A dual-functional chemosensor for fluorescent on-off and ratiometric detection of Cu2+ and Hg2+ and its application in cell imaging

Article abstract of DOI:10.1016/j.dyepig.2019.107642

A dual-functional chemosensor BHMB was synthesized and characterized. It showed highly selectivity through significant fluorescence turn-off and obvious color change towards Cu²⁺ and Hg²⁺, respectively. The binding ratios of BHMB to

Full text of DOI:10.1016/j.dyepig.2019.107642

Accepted Manuscript
A dual-functional chemosensor for fluorescent on-off and ratiometric detection of
2+
2+
Cu and Hg and its application in cell imaging
Shuang Zeng, Shi-Jie Li, Xue-Jiao Sun, Ting-Ting Liu, Zhi-Yong Xing
PII:
S0143-7208(19)30884-8
DOI:
https://doi.org/10.1016/j.dyepig.2019.107642
Article Number: 107642
Reference:
DYPI 107642
To appear in: Dyes and Pigments
Received Date: 20 April 2019
Revised Date: 4 May 2019
Accepted Date: 13 June 2019
Please cite this article as: Zeng S, Li S-J, Sun X-J, Liu T-T, Xing Z-Y, A dual-functional chemosensor for
2+
2+
fluorescent on-off and ratiometric detection of Cu and Hg and its application in cell imaging, Dyes
and Pigments (2019), doi: https://doi.org/10.1016/j.dyepig.2019.107642.
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ACCEPTED MANUSCRIPT

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A dual-functional chemosensor for fluorescent on-off and ratiometric
2
+
2+
detection of Cu and Hg and its application in cell imaging
a, 1
b, 1
a
a
a,
Shuang Zeng , Shi-Jie Li , Xue-Jiao Sun , Ting-Ting Liu , Zhi-Yong Xing *
a
Department of Applied Chemistry, College of Science, Northeast Agricultural University, Harbin
150030, PR China
b
College of Life Science, Northeast Agricultural University, Harbin 150030, PR China
∗
Corresponding author at: Department of Applied Chemistry, College of Science, Northeast
Agricultural University, Harbin 150030, PR China
E-mail addresses: zyxing@neau.edu.cn, Tel: +86 451 5519 1810, Fax: +86 451 5519 0317
1
These authors contributed equally to this work and should be considered co-first authors.
Abstract: A dual-functional chemosensor BHMB was synthesized and characterized. It showed
2
+
highly selectivity through significant fluorescence turn-off and obvious color change towards Cu
2
+
2+
2+
and Hg , respectively. The binding ratios of BHMB to Cu and Hg confirmed by job’ plot were
2
+
2+
2
:1 and 1:1, respectively. Moreover, the detection of limit of BHMB to Cu and Hg was
-8
-7
confirmed as 4.47×10 M and 6.72×10 M, respectively. Especially, BHMB was successfully
2
+
2+
used in test paper for fast identification of Cu and Hg , logic gate construction and cell imaging
in human stromal cell (HSC).
2
+
2+
Key Words: benzothiazole; Cu ; Hg ; ratiometric chemosensor; cell imaging
1

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1
. Introduction
The issue of heavy metals pollution, especially mercury pollution, which is a fatal hazard to
our environment and human health, has been a hot topic on its qualitative and quantitative analysis.
2
+
As is well known, Hg is easily accumulate in human body and the profound toxicity lies in its
high binding ability with thiols and amino groups in proteins and enzymes, which may further
induce some diseases such as kidney failure, motor disorders and tumor formation [1-4]. On the
2
+
other hand, Cu , the third most abundant and necessary trace element in the human body, plays a
closely associate with biological processes including cellular respiration, connective tissue
2
+
development and bone formation [5-8]. However, either deficiency or overloading of Cu will
increase the risk of a number of diseases such as heart disease, iron-deficient anemia, Alzheimer's,
Parkinson's, Menkes and Wilson's diseases, metabolic disorders and cancer [9-13]. According to
the requirement of Environmental Protection Agency (U.S. EPA), the maximum permitted limits
2
+
2+
of the Hg and Cu ion in drinking water are less than 2 ppb (2 ng/L) and 1.3 ppm (1.3 mg/L),
respectively. Hence, it is of great importance for the development of efficient method for
2+
2+
monitoring of Hg and Cu in the water sources and biological environments.
Fluorescence-based probes, which display a lot of virtues such as high sensitivity and
selectivity, rapid response and simplicity for heavy and transition metal ions in environmental and
biological detection, had received considerable attention among researchers on its development
2
+
2+
[
14, 15]. Many fluorescent probes were developed for the detection of Hg [16-22] or Cu
[23-29], but most of them were one probe for one analyte. Recently, the designing idea that one
probe for muti-target had gained more and more researchers’ interesting due to its high efficiency
and cost reduction. Although a lot of multifunctional probes were successfully developed for
2

