Selective adsorption of proteins on single-wall carbon nanotubes by using a protective surfactant

Article abstract of DOI:10.1002/chem.201101182

The dispersion of highly hydrophobic carbon materials such as carbon nanotubes in biological media is a challenging issue. Indeed, the nonspecific adsorption of proteins occurs readily when the nanotubes are introduced in biological media; therefore, a methodology to control adsorption is in high demand. To address this issue, we developed a bifunctional linker derived from pyrene that selectively enables or prevents the adsorption of proteins on single-wall carbon nanotubes (SWNTs). We demonstrated that it is possible to decrease or completely suppress the adsorption of proteins on the nanotube sidewall by using proper functionalization (either covalent or noncovalent). By subsequently activating the functional groups on the nanotube derivatives, protein adsorption can be recovered and, therefore, controlled. Our approach is simple, straightforward, and potentially suitable for other biomolecules that contain thio or amino groups available for coupling.

Full text of DOI:10.1002/chem.201101182

FULL PAPER
DOI: 10.1002/chem.201101182
Selective Adsorption of Proteins on Single-Wall Carbon Nanotubes by Using
a Protective Surfactant
Anton Knyazev,*[a] Loꢀc Louise,[a] Michꢁle Veber,[a] Dominique Langevin,[a]
Arianna Filoramo,[b] Adriele Prina-Mello,[c] and Stꢂphane Campidelli*[b]
Abstract: The dispersion of highly hy-
drophobic carbon materials such as
carbon nanotubes in biological media
is a challenging issue. Indeed, the non-
specific adsorption of proteins occurs
readily when the nanotubes are intro-
duced in biological media; therefore, a
methodology to control adsorption is
in high demand. To address this issue,
we developed a bifunctional linker de-
rived from pyrene that selectively ena-
bles or prevents the adsorption of pro-
teins on single-wall carbon nanotubes
(SWNTs). We demonstrated that it is
possible to decrease or completely sup-
press the adsorption of proteins on the
nanotube sidewall by using proper
functionalization (either covalent or
noncovalent). By subsequently activat-
ing the functional groups on the nano-
tube derivatives, protein adsorption
can be recovered and, therefore, con-
trolled. Our approach is simple,
straightforward, and potentially suita-
ble for other biomolecules that contain
thio or amino groups available for cou-
pling.
Keywords: adsorption
nanotubes proteins
probe microscopy
·
carbon
scanning
·
·
Introduction
overall toxicity of the nano-object.[4–6] Single-wall carbon
nanotubes (SWNTs) are peculiar nano-objects that show a
high aspect ratio, low reactivity under standard conditions,
metallic or semiconducting character according to their geo-
metrical features,[7] and, more interestingly, a great capabili-
ty of penetrating cell membranes.[8,9] The penetration of cell
membranes has been related to their high aspect ratio[10]
and their surface groups after chemical treatments and func-
tionalization.[11–13]
It is noteworthy that over the last few years the issue of
the cytotoxicological effect of carbon nanotubes has gener-
ated heated debate among scientists as evidence for and
against the hypothesis of their toxicity has piled up.[9,14–16]
Although there is no full consensus yet, from the results re-
ported so far we feel that the question needs to be handled
with care because carbon nanotubes are a diverse group of
materials and their toxicity level seems to depend on nano-
tube type (i.e., diameter and length),[15,17] cell type,[18] test
system,[19] method of delivery (i.e., aerosol or solvent/surfac-
tant versus aggregation),[20–23] nanotube purification,[24,25] and
functionalization.[26–28]
Control over the assembly of proteins or lipidic corona
around nano-objects is of primordial importance for finding
new opportunities in biomedicine and materials science.[1–3]
In biological media, such as protein serum or cell media, the
phenomenon of physisorption occurs often and readily when
nano-objects are incorporated. The nature and lifetime of
the protein corona formed at the nanotube surface can
affect the way in which the cells interact with the nano-
object, and therefore influence both the functionality and
[a] Dr. A. Knyazev,+ L. Louise, Dr. M. Veber, Dr. D. Langevin
Universitꢀ Paris 11
Laboratoire de Physique des Solides
UMR 8502, 91405 Orsay (France)
[b] Dr. A. Filoramo, Dr. S. Campidelli
CEA, IRAMIS
Laboratoire d’Electronique Molꢀculaire
SPEC, 91191 Gif sur Yvette (France)
E-mail: stephane.campidelli@cea.fr
Herein, we have not explored SWNT toxicology; howev-
er, we were concerned with the appropriate functionaliza-
tion to realize a controlled protein corona on SWNTs.
