Non-Covalent Attachment of Silver Nanoclusters onto Single-walled Carbon

Nanotubes with Human Serum Albumin as Linking Molecule.

Andrès Rodríguez-Galvána,c*, Alejandro Herediaa, Oscar Amelines-Sarriab, Margarita

Riverab, Luis A. Medinac, Vladimir A. Basiuka.
a
Instituto de Ciencias Nucleares, Universidad Nacional Autónoma de México, Circuito
Exterior C.U., 04510 México D.F., México
b
Instituto de Física, Dpto. Materia Condensada, Universidad Nacional Autónoma de
México, Coyoacán, 04510 México D.F., México
c
Instituto de Física, Universidad Nacional Autónoma de México, México, DF 04510,
México. Unidad de Investigación Biomédica en Cáncer INCan-UNAM, Instituto
Nacional de Cancerología, México, DF 14080, México

∗ Corresponding author at: Instituto de Ciencias Nucleares, Universidad Nacional

Autónoma de México, Circuito Exterior C.U., 04510 México D.F., México
Phone: +52-55-5622-4739, Ext. 2224,
+52-55-5622-4675,
Fax: +52-55-5622-4693
E-mail addresses: andres.rodriguez@nucleares.unam.mx
Abstract

The attachment of silver nanoclusters (AgNCs) onto Single-walled carbon nanotubes

(SWNTs) for the formation of integrated fluorescence sites has attracted much attention

due their potential applications as biological probes and nanovectors in theragnosis.

Here, we report the preparation through assembly of fluorescent quasi 1-D nanomaterial

based on SWNTs and silver nanoclusters (AgNCs) non-covalently attached to human

serum albumin as biological linker. The fluorescent SWNT-AgNCs-HSA conjugates

were characterized by atomic force microscopy, high-resolution transmission electron

microscopy (HRTEM), high angle annular dark field scanning TEM (HAADF-STEM),

fluorescent and UV-Vis spectroscopy. The above techniques confirmed that AgNCs

were non-covalently attached onto the external surface of SWNTs. In addition, it was

observed that the modification did not affect the optical properties of the synthesized

AgNCs since the absorption spectra and fluorescence under UV irradiation (λ = 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.

Keywords

Single-walled carbon nanotube; silver nanoclusters; human serum albumin; hybrid

nanomaterials
1. Introduction

Single-walled carbon nanotubes (SWNTs) are quasi one-dimensional (1-D)

nanomaterials with unique physical and chemical properties that can be used for

applications in life sciences and medicine [1]. Particularly, the fluorescence of SWNTs

(in the near-infrared spectrum) is a promising dye in biological applications and for the

design of optical biomedical sensors [1–5]. 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 fluorescent organic dyes onto CNTs, SWNTs

become biocompatible and thus, an excellent alternative as fluorescent materials [7–9].

Another useful modification although less explored with great potential, is the

attachment of metallic nanoclusters (NCs) onto SWNTs for the fabrication of

fluorescent nanotubes [10]. NCs have been of particular interest because, in addition

their good photostability, stability and solubility properties, they exhibit low toxicity for

biological systems [11–14].

The NCs are constituted from tens to hundreds of atoms, and due to their small size (< 2

nm) their optical properties are intermediate between molecules and nanoparticles [14].

For biological applications, the fluorescence and related properties such as high

photostability, broad absorption band and a high Stokes shift are of particular interest

[13]. Different types of capping ligands ranging from small biomolecules like DNA to

large molecular weight proteins such as bovine serum albumin (BSA) have been used to

synthesize NCs [13]. Particularly, protein-stabilized silver nanoclusters (AgNCs) are

receiving increasing attention due their strong and tunable fluorescence properties in the

visible spectrum [15]. Among them, globular proteins as human serum albumin (HSA)

has been extensively used as templates for the synthesis of AgNCs because of their

capability to interact and trap metal ions, and to prevent the NCs aggregation [16–19].

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 [22–24]. Furthermore, HSA has been used as linking

molecules between AgNPs and multi-walled carbon nanotubes (MWNTs) to form

hybrid materials under mild conditions [23].

