A dual functional probe: Sensitive fluorescence response to H2S and colorimetric detection for SO32-

Article abstract of DOI:10.1039/c6ra15065a

Hydrogen sulfide (H₂S) and sulfite (SO₃^2-) are two important sulfur-containing species that play different and important roles in industrial and biological processes. Accordingly, the development of efficient methods for simple, rapid, sensitive and selective monitoring of H₂S and SO₃^2- is of the utmost importance in both environmental and biological sciences. In this study, we developed a new dual functional probe NIR-DNP for discriminative detection of H₂S and SO₃^2-. This probe can sense H₂S and SO₃^2-via two different approaches, a significant near-infrared fluorescence enhancement and color change from purple to cyan induced by H₂S as well as a visible color change from purple to colorless caused by SO₃^2-. The detection limits of the probe NIR-DNP for H₂S and SO₃^2- in aqueous solutions were 36.53 nM and 33.33 nM, respectively. Competitive experiments demonstrated that the probe NIR-DNP had a high fluorescence selectivity for H₂S and excellent colorimetric selectivity for SO₃^2- over other analytes. The sensing mechanism of the probe toward H₂S and SO₃^2- was based on the H₂S-induced thiolysis of dinitrophenyl ether and SO₃^2--induced nucleophilic addition, respectively. Further investigation showed that the probe NIR-DNP could be used to develop an easy-to-prepare and easy-to-detect paper-based test strip for cheap and effective detection of SO₃^2-. Also, the probe NIR-DNP has the potential to image exogenous and endogenous H₂S in living cells.

Full text of DOI:10.1039/c6ra15065a

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PAPER
A dual functional probe: sensitive fluorescence response to H S
2
2-
and colorimetric detection for SO †
3
Received 00th January 20xx,
Accepted 00th January 20xx
a
b
a
a
a
a
Sheng Chen, Chao Ma, Mao-Sen Yuan, Wenji Wang, Dong-En Wang, Shu-Wei Chen and Jinyi
a,b
Wang *
DOI: 10.1039/x0xx00000x
2
-
Hydrogen sulfide (H
2 3
S) and sulfite (SO ) are two important members of sulfur-containing species, which play different and
www.rsc.org/
important roles in industrial and biological processes. Accordingly, the development of efficient methods for simple, rapid,
2
-
sensitive and selective monitoring of H
2 3
S and SO is of utmost importance in both environmental and biological science.
2
-
In this study, we developed a new dual functional probe NIR-DNP for discriminative detection of H
2
S and SO
3
. This probe
2
-
could sense H
change from purple to cyan induced by H
detection limits of the probe NIR-DNP for H
Competitive experiments demonstrated that the probe NIR-DNP had high fluorescence selectivity for H
2 3
S and SO
by two different approaches, a significant near-infrared fluorescence enhancement and color
2-
2
S, as well as a visible color change from purple to colorless caused by SO
3
. The
2
-
2
S and SO in aqueous solutions were 36.53 nM and 33.33 nM, respectively.
3
2
S and excellent
2
-
2-
3
colorimetric selectivity for SO
respectively based on H S-induced thiolysis of dinitrophenyl ether and SO
investigation showed that the probe NIR-DNP could be used to develop an easy-to-prepare and easy-to-detect paper-
3
over other analytes. The sensing mechanism of the probe toward H
2
S and SO
were
2
-
2
3
-induced nucleophilic addition. Further
2
-
based test strip for cheap and effective detection of SO
and endogenous H S in living cells.
3
. Also, the probe NIR-DNP has the potential to image exogenous
2
environmental and biological science.
Introduction
Currently, several techniques such as electrochemical
1
2
13
14
2
ꢀ
assay, gas chromatography and sulfide precipitation have
Hydrogen sulfide (H S) and sulfite (SO ) are two important
members of sulfurꢀcontaining species, which play vital roles in
industrial, environmental and biological processes. H S has
been the third endogenous gaseous signaling molecule
following nitric oxide and carbon monoxide. Physiological
levels of endogenous H S have been known to be involved in a
variety of physiological processes such as antiꢀinflammation,
neuromodulation and apoptosis. Abnormal levels of H S are
closely linked with various diseases such as liver cirrhosis and
Alzheimer’s disease. Sulfite (SO ), because of its antioxidant
and preservative properties, is widely used to preserve foods,
beverages and pharmaceutical products from oxidation and
microbial reactions.
exposure to high doses of sulfite can cause adverse reaction and
acute symptom, including dermatitis, urticaria, flushing,
hypotension, abdominal pain and diarrhea. The development
of efficient methods for simple, rapid, sensitive and selective
2
3
2
ꢀ
2 3
been developed for the detection of H S and SO . However,
1
ꢀ3
these methods often require tedious sample and reagent
preparation or complicated instruments, and therefore are not
suitable for routine laboratory and onꢀsite analysis. Fluorescent
and colorimetric means provide good alternatives due to their
low cost, easyꢀoperation, simple instrumental implementation,
fast response and excellent selectivity. Much effort has been
devoted to the development of fluorescent and colorimetric
2
4
2
5
6
7
2
8
2
ꢀ
9
2ꢀ
probes for H
2
S and SO
3
. To date, several probes have been
3
designed for H
2
S detection by taking the advantages of some
specific chemical reactions, including reduction of azides/nitro
1
5,16
17
1
0
groups,
demetallation of macrocyclic Cu(II) complexes,
However, longꢀterm and frequent
1
8
19
nucleophilic reaction and thiolysis of dinitrophenyl ether.
