Recognition of copper and hydrogen sulfide in vitro using a fluorescein derivative indicator

Article abstract of DOI:10.1039/c2dt12462a

We report the development of a fluorescein-based chemosensor (L1) for monitoring ions or micromolecules (H₂S). Copper ions are known to be toxic at high concentrations and hydrogen sulfide induces various problems. Herein we develop a simple me

Full text of DOI:10.1039/c2dt12462a

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PAPER
Recognition of copper and hydrogen sulfide in vitro using a fluorescein
derivative indicator†
Fengping Hou,a Ju Cheng,b Pinxian Xi,a Fengjuan Chen,a Liang Huang,a Gouqiang Xie,a Yanjun Shi,a
Hongyan Liu,a Decheng Bai*b and Zhengzhi Zeng*a
Received 21st December 2011, Accepted 26th February 2012
DOI: 10.1039/c2dt12462a
We report the development of a fluorescein-based chemosensor (L1) for monitoring ions or
micromolecules (H2S). Copper ions are known to be toxic at high concentrations and hydrogen sulfide
induces various problems. Herein we develop a simple method for detecting Cu(II) and H2S with high
selectivity and sensitivity. The chemosensor L1 displays on–off–on type fluorescence change with
alternately added Cu(II) and H2S to the media along with reversible forming–separating of the complex.
The potential biomedical relevance of the chemical mechanisms involved in the detection of L1 is
described.
show that hydrogen sulfide (H2S), like nitric oxide and carbon
Introduction
monoxide, may act as a gaseous signalling molecule in physio-
logical systems.7 H2S appears to be involved in various physio-
Indicating and eliminating all kinds of micromolecules and ions
logical processes, including mediation of neurotransmission,8
is an important task for chemical researchers. Heavy metal ions
relaxation of vascular smooth muscles,9 mediation of respiratory
are of great concern, in spite of the fact that some heavy metal
system and inhibition of apoptosis.10 Therefore, it must be con-
ions play important roles in living systems, they are very toxic
and hence capable of causing serious environmental and health
problems.1 Copper is the third-most abundant transition metal in
the body and in the brain,2 with average neural copper concen-
trations in the order of 0.1 mM.3 Even though a variety of
quantification techniques have been developed, fluorescent tech-
niques have become the base standard for Cu(II) sensing because
of their sensitivity and specificity, and real-time monitoring with
fast response time.4 Cu(II) is a typical ion that leads to decreased
fluorescent emissions owing to quenching of the fluorescence by
mechanisms inherent to the paramagnetic species.5 Interestingly,
the resultant Cu(II) complex may be a probe for other substances.
As is known to all, hydrogen sulfide (H2S) is a poisonous gas
that smells like rotten eggs. Nevertheless, this dogma was found
to be no longer valid as a result of the observation that the H2S-
generating enzymes exist in the body, which lead to detectable
levels of H2S from endogenous sources.6 Recent investigations
sidered a biologically important molecule in both health and
disease. It appears that hydrogen sulfide shows more beneficial
effects than NO as the former do not generate toxic reactive
oxygen species (ROS). Viewed in this light, it is particularly
important to develop new methods for monitoring hydrogen
sulfide, especially under physiological conditions.
To the best of our knowledge, fluorescence probes are a sensi-
tive and real-time method for indicating micromolecules and
ions. Recently, metal-complex based luminescent chemosensors
have attracted great attention.11 For example, the Lippard group
has reported a lot of work based on fluorescein and its com-
plexes,12 as fluorescein is a strong fluorophore and can be used
for labelling in organisms. Fluorescent sensors mostly involve
ions, but for hydrogen sulfide there are relatively few,13
especially in vivo and in vitro. Recently several fluorescent
probes for hydrogen sulfide were reported and these sensors
could be applied in fluorescence imaging in living cells.14
With this in mind, we synthesized compound L1 based on flu-
orescein. Compound L1 displayed obvious fluorescence quench-
ing after the addition of Cu(II). Once interacted with Cu(II) to
form complex L1Cu, the new complex showed high specificity
for hydrogen sulfide, meaning the target molecule reacted with
the complex and liberated the dye. The signalling was accom-
plished by an inorganic molecule-induced dye release method.
