 8, 2010 Published on Web Date: April 14, 2010
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within the approximately (2% statistical error) water diffusion coefficients in all measured solutions (for more details, see Experimental Methods section). The validity of the assumption of fast exchange was confirmed by comparing the BSA spectral integrals obtained in conventional single-pulse experiments for solution A on one hand and those for solutions B1 and B2 on the other hand. In the case of slow exchange, proteins binding to the NTs would experience very slow tumbling that would lead to the loss of their signal because of the very large broadening. However, no such reduction in the signal could be found, and the assumption of fast exchange should therefore hold for this system. (Alternatively, if there were slow exchange, the fraction of NT-bound BSA should be lower that the 2-3% accuracy of the NMR intensity measurements). As an additional indication, the BSA line widths were slightly larger in solutions B1 and B2 as compared to those in solution A. This behavior is as expected for BSA molecules in fast exchange between sites with slow (in solution) and fast (bound to NTs) transverse relaxation. In fast exchange, the observed BSA diffusion coefficient (Dobs) in solutions B1 and B2 becomes the population average of the diffusion coefficients in the bound and free states.42 Dobs ¼ Dfree 3 pfree þ Dbound 3 ð1 - pfree Þ ð1Þ
BSA in an aqueous solution has been found to adsorb to CNTs.36 The proposed mechanism is that the protein partly unfolds and exposes some of its hydrophobic amino acids, which interact with the surface of the CNTs.37,38 The binding of protein to the surface introduces steric repulsion and will disperse CNTs. In this paper, we show that in the dispersed state, the protein molecules cover only a very small fraction of the nanotube surface and exchange quickly with the pool of unadsorbed proteins. Samples were prepared by mixing protein and nanotubes, sonicating and extracting the supernatant by centrifugation (for details, see Supporting Information). The concentrations of NTs in the dispersion were determined by UV-vis-based chemometric techniques22 and are presented in Table 1 along with the initial mixing concentrations. As concerns the protein component, BSA concentrations as provided by NMR spectral intensities of BSA were identical (within the experimental error of a few percent) before and after centrifugation. We first present the results of our self-diffusion measurements and then relate to our cryo-TEM micrographs, including calculations of area per adsorbed protein molecule. Self-Diffusion Study. Previous studies of self-diffusion of NT systems concerned the diffusion of gases and liquids confined inside of the NTs.39,40 Our use of the molecular diffusion here is entirely different since we exploit self-diffusion NMR to report on the ratio of BSA bound to CNTs versus that free in the solution. It is assumed that these are the only states that the protein takes and also that the exchange between the two states is fast on the NMR time scale. A third assumption is that when the protein is bound to CNTs, its diffusion becomes negligible. This is reasonable considering that the NTs are 100-1000 times larger than the proteins, and their movement should therefore be correspondingly slower. Finally, we assume that the obstruction41 on diffusion by the NTs is negligible, which is validated by the identical (1.90 10-9 m2/s
Considering that the diffusion of BSA in its NT-bound state is negligibly small, Dfree . Dbound, this expression simplifies to Dobs = Dfree 3 pfree, from which the fraction of bound BSA molecules can be estimated as Dobs ð2Þ pbound ¼ 1 - pfree ¼ 1 Dfree The fractions and resulting absolute concentrations Cbound of NT-bound BSA as provided by eq 2 are presented in Table 1. For both SWNTs and MWNTs, we find that the bound fractions are small, on the order of only a few percent. Unfortunately, this and the comparable magnitude of the precision of the diffusion data also mean that the obtained relative error of pbound is high. We use the obtained Cbound and the total dispersed concentration of NT, CNT, from chemometric measurements22 to calculate the area per one BSA molecule on NT, ABSA(NT). First, the number of bound BSA molecules per volume unit is obtained as Cbound NA ð3Þ NBSA ¼ MWBSA
Figure 1. The dispersant-dispersant distance (L) in the arrangement of dispersant molecules (dotted) adsorbed on a nanotube.
Table 1. Initial NT and BSA Mixing Weights and Final (for solutions B1 and B2) NT Concentrations after Sonication and Centrifugationa sample
compound
initial conc. [mg/mL]
CNT [mg/mL]b
Dobs [10-11 m2/s]
Pboundc
Cboundd [mg/mL]
A
BSA
4
5.22 ( 0.13
B1
BSA
4
5.03 ( 0.05
0.04 ( 0.03
0.16 ( 0.12
SWNT
2
BSA
4
4.74 ( 0.13
0.09 ( 0.05
0.36 ( 0.20
MWNT
2
B2
0.24 ( 0.01 0.02
a
NT concentrations were obtained by chemometric-based experiments on UV-vis spectroscopy,22 and NMR spectral intensities indicate identical BSA concentrations before and after centrifugation. b Dispersed concentration in the supernatant. c Fraction of NT-bound BSA. d The concentration of NT-bound BSA.
