A PET-based fluorometric chemosensor for the determination of mercury(II) and pH, and hydrolysis reaction-based colorimetric detection of hydrogen sulfide

Article abstract of DOI:10.1039/c6dt00147e

A simple fluorescent chemosensor 1 for the detection of Hg²⁺ and pH was developed by a combination of 2-aminoethyl piperazine and 4-chloro-7-nitrobenz-2-oxa-1,3-diazole. The sensor 1 showed OFF-ON behavior for different colors of fluorescence in the presence of Hg²⁺ and under acidic conditions, respectively, in a near-perfect aqueous solution. The turn-on fluorescence caused by inhibition of photoinduced electron transfer was explained by theoretical calculations. 1 could be used to quantify Hg²⁺ in water samples, and its in vitro studies with HeLa cells showed fluorescence in the presence of Hg²⁺. In addition, 1 could selectively detect S^2- by changing its color from orange to pink in a near-perfect aqueous solution. Moreover, 1 could be used as a practical, visible test kit for S^2-.

Full text of DOI:10.1039/c6dt00147e

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DOI: 10.1039/C6DT00147E
A PET-based fluorometric chemosensor for determination of mercury (II) and pH, and
hydrolysis reaction-based colorimetric detection of hydrogen sulfide
Jae Jun Lee,a Yong Sung Kim,a Eunju Nam,b Sun Young Lee,a Mi Hee Lim,b* Cheal Kima*
aDepartment of Fine Chemistry and Department of Interdisciplinary Bio IT Materials, Seoul
National University of Science and Technology, Seoul 139-743, Republic of Korea. Fax: +82-
2-973-9149; Tel: +82-2-970-6693; E-mail: chealkim@seoultech.ac.kr
bDepartment of Chemistry, Ulsan National Institute of Science and Technology (UNIST),
Ulsan 44919, Republic of Korea. Fax: +82-52-217-5409; Tel: +82-52-217-5422; E-mail:
mhlim@unist.ac.kr
Abstract
A simple fluorescent chemosensor 1 for detection of Hg2+ and pH was developed by
combination of 2-aminoethyl piperazine and 4-chloro-7-nitrobenz-2-oxa-1,3-diazole. The
sensor 1 showed ‘OFF-ON’ different colors of fluorescence in presence of Hg2+ and under
acidic conditions, respectively, in a near-perfect aqueous solution. The turn-on fluorescence
caused by inhibition of photoinduced electron transfer was explained by theoretical
calculations. 1 could be used to quantify Hg2+ in water samples, and its in vitro studies with
HeLa cells showed fluorescence in the presence of Hg2+. In addition, 1 could selectively detect
S2- by changing its color from orange to pink in a near-perfect aqueous solution. Moreover, 1
could be used as a practical, visible test kit for S2-.
Keywords: fluorescence, colorimetry, mercury, hydrogen sulfide, pH indicator, cell imaging
1

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1. Introduction
There is a considerable concern over the heavy metal ions from environment, food and
products. In particular, mercury is one of the most concerned metal ions because it could cause
severe toxicity in vivo.1,2 Nevertheless, mercury is widely used in modern life, such as batteries,
paints and electronic equipment. Owing to these wide usages, human body can be exposed to
mercury through the skin and cell, respiratory and cell membranes, leading to digestive, cardiac,
kidney and DNA damage, mitosis impairment and especially permanent damage to the central
nervous system.3-8 Therefore, the detection and control of mercury is important for human
health and environmental protection.9-13
pH plays an important role in many processes in agriculture, industry and human
health.14-16 Many analytical methods for detection of pH have been extensively explored.
Especially, fluorescent pH indicators have been considerably attractive, owing to high
sensitivity, real-time visualization and quick response time compared to traditional glass pH
electrodes.17 Nevertheless, the fluorescent pH indicators reported to date have limitations such
as complicated synthetic procedures, high detection limits and use of organic solvents.18,19 Thus,
the development of a new water-soluble fluorescent pH indicator by a simple synthetic method
is of great interest.
Hydrogen sulfide (H2S) is considered as the third gasotransmitter, together with carbon
monoxide (CO) and nitric oxide (NO).20-22 Most endogenous hydrogen sulfides have been
devoted to its numerous physiological functions, such as modulation of blood pressure,
mediation of neurotransmission, regulation of inflammation and suppression of oxidative
stress.23-25 However, hydrogen sulfide was also known as a poisonous gas in nervous system
of human being, which could cause serious diseases such as hyperglycemia, Down’s syndrome
and Alzheimer’s disease.26-29 In addition, inhalation of high concentration of hydrogen sulfide
causes various physiological and biochemical problems. It can irritate mucous membranes and
even cause unconsciousness and respiratory paralysis.30-32 Thus, it is important to develop an
efficient method for the detection of H2S in physiological media.