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simultaneous detection of various analytes [30-38], only a few of them were of qualitative and
2
+
2+
quantitative analysis for Hg and Cu simultaneously based on different fluorophores such as
rhodamine [39, 40], benzothiazole [41], pyrene [42], dansyl [43, 44], BODIPY [45], and
ferrocenyl–naphthalimide [46]. So, it is still full of demand to develop muti-functional
2
+
2+
chemosensors for the detection of Hg and Cu .
2
-(2’-Hydroxyphenyl) benzothiazole (HBT), an excellent fluorophore basing the excited state
intramolecular proton transfer (ESIPT) mechanism, is usually employed in construction
chemosensors due to its excellent properties in the fields of photophysics and photochemistry [41,
4
7]. Herein, we designed and synthesized HBT-based chemosensor BHMB through condensation
of 3-(benzo[d]thiazol-2-yl)-2-hydroxy-5-methylbenzaldehyde and 4-methylbenzenesulfono-
2
+
2+
hydrazide. Chemosensor BHMB displayed high selectivity to Hg and Cu through different
signal response in DMF-H O (3/7, v/v, 0.01M HEPES, pH=7.0) medium. Moreover, the
2
application of BHMB in real samples, logic gate construction and cell imaging were all
investigated.
2
. Experimental
2
.1. Materials and instruments
All chemicals were analytical or spectroscopic grade which were obtained commercially and
used without further purification. All metal ion solutions were prepared by its perchlorate or
nitrate salts. Buffer solution was prepared by hydroxyethyl piperazine ethanesulfonic acid
(HEPES) and NaOH.
Melting point was recorded on a Beijing XT4-100X microscopic melting point apparatus.
1
FT-IR spectra were measured by SEWERE ALPHA-T (Bruker Company, DEU). H NMR spectra
3

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13
and C NMR spectra were recorded on a Bruck AV-600 spectrometer using DMSO-d as the
6
solvent. The model of PHS-3C meter (Shanghai, China) was used for the pH measurement.
Absorption spectra were recorded on a Shimadzu UV-2700 UV–vis spectrometer at 25 ℃.
Fluorescence measurements were measured on a Perkin Elmer LS55 fluorescence spectrometer.
Mass spectra were determined on a Waters Xevo UPLC/G2-SQ Tof MS spectrometer.
2
.2. Synthesis
Compounds 1-2 were prepared according to our previous reported procedure [47].
2
.2.1 Synthesis of Sensor BHMB
Compound 2 (102 mg, 0.38 mmol) and 4-Methylbenzenesulphonyl hydrazide (73 mg, 0.39
mmol) were dissolved in ethanol (10 mL), and the mixture was refluxed 6 h. After the complete
consumption of the starting material monitored by TLC, the mixture was cooled to room
temperature. After which was poured into water (20 mL), the solid was collected by filtration and
1
washed 3 times with distilled water and dried to give BHMB. Yield: 82.4%. m.p.:231-233 °C. H
NMR (600 MHz, DMSO-d6) (Fig. S1) δ (ppm) 12.43 (s, 1H), 11.77 (s, 1H), 8.29 (s, 1H), 8.17 (d,
J = 7.8 Hz, 1H), 8.07 (d, J = 8.0 Hz, 1H), 7.90 (s, 1H), 7.80 (d, J = 8.2 Hz, 2H), 7.57 (t, J = 7.6 Hz,
1
3
1
H), 7.51(s, 1H), 7.48 (t, J = 7.8 Hz, 1H ) 7.45 (d, J = 8.4 Hz, 2H), 2.37 (s, 3H), 2.33 (s, 3H). C
NMR (151 MHz, DMSO-d ) (Fig. S2) δ (ppm) 166.23, 153.81, 151.56, 145.43, 144.23, 136.37,
6
1
34.00, 131.29, 130.95, 130.34, 129.43, 127.59, 127.29, 126.10, 122.69, 122.62, 120.93, 118.61,
+
+
2
1.48, 20.39. HRMS (m/z) (TOF MS ES ) (Fig. S3): calcd for C H N O S : 438.0946 [M+H] ,
22 19 3 3 2
found: 438.0933.
4