Similarly, many probes for sulfite have been also designed on
2
0
1
1
the basis of selective deprotection of levulinate groups,
2
1
22
reaction with aldehyde groups, Michaelꢀtype additions, as
2
3
2ꢀ
2
ꢀ
well as complexation with amines in the presence of SO
3
.
ꢀ
monitoring of H S and SO
is very important in both
2
3
2
However, most of these probes could only sense one of SO
and H S. One probe that could simultaneously detect for SO
and H
3
3
2
ꢀ
2
a.
College of Science, Northwest A&F University, Yangling, Shaanxi 712100, P. R.
China..
College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi
2
S remains relatively few due to some of their common
features such as nucleophilic and reducing properties. In
2
4
b.
7
12100, P. R. China.
Electronic Supplementary Informaꢀon (ESI) available: Synthesis, additional
addition, most of the existed probes for H S have emissions in
2
†
absorption and fluorescence spectral details, calculation of the detection limit,
data for investigation of sensing mechanisms, effect of pH, cytotoxicity of various imaging is easily interfered by cell autoꢀfluorescence. As we
concentrations of the probe to cell, and H- NMR, C- NMR, HRMS, ESI-MS, FT-IR
spectra. See DOI: 10.1039/x0xx00000x
the ultraviolet or visible region, in which the fluorescence
2
5
1
13
known, nearꢀinfrared (NIR, 650ꢀ900 nm) fluorescent probes
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Scheme 1 Synthesis of probe NIR-DNP. Reagents and conditions: (a) 1,3-propane sultone, toluene, reflux, 24 h, 89%; (b) DMF, CH
acetic anhydride, 70 °C, 1 h, 80.8%; (d) resorcinol, triethylamine, DMF, N atmosphere, 50 °C, 4 h, 46.6%; (e) 1-fluoro-2,4-dinitrobenzene, K
7%.
2
Cl
2
, POCl
3
, reflux, 3 h, 73.9%; (c) sodium acetate,
2
2 3 2
CO , DMF, N atmosphere, 50 °C, 5 h,
6
have unique advantages for tracing molecules in vitro and in purchased from Fisher Scientific (Pittsburgh, PA). Dry solvents
vivo, such as minimum photoꢀdamage to biological samples, used in the synthesis were purified using standard procedures.
deep tissue penetration, and minimum interference from All other chemicals were obtained from qualified reagent
background autoꢀfluorescence by bioꢀmolecules in living suppliers with analytical reagent grade.
2
5ꢀ27
systems.
Therefore, it is necessary to develop new types of
Instruments
probes that can be used for simultaneous detection of H
2
S and
2
ꢀ
1
13
SO
3
, preferably with emission located in the NIR region.
Hꢀnuclear magnetic resonance (NMR) and CꢀNMR spectra
In this work, we design and synthesize a new NIR of the products were recorded on a Bruker 500 MHz/125 MHz
fluorescence probe NIR-DNP (Scheme 1) which can NMR spectrometer using tetramethylsilane (TMS) as the
2
ꢀ
distinguish H
2
S and SO
3
by naked eyes. The probe was internal standard (chemical shifts in ppm). Electrospray
constructed by connecting 2,4ꢀdinitrophenyl to hemicyanine ionization mass spectroscopy (ESIꢀMS) data were obtained
skeleton, in which a propanesulfonate residue was introduced to using a Thermo Scientific LCQ FLEET mass spectrometer.
2
8
indolium group to improve its water solubility. The probe Highꢀresolution mass spectra (HRMS) data were recorded on a
®
displayed a selective recognition of H
change from purple to cyan and a significant fluorescence Manchester, UK) equipped with a Zpray
enhancement in NIR region over other relevant species, and operating in the negative ion mode. Fourierꢀtransform infrared
2
S via a visible color Waters Xevo G2ꢀXS QTOF mass spectroscopy (Waters,
TM
ESI source
2
ꢀ
also exhibited a sensitive response to SO
3
through a visible spectroscopy (FTꢀIR) was recorded on a Nicolet 5700
−
1
−1
color change from purple to colorless. In addition, the further spectrometer at wavelengths of 400 cm to 4000 cm and a
applications of NIR-DNP in paperꢀbased test strip for SO , as resolution of 3 cm over 32 scans. The pH values were
3
2
ꢀ
−1
well as imaging of exogenous and endogenous H
cells were studied.
2
S in living measured using a digital pH meter (PHSꢀ3C, Leiꢀci, Shanghai,
China). The UVꢀVis absorption spectra were measured by
using Shimadzu UV1780 spectrometer (Shimadzu, Japan). The
fluorescence spectra measurements were performed using
Shimadzu RFꢀ5301 fluorescence spectrometer (Shimadzu,
Japan). The fluorescence images of cells were taken using an
inverted microscope (Olympus CKX41, Japan).
Experimental section
Materials
2
,3,3ꢀTrimethylindolenine was purchased from J&K Chemical
Technology (Shanghai, China). Cyclohexanone, resorcinol and
,3ꢀpropanesulfonate were obtained from Aladdin (Shanghai, The probe NIR-DNP was prepared as depicted in Scheme 1.
