The influences of different substituents on spectral properties of rhodamine B based chemosensors for mercury ion and application in EC109 cells

Article abstract of DOI:10.1139/cjc-2017-0017

Six rhodamine-based "turn-on" fluorescence chemosensors (L₁-L₆) with different substituents for mercury (Hg²⁺) were readily synthesized and investigated; they displayed high selectivity and chelation enhanced ratiometric f

Full text of DOI:10.1139/cjc-2017-0017

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The influences of different substituents on spectral properties of
Rhodamine B based chemosensors for mercury ion and application in
EC109 cells
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Wenqi Du[a], Yu Cheng[a],Weixin Shu[a], Baoying Wu[a], Zhineng Kong[a], Zhengjian
Qi*[a]
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[a]College of Chemistry and Chemical Engineering,Southeast University Road 2,
Jiangning District 211189, Nanjing, Jiangsu Province, P.R. China
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Email:qizhengjian506@163.com
*For correspondence
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Abstract: Six rhodamine-based “Turn-on” fluorescence chemosenors(L1~6) with
different substituents for Hg2+ were readily synthesized and investigated, which
displayed high selectivity and chelation enhanced ratiometric fluorescence change and
colorimetric change with Hg2+ among the metal ions examined. Based on UV spectral
data and fluorescence spectral data, the effect of different substituents on spectral
properties of the probes were presented and discussed. The detection limit of Hg2+ to
probe L1 was as low as 50 nM, which attribute to its electron donating group.
Theoretical calculation also support the process of reaction. Confocal laser scanning
microscopy experiments showed that probe could be usedto detect Hg2+ in living
cells.
Keywords: Rhodamine B; Fluorescent chemosesor ; Sensing mercury ion; Living
cells.
1.Introduction
Various transition-metal ions are essential to the life of creatures. Mercury is a
dangerous and hazardous toxic which has posed a great threat to our environment1-4.
Mercury is heavy metal, which can be dispersed in the atmosphere, the ocean, and
the soil. Different speciation mercury can penetrate our environment by various ways,
such as methyl mercury produced by aquatic microbes which bioaccumulates
through the food chain and oxidation of mercury vapor in atmosphere to
water-soluble Hg2+ ions5-7. After long-term exposure in the environment, mercury ion
accumulate in organisms which can cause nausea, vomiting, abdominal pain, renal
dysfunction and other diseases.
At present, UV spectrophotometry, electrochemical methods, ionization coupled
plasma mass spectrometry, ionization coupled plasma atomic emission spectrometry,
atomic absorption spectroscopy and other analytical methods can be used to identify
and detect metal ions8-12. However, these methods have some disadvantages in the
application, such as high detection costs, narrow range of applications, and can’t
achieve real-time detection. In contrast, the fluorescent molecular probe has no

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damage to the sample, and gives its priority to easy operability and high sensitivity,
so fluorescence analytical methods are effective and efficient ways to detect ions13-15
.
Compared with other fluorescent dyes, rhodamine dyes have some excellent
photophysical properties, such as high fluorescence quantum yield, pH insensitive,
high molar absorptivity and high extinction coefficient, particularly in its nucleotide
and nucleic acid conjugates16. Some rhodamine-based chemosensors for Hg2+ ions
have been reported17-22, but dual colorimetric and fluorescent chemosensors for
mercury ion were still rare23 and some of them were not efficient enough to be
selective toward mercury ion.
Here, we report six rhodamine-based caprolactam derivatives as a sensor for
Hg2+, when binding phenomena could be probed through binding-induced changes in
an electronic spectral pattern. The difference between these probes are that we
selected different substitutional group such as methyl group, nitro group, aldehyde
group, halogen group, phenyl group. These rhodamine-based derivatives that
behaves as a fluorescent Hg2+ sensor with high emission sensitivity and selectivity,
which exhibited sensitive detection toward Hg2+ via significant fluorescence
enhancement in solution, and in the meantime they showed a significant color
change from colorless to rose red. We have designed some similar structures to
compounds L1~6 and conduct a series of experiments to explore their properties
which have the correlations of nature of substituent group. After comparison of
optical properties,probe L1 showed a good performance in the recognition toward
Hg2+,which indicated that the different electronic distribution among the senors’
structures make an influence on their properties. For this reason, we selected L1 as a
typical example to expound in the following discussion.
2. Experimental
2.1 Apparatus and Materials
Fluorescence spectra measurement were performed on Horiba Jobin Yvon Inc.
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Fluorolog 3-TSCPC. H NMR spectrum was run on a Bruker 300 MHz spectrometer
using TMS as the internal standard. Mass spectrum was recorded with a VG ZAB-HS
double focusing mass spectrometer. Absorption spectra were measured on a UV-2201

