A fully-aqueous red-fluorescent probe for selective optical sensing of Hg2+ and its application in living cells

Article abstract of DOI:10.1016/j.dyepig.2016.03.028

A new red-fluorescent mercury ion sensor material is designed and synthesized, which is composed of a tweezer-shaped hydrophilic probe containing bifurcated soft-base atoms N and S coupled with 2-dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran (TCF) unit. By virtue of the strong electron-accepting nature of TCF unit (as a push-pull chromophore), this designed sensor material can selectively detect Hg²⁺ over various tested metal ions in a 100% aqueous medium via naked-eye and photoluminescence (PL) observations. Theoretical and time-resolved photoluminescence measurements further confirmed the selectivity and reversibility of the probe towards Hg²⁺ via intramolocular charge transfer mechanism in this sensor material. Moreover, the living cell tests by confocal fluorescence images of this sensor material towards Hg²⁺ were also investigated. Finally, distinguished absorption changes and fluorescence quenching spectral appearances allowed us to present the selective optical indicator of Hg²⁺ via TCF moiety for the first time.

Full text of DOI:10.1016/j.dyepig.2016.03.028

Accepted Manuscript
2+
A fully-aqueous red-fluorescent probe for selective optical sensing of Hg and its
application in living cells
Murali Krishna Pola, Mandapati V. Ramakrishnam Raju, Chein-Ming Lin, Raghunath
Putikam, Ming-Chang Lin, Chandra Prakash Epperla, Huan-Cheng Chang, San-Yuan
Chen, Hong-Cheu Lin
PII:
S0143-7208(16)30087-0
10.1016/j.dyepig.2016.03.028
DYPI 5153
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 21 January 2016
Revised Date: 13 March 2016
Accepted Date: 14 March 2016
Please cite this article as: Pola MK, Ramakrishnam Raju MV, Lin C-M, Putikam R, Lin M-C, Epperla CP,
Chang H-C, Chen S-Y, Lin H-C, A fully-aqueous red-fluorescent probe for selective optical sensing of
2+
Hg and its application in living cells, Dyes and Pigments (2016), doi: 10.1016/j.dyepig.2016.03.028.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical Abstract

ACCEPTED MANUSCRIPT
2
+
A fully-aqueous red-fluorescent probe for selective optical sensing of Hg and its
application in living cells
a
a
a
b
Murali Krishna Pola, Mandapati V. Ramakrishnam Raju, Chein-Ming Lin, Raghunath Putikam,
b
c
c
a
a,*
Ming-Chang Lin, Chandra Prakash Epperla, Huan-Cheng Chang, San-Yuan Chen, Hong-Cheu Lin
a
Department of Materials Science and Engineering, National Chiao Tung University, Hsinchu 300,
Taiwan.
b
Center for Interdisciplinary Molecular Science, Department of Applied Chemistry, National Chiao
Tung University, Hsinchu 300, Taiwan.
c
Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei 106, Taiwan.
Supporting Information Placeholder
KEYWORDS: aqueous medium; push-pull chromophore; ICT, living cell.

ACCEPTED MANUSCRIPT
Abstract
A new red-fluorescent mercury ion sensor material is designed and synthesized, which is composed of a
tweezer-shaped hydrophilic probe containing bifurcated soft-base atoms N and S coupled with 2-
dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran (TCF) unit. By virtue of the strong
electron-accepting nature of TCF unit (as a push-pull chromophore), this designed sensor material can
2
+
selectively detect Hg over various tested metal ions in a 100% aqueous medium via naked-eye and
photoluminescence (PL) observations. Theoretical and time-resolved photoluminescence measurements
2
+
further confirmed the selectivity and reversibility of the probe towards Hg via intramolocular charge
transfer mechanism in this sensor material. Moreover, the living cell tests by confocal fluorescence
2
+
images of this sensor material towards Hg were also investigated. Finally, distinguished absorption
changes and fluorescence quenching spectral appearances allowed us to present the selective optical
2
+
indicator of Hg via TCF moiety for the first time.
2

ACCEPTED MANUSCRIPT
1
. Introduction
Despite the vital roles of many metal ions in sustaining life, it is required to have precise control
to maintain an ecological balance in living cells. However, the accumulations of heavy metal
ions in environment within the approved environmental protection agency limits are highly
2
+
challenging. Among all heavy metal ions, Hg is extremely toxic owing to its lipophilic nature
of organo-mercury (CH HgX) under environmental aqueous media [1-3]. Mercury ingestion
3
could cause fatal damage to human central nervous and endocrine systems [4-5]. Due to its
unique chemical properties and lethal effects on environment along with living organism, in
recent years rapid, selective and sensitive detections of mercury ions are intriguing targets [6-10].
2
+
Consequently, myriads of chemosensors towards sensitive Hg detections based on chelation-
enhanced fluorescence [11-20] and fluorometric chemodosimeter [21-30] approaches have long
been designed and discussed. However, conventionally most of those chemosensing probes in
2
+
selective fluorometric detections of Hg are suffered owing to a fact that fluorescence intensities
of probe molecules could be altered by photo bleaching, concentration variations of probe
2
+
ensembles and heterogeneities of surrounded microenvironments. Thus, developing Hg
selective optical indicators are highly demanding.
An exceptional electron-accepting capability of 3-cyano-4,5,5-trimethyl-2,5-dihydrofuran
TCF) make it as an excellent moiety for the applications of nonlinear optoelectronics [31-33].
(
However, its unique push-pull chromophoric nature stimulated scientists recently in designing
novel red fluorophores for the applications of bioimaging- [34-37], pH sensing- [38,39], and
chemosensing- [40-43] platforms, as well as dye sensitized solar cells (DSSCs) [44]. Liao et al.
have demonstrated negative photo-chromism of a TCF chromophore [45]. Recently, Yu et al.
have presented a near-infrared TCF-based probe for the colorimetric and ratiometric detection of
3

