A novel NIR fluorescent probe for the double-site and ratiometric detection of SO2 derivatives and its applications

Article abstract of DOI:10.1039/c9nj03997j

A feasible double-site near-infrared (NIR) fluorescent probe Q5 based on xanthenes was developed. Probe Q5 showed clearly HSO₃^- induced changes in the fluorescence ratio of two well-separated NIR and VIS peaks, showing excellent selectivity compared with other analytes. And the detection limit of the probe for HSO₃^- was found to be 89 nM. Furthermore, fluorescence imaging experiments of HSO₃^- in CEM cells revealed that the probe has potential application value in biological systems.

Full text of DOI:10.1039/c9nj03997j

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March 2018
Pages 3963-4776
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Chechia Hu, Tzu-Jen Lin et al.
Yellowish and blue luminescent graphene oxide quantum dots
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DOI: 10.1039/C9NJ03997J
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A novel NIR fluorescent probe for double-site and ratiometric
detection of SO derivatives and its application
2
Received 00th January 20xx,
Accepted 00th January 20xx
a
a
b*
a
a
c,*
a
Jianming Zhu , Fengyun Qin , Di Zhang , Jun Tang , Wenya Liu , Wenbo Cao , YongYe
DOI: 10.1039/x0xx00000x
A feasible double-site near-infrared (NIR) fluorescent probe Q5 based on xanthenes was developed. Probe Q5 exhibited
-
clearly HSO
3
induced changes in the fluorescent ratio of two well-separated NIR and VIS peaks, showing excellent
-
selectivity compared with other analytes. And the detection limit of probe for HSO
3
was found to be 89 nM. Furthermore,
-
fluorescence imaging experiments of HSO
systems.
3
in CEM cells revealed the probe has potential application value in biological
synthesis route of these probe is long or complex, due to two
fluorophores needed.
NIR fluorescent probes have become a research hotspot in the
field of fluorescent probes, due to the advantages of strong
Introduction
2
SO and its derivatives play an important physiological role in
maintaining normal cardiovascular function. SO
2
has some
peculiar biological activities, such as vasodilation and anti-
4
3-47
oxidation.1 SO
-3
tissue permeability and less damage to tissues or cells.
So
2
is widely used as antibacterials, enzyme
-
4
,5 far, many NIR fluorescent probes for the detection of HSO
3
inhibitors and antioxidants in foods, drinks and beverages.
However, beyond a certain concentration, SO can cause
damage to the skin, respiratory tract and gastrointestinal tract.
Inhalation of SO causes respiratory diseases and cancer and
4
8-51
have been developed.
Nevertheless, few probes have been
2
-
reported for double-site detection of HSO
3
. Herein, a new
xanthene-based NIR ratio fluorescence probe Q5 which could
2
-
6
,7
be used for detecting HSO
3
in double-sites under neutral and
has been extensively studied in toxicology. Therefore, there
is a great need to develop reliable detection methods for
alkaline conditions was developed and synthesized. Ratio
fluorescence imaging experiments in CEM cells has revealed its
potential application in biological systems.
monitoring SO
SO in aqueous environments is the precursor of bisulfite
HSO -
2
and its derivatives. The main chemical state of
2
2-
8
(
3 3
) and sulfite (SO ). Up to now, various analytical means
Experimental
chemicals and instruments
have been designed and fluorescent analysis methods have
received considerable attention because of their advantages of
simple operation, high sensitivity and real-time sensing
Cyclohexanone, 3-(diethylamino)phenol, 1,4-Phthalaldehyde
and other reagents were purchased from regular sales
channels, without special explanation, and were directly used
in the experiments. The reagents used in the analysis were all
pure analytical grade, and can be directly used without any
treatment. The deionized water was purified by Milli-Q. The
capability.9 Fluorescence analysis, as an important analytical
-11
1
2-15
method, is currently widely used in biological research.
Currently, the fluorescent probes of SO designed and
synthesized in the reported literature have been mainly based
2
1
6-31
on the nucleophilic nature of SO2.