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double-beam UV/VIS spectrometer. Melting point was determined using an SGW X-4
digital melting point apparatus.
All the materials for synthesis were purchased from Sinopharm Chemical Reagent
Co.,Ltd (Shanghai, China) and used without further purification. Metal solutions
were prepared from their analytical grade nitrate salts. The metal ions were prepared
as 0.2 mM in water solution.
2.2 Syntheses
As shown in Scheme 1, the compounds L1~L6 were prepared by reacting RBH with
aromatic aldehyde. RBH was synthesized according to literature.
2.2.1 Synthesis of RBH
As for the synthesis of RBH, sereval different procedures have been reported24-26
In this work, RBH was synthesized by a modified method according to Xiang27. m/z:
.
－
1
457.2265 ([M+H]+); M+ calculated 456.2265. IR(KBr,cm ):ν= 3428, 2987，2926,
1689, 1614, 1514, 1380, 1218, 1117, 818, 767. 1H NMR (300 MHz, CDCl3, δ ppm), δ
(ppm):1.17 (t, 12H), 3.34 (q, 8H), 3.62 (bs, 2H), 6.29 (dd, 2H), 6.43(d, 2H),6.45 (d,
2H), 7.11 (dd, 1H), 7.42 (d, 1H), 7.44(d, 1H), 7.93 (dd, 1H). (Fig.S1~S3)
2.2.2 Synthesis of L1
RBH ( 0.5 mmoL, 0.24 g) was dissolved in 30 mL ethanol, and then p-tolualdehyde
(0.5 mmoL, 0.12 g) was slowly added. The mixture was stirred and refluxed for 12 h
at 80 ℃. After distillation in vacuum, the residue was recrystallized with methanol
and water to give the final product L1 in yield of 79.8%. m/z: 559.3020([M+H]+);
1
M+calculated 558.3020. H NMR (300 MHz, CDCl3, δ ppm): 8.64 (s, 1H),7.97 (m,
1H), 7.49 (d, 2H), 7.45 (m, 2H),7.26 (d, 1H), 7.09 (m, 2H), 6.54 (d, 2H), 6.43 (s, 2H),
6.26 (d, 2H), 3.33 (q, 8H), 2.29 (s, 3H), 1.15(t, 12H). (Fig.S4~S6)
2.2.3 Synthesis of L2~6
Compound L2~6 was prepared using a general procedure which is essentially similar
to that used for L1. (Fig.S7~S21)
3. Results and Discussion
1
The structure of compounds L1~6 were characterized by H NMR, HR-MS. The
results were in good agreement with the structure showed in Scheme 1. Fluorescence