Herein, we report the synthesis, X-ray crystal structure, photo-
physical characterization, and cellular imaging experiments of
the fluorescein-based probe L1 which is selective for copper
ions, the subsequent complex L1Cu displays characteristic ‘‘off–
on’’ behaviour for hydrogen sulfide.
aKey Laboratory of Nonferrous Metals Chemistry and Resources
Utilization of Gansu Province, State Key Laboratory of Applied Organic
Chemistry and College of Chemistry and Chemical Engineering,
Lanzhou University, Lanzhou 730000, PR China. E-mail:
zengzhzh@lzu.edu.cn, zengzhzh@yahoo.com.cn; Fax: +86 931
8912582; Tel: +86 931 8912596
bSchool of Basic Medical Sciences, Lanzhou University, Lanzhou
730000, PR China. E-mail: bdc@lzu.edu.cn
†Electronic supplementary information (ESI) available: Instruments and
some experimental methods, characterisation data, supplementary
spectra; NMR, MS spectra, crystal data. CCDC reference number
854854. For ESI and crystallographic data in CIF or other electronic
format see DOI: 10.1039/c2dt12462a
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Fig. 1 ORTEP diagram of the compound L1 (50% probability level
for the thermal ellipsoids).
Fig. 3 Job plot for determining the stoichiometry of L1 and Cu(II) in
HEPES–CH3CN (pH 7.0, 6 : 4, v/v) (the total concentration of L1 and
Cu(II) is 5 μM, χCu = [Cu2+]/([Cu2+] + [L1]).
Fig. 2 Fluorescence spectra of L1 (10 μM) in HEPES–CH3CN (pH
7.0, 6 : 4, v/v) in the presence of different concentrations of Cu(II) (0–1.5
equiv.). λex = 494 nm. Insert: fluorescence intensity at 523 nm of L1 as
a function of Cu(II) concentration.
Results and discussion
Fig. 4 UV-vis absorbance of L1 (10 μM) upon the addition of different
concentrations of Cu(II) (0–1.5 equiv.) in HEPES–CH3CN (pH 7.0,
6 : 4, v/v.)
For the synthesis of compounds, intermediate product 2-(5-
formyl-6-hydroxy-3-oxo-3H-xanthen-9-yl) benzoic acid (FA)
was synthesized following the reported method.15 Then the com-
pound FA reacted with benzoyl hydrazine to get L1 in 65%
yield. The new compound was fully characterized by NMR,
ESI-mass spectroscopy and X-ray crystallography (Fig. 1). The
1H NMR, 13C NMR, and ESI-MS were reported in ESI†
(Fig. S11, S14 and S15). The complex L1Cu was also obtained
from L1 and CuCl2·2H2O, which was certain by ESI-MS
(Fig. S12†), element analysis, UV-vis spectrum, and supported
by DFT calculation (Fig. 5).
The fluorescence spectra were obtained by excitation of the
fluorophore at 494 nm in HEPES–CH3CN (pH 7.0, 6: 4, v/v). In
the absence of metal ions, a strong emission peak was observed
at 523 nm, after adding 1 equiv. of Cu(II), it displayed 28 fold
fluorescence quenching (Fig. 2). The data were recorded 1 min
after copper ion was added. In addition, we investigate the time
course of the response of L1 to Cu(II) (Fig. S4†), and the results
showed that the reaction between L1 and Cu(II) finished within
1 min and remained unchanged over 5 hours. The linear relation-
ship of the fluorescence titration showed that chemodosimeter
L1 responded to Cu(II) in 1 : 1 stoichiometry (Fig. 1, inset).
Besides, the Job plot was a definitive demonstration for the 1 : 1
stoichiometry (Fig. 3). The association constant for Cu(II) was
estimated to be 4.38 × 103 M−1 in the media (Fig. S1†) on the
basis of linear fitting of the titration curve assuming a 1 : 1
stoichiometry.
