A new 1,2,3-triazole and its rhodamine B derivatives as a fluorescence probe for mercury ions

Article abstract of DOI:10.1016/j.ab.2020.113690

A newly synthesized compound, 5-methyl-1-phenyl-1H-1,2,3-triazole-4- carboxylic acid (MPC) was analyzed for its quantum chemical parameters and theoretical spectrum by computational chemistry. The calculated spectrum was in accord with the experimental me

Full text of DOI:10.1016/j.ab.2020.113690

AnalyticalBiochemistry598(2020)113690
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Analytical Biochemistry
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A new 1,2,3-triazole and its rhodamine B derivatives as a fluorescence probe
for mercury ions
Wenying Hea,∗, Rongqiang Liua, Yuanhao Liaob, Guohua Dingb, Jianling Lib, Wei Liua,
Luyong Wub, Huajie Fengc, Zaifeng Shia, Mengxiong Hec
a Key Laboratory of Water Pollution Treatment & Resource Reuse of Hainan Province, Haikou, 571158, China
b Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, Haikou, 571158, China
c College of Chemistry and Chemical Engineering, Hainan Normal University, Haikou, 571158, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
A newly synthesized compound, 5-methyl-1-phenyl-1H-1,2,3-triazole-4- carboxylic acid (MPC) was analyzed for
its quantum chemical parameters and theoretical spectrum by computational chemistry. The calculated spec-
trum was in accord with the experimental measurements in a great degree. Then MPC was successfully designed
and synthesized to a novel rhodamine B derivative RMPC. The RMPC exhibited about a 4000-fold increase in
fluorescence intensity in the presence of Hg2+ ions over most other competitive metal ions. The triazole ap-
pended colorless chemodosimeter RMPC turns to pink upon the complex formation only with Hg2+ ions as a 1:
2 M ratio and enables naked-eye detection. The coordination mechanism of turning on/off fluorescence for Hg2+
ions were well proposed by explaining Hg2+ inducing the ring-opened rhodamine B moiety. The fluorescence
imaging experiments of Hg2+ in HeLa cell demonstrated that the probe was labeled and it could be used in
biological systems.
5-methyl-1-phenyl-1H-1,2,3-triazole-4-
carboxylic acid
Structural properties
Rhodamine B
Colorimetric
Hg2+ ion
Cell imaging
1. Introduction
system and central nervous system [9]. How to recognize mercury ions
sensitively and efficiently has drawn the more and more interest of
Nitrogen-rich azole heterocycles are promising compounds that
fulfill many requirements in many fields with the development of click
chemistry, including energetic materials, chemical, biological, macro-
molecular materials, and pharmaceutical sciences. Owing to less toxic
effects, good biological activity, high positive heats of formation re-
sulting from the large number of N–N and C–N bonds and the high level
of environmental compatibility, triazole, and tetrazole compounds have
been studied over the last couple of years with growing interest [1–3].
Several researches have been reported that 1,2,3-triazoles can also
serve as ligands capable of binding to various metal ions and have
shown distinct pharmacological activity because of their satisfactory
coordination abilities [4–7]. However the overdose or lack of metal ions
will harm the human body and organisms. Especially, the contamina-
tion of heavy metal ions such as Hg2+, As2+, Pb2+, and Cd2+ has been
becoming one of the most serious problems in the environment because
of the increasing industrial and agricultural activities as well as the
improper release of metal ions from waste water and domestic effluents
[8]. For example, mercury ion (Hg2+) is one of the most harmful
substances to human beings because of its combination with sulfhydryl
groups of proteins in various organs including kidneys, brain, immune
researchers. Many studies have been reported on exploiting a wide
variety of analytical methods for detecting Hg2+ [8–12].
Recently, some techniques such as electrochemistry [12], fluores-
cence spectroscope [13], atomic absorption spectroscopy(AAS) [14]
and inductively coupled plasma mass spectrometry (ICP-MS) [15] have
been developed for detection of metal ions. Accordingly, considerable
efforts have been devoted to developing fluorescent probes for Hg2+
because fluorescent detection is the most efficient method, operational
simplicity, low cost, real-time monitoring, and high sensitivity [16].
Especially the chromogenic reaction is usually applied to detect and
probe analytical species due to it is extremely simple and practical
feature. So the colorimetric sensors using the fluorescence analysis
method have drawn more and more attention because of many ad-
vantages such as fast response, signal visibility, easy operation, and
high throughput analysis [17,18]. Among various fluorescent, colori-
metric probes, some fluorophores employed as signal reporters of these
probes mainly focused on rhodamine, which exhibited the excellent
photophysical properties such as high fluorescence quantum yield,
large extinction coefficient, long absorption, emission wavelength and
high stability against light [19–21]. Generally, rhodamine with a
∗ Corresponding author. Key laboratory of Water Pollution Treatment & Resource Reuse of Hainan province, Hainan Normal University, Haikou, 571158, China.
E-mail address: hewenying@hainnu.edu.cn (W. He).
https://doi.org/10.1016/j.ab.2020.113690
Received 27 October 2019; Received in revised form 2 January 2020; Accepted 12 March 2020
Availableonline04April2020
0003-2697/©2020ElsevierInc.Allrightsreserved.

