A novel FRET-based ratiometric fluorescent probe for highly sensitive detection of hydrogen sulfide

Article abstract of DOI:10.1039/c4ra16578k

A novel FRET-based ratiometric fluorescent probe H₂S-CR for the quantitative detection of H₂S was designed and synthesized. It exhibits a response time of 20 min, a considerable fluorescence signal enhancement (15 fold), and an extremely low detection limit (19 nM). It can be successfully applied to imaging H₂S in living cells. This journal is

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A novel FRET-based ratiometric fluorescent probe H2S-CR of dinitrophenyl ether20 as well as specific Michael addition reaction
for the quantitative detection of H2S was designed and towards unsaturated double bonds.21 Besides, as far as we know, the
synthesized. It exhibits a response (20 min), a considerably probes utilizing the unique dual nucleophilic character of H2S
fluorescence signal enhancement (15 folds), and an extremely exhibited superior selective advantage because the potential
low detection limit (19 nM). It can be successfully applied to interference from bio-thiols can be well excluded.15 Usually, such
imaging H2S in living cells.
fluorescent probes contain a potential fluorescent group and a
specific H2S trap group with two nucleophilic reaction sites.
As a member of the reactive sulfur species (RSS) family, hydrogen
sulfide (H2S) has been regarded as a toxic pollutant with the typical
smell of unpleasant rotten eggs for a long time. However, more
recent studies on endogenous H2S have challenged this traditional
view of H2S as a toxin and suggested that H2S is the third member of
the gasotransmitter family, in addition to nitric oxide (NO) and
carbon monoxide (CO), existing in the human body and other
biological systems.1 Furthermore, H2S at physiological concentration
appears to be involved in various physiological processes, including
regulation of cell growth,2 stimulation of angiogenesis,3 modulation
of neuronal transmission,4 the anti-inflammation effect and etc.5
Concurrently, abnormal H2S levels are engaged in diseases such as
Alzheimer’s disease,6 Down’s syndrome,7 gastric mucosal injury,8
diabetes,9 liver cirrhosis, and etc.10 Therefore, highly sensitive and
selective detection of H2S and direct indication of its concentration
in living systems are crucial for the better understanding of its
related physiological and pathological functions.
Although these probes utilizing the unique dual nucleophilic
character of H2S are innovative and effective, some improvements
are still needed. One of the main concern using this strategy is how
to avoid the probe consumption by biothiols,15b which would
otherwise lead to high probe loading and low sensitivity. Another
concern is that most of these probes exhibit a response to H2S with
changes only in fluorescence intensity at a single wavelength,15(a, c, d)
and single increase or decrease emission detection is sometimes
problematic for precise fluorometric analysis because fluorescence
intensity can be affected by variables such as excitation intensity
variations, environmental factors, light scattering, p robe
concentrations, and etc. Moreover, the development of novel
fluorescent probes for H2S detection with better optical properties
and higher sensitivity is of pressing need. Herein, we reported the
design and synthesiz e of a novel ratiometric fluorescence probe H2S-
CR (Scheme 1) based on a H2S induced M ichael addition-
In the past decades, the detection of H2S has attracted a wide
research interest, and some strategies including metal-induced
sulfide precipitation,11 colorimetric assays,12 electrochemical
analysis13 and gas chromatograph14 have been developed. Among
them, fluorescence-based detection with a fluorescent probe is
extremely attractive because of its simplicity, real-time imaging, and
nondestructive detection of intracellular biomolecules. Recently,
a number of fluorescent probes for H2S have been constructed using
the special properties of H2S, such as its dual nucleophilicity,15 good
reducing property toward azides,16 nitros17 and hydroxyl amines,18
high binding affinity with copper and zinc ions,19 efficient thiolysis
Scheme 1 Theproposed mechanism of ratiometric fluorescent probe
H2S-CRfor H2S detection.
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prepared from 4-(diethylamino)-2-hydroxybenzaldehyde by a four-
step procedure under mild conditions with a good yield. The
coumarin donor building block 7 and rhodamine/fluorescein
acceptor building block 8 were synthesized by a reported synthetic
procedure.23 The FRET dyad CR was prepared by the condensation
of carboxyl compound 7 with amide 8. Finally, we transformed the
compound CR to probe H2S-CR. The structural characterization of
the probe was characterized by standard 1H NMR, 13C NMR, HRMS,
and etc (Fig. S3).
We first evaluated the effect of buffer solution to the fluorescence
of the probe. As shown in Fig. S5, the probe could work well in
phosphate buffered solutions. The photophysical properties of the
probe (10 μM) were investigated under simulated physiological
conditions (30 mM, pH 7.4, 1:9 v/v CH3CN/PBS). Prior to reaction
with NaHS, probe presented a fluorescence maximum at 470 nm
(Fig. 1A) with a corresponding major absorption band centered at
408 nm (Fig. 1B) (Φ = 0.132). With the addition of NaHS (80 μM)
into the solution of probe, the fluorescence emission intensity at 470
nm gradually decreased within 12 min, along with a time-dependent
fluorescence emission intensity increase centred at 541 nm (Fig. 1A)
(Φ = 0.100). Simultaneously, there emerged a new absorption peak
at 511 nm, accompanied by a dramatic change in the probe solution
from colorless to bright orange. Furthermore, we performed the
time-dependent fluorescent spectra studies. As shown in Fig. 2,
although 80 μM NaHS could cause the reaction to be completed
within 12 min, the low concentrations of NaHS needed the longer
reaction time (20 min) to reach the fluorescence intensity ratio
(I541/I470) saturation. Thus, the time point after the addition of NaHS
was selected to be 20 min in the subsequent experiments.
Scheme 2 Thesynthesis of the probe H2S-CR.
cyclization cascade reaction and the FRET modulated fluorescence
process. It exhibits a response time (20 min), a considerably
fluorescence signal enhancement (15-fold), and an extremely low
detection limit (19 nM) toward H2S. Moreover, we successfully
applied it to image thechange of H2S level in living cells.
As shown in Scheme 2, probe H2S-CR can be conveniently
To gain insight into potential of H2S-CR as a probe for H2S, a
titration experiment was performed under simulated physiological
conditions. Accordingly, as shown in Fig. 3A, upon excitation at 414
nm, the fluorescence intensity around 470 nm decreases along with
the incremental addition of NaHS, and simultaneously a new
emission band around 541 nm gradually increased. The fluorescence
Fig. 1 (A) Fluorescence responses of 10 μM probe to 80 μM NaHS.
(B) Time-dependent absorption spectra of probe (10 μM) in the
presence of 80 μM NaHS. Conditions: excitation wavelength is 414
nm, acetonitrile-PBS buffer solution (30 mM, pH 7.4, 1:9 v/v) at 25
oC.
Fig. 2 Time-dependent fluorescence intensity changes of probe (10
μM) at 541 nm upon addition of varied concentrations of NaHS.
Conditions: excitation wavelength is 414 nm, acetonitrile-PBS
buffer solution (30 mM, pH 7.4, 1:9 v/v) at 25 oC.
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and reaction product coumarin-rhodamine/fluorescein and
cyclization product were confirmed by HPLC-MS experiments (Fig.
S6).
To study the specificity of H2S-CR towards NaHS, an important
test was performed to determine whether biological species other
than NaHS could potentially introduce signal response (Fig. 4). As
expected, H2S-CR was considerably inert to the common cations
and anions, such as Na+, Mg2+, Ca2+, Zn2+, Cu2+, Cu+, K+, Fe3+, and