Herein, by using the dual hydrophobic-hydrophilic 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 first assemblies SWNTs with fluorescent AgNCs wrapped up

with the HSA as linking molecule. The method employed demonstrated the

combination of unique specific molecular properties of the three nanomaterials,

resulting in an interesting molecular aggregation with potential applications. The

conjugates were characterized by UV-Vis spectroscopy and microscopy techniques such

as atomic force microscopy, high-resolution transmission electron microscopy and dark-

field 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.

2. Materials and Methods

2.1 Materials

Silver nitrate (AgNO3), HSA (MW ~67 KDa), sodium borohydride (NaBH4) and

potassium hydroxide (KOH) were purchased from Sigma-Aldrich. SWNTs (95% of

purity) were purchased from ILJIN Nanotech Co., Korea. All reagents were used as

received, and for all the experiments deionized water (18.2MΩ.cm) was used.

2.2 AgNC synthesis with HSA

AgNCs/HSA were synthesized by the method reported by Mathew and coworkers [15].

In brief, 100 μL of HSA (50 mg/mL) were incubated with 100 μL of 10 mM AgNO3 for

5 min. Afterwards 5 μL of 1M of NaOH and left for 5 min. Finally, 4 μL of 10 mM of

NaBH4 as reducing agent was added.

2.3 Decoration of SWNTs with AgNCs to form the conjugates

For the preparation of SWNTs-AgNCs-HSA (referred to as “conjugates” hereafter), 1

mL of oxidized SWNTs (0.5 mg/mL) in water solution was mixed with 1 mL of AgNCs

(50 mg/mL) synthetized using HSA as template and incubated for 15 min under slow

stirring. In addition, two control samples were prepared as follows:

(a) 1 mL of SWNTs (0.5 mg/mL) was mixed with 1mL of HSA (50 mg/mL) and

sonicated for 20 min in an ultrasonic bath, then 100 µL of AgNO3 (1 M) was added

gently and the pH was adjusted to 12 with KOH. Finally 100 µL of NaBH4 (10 mM)

was added as reducing agent.

(b) The second sample was prepared in a similar way as the above, however the pH was

kept neutral.

Each sample was dialyzed in PBS to eliminate the excess of AgNO3 and NaBH4 and

centrifuged at 16000 g for 20 min.

2.4 Effect of the conjugates on the integrity of red blood cells (RBC)

For the blood cells, native whole blood was collected from consenting informed healthy

volunteer donors. The blood was collected into vacutainer tubes containing 3 % of

sodium citrate solution to obtain anticoagulant blood samples for uses in blood exposure

assays; the volume ratio of blood to sodium citrate solution was 9:1. The procedure for

RBCs extraction and for the test were followed as previously reported [25]. SEM

characterization was carried out using a JSM-6510LV (JEOL, Japon) instrument

operating at 20 kV.

2.5 Characterization

2.5. 1 Ultraviolet-visible and Fluorescence spectroscopy

UV-Vis spectroscopy was carried out using a Cary 100 UV-Vis spectrophotometer

(Varian, USA) in a range between 200 and 700 nm. Fluorescence analysis was
performed in a LS-55 luminescence Spectrometer (PerkinElmer, USA). The emission

spectra were recorded in a range of 500-700 nm upon excitation at 510 nm.

2.5.2 High-resolution transmission electron microscopy and dark-field scanning

transmission electron microscopy

The surface morphology and chemical composition were investigated with HAADF-

STEM and HRTEM. The images were obtained with a JEM-2010F FASTEM

instrument (JEOL, Japon) coupled to a NORAN energy dispersive spectrophotometer

(EDS) operating at 20 kV. The samples were prepared by casting 5μL of the samples

onto carbon coated copper grids (Ted Pella). The solution excess was then removed

with filter paper and dried at room temperature in the overnight. The particle/nanotube

dimensions were measured using the ImageJ version 1.40 software (NIH, Wayne

Rasband).

2.5.3 Atomic force microscopy

The AFM images were recorded in tapping mode under ambient conditions on a JSPM-

5200 (JEOL, Japan) microscope. 50 µL of sample was deposited onto freshly cleaved

mica substrate, incubated 15 min, washed with 2 mL of deionized water and dried at

room temperature for 2 h. The images and profile analysis wereas performed with the

Gwyddion 2.36 software (Czech Republic).