Carbon nanotube/protein assemblies have been extensively
studied since the initial report of Mioskowski and co-work-
ers[29] for several reasons, namely, the conjugation of pro-
teins with nanotubes have a great potential in sensing appli-
cations[30] and vectorisation[8,31,32] or simply for the disper-
sion of nanotubes.[33–35] Although it has been demonstrated
that most proteins can adsorb spontaneously onto nano-
tubes,[32,36] the control of the assembly of proteins on the
nanotube surfaces could be crucial. For that reason, the
[c] Dr. A. Prina-Mello
Trinity College Dublin
CRANN and NanoMedicine and Molecular Imaging Group
Dublin 2 (Ireland)
[+] Present address:
Ecole Polytechnique Fꢀdꢀrale de Lausanne
Laboratoire de Physique de la Matiꢁre Complexe
1015 Lausanne (Switzerland)
E-mail: anton.knyazev@epfl.ch
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201101182, in which techniques,
experimental details, and characterization data for the synthesis of
the pyrene derivatives are given.
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14663

combination of proteins on nanotubes through covalent[37–40]
or noncovalent interactions[41,42] have been reported. Herein,
we exposed different nanotube derivatives to bovine serum
albumin (BSA), which was used as the reference protein.
The nanotube properties were tuned to prevent the forma-
tion of protein (or lipidic) corona when introduced in bio-
logical media or, on the contrary, to mimic this protein
corona in vitro. For such purposes, BSA was introduced
onto the nanotube surfaces either by covalent or noncova-
lent means. The nanotube derivatives were characterized by
Raman, UV/Vis, and NIR spectroscopy and the protein-
based assemblies were visualized by atomic force microsco-
py (AFM). The aim of this work is the realization of stable
and soluble SWNT hybrids that could be potential vessel
candidates for drug transportation in living organisms.
Scheme 1. Synthesis of amphiphilic pyrene derivative 1. DCC= dicyclo-
hexylcarbodiimide, DMAP=N,N-dimethylaminopyridine.
Results and Discussion
To obtain a selective adsorption of proteins onto nanotubes,
we designed and compared two systems in which the BSA
was linked to the SWNTs either by covalent or noncovalent
means. The noncovalent approach was based on the immo-
bilization of an amphiphilic pyrene unit that contained a re-
active terminal moiety for protein attachment, whereas the
covalent approach was based on the modification of SWNTs
with diazonium salts and the subsequent attachment of
BSA.