W. He, et al.
AnalyticalBiochemistry598(2020)113690
particular spirolactam structure is colorless and nonfluorescent. Metal
ion can stimulate the spirolactam form to turn into the open-ring forms
with pink color and strong fluorescence [22]. The rhodamine B deri-
vatives can exhibit apparent fluorescence enhancement and color
changes from the closed-loop state to the open-loop state when binding
to various metal ions, which have been broadly utilized to design as
“off-on” fluorescent probes for detecting these metal ions [23]. Recent
studies on rhodamine-triazoles systems have shown that these com-
(Shimadzu, Japan) and a F-7000 Spectro fluorophotometer (Hitachi,
Japan) equipped with a xenon lamp source and a water bath. The
UV–vis absorbance spectra were measured on a Hitachi U3900/3900H
spectrophotometer (Hitachi, Japan) equipped with 1.0 cm quartz cells.
ESI-MS spectra were recorded on an Agilent 1100-Bruker Esquire HCT
at room temperature. The compound was ionized in the electrospray
ionization (ESI) and operated in negative mode.
plexes could specifically recognize many metal ions such as Cu2+
、
2.2. Synthesis
Fe3+、Sn2+、Hg2+、Zn2+、Al3+ etc [24–31], which all indicated as a
monofunctional probe with different selective and sensitive colori-
metric assays.
As shown in Scheme 1, the first step is to synthesize rhodamine B
hydrazide (1) according to the reported reference [48]. Rhodamine B
(1.0 g, 2.1 mmol) and 80% hydrazine hydrate (2 mL) were mixed and
stirred in 20 mL ethanol at room temperature. Then the mixture was
heated to reflux for 3 h and extracted with ethyl acetate (50 mL) for
three times. The supernatant was dried over anhydrous sodium sulfate
and filtered, obtaining intermediate 1 as a yellow solid (0.52 g,
54.74%); 1H NMR (400 MHz, CDCl3):δ (ppm) = 1.16 (t, J = 6.8 Hz,
12H),3.34 (q, J = 7.2 Hz, 8H), 3.61 (s, 2H), 6.30 (m, 2H), 6.45 (m, 4H),
7.1 (m, J = 2.64 Hz, 1H), 7.44 (m, 2H), 7.94 (m, 1H); 13CNMR
(400 MHz, CDCl3) δ (ppm) = 166.03, 153.75, 151.45, 148.78, 132.39,
129.91, 128.00, 127.95, 123.71, 122.83, 107.94, 104.47, 97.89, 77.32,
77.00, 76.68, 65.82, 44.25, 12.50.
The different fluorescence probes for mercury ion detection have
been reported in a large number of the existing literatures. In con-
sideration of these chemosensors for Hg2+ ions based on triazole de-
rivatives, there are generally two different modes. On the one hand,
these compounds bearing triazole units for recognition of Hg2+ ions
exhibited the fluorescence enhancement or quenching without any
variation in colour [32–38]. On the other hand, some probes showed a
highly selective and sensitive response as fluorometric and colorimetric
sensors towards Hg2+ ion accompanied by distinct color changes from
colorless to pink [39–41], or from yellow to purple [42,43], or from
yellow to brownness [44,45], or from yellow to green [46] etc, all of
which provided “naked eye” detection of Hg2+. Above all, there are
several similar aspects such as enhancing the fluorescence signal in
methanol or weakly neutral aqueous solution. Also the stoichiometry of
the complex formed between Hg2+ and triazoles is 1:1 or 2: 1 ratio in
most cases. The similar results could also be found in our previous
The next step is to synthesize RMPC as follows: A mixture of MPC
(100 mg, 0.49 mmol) and a solid of SOCl2 (2.3 mL) in a 10 ml round-
bottomed flask was heated to reflux for 3 h. Then the distillation device
substituted the reflux device and the residuary SOCl2 was distilled out.
The product 2 was obtained and dissolved in 10 mL dichloromethane.
Also it was mixed with rintermediate 1 (230 mg, 0.50 mmol) and 1 mL
ethylenediamine. Then the mixture was stirred at room temperature for
12 h and extracted with H2O and ethyl acetate. The organic phase was
washed for three times with water and dried over Na2SO4. The solvent
was removed by evaporation and the crude product was dried. After
drying, the crude product was purified on a silica column using pet-
roleum ether: ethyl acetate (2:1, v/v) as the eluent to afford product
RMPC(130.2 mg, 41.21%). 1H NMR (400 MHz, CDCl3) δ 8.40 (s, 1H),
7.98 (d, J = 6.4 Hz, 1H), 7.56–7.43 (m, 5H), 7.38–7.35 (m, 2H), 7.12
(d, J = 6.6 Hz, 1H), 6.78 (d, J = 8.5 Hz, 2H), 6.36 (s, 3H), 6.33 (s, 1H),
studies, which revealed
a series of new 1,2,3-triazole appended
rhodamine chemosensors for only selective detection of Hg2+ ions and
again proved in the present study [30,31]. Herein, it is indicated that
the probe RMPC showed a high selectivity for Hg2+ ions but a narrower
linear range and a lower sensitivity. However, RMPC is an advancement
for the application of 1,2,3-triazole compound and provides guidance
for using simple and high-selectivity Hg2+ probes in aqueous solutions
under physiological conditions (pH 7.40).
