Journal of Medical and Biological Engineering, 33(1): 23-28
23
Facile Method for Gold Nanocluster Synthesis and
Fluorescence Control Using Toluene and Ultrasound
Jimmy Kuan-Jung Li1,2
Zhi-Hua Cai1
Cherng-Jyh Ke1
Ching-Yun Chen1
Cheng-An J. Lin1,3
Walter H. Chang1,3,*
1
Department of Biomedical Engineering, Chung Yuan Christian University, Chungli 320, Taiwan, ROC
2
Medical Device Innovation Center, National Cheng Kung University, Tainan 704, Taiwan, ROC
3
Center for Nano Bioengineering, Chung Yuan Christian University, Chungli 320, Taiwan, ROC
Received 6 Oct 2010; Accepted 20 Oct 2011; doi: 10.5405/jmbe.874
Abstract
This study develops a method for the synthesis of environmentally safe, non-toxic, and color-tunable fluorescent
nanomaterials using AuCl3 and toluene without additional surfactants or reduction agents. A facile method for the
fluorescence control of gold nanoclusters using simple ultrasonic irradiation is reported. The cluster composition, size,
and emission wavelength can be controlled by the ultrasonic irradiation time. There is low photobleaching and the
quantum yields are sufficient for optical devices and biosensors. The proper control of ultrasonic conditions such as
exposure time, temperature, frequency, duty cycle, and intensity allows the composition and color of fluorescent gold
nanoclusters to be adjusted. The obtained materials will be very useful for multicolor molecular probe design in the
future.
Keywords: Ultrasound, Au, Nanoclusters, Composition, Fluorescence
1. Introduction
Research on fluorescent semiconductor nanocrystals, also
known as quantum dots, has shown tremendous advances over
the past ten years in areas ranging from pure materials science
to biological applications. However, most quantum dots are
made of cadmium-based or lead-based materials, which contain
toxic heavy metals and are therefore potentially harmful to the
environment [1]. Artificial fluorescent nanoparticles are
promising candidates for more environmentally safe quantum
dots. Investigations have been conducted on semiconductor
alternatives [2,3], and metallic [4-7], carbon-based [8], and
silicon nanoparticles [9-11].
Small metal nanoclusters (NCs) have sizes comparable to
the Fermi wavelength of electrons (ca. 0.7 nm), which gives
them molecule-like properties, such as size-dependent
fluorescence [12,13] and discrete electronic states [14,15]. The
fluorescence properties and non-toxicity of Au NCs (AuNCs)
make them potential labels for biological experiments [16,17].
The two major synthetic strategies for fabricating fluorescent
AuNCs are the template-based and monolayer-protected NC
methods. For the template-based method, Zheng et al. and
* Corresponding author: Walter H. Chang
Tel: +886-3-2654503; Fax: +886-3-2654581
E-mail: [email protected]
Garcia-Martinez et al. studied color-tunable fluorescent AuNCs
using poly(amidoamine) dendrimers [12,18], and Duan et al.
proposed etching methods with hyperbranched and multivalent
polymers [19]. For the monolayer-protected NC method,
fluorescent AuNCs have been synthesized using glutathione,
tiopronin, meso-2,3-dimercaptosuccunic acid, phenylethylthi
olate, dodecanethiol, mercaptoundecanol, and bovine serum
albumin, [17]. Recently, our group developed a
precursor-induced nanoparticle etching technique with reduced
lipoic acid as a protected ligand for red-luminescent AuNC
synthesis [17].
Ultrasound is considered a ‘green’ technology due to its
high efficiency, low instrumental requirements, significantly
reduced process time compared with other conventional
techniques, and its low cost [20]. It can be considered a
combination of chemical reactions that utilizes the formed
radicals and physical effects that are associated with an increase
in temperature (pyrolysis and combustion) and shearing. The
sonochemical synthesis of various types of nanoparticle and
nanostructured material composed of noble metals [21,22],
transition metals, semiconductors, carbon materials, and
polymeric materials has received a lot of attention in recent
years [23]. The size and shape of gold nanoparticles vary
according to reaction temperature, type of surfactant, thiolated
ligand, and ultrasound parameters such as frequency, intensity,
duty cycle, and exposure time [23,24]. There have been no
previous studies on the fluorescence control of AuNCs under
24
J. Med. Biol. Eng., Vol. 33 No. 1 2013
ultrasound irradiation. In this study, an environmentally friendly
and facile method of tunable fluorescent AuNC synthesis and
control using toluene and ultrasound irradiation is presented.
