Advances in Materials Physics and Chemistry, 2013, 3, 307-313
Published Online December 2013 (http://www.scirp.org/journal/ampc)
Open Access AMPC
A Fluorescence Ratiometric Probe for Detection of
Cyanide in Water Sample and Living Cells
Lingliang Long*, Lin Wang, Yanjun Wu
Functional Molecular Materials Research Centre, Scientific Research Academy & School of Chemistry and Chemical Engineering,
Jiangsu University, Zhenjiang, China
Received October 30, 2013; revised November 28, 2013; accepted December 2, 2013
Copyright © 2013 Lingliang Long et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In accor-
dance of the Creative Commons Attribution License all Copyrights © 2013 are reserved for SCIRP and the owner of the intellectual
property Lingliang Long et al. All Copyright © 2013 are guarded by law and by SCIRP as a guardian.
In the present work, Compound 1 has been synthesized as a novel fluorescence ratiometric probe for CN−. Upon treat-
ment with CN−, Probe 1 exhibited a fluorescence ratiometric response, with the emission wavelength shift from 570 nm
to 608 nm. When 90 μM CN− was introduced, the emission ratios (I570/I608) of the probe changed dramatically from
0.52156 to 4.21472. The detection limit was also measured to be 0.24 μM (S/N = 3). In addition, Probe 1 had a selective
response to CN−, while other anions caused nearly no interference. The sensing reaction product of Probe 1 with CN−
was characterized by 1H NMR spectra and ESI Mass spectrometry. Furthermore, Probe 1 has been successfully applied
to detect CN− in natural water samples. The fluorescence imaging experiments in living cells also demonstrated that
Probe 1 could monitor CN− in biological samples.
Keywords: Organic Fluorescence Materials; Fluorescent Probes; Cyanide; Fluorescence Imaging
Anion recognition has received intense attention due to
its important role in an extensive range of environmental,
clinical, chemical and biological applications . Cya-
nide (CN−) is one of the most important anions, and it has
been widely used in various industrial fields such as gold
mining, electroplating, metallurgy, synthetic fibers and
resins . But unfortunately, the cyanide is extremely
detrimental to the living organism; it can inhibit the cel-
lular respiration upon interacting strongly with the heme
unit at the active site of cytochrome a3 . Uptake of the
toxic cyanide could occur through absorption by lungs,
exposure to skin, and also from contaminated food and
polluted drinking water [4-6]. Therefore, it is very im-
portant to develop an efficient method to detect cyanide
concentration in natural water sample and biological
Among various methods for measurement of CN−, the
fluorescence method based on fluorescent probe is more
attractive due to its desirable features including high sen-
sitivity, simplicity, and potential for in vivo imaging .
Accordingly, in the past decade, a large number of fluo-
rescent probes for detection of CN− have been reported in
the literature . Whereas many of them only utilized the
changes in emission intensity as detecting signals. A
major limitation of the intensity-based fluorescent probe
is that the signal output could be interfered by the factors
such as environmental conditions, probe distribution, and
instrumental efficiency [9,10]. By contrast, a ratiometric
measurement, employing the ratio of two emissions at
different wavelengths as the detecting signal, could pro-
vide a built-in correction for the above mentioned factors
and thus allow more accurate analysis [11,12]. However,
there are only very few fluorescence ratiometric probes
that have been applied to monitor CN− concentration in
water samples or biological samples [13-18].
Encouraged by these considerations, we developed
Compound 1 as a novel fluorescence ratiometric probe
for CN− in this work. Upon treatment with CN−, the
probe showed ratiometric response. In addition, the probe
has been successfully applied to detection of CN− level in
natural water samples and living cells.
L. L. LONG ET AL.
2. Experimental Section
2.1. Reagents and Apparatus
Unless otherwise stated, all reagents were purchased
from commercial suppliers and used without further pu-
rification. Solvents were purified by standard methods
prior to use. Twice-distilled water was used throughout
Mass spectra were recorded on a LXQ Spectrometer
(Thermo Scientific) operating on ESI. 1H NMR and 13C
NMR spectra were recorded on a Bruker Avance 400
spectrometer operating at 400 MHz and 100 MHz re-
spectively. Elemental (C, H, N) analysis were carried out
using Flash EA 1112 analyzer. Electronic absorption
spectra were obtained on a SHIMADZU UV-2450 spec-
trometer. Fluorescence spectra were measured on a Pho-
ton Technology International (PTI) Quantamaster
fluorometer with 3 nm excitation and emission slit
widths. Cells imaging were performed with an inverted
fluorescence microscope (Carl Zeiss, Axio Observer A1).
All pH measurements were performed with a pH-3c dig-
ital pH-meter (Shanghai ShengCi Device Works, Shang-
hai, China) with a combined glass-calomel electrode.