Based on the favorable biological activity and chemical versatility of
1,2,3-triazoles, there is still a forever demand to exploit and utilize new
1,2,3-triazoles for highly selective and sensitive probing Hg2+ ions in
actual samples. In present work, 5-methyl-1-phenyl -1H-1,2,3-triazole-
4-carboxylic acid (MPC, Scheme 1) was a novel compound generated
from copper-catalyzed oxidative reactions [47]. The structural proper-
ties for MPC such as electron affinity, ionization potential, molecular
orbitals, and predicted spectra had been performed by TD-DFT method
with HF/6-31G(d) and B3LYP/6-31G(d) basis set. Moreover MPC was
designed and synthesized with rhodamine B to produce a new deriva-
tive called RMPC, which showed excellent selectivity to Hg2+ with
colorimetric reaction over other metal ions.
3.34 (q, J = 7.0 Hz, 8H), 2.49 (s, 3H), 1.16 (t, J = 7.0 Hz, 12H);13
C
NMR (100 MHz, CDCl3) δ 165.14, 158.87, 153.66, 152.36, 149.01,
137.82, 136.95, 135.51, 133.15, 129.98, 129.65, 129.26, 128.69,
128.16, 125.27, 124.03, 123.50, 108.17, 104.39, 97.89, 77.40, 77.08,
76.76, 66.04, 44.36, 12.68, 9.77. HRMS (ESI) (m/z): [M+H]+ calcd for
C
38H40N7O3, 642.3193; found, 642.3199.
2.3. Computational study
All quantum chemical calculations were performed by using the
GaussView5.0 program [49] in combined with Gaussian 09 software
[50]. The molecular structures of MPC in the ground state were opti-
mized by the GaussView5.0 program at Hartree-Fock Restricted algo-
rithm level and the time-dependent density functional theory (TD DFT)
including the CIS/6-31G(d) and B3LYP/6-31G(d) methods, respec-
tively. The molecular orbital was analyzed using the HF/6-31G (d)
method. The structural properties of MPC such as frontier molecular
orbital LUMO, HOMO energies, and their band gap were calculated to
exhibit the influence on the electron density transfer within the mole-
cule. Moreover the values of electron affinity and ionization potential of
MPC were carried out through DFT method at the DFT//B3LYP/6-31G*
basis set [50]. The predicted absorption spectra and fluorescence
emission spectra of MPC were calculated by TD DFT method [49–51].
The configurations of the lowest excited state of MPC was fully opti-
mized by the Configuration Interaction with Single excitations (CIS)
method. Also, the molecular structure of the compound RMPTC was
fully optimized by B3LYP method based on Gaussian 09 program
package. Then the structural energy in terms of LANL2TZ basis set for
2. Experimental section
2.1. Materials and instrumentation
MPC was provided by Organic Chemistry Laboratory of Hainan
Normal University, China. The stock solution of MPC (5.0 × 10−4 mol/
L) was prepared in methanol. The tris buffer solution (pH 7.4) was
selected to keep the pH values of the systems. Sodium hydroxide and
chloride salts such as Mg2+, Mn2+, K+, Na+, Co2+, Ca2+, Cd2+
,
Pb2+,Cu2+, Ni2+, Fe3+, Zn2+, Ag+, Hg2+ and Li+ were purchased
from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). All other
reagents were of analytical reagent grade and obtained commercially
without further purification. Doubly distilled water was used
throughout the experiment.
NMR spectra were recorded on a Bruker Avance 400 MHz spectro-
meter at 400 MHz in CDCl3 at room temperature. Fluorometric ex-
periments were performed on a RF-5301PC spectrofluorophotometer
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AnalyticalBiochemistry598(2020)113690
Scheme 1. The synthesis route of RMPC.
Hg and 6-311G(d) basis set for H, C, N, O, and F were calculated [52].
3. Results and discussion
3.1. Geometry optimization of MPC
2.4. Spectra measurements
The theoretical quantum chemistry methods based on HF and DFT
are widely used for the calculation of optimized geometry, absorption
spectrum, UV, IR, and NMR spectra of the organic molecules [53]. In
the present work, the conformational analysis for the molecule MPC
was performed to reach the most stable conformations. Then, the most
stable conformations of MPC was fully optimized, and the corre-
sponding quantum chemical calculations were obtained by using the
GaussView5.0 program at Hartree-Fock Restricted algorithm level and
the time-dependent density functional theory (TD DFT) including HF/6-
31G(d) and B3LYP/6-31G(d) basis sets respectively (as shown in
Fig. 1A). From Fig. 1A, it can be seen that the structure of MPC is
nonplanar with various groups. The compound MPC contains 24 atoms,
and the geometrical parameters of MPC including bond length and
bond angle are listed in Table S1 by using HF/6-31G(d), CIS/6-31G(d),
and B3LYP/6-31G(d) basis sets respectively. It is noticed that the cal-
culated values of bond angles and bond lengths are semblable in all
levels of calculations. The bond angle changed from 103.329 to
133.110°, and the bond length changed from 0.9497 Å to 1.4976 Å.
However, the dihedrals appeared somewhat different because of dif-
ferent computing methods. Specifically, the C–C bond length in benzene
ring is between 1.3544 and 1.4651 Å at three levels, which are much
shorter than that of the classic C–C single bond (1.54 Å), shorter than
that of the typical C–N bond (1.47 Å) and longer than that of the normal
C]C double bond (1.34 Å) [54].