Support for this conclusion was gained from the UV-vis spec-
trum (Fig. 4). The absorption of L1 in the same media from 250
to 750 nm increased with the addition of Cu(II) until the concen-
tration of Cu(II) reached 10 μM. As shown in Fig. 4, the absorp-
tion band displayed a tiny blue shift, and the whole spectra
presented an ascendant trend. When the equivalent of copper
was added, the absorption peak did not change, which confirmed
the 1 : 1 stoichiometry between L1 and Cu(II) and the main exist-
ence form of L1 is open loop control.
In addition, to further verify the configuration of L1Cu, we
carried out density functional theory (DFT) calculations with
B3LYP by using the Gaussian 09 package. The 6-31G basis set
5800 | Dalton Trans., 2012, 41, 5799–5804
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was used for the H, C, N, O atoms; the exception was for the Cu
atom and Cl atom, where the LANL2DZ effective core potential
(ECP) was employed. The optimized configuration is shown in
Fig. 5, which shows that the Cu(II) ion binds to L1 very well
through four coordination sites, and the whole molecular system
forms a nearly planar structure. The Cu–O bond lengths are
2.26 Å (Cu–Oester-oxygen) and 1.63 Å (Cu–Oxanthane), and Cu–N
bond length is 1.84 Å (Cu–Nschiff base-nitrogen), and Cu–Cl bond
length is 2.20 Å (Cu–Cl) respectively. These data indicate that
L1 could provide a suitable space to better accommodate corre-
sponding ions.
We evaluated the response of L1 to other metal ions and
found that only Cu(II) caused beyond 28-fold fluorescence
quenching. K(I), Na(I), Ag(I), Ca(II), Cd(II), Cu(I), Mg(II), Zn(II),
Mn(II), Hg(II), Cr(III), Ni(II), Fe(III) ions were used to evaluate the
metal ions binding properties of the probe (10 μM) in HEPES–
CH3CN (pH 7.0, 6 : 4, v/v) (Fig. 6). Except Fe(III) and Ni(II)
partly decreased the fluorescence intensity, other ions either did
not influence or enhance the fluorescence intensity. After added
Cu(II) in the presence of 2 equiv. of other cations, the fluorescence
quenched the same as without other cations, so the binding of the
sensor and Cu(II) wasn’t affected by concomitant ions. The detec-
tion limit could reach up to 1.08 × 10−5 M (Fig. S2†). Also, the
results of a pH titration experiment revealed that L1 is stable over
a relatively wide pH range (5–11) (Fig. S3†).
Thus, the resultant product L1Cu displayed high specificity
for H2S. In these experiments, NaHS was used as the equivalent
of H2S. It is known that in aqueous state under the physiological
pH of 7.4, the major form of H2S exists as HS−; the ratio of
HS− : H2S is approximately 3 : 1.16 Then we investigated the
emission intensity of sensor L1Cu against pH in the range of 4
to 12 (Fig. S5†, λem = 523 nm). The data showed that L1Cu was
stable over a wide pH range from 4 to 12, and the response to
hydrogen sulfide wasn’t affected in the pH range 5–11. On the
other hand, the sensor L1Cu displayed 25–30 fold fluorescence
enhancement within 1 min after adding 20 μM H2S to the media.
And the fluorescence intensity remained almost unchanged for
10 min (Fig. S6†). Also, we found that the fluorescence intensity
was still unchanged after 2 hours when we took photographs.
Therefore, the chemosensor is a real-time one and has a low
detection limit at 1.7 μM (Fig. S7†).
Fig. 5 Calculated energy-minimized structure of L1Cu.
Fig. 6 Fluorescence emission spectra of L1 (10 μM) and different cations (20 μM) in HEPES–CH3CN (6 : 4, v/v, pH = 7.0) in the absence (black
bar) and presence (red bar) of Cu(II) (20 μM). Excitation wavelength: 494 nm.