China). 1ꢀFluoroꢀ2,4ꢀdinitrobenzene was purchased from Xiya For the detailed information on the synthesis of the
Reagent (Shandong, China). Whatman No. 1 filter paper was intermediates
see the electronic supplementary
Synthesis of the probe NIR-DNP
1
1–4,
2
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information (ESI†). The probe NIR-DNP was synthesized by (DMEM, pH 7.4) supplemented with 10% fetal bovine serum
the nucleophilic substitution reaction between compound and (FBS, Gibco Invitrogen, CA, USA), penicillin (100 U/mL), and
ꢀfluoroꢀ2,4ꢀdinitrobenzene. To a solution of (24.58 mg, 0.05 streptomycin (100 U/mL) at 37°C in 5% CO atmosphere. To
4
1
4
2
mmol) in N,Nꢀdimethylformamide (DMF, 2 mL) was added maintain cells in the exponential growth phase, they were
potassium carbonate (13.8 mg, 0.1 mmol) and the mixture was normally passaged at a ratio of 1:3 every 72 h. Before using, Uꢀ
stirred for 30 min under N
dinitrobenzene (18.6 mg, 0.1 mmol) dissolved in DMF (2 mL) reꢀseeded on 24ꢀwell plates at a density of 5 ×10 cells/mL and
was add to the solution. The resulting mixture was stirred for 5 cultured for 24 h. For exogenous H S imaging, the culture
h at 50 C. The solvent was evaporated and the crude product medium (the DMEM supplemented with 10% FBS) was
was purified by column chromatography on silica gel (CH Cl removed and the cells were washed twice with PBS (0.01
CH OH = 20:1, v/v) to afford the probe NIR-DNP as a blueꢀ mol/L, pH 7.4). Then, the cells were treated with NIR-DNP (50
purple solid (22 mg, 67% yield). H NMR (500 MHz, µM) at 37 ºC for 60 min. After washing with PBS, the cells
CDCl /CD OD): 8.93 (d, = 2.7 Hz, 1H), 8.74 (d, = 15.1 were incubated for another 60 min in the presence of Na S (500
Hz, 1H), 8.49 (dd, = 9.2, 2.8 Hz, 1H), 7.65 (d, = 5.8 Hz, ꢁM). The controls were simultaneously performed by treating
H), 7.59 – 7.53 (m, 4H), 7.48 (t, = 7.4 Hz, 1H), 7.33 (d, the cells only with the probe NIR-DNP for 60 min at 37 ºC.
.2 Hz, 1H), 7.26 – 7.24 (m, 2H), 7.06 (dd, = 8.5, 2.2 Hz, After washed twice with PBS, the cells were incubated with 4%
H), 6.87 (d, = 15.1 Hz, 1H), 4.65 (t, 2H), 3.02 – 2.99 (m, (m/v) paraformaldehyde aqueous solution for 20 min at room
H), 2.82ꢀ2.79 (m, 4H), 2.37 – 2.31 (m, 2H), 2.00 – 1.93 (m, temperature. Then, Hoechst 33258 (0.5 ꢁg/mL) in PBS was
2
atmosphere. Then, 1ꢀfluoroꢀ2,4ꢀ 251 cells in the exponential growth phase were collected and
4
2
o
2
2
:
3
1
3
3
δ
J
J
2
J
J
1
J
J =
9
1
2
2
1
1
1
1
2
2
1
1
J
J
1
3
H), 1.81 (s, 6H) ppm; C NMR (125 MHz, CDCl
79.00, 159.80, 156.18, 154.38, 153.91, 147.83, 146.89, taken after washing the cells with PBS twice. For endogenous
42.67, 142.28, 141.02, 140.24, 130.73, 130.55, 129.33, S imaging, cells were first incubated with thiols (Cysteine,
29.12, 128.01, 122.46, 121.96, 120.16, 120.07, 116.59, 500 µM) for 60 min at 37 ºC. After washing, the cells were then
15.92, 113.21, 107.65, 106.17, 51.06, 47.27, 44.16, 29.26, incubated with NIR-DNP (50 µM) for another 60 min. The
7.46, 23.91, 23.57, 20.12 ppm; IR (KBr) ν 3040.96, 2923.43, medium was replaced by PBS and cells were imaged
854.68, 1603.70, 1578.15, 1534.07, 1499.74, 1455.64, immediately.
3
/CD
3
OD):
δ
added to stain the nuclei for 10 min. Fluorescence imaging was
H
2
346.55, 1319.87, 1260.96, 1207.56, 1152.62, 1115.21,
061.72, 1034.94, 966.35, 922.66, 833.94, 761.06 cm ; HRMS
ꢀ1
+
Results and discussion
31 3 9
(ESI, m/z): Calcd. for [C34H N O S + H] , 658.1815; Found,
658.1951.
Synthesis of probe NIR-DNP
General procedure for spectra measurement
The synthesis of the probe NIR-DNP is outlined in Scheme 1.
The stock solution of the probe NIR-DNP was prepared at 1.0 The intermediates
1 and 2 were first prepared, commencing
mM in DMSO. Stock solutions of various testing species in from 2,3,3ꢀtrimethylindolenin and cyclohexanone according to
3
0,31
water or phosphate buffer saline (PBS) were prepared from the literature procedures.
