A new fluorescent "turn-on" chemodosimeter for the detection of hydrogen sulfide in water and living cells

Article abstract of DOI:10.1039/c3ra45822a

A new fluorescent turn-on probe for the selective detection of hydrogen sulfide in water and living cells based on a 8-hydroxyquinoline fluorophore functionalized with a 2,6-dinitrophenyl ether moiety has been developed.

Full text of DOI:10.1039/c3ra45822a

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A new fluorescent “turn-on” chemodosimeter for the
detection of hydrogen sulfide in water and living cells†
Cite this: RSC Adv., 2013, 3, 25690
Sameh El Sayed,abc Cristina de la Torre,abc Luis E. Santos-Figueroa,abc
d
abc
abc
*
*
´
´
´
´
´~
´ ´
Felix Sancenon,
Enrique Perez-Paya,‡ Ramon Martınez-Manez,
Received 14th October 2013
Accepted 23rd October 2013
ae
Margarita Parraae and Salvador Gilae
*
Ana M. Costero,
DOI: 10.1039/c3ra45822a
www.rsc.org/advances
A new fluorescent turn-on probe for the selective detection of hydrogen which sulde changes the functions of a wide range of
sulfide in water and living cells based on a 8-hydroxyquinoline cellular proteins and enzymes. Moreover, abnormal levels of
fluorophore functionalized with a 2,6-dinitrophenyl ether moiety hydrogen sulde are associated with various diseases, such as
has been developed.
Alzheimer's11 and Down syndrome.12
Based on these concepts and given the important roles that
hydrogen sulde plays in biological processes, growing interest
has been shown in the development of reliable selective and
sensitive detection systems for this chemical. Current tech-
niques for sulde detection, such as colorimetric,13–15 electro-
chemical assays,16 gas chromatography,17 and metal-induced
sulde precipitation,18 are oen not suitable for monitoring
sulde levels in biological living environments. As an alterna-
tive to these methods, the design of uorogenic probes has
recently become an important focal point. In particular,
reduction reactions of azide or nitro groups to amines coupled
with emission changes,19–22 and demetallation of macrocyclic
Cu(II) complexes,23,24 have been used for the development of
highly sensitive probes for sulde detection.
Another alternative to the above-mentioned protocols, the
thiolysis of 2,4-dinitrophenyl ethers, has also been used as a
mechanism in the preparation of HSꢀ turn-on uorescent
probes. In particular, Lin and co-workers synthesized a NIR
uorescent chemodosimeter for the detection of HSꢀ in
solutions and living cells. This probe is based on a BODIPY
dye functionalized with a 2,4-dinitrophenyl ether moiety that
is non-uorescent in PBS–ethanol 9 : 1 v/v mixtures. Addition
of HSꢀ induces the hydrolysis of the ether with subsequent
emission enhancement.25 Very recently, Xu and co-workers
synthesized a 1,8-naphthalimide probe also containing a
2,4-dinitrophenyl ether group which was utilized for the
uorescent detection of HSꢀ anion in PBS–acetonitrile 9 : 1 v/v
solutions.26
Hydrogen sulde has traditionally been considered a highly
toxic gas. However, recent studies have demonstrated that H2S
is an endogenously generated gaseous signaling molecule with
very potent cytoprotective properties similar to those found for
the two well-known endogenous nitric oxide (NO) and carbon
monoxide (CO) gasotransmitters.1 Hydrogen sulde may
interact with downstream proteins by the post-translational
cysteine sulydration2 and binding to iron centers,3 which
regulate various physiological processes, including ischemia
reperfusion injury,4 vasodilation,5 apoptosis,6 angiogenesis,7
neuromodulation,8 inammation,9 and insulin signaling.10 The
exact hydrogen sulde mechanisms of action are still being