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Scheme 1 Graphic of proposed mechanism of the monitoring system.
Fig. 7 Fluorescence emission spectra of L1Cu (10 μM) in HEPES–
CH3CN (pH 7.0, 6 : 4, v/v) in the presence of (a) different anions
(40 μM) and (b) RO(N)S: (1)200 μM H2O2, (2)200 μM ClO−,(3)
200 μM 1O2,(4)200 μM ONOO−, (5)200 μM TBHP, (6)200 μM OH˙,
t
(7)200 μM NO˙,(8)200 μM BuO˙,(9)20 μM NaHS. Excitation wave-
length: 494 nm.
Fig. 9 Confocal fluorescence images in Hep G2 cells (Zeiss LSM
510META confocal microscope, 40× objective lens). (a) Hep G2 cells
without adding indicator. (b) Fluorescence image of Hep G2 cells incu-
bated with L1 (10 μM). (c) Darkfield and (d) brightfield of cells sup-
plemented with 10 μM L1 in the growth media for 60 min at 37 °C and
then incubated with 15 μM CuCl2 for 30 min at 37 °C. Cells incubated
with L1 and CuCl2 and (e) 200 μM, (f) 400 μM NaHS for another
30 min.
Sensor L1Cu also showed high selectivity over other inor-
ganic sulfur compounds (Na2SO4, Na2SO3, Na2S2O3, K2S2O8,
KHSO4, and sulfoacetic acid disodium salt (SAA)), and a redu-
cing reagent, for instance L(+)-ascorbic acid (SA), and other
common anions (Fig. 7a and S8†). Furthermore, the sensor did
not show any obvious fluorescence enhancement in response to
reactive oxygen species ROS or reactive nitrogen species (RNS),
such as hydrogen peroxide (H2O2), hypochlorite (ClO−), singlet
oxygen (1O2), peroxynitrite (ONOO−), tert-butyl hydroperoxide
(TBHP), hydroxyl radical (˙OH), nitric oxide (˙ON) and tert-
butyl peroxyl radical (˙OtBu) (Fig. 7b). Even though the strong
chelating agent EDTA scarcely influenced the complex L1Cu,
Fig. 8 Fluorescent intensity of L1 (10 μM) in HEPES–CH3CN (pH
7.0, 6 : 4, v/v) upon the alternate addition of Cu(II)–NaHS with several
concentrations ratio (0 : 0, 15 : 0, 15 : 20, 35 : 20, 35 : 40, 60 : 40, 60 : 80,
90 : 80, 90 : 130, and 140 : 130 μM, respectively). Excitation at 494 nm.
Inset: the fluorescence profiles of L1 (20 μM), L1Cu (20 μM), and
L1Cu (20 μM) with 40 μM NaHS. Inset: photographic comparison
under UV radiation. [L1] = 20 μM, [Cu(II)] = 30 μM, [NaHS] = 60 μM.
5802 | Dalton Trans., 2012, 41, 5799–5804
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those that have been used to elicit physiological responses of
H2S to be 10–600 μM.9,14,17 Strong fluorescence in the cells was
observed after 30 min. However, no significant fluorescence
enhancement was observed in the presence of 400 μM NaHS
compared with 200 μM NaHS. It is suggested that 200 μM
NaHS could completely react with L1Cu under these conditions,
so the fluorescence showed little change between the two
concentrations. Thus, we reach the conclusion that probe L1 can
be used for the detection of Cu(II) in cultured cells and the
complex L1Cu would be an excellent sensor for hydrogen
sulfide in living cells. What’s more, the results of the MTT assay
showed that the sensors exhibited no cytotoxicity at concen-
trations up to 200 μM (L1), and 80 μM (L1Cu) (Fig. 10 and
S10†). Therefore, L1 and L1Cu have potential value in biomedi-
cal systems.
Fig. 10 MTT assay of L929 cells cultured for 24 hours in media con-
taining various concentrations of L1.