3 Results and discussion

3.1 Decoration of SWNTs with AgNCs

Silver nanoclusters synthesized using HSA as biological template (Figure 1) were

incubated with SWNTs (Figure 2a). The UV-Vis absorption spectra of the conjugates

showed two absorption peaks, at 280 nm and 450 nm, assigned as HSA and AgNCs,

respectively (Figure 3a) [15]. The absorption of HSA is attributed to the π→π*

transition of aromatic aminoacids. At the same time, it is well known that the UV-Vis

absorption spectra of nanoclusters exhibit molecule-like optical transition with

absorbance bands due to the quasi-continuous electronic energy band structure and

quantum confinement 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 Materials and Methods section, Figure

2b) of silver nanolcusters 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

synthesis under protein control [16]; and (b) at pH 7, since the deposition of silver

nanoparticles using HSA onto SWNTs has been reported under neutral conditions [23].

The UV-Vis spectra of the two control samples were similar and displayed the lack of

the AgNCs absorption band, in contrast they exhibited one peak at 420 nm (Figure 3a),

that is well known to correspond to the surface resonance plasmon of silver

nanoparticles (particles with a size > 2 nm). The Physisorption of HSA onto the SWNTs

surface might explain the observed, this might restrict the native protein dynamic for the

correct interaction between lateral chains and silver ions, and furthermore this might
restrict the confinement 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 (insert in Figure 3c). The Figure 3c shows the fluorescence

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 maximum

around 605 nm upon excitation at 480 nm. The emission is known to be originated from

the inter-band transitions from the submerged and quasi-continium 5d band to the

lowest unoccupied conduction band of Ag nanoclusters [26]. The conjugates showed a

good stability since the fluorescence intensity was kept without changes for some hours.

We observe the successful formation of conjugates needed an excess of AgNCs

(SWNTs: AgNCs; 1:100), since when the relative SWNTs concentration increased the

fluorescence intensity of the conjugates decreased drastically (Figure 3d). The SWNTs

have a quenching capability for a variety of fluorophores, such as pyrenes, porphyrins,

and chromophores, and it is thought that either energy transfer or electronic transfer

might contribute to the quenching effect [16]. In this work the interaction between the

nanoclusters and the surface of SWNTs might not be an explanation for the

fluorescence quenching, we inferred the protein shell avoided the direct interaction.

However, it was reported the interaction between metallic ions with the organic shell of

protect silver nanoclusters could promote the emission quenching [26]. Due to the lack

of information about the Ag nanoclusters at present other factor as impurities

(amorphous carbon and graphite) should be considered [27].

3.2 Microscopic characterization

Atomic force microscopy was used to confirm the deposition of silver nanoclusters onto

SWNTs, since it is a powerful tool for the characterization of nanomaterials. By

comparing typical topography AFM images of bare SWNTs (Figure 4a, b) with images

of the conjugates (Figure 4c, d), it is evident the deposition of oblong particles (high

blue particles in the images) along SWNTs after the incubation with AgNCs. The

oblong particles were observed on all the carbon nanotube samples, showing high

conjugation. Comparative transversal cross sections analysis of the bare nanotubes and

their conjugates (Figure 4b,d) confirmed the deposition. The carbon nanotubes

exhibited heights around 4 nm and a flat longitudinal profile (Figure 4e, f). In contrast,

the conjugates showed transversal profiles with an increase of 3 nm and a longitudinal

profile with peaks and valleys with heights of 2-3 nm (Figure 4e, f), which correspond

to the nanocluster-covered and bare surfaces, respectively [6].

Figure 5 shows HRTEM images of pristine SWNTs, HSA-decorated nanotubes and

conjugates (5a, b and c, respectively). The conjugates showed few black spots, with a

size of approximately 2 nm, embedded into a protein film that was covering the

nanotube sidewalls (Figure 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 exhibited black spots and showed the absence of silver signals in EDS. A HAADF-

STEM analysis was performed and interestingly contrasted with the observed in

HRTEM, the images showed a big number of AgNCs along the SWNTs surface (bright

spots in Figure 5d). These 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’s important to remark the formation of fluorescent quasi 1-

D nanomaterials was successful due the use of the SWNTs as template, since without

them the AgNCs showed a random arrangement (Figure 5e).