The intermediate
by condensation reaction of intermediates
anhydride. The intermediate was subsequently synthesized by
with resorcinol in the presence of triethylamine at 50
S source in all experiments, °C for 4 h. Finally, under basic condition, intermediate was
treated with 1ꢀfluoroꢀ2,4ꢀdinitrobenzene to give the target probe
A typical test solution was prepared by placing 20.0 NIR-DNP through nucleophilic substitution reaction. The
NIR-DNP stock solution (1.0 mM), 180.0 ꢁL DMSO and chemical structures of the compounds were well characterized
3
was obtained
2
Na
NaN
NaHCO
aqueous solution was used as a H
and Na SO (50 mM) aqueous solution was used as a SO
2
S, Na
, Na
, NaH
2
SO
3
, CaCl
, Na
PO
2
, KCl, NaCl, NaF, NaBr, NaNO
•5H O, CH COONa, Na CO
, Cysteine and glutathione. Na
3
, NaNO
2
,
,
1
and in acetic
3
2
SO
4
2
S
2
O
3
2
3
2
3
, H
2
O
2
4
S (50 mM) treating
3
3
2
3
2
4
2
2
ꢀ
2
7,29
3
3
1
source.
ꢁ
L
1
13
appropriate aliquot of each analyte stock solution into a 5.0ꢀmL by H NMR, C NMR, HRMS and FTꢀIR. The detailed
centrifugal tube, and diluting the solution to 2.0 mL with PBS synthetic procedures and relevant spectral data are given in the
(20 mM, pH 7.4). For the detection of H
2
S, the resulting experimental section and electronic supplementary information
solution was incubated with appropriate testing species at (ESI†).
ambient temperature for 45 min before recording the UVꢀVis
2
ꢀ
2
Sensing property of probe NIR-DNP for H S
absorption and fluorescence spectra. For the detection of SO
3
,
the resulting solution was incubated with appropriate testing The sensing ability of probe NIR-DNP for
2
H S was
species for 3 min before recording UVꢀVis absorption spectra. investigated in PBS (20 mM, pH 7.4) containing 10% DMSO
The 650ꢀnm excitation wavelength was used for all the (v/v). NIR-DNP (10 ꢁM) showed a maximum absorption at
2
6
4
−1
−1
2
measurements. The excitation and emission slit widths were 595 nm (ε = 2.96 ×10 M cm ). Upon the addition of Na S
all 5 nm.
(50.0 equiv) to NIR-DNP solution, the absorbance at 595 nm
decreased notably, whereas a new absorption peaked at 685 nm
appeared and gradually increased against time, as well as a
Cell culture and fluorescence imaging
Human glioblastoma (Uꢀ251) cells were obtained from the distinct color change from purple to cyan (Fig. 1A and inset).
Chinese Academy of Sciences (Shanghai, China). Uꢀ251 cells And the UV spectrum was close to the characteristic peak of
4
−1
−1
routinely cultured with Dulbecco’s modified Eagle’s medium compound
4
(
ε
= 2.95 ×10 M cm ) (Fig. S1A, ESI†). The
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results showed that the probe allowed colorimetric detection of spectra of NIR-DNP in the presence of different concentrations
S by ‘naked eyes’. Fluorescence analysis (Fig. 1B) exhibited of Na S were also investigated (Fig. 1C and S3, ESI†). Upon
that NIR-DNP solution was almost nonꢀfluorescence (Φ= treatment with increasing concentrations of Na S,
.007, Table S1, ESI†). However, upon the addition of Na S, a characteristic fluorescence emission at 707 nm was observed
large fluorescence enhancement at 707 nm was observed, and and enhanced gradually. The fluorescent intensity reached
the resulting fluorescence spectrum was consistent with stability when the amount of Na S was more than 200 ꢁM (Fig.
compound (Φ= 0.031) (Fig. S1B and Table S1, ESI†). Kinetic S3, ESI†). Further analysis showed that the fluorescent
studies of probe NIR-DNP (10 ꢁM) with the addition of Na intensity at 707 nm increased linearly with the concentration of
50.0 equiv) showed that the emission intensity at 707 nm Na S ranging from 0 to 7.5 ꢁM ( = 0.9993) (Fig. 1C, inset).
gradually increased against time until it reached a plateau at The detection limit of NIR-DNP for H S was experimentally
about 45 min (t1/2 ≈ 7 min) under the test conditions (Fig. 1B, determined to be 36.53 nM based on signalꢀtoꢀnoise ratio (S/N)
inset). The observed first order rate constant obs was = 3 under the test conditions, which was much lower than those
H
2
2
2
a
0
2
2
4
2
S
(
2
R
2
k
−
1
determined to be about 0.0718 min (Fig. S2, ESI†) and the obtained by most of the existing smallꢀmolecule fluorescent
emission intensity was found to increase about 53ꢀfold at 707 probes for H S (Table S2, ESI†). This result further confirmed
2
nm. The relatively fast and distinct fluorescence signal change that NIR-DNP is highly sensitive to H S. Also, the result
2
in NIR region indicated that probe NIR-DNP can be used as a indicated that the probe NIR-DNP was favorable for imaging
sensitive NIR fluorescent sensor for ‘turnꢀon’ detection of H2S intracellular H S whose physiological relevant concentrations
2
in aqueous solutions.