Conclusions
In this study, a fluorescein-based fluorescence probe L1 was syn-
thesized and characterized. The chemosensor L1 displayed high
selectivity and sensitivity for copper ions based on cation-
induced inhibition of the resonance process. The consequent
product of L1 and Cu(II), L1Cu, was an excellent indicator for
hydrogen sulfide for depriving Cu(II) from the complex L1Cu.
The chemosensor L1 exposed green fluorescence, which was
quenched by copper ions, then recovered with the addition of
hydrogen sulfide. Therefore, that constituted an on–off–on type
fluorescence monitoring system, meaning that the process was a
reversible one. Furthermore, we carried out live-cell imaging
using fluorescence confocal microscopy, and the results revealed
that the sensor can be utilized in living cells for monitoring both
Cu(II) and hydrogen sulfide.
H2S can induce 27-fold fluorescence enhancement (Fig. S8†).
Besides, the amino acid containing sulfur, for example, L-meth-
ionine (Met), did not enhance fluorescence, however, the
common organic substance mercaptoacetic acid (MAP)
(100 μM) enhanced 22% and L-cysteine (Cys) (200 μM) 62%
respectively relative to that H2S did. As presented in Fig. 8, the
alternate addition of Cu(II) and H2S to the solution of L1 gave
rise to a switchable change in the fluorescence intensity at
523 nm.
Such a reversible interconversion of L1–L1Cu can be repeated
more than 10 times by the modulation of Cu(II)/H2S added, indi-
cating that L1 can be developed as a reversible fluorescence on–
off–on probe for Cu(II) and H2S. The distinct fluorescent color
changes from green to dark then to green under ultraviolet lamp
are also displayed in Fig. 8 inset. Moreover, the fluorescence
titration spectra of H2S (Fig. S9†) and Cu(II) (Fig. 2) were very
similar, so the fluorescence intensity recovered totally.
Experimental
General
As shown in Scheme 1, the compound L1 exists as two
isomers, that is, the spirocyclic form (Fig. 1) in methanol and
acetone mixture solution and open loop control in HEPES–
CH3CN (pH 7.0, 6 : 4, v/v). The conversion of L1–L1Cu was
also verified from ESI-MS. The peak at m/z 574.3 corresponding
to [L1Cu + Cl − 2H]− (calcd m/z 576.01) was found after 1
equiv. of CuCl2 was added to the solution of L1 (Fig. S12†). On
this basis, the peak at m/z 479.3 corresponding to [L1 + H]+
(calcd m/z 478.12) was observed and the peak at m/z 574.3 dis-
appeared upon the addition of 2 equiv. of NaHS (Fig. S13†).
Thus, ESI-MS results firmly support the conclusion that the
interconversion of L1–L1Cu can be modulated by the decom-
plexation–complexation interaction.
All the materials for synthesis were purchased from commercial
suppliers and used without further purification. Acetonitrile for
spectra detection was HPLC reagent without fluorescent impur-
1
ity. H and 13C NMR spectra were taken on a Varian mercury-
400 spectrometer with TMS as an internal standard and DMSO
as solvent. Absorption spectra were determined on a Varian
UV-Cary100 spectrophotometer. Fluorescence spectra measure-
ments were performed on a Hitachi F-4500 spectrofluorimeter.
All pH measurements were made with a pH-10C digital pH
meter. ESI-MS spectra were determined on a Bruker Daltonics
Esquire 6000 spectrometer.
