Engineered Protein-Suspended Single-Walled Carbon Nanotubes (SWNTs) in Water

Qi Xu, Xin Ai,* Qing Song, Timothy J. McDonald, Hai Long, Shi-You Ding, Michael. E. Himmel and
Garry Rumbles*
National Renewable Energy Laboratory, 1617 Cole Blvd, Golden, CO 80401-3393.
RECEIVED DATE (automatically inserted by publisher); E-mail: xin_ai@nrel.gov; garry_rumbles@nrel.gov

The unique electronic, optical, and mechanical properties1 of

SWNTs make them potential candidates for a variety of
applications, including photovoltaic, hydrogen storage, drug
delivery, biosensing and biotherapy.2-5 However, due to a
propensity to bundle together through strong non-covalent
interactions between SWNTs, poor solubility in both aqueous and
non-aqueous solutions is the major obstacle for separation of
SWNTs, a critical step for further characterization and
applications. Although chemical modification of SWNTs via
covalent approaches6 can improve the SWNTs solubility, the
intrinsic properties of SWNTs may be altered. An alternative
approach is a non-covalent adsorption of various functional
molecules around SWNTs, including surfactants,7 polymers,8
carbohydrates,9 DNA and RNA, 10-12 and peptides or proteins.13-19
In case of proteins, naturally occurred proteins13-16 and artificial
peptides18-20 have been used to suspend SWNTs. However, so far
there are no reports on engineered or recombinant proteins that
can effectively suspend SWNTs. Compared to naturally occurred
proteins, engineered proteins are the proteins that can be
overexpressed in an expression system, and have several potential
advantages in various SWNT applications: (1) It is possible to
develop a cheap and large-scale production to isolate and disperse
SWNTs, if protein genes have been identified and their optimal
overexpression conditions have been established already. (2) By
rotational design of protein structures via modern gene
engineering technology, it becomes feasible in general to
elucidate the mechanism on how proteins bind to SWNTs.
Therefore, the binding specificity, affinity and strength between
proteins and SWNTs may be understood and improved. (3)
Through the specific surface functionalization of the engineered
protein, the protein/SWNT conjugates may readily be further
functionalized, for example, grafting antibodies or drugs for
complicated biotargeting and biotheraphy, or introducing
quantum dots (QDs) for dual biolabelling, bioimaging and solar
energy conversion.
Here, we first report using an engineered CarbohydrateBinding Modules (CBMs) protein, CtCBM4, to successfully
suspend and debundle SWNTs in aqueous solution. The CtCBM4
in this work is from one of the natural proteins (Celk gene derived
from Clostridium thermocellum), and overexpressed in the E. coli
expression system, the easiest and the most efficient expression
system. Briefly, the raw SWNTs were dispersed in CtCBM4
protein buffer solution with the assistance of ultrasonication.21
The suspension was centrifuged at 17000g for 60 min to remove
the bundled nanotubes. The supernatant was collected, and was
centrifuged again at 22000g for 15 min. The upper supernatant
containing CtCBM4/SWNTs was collected and stored at 4 C for
further characterization. It is noted that this CtCBM4/SWNTs
solution is stable up to several months.
Figure 1a is a photograph showing a comparison of SWNTs
dispersed by CtCBM4 and by sodium dodecylbenzene sulfonate

Figure 1. (a) A photography of CtCBM4/SWNTs and SDBS/SWNTs

suspensions. (b) HRTEM image of CtCBM4/SWNTs.

(SDBS) under the same experimental conditions. If we assume

that the color of the dispersion is an indicator of the concentration
of suspended nanotubes, the CtCBM4/SWNTs suspension has a
relatively high content of SWNTs, even though it is still not as
high as in SDBS, which has been proved to be the most effective
surfactant for dispersing SWNTs.7 A quantitative analysis of the
SWNTs solubility was performed.21 After the content of CtCBM4
was estimated and subtracted accordingly, the SWNTs solubility
in CtCBM4 solution was determined to be about 1.50 0.38
mg/mL. Figure 1b is a representative high-resolution transmission
electron microscopy (HRTEM) image of CtCBM4/SWNTs.21
Clearly, there exist both individual SWNTs and some small
bundles in the sample.21 Also, due to the solvent evaporation,
some networks of nanotubes are formed from the individual
CtCBM4/SWNTs overlapping on the TEM copper grid.
The linear absorption spectrum of CtCBM4/SWNTs
suspension is illustrated in Figure 2a (blue line). The welldefined van Hove features are identical to those of SDBS/SWNTS
suspension in Figure 2a (red line). The similarity on the fine
structures in spectra indicates that the effectiveness and quality of
debundle and dispersion of SWNTs in CtCBM4 suspension are
compatible with those in SDBS suspension. Furthermore, all
peaks in the absorption spectrum from CtCBM4-SWNTs solution
are red-shifted, and the reason will be discussed below.
The two-dimensional (2D) photoluminescence (PL) spectra
were measured by a customized Thermo-Electron FT960 Raman
spectrometer with a cooled (77K) Ge detector that has been
described previously.22 All collected spectra were corrected for
variations in the lamp excitation spectrum as well as for the
responses of the collection system and detector. Figure 2b shows
the 2D-PL contour map of CtCBM4/SWNTs suspension. The
individual bright spot can be assigned to emission from a specific
type of (n,m) nanotube based on the work of Weisman et al.,23
comparable to the isolated nanotubes shown on the 2D-PL spectra
of SDBS/SWNTs.21 The ratio of the maximum intensity of each
individual (n,m) type of tube from CtCBM4/SWNTs sample over
the maximum intensity of the counterpart from SDBS/SWNTs
sample is roughly the same for all measurable features on the 2D-