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Scheme 1. Synthesis of chemosensor BHMB
.
2
.2. General information
The stock solution of BHMB (0.1 mM) was prepared in DMF. The stock solutions (10 mM)
+
+
2+
2+
2+
3+
2+
2+
3+
2+
2+
2+
of the cationic salts including Na , K , Mg , Ca , Ba , Cr , Mn , Fe , Fe , Co , Ni , Cu ,
+
2+
2+
2+
3+
2+
Ag , Zn , Cd , Hg , Al and Pb were prepared with ultrapure water, respectively. The test
solution were prepared by adding appropriate stock solution of BHMB (0.1 mM) and DMF using
pipette and diluted with HEPES buffer (10 mM) for the measurement of UV-vis absorption and
fluorescence spectra. The excitation was set at 340 nm for the measurement of fluorescence, and
the excitation and emission slit widths were set at 5 nm and 10 nm, respectively.
2
+
2+
2
.3. Preparation of [BHMB-Cu ] and [BHMB-Hg ]
The compound BHMB (10 mg, 0.023 mmol) and Cu(ClO ) ·6H O (17..2 mg, 0.046 mmol)
4
2
2
or HgCl (6.3 mg, 0.023 mmol) were dissolved in ethanol (10 mL), respectively. Then the mixture
2
was refluxed under stirring. After the consumption of BHMB monitored by TLC, the mixture was
cooled to room temperature. Then the complexes were obtained by removing the solvent under
reduced pressure.
2
.4. Cell culture and staining
Human stromal cell line (HSC), a fibroblast cell line which was purchased from ATCC
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2
+
(
CRL-4003), has been used to evaluate whether BHMB could detect Cu in cells. HSC were
routinely cultured in mixture medium (DMEM:F-12=1:1), which was supplemented with 10%
heat-inactivated FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 ℃ , 5% CO . HSC
2
5
were planted into 6-well plates with sterile cover glass at concentration of 10 cells/well. After 48
2
+
hours, the media contained Cu at concentration of 0, 10 and 100 µM and without FBS was used
to incubate cells 2 hours for chemical treatment. Then fibroblast cells were fixed by using a
standard paraformaldehyde fixation protocol and were rinsed with DMF: H O = 3:7 mixture
2
solutions. Then cells were stained with BHMB (10 µM) for 2 hours. Lastly, the cover glass were
mounted over slide glass with anti-fluorescence quenching agent and imaged by fluorescence
microscope.
3
. Results and Discussion
3
.1. Spectrum studies of BHMB with different metal ions
The UV-visible absorbance of chemosensor BHMB were firstly measured upon addition of
+
+
+
2+
2+
2+
2+
2+
2+
2+
2+
2+
2+
different metal ions (Na , K , Ag , Mg , Ba , Ni , Ca , Mn , Fe , Co , Cu , Zn , Hg ,
2
+
2+
3+
3+
3+
Cd , Pb , Fe , Cr and Al ) in the solution of DMF-H O (3/7, v/v, 0.01M HEPES, pH = 7.0)
2
2
+
(
Fig. 1a), the result showed that the addition of Cu caused a significant change in which the
absorbance of BHMB centered at 365 nm disappeared while a new peak at 434 nm formed. As for
2
+
the addition of Hg , which caused the absorbance spectrum of BHMB centered at 365 nm
changed broader and the maximum absorbance peak slightly took red-shift to 376 nm. The above
2
+
2+
phenomenon indicated that the interaction of BHMB with Cu /Hg occurred and further
2
+
2+
enhanced the degree of conjugation of BHMB-Cu /Hg system which caused the red-shift of
absorbance spectrum of BHMB. Moreover, the fluorescence spectrums of BHMB before and after
6