Fe2+ (4 mM for each) (Fig. 4A); F, Cl, I, AcO, N3 , CO3, NO3 ,
NO2 , SO4, and HCO3 (4 mM for each) (Fig. 4A). The results
revealed that the NaHS-induced ratiometric fluorescence response is
hardly affected in response to reactive oxygen/nitrogen species


(ROS/RNS), such as H2O2, NO, ROO·, ·O, ClO, ·OH, O2, and
1
CNOO (4 mM for each) (Fig. 4B), reducing condition, such as
sodium ascorbate (4 mM) (Fig. 4B), and non-thiol amino acids, such
as Lys, Ser, Glu, Pro, Phe, Arg, Thr and Asn (4 mM for each) (Fig.
2

4B). Moreover, CN (4 mM) (Fig. 4B), reactive sulfur species (SO3
HSO3 , and S2O3  (0.8 mM for each)) (Fig.4B) and biothiols(GSH,
,
2

Cys, and Hcy (0.8 mM for each)) (Fig. 4B) underwent limited
fluorescence response. However, the fluorescence ratio (I541/I470
)
increase was far weaker than that caused by NaHS. By contrast,
NaHS induced a robust increase in the fluorescence intensity ratio
Fig. 3 (A) Fluorescence response of 10 μM probe in the presence of
0-10 equiv of NaHS. (B) Fluorescence ratio (I541/I470) of probe (10
μM) in the presence of 0-10 equiv of NaHS. Conditions: excitation
wavelength is 414 nm, acetonitrile-PBS buffer solution (30 mM, pH
7.4, 1:9 v/v) at 25 oC for 30 min.
intensity could reach saturation when the addition amount of NaHS
reached 9 equiv. of the probe (Fig. 3B). At this amount, the ratio of
the fluorescence emission intensities at 541 and 470 nm (I541/I470
)
exhibited a drastic change from 0.15 in the absence of NaHS to 2.3
after complete conversion, a 15-fold enhancement in the I541/I470
ratios. This suggested that the excitation energy of the coumarin
donor is efficiently transferred to the rhodamine/fluorescein
acceptor. The intramolecular energy transfer efficiency from the
coumarin donor to the rhodamine/fluorescein acceptor in H2S-CR
was calculated to be 70.7% (in supporting information). The
detection limit (S/N=3) of the ratiometric probe was determined to
be 19 nM (in supporting information). Additionally, a standard curve
between emission intensity ratio (I541/I470) and NaHS concentration
was set up. To our satisfaction, as shown in Fig. 3B, a good linearity
between the fluorescence intensity ratio (I541/I470) and the NaHS
concentration in the range of 0-50 μM was observed, suggesting that
H2S-CR is potentially useful for quantitative determination of NaHS.
Based on the well-established dual nucleophilicity mechanism, the
mechanism has been proved by reaction between H2S-CR and NaHS,
Fig. 4 Fluorescence ratio (I541/I470) of 10 μM probe in an acetonitrile-
PBS buffer solution (30 mM, pH 7.4, 1:9 v/v) towards hydrosulfide
and potential interferences for 30 min. Bars represent fluorescence
ratio (I541/I470) to each compound. (A) The fluorescence emission of
probe spiked with selected cations, such as Na+, Mg2+, Ca2+, Zn2+,
Cu2+, Cu+, K+, Fe3+, and Fe2+ (4 mM for each) and selected anions,