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and UV–vis studies were performed using a 20 µM solution of L1~6 in a CH3CN/H2O
(1:1, v/v) solution with appropriate amounts of metal ions.
3.1 Comparison of six rhodamine B based senors
Under the same conditions,we explore the sensitivity measurement of ion probes.
As shown in Fig.1, rhodamine B derivatives with methyl exhibits excellent sensitivity
towards mercury ion. Derivatives with halogen group also show good sensitivity
owing to its electron donating groups. Based on these phenomena, it suggested that
chelation-enhanced fluorescence appears when compounds with electron donating
groups are complexed with Hg2+. In order to further explore the affinity capacity
between probes and metal ions, association constants were calculated to verify the
experiment we have conducted. It is indicated in the Scheme 2, the numerical size of
association constants minished in order, which in accordance with the results of
fluorescence intensity when different substituents with rhodamine B binds to mercury.
After comparison between these probes, we can come to conclude that methyl
group, halogen group and phenyl group belong to electron donating group while nitro
group, aldehyde group belong to electron-attracting group. When interacting with
mercury icon, electron donating group provide electron to coordination site which
make it easier to combine. On the contrary, electron-attracting group make it more
difficult for probe to interact with mercury icon. So, we prefer to select electron
withdrawing substituents to detect metal ions which reveals high sensitivity. To
investigate photoelectric properties of these probes further,we selected L1 as an
typical example to expound in the discussion.
3.2 Metal ion selectivity and competition experiments
As shown in Fig.2, UV–vis spectrum of compound L1 (20µM) exhibited only very
weak bands over 450 nm, which could be attributed to the presence of a trace amount
of the ring-opened form of compounds. On addition of 10 equiv. Hg2+ into solution,
L1 immediately resulted in a significant enhancement of absorbance at about 556 nm
simultaneously the color changed into rose red. Other metal ions such as Zn2+, Mg2+,
Ca2+, Cd2+, Cu2+, Pb2+, Ba2+, Ni2+, Mn2+, K+, Li+, Ag+, Co2+, except for Fe3+, did not
show any significant color and spectral change under any significant color and

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spectral change under identical conditions. These phenomena suggest that these
compounds can serve as a “naked-eye” chemosensor for Hg2+. To further investigate
the interaction of Hg2+ and the probe, the Hg2+ binding stoichiometry of the probe can
be determined from titration and the Job plot28, 29. As shown in Fig.3, it is obvious that
the fluorescence intensity reached a maximum when the ratio was 0.5, which
suggesting that a 1:1 stoichiometry of the Hg2+ to the probes respectively in the
complex. Furthermore, we also conducted the competitive experiments which
indicates the background metal ions showed very low interference with the detection
of Hg2+ in the water solutions (Fig.4.).
3.3 Emission spectra and detection limit of sensor
A fluorescence titration experiment was carried out in order to further explore the
interaction of probes and Hg2+. On addition and gradual increase concentration of
mercury ions to the L1 in CH3CN/H2O(V:V=1：1), the fluorescence intensity gradually
increased and the solutions turned colorless to rose red. Limit detection of metal ions
plays an important role in evaluating fluorescence chemosensor. As for probe L1, the
linear response for the fluorescence intensity response of compound L1 was between 0
and 140 µM (Fig. 5), and the detection limit of Hg2+ was measured to be 0.05 µM.
The association constant K of the complex L1–Hg2+ was then calculated to be
3.24×103 M−1 with a linear relationship (Fig. 6) by Benesi–Hildebrand method, Eq.
(1)30, 31
1
1
1
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=
+
F
F − F0 K(F − F )[Hg2+ ]
− F
Max
Min
Max
Min
(1)
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As shown in the Fig.7, probe L1 show a weak fluorescence in the absence of metal
ions. On the addition of 10 eqiv. metal ions, the fluorescence intensity has changed. It
is obvious that when 10 eqiv. Hg2+ was introduced into a solution of L1 in
CH3CN/H2O (1:1, v/v), the fluorescence intensity increased sharply. Under this
conditions, Fe3+ induce a mild fluorescence enhancement while Zn2+, Mg2+, Ca2+,
Cd2+, Cu2+, Pb2+, Ba2+, Ni2+, Mn2+, K+, Li+, Ag+, Al3+, Co2+ and Na+ did not show
remarkable changes in fluorescence intensity and the color.
3.4 Time-dependence and Fluorescent lifetime

