ANALYTICAL SCIENCES JUNE 2011, VOL. 27
2011 © The Japan Society for Analytical Chemistry
Ultra-small gold nanoparticles (Au NPs) from subnanometer
scale to approximately 2 nm (also called gold nanoclusters
[Au NCs]) have recently attracted much attention in the fields of
physics, chemistry, material science, and biosciences because of
the relative ease of their synthesis by chemical reduction of their
salts in solution. This is done using stabilizing agents such as
thiol and phosphine compounds.1–9 The specific characteristics
of Au NCs are attributed to the ultra-small size of the NPs
around 1 nm. In contrast to Au NPs with sizes of more than
3 nm, the Au NCs show no localized surface plasmon resonance
(LSPR) band in the optical absorbance spectra. They display
various interesting molecular-like properties such as discrete
electronic states and size-dependent fluorescence, and have a
variety of applications in the field of catalysis, chemical sensing,
electronic devices, optics, and biomedics.10–25
Recently, there has been increasing interest in the development
of biological synthesis for Au NPs because of the need for an
environmentally acceptable solvent system and eco-friendly
reducing and capping agents.26–28 Xie et al. first demonstrated
the synthesis of highly fluorescent protein-stabilized Au NCs
using a bovine-serum alubumin (BSA)-templated method.22
The as-prepared Au NCs consisted of 25 gold atoms (Au25) with
a red emission at 640 nm. It has been suggested that the surface
of Au NCs are stabilized with Au+, which were further utilized
for luminescence sensing of Hg2+ ions through fluorescence
quenching by Hg2+-Au+ interaction.29 Although it is not yet
clear how the protein-stabilized Au NCs are formed in the
solution synthesis, it has been suggested that rich tyrosine (Tyr)
and cystein (Cys) residues (34 Cys and 21 Tyr) in BSA are
important to produce the protein-stabilized Au NCs. This is
because Cys residues, similar to thiol-protected Au NCs, are
able to stabilize Au NCs, and Tyr residues can reduce Au(III)
ions in alkaline pH above the pKa of Tyr (~10).22
On the basis of these previous studies, proteins with rich Cys
and Tyr sequences are considered to be strong candidates for the
synthesis of protein-stabilized Au NCs. Thus, there are many
natural proteins that are highly likely to produce such Au NCs,
and the use of various proteins for protein-stabilized Au NCs
may lead to the production of nanomaterials with highly specific
or multiple functions, or protein-mediated self-assembly.
However, so far only a few protein systems (i.e., BSA and
lysozyme) are available for protein-stabilized fluorescent
In the present paper, we report on the synthesis of
trypsin-stabilized fluorescent Au NCs with a red emission by
mixing trypsin and HAuCl4 at pH 12. Trypsin is a serine
protease found in the digestive system of many vertebrates,
where it hydrolyses proteins. Trypsin is also a strong candidate
for the synthesis of protein-stabilized Au NCs, since trypsin
includes rich amino acid residues with 7 Cys and 10 Tyr. The
photostable properties of the trypsin-stabilized Au NCs and the
effectiveness of fluorescent-based heavy metal ion sensing were
examined. We found that the fluorescence was particularly
quenched by Hg2+, and therefore, the Au NCs can be used as
sensors for sensitive and selective Hg2+ detection to a
detection limit of 50 ± 10 nM. The quantitative detection of
Hg2+ was possible over the wide and low concentration range of
50 – 600 nM.
HAuCl4·4H2O (99.9%) as a source of gold atoms was obtained
from Wako Chemical Co., from where we also purchased
trypsin. Standard heavy metal solutions (CaCO3 (1002 mg/l),
Cd(NO3)2 (1001 mg/l), Co(NO3)2
(1000 mg/l), Mg(NO3)2 (1001 mg/l), Ni(NO3)2 (1001 mg/l),
Pb(NO3)2 (997 mg/l), Zn(NO3)2 (1005 mg/l)) were obtained
from Wako Chemical Co. The ultra-pure water used throughout
(1001 mg/l), HgCl2
† To whom correspondence should be addressed.
Trypsin-Stabilized Fluorescent Gold Nanocluster for Sensitive and
Selective Hg2+ Detection
Hideya KAWASAKI,† Kouta YOSHIMURA, Kenji HAMAGUCHI, and Ryuichi ARAKAWA
Department of Chemistry and Materials Engineering, Faculty of Chemistry, Materials and Bioengineering,
Kansai University, 3-3-35 Yamate, Suita, Osaka 564–8680, Japan
We report on trypsin-stabilized fluorescent gold nanoclusters (Au NCs) for the sensitive and selective detection of Hg2+
ions. The Au NCs have an average size of 1 nm and show a red emission at 645 nm. The photostable properties of the
trypsin-stabilized Au NCs were examined, and their photochemical stability was found to be similar to that of CdSe
quantum dots. The fluorescence was particularly quenched by Hg2+, and therefore the Au NCs can be used as fluorescent
sensors for sensitive and selective Hg2+ detection to a detection limit of 50 ± 10 nM and the quantitative detection of Hg2+
in wide and low concentration range of 50 – 600 nM.
(Received April 1, 2011; Accepted May 3, 2011; Published June 10, 2011)
596 ANALYTICAL SCIENCES JUNE 2011, VOL. 27
of trypsin on the Au surfaces. Moreover, by direct interaction
with the Au surfaces, Hg2+ spontaneously reacts with Au to form
a Au amalgam, resulting in fluorescence quenching of the
trypsin-stabilized Au NCs. Based on these results, we consider
that trypsin-stabilized Au NCs can selectively detect Hg2+ with a
relatively high limit of detection via the specific interaction
between trypsin-stabilized Au NCs and Hg2+.
Trypsin-stabilized fluorescent Au NCs with a red emission of
640 nm in basic aqueous solution were synthesized using trypsin
as a reducing and stabilizing agent. The trypsin-stabilized
Au NCs are approximately 1 nm in size. The CD spectroscopy
of trypsin-stabilized Au NCs showed a large conformational
change by the encapsulation of Au NCs in trypsin, which
contrasted with the case of BSA-Au NCs, which had little effect
on the structure of the BSA scaffolds. The photostability of
trypsin-stabilized Au NCs was similar to that of CdSe QDs,
while it was no better than that of DMF-protected Au NCs. The
fluorescence was particularly quenched by Hg2+, and therefore
the Au NCs can be used as sensors for sensitive and selective
Hg2+ detection to a detection limit of 50 ± 10 nM and the
quantitative detection of Hg2+ in the wide and low concentration
range of 50 – 600 nM.
We thank Dr. Y. Hagihara and Dr. Y. Shigeri at the National
Institute of Advanced Industrial Science and Technology (AIST)
for measurements of the CD spectra, Dr. Takahasi and
Dr. Yamazaki at Kobelco Research Inst. for measurements of
the XPS spectra and TEM images. This study was partially
supported by a Grant-in-Aid for Scientific Research (B)
(Nos. 23360361 (to H. K.) and 22350040 (to R. Y.)) from the
Japan Society for the Promotion of Science (JSPS). This study
was supported by the “Strategic Project to Support the Formation
of Research Bases at Private Universities”: Matching Fund
Subsidy from MEXT.
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