oxygen atoms in p-NT are present as hydroxy, ketone, and
carboxylic functional groups.[58,59] The XPS spectrum of the
purified nanotubes is shown in Figure 1a; the spectrum
showed only the presence of carbon and oxygen atoms in
the sample. The deconvolution of the high-resolution XPS
spectrum of carbon was performed with four symmetrical
peaks at 284.2, 284.9, 286.0, and 288.7 eV (Figure 1b); the
2
À
peaks C1, C3, and C4 were attributed to sp , C OH, and
COOH carbon atoms, respectively. The origin of the peak at
284.9 eV is attributed to defects in the sp2 carbon
atoms.[60–62] For oxygen, the peaks O1 and O2 at 531.8 and
Noncovalent approach: The noncovalent functionalization,
based on the p stacking of pyrene derivatives on carbon
nanotubes, has been widely used to disperse nanotubes[43,44]
and/or combine the nanotubes with photoactive/electroac-
tive species[45–47] or biomolecules.[41,48–52] Herein, we designed
a new pyrene-based surfactant 1 that contained a tetraethy-
lene glycol polar chain modified with an amine group. In
this molecule, the apolar pyrene moiety ensures the interac-
tion with the nanotubes, whereas the polar oligoethylene
glycol chain ensures solubility in biological media and pre-
vents the undesirable attachment of proteins onto the nano-
tube sidewalls.[42,53] The presence of the amine group on the
ethylene glycol chain allows us to introduce a maleimide
group that can react with the thiol functional group of a
BSA cysteine residue.
The synthesis of the pyrene derivative 1 is depicted in
Scheme 1. 1-Amino-11-azido-3,6,9-trioxaundecane was syn-
thesized according a reported procedure[54] and was allowed
to react with 1-pyrene butyric acid in the presence of DCC
and DMAP. The azido pyrene derivative 2 was reduced by
hydrogen in the presence of Pd on carbon (Pd/C) to give 1.
As an interesting side result, this pyrene derivative possesses
original surfactant properties.[55] Pristine SWNTs, produced
by laser ablation,[56] were purified by following previously
reported protocols[57] and characterized by X-ray photoelec-
tron spectroscopy (XPS). The amount of oxygen in the puri-
fied nanotubes (p-NT) was estimated to be approximately
8% (atomic percentage) by XPS. The above-mentioned
À
533.2 eV, respectively, are attributed to C=O and C O
carbon atoms, respectively (Figure 1c). The spectrum is in
accordance with the XPS characterization described by
Hirsch and co-workers.[59] We were concerned about the fact
that BSA could eventually interact with the oxygen-contain-
À
ing functional groups (i.e., OH, C=O, or COOH). Actually,
electrostatic interactions with carboxylic functional groups
are unlikely because the isoelectric point of BSA is approxi-
mately 5.3; therefore, at neutral pH the BSA surface is glob-
ally negative and BSA is repelled by the carboxylic func-
tional groups. The other oxidized carbon-containing groups
are less hydrophobic than pure carbon, and therefore will be
less effectively coupled to the protein through hydrophobic
interactions. The dispersion of purified nanotubes was pre-
pared by dispersing SWNTs in a solution of 4-(2-hydroxy-
AHCTUNGTREGeNNNU thyl)-1-piperazineethanesulfonic acid (HEPES) buffer
(10 mm). After the sonication and centrifugation processes,
the final concentration of the SWNT solution was
70 mgmLÀ1 (see Experimental Section for details). The
nanotube solution was mixed with an aqueous solution of 1
(5 mgLÀ1, 11 mm) to give Pyr-NT. For specific anchoring of
BSA, a solution of Mal-Pyr-NT was prepared by mixing p-
NT with pyrene-containing maleimide (Mal-Pyr) and treated
with a solution of BSA (14 mm) in HEPES to give BSA-Mal-
Pyr-NT. The resulting dispersions were characterized by
Raman, absorption, and emission spectroscopic analysis.
The Raman spectra of purified SWNTs (p-NT), SWNT/
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Controlled Adsorption of Proteins on Carbon Nanotubes
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Figure 1. a) XPS spectrum of the purified SWNTs p-NT. b) High-resolu-
tion spectrum of carbon (C 1s) deconvoluted with four peaks at 284.2,
284.4, 286.0, and 288.7 eV. c) High-resolution spectrum of oxygen (O 1s)
deconvoluted with two peaks at 531.8 and 533.2 eV.
pyrene (Pyr-NT), and SWNT/pyrene/BSA (BSA-Mal-Pyr-
NT) derivatives (Figure 2a) show the G and radial breathing
mode (RBM) bands characteristic of the nanotubes. It is of
interest to note that the presence of pyrene and BSA does
not have significant influence on the signal of the nanotubes.