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Time-dependence for binding of the probe L1 with Hg2+ is given in Fig.8.
Following the addition of 10 equiv. Hg2+ ion to 20.0 µM probe L1, the fuorescence
intensity of probe L1 was turn on rapidlly and reached a stable value within 2 min.
The fluorescence lifetime was measured at an excitation 460 nm of the NanoLED
source. The decays of probes were found to be monoexponential. The lifetime decays
in the absence of Hg2+ and in the presence of Hg2+ are shown in Fig.9. The average
lifetime of L1 was 1.87 ns and 5.00 ns (XSQ = 1.06) while the lifetime of L1 + Hg2+
was 1.91 ns (XSQ = 1.14). Double exponential fitting equation was used to describe
the fluorescence lifetime of probe itself , because xanthene was a chromophore which
formed a conjugated system. The upper part of structure formed a plane and
orthogonalized with the ring of xanthene which also can be seen as a chromophore.
After adding metal ions，the upper part including carbanyl group and phenyl group do
not conjugate with xanthene,and the stereo-hindrance effect of carbanyl group limits
the rotation of the benzene ring. So the major chromophore is still xanthene which
used single exponential fitting equation.
3.5 Application
3.5.1 Test papers
To demonstrate the practical application of our sensor, we prepared the test papers
of sensor L1. As depicted in Fig.10, the color of the test paper changed from colorless
to purple and deepened gradually with the increasing of Hg2+ concentration. These
paper-made test kits may be used as a simple tool for detecting Hg2+ in environmental
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samples.
3.5.2 Fluorescence imaging for EC109 cells
Owing to its favourable spectroscopic properties of the response to Hg2+, L1 should
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be suited for fluorescence imaging in living cells. Laser scanning confocal
microscopy was used to investigate this proposition. As determined by laser
scanningconfocal microscope, EC109 cells with a 10ꢀM solution of probe L1 in
CH3CN–H2O (1:1, v/v) 30 min at 25 ◦C led to no intracellular fluorescence(Fig.11b.).
After washed with PBS three times, the staining cells were then supplemented with 10
ꢀM of Hg2+ under the same condition for 30 min, a significant increase of

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fluorescence from the intracellular area was observed ( Fig.11c).
3.6 Mechanism
To investigate the interaction of probes and metal ions, probe L1 was selected as an
example to explain mechanism. IR spectra of L1 and L1+Hg2+ were taken in KBr disks
(Fig.S22). It can be seen that the peak at 1698 cm-1 which relates to the amide
carbonyl absorption disappeared upon with the addition of Hg2+. To further
investigate the mechanism of the reaction,we selected L1 as an typical example to
explain the phenomena. In addition，EDTA-adding experiment has been conducted to
confirm the reversibility of reaction and the result was shown in the Fig.12,when the
30 eqiv. EDTA was added into L1+ Hg2+ in CH3CN/H2O solution, the fluorescence
intensity was decreased rapidly(green line).Then after adding the 30 eqiv. Hg2+, the
fluorescence intensity was recovered（red line）. This phenomenon indicates that the
reaction between the compound and the metal ion are reversible.
As shown in the Fig.13, it indicates that these sereval chemosenors exhibit
reversible and sensitive detection to Hg2+. While hydrolytic action of compounds
might be happen after a long-term interaction with metal ions. Theoretical calculation
has been conducted to verify the stability of compound L7, we assume the structure L7
was existed after interacting with Hg2+. Frequency calculation successfully provide
the evidence for the existence of the stable structure. To investigate the possible
mechanism of the absorption and fluorescent emission of L1, theoretical calculation of
L1 which based on the optimized structures of the ground S0 state and the first
excited S1 state were performed in Fig.14. After adding Hg2+, the energy gap between
the HOMO and LUMO is greatly decreased32.
4. Conclusions
In summary, we reported the design and synthesis of six new rhodamine-based
derivatives L1~6 used as sensitive and selective chemosenor, which could specifically
recognize Hg2+ ion in solutions by naked eyes. These six probe display 1:1 complex
formation with Hg2+ which could be monitored by the spectral changes as well as
color changes. Based on the experiment above, after a series comparison of these
probes with different substituent group, L1 shows better optical properties than others

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owing to its electron donating group and excellent properties. Confocal laser scanning
microscopy experiments showed that L1 could beused to detect Hg2+ in living cells.
Futhermore, these six new probes show a great potential for biotic environmental
detection.
Supplementary Information (SI)
The structure of compounds RBH, L1~6 were characterized by 1H NMR, HR-MS,IR in
Supplementary Information. Supplementary Information is available at
www.ias.ac.in/chemsci.
Acknowledgements
This work was supported by the Fundamental Research Funds for the Central
Universities (KYLX15_0125) and National Major Scientific Instruments and
Equipment Development Projects (2014YQ060773) and A Project Funded by the
Priority Academic Program Development of Jiangsu Higher Education Institutions
(1107047002) and Policy Guidance Program (Research Cooperation)--Prospective
Joint Research Project (BY2016076-02).
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Scheme 1. Synthetic route of target compounds.

