Noncovalent functionalization of carbon nanotubes by fluorescent polypeptides: Supramolecular conjugates with pH-dependent absorbance and fluorescence

Article abstract of DOI:10.1166/jnn.2013.7851

Fluorescent cut single-walled carbon nanotube (CSWCNT) were prepared by simply mixing CSWCNT with water soluble rhodamine 6G (Rh6G) conjugated poly(3,4-dihydroxyphenylalanine) and poly(L-tyrosine) to form highly stable product with good dispersity in buffer solution. The optical absorbance and fluorescence spectra of the resulting fluorescent CSWCNT display interesting pH-dependent optical properties, emitting strong fluorescence only in acidic environment. Considering the extracellular pH of tumor tissue is acidic, the pH-sensitive conjugates have advantages to sense tumor cells selectively, enabling it to be utilized as a biosensor for detecting cancer cells. The protocol employed to functionalize the CSWCNT with Rh6G conjugated polypeptides in aqueous solution is proven to be direct, fast and easily scalable. Copyright

Full text of DOI:10.1166/jnn.2013.7851

Copyright © 2013 American Scientific Publishers
All rights reserved
Printed in the United States of America
Journal of
Nanoscience and Nanotechnology
Vol. 13, 7406–7412, 2013
Noncovalent Functionalization of Carbon Nanotubes by
Fluorescent Polypeptides: Supramolecular Conjugates
with pH-Dependent Absorbance and Fluorescence
Xin Hua Huang, Renjith P. Johnson, Song I. Song, and Il Kim∗
WCU Centre for Synthetic Polymer Bioconjugate Hybrid Materials, Department of Polymer Science and Engineering,
Pusan National University, Pusan 609-735, Korea
Fluorescent cut single-walled carbon nanotube (CSWCNT) were prepared by simply mixing
CSWCNT with water soluble rhodamine 6G (Rh6G) conjugated poly(3,4-dihydroxyphenylalanine)
and poly(L-tyrosine) to form highly stable product with good dispersity in buffer solution. The opti-
cal absorbance and fluorescence spectra of the resulting fluorescent CSWCNT display interesting
pH-dependent optical properties, emitting strong fluorescence only in acidic environment. Consid-
ering the extracellular pH of tumor tissue is acidic, the pH-sensitive conjugates have advantages to
sense tumor cells selectively, enabling it to be utilized as a biosensor for detecting cancer cells. The
protocol employed to functionalize the CSWCNT with Rh6G conjugated polypeptides in aqueous
solution is proven to be direct, fast and easily scalable.
Keywords: Carbon
Nanotube,
Fluorescence,
Poly(3,4-dihydroxyphenylalanine),
Poly(L-tyrosine), Rhodamine 6G, pH Sensitivity.
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Copyright: American Scientific Publishers
1. INTRODUCTION
SWCNT and thus undesirably compromises the electronic
and mechanical properties.8ꢀ9 Moreover, the utilization of
covalent-modified SWCNT may lead to the non-uniform
coverage of additional components that tend to attach at
the ends and defective sites of SWCNT, where the con-
centration of functional moieties are the largest. To this
end, liable fabrication of SWCNT-based heterogeneous
nanohybrids via non-covalent approaches have attracted
immense interest.
In a typical non-covalent process, easily dispersible
SWCNT sols can be achieved by deposition of functional
molecules onto SWCNT sidewalls through ꢁ–ꢁ stacking
and/or wrapping interactions. Mediated by the deposited
molecules, these surface-modified SWCNTs can be fur-
ther assembled with a variety of nanoparticles or inorganic
oxide materials by means of in situ synthesis technique,
yielding SWCNT-based hybrids that exhibit tailored prop-
erties while still reserving nearly all the intrinsic properties
of SWCNT.5 In these non-covalent processes, the sidewall
modifier of SWCNT acts as a “bridge” to integrate func-
tional components onto SWCNT sidewalls. Therefore, the
molecular composition and structure of modifiers directly
decide the efficiency and diversity of the fabrication of
novel SWCNT-based nanohybrids.