2. Experiments
Three experiments were conducted. For the first
experiment, gold trichloride (AuCl3) powders were dissolved in
five solvents for fluorescence testing. 4 ml of solvent (acetone,
chloroform, water, methanol, or toluene) (Aldrich) was added
into 30 mg of AuCl3 powders (Sigma) under 3 min of vigorous
stirring (Vortexer Genie 2 G-560, Scientific Industries, USA) or
60 min of ultrasound irradiation (30 W, 40 kHz,Bransonic 3510,
Branson Ultrasonic, The Netherlands) and then centrifuged at
4000 rpm for 5 min (Allegra X-30, Beckman Coulter, USA).
The sample vial was held with styrofoam in the middle of the
tank and irradiated by ultrasound through the water bath. The
fluorescence of each supernatant was then analyzed by
photoluminescence (PL) spectroscopy (FP-6200, Jasco, Japan).
For the second experiment, three Au compounds (AuCl3,
HAuCl4, and AuBr3) (Sigma) were dissolved in toluene for
fluorescence testing. A given Au compound was mixed with
toluene (Sigma) as a 25-mM solution under 3 min of vigorous
stirring or 60 min of ultrasound irradiation, and then
centrifuged at 4000 rpm for 5 min. The fluorescence of each
supernatant was then analyzed by PL spectroscopy.
In the third experiment, the supernatants of AuCl3 in
toluene (AuCl3@toluene) solutions were treated for various
ultrasound irradiation times. 25-mM AuCl3 solutions were
prepared from AuCl3 powders and toluene with 3 min of
vigorous stirring. The supernatants were harvested after 5 min
of centrifuging (4000 rpm). The supernatants were treated for
various periods (0, 30, 60, 120, and 180 min) of ultrasound
exposure (30 W, 40 kHz), and then centrifuged at
4000 rpm for 5 min to be harvested as the test samples. The
photo-characteristics of the supernatants were analyzed by
UV-vis absorption spectroscopy (Cary 50, Varian, USA) and PL
spectroscopy, and their photo-stabilities were tested under
continuous 320-nm UV irradiation. The size and composition
of samples were then evaluated using high-resolution
transmission electron microscopy (HRTEM, JEM-2100, JEOL,
Japan), atomic force microscopy (AFM, JSPM-5200, JEOL,
Japan) and electrospray ionization (ESI) mass spectrometry
(LC-ESI-Mass, Mass, USA).
Figure 1. PL spectra of supernatants of five kinds of mixture
(AuCl3@acetone, chloroform, water, methanol, or toluene)
after 3 min of vigorous stirring and 5 min of centrifuging.
Figure 2. PL spectra of supernatants of five kinds of mixture
(AuCl3@acetone, chloroform, water, methanol, or toluene)
after 60 min of ultrasound irradiation and 5 min of
centrifuging.
3. Results and discussion
Figure 1 shows that the supernatant of AuCl3@toluene has
a broad fluorescence spectrum, whereas those of the other
tested solutions (AuCl3@acetone, chloroform, water, or
methanol) do not. After 60 min of ultrasound irradiation, an
obvious fluorescence peak appears for supernatant of AuCl3@toluene; there is no response for the other tested solutions
(Fig. 2). After vigorous stirring, only the supernatant of
AuCl3@toluene exhibits fluorescence (Fig. 3).
Figure 3. PL spectra of supernatants of three kinds of mixture (HAuCl4,
AuBr3, or AuCl3@toluene) after 3 min of vigorous stirring
and 5 min of centrifuging.