Reaction-based chemosensors for H2S have been studied intensively, owing to their
high sensitivity and selectivity.33-36 Especially, electro-poor aromatic groups can be used for
detection of the nucleophile H2S by the nucleophilic aromatic substitution reaction.37 It shows
2

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fast reaction time and strong colorimetric response, compared with other reported reaction-
based chemosensors.38,39 Nevertheless, a few reports on this reaction were only recently
reported. For example, 4-chloro-7-nitrobenz-2-oxa-1,3-diazole (NBD chloride) has an
electron-poor carbon, which can undergo the nucleophilic aromatic substitution reaction with
sulfide, resulting in 7-nitrobenzo-2,1,3-oxadiazole-4-thiol (NBD-SH).37-39
Photoinduced electron transfer (PET) has been widely applied in the design of the
fluorescence sensors for protons and metal ions.39 Generally, PET takes place from lone pair
of the coordinating atoms (N, O, S, and P) in receptor to the HOMO of the excited
fluorophore.40 The presence of a coordinated metal ion or protonation in receptor lowers the
energy of the lone pair preventing the PET, thus causing the switch-ON of the fluorescence. As
for a fluorophore, NBD has been widely used, because its chloride and fluoride derivatives
easily react with amino and thiol groups, and because it has also a high quantum yield in organic
solvent or membrane. Several NBD-based chemosensors have been reported for the detections
of various transition metal ions, being a useful candidate in PET-based chemosensor.41-44 As
for a receptor, 2-aminoethyl piperazine offers a good possibility to chelation with transition
metal ions, and also acts as a feasible acceptor for proton.45,46 In this regard, we expected that
NBD bridging with 2-aminoethyl piperazine (fluorophore-receptor) might be a good system
for controlling PET process for the detection of metal ions and proton.
Herein, we report on a simple chemosensor 1 with 2-aminoethyl piperazine and NBD
moieties, which was designed and synthesized as a PET-based fluorescent probe for Hg2+ and
pH. The receptor 1 exhibited turn-on fluorescent properties toward Hg2+ and under acidic
conditions via different fluorescent color changes, respectively, in a near-perfect aqueous
solution. The sensing mechanism was explained by theoretical calculation. Moreover, 1
displayed highly selective colorimetric recognition toward S2- by color change from orange to
pink in a near-perfect aqueous solution. Additionally, colorimetric test kit for S2-, and the
determination of Hg2+ in living cells and water samples showed that 1 could be used as a
practical chemosensor.
2. Experimental section
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2.1. Materials and equipment
All the solvents and reagents (analytical grade and spectroscopic grade) were obtained
1
from Sigma-Aldrich and used as received. H NMR and 13C NMR measurements were
performed on a Varian 400 MHz and 100 MHz spectrometer, respectively, and chemical shifts
were recorded in ppm. Electrospray ionization mass spectra (ESI-MS) were collected on a
Thermo Finnigan (San Jose, CA, USA) LCQTM Advantage MAX quadruple ion trap instrument.
Elemental analysis for carbon, nitrogen, and hydrogen was carried out by using a Flash EA
1112 elemental analyzer (thermo) in Organic Chemistry Research Center of Sogang University,
Korea. UV-vis spectra were recorded at room temperature using a Perkin Elmer model Lambda
25 UV/Vis spectrometer. The emission spectra were recorded on a Perkin-Elmer LS45
fluorescence spectrometer.
2.2. Synthesis of receptor 1
2-Aminoethyl piperazine (200 μL, 1.5 mmol) was dissolved in 25 mL of methylene
chloride and NBD chloride (0.31 g, 1.5 mmol) was added into the solution over 15 min.
Triethylamine (1 equiv) was added to remove HCl from the reaction. The solution was stirred
for 1 h at room temperature. The dark orange product was filtered and washed with ethanol.
1
The yield: 0.39 g (89 %). H NMR (400 MHz, DMSO-d6) δ 8.50 (d, J = 9 Hz, 1H), 7.87 (s,
2H), 6.71 (d, J = 9 Hz, 1H), 4.16 (t, J = 5 Hz, 4H), 2.97 (t, J = 6 Hz, 2H), 2.68 (t, J = 5.0 Hz,
4H), 2.60 (t, J = 6 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ 145.28 (1C), 144.84 (1C), 144.77
(1C), 136.28 (1C), 121.10 (1C), 103.60 (1C), 53.59 (1C), 51.95 (2C), 49.23 (2C), 35.65 (1C).
LRMS (ESI): m/z calcd for C12H16N6O3+H+ ([M+H+]), 293.136; found, 293.135. Anal. calcd
for C12H16N6O3 (292.30): C, 49.31; H, 5.52; N, 28.75; Found: C, 49.14; H, 5.71; N, 28.47.
2.3. Fluorescence titration measurement
Receptor 1 (0.9 mg, 0.003 mmol) was dissolved in dimethyl sulfoxide (DMSO, 1 mL)
and 10 μL of the receptor 1 (3 mM) was diluted to 2.990 mL bis-tris buffer solution (10 mM,
pH 7.0) to make the final concentration of 10 μM. Hg(NO3)2·H2O (3.4 mg, 0.01mmol) was
dissolved in 1 mL bis-tris buffer solution (10 mM, pH 7.0). 4.5 - 63 μL of the Hg2+ solution (10
mM) was transferred to receptor 1 solution (10 μM) prepared above. After mixing them for a
few seconds, fluorescence spectra were taken at room temperature.
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2.4. UV-vis titration measurements
For Hg2+, receptor 1 (0.9 mg, 0.003 mmol) was dissolved in DMSO (1 mL) and 10 μL
of the receptor 1 (3 mM) was diluted to 2.990 mL bis-tris buffer solution (10 mM, pH 7.0) to
make the final concentration of 10 μM. Hg(NO3)2·H2O (3.4 mg, 0.01mmol) was dissolved in 1
mL bis-tris buffer solution (10 mM, pH 7.0). 3 - 30 μL of the Hg2+ solution (10 mM) was
transferred to receptor 1 solution (10 μM) prepared above. After mixing them for a few seconds,
UV-vis spectra were taken at room temperature.