Single-walled carbon nanotubes (SWCNTs) continue to
gain tremendous interest in recent decades owing to their
unique physical, chemical and physiological properties,
thus enabling them for use in many applications in the field
of nanoscience, nanotechnology and biotechnology.1–4
Nowadays, two of the most investigated SWCNT-based
nanohybrids were prepaeared from the deposition of flu-
orescein and fluorescent hybrids onto the SWCNT side-
walls, which exhibit interesting pH-dependent optical
properties, such as pH-influenced fluorescent selectivity
with increased fluorescence and absorption intensity only
in acidic environment.5ꢀ6
To efficiently synthesize SWCNT-based fluorescent
materials, it is vital to activate the graphitic surface of
SWCNT that tends to be chemically inert and shows
solubility.5 In this direction, the removal of the toxic
metal ions, the decrease of SWCNT length and the cova-
lent decoration of SWCNT sidewall with molecular or
polymeric entities provide a direct and effective route
to fabricate surface-modified SWCNT derivatives.7 How-
ever, the covalent functionaliztion of SWCNT unavoid-
ably causes the destruction of the band structures of
In this work, water soluble fluorescent rhodamine
6G (Rh6G) conjugated poly(3,4-dihydroxyphenylalanine)
∗Author to whom correspondence should be addressed.
7406
J. Nanosci. Nanotechnol. 2013, Vol. 13, No. 11
1533-4880/2013/13/7406/007
doi:10.1166/jnn.2013.7851

Huang et al.
Noncovalent Functionalization of Carbon Nanotubes by Fluorescent Polypeptides
[p(DOPA)] and poly(L-tyrosine) [p(Tyr)] were chosen as
maker for further modification of cut single-walled carbon
nanotube (CSWCNT) to preparae a new hybrid material.
We present a facile protocol to fabricate the CSWCNT
based on a superficially modification of CSWCNT via
a non-covalent function with p(DOPA)-Rh6G [or p(Tyr)-
Rh6G] in a one-pot reaction. The properties of the resul-
tant fluorescent hybrid materials were studied in deepth
and the detailed mechanism was also investigated and
discussed.
further purification, dimethylformamide (DMF), tetrahy-
drofuran (THF) and water were purified according to gen-
eral procedures before use.
2.2. Characterizations
1H NMR and 13C NMR spectra were recorded on
1
400 MHz H (100 MHz 13C) spectrometer (Varian Unity
Plus), and the fast-atom bombardment mass spectrum
(FAB-MS) was recorded on a Jeol JMS DX300 appa-
ratus (Jeol, Tokyo, Japan). Molecular weight (MW) and
polydispersity index (PDI) were measured by using a HP
1100 series gel permeation chromatograph (GPC) with
polystyrene as the standard, using DMF as the eluent at
a flow rate of 1 mL min−1. Samples for transmission
electron microscopy (TEM) were deposited onto carbon
coated copper electron microscope grids and dried in air.
The size, shape and fine structures of nanoparticles (NPs)
were investigated using a Hitachi MODEL H-7600 (Nissei,
Japan) low resolution and a Jeol 1200 EX II high res-
olution TEMs. Fourier transform infrared (FTIR) spectra
were obtained at a resolution of 1 cm−1 with a Shimadzu
IR Prestige–21 (Kyoto, Japan) spectrophotometer between
2. EXPERIMENTAL DETAILS
2.1. Materials
Rh6G (98%), isophorone diisocyanate (IPDI, 99%),
sodium hydroxide (NaOH, 99%), ethylenediamine (96%),
tin octoate (98%), DOPA (99%), L-tyrosine (99%), HBr/
acetic acid (30%), trifluoroacetic acid (98%), triphosgene
(98%), SWCNT (97%), sulfuric acid (H2SO4, 98%), nitric
acid (HNO3, 65%), benzyl chloroformate (CBz-Cl, 98%)
and phosphorus pentachloride (PCl5, 99%) were obtained
from Sigma Aldrich, and all of them were used without
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Scheme 1. Synthesis procedures of Rh6G conjugated p(DOPA) and p(Tyr): (i) NaOH (30%); (ii) IPDI, THF, 40 ꢀC, tin octoate, 4 h; (iii) DMF, r,t.,
ethylenediamine; (iv) and (vi) DMF, 40 ꢀC, 40 h; and (v) HBr/acetic acid, CF3COOH, CH2Cl2.