ACCEPTED MANUSCRIPT
2
+
2
SO [46]. Cho and co-workers have reported a selective optical TCF-based indicator for Hg via
2
+
an Hg mediated intramolecular cyclization reaction of ethynyl phenols [47]. However, to the
best our knowledge the selective and sensitive optical indicator based on the TCF moiety
2
+
towards Hg via chelation induced large hypsochromic shifted chemosensing ensemble has
never been reported.
2
+
Herein, we demonstrate the first selective optical indicator T2 towards Hg , which was
constructed by bridging an electron-withdrawing TCF moiety with a water soluble and well-
known thiophilic ligand [48,49]. The utilization of the TCF moiety in this work as a strong
electron-delocalizing unit via its flexible push-pull feature leads to an unprecedented sensing
ensemble T2. It displays a 100% water solubility with surprisingly large blue-shifted
2
+
photophysical properties of the selective detection towards Hg via a H-type self-association
and alterable ICT of the TCF moiety and yields a better live cell permeability as shown in
2
+
scheme 1. Prominently, the selective detection of probe T2 towards Hg was completely
reversible upon the addition of EDTA, which proves its practical utility.
2
. Experimental Section
2
.1. Materials
All chemicals and solvents were used are reagent grades and HPLC grades respectively and
were purchased from Aldrich, ACROS, Fluka, TCI, TEDIA, and Lancaster Chemical Co. All
chemicals were used without any further purification. Anhydrous solvents were obtained by
passing through activated alumina column purification system, further dried by standard drying
procedures. Solvents were degassed by freeze/thaw/pump cycle technique prior to use. Thin
layer chromatographies (TLC) were performed on glass plate coated with silica 60 F24 (Merck).
4

ACCEPTED MANUSCRIPT
2
The plates were visualized using ultra-violet light (256 nm) and developed using I chamber.
Flash chromatographies were performed on Merck silica gel 60 (230-400 mesh) under pressure
using desired solvents.
2
.2. General Characterization Methods
All reactions and operations were carried out under an atmosphere of inert argon or nitrogen
1
13
using Schlenk techniques unless otherwise stated. H NMR and C NMR spectra were recorded
on Bruker DRX-300 Avance series or on a Varian Inova 400, Inova 500, and Inova 600 Series
1
13
(
H: 300, 400, 500, and 600 MHz; C: 75, 100, 125, and 150 MHz) at a constant temperature of
2
98 K. Chemical shifts were reported in parts per million from low to high field and referenced
1
13
to residual solvent (CDCl , DMSO-d : H δ = 7.26, 2.49 ppm and C δ = 77.23, 39.52 ppm,
3
6
respectively). Coupling constant (J) were reported in Hertz (Hz). UV-Vis spectra were recorded
on the Jasco UV-600 spectrophotometer using 1 cm quartz cuvette. Fluorescence measurements
were conducted with HITACHI 7000 Series Spectrophotometer. All emission and excitation
spectra were corrected for the detector response and the lamp output. Melting points were
determined using a Fargo MP-2D apparatus and are uncorrected. Elemental analyses were
conducted on HERAEUS CHN-OS RAPID elemental analyzer. Time resolved
photoluminescence (TRPL) spectra were measured using a home built single photon counting
system with excitation from a 400 nm diode laser (Picoquant PDL-200, 50 ps fwhm, 2 MHz).
The signals collected at the excitonic emissions of all sample solutions were connected to a time-
correlated single photon counting card (TCSPC, Picoquant Timeharp 200). The emission decay
data were analyzed for T2 and complex T2-Hg with biexponential kinetics, from which two
decay components were derived; the lifetime values of (τ1, τ2) and pre-exponential factors (A1,
A2) were determined. Confocal imaging was carried out using Leica TCS SP8 confocal
5

ACCEPTED MANUSCRIPT
fluorescence microscope, confocal fluorescence imaging with using 60 × times oil objective.
Semi-empirical PM3 calculations were calculated using Gaussian-09 suite.
2
.3. Cell Culture and Imaging
The human cervical cancer cell line (HeLa cells) were seeded onto cover slips at a
concentration of (2 × 105 cells/mL) and cultured in Dulbecco’s Modified Eagle’s Medium
o
(
DMEM) and 10% fetal bovine serum in an incubator (37 C, 5% CO , and 25% O ). After 30 h,
2
2
the cover slips were rinsed slightly 3 times with PBS to remove the media and then cultured in
PBS for later use. In view of imaging procedure, initially cells were incubated with 5 µM of
probe T2 alone for 20 min at 37°C and observed under microscope and then again the samples
were treated with HgCl (10 µM) incubated for 20 min and then again observed under
2
microscope and then the samples were treated with EDTA (10 µM) incubated for 20 min and
moved to the confocal stage. All the samples were slightly rinsed for 3 times with PBS buffer
before observing them under the microscope. All the cell images were obtained with Leica TCS
SP8 confocal fluorescence microscope using 60 × times oil objective.
2
.4. Stock Solutions
2
Standard solution of probe T2 (100 mM) were prepared in double distilled water and HgCl ,
other metal ions stock solutions with concentration of (10 mM) were prepared, respectively in
water. Before the titrations probe and analytes were diluted to their desired volumes.
Synthesis
of
Diethyl2,2'-((((4-formylphenyl)azanediyl)bis(ethane-2,1-
diyl))bis(sulfanediyl))diacetate-(4). In a 25 mL dried round bottomed flask, sodium (0.48 g, 20
mmol) was added to anhydrous ethanol (12 mL). After sodium was dissolved completely, ethyl-
2
4
-mercaptoacetate (2.40 g, 23 mmol) was added dropwise. The mixture was stirred for 2 h at
0°C. Then a solution of 4-(bis (2-chloroethyl) amino) benzaldehyde (3) (2.24 g, 9 mmol) in
6