For S2-, receptor 1 (0.9 mg, 0.003 mmol) was dissolved in DMSO (1 mL) and 10 μL
of the receptor 1 (3 mM) was diluted to 2.990 mL bis-tris buffer solution (10 mM, pH 7.0) to
make the final concentration of 10 μM. Sodium sulfide nonahydrate (Na2S·9H2O, 2.5 g, 1.0
mmol) was dissolved in 5 mL bis-tris buffer solution (10 mM, pH 7.0). 1.2 - 24.8 μL of the S2-
solution (200 mM) was transferred to receptor 1 solution (10 μM) prepared above. After mixing
them for a few seconds, UV-vis spectra were taken at room temperature.
2.5. Job plot measurements
For Hg2+, 1 (2.9 mg, 0.01 mmol) was dissolved in DMSO (1 mL). 3, 2.7, 2.4, 2.1, 1.8,
1.5, 1.2, 0.9, 0.6, 0.3 and 0 µL of the 1 solution were taken and transferred to vials. Each vial
was diluted with bis-tris buffer solution (10 mM, pH 7.0) to make a total volume of 2.997 mL.
Hg(NO3)2∙H2O (3.4 mg, 0.01 mmol) was dissolved in DMSO (1 mL). 0, 0.3, 0.6, 0.9, 1.2, 1.5,
1.8, 2.1, 2.4, 2.7, and 3 µL of the Hg(NO3)2∙H2O solution were added to each diluted 1 solution.
Each vial had a total volume of 3 mL. After reacting them for a few seconds, fluorescence
spectra were taken at room temperature.
For S2-, 1 (5.9 mg, 0.02 mmol) was dissolved in DMSO (1 mL). 3, 2.7, 2.4, 2.1, 1.8,
1.5, 1.2, 0.9, 0.6, 0.3 and 0 µL of the 1 solution were taken and transferred to vials. Each vial
was diluted with bis-tris buffer solution (10 mM, pH 7.0) to make a total volume of 2.997 mL.
Na2S·9H2O (4.9 mg, 0.02 mmol) was dissolved in DMSO (1 mL). 0, 0.3, 0.6, 0.9, 1.2, 1.5, 1.8,
2.1, 2.4, 2.7, and 3 µL of the Na2S·9H2O solution were added to each diluted 1 solution. Each
vial had a total volume of 3 mL. After reacting them for a few seconds, UV-vis spectra were
taken at room temperature.
2.6. Competitive experiments
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For Hg2+, receptor 1 (0.9 mg, 0.003 mmol) was dissolved in DMSO (1 mL) and 10 μL
of the receptor 1 (3 mM) was diluted to 2.990 mL bis-tris buffer solution (10 mM, pH 7.0) to
make the final concentration of 10 μM. MNO3 (M = Na, K, 0.01 mmol), M(NO3)2 (M = Mn,
Ni, Co, Zn, Fe, Cd, Mg, Ca, Pb, 0.01 mmol), or M(NO3)3 (M = Al, Ga, In, Fe, Cr, 0.01 mmol)
was separately dissolved in 1 mL bis-tris buffer solution (10 mM, pH 7.0). 60 μL of each metal
solution (10 mM) was taken and added into 3 mL of each receptor 1 solution (10 μM) prepared
above to make 20 equiv. Then, 60 μL of Hg(NO3)2∙H2O solution (10 mM) was added into the
mixed solution of each metal ion and receptor 1 to make 20 equiv. After mixing them for a few
seconds, fluorescence spectra were taken at room temperature.
For S2-, receptor 1 (0.9 mg, 0.003 mmol) was dissolved in DMSO (1 mL) and 10 μL
of the receptor 1 (3 mM) was diluted to 2.990 mL bis-tris buffer solution (10 mM, pH 7.0) to
make the final concentration of 10 μM. Tetraethylammonium (TEA) salts of CN-, F-, Cl-, Br-,
-
and I-, and tetrabuthylammonium (TBA) salts of H2PO4 and OAc-, and sodium salts of SCN-,
-
-
-
N3 , NO2 and HSO4 (0.20 mmol) were separately dissolved in 1 mL bis-tris buffer solution
(10 mM, pH 7.0). 22.5 L of each anion solution (200 mM) were taken and added into 3 mL
of the solution of receptor 1 (10 M) to give 150 equiv of anions. Then, 22.5 L of Na2S·9H2O
solution (200 mM) were added into the mixed solution of each anion and 1 to make 150 equiv.
After mixing them for a few seconds, UV-vis spectra were taken at room temperature.
2.7. pH titration of 1
Receptor 1 (0.9 mg, 0.003 mmol) was dissolved in DMSO (1 mL) and 10 μL of the
receptor 1 (3 mM) was diluted to twenty-three 2.990 mL bis-tris buffer solution (10 mM) with
different pH values (1.4, 1.8, 2.2, 2.6, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7, 7.5, 8, 8.5, 9, 9.5,
10, 10.5, 11, 11.5 and 12) to make final concentration of 10 M. After mixing them for a few
second, fluorescence spectra were taken at room temperature. pKb value was calculated by a
nonlinear fitting method according to Dose Response (DoseResp) fitting function.