2.2.1. Synthesis of Compound 2
The synthetic procedures were showed in Scheme 1. Un-
der the N2 atmosphere, a solution of 4-Diethylamino-
salicylaldehyde (2.90 g, 15 mmol), diethylmalonate (4.8
g, 30 mmol) and piperidine (1 mL) in absolute ethanol
(40 mL) was heated under reflux overnight. The ethanol
was evaporated under reduced pressure, and then con-
centrated HCl (20 mL) and glacial acetic acid (20 mL)
were added to hydrolyze the reaction with stirring for
another 6 hours. The solution was cooled to room tem-
perature and poured into 150 mL ice water. NaOH solu-
tion (40%) was added dropwise to modulate pH of the
solution to 5, and a pale precipitate formed immediately.
After stirring for 30 min, the mixture was filtered,
washed with water, dried, then recrystallized in toluene
to give 2 (2.70 g, yield 83%). 1H NMR (CDCl3, 400
MHz) δ (ppm): 7.55 (d, J = 9.3 Hz, 1H), 7.23 (d, J = 8.7
Hz, 1H), 6.59 (dd, J = 8.7 Hz, J = 2.4 Hz, 1H), 6.51 (d, J
= 2.4 Hz, 1H), 6.06 (d, J = 9.3 Hz, 1H,), 3.42 (q, J = 7.2
Hz, 4H), 1.21 (t, J = 7.2 Hz, 6H). MS (m/z): 218.4
[M+H]+; Anal. calcd for C13H15NO2: C 71.87, H 6.96, N
6.45; found C 71.78, H 6.70, N 6.41.
2.2.2. Synthesis of Compound 3
Fresh distilled DMF (6.5 mL) was added dropwise to
POCl3 (6.5 mL) at 20˚C - 50˚C with N2 atmosphere and
stirred for 30 minutes to yield a red solution. This solu-
tion was added to a solution of 7-diethylaminocoumarin
(4.50 g, 20.7 mmol) in 30 mL DMF to allow a scarlet
Scheme 1. (a) the sensing reaction of Probe 1 with CN−; (b)
the synthetic procedure of Probe 1.
suspension. The mixture was stirred at 60˚C overnight
and then poured into 300 mL of ice water. NaOH solu-
tion was added to adjust the pH = 5.0 of the mixture to
yield large amount of precipitate. The crude product was
filtered, thoroughly washed with water, dried and recrys-
tallized in absolute ethanol to give 3 (3.67 g, yield
72.3%). 1H NMR (CDCl3, 400 MHz) δ (ppm): 10.13 (s,
1H), 8.26 (s, 1H), 7.41 (d, J = 8.8 Hz, 1H), 6.64 (dd, J =
2.4 Hz, 8.8 Hz, 1H), 6.49 (d, J = 2.4 Hz, 1H), 3.48 (q, J =
7.2 Hz, 4H), 1.26 (t, J = 7.2 Hz, 6H); MS (m/z): 246.1
[M+H]+; Anal. calcd for C14H15NO3: C 68.56, H 6.16, N
5.71; found C 68.49, H 6.20, N 5.68.
2.2.3. Synthesis of Probe 1
A solution of 3 (246 mg, 1 mmol), acetophenone (240
mg, 2 mmol) and pyrrolidine (4 drops) in 10 ml CHCl3
was stirred overnight at room temperature. The solvent
was removed, and the residue was purified by column
chromatography on silica gel (eluent: CH2Cl2) to afford
Probe 1 as orange solid (236 mg, 68%). 1H NMR (CDCl3,
400 MHz) δ (ppm): 8.25 (d, J = 15.2 Hz, 1H), 8.11 (m,
2H), 7.80 (s, 1H), 7.66 (d, J=15.2 Hz, 1H), 7.57 (m, 1H),
7.51 (m, 2H), 7.35 (d, J = 8.8 Hz, 1H), 6.63(dd, J = 2.4
Hz, 8.8 Hz, 1H), 6.53 (d, J = 2.4 Hz, 1H), 3.46 (q, J = 7.2
Hz, 4H), 1.26(t, J = 7.2 Hz, 6H); 1H NMR (CDCl3, 100
MHz) δ (ppm): 190.8, 160.2, 156.6, 151.9, 146.1, 139.8,
138.4, 132.6, 130.0, 128.6, 128.5, 128.1, 123.0, 115.1,
109.5, 108.9, 97.0, 45.0, 12.5; MS (m/z): 348.1 [M+1]+;
Anal. calcd for C22H21NO3: C 76.06, H 6.09, N 4.03;
found C 75.97, H 6.11, N 4.00.