J. Nanosci. Nanotechnol. 13, 7406–7412, 2013
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Noncovalent Functionalization of Carbon Nanotubes by Fluorescent Polypeptides
Huang et al.
4000 and 400 cm−1. The IR measurements of the pow-
der samples were performed in the form of KBr pellets.
UV-Vis absorption spectra of the samples were recorded
at room temperature on a Shimadzu UV-1650PC spec-
trometer (Kyoto, Japan). All the fluorescence spectra were
obtained using a Hitachi spectrofluorometer model F-4500.
Zeta potential was conducted using a Malvern Zetasizer
3000 particle size and zeta potential analyzer (Worcester-
shire, UK) at room temperature.
(CBz) protected DOPA NCA (400 mg, 8ꢅ15 × 10−4 mol)
were added into DMF (10 mL) solution. The mixture
ꢀ
was stirred at 40 C for 2 days. The residue was puri-
fied by precipitation and the CBz groups were deprotected
under HBr/acetic acid condition. The resulting water solu-
ble Rh6G conjugated poly(DOPA) [p(DOPA)-Rh6G] was
ꢀ
1
subsequently dried for 10 h at 50 C under vacuum. H
NMR (D2O, 400 MHZ) ꢂ 7.90, 7.54, 7.21, 6.74, 6.71, 6.57
(phenyl ring), 4.59 (OH), 3.52–2.56 (CH2 and CH derived
from p(DOPA)-Rh6G backbone), 2.06–1.81 (CH2 derived
from p(DOPA)-Rh6G backbone), 1.21–0.99 (CH3ꢃ, num-
ber average molecular weight (Mnꢃ = 5500, PDI:1.03.
The water soluble Rh6G conjugated poly(L-tyrosine)
[p(Tyr)-Rh6G)] was prepared by the ROP of L-tyrosine
NCA: 1H NMR (D2O, 400 MHZ) ꢂ 7.81, 7.05, 6.93, 6.75,
6.50 (phenyl ring), 4.40 (OH), 4.02, 3.83, 3.396, 3.08–
2.62 (CH2 and CH derived from p(Tyr)-Rh6G backbone),
1.90 (CH2 derived from p(Tyr)-Rh6G backbone), 1.10–
1.04 (CH3ꢃ, Mn = 7500, PDI = 1ꢅ01.
2.3. Synthesis of Rh6G Amine
The primary amine functionalized Rh6G-NH2 was syn-
thesized following the procedure in Scheme 1 to obtain
a pink solid ꢀafter vaccum drying. Yield: 86.5%. Melting
1
point = 165 C, H NMR (DMSO-d6, 400 MHZ): ꢂ 1.19
(m, 24 H, –CH3ꢃ, 1.83 (m, 10 H, –CH2–), 2.10 (m, 3
H, –CH3), 2.91 (t, 2 H, –CH2–), 3.10 (m, 4 H, –CH2–),
3.15 (s, 1 H, CH) 5.03 (s, NH), 6.06 (s, 2 H, ArH), 6.25
(s, 2 H, ArH), 6.93 (1 H, t, ArH), 7.45 (d, 2 H, ArH),
7.74 (d, 1 H, ArH). 13C NMR (DMSO-d6, 100 MHZ):
ꢂ 167.4, 154.0, 151.4, 148.0, 132.0, 131.0, 128.6, 128.0,
124.0, 122.7, 118.6, 105.2, 96.1, 64.6, 43.9, 37.9, 17.4,
14.6. IR (KBr) 3437, 3370, 2963, 2926, 2852, 1682, 1637,
1607, 1518, 1281, 1222, 1007, 815, 748 cm−1. FAB-Mass
m/z (%) 726.1 (100) [M+H]+.