2.8. NMR titrations
For Hg2+, five NMR tubes of receptor 1 (2.9 mg, 0.01 mmol) dissolved in DMSO-d6
were prepared and then five different concentrations (0, 0.002, 0.005, 0.01 and 0.015 mmol) of
Hg(NO3)2∙H2O dissolved in DMF-d7 were added to each solution of receptor 1. After shaking
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them for a minute, 1H NMR spectra were taken at room temperature.
For S2-, three NMR tubes of receptor 1 (2.9 mg, 0.01 mmol) dissolved in DMSO-d6
were prepared and then three different concentrations (0, 0.01 and 0.03) of Na2S·9H2O
dissolved in D2O were added to each solution of receptor 1. After shaking them for a minute,
1H NMR spectra were taken at room temperature.
2.9. Imaging experiments in living cells
HeLa cells (ATCC, Manassas, USA) were maintained in media containing DMEM,
10 % fetal bovine serum (FBS, GIBCO, Grand Island, NY, USA), 100 U/ml penicillin (GIBCO),
and 100 mg/ml streptomycin (GIBCO). The cells grew in a humidified atmosphere with 5 %
CO2 at 37 °C. Cells were seeded onto 6 well plates (SPL Life Science Co., Ltd., South Korea)
at a density of 150,000 cells for 1 mL and then incubated at 37 °C for 24 h. Cells were treated
with 1 (dissolved in DMSO; 0.5 % v/v final DMSO concentration; 5 M; at room temperature)
for 5 min. Prior to addition of various concentrations of mercury nitrate (dissolved in DMF;
0.5 % v/v), cells were washed with 2 mL of 10 mM bis-tris buffer (pH 7.4, 150 mM NaCl).
Imaging was performed with an EVOS FL fluorescence microscope (Life technologies) using
a GFP light cube (excitation 470 (± 11) nm; emission 510 (± 21) nm).
2.10. Theoretical calculation methods
All theoretical calculations were performed by DFT/TD-DFT method with the hybrid
exchange-correlation functional B3LYP47,48 applying without any symmetry restrictions in the
gas phase. The 6-31G** basis set49,50 was used for the main group elements, whereas the
Lanl2DZ effective core potential (ECP) was employed for Hg.51-53 To understand the PET
mechanism, receptor 1 was calculated with two separated species, 2-aminoethyldiethylamine
(R) and 4-dimethylamino-7-nitrobenz-2-oxa-1,3-diazole (F). The energy-minimized structures
of R and F species were obtained in various geometric forms. On the basis of their energy-
minimized structures, the optimized structures of 1 and 1-Hg2+ complex were also obtained. In
vibrational frequency calculations, there was no imaginary frequency for the optimized
geometries of 1 and 1-Hg2+ complex, suggesting that these geometries represented local
minima. For all calculations, the solvent effect of water was considered by using the Cossi and
Barone’s CPCM (conductor-like polarizable continuum model).54,55 In order to investigate the
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transition energies for the optimized structures of 1 and 1-Hg2+ complex, we caDlcOuI:l1a0.t1e0d39/tCh6eDT00147E
lowest 25 singlet-singlet transitions using their ground state geometry (S0) with TD-DFT
(B3LYP) method. The GaussSum 2.1 was used to calculate the contribution of molecular
orbital in electronic transitions.56 All the calculations were performed with Gaussian 03 suite.57
2.11. Determination of Hg2+ and S2- in water samples
For Hg2+, fluorescence spectral measurement of water samples containing Hg2+ was
carried out by adding 10 μL of 3 mmol/L stock solution of 1 and 0.60 mL of 50 mmol/L bis-
tris buffer stock solution to 2.390 mL sample solutions. After well mixed, the solutions were
allowed to stand at 25 ◦C for 2 min before the test.
For S2-, UV-vis spectral measurement of water samples containing S2- was carried out
by adding 10 μL of 3 mmol/L stock solution of 1 and 0.60 mL of 50 mmol/L bis-tris buffer
stock solution to 2.390 mL sample solutions. After well mixed, the solutions were allowed to
stand at 25 ◦C for 2 min before the test.
2.12. Colorimetric test kit for S2-
Receptor 1 (0.9 mg, 0.009 mmol) was dissolved in methanol (3 mL). Receptor 1-test
kits were prepared by immersing filter papers into receptor 1 solution (3 mM), and then dried
-
in air. TEA salts of CN-, F-, Cl-, Br-, and I-, and TBA salts of H2PO4 and OAc-, and sodium
-
-
-
salts of SCN-, N3 , NO2 and HSO4 (0.20 mmol) were separately dissolved in bis-tris buffer (1
mL, 200 mM, pH 7.0). The test kits prepared above were added into different anion solutions
(50 μM), and then dried at room temperature.
3. Results and discussion
The receptor 1 was obtained by coupling 2-aminoethyl piperazine and 4-chloro-7-
nitrobenz-2-oxa-1,3-diazole with 89 % yield in methylene chloride (Scheme 1) and analyzed
by 1H NMR, 13C NMR, ESI-mass spectrometry and elemental analysis.