However, there are still
-
-
-
analytes ofbiothiols (Cys, Hcy, and GSH), anions (HSO
3
, HS , F ,
many problems need to be solved, such as background
fluorescence interference, unsatisfactory detection limit, long
response time, and suffering interference from other active
In order to reduce the interference of
background fluorescence and auto-fluorescence, some ratio
probes, which based on FRET or TBET were developed. But the
-
-
-
3-
2-
2-
-
−
−
2-, S
2-
Cl , Br , I , PO
4
, HPO
4
-
,CO
3
, HCO
3
2-
, NO
2
, NO
3
, SO
4
2
O
8
,
,
2
-
, OAc-
2-
2-,S
-, H
S O
2 5
,
S , SCN , S O
2 3
O
2 4
,), ROS (HClO, ClO
4
2
O
2
sulfur molecules.3
2-42
2
KO ) and potassium or sodium salts of anions were prepared
as 10.00 mM in water solution, which were used to sense
objects of Q5.
Absorption spectra were accurately measured on a Lambda
3
5 UV/VIS spectrometer, Perkin Elmer precisely. Fluorescence
a. Phosphorus Chemical Engineering Research Center of Henan Province, the College
of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou
450052, China. E-mail: yeyong@zzu.edu.cn.
Institute of Agricultural Quality Standards and Testing Technology, Henan
Academy of Agricultural Sciences, Zhengzhou 450002, China. E-mail:
spectra were measured on the F-4500 FL Spectrophotometer,
and The EXSlit and EM Slit were both set at 10.0 nm. The pH
1
b.
was measured by a Model pHs-3Cmetr (Shanghai, China). H
1
3
NMR and C NMR spectra were measured on a Bruker DTX-
00 spectrometer using TMS as internal reference. ESI mass
pandy@163.com.
4
c.
School of Basic Medical Science, Zhengzhou University, Zhengzhou 450001, China.
Electronic Supplementary Information (ESI) available. Structure characterization of spectra were performed on a HPLC Q-Tof HR-MS spectrometer
Q5, HR-MS spectrum of Q5 additional spectroscopic data. See
DOI: 10.1039/x0xx00000x
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by using methanol as mobile phase. Cells were imaged on a curve of the probe for various analytes. Fig. 1 showed Q5 (10
LEICA TCS SP8 laser scanning confocal microscope.
Synthesis of probe Q5
Compound a. 3-(diethylamino)phenol (6.27 g, 38 mmol) and SO
phthalic anhydride (5.6 g, 38 mmol) were dissolved in toluene, HS , S , SCN , S
and the reaction mixture was refluxed for 5-6 h. After the major absorption peak at 575 nm, because of the structural
reaction was completed, it was filtered, washed with methanol. conjugation. In addition, Q5 also had some absorption peaks
The obtained solid was not further purified and used for the between 250-400 nm. After addition of HSO
next reaction.
Compound b. Cyclohexanone (0.66 ml, 6.37 mmol) was added obvious decreased, the absorption peak at 375 nm and 260 nm
dropwise to concentrated sulfuric acid (7 mL) at 0 °C, then was increased significantly. This is because that the 1,4 -
compound a (1.0 g, 3.2 mmol) was added to the vigorously addition reaction occurred between Q5 and HSO
stirring mixture solution. The mixture was then heated to 90 °C broken into two parts, so that there were two obvious
and the temperature was maintained for 1-1.5 h. After the absorption peaks after that, allowing colorimetric detection of
reaction finished, the reaction mixture was cooled to room HSO
temperature, and the reaction mixture was poured into a addition reaction with the aldehyde group of Q5. The color
suspension containing 30 g of ice water with stirring, and 0.7 change of probe solution can be recognized by naked eyes (Fig.
mL of 70% perchloric acid was added under stirring, resulting 1 inset). However, when other analytes were added, there
in a large amount of red precipitation immediately. The solid were almost no obvious change in the UV absorption curve of
was washed with 20 mL ice water and dried to obtain red solid the probe Q5. The results of data analysis showed that the Q5
for further reaction.