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addition of above-mentioned metal ions were measured, respectively (Fig. 1b). BHMB itself
displayed the strong fluorescence centered at 540 nm which was attributed by the keto emission
generated from the excited-state intramolecular proton transferred process [41] illustrated in Fig.
2
+
S4. However, the addition of tested metal ions to the solution of BHMB showed that Hg ,
2+
especially the Cu completely quenched the fluorescence of BHMB. This result might attributed
2
+
2+
to co-contribution effect of the heavy atom effect of Cu /Hg and the inhibition of ESIPT
2
+
2+
process after coordination of phenolic hydroxyl group with Cu /Hg that previously reported by
other researchers [37, 48, 49], and also indicated the existence of interaction between the
2
+
2+
chemosensor BHMB and Cu /Hg .
Fig.1 (a) Absorption spectra of BHMB (10 µM) recorded without and with different metals (5.0 equiv.) in
DMF-H O (3/7, v/v, 0.01M HEPES, pH=7.0). (b) Fluorescence spectra of BHMB (10 µM) without and with
2
different metals (5.0 equiv.) in DMF-H O (3/7, v/v, 0.01M HEPES, pH=7.0).
2
In order to investigated the detailed variation of BHMB upon the addition of different
2
+
2+
equivalent Cu /Hg , the UV-visible and fluorescence titration of BHMB were measured,
2
+
respectively. Upon gradual addition of Cu , the absorbance centered at 365 nm of BHMB was
decreased and meanwhile absorbance centered at 434 nm was increased (Fig. 2a), a clear isobestic
2
+
point at 388 nm was formed, all of these indicating the complex formation of BHMB with Cu .
The good relationship between the absorbance intensity ratio (A434/A365) versus the concentration
2+
of Cu (0-5 µM) was detected (Fig. S5), which indicated that BHMB could be used as a
7

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2
+
ratiometric chemosensor for the detection of Cu . The limit of detection was calculated as
-8
4
.47×10 M (according to the 3σ/k, where σ is the standard deviation of the blank measurements,
and k is the slope of the intensity ratio versus sample concentration plot) [41, 50]. With regarding
2
+
to the addition of Hg to the solution of BHMB (Fig. 2b), absorbance at 311 nm was decreased
gradually and a slight red-shift from 365 nm to 375 nm was found with an isobestic point at 372
2
+
nm, all these results indicated the happens of interaction between BHMB and Hg . The LOD was
-7
determined as 6.72×10 M according to the good linear relationship between the absorbance
2
+
intensity at 311 nm and the concentration of Hg (0-5 µM) using above mentioned method (Fig.
S6).
2+
Fig. 2 (a) Absorption titration of BHMB (10 µM) upon addition of Cu (0-0.5 equiv.) in DMF-H O (3/7, v/v,
2
2+
0
.01M HEPES, pH = 7.0), Insert: plot of absorbance intensity at 365 nm and 434 nm of BHMB versus Cu (0-5.0
2+
equiv.) ; (b) Absorption titration of BHMB (10 µM) upon addition of Hg (0-0.5 equiv.) in DMF-H O (3/7, v/v,
2
2+
0
.01M HEPES, pH = 7.0), Insert: plot of absorbance intensity at 311 nm of BHMB versus Hg (0-0.5 equiv.).
2
+
2+
On the other hand, fluorescence titrations of BHMB to Cu /Hg were depicted in Fig. 3,
and the results showed that the fluorescence intensity of BHMB was gradually decreased
2
+
2+
2+
2+
whatever the addition of metal ions was Cu or Hg . Moreover, the addition of Cu and Hg
induced the color of BHMB solution changed from bright green to dark green and khaki,
2
+
2+
respectively. This result made it possible to distinguish Cu and Hg , and further realized the
2
+
2+
dual function of BHMB to detect Cu and Hg . According to the recorded fluorescence intensity
8