3.1. Fluorometric determination of Hg2+ and pH
To examine the fluorometric properties of 1, the emission was measured with various
metal ions (Na+, K+, Mg2+, Ca2+, Al3+, Ga3+, In3+, Cr3+, Mn2+, Fe2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+
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Cd2+, Hg2+ and Pb2+) in bis-tris buffer (10 mM, pH 7.0). As shown in Fig. 1, receptor 1 alone
has a weak fluorescence emission (max = 542 nm and ex = 470 nm) due to the PET process
from the amine moieties of ethylenediamine to 4-dimethylamino-7-nitrobenz-2-oxa-1,3-
diazole. When 18 equiv of metal ions such as Na+, K+, Mg2+, Ca2+, Al3+, Ga3+, In3+, Cr3+, Mn2+,
Fe2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+ Cd2+ and Pb2+ were added to the receptor 1, it was found that
the solution of 1 exhibited either no or a small increase of the fluorescence. In contrast, the
addition of Hg2+ into 1 showed a significant yellow fluorescence enhancement (7 folds) at 542
nm. The fluorescence enhancement might be originated from blocking the PET process, which
was induced by the chelation of Hg2+ with ethylenediamine moiety of 1.39 These results
indicated that the receptor 1 could be used as a fluorescence chemosensor for Hg2+. On the
other hand, the colorimetric sensing abilities of 1 with various metal ions were tested, but 1 did
not show any color changes.
To further understand the sensing properties of 1 toward Hg2+, fluorescence titration
of the receptor 1 with Hg2+ ion was performed (Fig. 2). The emission intensity of 1 at 542 nm
gradually increased until the amount of Hg2+ reached 20 equiv. The binding properties of 1
with Hg2+ were further studied by UV-vis titration (Fig. S1). Upon the addition of Hg2+ to a
solution of 1, the absorption band at 495 nm exhibited a small decrease and reached minimum
at 9 equiv of Hg2+. There was a clear isosbestic point at 332 nm, demonstrating that only one
product was generated from the interaction of 1 with Hg2+. Based on the UV-vis titration of 1
with Hg2+, the association constant (K) of 1 with Hg2+ was calculated by the non-linear fitting
method (Fig. S2).58,59 The K value was determined to be 2.3 x 103, which was within a range
of those previously reported for Hg2+ chemosensor.60-64
Job plot65 referred to a 1:1 stoichiometry between 1 and Hg2+ (Fig. S3), which was
further confirmed by positive ion ESI-mass spectrometry analysis (Fig. S4). A peak at m/z
606.867 was assigned to [1 + Hg2+ + Cl- + solvent]+ (calcd. 607.081).
1
To further get information for the binding mode of 1 with Hg2+, H NMR titrations
were performed (Fig. 3). Upon the addition of 1 equiv of Hg2+ to 1, the NH2 (Ha) of 2-
aminoethyl piperazine at 7.87 ppm and the Hc showed significant and slight upfield shifts,
respectively. The Hb, Hd, Hg, and Hf showed down-field shift. The peaks didn’t change upon
further addition of Hg2+. These results suggested that the NH2 and tertiary amine might
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coordinate to Hg2+ as shown in Scheme 2.
To check the practical applicability of receptor 1 as a Hg2+ selective sensor, the competition
experiments were conducted (Fig. 4). Upon treatment with 20 equiv of Hg2+ in the presence of
the same concentration of other metal ions, there were no obvious interferences, while Fe2+ and
Fe3+ showed slightly interference. Thus, 1 could be as an excellent fluorometric sensor for
detection of Hg2+ in the presence of most competing metal ions.
We constructed a calibration curve for the determination of Hg2+ by 1 (Fig. S5). A
good linear relationship was observed between the fluorescence intensity of 1 and Hg2+
concentration (0.00-16.66 μM) with a correlation coefficient of R2 = 0.99846 (n = 3). This
result indicates that 1 is suitable for quantitative detection of Hg2+. The detection limit was
calculated as 0.57 μM based on the definition by IUPAC (CDL =3σ/k). To confirm the practical
applicability of 1 with Hg2+, 1 was applied for the determination of Hg2+ in both tap water and
water samples. As shown in Table 1, a suitable recovery and R.S.D. values from the tap water
samples were obtained. We also prepared artificially polluted water samples by adding various
metal ions known as being involved in industrial processes into deionized water. Suitable
recovery and R.S.D. values from the water samples were obtained. These results suggest that
the receptor 1 could be useful for the measurement of Hg2+ in chemical and environmental
applications.
For biological application, the pH dependence of 1 was examined at various pH.
Interestingly, the significant increase of a green fluorescence intensity (λmax = 528 nm) of 1 was
observed below pH 4 (Fig. 5). To understand these results in details, the pKb of 1 was calculated,
based on pH titration (Fig. S6). The value was found to be 3.57, which is close to that of tertiary
amine reported in the literature.66,67 These results indicated that the protonation of tertiary
amine of 1 could induce the green fluorescence enhancement by inhibition of PET process
below pH 4. Thus, the receptor 1 could be used as a fluorescent pH indicator under acidic
condition (below pH 4). Moreover, the pH dependence of 1 in presence of Hg2+ showed the
yellow fluorescence (λmax = 542 nm) between pH 7 and 10, and the green fluorescence (λmax
=
528 nm) between pH 2 and 4 (Fig. 5). To demonstrate these results, we proposed the three-step
‘ON-OFF-ON’ mechanism in Scheme 3. From pH 7 to 10, the yellow fluorescence was stable
owing to the formation of 1-Hg2+ complex. From pH 5 to 6, the protonation of primary amine
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induced demetallation of Hg2+ from 1-Hg2+ complex, resulting in low fluorescence. Below pH
4, the protonation of tertiary amine of 1 showed green fluorescence. Therefore, 1-Hg2+ complex
could be used as a metal-based pH indicator as shown in Scheme 3. To the best of our
knowledge, this is the first pH indicator by using H+-induced demetallation of mercury
complex.