View Article Online
DOI: 10.1039/C9NJ03997J
-
μM) had clearly specific response to HSO
3
with 26 other
-
-
-
-
3-
2-
2-, HCO -
, KO ,Cys, Hcy, GSH,
2 4
and S O ). The single probe Q5 showed a
−
−
analytes (F , Cl , Br , I , PO
4
, HPO
4
,CO
3
3 2 3
, NO , NO ,
2
-
2-
2-
-
-
4
-
, S O
2 8
, S
O
2 5
, OAc , HClO, ClO
2-
4
, H
2
O
2
2
2-
-
2-
O
2 3
-
(100 μM), the
0
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3
absorption peak of Q5 at 575 nm and 290-340 nm were
-
3
. Q5 was
-
-
3
. Meanwhile, HSO
3
might also happen nucleophilic
-
could identify HSO
3
specifically in PBS (pH = 7.40, 10 mM,
b (376 mg, 1 mmol) and containing 30% MeOH), and could achieve the effect of naked
Compound Q5. Compound
terephthalaldehyde (402 mg, 3 mmol) were dissolved in acetic eye recognition.
acid (19 mL), and the reaction mixture was heated to 90-
1
00 °C and further stirred at 90-100 °C for 2-5 h, before cooling
to room temperature. Decompression was performed to
remove the solvent to obtain the crude product, which was
purified by silica gel flash chromatography using CH
2 2
Cl / MeOH
(40:1) as eluent to obtain compounds Q5 as purple solid (394
mg, 80.1%).
1
3
H NMR (400 MHz, CDCl , ppm) δ: 1.17 (t, 6 H, J = 7.04 Hz),
1
.66 (m, 3 H), 2.07 (m, 1 H), 2.65 (m, 1 H), 2.79 (m, 1 H), 3.36
(q, 4 H, J = 7.08 Hz), 6.35 (dd, 1 H), 6.42 (d, 1 H, J = 2.48 Hz),
6
7
8
.48 (d, 1 H, J = 8.92 Hz), 7.22 (d, 1 H, J = 7.64 Hz), 7.42 (s, 1 H),
.56 (t, 3 H, J = 8 Hz), 7.65 (t, 1 H, J = 7.44 Hz), 7.88 (d, 2 H, J =
.2 Hz), 7.95 (d, 2 H, J = 7.6 Hz), 10.02 (s, 1 H); C NMR (100
1
3
Fig. 1.UV-vis absorbance spectra of Q5 (10 μM) in the absence and addition of
MHz, CDCl3, ppm) δ: 12.7, 22.4, 23.0, 27.4, 44.4, 86.3, 97.2, 10 eq. other analyses in PBS (pH = 7.40, 10 mM, containing 30% MeOH). Inset:
-
1
1
1
4
04.6, 108.8, 109.4, 123.5, 124.0, 125.0, 127.5, 128.6, 129.3, solution from purple to colorless before and after the addition of HSO
29.6, 130.0, 133.2, 134.6, 134.7, 143.7, 146.4, 149.4, 152.1, observed by the naked eye.
52.5, 170.0, 191.8; HR-MS m/z: Calcd for C32
92.2169, found 492.2175. (Supporting Information, Fig. S1-S3)
3
to Q5
+
H30NO
4
[M]
To further make an investigation on the relationship
-
between HSO
3
and Q5, an ultraviolet titration experiment was
-
performed (Fig. S4). When the concentration of HSO
3
was
O
O
COOH
COOH
increased, a new 375 nm-centered absorption peak was
emerged and the 575 nm-centered one was disappeared,
induced the destruction
of the π-conjugated structure of probe Q5 through Michael
+
O
O
N
OH2
O
N
OH
H2SO4
Et2N
O
b
-
which could be attributed to the HSO
3
a
COOH
addition. There was a good linear relationship between UV
CHO
CHO
COOH
CH3COOH
-
peak of Q5 at 550 nm and HSO
3
consistence (Fig. S4 inset).