To further evaluate the capability of NIR-DNP for highly levels.
are estimated to be in the range from nanomolar to millimolar
3
2
sensitive detection of H S, the changes of the fluorescence
To investigate the specificity of the probe to H S, the effects
2
2
Fig. 1 (A) Time-dependent absorbance spectra of probe NIR-DNP (10 μM) upon the addition of Na
2
S (500 μM) in PBS (20 mM, pH 7.4) containing 10% DMSO (v/v). Inset: plot of
S (500 μM) under visible light. (B)
S (500 μM) in PBS (20 mM, pH 7.4) with 10% DMSO (v/v). Inset: plot of the emission intensity at
07 nm as a function of time. (C) Fluorescence spectra of NIR-DNP (10 μM) upon addition of Na S (0-7.5 μM). Inset: plot of the emission intensity at 707 nm as a function of the
S. (D) Fluorescent intensity of NIR-DNP (10 µM) at 707 nm in the presence of various species (1 mM Cys, 5 mM GSH and 500 μM others).
the absorbance at 595 nm and 685 nm as a function of time; the color change of NIR-DNP (10 μM) before (i) and after (ii) the addition of Na
2
Time-dependent fluorescent spectra of NIR-DNP (10 μM) upon addition of Na
2
7
2
concentrations of Na
2
4
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of various analytes including typical anions, cations and other shown in Fig. S5A (ESI†), most of the analytes did not produce
2
ꢀ
biologically relevant species on the fluorescent intensity of an obvious change in the fluorescence intensity except for SO
3
2
ꢀ
3
NIR-DNP were tested (Fig. 1D and S4, ESI†). The fluorescent . When competing with SO , the fluorescence intensity was
intensity of the probe NIR-DNP did not show observable about 60% lower than that in its absence. Moreover, biothiol
change upon the addition of various analytes including anions (Cys or GSH) did not show significant influence on the
ꢀ
ꢀ
ꢀ
ꢀ
2ꢀ
ꢀ
ꢀ
2ꢀ
ꢀ
(
F , Cl , Br , N
3
, CO
3
, HCO
3
, CH
3 4 2 4 2
COO , SO , H PO ), detection of H S, even its concentration was in millimolar range
+
+
2+
cations (Na , K , Ca ), as well as reactive oxygen species (Fig. S5B, ESI†). All these results indicated that probe NIR-
ꢀ
ꢀ
2ꢀ
(
S
H
2
O
2
), nitrogen species (NO
3
, NO
). However, after reacted with Na
intensity of the probe NIR-DNP enhanced remarkably (~ 47ꢀ
fold). It is worth mentioning that although the addition of In the selectivity experiments of probe NIR-DNP to H
millimolar concentration of the biothiols (i.e., 1 mM for Cys or found that the color of the probe solution in the presence of
2
) and sulfur species (SO
3
,
2
DNP had high selectivity to H S.
2
ꢀ
2
O
3
2
S, the fluorescent
2
-
Sensing property of probe NIR-DNP for SO3
2
S, we
2
ꢀ
5
mM for GSH) to the probe gave a limited increase in the SO3 changed from purple to colorless. This phenomenon
fluorescence intensity, it was far weaker than that caused by inspired us that probe NIR-DNP may have the ability to sense
2
ꢀ
Na S. The high selectivity of the probe NIR-DNP to H S could H S and SO3 by different approaches. As we know, single
2
2
2
2
ꢀ
be attributed to the obvious distinction in terms of size and pKa probe that could simultaneously detect for SO3 and H2S
To check the practical remains relatively few (Table S2, ESI†). Therefore, the sensing
ability of NIR-DNP as a H S selective fluorescent probe, ability of the probe NIR-DNP for SO3 was also investigated
3
3,34
values between H S and biothiols.
2
2
ꢀ
2
competition experiments were also carried out by adding Na2S in PBS (20 mM, pH 7.4) containing 10% DMSO (v/v). Firstly,
to NIR-DNP solution in the presence of above analytes. As the timeꢀdependent absorption spectra of NIR-DNP (10 ꢁM) in
2
-
Fig. 2 (A) Time-dependent absorption spectra of NIR-DNP (10 μM) upon addition of SO
95 nm and 375 nm as a function of time; the color change of NIR-DNP (10 μM) before (i) and after (ii) the addition of Na
of NIR-DNP (10 μM) upon addition of Na SO (0-125 μM). Inset: plot of the A375/A595 ratio as a function of the concentrations of SO
upon addition of various species (1 mM Cys, 5 mM GSH and 500 μM others). (D) Absorption ratio A375/A595 of NIR-DNP (10 μM) in the presence of various species.