To demonstrate the potential of 1 to detect Hg2+ in living metrics, fluorescence imaging
experiments were carried out in living cells (Fig. 6). HeLa cells were first incubated with 1
solution (5 μM) for 5 min and then exposed to various concentrations of Hg2+ solutions (0, 10,
20, 30, 40 and 50 μM) before imaging. The HeLa cells which were cultured with both 1 and
Hg2+ exhibited fluorescence, while those cells cultured without Hg2+ or 1 didn’t showed any
fluorescence. Thus, these results confirmed that 1 could be a suitable sensor to detect Hg2+ in
living cells.
The reversibility of the chemosensor is one of the essential aspects for sensor
applications. Therefore, we performed the reversibility of receptor 1 by the alternate addition
of Hg2+ and ethylenediaminetetraacetic acid (EDTA). The emission band of the tested solution
at 542 nm showed the alternate increasing and decreasing processes for several cycles (Fig.S7).
To further understand the sensing mechanism of Hg2+ with 1, theoretical calculations
were performed in parallel to the experimental studies. 1 was separated into two moieties, R
(2-aminoethyldiethylamine) and F (4-dimethylamino-7-nitrobenz-2-oxa-1,3-diazole) based on
the previously reported literature,68 which were optimized with DFT/B3LYP/6-31G**. 1-Hg2+
complex was optimized with a diamagnetic character (S=0, DFT/B3LYP/main group atom: 6-
31G** and Hg: Lanl2DZ/ECP), based on Job plot, ESI-mass analysis and 1H NMR titrations.
The significant structural properties of the energy-minimized structures of R, F, 1 and 1-Hg2+
complex were shown in Fig. S8. Based on the energy-minimized structures, time-dependent
density functional theory (TD-DFT) calculations were performed to understand their electronic
transitions. The molecular orbital (MO) diagrams of 1 and 1-Hg2+ complex were compared
with those of R and F. The MOs of R, F and 1 are shown in Fig. 7a. The radiative transition
could be observed when the transition state of HOMO (MO 54 of F) to LUMO (MO 55 of F)
in F was excited by photon. The main MOs contributions of the first lowest excited state of 1
were determined for HOMO → LUMO and HOMO - 1 → LUMO (460.48 nm, Fig. S9).
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HOMO of 1 was composed of MO 25 of R and MO 54 of F, and HOMO - 1 of 1 was composed
of MOs 24 and 25 of R and MO 54 of F. Also, LUMO of 1 was composed of MO 55 of F. The
composition of MO 25 of R (-5.766 eV) in both HOMO and HOMO - 1 of 1 indicated higher
energy than that of MO 54 of F (-6.026 eV). Thus, the transitions of HOMO → LUMO and
HOMO - 1 → LUMO showed PET process from receptor (MO 25 of R) to fluorophore (MO
54 of F), which indicated the fluorescence quenching. In case of 1-Hg2+ complex, the main
MOs contribution of the first lowest excited state was determined for HOMO → LUMO
(432.51 nm, Fig. S10). As shown in Fig. 7b, the chelation of Hg2+ with 1 induced the
stabilization of MO 25 of R, which showed the inhibition of PET process from MO 25 of R to
MO 54 of F. In addition, the transition of HOMO → LUMO in 1-Hg2+ complex indicated the
radiative transition of F (MO 54 of F → MO 55 of F). Thus, the sensing mechanism of 1 with
Hg2+ was explained by inhibition of PET process of 1, controlling its MO’s energy by the
chelation of Hg2+.
Based on the Job plot, ESI-mass spectrometry analysis, previously reported similar
crystal structure of Hg2+-complex,69 and theoretical calculations, a square planar structure for
1-Hg2+ complex was proposed (Scheme 2).
3.2. Colorimetric detection of S2-
The colorimetric sensing abilities of receptor 1 with various anions (CN-, OAc-, F-, Cl-,
-
-
-
-
2-
-
3-
2-
Br-, I-, H2PO4 , BzO-, SCN-, N3 , NO2 , HSO4 , SO3 , HSO3 , PO4 , CO3 and S2-) in bis-tris
buffer (10 mM, pH 7.0) were monitored by UV-vis absorption spectrometry. Upon the addition
of 150 equiv of each anion, 1 showed almost no change in UV-vis spectra (Fig. 8a). In contrast,
only S2- showed both a distinct UV-vis change with a significant bathochromic shift and a color
change from orange to pink (Fig. 8b). Thus, 1 could be a suitable chemosensor for detection of
S2- by colorimetric change. On the other hand, when the anions were added to the receptor 1,
it was found that the solution of 1 exhibited either no or very weak fluorescence. In addition,
it is worthwhile to mention that 1 showed the fluorescent response to the specific cation species
Hg2+, while the specific anion species S2- was responded colorimetrically to 1.