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2
+
2+
of BHMB with the added amount of Cu and Hg , it could be found that the fluorescence
2
+
2+
intensity of BHMB almost reached a platform when the amount of Cu and Hg added was 5
2
+
2+
µM and 10 µM, respectively. This result indicated the binding ratio of BHMB to Cu and Hg
2
+
2+
was 2:1 and 1:1, respectively. The detection limit of BHMB to Cu and Hg was determined as
-8
-7
8
.14×10 M (Fig. S7) and 1.29×10 M (Fig. S8), respectively.
2+
Fig. 3 (a) Fluorescence titration of BHMB (10 µM) upon addition of Cu (0-1.0 equiv.) in DMF-H O (3/7, v/v,
2
2+
0
.01M HEPES, pH = 7.0), Insert: plot of fluorescence intensity at 540 nm of BHMB versus Cu (0-0.5 equiv.)
2+
and color change, E = 340 nm; (b) Fluorescence titration of BHMB (10 µM) upon addition of Hg (0-1.0 equiv.)
x
in DMF-H O (3/7, v/v, 0.01M HEPES, pH = 7.0), Insert: plot of fluorescence intensity at 540 nm of BHMB versus
2
2+
Hg (0-0.5 equiv.) and color change, λex = 340 nm.
Furthermore, in order to verify the resistance ability of interference which might come from
2+
2+
other co-existence metal ions, competition experiments of BHMB to Cu and Hg were carried
out by adding other tested metal ions (Fig. 4) , respectively. The results showed that BHMB had
2
+
2+
high selectivity to Cu / Hg without disturbance of tested metal ion and could be competent for
2
+
2+
the qualitative detection of Cu / Hg in relative complex surroundings.
9

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2+
2+
Fig. 4 Competition experiments of BHMB toward Cu (a) and Hg (b) in the presence of 5 equiv. of other metal
2+
2+
n+
cations. [BHMB]=10 µM, [Cu ]=50 µM, [Hg ]= 50 µM, and [X ]=50 µM in DMF/H O (3/7, v/v, 0.01M
2
HEPES, pH = 7.0). λ = 340 nm.
ex
3
.2. pH profiles of compound BHMB
In order to evaluate the practical applicability of BHMB, the compatible pH range for the
2
+
2+
detection of Cu and Hg were determined by fluorescence spectrum, respectively. As shown in
Fig.5, chemosensor BHMB itself showed lower fluorescence intensity (recorded at λ =540 nm)
em
at pH range of 2-5, while it significantly increased when the pH value was more than 5, and then
the fluorescence intensity was almost kept constant in the range of pH 7-13, showing excellent
stability of chemosensor BHMB in the wide range from neutral to alkaline environment. Upon the
2
+
addition of Cu , the fluorescence intensity was almost same as that of BHMB itself in the range
of pH = 2-4. Interestingly, a significant distinction in fluorescence intensity of pH ranging from 6
to 13, especially in the range of pH = 7-9 (Fig. 5a). This result showed that BHMB was an
2
+
excellent candidate probe for the detection of Cu in biological applications. On the other hand,
2
+
the fluorescence intensity of BHMB-Hg system was recorded in different pH medium (Fig. 5b).
2
+
The result showed that the optimum condition for the detection of Hg was pH = 7-8, which was
2
+
also adequate for Hg detection in biological surrounding.
1
0