+
N
O
According to the UV absorption changes of the probe Q5,
410 nm and 580 nm as the excitation wavelengths was used in
fluorescence spectroscopy, respectively. The fluorescence
selectivity of Q5 to various analytes was further explored. Fig.
demonstrated the single probe Q5 showed an emission peak
at 650 nm ascribe to the entire conjugate structure of Q5 and
N
O
b
Q5
CHO
Scheme 1. Synthetic route of Q5.
2
Results and discussion
Spectroscopic responses of probe Q5 to HSO
First, ultraviolet spectrometer was used to determine the UV
absorption curve of the single probe and the UV selectivity
-
3
another one at 464 nm ascribe to xanthene moiety in the PBS
pH = 7.40, 10 mM, containing 30% MeOH). A significant
(
2
| J. Name., 2012, 00, 1-3
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increment of particular fluorescence of xanthene fluorophore
moiety was appeared at 485 nm (about 6 times enhanced) of HSO
with 10 eq.HSO
nm was obvious decreased. As a result of the 1,4-addition HSO
reaction was occurred between probe Q5 and HSO
fluorescence of xanthene moiety was released. It was (19) H
remarkable that the difference between the two emission
wavelength is quite large (almost 165 nm), caused a large
ratiometric value. Simultaneously, under the same condition,
no significant changes could be observed after adding the interaction between the probe Q5 and HSO
same number of other analyses. The fluorescence emission this new probe for HSO
peak at 650 nm of Q5 could be seen more clearly when excited experiments at different excitation wavelengths (410 nm, 580
at 580 nm (Fig. S5). According to the above results, probe Q5 nm) were studied, respectively. The concentration of Q5 was
could specific identify HSO
In addition, Fig. 3 showed that the free probe Q5 exhibited μM) was added continually. As shown in Fig. 4, with the
very low fluorescence intensity ratiometric value in the increase of the concentration of HSO
Fig. 3. Fluorescence intensity ratio (F485/F650) of Q5 (10 μM) aVftieewr tAhrteicaledOdintliionne
-
DOI: 10.1039/C9NJ03997J
3
(100 μM) and background analytes (100 μM) in PBS (pH = 7.40, 10 mM,
-
3
, and the fluorescence emission peaks at 650 containing 30% MeOH). (ex = 410 nm,λem= 485 nm ，650 nm ).(1) Probe, (2)
-
-
-
-
-
3-, (8) HPO
2-, (9) CO
2-, (10) HCO
-, (11)
-,
3
, (3) F , (4) Cl , (5) Br , (6) I , (7) PO
-, (12) NO -, (13) SO 2-, (14) S
,(20) O
(28) S
2-.
4
4
3
3
-
2-, (15) S
2-
-
-
3
, the NO
3
2
4
2
O
8
2
O
5
, (16) ACO , (17) ClO ,(18) ClO
4
.-
-
2-
-
2
O
2
2
,(21) GSH, (22) Hcy,(23) CYS, (24) HS , (25) S , (26) SCN , (27)
2
-
2
S O
3
,
2 4
O
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To further verify the quantitative relationship of the
-
3
, and the DL of
two fluorescence titration
-
3
,
-
-
3
as a ratio probe.
3
kept constant and the increasing concentration of HSO (0-150
-
3
(ex = 410 nm), the
present of background analytes, while this value was fluorescence emission peaks at 465 nm was gradually shift to
significantly enhanced (about 10 times enhanced) with 10 eq. 485 nm due to 1,4-addition reaction between probe Q5 and
-
-
HSO
HSO
3
. Therefore, other analytes had low interference for HSO
in PBS (pH = 7.40, 10 mM, containing 30% MeOH). These The fluorescence emission intensity at 650 nm was gradually
3
, and the fluorescence intensity was constantly enhanced.
