A highly selective and ratiometric fluorescence probe for the detection of Hg2+ and pH change based on coumarin in aqueous solution

Article abstract of DOI:10.1016/j.tet.2014.09.082

A simple but highly selective coumarin-based fluorescence probe 1, 8-(1,3-dithiane)-7-hydroxycoumarin was designed and synthesized for both the ratiometric detection of Hg²⁺ and the on-off response to pH change in aqueous solution. The sensor detected Hg²⁺ selectively via Hg²⁺-promoted thioacetal deprotection reaction within five minutes and reflected pH in the range from 7.8 to 11.9 as a result of the equilibrium between weak-fluorescent acid form and strong-fluorescent base form. In addition, the probe has an excellent selectivity towards Hg²⁺ over other competitive metal ions for biomedical and environmental applications. The sensing behavior of our probe was studied by UV-visible absorption spectra and fluorescence spectra.

Full text of DOI:10.1016/j.tet.2014.09.082

Tetrahedron 70 (2014) 8914e8918
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Tetrahedron
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A highly selective and ratiometric fluorescence probe for the
detection of Hg2þ and pH change based on coumarin in aqueous
solution
,
Shouhai Yang a, Wenge Yang b, Qirun Guo a, Tuan Zhang a, Keyi Wu a, Yonghong Hu a
*
a College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, No. 200, North Zhongshan Road, Nanjing 210009, China
b Jiangsu Engineering Technology Research Center of Polypeptide Pharmaceutical, Nanjing 210038, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 July 2014
Received in revised form 13 September 2014
Accepted 26 September 2014
Available online 2 October 2014
A simple but highly selective coumarin-based fluorescence probe 1, 8-(1,3-dithiane)-7-hydroxycoumarin
was designed and synthesized for both the ratiometric detection of Hg2þ and the oneoff response to pH
change in aqueous solution. The sensor detected Hg2þ selectively via Hg2þ-promoted thioacetal de-
protection reaction within five minutes and reflected pH in the range from 7.8 to 11.9 as a result of the
equilibrium between weak-fluorescent acid form and strong-fluorescent base form. In addition, the
probe has an excellent selectivity towards Hg2þ over other competitive metal ions for biomedical and
environmental applications. The sensing behavior of our probe was studied by UVevisible absorption
spectra and fluorescence spectra.
Keywords:
Coumarin
Thioacetal
Ó 2014 Elsevier Ltd. All rights reserved.
Fluorescence quenching
Hg2þ
pH
1. Introduction
photostability, and derivatizable backbone.7 It was reported that the
electrondonatinggroupsin the 7-position and electron-withdrawing
The detection of ions and molecules by chemosensors is sig-
nificant for medical diagnostics, analytical chemistry, and bio-
technology.1 Hg2þ is known as a severe environmental pollutant
because of its durability, which can transfer through biological
membranes and accumulate in body, causing damage to health.2
Considerable efforts have been devoted to the development of
Hg2þ-selective chemosensors. Among the various chemosensors
for Hg2þ, fluorescent chemosensors present more advantages: high
sensitivity, low cost, and easy detection.3
The detection of pH is essential for many processes in agricul-
ture, industry, and human health.4 As common cations, protons
play key roles both in metabolism and cellular events, such as cell
growth, calcium regulation, enzyme activities, chemotaxis, and so
on.5 In addition, certain diseases, such as cancers and Alzheimer’s
disease, have been related to abnormal pH values.6 Therefore, it is
important to be able to measure the intracellular pH promptly and
precisely. Interestingly, probe 1 is also sensitive to pH, thus it can be
used as a fluorescence probe to measure intracellular pH.
groups in the 3-position can be able to enhance the fluorescence in-
tensity of coumarins based on an intramolecular charger transfer
(ICT) process.8 In another word, inhibition of such an ICT process may
quench the fluorescence.9 This fluorescence quenching mechanism is
involved in our design strategy for the detection.
To the best of our knowledge, there were really few probes with
two functions for both metal ions and pH changes.10 In this work, we
described a new probe for rapid, sensitive, selective detection of Hg2þ
and pH. Our design strategy for the detection took advantage of the
well-known specific Hg2þ-promoted thioacetal deprotection re-
action,11 which is also a typical off-on emission mode manipulated by
acid/base. Thus, probe 1 can be used as one excellent chemosensor for
the ratiometric detection of Hg2þ and the oneoff response to pH.
2. Experimental
2.1. Materials and instruments
Coumarin is one of the most favorable fluorophore groups to de-
velop fluorescent probes because of its high quantum yields, high
7-Hydroxycoumarin was purchased from Aladdin Reagent Co.,
Ltd and used without purification. Organic solvents purchased from
Shanghai Lingfeng chemical reagent Co., Ltd. were all analytical
reagent grade. The salts used in stock solutions of metal ions were
NaNO3, Ca(NO3)2, CdCl2$H2O, Co(NO3)2$6H2O, Mg(NO3)2, CuSO4,
* Corresponding author. Tel./fax: þ86 25 83587108; e-mail addresses:
yonghonghu11@126.com, 351417841@qq.com (Y. Hu).
http://dx.doi.org/10.1016/j.tet.2014.09.082
0040-4020/Ó 2014 Elsevier Ltd. All rights reserved.