such as F, Cl, I, AcO, N3 , CN, CO3, NO3 , NO2 , SO4, and

HCO3 (4 mM for each). (B) Fluorescence responses of probe
supplemented with reactive oxide species, such as H2O2, ROO·, ·O2,
ClO, ·OH, and 1O2 (4 mM for each), reactive nitrogen species, such
as NO and CNOO (4 mM for each), reactive sulfur species, such as


SO3, HSO3 , and S2O3 (0.8 mM for each), sodium ascorbate (4
mM ) non-thiol amino acids, such as Lys, Ser, Glu, Pro, Phe, Arg,
Thr and Asn (4 mM for each) and biothiols, such as GSH, Cys, and
Hcy (0.8 mM for each).
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(I541/I470), and only the probe solution turned from colorless to bright
orange when treated by NaHS in the selective experiment. High H2S, we have developed
In conclusion, with recognition of the biological significance of
unique FRET-based ratiometric
a
selectivity toward H2S in the presence of other competitive species is fluorescence probe H2S-CR. Based on a H2S induced Michael
a very important feature to evaluate the performance of the addition-cyclization cascade reaction, the probe exhibited a high
fluorescent probe. Therefore, the competition experiments were also selectivity and sensitivity for H2S over other biologically relevant
conducted when CN/biothiols/(reactive sulfur species) and NaHS species, a 15-fold fluorescence signal enhancement, and an obvious
co-existed in the system. To our delighted, when NaHS and these colour change from colourless to bright orange. Moreover, H2S-CR
competitive species coexisted, almost the same I541/I470 signal can detect H2S quantitatively with a low detection limit up to 19 nM.
enhancement was observed as that only treated by NaHS (Fig. S7). Fluorescent inverted microscope images indicated that this probe can
Taken together, H2S-CR can selectively respond to NaHS detect the level changes of H2S in living cells. In addition, this
independently of negligible disturbance from the interference of ratiometric fluorescence probe has the potential to be a useful tool
other biological species, and it can serve as a “naked-eye” probe for for the fast and real-time detection of H2S in more types of
colorimetric detection of H2S (Fig. S7).
biological samples.
To verify whether the probe is suitable for the physiological
detection, we evaluated the effect of pH on the fluorescence of the
probe. As shown in Fig. S8, in the absence of NaHS, almost no
change in fluorescence ratio (I541/I470) was observed in the free probe
over a wide pH range of 2-11 indicating excellent pH stability.
Furthermore, upon treatment with NaHS, the maximal fluorescence
ratio (I541/I470) displayed constant in the pH range of 6-11. Thus, the
observation that H2S-CR had the maximal sensing response at
physiological pH, suggested that H2S-CR is promising for biological
applications.
Acknowledgements
We are grateful to National Natural Science Foundation of China
(81271634), Doctoral Fund of Ministry of Education of China (No.
20120162110070), the Fundamental Research Funds for the Central
Universities, and Hunan Provincial Natural Science Foundation of
China (12JJ1012) and Hunan Provincial Innovation Foundation for
Postgraduate (CX2014B120).
Notes and references
Having demonstrated the selectivity and sensitivity of H2S-CR for
H2S in vitro, we next evaluated the potential utility of H2S-CR as a
probe for H2S within living cells (Fig. 5). H9C2 cells were incubated
School of Pharmaceutical Sciences, Central South University, 172
Tongzipo Road, Changsha, 410013, P. R. China. *Author of
correspondence: Prof. Dr. Wenbin Zeng, Tel/Fax: (86)731-8265-
0459; Email: wbzeng@hotmail.com.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/c000000x/
o
with 10 μM H2S-CR for 20 min at 37 C. After washed three times
with physiological saline to remove the remaining probes, the cells
were then incubated with buffer containing different concentrations
of NaHS (10, 20, 30, 40 and 80 μM) for 30 min. As for the control
experiment, the cells untreated with NaHS were examined. The
optical imaging was carried out by a fluorescent inverted microscope.
A faint fluorescence was observed in the control experiments, and
the lever changes were depended on the concentration of NaHS (Fig.
5). It is worth noting that the inverted fluorescence images grew
brighter as the concentrations of NaHS increased from 10 to 80 M
(Fig. 5 E-A). These results demonstrated that H2S-CR is cell
membrane permeable and has potential in visualizing H2S levels
change of living cells.
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