The absorption and emission spectra of the Pyr-NT solution
show the presence of peaks that correspond to pyrene deriv-
ative 1 (Figure 2band c). To demonstrate the effect of the
pyrene derivative 1 against protein aggregation, we per-
formed a series of experiments in which either bare purified
nanotubes p-NT or nanotubes covered with 1, that is, Pyr-
NT, were exposed to BSA. The resulting biohybrids were in-
vestigated by using tapping mode AFM and compared with
the nanotubes functionalized with BSA through the pyrene/
maleimide linker (BSA-Mal-Pyr-NT). For each sample, the
AFM measurements were repeated several times based on
three separate batch preparations (that is, a different reac-
tion took place in each batch). The synthetic pathway for
the preparation of the nanotube/BSA biohybrids is given in
Scheme 2. First, a solution of purified SWNTs p-NT was
Figure 2. a) Raman spectra with excitation at l=457.9 nm of the purified
SWNTs p-NT, Pyr-NT, and biohybrids BSA-Mal-Pyr-NT. b) Absorption
and c) emission spectra of Pyr and Pyr-NT in HEPES (10 mm), respec-
tively.
mixed with a solution of BSA (14 mm) in HEPES (Scheme 2,
middle). The resulting NT/BSA solution was deposited on a
polylysine-treated mica surface. The AFM pictures showed
a net increase in diameter distribution for the nanotubes
after exposure to BSA. The p-NT nanotubes have an aver-
age profile height of approximately 0.9–1.2 nm according to
the sample (Figure 3a), whereas the nanotubes exposed to
BSA (i.e., NT/BSA) show an average diameter of about
5.8 nm (Figure 3b). This observation is consistent with the
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S. Campidelli, A. Knyazev et al.
Scheme 2. Representation of the noncovalent functionalization of carbon nanotubes with BSA.
presence of BSA on the nanotube sidewalls because the
BSA adsorbs spontaneously through hydrophobic interac-
tions. The average diameter of free BSA at neutral pH is
known to be about 5.3 nm.[35,63]
(14 mm) in HEPES, average diameters of roughly 8 nm were
observed for the BSA-Mal-Pyr-NT biohybrids (Figure 3e)
instead of 3.5 nm for Pyr-NT. This observation is in agree-
ment with the coating of the nanotube sidewalls with BSA.
These results confirm that pyrene derivatives are a very
useful and powerful class of material for nanotube function-
alization. Theoretical studies demonstrated the affinity of ar-
omatic molecules for the nanotube sidewalls,[64–66] and, in
particular, pyrene derivatives are known to bind strongly
onto nanotube sidewalls, with a binding energy of approxi-
mately 0.45 eV (ca. 43 kJmolÀ1).[67] However, we recently
demonstrated that pyrene molecules located on the nano-
tube sidewalls could be displaced.[52] Therefore, to obtain in-
creased stability for the biohybrids, we investigated the pos-
sibility of covalently introducing BSA onto SWNTs.
The “protective” character of pyrene 1 was verified by ex-
posing SWNT/pyrene to BSA. A solution of Pyr-NT was
mixed with a solution of BSA (14 mm) in HEPES (Scheme 2,
left) and the two solutions (i.e., Pyr-NT and Pyr-NT/BSA)
were investigated by AFM. First, the dispersed nanotubes
(pyrene 1) possess a diameter of about 3.5 nm (Figure 3c).
The difference between the diameters of p-NT and Pyr-NT
is due to the presence of pyrene 1 onto the nanotube side-
walls, thus representing an increase of about 2.3–2.6 nm.
This outcome is in accordance with the coating of the nano-
tubes by the pyrene derivative (in fully extended conforma-
tion the length of the PEG-treated chain of 1 is 2.0 nm).