The solutions of RMPC and Hg2+ solutions of different concentra-
tion were prepared by stepwise dilution of a stock solution (0.5 mM for
RMPC and 5 mM for Hg2+, respectively) in water and acetonitrile, re-
spectively. The stock solution of RMPC(60 μL)was added into a quartz
cell, then various metal ions of 50 μM were added respectively. The
solution was diluted with DMF-water (v/v, 1:1, pH 7.4) to obtain a
concentration of 10 μM for RMPC. The fluorescence and UV–vis ab-
sorption spectra titration were carried out by increasing the volume of
different metal ions solution to a solution of RMPC. The excitation
wavelength of the fluorescence experiment is set at 563 nm and that of
emission wavelength at 584 nm, respectively. In order to evaluate the
selectivity of RMPC for Hg2+ detection, 16 kinds of competitive metal
ions, including Pb2+, Mn2+, K+, Na+, Ag+, Ca2+, Cd2+, Co2+, Cu2+
,
Fe3+, Zn2+, Ni2+, Hg2+, Li+ and Mg2+ were measured under the same
experimental condition with a final concentration of 40 μM. The
fluorescence and UV–vis absorbance titrations were repeated for three
times until the reasonable values were obtained.
2.5. Cell culture and fluorescence imaging
HeLa cells were cultured on a 6-well plate with a density of 2 × 10
3 cells per well in cell incubator at 37 °C with a humidified atmosphere
for 12 h before staining. After removing culture media, cells were ad-
ditionally washed with PBS buffer for three times. Then HeLa cells were
incubated with chemosensor RMPC(10 μM) in the culture media con-
taining ethanol for 30 min at 37 °C. The experiments to assess Hg2+
uptake were performed in the same culture media supplemented with
25 μM Hg2+ for 30 min. Moreover the fluorescence images were ac-
quired through fluorescence microscopy.
3.2. Frontier molecular orbital analysis of MPC
Frontier molecular orbitals (FMO's) is one of the most useful and
crucial tools to analyze the highest occupied molecular orbital (HOMO)
and lowest unoccupied molecular orbital (LUMO) in quantum
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AnalyticalBiochemistry598(2020)113690
0
Fig. 1. (A) The optimized structure of MPC; (B1) The frontier molecular orbital
of ground state on HOMO; (B2) The frontier molecular orbital of ground state
on LOMO.
chemistry. Based on the FMO's theory, the interaction between HOMO
and LUMO levels of the reacting subjects can lead to the formation of a
transition state. Generally, LUMO can be thought as the innermost or-
bital containing free places and reflect the ability to accept an electron,
while HOMO can be considered as the outermost orbital which contains
electrons reflecting the ability to donate an electron. The lower energy
of the LUMO means it is easier to accept electrons from some electron-
donating groups. It is reported that the properties of HOMO have
mainly impact on the ionization potential, and the properties of LUMO
have directly impact on the electron affinity [55,56]. Fig. B1, and Fig.
B2 exhibited the LUMO value of MPC (0.10848 ev) and the HOMO
value of MPC (−0.35175 ev) respectively, which indicated that it is
harder for MPC to accept electrons from other electron-donating group.
The energy difference between the LUMO and HOMO orbitals is
named the energy gap, which acts as a crucial stability factor for the
compound [57]. Herein the value of energy gap for MPC is 0.4602 ev,
which indicated that the molecule has a stable structure but lower gap.
A molecule with a small energy gap means more polarizable and is
generally related to high chemical reactivity, low kinetic stability and is
termed as soft molecule [58,59]. So it indicated that the electrons are
more easily to be excited from the ground state to the excited state
because of lower the energy gap, which is confirmed by the following
results obtained from the spectral experiment. Additionally, the diffuse
functions B3LYP/6-311G+(d) at the DFT//B3LYP/6-31G* algorithm
level were applied to calculate some physicochemical values such as
adiabatic electron affinity (1.65 eV), vertical electron affinities
(1.27 eV), adiabatic ionization potential (6.69 eV) and vertical ioniza-
tion potential (7.07 eV). The obtained parameters implied that the
electron accepting ability of MPC is very weak [60].
Fig. 2. (A) The calculated fluorescence emission spectra of MPC. inset: (a) the
experimental fluorescence emission spectra of MPC (60 μM) in methanol; (b)
the experimental fluorescence excitation spectra of MPC (60 μM) in methanol.
(B) The calculated absorption spectra of MPC, inset: the experimental absorp-
tion spectra of MPC (60 μM) in methanol.
Fig. 2A exhibited the emission spectra calculated by TD DFT methods
and that of measured in methanol (plot “a” in the illustration of
Fig. 2A). The calculated spectra displayed the maximum fluorescence
emission peak at 348 nm, which is different from the experimental
spectra measured at 330 nm in the plot “a” of Fig. 2A (inset). The ra-
tional explanation is that the conjugated aromatic compound MPC led
to a π→π* transition from the ground state to an excited state. Owing to
the solvent effect, the polarity of the ground state is weaker than that of
excited state, and a stronger polar solvent can have more influence on
stabilizing the excited state, which results in the position of maximum
fluorescence emission peak shifting towards the shorter wavelength
side [62]. Therefore, methanol, as a higher polar solvent, brought about
the blue-shift effect with the shift of the maximum peak of fluorescence
emission from 348 nm to 330 nm.
The computed electronic absorption spectra of MPC is shown in
Fig. 2B, in which the illustration is the absorption spectra of MPC ob-
tained from the experimental measurement in methanol. From Fig. 2B,
it showed that both of the two graphs showed the distinguishing pro-
files with a broad peak for the computed spectra and a single peak for
the measured spectra. The range of maximum absorption bands for the
computed spectra is from 189 nm to 233 nm, and that of the measured
spectra is only at 205 nm. In theory, the absorption peak at about
203 nm was attributed to E absorption band of π-π* transition, which is
a typical spectral signal for aromatic compounds [63]. However, it is
noticed that the maximum absorption peak is at 205 nm for the mea-
sured spectra, which is in good agreement within the range of the
strong absorption peak obtained from the predicted spectra of MPC.