supported by another study,13 in which several natural proteins

with similar content of aromatic groups were investigated, but
only certain type(s) of proteins can debundle SWNTs. Further
work attempting to reveal the mechanism on how CtCBM4 binds
to SWNTs is undergoing in our laboratory.
In summary, we have demonstrated that engineered protein
CtCBM4 is able to effectively debundle and suspend SWNTs.
Characterizations by HRTEM, absorption and emission spectra
confirmed a good dispersion of SWNTs at individual level in
CtCBM4 solution. Possible factors that may determine CtCBM4
binding to SWNTs were discussed. Both primary and the higherorder structures of protein are proposed to play an important role
in interaction. By using engineered protein, this work opens up
new avenues to effectively separating SWNTs and further
functionally modifying SWNTs for various applications.
Acknowledgment. The work was supported by the U. S
Department of Energy under contract No. DE-AC36-99GO10337
with NREL, and the NREL Laboratory Directed Research and
Development (LDRD) program.
Supporting Information Available: Experimental details, and
2D-PL of SDBS/SWNTs and a comparison of emission spectra
between CtCBM4/SWNTs and SDBS/SWNTs.
This material is
available free of charge via the Internet at http://pubs.acs.org.
Figure 2. Two-dimensional (2D) excitation versus emission
photoluminescence contour map of CtCBM4/SWNTs suspension.

PL spectra.21 Assuming SDBS molecules bind equally to all

kinds of nanotubes in the solution, the similar ratio indicates that
CtCBM4 binds to SWNTs without a specific selectivity.
In addition, comparing the emission and excitation spectra of
each individual nanotube in two samples, the CtCBM4/SWNTs
show red-shifted peaks21 in both PL and excitation spectra
relative to those in SDBS/SWNTs spectra. The shifted absorption
and emission spectra of SWNTs in different medium have been
reported previously.7 It can be attributed to the solvatochromism
due to the different dielectric properties of the medium around the
SWNTs,24 which has also been observed in QDs.25 Because the
protein molecules possess charged residues and associated water
molecules, it is not surprise to anticipate a higher dielectric
constant of the surrounding medium in CtCBM4/SWNT
suspension, leading to the peaks redshifted in absorption and
emission spectra.
Although the binding mechanism of CtCBM4/SWNTs is not
clear yet at this moment, we believe that both the primary and
high-order structures of the protein are critical in CtCBM4
binding SWNTs. In a previous study investigating the artificial
short helix peptides, aromatic content in peptide is shown to
play an important role in -stacking protein/nanotube
interactions.19, 20 Naturally, CBMs can specifically bind to various
carbohydrates based on their diversity of structures.26 The binding
and recognition mechanism of which the CBMs bind to
carbohydrates is the interaction of aromatic amino acid side
chains (including tryptophan, tyrosine, phenylalanine, and
possibly proline) of CtCBM4 with pyranose ring(s) of
carbohydrates. These aromatic side chains form the hydropholic
platform in CBM-binding sites, and acclimatize their directions to
be planar, twisted or sandwich-structured to bind to the insoluble
crystalline cellulose, polysaccharides and oligosaccharides.
However, not every CBM protein tested in our experiments shows
the binding ability to SWNTs. Therefore, besides aromatic
residues, higher-order protein structure could also play a key role
in debundling and suspending SWNTs. This hypothesis is

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The interactions between proteins and carbon nanotubes have been an intensively investigated topic recently due to the
various potential applications. Naturally occurred proteins and artificial short peptides have been used to suspend SWNTs in
water. However, so far there are no reports on engineered or recombinant proteins binding carbon nanotubes. Engineered
proteins are preferred due to the capability being engineered, such as specific surface functionalization or fusion on tags for
easy protein purification or connection to other functional group(s). Here, we first report using engineered protein CtCBM4, one
of Carbohydrate-Binding Modules (CBMs) to successfully suspend and debundle SWNTs at individual level in aqueous
solution. Characterizations by HRTEM, absorption and emission spectra confirm the effectiveness and quality of the
disperstion of SWNTs. This work opens up new avenues to effectively separating SWNTs and further modifying and
functionalizing SWNTs for diverse applications.