ACCEPTED MANUSCRIPT
DMF (5 mL) was added. The stirring was continued for another 3 h. Water (20 mL) was added to
the residual and extracted with dichloromethane (3 × 10 mL). The combined organic phase was
washed twice with water and dried over anhydrous magnesium sulfate. The solvent was removed
by evaporation and dried in vacuum, yielding a yellow oil of compound 4 (3.58 g, 95%). The
1
13
1
structure of 4 was confirmed by H NMR and C NMR, which are shown as follows: H NMR
500 MHz, CDCl3): δ 9.58 (s, 1H), 7.61 (d, J = 8.5 Hz, 2H), 6.63 (d, J = 9.0 Hz, 2H), 4.07 (q, J
14.2 Hz, 4H), 3.56 (t, J = 7.5 Hz, 4H), 3.17 (s, 4H), 2.76 (t, J = 7.5 Hz, 4H), 1.16 (t, J = 7.0
(
=
1
3
Hz, 6H); C NMR (125 MHz, CDCl3): δ 190.0, 170.1, 151.3, 132.0, 125.2, 110.7, 61.3, 50.23,
3
3.1, 28.9, 13.8.
Synthesis of (E)-Diethyl 2,2'-((((4-(2-(4-cyano-5-(dicyanomethylene)-2,2-dimethyl-2,5-
dihydrofuran-3-yl)vinyl)phenyl)azanediyl)bis(ethane-2,1-diyl))bis(sulfanediyl))diacetate-
(T1). Diethyl 2,2'-((((4-formylphenyl)azanediyl)bis(ethane-2,1-diyl))bis(sulfanediyl))diacetate-
(4) (1.24 g, 3 mmol), 2-(3-cyano-4,5,5- trimethylfuran-2(5H)-ylidene)propanedinitrile (TCF)
(0.6 g, 3 mmol), and ammonium acetate (0.23 g, 3 mmol) were stirred overnight in the dark at
room temperature in a mixture of THF (10 mL) and EtOH (2.5 mL). The solution rapidly turns
from pale yellow to reddish-orange. The mixture is diluted in water, extracted with ethyl acetate,
washed with brine and dried over anhydrous MgSO . Evaporation of the solvents under reduced
4
pressure yields a crude product that is purified by column chromatography on silica gel using
progressively more polar 2:1 to 3:1 ethyl acetate: hexanes as the mobile phase to afford the
1
desired T1 red solid flakes (1.28 g, 72%). The structure of T1 was confirmed by H NMR and
1
3
1
C NMR, which are shown as follows: H NMR (500 MHz, CDCl ): δ 7.58 (d, J = 16.0 Hz,
3
1
1
H), 7.54 (d, J = 8.5 Hz, 2H), 6.76 (d, J = 8.5 Hz, 2H), 6.74 (d, J = 16.0 Hz, 1H), 4.18 (q, J =
4.5 Hz, 4H), 3.67 (t, J = 7.0 Hz, 4H), 3.24 (s, 4H), 2.86 (t, J = 7.0 Hz, 4H), 1.73 (s, 6H), 1.26
7

ACCEPTED MANUSCRIPT
1
3
(
t, J = 7.0 Hz, 6H); C NMR (125 MHz, CDCl
3
): δ 176.1, 174.3, 170.1, 151.0, 148.0, 132.3,
1
22.6, 112.5, 112.3, 111.8, 111.2, 109.4, 97.0, 95.0, 61.5, 50.6, 33.5, 29.4, 26.6, 14.1; HRMS-
+
ESI (m/z) calcd for C H N O S : 595.2049 [M+H] ; found 595.2045; Anal. calcd for
3
0
35
4
5 2
C H N O S : C, 60.58; H, 5.76; N, 9.42; found C, 60.51; H, 6.22; N, 9.46 (%).
30
34
4
5 2
Synthesis of Lithium (E)-2,2'-((((4-(2-(4-cyano-5-(dicyanomethylene)-2,2-dimethyl-2,5-
dihydrofuran-3-yl)vinyl)phenyl)azanediyl)bis(ethane-2,1-diyl))bis(sulfanediyl))diacetate-
(T2). A solution of T1-ester (416 mg, 0.7 mmol) in a 1:1 tetrahydrofuran/methanol mixture (50
mL) under a nitrogen atmosphere was cooled to 0°C and treated with 15 equiv. of lithium
hydroxide. The resulting mixture was stirred for 2 days under nitrogen. Once hydrolysis was
completed based on TLC THF-MeOH, the solvent was evaporated under reduced pressure and
directly absorbed on short pad of silica and eluted in DCM-THF from 3:1 to 1:3 to get final T2
1
13
salt. (323 mg, 84%). The structure of T2 was confirmed by H NMR, C NMR, and elemental
1
analysis, which are shown as follows: H NMR (400 MHz, DMSO-d ): δ 7.90 (d, J = 16.0 Hz,
6
1
H), 7.75 (d, J = 9.2 Hz, 2H), 6.89 (d, J = 16.0 Hz, 1H), 6.85 (d, J = 8.8 Hz, 2H), 3.71 (t, J = 7.2
13
Hz, 4H), 3.27 (s, 4H), 2.81 (t, J = 7.2 Hz, 4H), 1.74 (s, 6H); C NMR (100 MHz, DMSO-d
6
): δ
1
5
77.3, 135.5, 151.5, 149.2, 133.1, 122.3, 113.5, 112.6, 112.2, 112.0, 108.6, 98.3 (2C), 92.5, 51.1,
-
0.2, 30.4, 28.7, 25.6; MS-ESI (m/z) calcd for C H N O S : 536.1 [M-2Li] ; found 536.7;
26
24
4
5 2
Anal. calcd for C H Li N O S : C, 56.73; H, 4.39; N,10.18; found C, 55.95; H, 4.44; N, 9.91
2
6
24
2
4
5 2
(%).
3
. Results And Discussion
3
.1. Synthesis
8

