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Laboratory, Quantitative Biology Center, Riken, Japan

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Aqueous synthesis of glutathione-coated PbS quantum dots with

tunable emission for non-invasive fluorescence imaging in the
second near-infrared biological window (10001400 nm)
Glutathione-coated PbS quantum dots with tunable emission in
the second near-infrared biological window can be synthesized in
the aqueous phase. This PbS quantum dots enable the noninvasive
fluorescence imaging of a lymph system and cancer tumor in living
mice.
See Takashi Jin et al.,
Chem. Commun., 2013, 49, 7584.

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Cite this: Chem. Commun., 2013,

49, 7584
Received 28th May 2013,
Accepted 22nd June 2013
DOI: 10.1039/c3cc44000a
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Aqueous synthesis of glutathione-coated PbS quantum

dots with tunable emission for non-invasive
fluorescence imaging in the second near-infrared
biological window (10001400 nm)
Yuko Nakane,a Yoshikazu Tsukasaki,a Takao Sakata,b Hidehiro Yasudab and
Takashi Jin*acd

Glutathione-coated PbS quantum dots with tunable emission were

synthesized in the aqueous phase and used for non-invasive tissue
imaging in the second near-infrared biological window.

Near-infrared (NIR) fluorescence imaging has become an

important modality for the non-invasive visualization of living
tissues because of its low absorption and scattering.1,2 To date,
conventional NIR imaging has been utilized at the 700 to
900 nm wavelength range, also known as the first NIR (1st-NIR)
biological window.1 A variety of NIR fluorescent probes (e.g. Cy 5.5
and ICG) are commercially available for imaging in this window.3
Recent advances in NIR fluorescence imaging have suggested that
an even better signal to background ratio of the fluorescence
image is possible in the second NIR (2nd-NIR) biological window
(10001400 nm),2 which should make in vivo tissue imaging at the
whole-body level more feasible.4 Here, we report the aqueous
synthesis of highly bright, 2nd-NIR emitting PbS quantum dots
(QDs) for tissue imaging in the 2nd-NIR window.
Although much attention has been given to in vivo fluorescence
imaging in the 2nd-NIR window, there are only a few 2nd-NIR
fluorescent probes available, all based on single-walled carbon
nanotubes5 and Ag2S QDs6 that give quantum yields of less than
6% in aqueous solution. Moreover, the emission wavelengths of
these probes cannot be tuned for the 2nd-NIR window. For
instance, single-walled carbon nanotubes have a broad emission
spectrum over the wavelength range from 1000 to 1500 nm.5
Although Ag2S QDs emit at 1200 nm, their emission peak

Laboratory for Nano-Bio Probes, Quantitative Biology Center, Riken,

Furuedai 6-2-3, Suita, Osaka 565-0874, Japan. E-mail: tjin@riken.jp;
Fax: +81-6-6155-0112; Tel: +81-70-6800-3896
b
Research Center for Ultra-High Voltage Electron Microscopy, Osaka University,
7-1, Mihogaoka, Ibaraki, Osaka 567-0047, Japan
c
Graduate School of Frontier Bioscience, Osaka University, Yamadaoka 2-1, Suita,
Osaka 565-0871, Japan
d
WPI Immunology Frontier Research Center, Osaka University, Yamadaoka 1-3,
Suita, Osaka 565-0871, Japan
Electronic supplementary information (ESI) available: Experimental details and
characterization data for GSH-coated PbS QDs. See DOI: 10.1039/c3cc44000a

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Chem. Commun., 2013, 49, 7584--7586