3. Results and Discussions
3.1. Optical Response to CN−
The sensing properties of Probe 1 in response to CN−
were investigated in 20 mM potassium phosphate buffer/
CH3CN (v/v 1: 4, pH 7.4) at room temperature. As
shown in Figure 1(a), in the absence of CN−, Probe 1
displayed fluorescence emission centered at 570 nm.
However, when increasing concentrations of CN− were
introduced, the emission at 570 nm gradually decreased.
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L. L. LONG ET AL. 309
Concomitantly, a new emission centered at 608 nm ap-
peared and increased, with a well-defined isoemission
point at 588 nm. The changes in fluorescence emission
spectra also elicited an obvious variation in emission
color. With the addition of CN−, the fluorescence color of
Probe 1 changed from green to red (Figure 1(a)). There-
fore, Probe 1 can be used as a nake eye indicator for CN−.
In addition, the emission ratio (I608/I570) of Probe 1 re-
sponse to CN− displayed a large increase from 0.52156 to
4.21472 after 90 μM CN− added (8-fold enhancement)
(Figure 2). The emission ratios (I608/I570) also showed a
good linearity with CN− concentration in the range of 0 -
30 μM (Figure 1(b)), indicating the probe can be poten-
tially used to quantitatively detection of CN−. The detec-
tion limit for CN− was estimated to be 0.24 μM (S/N = 3)
according to a reported procedure . The low detec-
tion limit together with the large emission ratio en-
hancement demonstrates that Probe 1 is highly sensitive
to CN−. The absorption spectra of Probe 1 in the nm.
Figure 1. Changes in fluorescence emission spectra (λex =
510 nm) of Probe 1 (5 μM) with various amount of CN− (0
to 90 μM), inset: visual fluorescence color changes of Probe
1 (5 μM) in the absence and presence of CN− (90 μM), the
photo was taken under illumination of a handheld UV lamp;
(b) Changes in fluorescence emission ratios (I608/I570) of
Probe 1 (5 μM) to various amount of CN− (0 to 30 μM).
Upon addition of increasing concentrations of CN−, pres-
ence of different amounts of CN− are shown in Figure 3.
Probe 1 itself exhibited absorption centered at 449 the
absorption peak at 449 nm decreased, and a new absorp-
tion peak at 506 nm appeared and increased. At the same
time, the solution color varied from yellow to red (Fig-
3.2. Selectivity Studies
As shown in Figure 4, Probe 1 response to other anions
was also investigated. The anions such as F−, Cl−, Br−, I−,
, CH3COO−, 4
SCN− exerted no visible effect on the fluorescence ratios
(I608 / I570) of Probe 1. Obviously, large fluorescence ratio
change was only observed for Probe 1 treated with CN−.
Moreover, the ratiometric responses of Probe 1 toward
CN− in the presence of other anions were examined.
Most of other anions gave nearly no influence on Probe 1
detection of CN− (Figure 4). These results demonstrated
that Probe 1 had selective response towards CN−.
Figure 2. Changes in fluorescence emission ratios (I608/I570)
of Probe 1 (5 μM) to various amount of CN− (0 to 90 μM),
λex = 510 nm.
Figure 3. Changes in absorption spectra of Probe 1 (5 μM)
with various amount of CN− (0 to 90 μM), inset: visible
color changes of Probe 1 (5 μM) in the absence and pres-
ence of CN− (90 μM).
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L. L. LONG ET AL.
Figure 4. Fluorescence ratiometric response of Probe 1 (5
μM) to various anions (90 μM) in the absence (blank bar)
and presence (red bar) of CN− (90 μM). 1) blank; 2) F−; 3)
Cl−; 4) Br−; 5) I−; 6) ; 7) CH3COO−; 8)
; 10) ; 11) ; 12) SCN−. Excitation
wavelength was 510 nm.
3.3. Response Time and Effect of pH
The kinetic studies of Probe 1 in the absence or presence
of CN− was investigated by fluorescence spectra. As dis-
played in Figure 5, in the absence of CN−, almost no
variation in emission intensity (at 608 nm) of Probe 1
was found, implying that Probe 1 was stable in the assay
condition. However, upon addition of CN− (90 μM), a
dramatic enhancement in emission intensity at 608 nm
was observed, denoting the rapid reaction of Probe 1
with CN−. And the emission intensity reached a plateau
after 30 min reaction. The responses of Probe 1 toward
CN− at different pH conditions were also conducted
(Figure 6). Probe 1 can be employed to detect CN− in the
pH range of 5.5 - 9.5, and function properly at physio-
logical pH. Thus, Probe 1 can be potentially utilized to
detect CN− in biological samples.