2.5. Cutting and Purification of SWCNT
The cutting and purification of SWCNT was essen-
tially carried out using modified literature procedures.11
Commercial SWCNT (500 mg) was added into a mixture
of H2SO4/HNO3 solution (v/v = 3:1, 200 mL) and exposed
to sonication at room temperature for 24 h. The result-
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ing CSWCNT was then thoroughly rinsed with ultrapure
water and filtered through a microporous filtration mem-
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2.4. Ring Opening Polymerization (ROP) of ꢄ-Amino
Copyright: American Scientific Publishers
brane (F = 0ꢅ22 mm). Obtained product was redispersed
in HNO3 (2.6 M, 200 mL) and heated at reflux for 24 h,
collected by vacuum filtration, and washed several times
with ultrapure water to neutrality. The product was then
Acid N-Carboxyanhydride (NCA) (Scheme 1)
DOPA NCA was synthesized in accordance with the
previous procedures.10 Typically, at nitrogen atmosphere,
Rh6G-NH2 (59 mg, 8ꢅ15 × 10−5 mol) and carboxybenzyl
ꢀ
dried under vacuum at 50 C for 24 h for further use.
Fig. 1. Organic functionalization of SWCNT. Pristine SWCNT can be treated with acid to cut and generate carboxylic groups at the tip and the
sidewall (CSWCNT), and then p(DOPA0-Rh6G [or p(Tyr)-Rh6G] conjugate mixed with CSWCNT to yield noncoordinatively solubilized hybrid in
water.
7408
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Huang et al.
Noncovalent Functionalization of Carbon Nanotubes by Fluorescent Polypeptides
minimize any adverse effects that might be caused by the
dye label.16
In principle, chemical modification of Rh6G may allow
this biomedically relevant chromophore to be readily incor-
porated into polymer chains, either as atom transfer radical
polymerization initiators or as a comonomer. The primary
amine functionalized Rh6G-NH2 has been prepared in high
yield by reacting Rh6G with IPDI, and ethylenediamine
(Scheme 1). The sterically unhindered primary aliphatic
amine groups of Rh6G-NH2 can initiate ROP of NCA to
yield polypeptide.17–19 The control of the degree of poly-
merization can easily be achieved by variation of the feed
ratio NCA/amine (i.e., monomer/initiator ratio). For the
cyclization of amino acids, an amino acid suspension was
treated in a suitable solvent with 1/3 eq. of triphosgene
Fig. 2. TEM images of (a) CSWCNT, (b) p(DOPA)-Rh6G-CSWCNT,
and (c) p(Tyr)-Rh6G-CSWCNT. Inset of each image illustrates the pho-
tograph of water dispersion of each sample.
2.6. Dispersion of CSWCNTs in Aqueous Solution
of p(DOPA)-Rh6G [or p(Tyr)-Rh6G)]
The CSWCNT (1.0 mg) was mixed with 5 mg of
p(DOPA)-Rh6G [or p(Tyr)-Rh6G)] in phosphate-buffered
saline (PBS; 0.01 M, pH 7.4, 10.0 mL). The mixture was
sonicated for 10 min, stirred overnight at room tempera-
ture, and dialyzed against ultrapure water using a dialysis
membrane (molecular weight cut off of 12 K) at room
temperature for 24 h to remove free polypeptides.
3. RESULTS AND DISCUSSION
Rhodamine dyes are generally more photostable than
fluorescein12 and are also relatively water soluble com-
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pared to naphthalimide-based dyes.
ifying the commercially available dye, Rh6G, because it
Copyright: American Scientific Publishers
has an exceptionally high quantum yield (> 0.9) compared
We focus on mod-
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to rhodamine B, which typically has quantum yields of
the order of 0.3–0.4.15 Moreover, given very high absorp-
tion coeficient of Rh6G (in excess of 100000 M−1 cm−1ꢃ,
only a minimal amount of label is necessary, which should
Fig. 3. UV/Vis spectra of CSWCNT, p(DOPA)-Rh6G, p(Tyr)-
Rh6G, p(DOPA)-Rh6G-CSWCNT and p(Tyr)-Rh6G-CSWCNT in PBS
(2.0 ꢆg mL−1ꢃ at pH 7.4.