does not change with the particle size from 5.4 to 10 nm.6
Semiconductor PbS QDs are alternative candidates as 2nd-NIR
fluorescent probes, because their band gap energy (0.41 eV)
corresponds to the energy gap in the 2nd-NIR window.7 Currently,
most PbS QDs are synthesized by thermally decomposing precursor
organometal compounds at high temperature.8 The resulting PbS
QDs show high brightness, but are not soluble in water. To resolve
this solubility issue, surface modifications of the QDs with amphiphilic compounds are needed.
Water-soluble PbS QDs can be prepared in the aqueous
phase using coating agents such as 1-thioglycerol/dithioglycerol,9
dihydrolipoic acid,10 L-cysteine,11 apoferritin,12 and luciferase.13
Among these coated QDs, only those coated with 1-thioglycerol/
dithioglycerol are emission tunable in the 2nd-NIR window.
However, PbS QDs coated with thiol compounds such as 1-thioglycerol and dithioglycerol are non-biocompatible and cytotoxic.
We therefore considered glutathione (reduced form, GSH) as a
coating agent for the preparation of biocompatible 2nd-NIR
emitting PbS QDs. GSH is a natural mono-thiol compound that
exists in most organs at the mM level and has no cytotoxicity.14
In addition, surface modifications of GSH-coated QDs with biomolecules are relatively easy, because GSH has two functional
groups, two carboxyl and one primary amino groups, which can
be conjugated to a biological compound such as an antibody.15
We synthesized GSH-coated PbS QDs by reacting Pb(CH3COO)2
and Na2S in the presence of GSH (Fig 1a). The emission and
absorption spectra of the 2nd-NIR emitting PbS QDs are shown in
Fig. 1b. When an aqueous solution of Na2S is added to a solution of
Pb(CH3COO)2 + GSH, the solution color immediately changes from
clear to brown, indicating the formation of PbS QD nanoparticles
(inset in Fig. 1b). The amount of Na2S significantly aects the
emission wavelength of the resulting PbS QDs (Fig. S1, ESI),
because the formation of the QD nanoparticles follows the
bimolecular reaction between the Pb2+ and S2 ions. Additionally, the pH and/or temperature of the solution of reactants
(Pb(CH3COO)2 + GSH) also aects the emission wavelength (Fig. 2).
A higher pH may increase the concentration of the complex
between Pb2+ and GSH, because the pKa of the sulfhydryl group
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Fig. 3 (a) TEM image of GSH-coated PbS QDs with 1000 nm emission peaks. The
inset shows the SAED pattern of the PbS QDs. (b) XRD pattern of GSH-coated
PbS QDs. (c) Histogram of the hydrodynamic diameter of GSH-coated PbS QDs
measured by dynamic light scattering.

Fig. 1 (a) Schematic of the aqueous synthesis of GSH-coated PbS QDs. (b) Fluorescence and absorption spectra of GSH-coated PbS QDs with emission peaks at
925 (red), 1050 (green), and 1200 nm (black), where the QDs were synthesized
at 5 1C (pH = 6), 25 1C (pH = 10), 50 1C (pH = 10), respectively. The inset graph
shows magnification of the absorbance spectra at 800 to 1200 nm. The inset
photograph shows GSH-coated PbS QDs (1050 nm emission peak) in water.

Fig. 2 Fluorescence emission peaks of GSH-coated PbS QDs versus pH and temperature of the aqueous solution of Pb(CH3COO)2 + GSH, where [Pb(CH3COO)2] =
3.6 mM and [Pb(CH3COO)2] : [GSH] : [Na2S] = 1 : 2 : 0.5.

of GSH is 9.65,16 to enhance the formation rate of GSH-coated PbS

QDs. At the same time, a higher temperature can also increase the
formation rate of the PbS QDs. Thus, by controlling the amounts of
Na2S and the pH and temperature of the solution of reactants, the
emission wavelength of GSH-coated PbS QDs can be tuned in the
2nd-NIR window. The GSH-coated PbS QDs are photostable against
the irradiation of 1st-NIR light (Fig. S2 in ESI).
A good fluorescence quantum yield (QY) of the NIR probes is
crucial for non-invasive tissue imaging with a high signal to
background ratio. The QY of GSH-coated PbS QDs was measured
using 780 nm-emitting QDs (CdSeTe/CdS)14,17 as a standard. QY
values for the PbS QDs were determined to be 16%, 12% and 6%
for QDs with emission peaks at 1000, 1100 and 1200 nm, respectively. These GSH-coated PbS QDs exhibit high brightness as well
as emission tunability in the 2nd-NIR window. Fig. 3a shows a
transmission electron microscope (TEM) image of the GSH-coated
PbS QDs with a 1000 nm emission peak. The selected area
diraction (SAED) pattern (inset of Fig. 3a) shows that the PbS
QDs are crystalline, and all diraction spots are assigned to a cubic
PbS phase. X-ray diraction (XRD) patterns of the PbS QDs also
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confirm that these QDs are nanometer-sized crystalline particles