ACCEPTED MANUSCRIPT
The synthetic route for T2 was demonstrated in Scheme 2. Compounds 2,2'-
phenylazanediyl)diethanol (2) [50], 4-(bis(2-chloroethyl)amino)benzaldehyde (3) [51], and 2-
3-cyano-4,5,5-trimethylfuran-2(5H) ylidene)malononitrile(TCF) [50] were prepared according
(
(
to literature procedures. Compound 3 was further reacted with 2-mercaptoacetic acid ethyl ester
to afford diester 4 in 95% yield. Coupling of 4 and TCF gave T1 in 72% yield. Ester hydrolysis
of T1 under basic conditions proceeds quantitatively to gave T2. The yielded compounds were
characterized by using different spectroscopic methods, and the spectral data was shown in
supporting information (Figs. S4-S9).
3
.2. UV-Visible and Fluorescence Measurements of Probe T2
Thanks to the lipophilic thiolate unit of T2, it possessed a complete water solubility, so we
primarily assessed spectroscopic properties of probe T2 (5 µM) in pure water, which showed a
strong UV-absorption band cantered at 575 nm with purple color and a red-fluorescent band at
6
50 nm as shown in Fig. S1a and S1b, respectively. Interestingly, T2 demonstrated excellent
2
+
sensor selectivity in both UV-Vis and photoluminescence (PL) spectra upon the addition of Hg ,
where the UV-absorption maximum was blue-shifted from 575 nm (purple color) to 405 nm
(color less) and the red-fluorescence of T2 was quenched as shown in Figures 1a and 1b,
respectively. Furthermore, we screened both UV-Vis and PL changes of probe T2 towards
+
+
+
+
+
+
2+
2+
various metal ions, all of the other metal ions, including Li , Na , K , Cs , Cu , Ag , Mg , Ca ,
2
+
2+
2+
2+
2+
2+
2+
2+
2+
2+
l3+
3+
3+
3+
Ba , Cu , Zn , Ni , Fe , Co , Pb , Cd , Mn , Ag , A , Cr , Fe , and Eu , could not
induce considerable fluorescent changes.
By considering the vivid spectroscopic clue and to evaluate quantitative analysis of probe T2,
we further measured both UV-Vis and PL changes of T2 (5 µM) by increasing the concentration
2
+
2+
of Hg from 0 to 10 µM. As shown in Fig. 2a, upon the addition of Hg a sharp decline in the
9

ACCEPTED MANUSCRIPT
absorption band at 575 nm and a simultaneous increase of newly blue-shifted band at 405 nm
were observed. However, the fluorescence emission band at 650 nm was gradually decreased
(
Fig. 2b) and the stern-volmer plot denoted a dynamic fluorescence quenching of probe T2
2
+
towards Hg . The unusual nature of absorption ratiometric calibration curve at A574/A405 and
the prominent hypsochromic shift in the absorption maxima of T2 could be ascribed to the H-
2
+
type self-association [53] of TCF moiety resulted from the coordination of Hg . Furthermore,
the observed isosbestic point at 468 nm clearly attested the aggregation of TCF moiety in the
aqueous medium owing to its strong electron-withdrawing nature. Conventionally, strong
electron acceptor dye molecules led to a depletion in quantum yields and imparts an unusual
7
-1
photophysical features [54,55]. The association constant was calculated to be 2.02 × 10 M and
1
:1 binding stoichiometry was estimated, which was based on the linear curve fitting for the
2
+
fluorescence emission changes of probe T2 supported by the Job’s plot in the presence of Hg
(
see Fig. 3).
.3 Screening of Other Metal Ions and Naked Eye Color Change
Moreover, the color changes of naked-eye observation and fluorescence emission of T2
towards metal ions are depicted in Fig. S2a and S2b. Moreover, to prove the specific selectivity
3
2
+
of probe T2 towards Hg by single and dual metal studies, the effects of commonly encountered
heavy metal ions on complex T2-Hg are evaluated as shown Fig. 4, where none of the tested
2
+
metal ions could interfere with the detection process of Hg . To gain the additional information
1
about the binding modes operating in these recognition processes, H NMR titration studies of
2
+
2+
probe T2 upon the addition of Hg as well as the reversibility of complex T2-Hg with EDTA
1
2+
are also performed. The stacked H-NMR spectra of probe T2, complex T2-Hg , and
2
+
T2+Hg(II)+EDTA are provided in Fig.5. Upon the addition of Hg to the solution of T2 in
1
0

ACCEPTED MANUSCRIPT
CD
3
CN-D
2
O, large down-field shifts and broadening of proton peaks are observed in the
aliphatic region of H , H , and H along with the aromatic region of H . These proton shifts of
a
b
c
d
2
+
2+
complex T2-Hg are attributed to the shielding effect of complexation of T2 and Hg .
1
However, a well-resolved spectrum of T2+Hg(II)+EDTA is almost identical with the H NMR
2
+
spectrum of T2 itself after the treatment of complex T2-Hg with EDTA. It confirms the
2
+
specific detection and reversibility of probe T2 towards Hg . To our delight, both on-off-on
fluorescence etiquettes and naked-eye color changes as shown in Fig. 6 were successfully
2
+
achieved via the alternative addition of Hg and EDTA up to 4 cycles.
3
.4. Time Resolved Photoluminescence Measurements
To substantiate the aforementioned photophysical observations, time resolved fluorescence
measurements were carried out for T2, T2-Hg, and recovered T2 probed at 650 nm (λ = 575
ex
nm). The original mono-exponential decay of T2 component with a life time of (τ1) 0.29 ns
greatly transformed [56,57] into a marked biexponential decay with life time values of τ1 = 0.32
2
+
ns (94.35%) and τ2 = 25.12 ns (5.65%) in the presence of Hg as shown in Fig. S3.
2
+
Biexponential life time components could be ascribed to the chelation of probe with Hg to
induce the intrinsic fluorescence quenching of TCF. However, the further addition of EDTA to
T2-Hg yielded a mono-exponential decay component with the life time value of 0.26 ns, which
evidenced the complete reversibility of probe T2.
3
.5. Theoretical Studies
To rationalize the coordination of Hg with T2 induced by ICT quenching process, it has been
2
+
carried out by using the density functional theory (DFT) method to further support the
experimental observation of ligand and metal interaction. All calculations were carried out using
the Gaussian 09 package [58]. The electronic ground state geometry optimization of T2 with
1
1