More interestingly, when this solution was exposed to BSA,
the average diameter of the nanotubes (Pyr-NT/BSA) did
not change, thus indicating that no protein was adsorbed on
the nanotubes (Figure 3d).
Covalent approach: The synthetic pathway for the elabora-
tion of the nanotube/BSA hybrids is depicted in Scheme 3.
First, purified SWNTs were modified with 4-(2-aminoethyl)-
benzenediazonium tetrafluoroborate[68] by using the diazoni-
um addition reaction[69] to give f-NT nanotube derivatives,
which were treated with 4-maleimidobutyric acid N-hydrox-
ysuccinimide ester to give Mal-NT. Solutions of both f-NT
and Mal-NT were incubated with a solution of BSA (14 mm)
in HEPES to give f-NT/BSA and Mal-NT/BSA, respectively.
The functionalized nanotubes were characterized by Raman,
In the last experiment, BSA was allowed to react with
Mal-Pyr-NT (Scheme 2, right). We observed that the pres-
ence of the maleimide unit on the pyrene 1 did not signifi-
cantly modify the average diameter of the nanotubes (Mal-
Pyr-NT) with respect to Pyr-NT (data not shown). However,
after exposure of the Mal-Pyr-NT to the solution of BSA
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Controlled Adsorption of Proteins on Carbon Nanotubes
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Figure 3. AFM images, profiles, and 3D representations of a) p-NT, b) p-NT/BSA, c) Pyr-NT, d) Pyr-NT/BSA, and e) BSA-Mal-Pyr-NT nanotube sys-
tems. The images showed in particular the evolution of the aspect of the nanotube derivatives as a function of the different treatments.
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S. Campidelli, A. Knyazev et al.
Scheme 3. Representation of the covalent functionalization of carbon nanotubes with BSA.
and absorption spectroscopic analysis and the nanotube/
BSA biohybrids were investigated by AFM. The Raman
spectra of the p-NT and f-NT nanotubes are shown in Fig-
ure 4a. The spectrum of the functionalized SWNTs f-NT
showed a typical increase of the D-band at about 1360 cmÀ1,
which is characteristic of the covalent functionalization of
SWNTs.[70] The absorption spectrum of f-NT showed a par-
tial loss of the transitions between the van Hove singulari-
ties when compared to that of purified SWNTs p-NT (Fig-
ure 4b). These observations are in accordance with the gen-
erally observed modifications that arise from the covalent
attachment of organic moieties to the sidewalls of
SWNTs.[70] As before, the different solutions of nanotubes
exposed to BSA were investigated by AFM. A typical image
of f-NT nanotubes is shown in Figure 5a; the functionalized
nanotubes exhibited an average height profile of about
1.5 nm. After exposure to BSA, the average diameter of the
nanotubes increased significantly (Figure 5b) and the pres-
ence of BSA was easily distinguishable on the nanotubes
and surfaces. Interestingly, the diazonium-functionalized
SWNTs f-NT were not fully coated with the proteins as p-
NT (Figure 3b). The presence of the functional groups dis-
turbed (but did not completely prevent) BSA adsorption.
After modification of the functionalized nanotubes with
maleimide and treatment with BSA, the diameters of the
Figure 4. a) Raman spectra with excitation at l=457.9 nm of the purified
SWNTs p-NT and covalently functionalized SWNTs f-NT (the spectra
were shifted for clarity). b) Absorption spectra of p-NT and f-NT nano-
tubes in HEPES (10 mm).