Fluorescence Control of Gold Nanoclusters Using Ultrasound
Figure 4 shows that the surface plasma resonance
(absorption peak at 520 nm) associated with Au nanoparticles
with a particle size > 2nm [25] does not appear for the
compounds tested here. Instead, an absorption peak at around
300 to 350 nm appears for all supernatants of AuCl3@toluene
after vigorous stirring or ultrasound irradiation. This finding is
in agreement with the results of previous studies [12,19], in
which they synthesized fluorescent AuNCs using templatebased methods. The absorbance increases and the absorption
peak red-shifts with increasing ultrasound irradiation time.
0 min
30 min
60 min 120 min
25
AuNCs derived via other synthesis routes (10-3-10-6) [7,15,17]
and are comparable to those reported by Zheng et al. (20~30%)
[12], Duan et al. (10~20%) [19], and Liu et al. (10~20%) [26].
Although the quantum yield values of the fluorescent AuNCs
reported here is only a quarter of those of traditional Cd-based
quantum dots, they are in the range required for optical devices
and biosensors. Figure 7 shows that there is low
photobleaching under 320-nm UV exposure for the fluorescent
AuNCs synthesized here. This result is similar to those
obtained for traditional semiconductor quantum dots
(CdSe/ZnS) and template-based NCs [12,19], but quite
different from those obtained for monolayer- protected AuNCs
[17].
180 min
(a)
0 min
30 min
60 min 120 min 180 min
(a)
(b)
Figure 4. (a) Photographs of samples harvested at ultrasonic exposure
times of 0, 30, 60, 120, and 180 min under daylight. (b)
UV-vis absorption spectra of the samples in (a).
Figure 5 shows the PL spectra of the samples obtained
under 350-nm light excitation. There is an obvious red-shift of
the fluorescence peak with increasing ultrasound irradiation
time. The intensity of the fluorescence peak initially increases
with ultrasound irradiation time (from 0 to 60 min), and then
decreases after longer exposures (120 and 180 min). Figure 6
shows the nonshifted emission peak that appears with the
change of the excitation wavelength for each sample. These
data give strong evidence that the observed emission is real
luminescence from the relaxed states rather than a scattering
effect. The wavelength difference between the emission and
excitation peaks widening with increasing ultrasound
irradiation time also supports this. Previous study suggests that
the fluorescence mechanisms in our samples are caused by the
excitation of inner electrons instead of the vibrations of the
energy band [26]. The increased size of the Stokes shift and
energy gap imply that the particle size of the fluorescent
AuNCs increases with ultrasound irradiation time.
Using quinine sulfate (Sigma) (quantum yield = 0.54 in
H2SO4) as the reference, the quantum yields of fluorescent
AuNCs with 0, 60, and 180 min of ultrasonic irradiation were
15  2.5%, 20.8  2.3%, and 17.4  4.2% in toluene,
respectively. These values are approximately 2 ~ 3 orders of
magnitude higher than the quantum yields of fluorescent
(b)
Figure 5. (a) Photographs of samples harvested at ultrasonic irradiation
times of 0, 30, 60, 120, and 180 min under UV excitation. (b)
PL spectra of the samples in (a).
Because of the resolution limit of TEM (about 1 nm), our
samples could not be detected in TEM images. This is
consistent with the results obtained for fluorescent AuNCs
synthesized by other groups [12,19,26]. AFM scans (Fig. 8)
reveal that only the sample treated with 180 min of ultrasonic
irradiation could be detected, with the largest cluster height
found to be 1.6 nm. The mean diameter was below 0.5 nm, but
the standard deviation was higher than 0.6 nm (beyond the
detection limitation of AFM). This indicates that even in the
group with 180 min of ultrasonic irradiation, most clusters are
smaller than 1.0 nm.