3
(500 μM) in PBS (20 mM, pH 7.4) with 10% DMSO (v/v). Inset: plot of the absorbance at
5
2
SO (500 μM) under visible light. (B) Absorption spectra
3
2-
2
3
3
. (C) Absorption spectra of NIR-DNP (10 μM)
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2
ꢀ
the presence of SO
3
(50.0 equiv) were studied (Fig. 2A). The
The changes of the absorption spectra of NIR-DNP with
2
ꢀ
2ꢀ
result showed that upon the addition of SO
3
(50.0 equiv), the SO
3
concentration increasing were further studied (Fig. 2B
absorption band at 595 nm decreased rapidly. Meanwhile, a and S7, ESI†). Upon the addition of increasing concentrations
2
ꢀ
new absorption band centered at 375 nm appeared, of SO
3
, the absorption intensity at 595 nm gradually
accompanied with the solution color changing from purple to decreased, while the absorption intensity at 375 nm gradually
2
ꢀ
colorless (Fig. 2A, inset). The isosbestic point at 419 nm increased. The A375/A595 ratio was linearly proportional to SO
indicated the formation of a new compound. Further study concentration in the range of 0ꢀ125 ꢁM ( = 0.9971) (Fig. 2B,
3
R
indicated that the absorption peak at 595 nm decreased rapidly inset). The detection limit was determined to be about 33.33
to the baseline over 0 to 180 seconds, and a new peak at 375 nM based on S/N = 3, suggesting that the probe was potentially
2
ꢀ
nm increased to the top of the wavelength range (Fig. 2A, useful for quantitative determination of SO
inset). The A595/A375 ratio varied from 7.37 to 0.078 after dynamic range and has high sensitivity.
3
in a large
complete conversion, which was about 95ꢀfold decrease (Fig.
S6, ESI†). Such a rapid and significant change in the signal samples, the selectivity of NIR-DNP toward SO3 was also
ratios at the two wavelengths indicated that probe NIR-DNP studied in the presence of other analytes including F , Cl , Br ,
Considering coexistence of other competing species in real
2
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
2ꢀ
ꢀ
ꢀ
ꢀ
2ꢀ
ꢀ
2ꢀ
could be used as a colorimetric sensor for high sensitive N , CO , HCO , NO , NO , S O , CH COO , SO ,
4
3
3
3
3
2
2
3
3
2
ꢀ
ꢀ
+
+
2+
detection of SO3
.
H PO , Na , K , Ca , Na S, Cys, GSH and H O . The results
2 4 2 2 2
Fig. 3 (A) The mechanism of probe NIR-DNP for discriminative sensing sulfite and sulfide. (B) Frontier molecular orbital plots of compound
molecular orbital plots of probe NIR-DNP in water. The result showed that the fluorescence emission of compound 4 is quenched by d-PET process.
4 in water. (C) Frontier
6
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Fig. 2C) showed that no obvious responses were observed in NIR-DNP for H
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(
2
S sensing was a thiolysis process of
the UVꢀvis spectra of NIR-DNP upon the addition of the above dinitrophenyl ether with the release of compound
4 (Fig. 3A).
2
ꢀ
species. However, the addition of SO
absorption band centered at 375 nm, accompanied by decrease
of the absorption band at 595 nm. Observation of the color The obvious blueꢀshifts in the absorbance spectra of NIR-DNP
3
induced a new
2
3
-
Sensing mechanism of NIR-DNP toward SO
2
ꢀ
2ꢀ
changes of the probe NIR-DNP before and after the addition of toward SO
3
are consistent with the addition of SO
3
to the
sulfite and other species under visible light showed that only electrophilic C=C bond in NIR-DNP, which lead to a short
2
ꢀ
SO
3
caused a marked change from purple to colorless (Fig. conjugation structure of the reaction product. To confirm the
1
S8A, ESI†). Quantitative analysis (Fig. 2D) exhibited that the formation of the addition product NIR-DNPꢀSO
3
Na, H NMR
375/A595 ratio of the probe was remarkably enhanced when titration experiment was carried out by addition of Na SO into
OD. The result (Fig. S12, ESI†) showed that
S could only induce a the resonance signal corresponding to the alkene proton H at
small decrease in the absorbance at 595 nm (Fig. 1A and 2C) 7.54 ppm disappeared after treated with Na SO , and a new
A
2
3
2
ꢀ
3 3
NIR-DNP was treated with SO . It is worth noting that during NIR-DNP in CD
2
ꢀ
the period (3 min) of SO
3
detection, Na
2
a
2
3
and a neglectable change of the A375/A595 ratio (Fig. 2D). peak at 3.82 ppm assigned to the proton Ha’ appeared. The
However, when the probe was treated with Na S for 45 min, the addition of Na SO to C=C resulted in the formation of a chiral
2
2
3
color of the probe solutions changed from purple to cyan (Fig. center of Ca’ with the nonequivalent protons of the methylene
S8B, ESI†). This result indicated that NIR-DNP could be used group at C . Therefore, the signal for H at 8.72 ppm shifted to
b
b
2
ꢀ
for distinguishing SO3 and H S by naked eyes in time a high field and appeared as two peaks at 2.48 ppm and 3.02
2
dimension. Moreover, the addition of biothiols (1 mM for Cys ppm, respectively, which was similar with those reported
2
2,35
and 5 mM for GSH) to the probe could not cause the change of previousely.
EIꢀMS analysis further confirmed this result. A
A375/A595 ratio, which might be ascribed to their steric effects peak appeared at m/z =760.24 in the mass spectrum, which is
3
4
and high pKa values. This result imply that the probe was nearly identical to the theoretical molecular mass of NIR-DNP
ꢀ
2
ꢀ
chemoselective for SO3 over these biothiols at physiological
2
ꢀ
pH. The detection of SO3 using NIR-DNP in the presence of
other analytes remain effective (Fig. S9, ESI†). All these
investigations clearly indicated that the probe NIR-DNP
2
ꢀ
possessed excellent selectivity for SO3
.