As the physiological adjustment of Cu(II) and H2S has been
confirmed, we then tested the abilities of L1 to visualize changes
in live-cell imaging mode using confocal microscopy. As shown
in Fig. 9, Hep G2 (liver cancer) cells were incubated with com-
pound L1 (10 μM) for 60 min, and the cells showed strong fluor-
escence. Then 15 μM CuCl2 were added. After another 30 min,
we did not observe any fluorescent cells. Last but not least,
various concentrations of NaHS (200, 400 μM) were added to
the cell media. These concentrations were within the range of
Syntheses
Syntheses of L1. Compound FA15 (360 mg, 1.0 mmol) was
dissolved in 50 mL ethanol, followed by addition of benzoyl
hydrazine (163 mg, 1.2 mmol). Then the mixture solution was
stirred at 50 °C for 6 hours. The cream-colored precipitates
formed were filtered and washed with ethanol and acetone. After
drying under vacuum, the compound L1 was obtained. Yield:
1
65%. H NMR (DMSO-d6, 400 MHz, TMS) δ (ppm): 12.761
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(s, 1H), 12.490 (s, 1H), 10.301 (s, 1H), 9.259 (s, 1H),
8.021–8.008 (t, J = 5.2 Hz, 3H), 7.830–7.795 (d, J = 7.2 Hz,
1H), 7.753–7.717 (t, J = 7.2 Hz, 1H), 7.681–7.645 (t, J = 7.2
Hz, 1H), 7.614–7.577 (t, J = 7.2 Hz, 2H), 7.344–7.325 (d, J =
Acknowledgements
This study was supported by the Fundamental Research Funds
for the Central Universities (Lzujbky-2012-65) and (Lzujbky-
2012-213). Thanks to Hongbo Wei for NMR measurements and
Xuefei Zhao for DFT calculations.
7.6 Hz, 1H), 6.750–6.682 (q, J = 7.0 Hz, 3H), 6.617 (s, 1H). 13
C
NMR (DMSO-d6, 100 MHz, TMS) δ (ppm): 169.04, 163.19,
160.41, 160.06, 152.68, 151.64, 150.00, 144.69, 136.16, 132.79,
132.72, 130.69, 129.11, 128.15, 126.50, 125.18, 113.76, 110.00,
109.88, 106.21, 102.53, 82.87. MS (ESI): found: m/z = 479.3
(M + 1)+, calcd C28H18N2O6 = 478.12.
Notes and references
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Preparation of L1Cu. The acetonitrile solution of
CuCl2·2H2O (17 mg, 0.1 mmol) was added to the DMSO and
ethyl acetate solution of L1 (24 mg, 0.05 mmol), and a dark
brown precipitate was produced. Then the mixture was stirred at
room temperature for 5 hours. The dark brown solid that had
formed was filtered, washed with water 5 times, and dried under
vacuum. MS (ESI): found: m/z = 574.3 (L1 + Cu2+ + Cl−
−
2H)−. Elemental analysis: Found (calculated) (%) for
C28H17N2O6CuCl: C, 57.88 (58.34); H, 3.42 (2.97); N, 4.56
(4.86).
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X-Ray crystallography of L1
X-Ray structure determinations: single crystal X ray diffraction
measurements were carried out on a Bruker SMA RT 1000 CCD
diffractometer operating at 50 kV and 30 mA using Mo-Kα radi-
ation (λ = 0.71073 Å). The selected crystal was mounted in-side
a Lindemann glass capillary for data collection using the
SMART and SAINT software. An empirical absorption correc-
tion was applied using the SADABS program. All the structure
were solved by direct methods and refined by full-matrix least
squares on F2 using the SHELXTL 97 program package.18 All
non-hydrogen atoms were subjected to anisotropic refinement,
and all hydrogen atoms were added in idealized positions and
refined isotropically. Crystal data for L1 (containing CH3OH):
ˉ
Mr = 510.49, yellow, block, triclinic, space group P1, a = 7.837
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17 K. Abe and H. Kimura, J. Neurosci., 1996, 16, 1066.
18 G. M. Sheldrick, SHELXS-97, Rel. 97-2, Program for the Solution of
Crystal Structures, University of Göttingen, Göttingen, Germany, 1997.
(4) Å, b = 12.342(6) Å, c = 13.048(7) Å, α = 108.194(5), β =
90.845(6), γ = 90.795(6), V = 1198.7(11) Å3, Z = 2 (Table 1,
ESI†). CCDC 854854 contains the supplementary crystallo-
graphic data for this paper†.
5804 | Dalton Trans., 2012, 41, 5799–5804
This journal is © The Royal Society of Chemistry 2012