ACCEPTED MANUSCRIPT
charge -2 and neutral complex T2-Hg were carried out by using the M06-2X method. The basis
set used in this calculation is Lanl2dz, which includes the D95 double-ξ basis set for Hg along
with Hay and Wadt’s effective core potential (ECP) and the 6-31G(d) basis set for main group
elements. We also calculated the frontier molecular orbitals at the same level of theory. As
shown in Fig. 7, the most stable optimized geometries of T2 and complex T2-Hg along with its
2
+
marked bond lengths were attested the strong Hg coordination within the lipophilic thiolated
ligand. As anticipated, both of the ground states HOMO and HOMO-1 molecular distributions of
T2 were mainly resided on thiophilic ligand part as shown in Fig. 8a. Whereas, both LUMO and
LUMO+1 molecular orbital distributions were mainly vested on TCF moiety (see Fig. 8b)
donated strong intramolecular charge transfer distribution between aniline thiophilic ligand to
2
+
TCF moiety. However, with the addition of Hg both ground state HOMO and LUMO level
distributions existed on TCF and π-bridge, and next excited state HOMO-1 and LUMO+1 were
2
+
shifted to Hg coordinated thiophilic ligand part, which clearly denoted the effective obstruction
of ICT within complex T2-Hg, as shown in Fig. 9a and 9b, respectively. Based on these concrete
2
+
experimental and theoretical results, we inferred that probe T2 could selectively detect Hg
2
+
without any interference from common heavy metals and act as a useful Hg selective optical
indicator.
3
.6. Confocal Imaging
We next sought to apply current probe T2 in living cells, stimulated by its described
photophysical characteristics. As shown in Figs. 10a-10c denoted red fluorescence, the cells
were incubated with probe T2 (5 µM) alone for 20 min at 37°C. However, a noticeable
2
+
fluorescence quenching in Figs. 10d-10f was monitored in the cells after the treatment with Hg
10 µM). As shown in Figs. 10g-10i, the red fluorescence was restored in the cells upon further
(
1
2

ACCEPTED MANUSCRIPT
treatment with EDTA. Noticeable changes signified that probe T2 was cell membrane permeable
2
+
and capable of selective imaging of Hg in the living cells.
4
. Conclusions
In conclusion, we have developed a selective and sensitive red fluorescent probe for the
2
+
detection of Hg based on thiophilic ligand connected to a push-pull active TCF moiety under
00% aqueous medium. Exclusively, probe T2 showed a selective colorimetric response with
unprecedented and exceedingly large hypsochromic shift (170 nm) in absorption of T2 induced
1
2
+
by the aggregation of TCF coordinated with Hg in aqueous medium. Current probe showed a
2
+
stable colorimetric and ratiometric response towards Hg over a range of tested transition and
other metal ions, where the UV-absorption maximum was blue-shifted from 575 nm (purple
color) to 405 nm (color less) and the red-fluorescence emission band at 650 nm was quenched,
respectively. Furthermore, the optimized geometries of T2 and T2-Hg ensembles via the energy
minimized and density functional theory (DFT) methods fortified the intramolecular charge
transfer (ICT) mechanism between the probe and TCF unit. Pivotal confocal imaging of T2 and
T2-Hg in living cells also demonstrated that probe T2 could be favorable for biological
applications. Besides, invincible absorption changes and fluorescence quenching spectral
2
+
appearances allowed us to present the selective optical indicator of Hg via H-type aggregation
of TCF moiety for the first time.
Acknowledgements
1
3

ACCEPTED MANUSCRIPT
We thank the Ministry of Science and Technology (MOST) in Taiwan for the financial supports
of this project through MOST 103-2113-M-009-018-MY3 and MOST 103-2221-E-009-215-
MY3.
References
[1] Nolan EM, Lippard SJ. Tools and tactics for the optical detection of mercuric ion. Chem Rev
2
008; 108: 3443–80.
[2] Boening DW. Ecological e ects, transport, and fate of mercury: a general review
Chemosphere 2000; 40: 1335–51.
[3] Ullrich SM, Tanton TW, Abdrashitova SA. Mercury in the aquatic environment: a review of
factors affecting methylation. Crit Rev Environ Sci Technol 2001; 31: 241–93.
[
4] Benoit JM, Fitzgerald WF, Damman AWH. The biogeochemistry of an ombrotrophic bog:
evaluation of use as an archive of atmospheric mercury deposition. Environ Res 1998; 78: 118–
3.
3
[5] Kuwabara JS, Arai Y, Topping BR, Pickering AIJ, George GN. Mercury speciation in
piscivorous fish from mining-impacted reservoirs. Environ Sci Technol 2007; 41: 2745–49.
[6] Desvergne JP, Czarnik AW. Eds. Chemosensors of ion and iolecule recognition. Kluwer:
Dordrecht; 1997.
[7] Czarnik AW. Eds. Fluorescent chemosensors for ion and molecule recognition. American
Chemical Society Washington DC; 1992.
1
4