3.4. Reaction Products of Probe 1 with CN−
In order to investigate the reaction product of Probe 1
with CN−, the product of the Probe 1 with CN− was iso-
lated and subjected to 1H NMR characterization. As
shown in Figure 7, the resonance signal of hydrogen (Hd)
at 4-position of the coumarin ring was completely disap-
peared in the isolated product of Probe 1 with CN−. This
observation clearly indicated that the hydrogen at the
4-position of the coumarin ring was substituted by CN−
and formed a 1-CN adduct. Moreover, the formation of
1-CN adduct was further confirmed by ESI Mass spec-
trometry, where a major peak at m/z 373.35 is assigned
to [1-CN+H]+ (Figure 8).Thus, we proposed a possible
reaction mechanism as shown in Figure 9. It included a
nucleophilic addition reaction of the CN− with the cou-
marin ring, and subsequent an elimination reaction. The
specific nucleophilic addition reaction renders Probe 1
selective response to CN−.
Figure 5. Time dependent fluorescence intensity (608 nm)
changes of Probe 1 (5 μM) in the absence (■) or presence (●)
of 90 μM CN−.
Figure 6. The variations of emission ratio (I608/I570) of Probe
1 (5 μM) in the absence (■) or presence (●) of CN− (90 μM)
as a function of pH.
3.5. Detection of Cyanide in Natural Water
The water resource may be contaminated by CN− from
the industrial waste. According to the World Health Or-
ganization, the maximum acceptable level of cyanide in
drinking water is 1.9 μM . Thus it is high importance
to monitor the level of CN− in water samples. The crude
water samples were obtained from Yangtzi River, pond
water and tap water, and were filtered through microfilm-
tration membrane before use. After the probe being
treated with the water samples, ratiometic values (I608/I570)
were determined. The CN− concentration in these water
samples was not detected. Next, the water samples were
spiked with standard CN− solutions and then analyzed
with Probe 1, the results are shown in Table 1. PROBE 1
was able to measure the concentrations of spiked CN−
with good recovery.
3.6. Fluorescence Imaging in Living Cells
To study the utility of Probe 1 detecting CN− in biologi-
cal sample, the Probe 1 was applied for fluorescence im-
aging in living cells. The pancreatic cancer cells was
incubated with Probe 1 (1 μM) for 30 min at 37˚C. After
washing with PBS buffer three times, the cells were used
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L. L. LONG ET AL.
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Figure 7. Partial 1H NMR (400 MHz) spectra of 1) Probe 1 and 2) the isolated product of Probe 1 + CN−.
Figure 8. The ESI-Mass spectra of the isolated product of Probe 1 + CN−.
Figure 9. The proposed reaction mechanism of Probe 1 with CN−.
L. L. LONG ET AL.
for fluorescence imaging. As shown in Figure 10, the
cells exhibited strong fluorescence in the green channel
(Figure 10 a)), and nearly no fluorescence in the red
channel (Figure 10 b)). These indicated that the probe
was cell membrane permeable. When the cell was pre-
treated with tetrabutylammonium cyanide (60 μM) for 10
min, and then further incubated with Probe 1 (1 μM) for
30 min, the cells exhibited strong fluorescence in the red
channel (Figure 10 d)), but almost no fluorescence in the
green channel (Figure 10 c)). These studies demon-
strated that Probe 1 could detect CN− in living cells.
A fluorescence ratiometric probe, Compound 1, for CN−
has been constructed. Upon treatment with CN−, the flu-
orescence of Probe 1 exhibited red shift from 570 nm
Table 1. Determination of CN− concentrations in natural
Sample CN− spiked
River 1 0 Not detected -
River 2 3.00 × 10−6 (3.13 ± 0.04) × 10−6 104.3
Pond water 1 0 Not detected -
Pond water 2 3.00 × 10−6 (3.13 ± 0.04) × 10−6 100.3
Tap water 1 0 Not detected -
Tap water 2 3.00 × 10−6 (3.13 ± 0.04) × 10−6 100.3
aRelative standard deviations were calculated based on three times of meas-
Figure 10. Fluorescence image of the pancreatic cancer cells
stained with Probe 1 (1 μm) for 30 min with emission at 530
± 10 nm a) and emission at 610 ± 10 nm b); fluorescence
image of the pancreatic cancer cells pre-treated with CN−
(60 μm) for 10 min, and then stained with Probe 1 (1 μm)
for 30 min with emission at 530 ± 10 nm c) and emission at
610 ± 10 nm d).
to 608 nm, with the emission ratio (I608/I570) changing
dramatically from 0.52156 to 4.21472. Probe 1 also had a
selective response towards CN−, while other anions gave
almost no influence on Probe 1 detection of CN−. Fur-
thermore, Probe 1 has been successfully applied to de-
tection of CN− in natural water samples and living cells.
This research was financially supported by National Na-
tural Science Foundation of China (21202063), the Natu-
ral Science Foundation of Jiangsu Province (BK2012281),
the China Postdoctoral Science Foundation (2012M511200),
and the Research Foundation of Jiangsu University
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