A near-infrared fluorescent sensor for detection of cyanide in aqueous solution and its application for bioimaging

Article abstract of DOI:10.1039/c0cc03398g

A new NIR fluorescent sensor based on an amine-substituted heptamethine cyanine dye displayed a highly selective fluorescence enhancement with cyanide in aqueous solutions, and was applied for the imaging of anthropogenic and biogenic cyanide.

Full text of DOI:10.1039/c0cc03398g

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A near-infrared fluorescent sensor for detection of cyanide in aqueous
solution and its application for bioimagingw
ab
a
Xiaoqiang Chen,z Seong-Won Nam,z Gun-Hee Kim,c Nari Song,d Yongsuk Jeong,d
Injae Shin,c Seog K. Kim,e Jinheung Kim,a Sungsu Park*a and Juyoung Yoon*ad
Received 22nd August 2010, Accepted 30th September 2010
DOI: 10.1039/c0cc03398g
A new NIR fluorescent sensor based on an amine-substituted
heptamethine cyanine dye displayed a highly selective fluores-
cence enhancement with cyanide in aqueous solutions, and was
applied for the imaging of anthropogenic and biogenic cyanide.
attracted special attention.7 We report here a novel NIR
fluorescent sensor containing an amine-substituted hepta-
methine cyanine dye and a copper complex moiety, which
selectively sense cyanide in aqueous solution. Furthermore,
studies of biological applications using Caenorhabditis elegans
demonstrated that this system can be employed for the
imaging of cyanide.
HCN produced by Pseudomonas aeruginosa (PA) is considered
to be involved in the pathogenesis of CF lung disease.1 The
imaging of cyanide in vivo systems can be a valuable tool for
elucidating the mechanism of the bacterial cyanogenesis as
well as assessing the detriment of HCN to CF patients.
However, monitoring the amount of cyanide that is produced
by the microorganism in the lungs is not an easy task because
sputum samples are not easily obtained from either pediatric
patients or small animal models such as mice. Therefore, it is
highly needed to develop a fluorogenic method,2 which is
simple and convenient to detect cyanide in vivo. As part of
this effort, optical probes that can detect cyanide with ease and
high sensitivity have recently been exploited.3 Surprisingly,
most of these probes were designed based on UV-visible
fluorophores or sensed cyanide only in organic solvents, which
limit their applications on fluorescence imaging for clinical
diagnosis.
For the synthesis of a sensor precursor, as shown in
Scheme 1, N,N,N0-tris(pyridin-2-ylmethyl)ethane-1,2-diamine
(2) was obtained firstly according to the similar methods
reported previously.8 2 was then reacted with IR-780 in
DMF to give 1 in 15% yield. Compound 1 comprises a
N5-donor coordination sphere bearing three 2-pyridylmethyl
moieties, which lead to strong affinity between compound
1 and Cu2+. This design also assures strong fluorescence
quenching in the presence of copper ion due to the short
distance between the fluorophore and metal binding site.
When the Cu2+ concentration increases, the peak intensity
of the absorption maximum at 718 nm of this sensor decreases
with the concurrent formation of a new peak at 743 nm (Fig. 1,
top). When 1 equiv. of copper ion was added to 1 in aqueous
solution, complete fluorescence quenching was also observed
(Fig. 1, bottom). The binding constant of 1 with Cu2+ was
determined to be 1.1 ꢀ 106 Mꢁ1 based on the fluorescence
titration experiments. Other metal ions, such as Na+, K+,
Near-infrared (NIR) dyes have received considerable
attention for fluorescent bioimaging due to their advantages
over UV and visible fluorophores.4 In 2005, Peng et al.
reported that amine-substituted tricarbocyanines that have
absorption maximum at a much shorter wavelength thus
lead to much larger Stokes shifts than those of oxygen/
sulfur-substituted cyanines.5 Since then, amine-substituted
heptamethine cyanine dyes have been developed as fluorescent
sensors for metal ions and pH value.6 Even now, no NIR
fluorescent probe for imaging cyanide has been reported.
Among the various systems designed to detect anions,
sensors utilizing the affinity of anions for copper have
Ca2+, Mg2+, Al3+, Cd2+, Co2+, Fe2+, Fe3+, Hg2+, Mn2+
,
Ni2+, Pb2+, Zn2+ and Ag+, induced negligible fluorescence
quenching effects, which implies that 1 has potential as a
Cu2+-selective NIR sensor (Fig. S1, see ESIw).
As complete quenching happened when 1 equiv. of copper ion
was added to 1, the sensor 1–Cu2+ was prepared in situ by mixing
compound 1 with Cu(ClO4)2 at a 1 : 1 ratio in aqueous solution.
The extinction coefficients of compound 1 and 1–Cu2+ were
calculated to be 1.12 ꢀ 105 Mꢁ1 cmꢁ1 and 1.16 ꢀ 105 Mꢁ1 cmꢁ1
.
Using the IR 125 as standard, the fluorescence quantum yields
a Department of Chemistry and Nano Science, Ewha Womans
University, Seoul 120-750, Korea. E-mail: jyoon@ewha.ac.kr,
nanopark@ewha.ac.kr; Fax: +82 2-3277-3419;
Tel: +82 2-3277-2400
b State Key Laboratory of Materials-Oriented Chemical Engineering,
College of Chemistry and Chemical Engineering, Nanjing University
of Technology, Nanjing 210009, China
c Department of Chemistry, Yonsei University, Seoul 120-749, Korea
d Department of Bioinspired Science, Ewha Womans University,
Seoul 120-750, Korea
e Department of Chemistry, Youngnam University, Seoul 133-791,
Korea
w Electronic supplementary information (ESI) available: Experimental
details and supplementary figures and characterization of compounds.
See DOI: 10.1039/c0cc03398g
z Contributed equally to this work.
Scheme 1 Synthesis of compound 1.
c
This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 8953–8955 8953