(Fig. 3b), while dynamic light scattering measurements show that
the PbS QDs are well-dispersed particles and have a hydrodynamic
diameter of 3.5 nm in water (Fig. 3c). Size dependence of the
emission wavelength of the PbS QDs is shown in Fig. S3 (ESI).
Also colloidal stability of the QDs is shown in Fig. S4 (ESI).
To check the biocompatibility of the GSH-coated PbS QDs,
their cytotoxicity was examined using HeLa cells. Cell viability did
not decrease up to QD concentrations of 70 mg mL 1, suggesting
high biocompatibility (Fig. S5, ESI). The utility of GSH-coated
PbS QDs as 2nd-NIR fluorescent probes was confirmed by the
fluorescence imaging of the mouse lymph node. An aqueous
solution of GSH-coated PbS QDs (3 mg mL 1) exhibiting 1100 nm
emission peaks was injected into the mouse footpad (Fig. 4a).
Fluorescence images in the 2nd-NIR window were taken using a
home-built 2nd-NIR fluorescence microscopy system and an
excitation wavelength of 785 nm. The lymph system was clearly
visualized by 2nd-NIR fluorescence after the injection of GSHcoated PbS QDs (Fig. 4b). The flow image of QDs from a lymph
vessel to a popliteal lymph node (P)18 is shown in Fig. 4c.
Next we examined the feasibility of GSH-coated PbS QDs for
the 2nd-NIR fluorescence imaging of breast cancer tumors.
As an active targeting probe for the tumor imaging, we prepared
anti-HER219 (human epidermal growth factor 2) antibody conjugated GSH-coated PbS QDs using carbodiimide chemistry. To
confirm the binding ability of the probe to breast cancer cells,

Fig. 4 (a) The mouse lymph system. P, popliteal lymph node; I, iliac lymph node.
The green arrow shows the position where GSH coated PbS QDs were injected.
(b) 2nd-NIR fluorescence imaging of the two lymph nodes, P and I. (c) Time
course of QD flow through a lymph vessel to the popliteal lymph node (P).

Chem. Commun., 2013, 49, 7584--7586

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Fig. 5 (a and b) 2nd-NIR fluorescence images (1100 nm) of KPL-4 cells after the
incubation of anti-HER2 antibody conjugated PbS QDs (top) and BSA-conjugated
PbS QDs (bottom). Scale bar: 1 mm. (c and d) Histograms of the fluorescence
intensities of anti-HER2 antibody conjugated PbS QDs (top) and BSA-conjugated
PbS QDs (bottom) in KPL-4 cells.

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antibody conjugated PbS QDs at the breast tumor (Fig. 6d and e).
Fluorescence signals of GFP were uniformly observed over the breast
tumor (Fig. 6d). Although the 2nd-NIR fluorescence signals were also
observed for the tumor, the fluorescence signals were not uniform
over the tumor (Fig. 6e). This indicates that the antibody-conjugated
PbS QDs passed through the capillary blood vessels of the tumor to
become inhomogeneously distributed inside of it.
In conclusion, we have described the aqueous synthesis of
GSH-coated PbS QDs for the fluorescence imaging of tissue in
the 2nd-NIR biological window and demonstrated the capability
of 2nd-NIR emitting GSH-coated PbS QDs of the fluorescence
imaging of tissues such as lymph nodes/vessels and breast
cancer tumors. Compared with single-walled carbon nanotubes
and Ag2S QDs, GSH-coated PbS QDs are emission tunable in the
2nd-NIR window. In addition, they can be used as bioimaging
probes as their cytotoxicity is suciently low. We therefore
expect GSH-coated PbS QDs to have great potential as 2nd-NIR
fluorescent probes for tissue imaging in living systems.
We are grateful to Peter Karagiannis (QBiC, Riken) for critical
reading of this manuscript, and Akihito Komatsuzaki for the
measurements of 2nd-NIR fluorescence and absorption spectra.

Notes and references

Fig. 6 2nd-NIR fluorescence images (1100 nm) of a breast cancer tumor 1 h

(a) and 48 h (b) after injection of anti-HER2 antibody conjugated PbS QDs. The
tumor location in (a) is indicated by the dotted circle. Scale bar: 10 mm. Ex vivo
images of the breast cancer tumor using (c) bright field microscopy, (d) fluorescence microscopy at a visible light wavelength (535 nm), and (e) fluorescence
microscopy at a 2nd-NIR wavelength (1100 nm). Scale bar: 2 mm.