The results of ESI mass spectrometry analysis show strong
evidence of composition control using ultrasonic irradiation
(Fig. 9). There are many kinds of cluster composition,
especially with mass-to-charge (m/z) ratios smaller than 382.31
(Fig. 9a). The two most abundant compositions are
Au11 (m/z = 359.20, m/e = 1.82) and Au16 (m/z = 382.31,
m/e = 1.94). After 60 min of ultrasonic irradiation, the
composition becomes purer, with the most abundance being
Au16 (m/z = 382.33, m/e = 1.94) (Fig. 9b). For a longer
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J. Med. Biol. Eng., Vol. 33. No. 1 2013
(a)
Figure 7. Photostability test results of samples with 0 min (black), 60
min (red), and 180 min (blue) of ultrasonic irradiation and an
organic blue fluorescent dye, quinine sulfate (dark cyan),
under continuous 320-nm excitation.
(b)
Figure 8. AFM images (0.5 μm × 0.5 μm) of a sample after 180 min of
ultrasonic irradiation. The highest spot is about 1.6 nm.
(c)
Figure 6. PL excitation spectra of samples with (a) 0 min, (b) 60 min,
and (c) 180 min of ultrasonic irradiation.
ultrasonic irradiation time (180 min) (Fig. 9c), the most
abundant composition is Au16 (m/z = 382.39, m/e = 1.94), and
more kinds of cluster with m/z ratios larger than 382.39 appear.
The fluorescence properties (wavelengths and quantum
yields) of the fluorescent AuNCs are similar to those of
PAMAM-encapsulated Au13 clusters [12] and PEI stabilized
clusters [19]. The ESI mass spectrometry data also show that
the fluorescent AuNCs harvested from the mixture of
AuCl3@toluene at the beginning mainly contain Au11 and
Au16 gold atoms. After 60 min of ultrasonic irradiation, the
cluster compositions become purer and larger, with a wider
Stokes shift and a higher quantum yield. Longer ultrasonic
irradiation (180 min) might induce larger cluster size formation
according to the results of the emission red shift (Fig. 5 and
Fig. 6) and increased number of compositions (Fig. 9).
From the above results, it could be hypothesized that the
decomposition of toluene might be induced by vigorous stirring
or ultrasound irradiation, the remains of which react with AuCl3
to form AuNCs. The cavitation effect of ultrasonic irradiation is
a potential mechanism of size and composition control of
AuNCs through the surfactant- and reduction-agent-free
method [27,28]. According to the cavitation-enhanced crystal
nucleation model proposed by Grossier et al. [29], ultrasonic
irradiation might induce an increase in the size of AuNCs via
the cavitation effect. During acoustic cavitation, free radicals
are formed due to the dissociation of vapors trapped in cavities
[20]. Free radicals of toluene decomposition produced by
vigorous stirring or ultrasound irradiation and their oxides
might act as surfactants of AuNCs and cause the variation of
cluster compositions.
Fluorescence Control of Gold Nanoclusters Using Ultrasound
27
4. Conclusion
(a)
Fluorescent AuNCs were tuned according to size and color
through a facile ultrasound irradiation method. This surfactantand reduction-agent-free method is more environmentally
friendly than other methods, which utilize inorganic fluorescent
nanoparticles. Ultrasonic irradiation can easily modulate cluster
composition, size, fluorescence color, quantum yield, and
Stokes shift via exposure time. The optical properties, namely
absorption, PL, PL excitation, and photostability characteristics,
of the AuNCs are similar to those reported in previous studies.
Furthermore, there is low photobleaching and the quantum
yields are sufficient for optical devices and biosensors.
Although the mechanism of this synthesis method for
fluorescent AuNCs still needs to be clarified, it is believed that
the proper control of ultrasonic irradiation conditions such as
exposure time, temperature, frequency, duty cycle, and intensity,
offers a good strategy for the composition and color control of
fluorescent AuNC synthesis. These materials will be very
useful for multicolor molecular probe design in the future.
Acknowledgements
We gratefully acknowledge the financial support via
research grants from the Taiwan National Science Council
(NSC 101-2627-M-033-001, NSC 101-2120-M-038-001, NSC
101-2628-E-033-001-MY3).
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Figure 9. ESI mass spectra of samples with (a) 0, (b) 60, and (c)
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