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Instituto de Ciencias Nucleares, Universidad Nacional Autnoma de Mxico, Circuito Exterior C.U., 04510 Mxico D.F., Mexico
Instituto de Fsica, Dpto. Materia Condensada, Universidad Nacional Autnoma de Mxico, Coyoacn, 04510 Mxico D.F., Mexico
c
Instituto de Fsica, Dpto. Fsica Experimental, Universidad Nacional Autnoma de Mxico, Coyoacn, Mxico, DF 04510, Mexico
d
Unidad de Investigacin Biomdica en Cncer INCan-UNAM, Instituto Nacional de Cancerologa, Mxico, DF 14080, Mexico
b
a r t i c l e
i n f o
Article history:
Received 18 June 2014
Received in revised form 17 October 2014
Accepted 25 October 2014
Available online 10 November 2014
Keywords:
Single-walled carbon nanotube
Silver nanoclusters
Human serum albumin
Hybrid nanomaterials
a b s t r a c t
The attachment of silver nanoclusters (AgNCs) onto single-walled carbon nanotubes (SWNTs) for the formation of integrated uorescence sites has attracted much attention due their potential applications as
biological probes and nanovectors in theragnosis. Here, we report the preparation through assembly of
uorescent quasi 1-D nanomaterial based on SWNTs and silver nanoclusters (AgNCs) non-covalently
attached to human serum albumin as biological linker. The uorescent SWNTAgNCsHSA conjugates were characterized by atomic force microscopy, high-resolution transmission electron microscopy
(HRTEM), high angle annular dark eld scanning TEM (HAADF-STEM), uorescent and UVvis spectroscopy. The above techniques conrmed that AgNCs were non-covalently attached onto the external
surface of SWNTs. In addition, it was observed that the modication did not affect the optical properties of the synthesized AgNCs since the absorption spectra and uorescence under UV irradiation
( = 365 nm) remain the same. The effect of the functionalized systems was tested on mammal red blood
cells (RBCs) and it was found that their structural integrity was compromised by the conjugates, limiting
their biological and medical applications.
2014 Elsevier B.V. All rights reserved.
1. Introduction
Single-walled carbon nanotubes (SWNTs) are quasi onedimensional (1-D) nanomaterials with unique physical and
chemical properties that can be used for applications in life
sciences and medicine [1]. Particularly, the uorescence of SWNTs
(in the near-infrared spectrum) is a promising dye in biological
applications and for the design of optical biomedical sensors
[15]. Fluorescent SWNTs has been recently explored as dyes for
targeting cancer cells and as contrast agents to enhance organs
visualization [1,2]. However, it is well known that biological
applications of bared SWNTs are restricted due to their toxicity
[6]. By using organic coatings such as chromophores containing
polymers and uorescent organic dyes onto CNTs, SWNTs become
biocompatible and, thus, an excellent alternative as uorescent
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It is well known HSA protein is ideal to disperse carbon nanotubes in solution because of its strong adsorption onto hydrophobic
surfaces [20,21]. The ease formation of conjugated SWNTs and HSA
has been reported [21]. The adsorption onto the SWNTs sidewalls
occurs spontaneously due to Van der Waals interactions between
the protein and the carbon nanotubes [2224]. Furthermore, HSA
has been used as linking molecules between AgNPs and multiwalled carbon nanotubes (MWNTs) to form hybrid materials under
mild conditions [23].
Herein, by using the dual hydrophobichydrophilic capability
of HSA to act as template in the molecular coupling to synthesize the AgNCs and to be easily adsorbed onto SWNTs we obtained
the rst assemblies SWNTs with uorescent AgNCs wrapped up
with the HSA as linking molecule. The method employed demonstrated the combination of unique specic molecular properties
of the three nanomaterials, resulting in an interesting molecular
aggregation with potential applications. The conjugates were characterized by UVvis spectroscopy and microscopy techniques such
as atomic force microscopy, high-resolution transmission electron microscopy and dark-eld scanning transmission electron
microscopy. Additionally, by using scanning electron microscopy
(SEM), the functionalized SWNTs were used to test the structural
integrity of the membrane of red blood cells (RBCs), in order to
explore the toxicity of these materials.
Silver nanoclusters synthesized using HSA as biological template (Fig. 1) were incubated with SWNTs (Fig. 2a). The UVvis
absorption spectra of the conjugates showed two absorption peaks,
at 280 nm and 450 nm, assigned as HSA and AgNCs, respectively
(Fig. 3a) [15]. The absorption of HSA is attributed to the *
transition of aromatic amino acids. At the same time, it is well
known that the UVvis absorption spectra of nanoclusters exhibit
molecule-like optical transition with absorbance bands due to the
quasi-continuous electronic energy band structure and quantum
connement effects [15]. The absorption spectrum of AgNCs was
broad and low, from 400 to 500 nm, since the optical properties
of the nanoclusters were apparently affected by the protein shell
[15]. Furthermore, a different method for the formation (referred
to as control samples in Section 2, Fig. 2b) of silver nanoclusters was tested. The protein coating deposited onto the side-walls
of SWNTs was used as template for the deposition of silver ions
and then reduced with sodium borohydride. The reduction process was performed under two different pH conditions: (a) at
pH 12, since an alkaline media is required for the nanoclusters
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Fig. 1. Representative 3D AFM image of synthetized nanoclusters deposited on mica (a) and corresponding particle size histogram (c). HRTEM image of nanoclusters (b) and
corresponding particle size histogram (d). EDS spectrum of silver nanoclusters (inset in (b)).
dynamic for the correct interaction between lateral chains and silver ions, and furthermore this might restrict the connement effect
that is known to abolish the growing process towards nanoparticles
[22,23].