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it seems that the surface density should, if anything, be higher for MWNTs than that for SWNTs and that low values of R provide peculiarly high surface density values (more than monolayer coverage, under the assumption of adsorption on the external surface of the outermost tube). Electron Microscopy Study. The BSA molecules have insufficiently low contrast in cryo-TEM measurements (Figure 2A). To enhance the contrast, we attached an 8 nm gold nanoparticle (GNP) to the individual BSA molecules (Figure 2B). We have previously shown that the GNP-BSA complex disperses the SWNTwithout changing the conformation of the protein.36 The measured distribution of the dispersantdispersant distance (L; see Figure 1) for the BSA-GNP complex as imaged by cryo-TEM indicates an average distance of ÆLæ=77 ( 55 nm for SWNT solutions36 (Figure 2B) in order-ofmagnitude agreement with the NMR findings above. In the cryo-TEM image (Figure 2B), L has a rather wide polydispersity. Similar images in the corresponding MWNT system demonstrate binding, but we refrain from a quantitative analysis (see discussion above). The area per adsorbed BSA molecule, ABSA(NT), is obtained via eq 6 as ABSA(SWNT) = 360 ( 260 nm2.36 Since the concentration of the GNP-BSA was lower than the BSA concentration in the NMR experiments (see discussion above), this value is consistent with that derived from the NMR experiments. First, it should be noted that due to the low BSA contrast in TEM, the NMR and cryo-TEM measurements are not conducted on exactly the same system; (1) the GNP introduced into the cryo-TEM preparation are not added in the NMR experiments, and (2) the BSA concentration in the cryo-TEM measurement is much lower than that in the NMR experiments (see Experimental Methods section). Therefore, in Figure 2, we mostly see GNP-BSA that are bound (in close proximity) to the SWNT, which is in contrast to the NMR finding of most BSA being not bound. This contradiction can be resolved by considering the low GNP-BSA concentration. Here, the NMR findings can be considered as quantitatively accurate, while the cryo-TEM observations can primarily be seen as rough verifications of the finding of the small amount of CNT-bound protein. In this context, there is no easy way to perform the NMR and cryo-TEM experiments on the same system because binding the gold nanoparticles broadens the NMR signal to close undetectability. Even if the 1H NMR signal from GNP-BSA were detectable, the diffusion coefficient for them in the free state would be much lower, which would decrease the sensitivity of NMR experiments to binding. Since our current sensitivity is barely enough to detect binding, diffusion experiments with GNP-BSA are not feasible. Despite all possible shortcoming of our experiments, our current observations together with some earlier ones36 provide two significant findings. First, the most striking feature is that only a very small fraction of the dispersing molecules (a few percent; see Table 1) are actually adsorbed on the NT surface at a given instance. This is true for both the SWNTand MWNT (Table 1). However, if one attempts to disperse the NTs by initially adding only that much BSA that corresponds to the pbound (that is, for example, preparing a SWNT dispersion by
Figure 2. Cryo-TEM micrographs of SWNTs (A,B) and MWNTs (C,D) dispersed by unlabeled BSA (A,C) and by BSA labeled by 8 nm gold nanoparticles (B,D). It is impossible to see any unlabeled adsorbed BSA molecules. The spots in A and C marked by arrows are due to surface contamination during vitrification and transfer to the electron microscope. Bar = 100 nm.
where MWBSA=67100 Da is the molar mass of BSA and NA is the Avogadro number. The total surface area of NTs (per volume) in the solution, ANT, is obtained as ð4Þ ANT ¼ CNT 3 SSA where SSA is the reported specific surface area of the NTs. For SWNT, available values are SSA(SWNT) = 24843,44 or 283 m2/g.45 Hence, the area per adsorbed BSA molecule is ANT ¼ 80 ( 60 nm2 ð5Þ ABSA ðSWNTÞ ¼ NBSA This area yields, via ABSA ðNTÞ ¼ πDNT ÆLæ
ð6Þ
and the known diameter of SWNT (DNT=1.5 nm), the average dispersant-dispersant distance (L; see Figure 1) on the order of ÆLæ ≈ 20 nm, in agreement with reported values in the literature.31 The active surface area per weight (available for adsorption) for a MWNT is plausibly lower than the one obtained by BET measurements (233 m2/g46) if we assume that only the external surface of the outermost tube is accessible for adsorption of a protein molecule. Since (i) the reported values for the ratio of the active surface area/total surface area, R, for MWNTs are scattered within a very wide range of 0.1-0.001.47,48 and (ii) there is a large polydispersity in MWNT width and the number of layers, we cannot provide a surface density estimate for the MWNT solutions. However,
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Figure 3. Schematics of the dynamic equilibrium between adsorbed and free BSA molecules (red). According to our results, most BSA molecules are in the free state at any given instance, and the exchange is fast, on a time scale