3.3 Effect of conjugates on RBC integrity

Nanotubes with integrated fluorescence have been suggested for biological and medical

applications. However, for our known their impact on the integrity of red blood cells,

the main cellular component in the blood and principal oxygen carriers to body tissues,

has not been reported. Analysis by SEM was used to test the structural integrity of red

cells. Normal RBC typically displayed a biconcave disk-like appearance (Figure 6a).

After their incubation with the conjugates at a concentration of 0.025 mg/ml the RBC

exhibited a number of cells with aberrant morphology and structural damage (Figure

6c). Furthermore, increasing the concentration up to 0.25 mg/ml the aberrant

morphology and fragmentation became more evident (Figure 6e). SEM images of RBCs

incubated with naked SWNTs did not display noticeable damage or the transformation

of echinocytes (Figure 6g), discarding some notable effect from hydrophobic patches

form conjugates with an incomplete protein coat as was observed in the AFM analysis

(Figure 4d). An interesting fact resulted from the obtained SEM images of RBCs

incubated with the as-prepared AgNCs, they displayed clear cut fragmentation and high

transformation into echinocytes. In other words, the as-prepared AgNCs promotes the
erythrocyte damage and transformation. The interaction of silver nanoparticles with

erythrocytes recently has been studied and it was found silver nanoparticles interact

with the membrane of RBCs and induce a cellular damages since block the transport

channels [28], in particular the AgNPs produce an imbalance in the internal pH. It is

well known changes in the pH of RBCs to be related to the crenation phenomena [29].

Furthermore, the interaction of PEGylated gold nanoparticles with the membrane of

RBCs has been reported to promote the formation of echinocytes [30]. Here the SEM

images of RBCs incubated with the conjugates and with the as-prepared AgNCs showed

the presence of additional material onto the surface of the erythrocytes (Figure 6f).

Based in own results we suggest the interaction of the AgNCs with the RBCs membrane

promotes the structural damage in the cells.

Cytotoxic drugs are between the main drugs against malign cells, however, is well

known for some metabolic conditions a resistance to current drugs could be present

[31], as option the nanomaterials are emerged as option to overcome these drawback

[32]. In particular metallic nanoclusters are currently investigated as radiosensitizers for

cancer radiotherapy [33, 34]. The conjugates synthetized 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 fluorescent

AgNCs onto the SWCNTs. The conjugates exhibited a lack of hemo compatibility since

the integrity of RBCs was compromised. The SEM analysis showed a toxic effect

concentration-dependent and specifically that the AgNCs were the main toxic element.
Our results are not conclusive and suggest more experiments should be addressed to

study the hemocompatibility of the conjugates worked here and their possible use in

advance treatments.

5. 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). Andrés Rodríguez-Galván thanks to CONACyT for a PhD

fellowship. O. Amelines-Sarria acknowledges financial support of DGAPA

CJIC/CTIC/1334/2013. The authors thank to Fis. Roberto Hernández for TEM technical

assistance and to Dr. Victor Meza-Laguna for SEM technical assistance.

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Figures

(a) (b)

(c) (d)

Figure 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 (insert in

(b)).
Figure 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 first approach let the

formation of conjugates with AgNCs. In contrast the second way address the AgNPs

formation.
(a)
(b)

(c)
EX EM (d)

Figure 3: Low magnification (a) and high magnification (b) of UV–vis 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 2.5:2.5mg/mL,

purple, black and pink lines respectively).

 (a) (b)

(c) (d)

(e) (f)

Figure 4: Representative AFM images of pristine SWNTs (a, b) and conjugates (c, d)

deposited on mica, its cross section profiles are marked by arrows and identified by

colors (e, f).

Figure 5: (a) HRTEM image of pristine SWNTs, SWNTs decorated with HSA (b) and

conjugates (c, f). (d) HAADF-STEM image of conjugates and (e) AgNCs. The inset in

shows the EDS analysis of the conjugates (c).

 AgNCs SWNTs Conjugates Conjugates Control
0.25 mg/mL 0.25 mg/mL 0.25 mg/mL 0.025 mg/mL

(f)
(e)
(c)
(a)

(d)
(b)

Conjugates
(g)
AgNCs
Figure 6: Scanning electron microscopy images of erythrocytes (a) incubated with the
conjugates (b, c), SWNTs (d) and with AgNCs (e). SEM images showing agglomeration
of conjugates (f) and AgNCs (g) onto the membrane of RBCs. All the samples were
incubated at 37 ºC for 2 h.