3.3. Spectral characterization of MPC
According to the FMO's theory, the LUMO-HOMO gap is also an
important factor related to the luminescent properties [57,61]. The
computed results on the energy difference between the LUMO and
HOMO orbitals showed that the electron transition of MPC focused
mainly on HOMO→LUMO transition. It indicated that MPC had optical
properties, which was proved in the following experimental results.
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AnalyticalBiochemistry598(2020)113690
Based on the above fluorescence and absorption spectral analysis,
the theoretically calculated results by DFT method are consistent with
the experimental results in a great degree. According to the relevant
absorption theory, the molar absorption coefficient (ε = 2.126 × 105 L/
mol/cm) of MPC was determined from the plot of concentration vs.
absorbance (Fig. S1 in Supplemental Materials). Due to the special
structure of MPC, it is predicted that MPC should have higher fluores-
cence quantum efficiency because of its fluorescent emission peak at
330 nm and is a prospective blue light emitting fluorescent material in
the organic light-emitting diodes [64].
3.4. Synthesis of RMPC
Many studies reported that the triazole motifs as the platform had
been used to design and develop some new fluorescence chemosensors.
Several highly selective chemosensors for the detection of various metal
ions (such as Zn2+, Cu2+, Fe3+, Cd2+, and Hg2+ etc.) based on click
generated triazole have been synthesized and applied in different fields
[65–68]. In the present study, it is possible and feasible for MPC to
synthesize with rhodamine B hydrazide in principle due to the triazole
structure containing ester group [69]. By improving the experimental
conditions, a new rhodamine B derivative RMPC was synthesized by the
reaction between MPC and the rhodamine hydrazide (Scheme 1). The
structural characterizations of RMPC, including 1HNMR, 13C NMR, and
HRMS, were shown in Fig. S2, Fig. S3, and Fig. S4 respectively in
Supplemental Materials. The results can provide reasonable guidance
for a synthetic reaction between similar compounds containing 1,2,3-
triazole unit and rhodamine B.
3.5. Optimization of detection conditions
The selective recognition ability of RMPC for Hg2+ ions in various
solutions with different pH value was performed. The effect of pH on
the fluorescence intensity was measured between 3.0 and 10.0 for
RMPC (10 μM, plot “a”) and for RMPC-Hg2+ system with a con-
centration of Hg2+ fixed at 50 μM (plot “b”) in Fig. S5, respectively.
From Fig. S5, it can be seen that RMPC showed remarkable fluorescence
when the pH value is less than 4, illustrating that RMPC was a sensitive
acid-responsive probe. While there was not any distinct and specific
fluorescence for RMPC in the pH range from 4.0 to 10.0, which sug-
gested the formation of stable spirocyclic RMPC complex in the range of
the pH value. For RMPC-Hg2+ complex, the fluorescence intensity was
increased under the different pH value between 3.0 and 8.0, which
implied that the complex maintained in a “turn-on” state in this pH
range. However, it is very interesting to notice that the maximum
fluorescence response to Hg2+ was found at the pH value as 7.4. If the
pH value is higher than 7.4, there was no obvious fluorescence en-
hancement for RMPC. The rational explanation is that the metal ions
might bind to the OH− and form partial metal ion precipitation, which
might decrease the actual concentration of metal ions in the sample
solution and cause the decrease of fluorescence intensity. Taking the
optimized pH condition into consideration, the fluorescence and UV–vis
spectroscopic studies on RMPC and corresponding metal complexes
were evaluated in pH 7.4, which also indicated that RMPC was suitable
for detecting under physiological condition.
Fig. 3. The fluorescence spectra (A) and fluorescence intensities (B) at 584 nm
of RMPC (50 μM) in DMF-water (v/v = 1/1, Tris-HCl, pH 7.4) in the presence of
various cations (40 μM) (λex = 563 nm).
3.6. Fluorescence and UV–vis spectra of the interaction of RMPC with Hg2+
The binding behavior of RMPC towards various metal ions (Pb2+
Mn2+, K+, Na+, Ag+, Ca2+, Cd2+, Co2+, Cu2+, Fe3+, Zn2+, Ni2+
,
,
Hg2+, Li+ and Mg2+) was studied by fluorescence and UV–vis spec-
troscopy in DMF/H2O (v/v = 1:1, Tris-HCl, pH = 7.4) solutions.
Fig. 3A displayed the changes on the fluorescence spectra for RMPC in
the presence and absence of various metal ions. Free RMPC (50 μM)
solution is colorless and nonfluorescent, which indicated the rhodamine
moiety in ring-closed spirolactam form. Upon the addition of Hg2+ ion
(40 μM), a fluorescence emission band at 584 nm with excitation at
563 nm was observed, which further suggested ring-opened RMPC-
Hg2+ complex was generated. Besides that, the fluorescence emission
band almost had no changes while other metal ions were added at the
same condition. As shown the appended drawings in Fig. 3B, it ex-
hibited the photographs of the RMPC in the presence of all testing metal
ions. Correspondingly, it is only Hg2+ ion that changed the fluorescence
intensity and the color of RMPC solution from colorless to pink com-
pared to other metal ions. There were not any significant color and
spectral change under identical conditions in the presence of other
metal ions. Generally, rhodamine spirolactam derivatives are colorless
and nonfluorescent, whereas ring-opening of the relevant spirolactam
can cause stronger fluorescence emission and a pink color observed
[69]. The selectivity of RMPC to Hg2+ over other metal ions was
The effect of response time in the present system was measured, and
the results were shown in Fig. S6. It can be seen that the fluorescence
intensity of the RMPC-Hg2+ system at 584 nm gradually enhanced less
than 3 min and a plateau of fluorescence enhancement was achieved
within 60min, which suggested that RMPC reacted with Hg2+ was fast
and the reaction completed within 3 min. Thus the proper response
time is 3 min which can be applied in the related spectroscopic mea-
sures. Moreover RMPC could be used for the real-time detection of
Hg2+ in aqueous samples.