actively investigated. Some chemical and biochemical catabolic
reactions of H2S have been reported; for example, hydrogen
sulde can react readily with methemoglobin to form sule-
moglobin, which acts as the metabolic sink for sulde. As a
potential reductant, hydrogen sulde is likely to be consumed
by endogenous oxidant species, such as hydrogen peroxide,
superoxide, peroxynitrite, etc. This process is potentially
signicant because it provides a possible mechanism by
a
´
Centro de Reconocimiento Molecular y Desarrollo Tecnologico (IDM), Unidad Mixta
`
´
`
Universitat Politecnica de Valencia – Universitat de Valencia, Spain
b
´
`
´
Departamento de Quımica, Universitat Politecnica de Valencia, Camino de Vera s/n,
46022, Valencia, Spain. E-mail: rmaez@qim.upv.es
c
´
CIBER de Bioingenierıa, Biomateriales y Nanomedicina (CIBER-BBN), Spain
dLaboratorio de Peptidos
´
´
y Proteinas, Centro de Investigacion Principe Felipe,
Despite these interesting results, there is still room to
improve the behavior of probes based on the HSꢀ-induced
hydrolysis of 2,4-dinitrophenyl ethers. For instance, the exam-
ples reported by Lin and Xu, displayed sensing features in
media with a relatively large percentage (10%) of a non-aqueous
solvent and displayed an interesting turn-on uorescence
Autopista El Saler, Valencia, Spain
e
´
´
`
Departamento de Quımica Organica, Universitat de Valencia, Dr Moliner 50, 46100,
Burjassot, Valencia, Spain. E-mail: ana.costero@uv.es
† Electronic supplementary information (ESI) available: Experimental details,
synthesis, characterisation and sensing procedures. See DOI: 10.1039/c3ra45822a
‡ Deceased.
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enhancement of ca. 42- and 18-fold (upon the addition of 10 eq. values (from 2 to 9) with no signicant reduction in its sensing
of HSꢀ anion) which could, however, still be improved.
Bearing in mind our interest in developing molecular che-
behavior (see ESI†).
Aer assessing the highly selective turn-on response of probe
mosensors for anions of environmental and biological 1 to hydrogen sulde, the sensitivity of the probe was assessed
interest,27 we report herein the synthesis and sensing features by studies on the emission modulation of 1 in HEPES–DMSO
of uorescent probe 1 (see Scheme 1) for hydrogen sulde 99 : 1 v/v upon the addition of HSꢀ. Increasing the HSꢀ
detection in water and living cells. Probe 1 was readily concentration (up to 100 eq.) resulted in the progressive growth
prepared, with a good yield and in a one-step procedure, by a of uorescence intensity (see Fig. 2). From typical titration
nucleophilic aromatic substitution of 1-uoro-2,4-dinitroben- proles (see ESI†), a remarkable limit of detection (LOD) of 60
zene with 8-hydroxyquinoline (see ESI† for synthetic details nM was determined. 1 showed remarkably low sensitivity below
and characterization).
the hydrogen sulde concentration required to elicit physio-
The emission behavior of probe 1 in the presence of 10 logical responses (10–1000 mM).28
equivalents of selected anions (HSꢀ, OHꢀ, Fꢀ, Clꢀ, Brꢀ, Iꢀ, N3
,
The selectivity toward HSꢀ was ascribed to the HSꢀ-induced
ꢀ
CNꢀ, SCNꢀ, AcOꢀ, CO32ꢀ, PO43ꢀ), oxidants (H2O2), bio-thiols hydrolysis of the 2,4-dinitrophenyl ether moiety, which yielded
(Cys, Me-Cys, Hcy, GSH, lipoic acid) and reducing agents the highly uorescent 8-hydroxyquinoline group.29 This sensing
(SO42ꢀ, SO32ꢀ, S2O32ꢀ) was evaluated. HEPES (7 mM, pH 7.4)– mechanism was conrmed by isolating the reaction product
DMSO 99 : 1 v/v solutions of 1 are essentially non uorescent between 1 and the HSꢀ anion, which was unequivocally char-
(upon excitation at 450 nm). Of all the chemicals tested, only the acterized as 8-hydroxyquinoline.