S. Yang et al. / Tetrahedron 70 (2014) 8914e8918
8915
Zn(NO3)2$2H2O, Hg(ClO4)2, Ni(NO3)2$6H2O, Pb(NO3)2, AgNO3,
CrCl3$6H2O, and FeCl3. All of them were of analytical reagent grade
and used without purification. Water used in experiment was
double distilled water. The UVevisible spectra measurements were
performed with a UVevisible spectrophotometer (Unico UV-
2800H) equipped with 1.0 cm quartz cells. Fluorescence was
recorded on a spectrofluorimeter (Shimadzu RF-5301PC) equipped
with 1.0 cm quartz cell. pH values between 4.57 and 12.95 were
measured by a PHSJ-4A pH meter (Leichi Ltd., China).
prepared in ultra-pure water. Stock solutions of 1 (10
m
M) were
prepared in ethanol aqueous solution (EtOH/H2O¼8:2, v/v). Solu-
tions in the pH range of 4.57e12.95 were prepared by adding the
appropriate amounts of NaOH and H3PO4. The fluorescence spectra
were obtained 5 min after various analytes addition at room tem-
perature by excitation at 385 nm.
3. Results and discussion
3.1. Mechanism of probe 1
The thioacetal group in the 8-position, will be hydrolyzed into
aldehyde when Hg2þ was added into the system (Scheme 2). The
resulting aldehyde forms an intramolecular hydrogen bond with
neighboring hydroxyl, thus the original ICT process from the
7-position group to the 8-position group will be inhibited,12
quenching the fluorescence of probe 1. In addition, probe 1 with
pH-sensitive functional group exists in two forms in different pH
solutions: one is the weak-fluorescent acid form, and another is the
strong-fluorescent base form (Scheme 2). The equilibrium between
these two forms can be adjusted by changing the pH. We can take
advantage of the absorption and emission properties to judge the
pH value.
2.2. Synthesis of probe
2 . 2 . 1. S y n t h e s i s o f p r o b e
hydroxycoumarin). 1,3-Dimercaptopropane (108 mg, 1 mmol) and
boron trifluoride-diethyl etherate (20 L) were added to a solution
1 ( 8 - ( 1, 3 - d i t h i a n e ) - 7 -
m
of 2 in dichloromethane (5 mL). The yellow solution was stirred
overnight at room temperature. After reaction completed, the
crude product was purified by flash chromatography (petroleum/
ethyl acetate¼2:1, v/v) to afford 1 (85.7 mg, yield: 85%). 1H NMR
(400 MHz, CDCl3):
d
¼7.63 (d, J¼9.5 Hz, 1H), 7.57 (s, 1H), 7.34 (d,
J¼8.6 Hz, 1H), 6.87 (d, J¼8.6 Hz, 1H), 6.26 (d, J¼10.9 Hz, 2H),
3.31e3.09 (m, 2H), 2.93 (dt, J¼7.1, 3.8 Hz, 2H), 2.35e2.14 (m, 1H),
2.09e1.84 (m, 1H). 13C NMR (400 MHz, CDCl3)
d 159.75, 159.13,
150.70, 143.02, 128.25, 113.89, 111.65, 111.22, 109.99, 37.98, 30.21,
23.58. ESI-MS: [MþH]þ 281.04 (calcd¼281.03), [MþNa]þ 303.01
(calcd¼303.01). Elem. Anal. Calcd. for 1: C 55.69, H 4.31, O 17.12, S
22.87. Found: C 55.57, H 4.42, O 17.04, S 22.97.
Detailed procedures are described in Scheme 1 and character-
izations are shown in ESI.
3.2. Fluorescence detection of Hg2D
3.2.1. Fluorescence spectral properties. When the excitation was set
to 385 nm, probe 1 showed a maximum fluorescence emission peak
at 455 nm (Fig. 1). It is noticeable that the stokes shift reaches up to
70 nm, which can effectively overcome background fluorescence,
CHO
HO
O
O
HO
O
O
AcO
O
O
acetic anhydride
hexamethylenetetramine
TFA, reflux, 9h
pyridine, CH2Cl2
rt, 10h
3
4
2
S
S
1,3-dimercaptopropane
HO
O
O
BF3·Et2O, CH2Cl2
rt, overnight
1
Scheme 1. Synthesis of probe 1.
HgClO4
OH2
HgClO4
S
S
S
S
S
S
S
S
H
H+
Hg(ClO4)2
-O
O
HO
O
O
O
HO
HO
O
O
HO
O
O
OH-
base form
acid form
O
H
CHO
O
O
O
O
O
Scheme 2. The response mechanism of probe 1 to Hg2þ and pH.
2.3. Preparation of stock solutions
such as the auto-fluorescence from native cellular molecules. Probe
1 showed a maximum fluorescence emission peak at 455 nm
without Hg2þ. However, upon addition of different concentrations
of Hg2þ, the peak at 455 nm decreased with increasing the
Stock solutions (10 mM) of the anions of Naþ, Ca2þ, Cd2þ, Co2þ
,
Mg2þ, Cu2þ, Zn2þ, Hg2þ, Ni2þ, Pb2þ, Agþ, Cr3þ, and Fe3þ were