-
3
results indicated that probe Q5 had an excellent selectivity for decreased. The fluorescence intensity ratiometric value
-
HSO
3
in the presence of exogenous analyses of these tests.
(F485/F650) was found to be increased linearly with the
-
-
3 3
concentration of HSO and the DL (3σ/slope) of HSO was
calculated to be 89 nM (Fig. 4 inset). Comparison of reported
with Q5 (Table S1), Q5 has more
3
excellent sensitivity to HSO . Therefore, these results indicated
that the probe Q5 can act as a fluorescent sensor for high
. The change of the
fluorescence titration in the NIR region was clearer at the
excitation wavelength of 580 nm (Fig. S6). When the
was increasing, it was clear that the
fluorescence intensity at 650 nm was decreased, and there
-
fluorescence probe for HSO
3
-
-
sensitivity and selectivity detection of HSO
3
-
concentration of HSO
3
was a good decreased linear correlation between probe Q5
-
and HSO
3
(Fig. S6, inset).
Fig. 2. Fluorescence intensity of Q5 (10μM) after the addition of 10 eq. various
analytes in PBS (pH = 7.40, 10 mM, containing 30% MeOH). (λex= 410 nm, scan
range 430–750 nm).
Fig. 4. Fluorescence emission spectra of Q5(10μM) after the addition of various
-
concentrations of HSO
3
(0-15 equiv) in PBS (pH = 7.40, 10 mM, containing 30%
-
MeOH). Inset displayed the linear responses (F485/F650) of Q5 with HSO
3
concentrations (ex = 410 nm,λem= 485 nm，650 nm ) .
This journal is © The Royal Society of Chemistry 20xx
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In order to accurately detect analytes in different actual [MQ5'+H] (calcd = 656.1619). Both of the H NMR titration
environments. The effect of the different acid conditions to experiment and HR-MS results certified the proposed
the probe Q5 and Q5 toward HSO
showed that the ratiometric value (F485/F650) of the free probe Cellular imaging
was not obviously changed and was relatively stable under the
condition of pH<11 in PBS (10 mM, containing 30% MeOH), monitoring of HSO
While, in the presence of HSO
View Article Online
DOI: 10.1039/C9NJ03997J
-
3
were investigated. Fig. S7 mechanism shown in Scheme 2.
Fluorescence imaging applications of probe Q5 for
3
in living biological cells had also been
carried out. CEM cells were selected to co-cultured with probe
-
-
3
, the ratiometric value (F485/F650
)
was significantly enhanced between pH 7.0 and pH 12.0 Q5(10 μM) in PBS buffer for about 0.5 h at 37 ℃. Images were
neutral and alkaline) due to the occurrences of the 1,4 - photographed under natural channel, blue channel and red
0
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2
3
4
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1
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(
addition reaction and nucleophilic addition reaction between channel of the confocal microscope, respectively. The cells
Q5 and HSO
physiological environment in most organisms (pH=7.4). The washing with PBS buffer three times, then 20 uL response ion
results suggested that the probe Q5 can detect HSO
-
3
.
This pH span was conformed to the were observed only with red fluorescence (Fig. 5c). After
-
-
3
under HSO
3
(100 μM) were supplemented to the cells co-cultured for
about 0.5 h at 37 ℃. Images were photographed under natural
physiological conditions.
To examine the response time influence of the probe Q5 to channel, blue channel and red channel of the confocal
-
HSO
3
, the kinetic experiments and the photostability of the microscope, respectively. The cells were observed to show
probe Q5 (ex = 410 nm, λem= 485 nm, 650 nm) were blue fluorescence (Fig. 5e) and the red fluorescence
investigated, respectively. At the same excitation wavelength, disappeared (Fig. 5f). An image of cell incubated by Q5 and
-
-
1
3 3
0 eq. HSO was added, the fluorescence intensity changed HSO , under bright field, demonstrated that the cells were
with time at 485 nm and 650 nm, respectively. As shown in Fig. feasible in the whole experiments (Fig. 5a, 5d). The
S8, compared with the fluorescence intensity ratio F485/F650, it morphology of cells remained intact. These results of cell
could be seen that the photostability of the probe Q5 was experiments demonstrated that probe Q5 might successfully
excellent in 1 h. After the addition of HSO
was increased gradually. But there was no obvious maximum in living cells.