addition of HSꢀ induced the appearance of a strong emission
The selective emission enhancement of 1 in the presence of
band at 514 nm (lex ¼ 450 nm) with an impressive 345-fold HSꢀ and the poor response observed upon the addition of bio-
enhancement upon the addition of 10 equivalents of the anion thiols (GSH, Cys and Hcy) strongly suggest that the probe can be
(see Fig. 1). Besides, time-dependent studies have shown that a used for HSꢀ imaging in living cells. Based on these observations,
maximum change in emission was observed aer ca. 50 min the cytotoxicity of 1 was rst evaluated. HeLa cells were treated
upon the addition of HSꢀ (see ESI†). Moreover, uorescence with 1 at different concentrations over a 24-hour period and cell
studies at different pHs in water–DMSO 99 : 1 v/v solutions of 1 viability was determined by a WST-1 assay. The obtained results
in the absence and presence of 5 mM of HSꢀ anion have are shown in Fig. 3. As seen, probe 1 is essentially non-toxic in the
demonstrated that the probe can be used in a wide range of pH range of concentrations tested (5–50 mM).
In a second step, and in order to verify the feasibility of the
developed probe to detect HSꢀ in highly competitive environ-
ments, we prospectively used 1 for the uorescence imaging of
sulde in living cells. In a typical experiment, HeLa cells were
incubated in DMEM supplemented with 10% fetal bovine
serum. To conduct uorescence microscopy studies, HeLa cells
were seeded in 24 mm glass coverslips in 6-well plates and were
allowed to settle for 24 h. Cells were treated with 1 in DMSO
(1%) at a nal concentration of 30 mM. Aer 20 minutes, the
medium was removed and solutions of different concentrations
Scheme 1 Synthesis of chemodosimeter 1.
of NaHS in PBS were added (0, 100, 200 and 500 mM) and cells
were incubated for another 10-minute period. The results are
Fig. 1 Fluorescent intensity at 514 nm (excitation at 450 nm) of chemo-
dosimeter 1 (5.0 ꢁ 10ꢀ5 M) in HEPES (7 mM, pH 7.4)–DMSO 99 : 1 v/v upon the
addition of 10 equivalents of selected anions, biothiols, oxidants and reducing
agents after 50 min of the addition.
Fig. 2 Emission spectra of probe 1 (5.0 ꢁ 10ꢀ5 M) in HEPES (7 mM, pH 7.4)–
DMSO 99 : 1 v/v (excitation at 450 nm) upon the addition of increasing quantities
of HSꢀ anion (from 0 to 10 equivalents) and after 50 minutes of the addition.
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fellow. L.E.S.F. also thanks the Carolina Foundation and
UPNFM-Honduras for his doctoral grant. C.T. is grateful to the
Spanish Ministry of Science and Innovation for her grant.
Notes and references
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Fig. 3 Cell viability test of different concentrations of probe 1 at 24 h in HeLa
cells by a WST-1 assay.
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features of new uorescent probe 1. Probe 1 is easy to prepare
and is able to selectively detect the HSꢀ anion in HEPES–DMSO
99 : 1 v/v solutions via remarkable turn-on emission at 514 nm.
The observed uorescence enhancement is ascribed to a selec-
tive HSꢀ-induced hydrolysis of a 2,4-dinitrophenyl moiety in 1,
which gives a free 8-hydroxyquinoline uorophore. The probe
can selectively and sensitively detect HSꢀ anion in water over
other anions, biothiols and common oxidants. Moreover, real-
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Financial support from the Spanish Government (Project
MAT2012-38429-C04-01) and the Generalitat Valencia (Project 20 H. Peng, Y. Cheng, C. Dai, A. L. King, L. B. Predmore,
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