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AnalyticalBiochemistry598(2020)113690
Fig. 4. The absorption spectra of RMPC (50 μM) in the presence of different metal ions (40 μM) in DMF-water (v/v = 1/1, Tris-HCl, pH 7.4). Inset: The photographs
of the RMPC in the presence of Hg2+ and other metal ions under ultraviolet light.
measured by the fluorescence experiments(Fig. 3(B)). It can be seen
that the addition of Hg2+ to RMPC solution led to a 4500-fold increase
in fluorescence intensity at 584 nm. The result indicated that RMPC
could act as a sensor to detect Hg2+ through their distinct fluorescence
“off-on” responses by the naked eye. Moreover it is likely to form a
novel compound between RMPC and Hg2+ due to ring-opening from
the spirolactam (RMPC) to ring-opened amide (RMPC-Hg2+ system). So
it is demonstrated that the developed colorimetric sensor possesses high
selectivity for Hg2+ sensing, which may be due to the unique interac-
consequently became saturated when the concentration of Hg2+
reached 40 μM (Fig. 5A). The linear plot for fluorescence enhancement
of RMPC in the presence of Hg2+ was drawn in the range of
2.67 × 10−5-4.33 × 10−5M (R2 = 0.9511, Fig. S7 in Supplemental
Materials).
Fig. 5B showed the spectral changes of RMPC upon the gradual
addition of Hg2+ by the UV–vis titration experiments. An absorption
band centered at 565 nm appeared and gradually increased with the
successive increase of Hg2+ ion concentration. When the amount of
Hg2+ was increased from 2.33 × 10−5 to 3.67 × 10−5 M, the absorp-
tion intensity at 565 nm gradually enhanced. However, the observed
attainment of the plateau (the illustration in Fig. 5B) suggested sa-
turation in the interaction between the compound RMPC with the ad-
dition of Hg2+. A linear relationship was observed between the ab-
sorbance and the concentration of Hg2+ ions with a correlation
coefficient of R2 = 0.9463 (Fig. S8).
The above results from the fluorescence and UV–vis titration mea-
surements demonstrated that the dynamic response range is too
narrow. We think the most plausible reason is due to the triazole
structure of RMPC only containing an ester group, which led to a less
than ideal response ranges and detection limit. It is indicated that
RMPC was potentially applicable for qualitative analysis rather than
quantitative analysis of Hg2+ ion under physiological conditions (pH
7.40). Some other researches using biomolecular instead of organic
molecular as a probe showed better dynamic response ranges and de-
tection limit [70,71]. We will make attempts to use some biomolecular
materials instead of organic molecular as a biosensor for facile detec-
tion of metal ions in further research.
tion between RMPC and Hg2+
.
Fig. 4 is the UV–vis spectroscopy of RMPC in the absence and pre-
sence of different metal ions measured in DMF-H2O (v/v = 1:1, Tris-
HCl,pH7.40). It is noticed that there was a new absorption band at
565 nm for RMPC-Hg2+ complex with the increasing absorption in-
tensity. Other tested metal ions (such as Pb2+, Mn2+, K+, Na+, Ca2+
,
Cd2+, Co2+, Cu2+, Fe3+, Zn2+, Ni2+, Li+ and Mg2+ ions) did not
exhibit any new band formed under the same experimental conditions.
Similarly, the formation of RMPC-Hg2+ complex also brings about the
color change of the solution, as the same as shown in the photographs
of Fig. 4. The above results suggested that RMPC could be developed for
the noticeable naked-eye detection of Hg2+ ions, accompanied by an
obvious pink color appearing. The changes in color formation could be
explained by the metal-induced delactonization of rhodamine. Upon
RMPC binding with Hg2+ ions, colorless spirolactam form is turned into
its colored ring-opened amide form.
3.7. Sensitivity of RMPC to Hg2+ ions
The fluorescence and UV–vis titration measurements for RMPC with
the progressive addition of Hg2+ ions were carried out, as shown in
Fig. 5. The fluorescence intensity of RMPC (10 μM) at 584 nm was
gradually enhanced with the increasing concentration of Hg2+ ions and
3.8. The binding stoichiometry of RMPC and Hg2+ complex
Several studies showed that rhodamine B and triazoles appended
6

W. He, et al.
AnalyticalBiochemistry598(2020)113690
Fig. 6. (A) The working curve for determining the stoichiometry of RMPC and
Hg2+. (B)The optimized structure of RMPC binding to two of Hg2+ ions.