Mal-NT/BSA biohybrids were about 8 nm (Figure 5c). Al-
though the coating seems more homogeneous, we think that
a part of the BSA is physisorbed on the nanotubes (hydro-
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Controlled Adsorption of Proteins on Carbon Nanotubes
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Figure 5. AFM images, profiles, and 3D representations of a) f-NT, b) f-NT/BSA, and c) Mal-NT/BSA nanotube systems.
phobic interactions) and another part is chemisorbed (cova-
lent coupling through the maleimide groups). This behavior
constitutes the main differences with the noncovalent
system, which can be summarized as follows: 1) Unspecific
adsorption of protein is prevented in the noncovalent
system by the presence of the ethyleneglycol chain on the
pyrene unit and BSA adsorption is only possible through
chemical reaction when pyrene 1 is functionalized with mal-
eimide. 2) The presence of 4-(2-aminoethyl)phenyl groups
on the nanotubes in the covalent system does not provide
efficient protection against BSA adsorption (simply because
the protecting role is played by the ethyleneglycol chains)
and, in this case, nonspecific adsorption can occur. Finally,
when the maleimide linker is attached on the f-NT nano-
tubes BSA can adsorb following two different ways, that is,
specific adsorption through reaction with the maleimide
groups and nonspecific adsorption on the nanotube side-
walls. Unlike the noncovalent approach, the covalent graft-
ing of diazonium unit did not provide efficient protection
versus the hydrophobic aggregation of BSA proteins onto
the nanotube sidewalls. Such a system, in which a part of
the protein is covalently attached and therefore impossible
to remove from the nanotubes while another part is just
physisorbed, is valuable in the study of the dynamic of ex-
change of proteins on nano-objects (studies in this direction
are currently ongoing).
The two approaches described herein are complementary.
The noncovalent approach provides a tool for decorating
carbon nanotubes while preserving the sp2 framework integ-
rity. In addition, pyrene 1 is also prevents the physisorption
of proteins. The covalent approach provides permanent at-
tachment of BSA proteins onto the nanotubes. This advant-
age could be crucial for the realization of drug-delivery ves-
sels in which the risk of precipitation or aggregation in cells
is decreased due to the covalent shell of the proteins.
Conclusion
Herein, we elaborated carbon nanotubes with a tailored
core to shell architectures. The control of the formation of
carbon-nanotube-based biohybrids is one of the key steps
for the use of nanotubes in drug-delivery systems, imaging
in vivo, and understanding their toxicity. We demonstrated
that it is possible to decrease or suppress the undesirable ad-
sorption of proteins onto the nanotube sidewall by using
proper functionalization (either covalent or noncovalent).
Proteins can be subsequently attached to the nanotubes by
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S. Campidelli, A. Knyazev et al.
by the addition of diisopropylethylamine (DIEA). The nanotubes were
filtered, washed with water, and redispersed in HEPES (10 mm), and 4-
maleimidobutyric acid N-hydroxysuccinimide ester (1 mg) was added to
the reaction mixture. The resulting Mal-NT derivatives were exposed to
a solution of BSA (14 mm) in HEPES, thus yielding Mal-NT/BSA.
activating the functional groups already present on the
nanotube sidewalls. Our approach is simple, straightforward,
and potentially suitable for other biomolecules that contain
thio or amino groups available for coupling.
The systems that we have described herein can mimic the
formation of a protein corona. Therefore, several questions
arise from this behavior: Can a stable coating of appropriate
proteins make nano-objects invisible and/or nontoxic for
micro-organisms? Does the treatment facilitate dispersion
and prevent aggregation in vivo? Moreover, these systems
can be used to study the dynamics of the protein-corona for-
mation and the equilibrium between different proteins that
are assumed to exist in biological media. Indeed, Mal-NT/
BSA hybrids contain adsorbed and covalently linked BSA
on the nanotube sidewall; therefore, a part of the proteins
may be removed and replaced while another one will
remain on the nanotubes. All these questions are currently
under investigation.
Acknowledgements
The authors thank the European network STREP “Nanointeract”
(NMP4-CT-2006–033231) (A.K.) and the EC FP7 NAMDIATREAM
(NMP-2009–246479) research project (A.P.-M.) for financial support. E.