ACCEPTED MANUSCRIPT
[8] Mercury update: Impact on fish advisories, EPA fact sheetEPA-823-F-01-011, EPA, Office
of water. Washington DC; 2001.
[9] Kim YR, Mahajan RK, Kim JS, Kim H. Highly sensitive gold nanoparticle-based
colorimetric sensing of mercury (II) through simple ligand exchange reaction in aqueous media.
ACS Appl Mater Interfaces 2010; 2: 292–95.
[10] Hussain S, De S, Iyer PK. Thiazole-containing conjugated polymer as a visual and
fluorometric sensor for iodide and mercury. ACS Appl Mater Interfaces 2013; 5: 2234−40.
[11] Cheng X, Li Q, Qin J, Li Z. A new approach to design ratiometric fluorescent probe for
2
+
mercury (II) based on the Hg -promoted deprotection of thioacetals. ACS Appl Mater Interfaces
010; 2: 1066–72.
2
[12] Xin DJ, Jiuli Z, Qian L, Chang LS, Jian G, Gao TS, Lin JX. Synthesis of NIR fluorescent
2
+
thienyl-containing aza-BODIPY and its application for detection of Hg : Electron transfer by
2
+
bonding with Hg . Dyes and Pigments 2016; 125: 136-41.
[13] Srivastava P, Razi SS, Ali R, Gupta RC, Yadav SS, Narayan G, Misra, A. Selective naked-
2
+
eye detection of Hg through an efficient turn-on photoinduced electron transfer fluorescent
probe and its real applications. Anal Chem 2014; 86: 8693−99.
[14] Quang DT, Kim JS. fluoro- and chromogenic chemodosimeters for heavy metal ion
detection in solution and biospecimens. Chem Rev 2010; 110: 6280–301.
[15] Zhu M, Yuan M, Liu X, Xu J, Lv J, Huang C, Liu H, Li Y, Wang S, Zhu D. Visible near-
infrared chemosensor for mercury ion. Org Lett 2008; 10: 1481–84.
1
5

ACCEPTED MANUSCRIPT
[16] Yuan M, Li Y, Li J, Li C, Liu X, Lv J, Xu J, Liu H, Wang S, Zhu D. A colorimetric and
fluorometric dual-modal assay for mercury ion by a molecule. Org Lett 2007; 9: 2313–16.
[17] Neupane L, Park JY, Park JH, Lee KH. Turn-on fluorescent chemosensor based on an
amino acid for Pb (II) and Hg (II) ions in aqueous solutions and role of tryptophan for sensing.
Org Lett 2013; 15: 254–57.
[18] Satapathy R, Wu YH, Lin HC. Novel thieno-imidazole based probe for colorimetric
2
+
2+
detection of Hg and fluorescence turn-on response of Zn . Org Lett 2012; 14: 2564–67.
[
19] Nguyen KH, Nguyen CB, Nguyen TAN, Nguyen TT, Pham CN, Tran D, Jong SK, Duong
TQ. A highly sensitive fluorescent chemosensor for simultaneous determination of Ag (I), Hg
II), and Cu (II) ions: Design, synthesis, characterization and application. Dyes and Pigments
015; 116: 89–96.
(
2
[20] Mian W, Jing W, Zhaohai Q, Hongmei W. A new coumarinerhodamine FRET system as an
2
+
efficient ratiometric fluorescent probe for Hg in aqueous solution and in living cells. Dyes and
Pigments 2015; 120: 208–12.
[21] Du J, Fan J, Peng X, Sun P, Wang J, Li H, Sun SA. a new fluorescent chemodosimeter for
2
+
Hg : selectivity, sensitivity, and resistance to Cys and GSH. Org Lett 2010; 12: 476−79.
[
22] Lee H, Kim HJ. Ratiometric fluorescence chemodosimeter for mercuric ions through the Hg
II)-mediated propargyl amide to oxazole transformation. Bull Korean Chem Soc 2011; 32:
(
3
959–62.
[
23] Lee H, Kim HJ. Ratiometric fluorescence chemodosimeter for mercuric ions through the Hg
II)-mediated propargyl amide to oxazole transformation. Tetrahedron Lett 2011; 52: 4775–78.
(
1
6

ACCEPTED MANUSCRIPT
2
+
[
24] Zhou Y, Zhu CY, Gao XS, You XY, Yao C. Hg -selective ratiometric and “off−on”
chemosensor based on the azadiene−pyrene derivative. Org Lett 2010; 12: 2566–69.
[25] Zhao Q, Li FY, Huang C. Phosphorescent chemosensors based on heavy-metal complexes.
Chem. Soc Rev 2010; 39: 3007–30.
[26] Zhao Q, Huang C, Li FY. Phosphorescent heavy-metal complexes for bioimaging. Chem
Soc Rev 2011; 40: 2508–24.
[27] Ma X, Wang J, Shan Q, Tan Z, Wei G, Wei D, Du YA. “Turn-on” fluorescent
2
+
Hg chemosensor based on ferrier carbocyclization. Org Lett 2012; 14: 820–23.
[28] Yang MH, Thirupathi P, Lee KH. Selective and sensitive ratiometric detection of Hg (II)
ions using a simple amino acid based sensor. Org Lett 2011; 13: 5028–31.
2
+
[29] Sait M, Serkan E. A novel perylene-bisimide dye as “turn on” fluorescent sensor for Hg
ion found in DMF/H O. Dyes and Pigments 2015; 113: 763–69.
2
[30] Huan HW, Lin X, Cai LY, Yuan YQ, Hua J. Rhodamine-based fluorescent sensor for
mercury in buffer solution and living cells. Dyes and Pigments 2011; 91: 350–55.
[31] Cabanetos C, Bentoumi W, Silvestre V, Blart E, Pellegrin Y, Montembault V, Barsella A,
Dorkenoo K, Bretonnière Y, Andraud C, Mager L, Fontaine L, Odobel F. New cross-linkable
polymers with huisgen reaction incorporating high ꢀβ chromophores for second-order nonlinear
optical applications. Chem Mater 2012; 24: 1143−57.
1
7