8916
S. Yang et al. / Tetrahedron 70 (2014) 8914e8918
1000
950
900
850
800
750
700
650
600
550
455nm Emission
1000
800
600
400
200
0
500
0
10
20
30
40
300 350 400 450 500 550 600 650 700 750
Time (min)
Wavelength(nm)
Fig. 3. Fluorescence emission time-course of probe 1 (10
Hg2þ in EtOH/H2O (v/v¼8/2).
m
M) at pH¼7 with 1 equiv
Fig. 1. The fluorescence spectrum of probe 1 at pH¼7.
probe 1 toward Hg2þ is unaffected by the presence of the other
possible contaminating metal ions, demonstrating that probe 1
could meet the selective requirements for biomedical and envi-
ronmental applications.
concentration of Hg2þ (Fig. 2A). Moreover, there was a good line-
arity obtained between the fluorescence intensity (a.u.) with the
Hg2þ concentration ranging from (0e3)*10ꢀ5 mM (Fig. 2B). The
linear equation is y¼ꢀ185.416xþ850.33 (R2¼0.9957), where x, y
represents the concentration of Hg2þ (mM) and the fluorescence
intensity, respectively. All properties mentioned above indicate the
capability of probe 1 for quantitatively detecting Hg2þ by a ratio-
metric fluorescence method.
3.3. UVevisible and fluorescence response to pH
The UVevisible and fluorescence titrations were performed at
a probe concentration of 10
mM. As shown in Fig. 5A, probe 1
1000
1000
800
700
800
A
600
B
800
400
200
y=-185.416x+850.33
0
600
R2=0.9957
600
500
400
300
0
5
10
15
20
25
2+
Hg /uL
400
200
0
400
450
500
550
600
0.0
0.5
1.0
1.5
2+]
2.0
2.5
3.0
5
Wavelength (nm)
Hg /10 mM
Fig. 2. A: Fluorescence response (
l
¼455 nm) of 3 mL (10
m
M) probe 1 at pH¼7 to Hg2þ volume of 0, 0.15, 0.3, 0.6, 0.9, 1.2, 1.5, 1.8, 2.4, 3, 6, 9, 12, 15, 18, and 21
mL (10 mM) in EtOH/
H2O (v/v¼8/2), respectively. B: Fluorescence intensity as a function of Hg2þ concentration.
1200
1000
800
600
400
200
0
3.2.2. Influence of reaction time. In order to understand how 1 e
Hg2þ system changed with time, the time-dependent fluorescence
spectra of probe 1 in the presence and absence of Hg2þ were
studied. As shown in Fig. 3, upon addition of Hg2þ, the fluorescence
intensity (a.u.) reduced gradually along with the time consuming.
The minimum was reached after about 5 min and then the fluo-
rescence intensity (a.u.) remains stable. Thus, probe 1 could be used
Probe 1 + metal ion
Probe 1 + metal ion + Hg2+
for rapid detection of Hg2þ
.
3.2.3. Selectivity. In order to enable the probe to work in more
complex systems, we studied the selectivity and competition under
the same condition, which is one of the most important perfor-
mance indexes for the detection of Hg2þ 13 Naþ, Ca2þ, Cd2þ, Co2þ
,
.
Na Ca Cd Co Mg Cu Zn Hg Ni Pb Ag Cr Fe
Mg2þ, Cu2þ, Zn2þ, Hg2þ, Ni2þ, Pb2þ, Agþ, Cr3þ, and Fe3þ were se-
lected as competing ions and introduced into the detecting system.
In Fig. 4, the black bar portion shows that our proposed probe ex-
hibits high selectivity toward Hg2þ over other metal ions. Fur-
thermore, the red bar portion indicates that the response of the
Metal ions
Fig. 4. Fluorescence spectral data of 3 mL (10
m
M) probe 1 at pH¼7.0 with 4 equiv
metal ions Naþ, Ca2þ, Cd2þ, Co2þ, Mg2þ, Cu2þ, Zn2þ, Hg2þ, Ni2þ, Pb2þ, Agþ, Cr3þ, and
Fe3þ in EtOH/H2O (v/v¼8/2).