To study the colorimetric sensing properties, UV-vis titration experiments of 1 with S2-
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were performed (Fig. 9). On treatment with S2- to solution of 1, the absorption banDdOaI:t104.190739/nCm6DT00147E
gradually decreased, and a new band at 531 nm reached a maximum at 150 equiv of S2-. An
isosbestic point was observed at 517 nm, indicating that only one product was generated from
1 upon reacting with S2-. The binding mode between 1 and S2- was determined through Job plot
analysis (Fig. S11),65 which exhibited a 1:1 stoichiometry between 1 and S2-. Based on the UV-
vis titration of 1 with S2-, the association constant (K) of 1 with S2- was calculated to be 5.0 x
102 by Benesi–Hildebrand equation (Fig. S12).70
1
In order to further understand the sensing mechanism of S2- by 1, H NMR titrations
were performed (Fig. S13). Upon the addition of 3 equiv of S2- to 1, all protons gradually
disappeared and new peaks appeared in upfield, while Hf showed down-field shift. Importantly,
the new peaks are consistent with those of the compounds, 2-aminoethyl piperazine and NBD
sulfide. These results suggest that electron-poor NBD moiety of 1 underwent the nucleophilic
aromatic substitution reaction with S2- to produce NBD sulfide and 2-aminoethyl piperazine
species.
To confirm the nucleophilic aromatic substitution mechanism of 1 with S2-, positive
and negative ion ESI-mass spectrometry analyses were performed (Fig. S14). A peak of
positive ion mass spectrum at m/z 130.40 was assigned to the starting compound [1-(2-
aminoethyl)piperazine + H+]+ (calcd. 130.13, Fig. S14a), and a peak of negative ion mass
spectrum at m/z 196.40 was assigned to [7-nitrobenzofurazan-4-thiol - H+]- (calcd. 195.98, Fig.
1
S14b). These results are well consistent with the observations obtained in H NMR titration,
confirming that 1 undergoes the nucleophilic substitution reaction by S2- to produce 1-(2-
1
aminoethyl)piperazine and 7-nitrobenzofurazan. Based on Job plot, H NMR titration, ESI-
mass spectrometry analysis, we propose the plausible sensing mechanism of S2- by 1 (Scheme
4).
To check the practical applicability of receptor 1 as a S2- selective sensor, the
competition experiments were conducted (Fig. S15). Upon treatment with 150 equiv of S2- in
the presence of the same concentration of other anions, there were no obvious interferences,
-
-
while F-, I-, H2PO4 and N3 showed slightly interference. Nevertheless, they still kept
discernible colors by naked eye. Thus, 1 could be as an excellent colorimetric sensor for
detection of S2- in the presence of most competing anions.
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The effect of pH on the absorption response of receptor 1 to S2- was investigated in a
series of solutions with pH values ranging from 2 to 12 (Fig. S16). While the absorbance of 1
with S2- was very weak at pH 2 and 12, its stable and intense absorbance was observed in the
pH range of 3-11. These results indicated that S2- could be clearly detected by the naked eye or
UV-vis absorption measurements using 1 over the wide pH range of 3-11.
We also constructed a calibration curve for the determination of S2- by 1 (Fig. S17). A
good linear relationship was observed between the absorbance of 1 and S2- concentration (0-
600 μM) with a correlation coefficient of R2 = 0.9955 (n = 3). The calibration curve indicates
that 1 is suitable for quantitative detection of S2-. The detection limit was calculated as 46.97
μM based on the definition by IUPAC (CDL =3σ/k). The value is much lower than a Secondary
Maximum Contaminant Level (7.8 mM) by odor in drinking water, established by the EPA.71
Hence, receptor 1 could be used as a good detector for monitoring S2- ion in drinking water.
To confirm the practical applicability of 1 with S2-, 1 was applied for the determination
of S2- in both tap water and water samples. As shown in Table 2, a suitable recovery and R.S.D.
values from the tap water samples were obtained. The artificially polluted water samples also
showed a suitable recovery and R.S.D. values. The results indicate that the receptor 1 could be
useful for the measurement of S2- in chemical and environmental applications.
For the practical application of 1, test kits were prepared by immersing filter papers in
a solution of 1 and then drying air. These test kits were used to sense S2- ions among different
anions (Fig. 10). When the test kits coated with 1 were added into different anion solutions, the
obvious color change was observed only with S2- from orange to pink. Therefore, the test kit
coated with 1 was found to be convenient to detect S2- by the color change in the presence of
other anions.
4. Conclusion
NBD-based chemosensor 1 was simply developed for the fluorometric detection of
Hg2+ and the acidic condition in a near-perfect aqueous solution. 1 showed a highly selective
response to Hg2+, which showed turn-on fluorescence with yellow color. In below pH 4, 1
showed turn-on fluorescence with a green color. To the best of our knowledge, this is the first
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pH indicator by using H+-induced demetallation of mercury complex. The turn-on fDluOoI: 1r0e.s10c3e9n/Cc6eDT00147E
caused by inhibition of PET was explained by theoretical calculations. In addition, 1 could be
used to quantify Hg2+ ions in water samples and living cells. Moreover, 1 showed a selective
colorimetric detection of S2- among various anions with color change from orange to pink,
which could be used to quantify S2- in water samples. The detection limit (47 M) for S2- is
much lower than a Secondary Maximum Contaminant Level (7.8 mM) by odor in drinking
water. Importantly, 1 could be applied to detect S2- by a simple test strip in aqueous solution.
Therefore, these results indicate that receptor 1 can serve as a novel multifunctional
“fluorescence” chemosensor for the detection of Hg2+ and pH by different colors, and also a
“naked-eye” chemosensor for S2- in a near-perfect aqueous solution.
Acknowledgements
Financial support from Basic Science Research Program through the National
Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and
Technology (NRF-2014R1A2A1A11051794, NRF-2015R1A2A2A09001301 and NRF-
2014R1A2A2A01004877) are gratefully acknowledged. We thank Nano-Inorganic Laboratory,
Department of Nano & Bio chemistry, Kookmin University to access the Gaussian 03 program
packages.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://????.