ACCEPTED MANUSCRIPT
[32] Wu J, Bo S, Liu J, Zhou T, Xiao H, Qiu L, Zhen Z, Liu X. Synthesis of novel nonlinear
optical chromophore to achieve ultrahigh electro-optic activity. Chem Commun 2012; 48: 9637–
9.
3
[33] Yang Y, Xu H, Liu F, Wang H, Deng G, Si P, Huang H, Bo S, Liu J, Qiu L, Zhen Z, Liu X.
Synthesis and optical nonlinear property of Y-type chromophores based on double-donor
structures with excellent electro-optic activity. J Mater Chem C 2014; 2: 5124–32.
[34] Lord SJ, Conley NR, Lee HLD, Samuel R, Liu N, Twieg RJ, Moerner WE. A
photoactivatable push−pull fluorophore for single-molecule imaging in live cells. J Am Chem
Soc 2008; 130: 9204–05.
[35] Bouffard J, Kim Y, Swager TM, Weissleder R, Hilderbrand SA. A highly selective
fluorescent probe for thiol bioimaging. Org Lett 2008; 10: 37–40.
[36] Lee HLD, Lord SJ, Iwanaga S, Zhan K, Xie H, Williams JC, Wang H, Bowman GR, Goley
ED, Shapiro L, Twieg RJ, Rao J, Moerner WE. Superresolution imaging of targeted proteins in
fixed and living cells using photoactivatable organic fluorophores. J Am Chem Soc 2010; 132:
1
5099–10.
[37] Lee MK, Williams J, Twieg RJ, Rao J, Moerner WE. Chem Sci 2013; 4: 220–25.
[38] Jin Y, Tian Y, Zhang W, Jang SH, Jen AKY, Meldrum DR. Tracking bacterial infection into
macrophages by a novel red-emission pH sensor. Anal Bioanal Chem 2010; 398: 1375–84.
[
39] Ipuy M, Billon C, Micouin G, Samarut J, Andraud C, Bretonnière C. Fluorescent push–pull
pH-responsive probes for ratiometric detection of intracellular pH. Org Biomol Chem 2014; 12:
641–48.
3
1
8

ACCEPTED MANUSCRIPT
[40] Chen T, Zheng Y, Xu Z, Zhao M, Xu Y, Cui J. A red emission fluorescent probe for
hydrogen sulfide and its application in living cells imaging. Tetrahedron Lett 2013; 54: 2980–82.
[41] Gwon SY, Kim SH. Anion sensing and F-induced reversible photoreaction of D-p-A type
dye containing imidazole moiety as donor. Spectrochimica Acta Part A Molecular and
Biomolecular Spectroscopy 2014; 117: 810–13.
[
42] Park J, Kim H, Choi Y, Kim Y. A ratiometric fluorescent probe based on a BODIPY–
DCDHF conjugate for the detection of hypochlorous acid in living cells. Analyst 2013; 138:
368–71.
3
[43] Zhou X, Su F, Tian Y, Youngbull C, Johnson RH, Meldrum DR. A new highly selective
+
fluorescent K sensor. J Am Chem Soc 2011; 133: 18530–33.
[
44] Hao Y, Yang X, Cong J, Tian H, Hagfeldt A, Sun L. Efficient near infrared D–π–
A sensitizers with lateral anchoring group fordye-sensitized solar cells. Chem Commun 2009;
031–33.
4
[45] Peng P, Strohecker D, Liao Y. Negative photochromism of a TCF chromophore. Chem
Commun 2011; 47: 8575–77.
[46] Wu MY, Li K, Li CY, Hou JT, Yu XQ. A water-soluble near-infrared probe for colorimetric
and ratiometric sensing of SO2 derivatives in living cells. Chem Commun 2014; 50: 183–85.
[47] Atta AK, Kim SB, Heo J, Cho DG. Hg (II)-mediated intramolecular cyclization reaction in
aqueous media and its application as Hg (II) selective indicator. Org Lett 2013; 15: 1072–75.
1
9

ACCEPTED MANUSCRIPT
[48] Zhao Y, Lv X, Liu Y, Liu J, Zhang Y, Shi H, Guo W. The emission enhancement of the
2
+
NIR distyryl bodipy dyes by the indirect S0-S2 excitation and their application towards a Hg
probe. J Mater Chem 2012; 22: 11475–78.
[
49] Hiruta Y, Koiso H, Ozawa H, Sato H, Hamada K, Yabushita S, Citterio D, Suzuki K. Near
IR emitting red-shifting ratiometric fluorophores based on borondipyrromethene. Org Lett 2015;
7: 3022−25.
1
[50] Lin SW, Sun Q, Ge ZM, Wang X, Ye J, Li RT. Synthesis and structure–analgesic activity
relationships of a novel series of monospirocyclopiperazinium salts (MSPZ). Bioorg Med Chem
Lett 2011; 21: 940–43.
[51] Massin J, Dayoub W, Mulatier JC, Aronica C, Bretonniere Y, Andraud C. Near-infrared
solid-state emitters based on isophorone: synthesis, crystal structure and spectroscopic
properties. Chem Mater 2011; 23: 862–73.
[52] Zhang T, Guo KP, Qiu L, Shen Y. magnesium ethoxide as an effective catalyst in the
synthesis of dicayanomethylendihydrofurans. Synthetic Communications 2006; 36:1367–72.
[53] Zheng H, Zhang XJ, Cai X, Bian QN, Yan M, Wu GH, Lai XW, Jiang YB. Ratiometric
2
+
fluorescent chemosensor for Hg based on heptamethine cyanine containing a thymine moiety.
Org Lett 2012; 14: 1986–89.
[54] Gopalan P, Katz HE, McGee DJ, Erben C, Zielinski T, Bousquet D, Muller D, Grazul J,
Olsson Y. Star-shaped azo-based dipolar chromophores:ꢁ design, synthesis, matrix compatibility,
and electro-optic activity. J Am Chem Soc 2004; 126: 1741–47.
2
0