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2+
Fig. 5 (a) Plot showing the influence of BHMB (10 µM) in the absence and prescence of Cu in DMF/H O (3/7,
2
v/v) at different pH medium; (b) Plot showing the influence of BHMB (10 µM) in the absence and presence of
2+
Hg in DMF/H O (3/7, v/v) at different pH medium.
2
3
.3. Binding stoichiometry and sensing mechanism
2
+
2+
The binding ratio of BHMB to Cu and Hg were further determined by job’plot (Fig. 6),
respectively. The fitting results were illustrated in Fig. 6, and 2:1 and 1:1 stoichiometry were
2
+
2+
calculated for BHMB bonding to Cu and Hg , respectively. The ESI-MS spectrum of
2
+
BHMB-Cu (Fig. 7) showed peaks at 460.0755, 897.1677 and 958.0756 were assigned to the
+
+
+ +
+
2+
+ +
[
BHMB + Na ] , [2BHMB + Na ] and [2(BHMB - H ) +Cu + Na ] , respectively. Moreover,
2
+
peaks at 533.1292, 606.1688 and 729.1266 of complex of BHMB-Hg depicted in Fig. 7, were
+
+
+ +
+
2+
attributed to [BHMB + DMF + Na ] , [BHMB + 2DMF + Na ] , and [BHMB - H +Hg + DMF
+
+
H O] , respectively. According to the Benesi-Hilderbrand plot, the association constants of
2
2
+
2+
2
-1/2
4
-1
BHMB to Cu and Hg were counted to be 3.42×10 M and 5.11×10 M , respectively (Fig.
S9-S12).
11

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n+
n+
n+
n+
Fig. 6. Job plot of BHMB-X complex formation. {[ X ]/([ X ]+ [BHMB])} is the molar fraction of X ion. (a)
n+
2+
n+
2+
X
= Cu ; (b) X = Hg .
2+
2+
Fig. 7. ESI–MS spectrum of BHMB (10 µM) upon addition of Cu (a) and Hg (b) in DMF.
2
+
2+
In order to investigate the binding mode of BHMB to Cu /Hg , the FT-IR spectrum of
2
+
2+
BHMB in the absence and presence of Cu /Hg were measured, respectively. As for the
-1
-1
-1
-1
chemosensor BHMB itself, these peaks at 1094 cm , 1605 cm , 3049 cm , 3221 cm , were
attributed to the stretching vibration of S=O, C=N, N-H and O-H, respectively. Upon the addition
1
2

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2+
of Cu into the solution of compound BHMB (Fig. S13), the characteristic absorbance peaks of
-1
-1
-
OH and -NH were broadened and shifted to 3506 cm and 3147 cm , respectively. Moreover, the
-1
-1
characteristic frequency of the imine C=N band increased from 1605 cm to 1612 cm . These
obvious changes indicated that the hydroxyl oxygen atom and the imine nitrogen atom were
2
+
2+
involved in the coordination with Cu ion. While the addition of Hg into the solution of BHMB
(
Fig. S14), the correspondence characteristic frequency mentioned above including S=O, C=N,
-
1
-1
-1
-1
N-H and O-H were all decreased and shifted to 1093 cm 1597 cm , 3104 cm and 3183 cm ,
2
+
respectively. This suggested that compound BHMB might interact with Hg ions through the
hydroxyl oxygen atom and the imine nitrogen atom.
2
+
2+ 1
To further investigate the binding site of BHMB with Cu and Hg , HNMR titrations were
carried out (Fig. 8), respectively. The obvious change was that the proton signal of hydroxyl O-H
2
+
was gradually decreased upon the addition of Cu (Fig. 8a). Moreover, the similar phenomenon
2
+
was found in that of addition of Hg (Fig. 8b). These results strongly indicated the deprotonation
2
+
2+
of phenolic hydroxyl group upon the interaction with Cu or Hg , which also further confirmed
the inhibition of ESIPT process and resulted in the fluorescence quenching of BHMB mentioned
above.
1
3

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1
2+
2+
Fig. 8 H NMR spectra of BHMB with Cu (a) and Hg (b) in DMSO‑d6.
1
Taking above experimental results including job’ plot, HRMS, FT-IR and HNMR titration,
2
+
2+
the possible mechanism of the interaction of BHMB with Cu and Hg were illustrated in
Scheme 2.
1
4