fluorescence images of KPL-4 cells20 (HER2 overexpressing human

breast tumor cells) were taken after the incubation of anti-HER2
antibody conjugated PbS QDs and BSA (bovine serum albumin)conjugated PbS QDs (Fig. 5). Most KPL-4 cells were stained by the
anti-HER2 antibody conjugated PbS QDs, whereas only very weak
fluorescence signals were observed when stained with BSAconjugated PbS QDs. This result clearly shows the specific binding
ability of PbS QDs to HER2 surface receptors in KPL-4 cells.
In vivo imaging of a breast cancer tumor in a nude mouse
implanted with GFP (green fluorescent protein) labeled KPL-4 cells,21
where GFP expresses at the microtubules of cells, was conducted as
follows.22 An aqueous solution (200 mL) of anti-HER2 antibody
conjugated PbS QDs was injected into the tail vain, and fluorescence
images (ex: 785 nm, em: 1100 nm) of the mouse were taken (Fig. 6a
and b). Immediately after the injection of QDs, 2nd-NIR fluorescence
signals for the QDs were observed over the whole body with time:
after about 1 h QDs were detected in the liver (Fig. 6a) and after 48 h
the breast tumor was clearly detected (Fig. 6b). GFP fluorescence
signals (ex: 490 nm, em: 535 nm), however, could not be observed
due to strong absorption and scattering of the visible light by the
tissues. Ex vivo imaging confirmed the accumulation of anti-HER2
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1 R. Weissleder, Nat. Biotechnol., 2001, 19, 316317.

2 A. M. Smith, M. C. Mancini and S. Nie, Nat. Nanotechnol., 2009, 4,
710711.
3 C. L. Amiot, S. Xu, S. Liang, L. Pan and J. X. Zhao, Sensors, 2008, 8,
30823105.
4 Y. T. Lim, S. Kim, A. Nakayama, N. E. Stott, M. G. Bawendi and
J. V. Frangioni, Mol. Imaging, 2003, 2, 5064.
5 J. Crochet, M. Clemens and T. Hertel, J. Am. Chem. Soc., 2007, 129,
80588059.
6 Y. Zhang, G. Hong, Y. Zhang, G. Chen, F. Li, H. Dai and Q. Wang,
ACS Nano, 2012, 6, 36953702.
7 E. H. Sargent, Adv. Mater., 2005, 17, 515522.
8 M. A. Hines and G. D. Scholes, Adv. Mater., 2003, 15, 18441849.
9 X. Zhao, I. Gorelikov, S. Musikhin, S. Cauchi, V. Sukhovatkin,
E. H. Sargent and E. Kumacheva, Langmuir, 2005, 21, 10861090.
10 D. Deng, W. Zhang, X. Chen, F. Liu, J. Zhang, Y. Gu and J. Hong,
Eur. J. Inorg. Chem., 2009, 34403446.
11 Y. Yu, K. Zhang and S. Sun, Appl. Surf. Sci., 2012, 258, 71817187.
n, S. I. Molina,
12 B. Hennequin, L. Turyanska, T. Ben, A. M. Beltra
` and N. R. Thomas, Adv. Mater., 2008, 20,
M. Li, S. Mann, A. Patane
35923596.
13 N. Ma, A. F. Marshall and J. Rao, J. Am. Chem. Soc., 2010, 132,
68846885.
14 T. Jin, F. Fujii, Y. Komai, J. Seki, A. Seiyama and Y. Yoshioka, Int. J.
Mol. Sci., 2008, 9, 20442061.
15 (a) Y. Nakane, A. Sasaki, M. Kinjo and T. Jin, Anal. Methods, 2012, 4,
19031905; (b) D. K. Tiwari, S.-I. Tanaka, Y. Inouye, K. Yoshizawa,
T. M. Watanabe and T. Jin, Sensors, 2009, 9, 93329354.
16 R. M. C. Dawson, Data for Biochemical Research, Oxford University
Press, New York, 3rd edn, 1987, pp. 1617.
17 M. Hasegawa, Y. Tsukasaki, T. Ohyanagi and T. Jin, Chem. Commun.,
2013, 49, 228230.
18 Y. Inoue, S. Kiryu, M. Watanabe, N. Oyaizu and K. Ohtomo,
J. Fluoresc., 2010, 20, 599606.
19 L. Coussens, T. L. Yang-Feng, Y.-C. Liao, E. Chen, A. Gray, J. McGrath,
P. H. Seeburg, T. A. Libermann, J. Schlessinger, U. Francke, A. Levinson
and A. Ullrich, Science, 1985, 230, 11321139.
20 J. Kurebayashi, T. Otsuki, C. K. Tang, M. Kurosumi, S. Yamamoto,
K. Tanaka, M. Mochizuki, N. Nakamura and H. Soono, Br. J. Cancer,
1998, 79, 707717.
21 The GFP expressing KPL-4 cell was kindly provided by Prof. N. Ouchi
and K. Gonda (Tohoku University).
22 Q. Ma, Y. Nakane, Y. Mori, M. Hasegawa, Y. Yoshioka, T. M. Watanabe,
K. Gonda, N. Ohuchi and T. Jin, Biomaterials, 2012, 33, 84868494.

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