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[71] EPA, The Guidelines for the Health Risk Assessment of Chemical MixturesD,OUI: .1S0..10E39P/CA6D,T00147E
1986.
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Scheme 1. Synthesis of 1.
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Scheme 2. Proposed structure of 1-Hg2+ complex and sensing mechanism for the detection of
Hg2+ by 1.
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Scheme 3. Proposed mechanism for the detection of Hg2+ and pH by 1.
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Scheme 4. Proposed sensing mechanism of S2- by 1.
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Table 1. Determination of Hg2+ in water samples.
Sample
Hg2+ added
Hg2+ found
Recovery
(%)
R.S.D. (n = 3)
(μmol/L)
(μmol/L)
(%)
Tap water
0.00
5.00
3.00
0.00
4.91
2.85
98.2
95.0
4.7
4.0
Water
sample[a]
[a] Synthesized by deionized water, 3.00 μmol/L Hg2+, 5 μmol/L Cd2+, Pb2+, Na+, K+, Ca2+,
Mg2+. Conditions: [1] = 10 μmol/L in 10 mM bis-tris buffer-DMSO solution (999:1, pH 7.0).
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Table 2. Determination of S2- in water samples.
Sample
S2- added
S2- found
Recovery
(%)
R.S.D. (n = 3)
(μmol/L)
(μmol/L)
(%)
Tap water
0.00
80
0.00
82
102
105
3.6
4.5
Water
60
63
sample[a]
-
-
-
[a] Synthesized by deionized water, 60 μmol/L S2-, 70 μmol/L F-, Cl-, H2PO4 , HSO4 , NO2 ,
-
HCO3 . Conditions: [1] = 10 μmol/L in 10 mM bis-tris buffer-DMSO solution (999:1, pH 7.0).
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Figure Captions
Fig. 1 (a) Fluorescence spectral changes of 1 (10 μM) in presence of 18 equiv of various metal
ions in bis-tris buffer (10 mM, pH 7). Inset: Picture of the fluorescence corresponded with 1
and 1-Hg2+ (Excitation: 365 nm) (b) Bar graph representing the fluorescence intensity of 1 (10
μM, λmax = 542 nm, λex = 472 nm) upon treatment with various metal ions.
Fig. 2 Fluorescence spectral changes of 1 (10 μM) in the presence of increasing different
concentrations of Hg2+ (from 0 to 21 equiv) at room temperature. Inset: Plot of the fluorescence
intensity at 542 nm as a function of Hg2+ concentration.
Fig. 3 1H NMR titration of 1 with Hg2+.
Fig. 4 (a) Fluorescence spectral changes of 1 (10 μM) upon addition of Hg2+ (18 equiv) in the
absence and presence of 18 equiv of various metal ions in bis-tris buffer (10 mM, pH = 7.0).
(b) Bar graph representing the fluorescence intensity of 1 (10 μM, λmax = 542 nm, λex = 472
nm) with Hg2+ (18 equiv) in the absence and presence of 18 equiv of various metal ions in bis-
tris buffer (10 mM, pH = 7.0).
Fig. 5 (a) Fluorescence spectral changes of 1 (10 μM) with Hg2+ (18 equiv) at different pH (2-
12). (b) Fluorescence intensity (at 542 nm) of 1 (10 μM) and 1-Hg2+ (18 equiv) at different pH
(2-12). (c) Pictures of fluorescence color of 1 (10 μM, λex = 365 nm) upon addition of Hg2+ (18
equiv) at different pH (2-12).
Fig. 6 Fluorescent responses of 1 to Hg(II) in HeLa cells. Cells were preincubated with 1 for 5
min prior to addition of various concentrations of Hg(II). The top images were observed under
light microscope and the bottom images were recorded through a fluorescence microscope.
Conditions: [Hg(II)] = 0, 10, 20, 30, 40, and 50 μM; [1] = 5 μM; 37 °C; 5% CO2. The scale bar
is 50 μm.
Fig. 7 Molecular orbital diagrams of (a) 1 and (b) 1-Hg2+ with R and F, and the excitation
energies of 1 and 1-Hg2+ by TD-DFT methods.
Fig. 8 (a) Absorption spectral changes of 1 (10 μM) in presence of 150 equiv of various anions
in bis-tris buffer (10 mM, pH 7). (b) The color changes of 1 (10  in presence of 150 equiv
of various anions in bis-tris buffer (10 mM, pH = 7.0).
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Page 28 of 40
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DOI: 10.1039/C6DT00147E
Fig. 9 Absorption spectral changes of 1 (10 μM) in the presence of increasing different
concentrations of S2- (from 0 to 164 equiv) at room temperature. Inset: Plot of the absorbance
intensity at 531 nm as a function of S2- concentration.
Fig. 10 Photographs of the filter paper coated with 1 (3 mM) in the presence of various anions
(50 μM).
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Page 29 of 40
Dalton Transactions
View Article Online
DOI: 10.1039/C6DT00147E
(a)
(b)
700
600
500
400
300
200
100
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
+
+
+
+
+
+
+
+
+
+
+
+
+
+
K
+
+
+
2+
3+
3+ 3+
2+
3+
2+
3+
2+ 3+
2+ 2+
+
+
2+
2+
2+
Hg Al Ga In Zn Cd Fe Fe Mg Cr Co Ni Na
Ca Mn Pb
Fig. 1
29