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Fig. 2 (Top) Fluorescence changes of 1–Cu2+ (5 mM) with various
anions (100 equiv.) in 20 mM HEPES buffer (pH 7.4) (lex = 680 nm).
(Bottom) Fluorescence spectra of 1–Cu2+ (5 mM) with different
concentrations of CNꢁ (10, 20, 30, 40, 50, 60, 70, 80, 90, 100 equiv.)
in 20 mM HEPES buffer (pH 7.4, 0.5% CH3CN).
Fig. 1 (Top) Absorbance spectra of 1 (5 mM) with various concen-
trations of Cu2+ (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 mM) in 20 mM
HEPES buffer (pH 7.4, 0.5% CH3CN). (Bottom) Fluorescence spectra
of 1 (5 mM) with addition of various concentrations of Cu(ClO4)2 (0, 1,
2, 3, 4, 5, 6, 7, 8, 9, and 10 mM) in 20 mM HEPES buffer (pH 7.4, 0.5%
CH3CN) with an excitation at 680 nm. Inset: the plot of emission
intensity at 748 nm vs. [Cu2+]/[1].
was observed throughout the body of the nematodes (Fig. 3c).
This suggests that cyanide taken through the mouth can
diffuse easily across the intestine, whereas it is difficult for
it to diffuse across the cuticle layer on the epidermis that
protects the body from harsh environments. Interestingly,
NIR fluorescence was observed in the cytosol but not in the
nucleus, suggesting that once the toxin reaches the cell it
remains in the cytosol where the mitochondria, the target
organelle of cyanide, are present (Fig. 3d).
of compound 1 and 1–Cu2+ were calculated to be 0.026 and
0.005, respectively. When CNꢁ was added gradually to the
solution containing 1–Cu2+, the peak intensity of the emission
at 748 nm of this sensor increased, and the emission change
upon the addition of 100 equiv. cyanide was approximately
14-fold (Fig. 2, bottom). Adding 1000 equiv. CNꢁ to the
solution containing 1–Cu2+ (0.5 mM), the fluorescence can
be recovered completely (Fig. S2, see ESIw). Using 0.5 mM
1–Cu2+, the detection limit of the probe was evaluated to be
5 mM with a signal-to-noise ratio of 3 (Fig. S3, see ESIw). In the
presence of 100 mM Clꢁ or HCO3ꢁ, the fluorescence of 1–Cu2+
still increases remarkably in the presence of 100 equiv. CNꢁ (Fig. S4,
see ESIw). In addition, the fluorescence intensity at 748 nm has no
remarkable change in the pH 2.7–10.4 range (Fig. S5, see ESIw),
whichmeans that thedisturbancesofprotonandhighconcentration
of physiologically relevant anions are neglectable in the present
system.
PA is the most important pathogen causing chronic
infection in CF patients. It is able to synthesize HCN, a potent
inhibitor of cellular respiration. The present study investigated
whether cyanide is present in C. elegans infected with PA. For
the confirmation of bacterial infection in the intestine of the
nematodes, C. elegans fed on PA14 strain were labeled with
GFP (green fluorescent protein) before the imaging. As shown
in Fig. 4a, after C. elegans were fed with E. coli OP50 and then
exposed to the sensor, neither green nor NIR fluorescence was
observed in the nematodes. In contrast, both green and NIR
fluorescence were observed in the nematodes fed on PA14
and exposed to the sensor (Fig. 4b–d). Similarly with the
exogenous NaCN, the biogenic cyanide diffused across the
intestine and was thus observed throughout the body except