ACCEPTED MANUSCRIPT
[55] Liao Y, Bhattacharjee S, Firestone, KA, Eichinger BE, Paranji R, Anderson CA, Robinson
BH, Reid PJL, Dalton R. Antiparallel-aligned neutral-ground-state and zwitterionic
chromophores as a nonlinear optical material. J Am Chem Soc 2006; 128: 6847–53.
[56] Raju MVR, Lin HC. A novel diketopyrrolopyrrole (DPP)-based [2] rotaxane for highly
selective optical sensing of fluoride. Org Lett 2013; 15: 1274−77.
[57] Raju MVR, Prakash EC, Chang HC, Lin HC. A facile ratiometric fluorescent
chemodosimeter for hydrazine based on ingemanske hydrazinolysis and its applications in living
cells. Dyes and Pigments 2014; 103: 9–20.
[58] Frisch, M. J. et al, Gaussian, Inc: Wallingford CT; 2009.
2
1

ACCEPTED MANUSCRIPT
Schemes and List of Figures
2
+
Scheme 1. Proposed sensing mechanism of T2 towards Hg .
Scheme 2. Synthetic route of probe T2.
(
a) ClCH CH OH, CaCO , KI, H O, reflux 8 h, 86%; (b) POCl , dry DMF, 40°C, 8 h, 95%; (c)
2 2 3 2 3
ethyl 2-mercaptoacetate, NaOEt, DMF, 40°C, 6 h, 95%; (d) TCF, NH OAc, EtOH/THF, rt, 30 h,
4
7
2%; (e) LiOH, THF/MeOH, rt, 2 day, 84%.
Fig. 1. (a) UV-Visible and (b) PL spectra of T2 (5 µM) towards various metal ions in aqueous
2
+
solutions. Bottom red line: in the presence of Hg (10 µM) and the other metal ions (10 µM)
+
+
+
+
+
+
2+
2+
2+
2+
2+
2+
2+
2+
2+
2+
2+
Li , Na , K , Cs , Cu , Ag , Mg , Ca , Ba , Cu , Zn , Ni , Fe , Co , Pb , Cd , Mn ,
2
+
3+
3+
3+
3+
Ag , Al , Cr , Fe , and Eu , in aqueous solution. λ = 570 nm. Insets are naked eye and PL
ex
2
+
photographs of T2 (5 µM) in the absence and presence of Hg (10 µM).
Fig. 2. (a) UV-Visible and (b) PL spectra of T2 (5 µM) in aqueous solution upon the titration of
2
+
2+
Hg (0 to 10 µM). (Inset) Stern-Volmer plot of I/I0 (λ = 650 nm) vs. [Hg ]. λ = 570 nm.
em
ex
2
+
Fig. 3. Calculation of association constant for complex T2-Hg using Connors method.
Fig. 4. Emission intensity of T2 (5 µM) at 650 nm. Black bars: free probe T2, or treated with the
marked metal ions (20 equiv). Red bars: treated with the marked metal ions (20 equiv) followed
2
+
by 2 equiv Hg (λex = 570 nm).
1
2+
Fig. 5. H-NMR (300 MHz) spectral changes of probe T2, complex T2-Hg , and
2
+
T2+Hg +EDTA in CD CN-D O (1:1, v/v). Inset: proposed binding and recovery modes of
3
2
2
+
2+
complex T2-Hg and T2+Hg +EDTA, respectively.
2
2

ACCEPTED MANUSCRIPT
2
+
Fig. 6. (a) Absorption and (b) emission spectral changes of T2 (5µM), after addition of Hg (10
2
+
µM) and followed by EDTA (10 µM). (c) Naked eye color changes of T2 by adding Hg
followed by EDTA. (d) Fluorescence intensity changes of T2 (5 µM) at 650 nm under addition
2
+
of alternate compounds (Hg and EDTA) for 4 cycles (λ = 570 nm).
ex
Fig. 7. Optimized geometries of probe T2 anion and complex T2-Hg calculated using the
B3LYP/Lanl2dz level. Bond lengths are in angstroms.
Fig. 8. Frontier molecular orbital distributions of probe T2 caluculated by semi-empirical PM3
optimized (a) HOMO and (b) LUMO levels, respectively.
Fig. 9. Frontier molecular orbitals of complex T2-Hg based on optimal geometry, which is
calculated at the B3LYP/Lanl2dz+6-31G(d) level .
Fig. 10. Confocal fluorescence images of hela cells. Top, (a-c) cells were incubated with probe
2
+
T2 (5 µM) for 20 min. Middle, (d-f) cells were then further incubated with Hg (10 µM) for 20
min. Bottom, (g-i) cells were further treated with EDTA (10 µM) for 20 min. Bright field (a, d,
and g), fluorescence (b, e, and h), and merged field ( c, f, and i). λ = 570 nm.
ex
2
3

ACCEPTED MANUSCRIPT
Scheme 1.
2
4

ACCEPTED MANUSCRIPT
Scheme 2.
2
5

ACCEPTED MANUSCRIPT
Fig. 1.
Fig. 2.
2
6

ACCEPTED MANUSCRIPT
Fig. 3.
Fig. 4.
2
7

ACCEPTED MANUSCRIPT
Fig. 5.
2
8