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2+
2+
Scheme 2 Possible interaction mechanism of BHMB with Cu and Hg
3
.4. Reversibility studies
Reversibility was a key factor to evaluate a chemosensor in practical application such as logic
2
+
2+
gate construction. As shown in Fig. 9, the alternate addition of Cu (Fig. 9a) or Hg (Fig. 9b) and
EDTA to the solution of BHMB resulted in the variation in fluorescence intensity and color.
Although emission signal decay to some extent could be found for several cycles, the
BHMB-based colorimetric chemosensor showed good stability. This result indicated that the
2
+
2+
strong stability of complex of BHMB-Cu /Hg , and could be used in the construction of logic
gate through different output signals upon adding different materials.
1
5

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Fig. 9 (a) Reversible changes in color and fluorescence intensity of BHMB (10 µM) at 540 nm in DMF/H O (3/7,
2
2+
v/v, 0.01M HEPES, pH = 7.0) upon alternate addition of Cu and EDTA; (b) Reversible changes in color and
fluorescence intensity of BHMB (10 µM) at 540 nm in DMF/H O (3/7, v/v, 0.01M HEPES, pH = 7.0) upon
2
2+
alternate addition of Hg and EDTA.
4
. Applications
4
.1 Real sample detection
In order to investigate the applicability of the probe BHMB to the detection of Cu and Hg
in real water samples. We collected samples from the tap water in the Department of Chemistry
2
+
2+
2
+
2+
and the Songhua River in Harbin, and Cu or Hg with different concentrations (0, 1, 2, 3, 4 and
µM) were added to these corresponding water samples. The fluorescence responses of Probe
BHMB at 540 nm in those real water samples were determined, respectively. As shown in Fig. 10,
5
2
+
2+
we found that the detection of Cu (Fig. 10a) or Hg (Fig. 10b) in real water samples was similar
to the results detected in ultrapure water (Fig. S7-S8). In addition, the good linear relationships
(
Fig. S15-S18) were found in real water samples, which were all consistent with the linear
relationship previously obtained in ultrapure water. These results showed that the probe BHMB
1
6

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2
+
2+
had high recovery and precision for the detection of Cu and Hg in real water samples, and also
2
+
2+
indicated that it could be applied in the analysis of Cu and Hg in environmental water samples.
Fig. 10 Fluorescent detection of BHMB (10 µM) in “ultrapure water”, “tap water”, and “Songhua River” upon
2+
2+
addition of different concentration of (a) Cu and (b) Hg , respectively. The data was obtained at 540 nm.
4
.2 Application in test paper
Due to the good selectivity of the probe BHMB for Cu and Hg with the significant
2
+
2+
2
+
fluorescence color change for detection, the test strip experiments of the probe BHMB for Cu
2+
and Hg were examined, respectively. As shown in Fig. 11, with the increased concentration of
2
+
2+
Cu (Fig. 11a) or Hg (Fig. 11b), test papers (top and bottom) displayed a visible color change
from initial yellow to final bluish violet under 365 nm UV light, but the transitional colors of
2
+
2+
BHMB with different concentration of Cu was not same as that of Hg , which was another
2
+
2+
method to differentiate between Cu and Hg . The result also indicated that the probe BHMB
2
+
2+
could be applied to the fast detection of Cu and Hg by test strip.
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7