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Fig.1. Fluorescence intensity (at 580 nm) of L1~6 (20µM) in CH3CN/H2O (1:1, v/v) with the
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presence of Hg2+ (100 µM) (λex = 500 nm)

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Coordination constant
Br C6H6
-R
CH3
F
NO2
CHO
Coordination
3.24E3
2.67E3 2.38E3 2.27E3 3.70E2 2.56E2
constant/M−1
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Scheme 2.The coordination constant of six probes

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Fig. 2. Absorbance spectra of L1(20 µM) in CH3CN/H2O (1:1,v/v) solutions with the presence of
10 equiv. of various species.
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Fig.3. Job's plot of the complexation between L1 and Hg2+, the total concentration of L1 and Hg2+
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is 20.0 ꢀM .

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Fig.4. Fluorescence intensity (at 580 nm) of L1 upon the addition of 20 µM Hg2+ in the presence
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of 20µM background metal ions in CH3CN/H2O (1/1, v/v) (λex = 500 nm).

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Fig.5(a). The fluorescence intensity (at 580 nm) of compound L1 (20 µM) as a function of the
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Hg2+ concentration in CH3CN/H2O(1:1, v/v) solution (λex = 500 nm, slit = 3 nm)

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Fig.5(b). Linear relationship of L1 (20 µM) in CH3CN/H2O (1:1,v/v) upon addition of different
amounts of Hg2+ ions.
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Fig.6. Benesi–Hildebrand plot (λex = 500 nm) of L1, assuming 1:1 stoichiometry for association
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between L1 and Hg2+ in the solutions.

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Fig.7. Fluorescence spectra of L1 (20 µM) in CH3CN/H2O (1:1,v/v) with the presence of 10 equiv.
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of various metal ions (λex =500 nm).

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Fig. 8. Effects of time on the reaction of Hg2+ with L1
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Fig.9. Fluorescence decay curves of L1 and L1 + Hg2+ in solutions obtained at λex =500 nm.
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Fig. 10. Photographs of the test kits with L1 for detecting Hg2+ in(CH3CN/H2O = 1:1, v/v) solution
with different concentrations:(1) 0; (2) 1.0 × 10-5M; (3) 1.0 × 10-4M; (4) 1.0 × 10-3M;(5)1.0 ×
10-2M;(6)1.0 × 10-1M.
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Fig.11. Laser confocal scanning microscopy experiments of EC109 cells:（a）Bright-field
transmission image.（b）Cells with 10ꢀM solution of L1 in CH3CN–H2O (1:1, v/v) for 30 min at
25 ◦C.(c)The staining EC109 cells exposed to L1 (10ꢀM) for 30 min and then to a CH3CN–H2O
(1:1, v/v) solution of Hg2+ (10ꢀM) for 30 min.
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Fig.12. Fluorescence intensity (at 580 nm) of L1 (20 µM) to Hg2+ in CH3CN/H2O (1:1,v/v)
solutions (1) blue line: 10 µM L1 only; (2) red line: 20 µM L2 with 10 equiv. Hg2+; (3) green line:
20 µM L1 with 10 equiv. Hg2+ and then addition of 30 equiv. EDTA; (4) black line: 10 µM L1with
10 equiv. Hg2+ and 30 equiv. EDTA then addition of 10 equiv. Hg2+ (λex = 500 nm).
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O
O
R
N
Hg2+
N
N
N
R
N
O
N
N
O
N
L7
−Η2Ο
COOH
N
O
N
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Fig.13. Possible sensing mechanism of L1~6 with Hg2+

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Fig.14. Theoretical molding of the absorption of L1 without and with Hg2+ at the TDDFT level.