Sensing mechanism of NIR-DNP toward H2S
To confirm that the turnꢀon fluorescence response of NIR-DNP
was triggered by H S, the thiolysis product of NIR-DNP was
2
characterized by using NMR and EIꢀMS analysis, as well as
1
theoretically calculated. H NMR analysis showed that the
product of NIR-DNP after treated with Na S was identical to
2
the authentic compound
confirmed by EIꢀMS analysis. EIꢀMS showed a peak at m/z
490.32, which is the same with the theoretical molecular mass
4 (Fig. S10, ESI†), which was further
=
ꢀ
of compound
To better understand the photophysical properties of the probe
NIR-DNP and the thiolysis product , theoretical calculations
were also performed through the density functional theory
DFT) and timeꢀdependent DFT (TDDFT) by using Gaussian
4 (calcd for [M ꢀ 1] = 490.18) (Fig. S11, ESI†).
4
(
0
9 program (Fig. 3B, 3C, and Table S3, ESI†). For compound
4, the π electrons on both the HOMO and LUMO were
essentially distributed in the entire backbone of compound
By contrast, for NIR-DNP, the π electrons on HOMO and
LUMO+2 were primarily resided on the electronꢀdonating
4
.
2-
3
Fig. 4 (A) Detection of SO
in the absence and presence of other analytes by using
2-
3
paper-based test strips treated by probe NIR-DNP (100 μM): 1, 0 μM SO ; 2, other
2
3
-
2-
3
2-
3
2-
3
analytes mixture; 3, 10 μM SO
; 4, 50 μM SO
; 5, 100 μM SO
; 6, 200 μM SO
; 7,
2
3
-
2-
2-
2-
5
00 μM SO
; 8, 500 μM SO
3
and other analytes; 9, 1 mM SO
3
; 10, 5 mM SO
3
; 11,
moiety of compound
4, whereas, those on LUMO and
2-
2-
+
+
-
-
-
1
0 mM SO
3
; 12, 50 mM SO
3
. Other analytes mean a mixture of K , Na , F , Cl , Br ,
2-
LUMO+1 were mainly located on the electronꢀwithdrawing
group of 2,4ꢀdinitrobenzene. This result indicated that NIR-
DNP could bear efficient photoꢀinduced electron transfer (PET)
-
-
-
2-
4
2-
3
-
-
-
NO
3
, NO
2
, N
3
, SO
, S
2
O
3 3 2 4
, CO , HCO , H PO , AcO (500 μM of each analyte in PBS,
pH 7.4). (B) Fluorescence images of probe NIR-DNP in U-251 cells. The cell nuclei were
stained with Hoechst 33258. From left to right, cells were incubated with the probe
NIR-DNP (50 μM) for 60 min; cells were first incubated with the probe NIR-DNP (50
from compound
4 to the 2,4ꢀdinitrobenzene group, rendering
2
μM) for 60 min, and then incubated with Na S (500 μM) for another 60 min; cells were
the relatively weak fluorescence of the probe. By contrast, the
nearly complete overlap of electrons on the transition orbitals
may induce the strong fluorescence emission for compound 4.
first incubated with Cys (500 μM) for 60 min, and then incubated with the probe NIR-
DNP (50 μM) for another 60 min. From top to bottom, bright-field images;
fluorescence images; merged image of fluorescence and Hoechst 33258-stained
images.
All these investigations demonstrated that the mechanism of
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ꢀ
SO
3
Na (calcd. for [NIR-DNPꢀSO
3
Na–1] , 760.13) (Fig. S13, NIR-DNP (50 ꢁM) for 60 min at 37 °C showed no intracellular
S in cells, an
S was added to increase the intracellular H
The cells were treated with NIR-DNP (50 ꢁM) for 60
min, followed by Na S (500 ꢁM) for another 60 min. As a
result, red fluorescence was clearly observed in the cytoplasm,
suggesting that NIR-DNP could be used for exogenous H
ESI†). These results suggested that the mechanism of NIR- fluorescence. Owing to the low level of H
2
2
ꢀ
2ꢀ
DNP for SO
3
sensing was a SO
3
ꢀinduced nucleophilic exogenous Na
2
2
S
1
7,29
addition, with generating a short conjugation structure of the level.
addition product NIR-DNPꢀSO
3
Na (Fig. 3A).