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2+
2+
Fig. 11 The photographs of probe BHMB on test strips with different concentrations of (a) Cu and (b) Hg at
room temperature under 365 nm UV light, respectively.
4
.3 Logic gate construction
2
+
Thanks to the fluorescence quenching-restoration cycle of probe BHMB (10 µM) with Cu
2
+
(
10 µM) or Hg (10 µM) and EDTA (10 µM) (Fig. 12a, b), one logic gate can be implemented on
Boolean logic operations, which is a IMPLICATION logic gate and displaying memory unit with
2
+
2+
two inputs (In 1 and In 2) and one output(Fig. 12c, d). We set Cu or Hg and EDTA separately
as inputs In 1 and inputs In 2, whose presence and absence were itemized as 1 and 0. The output
signal is emission intensity at 540 nm and the threshold value is considered as 40 au. When the
fluorescence intensity is higher than the threshold value, the output signal is On (1). And when the
fluorescence intensity is lower than the threshold value, the output signal is Off (0). Based on the
2
+
2+
above basic logic gates, when the Cu or Hg and EDTA were all presence or absence, the output
signal is On (1). When inputs 1 and 2 were in a (1, 0) state, the output signal is Off (0). In addition,
in order to achieve reversible cyclic operation of logic gates, we further installed the memory
storage device to the logic circuit. When the output signal in a Off (0) state, the input signal can be
reset by the memory unit to return the output to the On (1) state. Thus, this logic circuit with
memory element can cause the entire process to be repeated several times.
1
8

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2+
Fig. 12 (a) bar diagram represents input (In 1 = Cu and In 2= EDTA ) and output emissions (at 40 au); (b) bar
2+
diagram represents input (In 1 = Hg and In 2= EDTA ) and output emissions (at 40 au); (c) IMPLICATION logic
gate with two inputs (In 1 and In 2) and one output; (d) corresponding truth table.
4
.4 cell imaging
To further investigate the practical applicability of probe BHMB in biological systems, the
cells imaging experiments were measured by fluorescence microscope. Compared to the complex
2
+
2+
of BHMB-Hg , the complex of BHMB-Cu has almost no fluorescence emission and can exist
in a wide pH range. Thus, we used the human stromal cells (HSC) that was incubated different
2
+
amounts of Cu and 10 µM of BHMB. As shown in Fig. 13a, when the cells was incubated 2 h
with BHMB (10 µM), the aquamarine fluorescence was observed under fluorescent microscope.
2
+
With increased concentration of Cu , the fluorescence intensity is gradually weakens and finally
2
+
quenched when 100 µM of Cu was incubated with cells (Fig. 13b, c). This result indicates that
2
+
the probe BHMB can be used to track Cu in biological systems.
1
9

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2
+
Fig. 13 (a) The cells was incubated 2 h with BHMB (10 µM); (b) cells was incubated 10 µM of Cu and 10 µM
2
+
of BHMB; (c) cells was incubated 100 µM of Cu and 10 µM of BHMB.
Conclusions
In conclusion, a dual-functional chemosensor BHMB was synthesized and characterized. It
2
+
2+
showed high selectivity to Hg and Cu through significant fluorescence turn-off and obvious
2+
2+
color change towards Cu and Hg in DMF-H O (3/7, v/v, 0.01M HEPES, pH=7.0) medium,
2
2
+
2+
respectively. The binding ratios of BHMB to Cu and Hg were determined as 2:1 and 1:1,
2
+
2+
respectively. Moreover, the detection of limit of BHMB to Cu and Hg was confirmed as
-8
-7
4
.47×10 M and 6.72×10 M, respectively. Especially, BHMB was successfully used in test paper
2
+
2+
for fast identification of Cu and Hg , logic gate construction and cell imaging in human stromal
cell (HSC).
Acknowledgements
This work was supported by the Postdoctoral Scientific Research Developmental Fund of
Heilongjiang Province (No. LBH-Q14023). We would like to thank Professor Jinlong Li (Qiqihar
University) for NMR and ESI-mass running.
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0

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Appendix A. Supplementary data
Supplementary data to this article can be found online at
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Highlights
●
Chemosensor BHMB with benzothiazole fluorophore was synthesized
and characterized.
●
Chemosensor BHMB exhibits highly selective fluorescence responses
2
+
2+
to Hg and Cu with significant fluorescence turn-off and color
change.
2
+
2+
●
●
The limit of detection (LOD) for Cu and Hg was reached the level
-8
-7
of 10 M and 10 M, respectively.
2
+
2+
BHMB was successfully applied in the detection of Cu and Hg in
test paper and cell imaging in HSC.