The selective response of the probe to cyanide was then
investigated. Various anions (100 equiv.), such as Fꢁ, Clꢁ,
Brꢁ, Iꢁ, NO3ꢁ, HSO4ꢁ, HCO3ꢁ, CH3COOꢁ, Pi (phosphate),
PPi (pyrophosphate), SCNꢁ and ClO4ꢁ, did not respond to
the probe (Fig. 2, top). In contrast, cyanide induced the revival
of fluorescence of the probe, resulting in ‘‘Turn-On’’ type
sensing of cyanide. As cyanide reacts with copper ions to form
stable [Cu(CN)x]nꢁ species, the quenched fluorescence of
system is then recovered upon the addition of cyanide.
The nematode C. elegans was used to assess its feasibility for
applications as a NIR sensor to imaging. It is an important
experimental model in various fields including microbiology9
and environmental toxicology.10 As shown in Fig. 3, NIR
fluorescence was not observed in C. elegans without the
pre-exposure to NaCN (Fig. 3a). When the nematodes were
exposed to 1 mM NaCN, NIR fluorescence was only observed
in the pharynx and intestine (Fig. 3b). However, when the
concentration of NaCN was increased to 100 mM, fluorescence
Fig. 3 NIR imaging of NaCN in C. elegans. The nematodes on NGM
plates were exposed for 4 h to various concentrations of NaCN; (a) no
cyanide, (b) 1 mM, (c) 100 mM. (d) The enlarged image of the vicinity of
the intestine in the nematode exposed to 100 mM NaCN. All the
exposed nematodes were then washed three times with NGM buffer
and incubated with the NIR chemosensor (final conc. 100 mM) for
20 min. The scale bars represent 20 mm (P = pharynx; I = intestine;
n = nucleus; c = cytosol).
c
8954 Chem. Commun., 2010, 46, 8953–8955
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PA14 strain was kindly provided by Prof. You-Hee Cho at
Seoul National University. C. elegans N2 strain was provided
by Caenorhabditis Genetic Center (Univ. of Minnesota,
Minneapolis, MN).
Notes and references
Fig. 4 NIR imaging of cyanide in C. elegans infected with a
P. aeruginosa strain (PA14) labeled with green fluorescent protein
(GFP). Before the imaging, the nematodes were fed on either
non-infectious E. coli OP50 (a) or GFP-labeled PA14 for 2 days
(b–d); (b) the anterior end, (c) the medial part, (d) the posterior end
of C. elegans. The scale bars represent 20 mm (IL = intestinal lumen;
I = intestine; E = eggs; PA = PA14-GFP; A = anus).
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In conclusion, we have shown that a new water-soluble and
NIR fluorescent sensor 1–Cu2+ effectively and selectively
recognizes cyanide in aqueous solution. This sensor was also
used to visualize the cyanide produced by P. aeruginosa in
C. elegans. These results suggested that the present system has
potential as a powerful tool for the imaging of anthropogenic
and biogenic cyanide.
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Fig. 5 Visualization of antibiotic efficacy against P. aeruginosa
infection in C. elegans with the NIR sensor. The nematodes were fed
on GFP-labeled P. aerugionosa strain (PA14) for 2 days. They were
then incubated with ceftazidime (200 mg mLꢁ1) for 2 h before the
in vivo imaging. The scale bars represent 20 mm.
c
This journal is The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 8953–8955 8955