Fig. 4. UV/Vis spectra of (a) p(DOPA)-Rh6G-CSWCNT and (b)
p(Tyr)-Rh6G-CSWCNT conjugates in PBS (2.0 ꢆg mL−1ꢃ at different
pH values.
J. Nanosci. Nanotechnol. 13, 7406–7412, 2013
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Noncovalent Functionalization of Carbon Nanotubes by Fluorescent Polypeptides
Huang et al.
ꢀ
at 40–50 C usually leads to a completely homogeneous
solution of the corresponding NCA within 1–3 h. Triphos-
gene undergoes nucleophilic attack at the carbonyl carbon;
the trichloromethoxy (Cl3CO−ꢃ leaving group dissociates
to a chloride anion and a molecule of phosgene, which
react immediately. Hence, there is no excess phosgene in
solution. Periodic sparging with nitrogen improves prod-
uct yields by driving the HCl evolved from the reaction
medium. The NCAs of CBz protected DOPA and Tyr were
prepared in good yield in dioxane as the solvent.
and purified CSWCNT is much more short in length (usu-
ally < 500 nm) and well separated, and formed very small
bundles as characterized by TEM observation. After this
process, many carboxylic groups are generated mainly
on the tip of CSWCNT, which futher enhanced the dis-
persibility of CSWCNT in buffer solution to form a stable
suspension.11
Figure 1 shows a schematic diagram for the prepara-
tion of the optical hybrid materials. Produced conjugates
exhibited ideal dispersity in buffer solution. The com-
bination of p(DOPA)-Rh6G and p(Tyr)-Rh6G molecules
with CSWCNT led to roughen the sidewalls of CSWCNT,
due to additional coating layers. The average length of
the CSWCNT conjugates is about 150 nm determined by
TEM observation in Figure 2(a). The p(DOPA)-Rh6G-
CSWCNT and p(Tyr)-Rh6G-CSWCNT suspension were
stable in PBS without aggregation even after one month
(see Figs. 2(b) and (c)). In addition, the zeta potential of
nascent CSWCNT suspension is −8ꢅ38 mV; however it
records to −14ꢅ6 mV and −13ꢅ7 mV for p(DOPA)-Rh6G-
CSWCNT and p(Tyr)-Rh6G-CSWCNT, respectively, indi-
cating that the positively charged polypeptides have been
successfully attached to the negatively charged CSWCNTs
due to electrostatic interactions in combination with ꢁ–ꢁ
stackings.20 When the Rh6G conjugated polypeptides are
added to the aqueous CSWCNT solution, non-covalent,
ROP of CBz protected DOPA NCA and Tyr NCA were
performed at 40 C in DMF for 2d by using Rh6G-NH2
ꢀ
as the initiator after controlling the monomer to initiator
ratio 100 (Scheme 1). The CBz groups in p(DOPA)-Rh6G
was deprotected under the HBr/acetic acid condition. The
Mn value of p(DOPA)-Rh6G and p(Tyr)-Rh6G conju-
gates was 5500 and 7500, respectively. In addition they
were characterized by very low PDI value less than 1.03,
demonstrating the resulting polymers are very uniform and
the initiation step is much faster than the growing step.
The pristine SWCNT tends to cluster into large bundles
so that they have very limited solubility in common sol-
vents. Moreover, some metal particles are usually included
in the bundles of SWCNT and thus can pose consider-
able biological toxicity. To eliminate these concerns, the
SWCNT used in this study was cut and purified. The cut
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Fig. 5. Photoluminescence spectra of (a) p(DOPA)-Rh6G-CSWCNT and (b) p(Tyr)-Rh6G-CSWCNT in PBS (20 ꢆg mL−1ꢃ at pH 7.4; photolumines-
cence spectra of (c) p(DOPA)-Rh6G-CSWCNT and (d) p(Tyr)-Rh6G-CSWCNT conjugates measured at different pH values.
7410
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Huang et al.
Noncovalent Functionalization of Carbon Nanotubes by Fluorescent Polypeptides
electrostatic (ion-dipole) interactions exist among the elec-
tropositive CSWCNTs and hydroxyl groups of inter- or
intrapolypeptide molecules. Such interactions force the
polypeptide molecules to be tightly trapped within the
voids of CSWCNT to form stable CSWCNT conjugates.