2
Effect of pH
2
S
The pH adaptability is very important for the practical imaging in Uꢀ251 cells. To further test whether NIR-DNP
applications of a new designed probe. In the current study, the could detect endogenous production of H S, cells were first
pH adaptability of the probe NIR-DNP was also investigated treated with Cys (500 ꢁM) for 60 min. The results of MTT
Figs. S14 and S15, ESI†). The result showed that the assays indicated that Cys did not show obvious cytotoxicity at
2
(
fluorescence intensity and the A375/A595 ratio of NIR-DNP had the experimental conditions (Fig. S20, ESI†). The cells were
no change in the pH range from 6.0 to 9.4, suggesting that the then treated with NIR-DNP (50 ꢁM). As shown in Fig. 4B, Cys
3
9,40
probe was stable and insensitive to pH changes in this range. induced the production enhancement of endogenous H
However, upon treatment with Na S (50 equiv), strong and clear fluorescence was observed, implying that NIR-DNP
fluorescence emission was observed in the pH range from 6.5 could be also used for endogenous H S imaging in Uꢀ251 cells.
to 9.4 (Fig. S14, ESI†), indicated that the probe NIR-DNP Moreover, probe NIR-DNP can be also used for H S imaging
could be used to recognize H S in physiological pH region. in murine hepatocytes (Fig. S21, ESI†). All the results above
Additionally, upon the addition of SO
enhancement of the A375/A595 ratio were observed in the pH with intracellular H
range of 6.75 to 8.5 (Fig. S15, ESI†), revealing that the probe imaging in cells with low cytotoxicity.
2
S,
2
2
2
2
2
−
3
(50 equiv), significant suggested that NIR-DNP was cellꢀpermeable and can react
S efficiently, which can be used for H
2
2
S
2
−
NIR-DNP could be also used to detect SO
physiological condition. The pH crossꢀtalk of probe in the “on”
state (compound ) was also studied. The results suggested
compound was a double NIRꢀemissive ratiometric fluorescent
3
under
Conclusions
4
4
In this study,
a dual functional probe NIR-DNP was
probe which exhibited pHꢀdependent optical responses (Fig.
S16 and S17, ESI†), similar with the studies reported
synthesized by introducing 2,4ꢀdinitrobenzene to hemicyanine
skeleton. The probe NIR-DNP exhibited colorimetric response
3
6,37
previousely.
was derived from the titration curve of emission ratio (
Fig. S18, ESI†).
The p
K
a
value was calculated to be 6.7 which
2ꢀ
to SO
3
along with a color change from purple to colorless, and
S along with a
color change from purple to cyan. The probe could be used to
I
706 669
/I )
NIR fluorescence enhancement response to H
2
(
2
ꢀ
sense H
presence of other biologically relevant species. The detection
limit of the probe was 36.53 nM for H S and 33.33 nM for
H NMR and ESIꢀMS analysis, as well as DFT and
TDFT theoretical calculation demonstrated that the sensing
2
S and SO
3
with a high selectivity, even in the
Paper-based test strips.
To demonstrate the practical application of the probe NIR-DNP
2
2
−
for the detection of SO
3
, a paperꢀbased test strip was
2ꢀ
1
3
SO .
developed by wetting a neutral filter paper with NIR-DNP
methanol solution (100 ꢁM). After drying in air, a blueꢀpurple
2ꢀ
mechanism of the probe toward
respectively based on H Sꢀinduced thiolysis of dinitrophenyl
ꢀinduced nucleophilic addition. Further
H
2
S
and SO
3
were
2
ꢀ
2
3 3
test paper was ready for use. For the detection of SO , SO –
2
contained solution was dropped on the test paper using a glass
capillary. As shown in Fig. 4A, a rapid color change occurred,
even in the presence of other analytes. However, the mixture of
2ꢀ
ether and SO
3
investigation showed that the probe NIR-DNP could be used to
develop an easyꢀtoꢀprepare and easyꢀtoꢀdetect paperꢀbased test
2
ꢀ
other analytes without SO
3
could not cause any detectable
2ꢀ
strip for cheap and effective detection of SO
3
. Also, the probe
changes. In addition, the test papers showed different color
NIR-DNP has the potential to image exogenous and
endogenous H S in living cells. Based on these results, we are
actively pursuing more sensitive and wideꢀranging analogues
2
ꢀ
changes to different concentrations of SO
3
ranging from 10
2
ꢁ
M to 50 mM. These results indicated that the probe NIR-DNP
could be used to develop an easyꢀtoꢀprepare and easyꢀtoꢀdetect
paperꢀbased test strip for a cheap and effective way to detect
2ꢀ
for simultaneous detection of SO
3
and H
2
S, as well as
fluorescence imaging of H
in our further works.
2
S in living cells, tissues and animals
2
ꢀ
SO
3
.
Cell imaging
To demonstrate the potential application of the probe NIR- Acknowledgements
DNP in fluorescence imaging of cells, live cell imaging
This study was supported by the National Natural Science
Foundation of China (Nos. 21375106 and 21202131), the Fund
of Youth Science and Technology Stars by Shaanxi Province
experiments were also performed using Uꢀ251 cells. Firstly, the
3
8
cytotoxicity of the probe was evaluated by MTT assay. The
result (Fig. S19, ESI†) showed that more than 80% cells were
viable when up to 80 ꢁM NIR-DNP was used, indicated that
the probe did not show significant cytotoxicity in the 0ꢀ80 ꢁM
range. As displayed in Fig. 4B, Uꢀ251 cells incubated with
(
2015KJXXꢀ15), and the Fundamental Research Funds for the
Central Universities (2014YB026).
8
| RSC Adv., 2016, 00, 1-3
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3
6 Q. Wan, S. Chen, W. Shi, L. Li and H. Ma, Angew. Chem. Int. Ed.
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Graphic Abstract
2
In this study, we developed a new dual functional probe NIR-DNP for discriminative detection of H S and
2
-
3
SO .
S1