S. Yang et al. / Tetrahedron 70 (2014) 8914e8918
8917
0.30
0.25
0.20
0.15
0.10
0.05
0.00
3.5
3.0
B
A
2.5
2.0
1.5
1.0
0.5
0.0
y=0.7930x-6.2822
2
R =0.9911
300
325
350
375
400
425
6
7
8
9
10
11
12
13
Wavelength(nm)
pH
Fig. 5. A: Changes of the UVevisible absorption spectra of probe 1 (10
probe 1 as a function of pH.
mM) with pH increase from 4.57 to 12.95 in EtOH/H2O (v/v¼8/2). B: Absorption intensity ratio A383/A328 of
1200
1200
1200
1000
800
A
B
1000
1000
800
600
400
200
0
600
400
800
200
0
4
6
8
10
12
14
pH
600
400
200
0
y=425.952x-3263.63
2
R =0.9823
400
450
500
Wavelength (nm)
550
600
8
10
pH
12
Fig. 6. A: Changes of the fluorescence intensity (a.u.) of probe 1 (10
a function of pH.
mM) with pH increasing from 4.57 to 12.95 in EtOH/H2O (v/v¼8/2). B: Fluorescence intensity (a.u.) of probe 1 as
displayed typical pH-dependent UVevisible absorption behavior.
As pH increased from 4.57 to 12.95, the absorption band centered at
328 nm decreased gradually while a new peak simultaneously
appeared at 383 nm. The obvious isosbestic point at 347 nm was
observed from Fig. 5A, which indicates the presence of acidebase
equilibrium between the two forms of probe 1 (Scheme 2). What’s
more, the ratio of fluorescence intensity between 383 nm and
328 nm (F383/F328) changes from 0.164 to 3.225 (R¼19.7 fold). There
was a linearity between the F383/F328 and pH ranging from 8.02 to
11.91, and the linear equation is y¼0.7930xꢀ6.2822 (R2¼0.9911),
where x, y represents pH and the fluorescence intensity, re-
spectively (Fig. 5B).
The fluorescence spectra of probe 1 at various pH values were
shown in Fig. 6A. With the pH value of test solution increasing,
a significant fluorescence turn-on response is noted in the emission
spectra. A good linearity (R2¼0.982) between fluorescence and pH
ranging from 7.84 to 9.95 was obtained according to fluorescence
titration (Fig. 6B). The analysis of fluorescence intensity changes as
a function of pH by using the HendersoneHasselbalch-type mass
action Eq. 1 yielded a pKa of 8.92:
advantages as follows: (1) Probe 1 can be used for the ratiometric
detection of Hg2þ within 5 min; (2) The stokes shift reaches up to
70 nm, which can effectively overcome background fluorescence; (3)
Probe 1 is highly selective for Hg2þ over the other competing metal
ions; (4) In addition to the detection of Hg2þ, it can also serve as an
outstanding pH probe (pH 7.8e11.9) that might work for basic
organelles.
Acknowledgements
This work was financially supported by the National High
Technology Research and Development Plan (2011AA100901), the
Science and Technology Support ProgramdAgriculture Part
(BE2013442), the Joint Innovation and Research Funding-
Prospective Joint Research Projects (SBY201320243), the Doctoral
Fund Class Topics (2011322111000).
Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.tet.2014.09.082.
lg½ðFmax ꢀ FÞ=ðF ꢀ FminÞꢁ ¼ pKa ꢀ pH
(1)
where F is the fluorescence emission intensity at 455 nm. It means
that probe 1 can serve as a functional pH probe that might work for
basic organelles.
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€
In summary, we have designed and synthesized a highly selective
and ratiometric dual-functional fluorescence probe
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