The as-prepared conjugates displayed a yellow color under
ambient light and orange color under the UV lamp (inset in Fig. 3c).
Fig. 3c shows the uorescence spectra of conjugates in aqueous
solution, as shown, they exhibit an intense absorption band centered at 480 nm and the emission spectrum displays an emission
Fig. 2. Schematic of the conjugation protocols for the non-covalent attachment of silver nanoclusters onto single-walled carbon nanotubes. The silver nanoclusters were
synthetized before their conjugation with SWNTs (a). In control samples the silver reduction was after the incubation of HSA with the SWNTs (b). The rst approach let the
formation of conjugates with AgNCs. In contrast the second way address the AgNPs formation.
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Fig. 3. Low magnication (a) and high magnication (b) of UVvis spectra of conjugates formed with AgNCs synthetized before their conjugation (purple line) and with silver
reduction was after the incubation at alkaline and basic pH (red and green line respectively). Absorption and emission spectra of conjugates formed with AgNCs synthetized
before their conjugation (c), the inserts are photographs of conjugates under ambient light and UV lamp. Emission spectra (d) of conjugates prepared at several concentrations
of SWNTs (SWNTs:AgNCs; 0.025:2.5, 0.25:2.5 and 2.5:2.5 mg/mL, purple, black and pink lines respectively). For interpretation of the references to color in this gure legend,
the reader is referred to the web version of this article.
all the carbon nanotube samples, showing high conjugation. Comparative transversal cross sections analysis of the bare nanotubes
and their conjugates (Fig. 4b and d) conrmed the deposition. The
carbon nanotubes exhibited heights around 4 nm and a at longitudinal prole (Fig. 4e and f). In contrast, the conjugates showed
transversal proles with an increase of 3 nm and a longitudinal
prole with peaks and valleys with heights of 23 nm (Fig. 4e and
f), which correspond to the nanocluster-covered and bare surfaces
respectively [6].
Fig. 5 shows HRTEM images of pristine SWNTs, HSA-decorated
nanotubes and conjugates (a, b and c, respectively). The conjugates showed few black spots, with a size of approximately 2 nm,
embedded into a protein lm that was covering the nanotube
sidewalls (Fig. 5f). The EDS analysis revealed silver peaks that suggested the black spots might be the AgNCs. In contrast, bare and
HSA-decorated SWNTs did not exhibit black spots and showed the
absence of silver signals in EDS. A HAADF-STEM analysis was performed and interestingly contrasted with that observed in HRTEM,
the images showed a big number of AgNCs along the SWNTs surface (bright spots in Fig. 5d). This difference is explained by the
high sensitivity of HAADF-STEM, it is based in atomic number (Z)
variations of the atoms in the sample, and the sensitivity lets contrast between the protein shell and the Ag nanoclusters core. The
atomic number (Z = 47) of silver is higher than the atomic components of proteins and carbon nanotubes, namely H (Z = 1), C (Z = 6),
N (Z = 7), O (Z = 8) and S (Z = 16) [10]. In addition, it is well know
that the protein shell does not let a high resolution of AgNCs by
HRTEM [16]. Finally, it is important to remark the formation of uorescent quasi 1-D nanomaterials was successful due the use of
the SWNTs as template, since without them the AgNCs showed a
random arrangement (Fig. 5e).
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Fig. 4. Representative AFM images of pristine SWNTs (a, b) and conjugates (c, d)
deposited on mica, its cross section proles are marked by arrows and identied by
colors (e, f). For interpretation of the references to color in this gure legend, the
reader is referred to the web version of this article.
Fig. 5. (a) HRTEM image of pristine SWNTs, SWNTs decorated with HSA (b) and conjugates (c, f). The inset shows the EDS analysis of the conjugates (c). (d) HAADF-STEM
image of conjugates and (e) AgNCs.
276
Acknowledgments
The work was supported by the National Autonomous University of Mexico (grant DGAPA-IN101313) and the National
Council on Science and Technology (grant CONACyT-127299).
Andrs Rodrguez-Galvn thank CONACyT for a PhD fellowship.
O. Amelines-Sarria acknowledges nancial support of DGAPA
CJIC/CTIC/1334/2013. The authors thank Fis. Roberto Hernndez for
TEM technical assistance and to Dr. Victor Meza-Laguna for SEM
technical assistance.
References
Fig. 6. Scanning electron microscopy images of RBCs (a) incubated with conjugates
(b, c), SWNTs (d) and AgNCs (e). SEM images showing agglomeration of conjugates
(f) and AgNCs (g) over the membrane of RBCs (red arrows). All the samples were
incubated at 37 C for 2 hFor interpretation of the references to color in this gure
legend, the reader is referred to the web version of this article.
resistance to current drugs could be present [31], as the nanomaterials emerged as option to overcome these drawbacks [32].
In particular metallic nanoclusters are currently investigated as
radiosensitizers for cancer radiotherapy [33,34]. The conjugates
synthesized here could be explored for their possible use in advance
treatments.
4. Conclusions
The approach ran here showed the capability for the formation
of a coat of uorescent AgNCs onto the SWCNTs using HAS protein
as template. The conjugates exhibited a lack of hemo compatibility
since the integrity of RBCs was compromised. The SEM analysis
showed a toxic effect concentration-dependency and specically
that the AgNCs were the main toxic element. Our results are not
conclusive and suggest more experiments should be addressed to
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