response platform in 1 h. With the same method, the
photostability of the probe Q5 and the response time to HSO
at the maximum emission wavelength 650 nm was measured
under the excitation wavelength of 580 nm (Fig. S9). When 10
-
-
3
, the ratio F485/F650 be used for detecting HSO
3
and realized fluorescence imaging
-
3
COOH
COOH
HSO3-
-
eq. HSO
3
was added, the fluorescence intensity achieved the
N
O
N
O
minimum response platform within 0.5 h. The results showed
that the probe Q5 can act as a probe to detect HSO
time.
The proposed mechanism of the probe Q5 towards HSO
We then explored the possible mechanism of the reaction
between probe Q5 and HSO
literature and the UV absorption spectra from Fig. 1. we
speculated that 1,4-addition reaction of HSO
bond of probe Q5 and a nucleophilic addition reaction of HSO
with aldehyde group took place. The reactions caused the
destruction of the conjugate structure and result in a change in
the fluorescence ratio. According to our knowledge,
recognition mechanism of Q5 to HSO
Scheme 2. In order to verify the mechanism, the H NMR
titration experiment of Q5 with HSO
were first investigated. The H
ppm and the H
Ha'
Ha
HO3S
-
Hb'
3
in real
O
OH
Hb
SO3H
-
3
Q5
Q5'
Scheme 2.The proposed mechanism of the response of Q5 to HSO
3
-.
-
3
. By consulting the relevant
-
3
with the double
-
3
a
-
3
was speculated in
1
-
3
in DMSO-d
6
(Fig. S10)
a
of the double bond at δ7.42
of aldehyde group at δ10.02 ppm of free
b
-
probe Q5 could be found. After adding 1-10 equivalent HSO
to Q5, both aldehyde hydrogen H
were gradually disappearing. At the same time, two new
3
a
and double bond hydrogen
Fig.5 Fluorescence microscopic images of CEM cells. CEM cells treated with Q5
H
b
o
(
10 μM) in PBS buffer for 0.5 h at 37 C, (a)and (d), bright channel, (b)and(e),
peaks Ha' and Hb' were appearing gradually at δ5.03 and δ5.40,
respectively. Then, the recognition mechanism of Q5 was also
verified by the HR-MS (Fig. S10). Q5 exhibited a specific peak
at m/z 492.2175 (Fig.S3). A mixture of Q5 and HSO
to HR-MS analysis and the peak at m/z 492.2175 was
disappeared. new significant peak at m/z 656.1622
blue channel, (c)and (f), red channel.
-
3
was added
Conclusions
In conclusion, a feasible double-site and ratiometric novel
NIR fluorescent probe Q5 based on xanthenes was developed.
A
distributed to Q5' was appeared (Fig.S11), which was basically
consistent with the value of the theoretical calculation
-
Probe Q5 could effectively detect HSO
3
in PBS solution
4
| J. Name., 2012, 00, 1-3
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compared with other coexistent analytes, and provided a
simple method with naked-eye recognition. The mechanism of
double-site recognition for HSO
titration and HR-MS. In addition, it had been used in CEM cell
imaging to demonstrate that it could act as a probe to monitor
3
the level of HSO in living biological cells with the low toxicity.
1
52, 8–13.
4 C. M. Yu, M. Luo, F. Zeng and S. Z. Wu, Anal. Methods,2012,
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Conflicts of interest
There are no conflicts to declare.
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View Article Online
DOI: 10.1039/C9NJ03997J
TOC (Table of Contents)
COOH
COOH
HSO3-
0
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N
O
N
O
Ha'
Ha
HO3S
Hb'
O
OH
Hb
SO3H
Q5
Q5'
A "naked-eye" fluorescent probe based on xanthenes was obtained.