Fig. 5. (A) The fluorescence spectrum of RMPC (10 μM) in DMF-H2O (v/v = 1/
1, Tris-HCl, pH 7.4) upon increasing concentration of Hg2+ ions ((2.67 × 10−5
-
formation of RMPC-Hg2+ complex as 1:2 stoichiometry was more ra-
tional and stable, which was verified theoretically in the following
experimental result. Fig. 6B depicted a reasonable structure of RMPTC-
Hg2+ complex as 1:2 stoichiometry, and the corresponding parameters
were showed in Table S2. As shown in Fig. 6B, the molecule of RMPC
seems like as two pairs of tongs, one pair of them is formed between one
Hg2+ ion and one nitrogen atom and oxygen atom from hydrazide unit,
another pair came from the ligand between another Hg2+ ion and one
nitrogen atom from triazoles group and one oxygen atoms from car-
bonyl group, respectively. The molecular modeling demonstrated that
RMPC could provide proper space to hold two Hg2+ ions with two
penta cyclic groups formed.
4.33 × 10−5m) (λex = 563 nm nm λem = 584 nm), inset: The plot of fluor-
escence intensity versus concentration of Hg2+ ions. (B) The absorption spec-
tral changes of RMPC (50 μM) at increasing concentrations of Hg2+
(2.33 × 10−5-3.67 × 10−5M), inset: The plot of absorption intensity against
the concentration of Hg2+ ions.
derivatives can bond with various metal ions by displaying a particular
color and the different complex ratio [58,66]. A plot of the fluorescence
intensity versus the molecular fraction of [RMPC]/([RMPC] +[Hg2+ ])
was provided in Fig. 6A by using the equimolar continuous variations
method [72]. The working curve showed that maximum emission in-
tensity was measured for a molar fraction of ca. 0.33, indicating the
binding stoichiometry between RMPC and Hg2+ was 1:2. This result
was also confirmed by quantum-chemistry calculation as follows.
Firstly, both molecular structures of complexes on RMPTC binding to
one and two Hg2+ ions were fully optimized, respectively. Then their
structural energies were determined by using B3LYP procedure in terms
of LANL2TZ basis set for Hg and 6-311G(d) basis set for H, N, C, F, and
O respectively. The calculated results were listed in Table 1 and Fig. 6B.
From Table 1, it can be seen that the formation of RMPC-Hg2+complex
as 1:2 stoichiometry generated the energy difference of ΔE(ev) and ΔE
(a.u.), which were smaller than that of as 1:1 stoichiometry. The values
of bond length for Hg–O in RMPC-Hg2+complex as 1:2 stoichiometry
was shorter than that of as 1:1 stoichiometry. The values of Mayer bond
order for Hg–O in RMPC-Hg2+complex as 1:2 stoichiometry was higher
than that of as 1:1 stoichiometry. These results indicated that the
3.9. Selectivity of RMPC to Hg2+ ions
In order to prove that RMPC was highly selective for Hg2+, the
competition experiments were carried out in the presence of Hg2+
(10 μM) mixed with the other metal ion (50 μM) at the same experi-
mental conditions (as shown Fig. 7). From Fig. 7, it can be seen that
there had not obvious influence on the fluorescence intensity of RMPC-
Hg2+ system by the coexistence of other tested metal ions at this con-
centration. Correspondingly, it is only Hg2+ ions that generated the
fluorescence turn-on responses, and all the measured coexistent metal
ions had no notable changes on the fluorescence intensity. The results
showed that these competitive metal ions have no interference on Hg2+
determination, suggesting RMPC can be applied to detect Hg2+ effec-
tively.
7

W. He, et al.
AnalyticalBiochemistry598(2020)113690
Table 1
Comparison on energy changes of RMPC binding to Hg2+ using the B3LYP functional in Gaussian 09.
RMPC:Hg2+
Energy change
ERMPC(a.u.)
EHg2+(a.u.)
ERMPC:Hg2+(a.u.)
ΔE(a.u.)
ΔE(ev)
1:1
1:2
−2080.90
−2080.90
−41.79
−83.59
−2123.14
−2164.98
−0.44
−0.49
−12.02
−13.42
Bond length (Å)
1:1
1:2
Hg–O
Hg–N
Hg–O
Hg–N
Hg(88)–O(29) 2.46
/
Hg(43)–O(42) 2.31
Hg(43)–N(29) 2.91
Hg(88)–O(41) 2.62
/
Hg(41)–O(40) 2.52
Hg(41)–N(36) 2.36
Mayer bond order
1:1
1:2
Hg–O
Hg–N
Hg–O
Hg–N
Hg(88)–O(29) 0.26
/
Hg(43)–O(42) 0.46
Hg(43)–N(29) 0.16
Hg(88)–O(41) 0.17
/
Hg(41)–O(40) 0.18
Hg(41)–N(36) 0.30
3.10. Proposed mechanism of RMPC response to Hg2+
be more rational and can be assigned to [RMPC+2Hg+Na]. The cal-
culated isotopic patterns (Fig. 8A, inset) matches those measured fairly
well.
Based on the above fluorescence, UV–visible spectral analysis, and
working curve, the proposed mechanism of detecting Hg2+ was shown
in Scheme 2. The compound RMPC is colorless and nonfluorescent,
which indicated the characteristic closed ring state for rhodamine B
domain due to the link to MPC. The addition of Hg2+ made the
spirocycle open via coordination with one molecule of RMP binding to
two molecules of Hg2+. It also exhibited that an obvious color change
from the colorless to pink associated with the reaction of RMPC with
Hg2+ is readily detectable visually, while no significant color changes
are promoted by other metal ions. So RMPC is high selectivity for the
qualitative detection of Hg2+ ions. This binding mode is also consistent
with the fluorescence enhancement changes observed as the induced
conformational changes of the triazole groups RMPC upon 1:2 com-
plexation, which was confirmed by ESI-MS measurement (Fig. 8A). As
shown in Fig. 8A, there is the strongest peak at m/z 902.4, which is
likely to assign to [RMPC+Hg+Na+Cl+2H] with less feasibility.