Doris, I. Lynch, D. Movia, B. Mustafa Mohamed, J. Conroy, Y. Volkov,
and K. Dawson are thanked for numerous fruitful discussions and P.
Jꢀgou for the XPS analyses.
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Experimental Section
Synthesis of Pyr-NT: Purified SWNTs were prepared following a protocol
described previously.[57] Briefly, pristine SWNTs prepared by laser abla-
tion were dispersed in HNO3 (35%) and heated at 1108C for 4 h. After
filtration and extensive washing with water, the nanotubes were redis-
persed in NaOH (2m), filtered, and washed with NaOH (1m), HCl (1m),
and water. Finally, the bucky paper (i.e., the thin film of carbon nano-
tubes obtained on the top of the filter after the filtration step) was dis-
persed in H2O2 (30%), heated at 1008C for 1 h, filtered, and extensively
washed with water. The purified SWNTs were dispersed in water or
10 mm HEPES solution (10 min at maximum of power of the sonication
bath and then 30 min at 40%) and centrifuged at 10000ꢃg for 15 min.
The supernatant (p-NT) was recovered and used without further treat-
ment. The final concentration of the solution of SWNTs in HEPES was
estimated by using UV/Vis absorption spectroscopy and comparing with
solutions of SWNTs at known concentrations. In this study, the solution
of nanotubes had a concentration of 70 mgmLÀ1 in HEPES. The solution
of p-NT in HEPES was mixed with an aqueous solution of 1 (1 mgmLÀ1
,
2 mm). The ratio used was 1:10 (v/v; Pyr/p-NT) for the concentration of
carbon nanotubes of approximately 70 mgmLÀ1. The resulting Pyr-NT so-
lution was tested for the adsorption of BSA: a solution of Pyr-NT was ex-
posed to a solution of BSA (1 mgmLÀ1, 14 mm) in HEPES (10 mm) in
ratio of 1:1 (v/v) and investigated by AFM.
Synthesis of BSA-Mal-Pyr-NT: 4-Maleimidobutyric acid N-hydroxysucci-
nimide ester (1 mg, 3.6 mmol) was dissolved in de-ionized water (1.5 mL)
and mixed with an aqueous solution of 1 (120 mL, 220 mm, 26 nmol). The
reaction mixture was stirred for an additional 15 min to give Mal-Pyr. A
solution of p-NT (100 mL , 40 mgmLÀ1) in HEPES (10 mm) was mixed
with the solution of Mal-Pyr (50 mL, 13 nmol) and stirred for 10 min. A
solution of BSA (60 mL, 14 mm) in HEPES was added and the mixture
was stirred for 45 min. The resulting solution was used for an AFM inves-
tigation on a mica substrate.
Synthesis of f-NT: Purified SWNTs (p-NT; 2 mg) were functionalized by
adding 4-(2-aminoethyl)benzenediazonium tetrafluoroborate (2 equiv) to
carbon nanotubes dispersed in an aqueous solution of sodium dodecylsul-
fate (SDS; 10 mm) at room temperature and stirred overnight. The nano-
tube derivatives were filtered and extensively washed with water and ace-
tone. The nanotubes were redispersed in SDS (10 mm), and the function-
alization procedure was repeated twice to reach maximum functionaliza-
tion. A portion of f-NT was redispersed in HEPES (10 mm) and exposed
to a solution of BSA (14 mm) in HEPES.
Synthesis of Mal-NT: The f-NT nanotubes (0.5 mg) were dispersed in N-
methyl-2-pyrrolidone (NMP) and the ammonium salts were neutralized
14670
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Chem. Eur. J. 2011, 17, 14663 – 14671

Controlled Adsorption of Proteins on Carbon Nanotubes
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Received: April 17, 2011
Revised: August 19, 2011
Published online: November 17, 2011
Chem. Eur. J. 2011, 17, 14663 – 14671
ꢂ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
14671