Once Rh6G conjugated polypeptide is conjugated
onto CSWCNTs, the interaction between two com-
ponents is concerted. In PBS at pH 7.4, absorption
peaks at 523 and 522 nm corresponding to p(DOPA)-
Rh6G and p(Tyr)-Rh6G, respectively, became nortably
broadened and shifted to 526 and 542 nm, respec-
tively, for p(DOPA)-Rh6G-CSWCNT and p(Tyr)-Rh6G-
CSWCNT (Fig. 3). Interestingly, decreasing pH values,
the UV-Vis absorption of p(DOPA)-Rh6G-CSWCNT and
p(Tyr)-Rh6G-CSWCNT conjugates became stronger with
a continuous red shift (Fig. 4).
successfully synthesized by the ROP of CBz protected
DOPA NCA and Tyr NCA, using Rh6G containing pri-
mary amine in structure as an initiator. Simple mix-
ing of the resulting p(DOPA)-Rh6G and p(Tyr)-Rh6G
conjugates with CSWCNT resulted in noncovalent inter-
ations between p(DOPA)-Rh6G or p(Tyr)-Rh6G and
CSWCNT to form highly stable product with good dis-
persity in buffer solution. The p(DOPA)-Rh6G-CSWCNT
and p(Tyr)-Rh6G-CSWCNT conjugates displayed interest-
ing pH-dependent optical properties emitting strong flu-
orescence only in acidic environment. Considering the
extracellular pH of tumor tissue is acidic, pH-sensitive
conjugates have advantages to sense tumor cells selec-
tively, which will be a fascinating topic for further
study.
It is also shown that the fluorescence of p(DOPA)-
Rh6G-CSWCNT and p(Tyr)-Rh6G-CSWCNT conjugates
is negligible in comparison to that of pure polypep-
tide excited at 523 nm at pH 7.4 (Figs. 5(a) and (b)).
These results suggest that there are strong interactions
between Rh6G conjugated polypeptide and CSWCNT.
As in the case of other noncovalently bonded prepara-
tions of fluorescent CSWCNTs,21ꢀ22 the energy and elec-
trons from the excitation of polypeptide might directly
flow into CSWCNTs and thus quench the fluores-
cence of polypeptide.5 Surprisingly, lowering the pH
Acknowledgments: This work was supported by
the Basic Science Research Program through the
National Research Foundation of Korea funded by
the Ministry of Education, Science and Technology
(2012R1A1A2041315).
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(d)). These results clearly indicate that p(DOPA)-Rh6G-
CSWCNT and p(Tyr)-Rh6G-CSWCNT conjugates are of
distinct pH dependence. The acidic solution might weaken
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sidewalls of CSWCNT at a very low pH. It can be vali-
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filtrate after the separation of p(DOPA)-Rh6G-CSWCNT
and p(Tyr)-Rh6G-CSWCNT conjugates at pH 2.0. Since
the Rh6G conjugated polypeptides and CSWCNT are
weak acid, the pH-dependent properties of p(DOPA)-
Rh6G-CSWCNT and p(Tyr)-Rh6G-CSWCNT conjugates
may result from the carboxylate ions and multi-hydroxyl
groups in the conjugates. Note that p(DOPA)-Rh6G and
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4. CONCLUSIONS
p(DOPA)-Rh6G and p(Tyr)-Rh6G with Mn value of 5500
(PDI = 1ꢅ03) and 7500 (PDI = 1ꢅ01), respectively, were
J. Nanosci. Nanotechnol. 13, 7406–7412, 2013
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Noncovalent Functionalization of Carbon Nanotubes by Fluorescent Polypeptides
Huang et al.
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Received: 30 April 2012. Accepted: 21 January 2013.
Delivered by Publishing Technology to: Chinese University of Hong Kong
IP: 204.12.196.98 On: Tue, 12 Jan 2016 05:43:21
Copyright: American Scientific Publishers
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J. Nanosci. Nanotechnol. 13, 7406–7412, 2013