However, a peak at m/z 1066.3 has stronger intensity, which appears to
In principle, to design a satisfactory fluorescence sensor systems
(the host) for probing small molecules (the guest), it is crucial to utilize
typical chemical reactions or host-guest interactions that lead to a
change in the optical characteristic of the system. If the binding of the
host to the guest is reversible and noncovalent, and the reaction can be
interrupted under certain conditions, the indicator is called as a che-
mosensor [73–75]. Many studies related to the fluorescent chemo-
sensors based on spiraling-opening reactions of rhodamine have been
reported in recent years [51,65,66,69]. If the interaction mode between
the guest and the host is based on an irreversible chemical reaction, the
indicator is referred to as a chemodosimeter. In view of chemodosi-
meters, it is important that there are at least two functional groups. One
of two groups is the reaction region, in which the host interacts with the
detection object. Moreover another group is responsible for a spectral
signal that relies on the reacting to the analyte. Both the interaction
between the detection object and the analyte and the alteration of the
Fig. 7. The fluorescent change of RMPC (50 μM)
toward Hg2+ over various competitive metal ions
(5.0 μM) in DMF-water (v/v = 1/1, Tris-HCl,
pH7.40). λex = 563 nm nm, λem = 584 nm. The
black bars represent the intensity of the solution
upon the addition of Hg2+(50 μM). The red bars
represent the intensity of RMPC in the presence of
other metal ions(50 μM) and Hg2+(50 μM). (For
interpretation of the references to color in this
figure legend, the reader is referred to the Web
version of this article.)
8

W. He, et al.
AnalyticalBiochemistry598(2020)113690
Scheme 2. The proposed recognition mechanism of RMPC towards Hg2+
.
detection signal are irreversible [74]. In general, rhodamine derivative
exhibits a red color variation and distinct fluorescence in acidic solu-
tions by activating a carbonyl group in a spirolactam or spironolactone
unit. Similarly, a proper ligand on a spironolactone ring can cause a
color change as well as a fluorescence change in the presence of some
metal ions, even though the extent of this reaction depended on the
chosen solvent system [69]. In the present work, the binding of RMPC
to Hg2+ could induce the formation of the ring-opened state of RMPC
from the spirolactam state. Therefore RMPC would act as a “naked-eye”
chemodosimeter targeted to Hg2+, similar to some researches on rho-
damine-appended fluorescence indicator for specific detection of metal
ions [76–78].
4. Conclusion
In summary, a novel 1,2,3-triazoles compound, 5-methyl-1-phenyl-
1H-1,2,3 -triazole-4-carboxylic acid (MPC), was studied on the struc-
tural feature, theoretical and experimental spectral characteristic by
quantum chemistry and spectroscopic methods, respectively. The
measured results are consistent with those theoretical calculations de-
rived from structural parameters, which guided for making full use of
MPC in the photophysical field due to its typical spectral property. Then
a new fluorescent chemodosimeter based on rhodamine derivative
RMPC for the detection of Hg2+ was synthesized. The probe displayed
high selectivity for Hg2+ over other metal ions in DMF-H2O (v/v = 1/
1, pH 7.4) solutions with color changes facilitating “naked-eye” re-
cognition for Hg2+. While the 1:2 stoichiometry of the complex formed
between chemodosimeter RMPTC and Hg2+ showed the potential for
3.11. Determination of Hg2+in HeLa cell
quantitative determination of Hg2+ ions. The fluorescence imaging of
2+
Based on the above, it indicated that a color change from the col-
orless to pink associated with the reaction of RMPC with Hg2+ is
readily detectable visually. In order to examine the practical properties
of RMPC in biological samples, the probe was applied for detecting
Hg2+ in HeLa cell by fluorescence microscopy. As depicted in Fig. 9, a
bright-field transmission image of cells with Hg2+ and RMPC confirmed
that the cells were viable throughout the imaging experiments, im-
plying the probe RMPC can penetrate the cell membrane and image
Hg2+ ions in HeLa cells, with the switching-on fluorescent signal.
Hg
in HeLa cells was successfully operated, demonstrating that
RMPC is of good membrane-permeable reagent for the biological ima-
ging applications.
Declaration of competing interest
The authors have declared no conflict of interest.
Fig. 8. (A) ESI-MS of RMPC in the presence of Hg2+ and trace amounts of Cl−. Inset: enlarged patterns for the [RMPC+2Hg+ Na]. (B) The calculated ESI-MS of
RMPC in the presence of Hg2+ and Cl−. Inset: calculated isotopic patterns for the [RMPC+2Hg+ Na].
9

W. He, et al.
AnalyticalBiochemistry598(2020)113690
Fig. 9. The fluorescence imaging of Hg2+ in HeLa cell with RMPC.
(a, c, e) The brightfield transmission images; (b) The images of cells incubated with the FBS culture solution as the sample blank; (d) The images of cells incubated
with RMPC(10 μM); (f) The images of cells incubated with RMPC(10 μM) and Hg2+ (20 μM).
Acknowledgments
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Appendix A. Supplementary data
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