A pH tuning single fluorescent probe based on naphthalene for dual-analytes (Mg2+and Al3+) and its application in cell imaging

Article abstract of DOI:10.1039/d0ra02101f

In this study, a naphthalene Schiff-basePwhich serves as a dual-analyte probe for the quantitative detection of Al³⁺and Mg²⁺has been designed. The proposed probe showed an ‘‘off-on’’ fluorescent response toward Al³⁺in etha

Full text of DOI:10.1039/d0ra02101f

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A pH tuning single fluorescent probe based on
naphthalene for dual-analytes (Mg2+ and Al3+) and
its application in cell imaging†
Cite this: RSC Adv., 2020, 10, 21399
ac
Chunwei Yu,a Shuhua Cui,b Yuxiang Ji,a Shaobai Wen,a Li Jiana and Jun Zhang
*
In this study, a naphthalene Schiff-base P which serves as a dual-analyte probe for the quantitative detection
of Al3+ and Mg2+ has been designed. The proposed probe showed an ‘‘off–on’’ fluorescent response toward
Al3+ in ethanol–water solution (1 : 9, v/v, pH 6.3, 20 mM HEPES) over other metal ions and anions, while the
detection by the probe could be switched to Mg2+ by regulating the pH from 6.3 to 9.4. The sensing
mechanisms of P to Al3+/Mg2+ are attributed to inhibition of the photo-induced electron transfer (PET)
process by the formation of 1 : 1 ligand–metal complexes. More importantly, the probe was applied
Received 5th March 2020
Accepted 27th May 2020
DOI: 10.1039/d0ra02101f
rsc.li/rsc-advances
successfully in living cells for the fluorescent cell-imaging of Al3+ and Mg2+
.
silent spectroscopic characteristics and poor coordination
ability.7 In particular, most of these reported probes focus on
the detection of single metal ions,8 so it will be complicated and
inconvenient to analyse some coexisting metal ions based on
different detection methods and/or experimental conditions.
For these reasons, much attention has been paid to single
probes for multiple analysis and they have been widely applied
in metal detection because they could determinate more than
one interesting ion, simultaneously. Some multi-target probes
such as Cr3+/Al3+,9 Bi3+/Zn2+,10 Zn2+/Mg2+,11 and Cu2+/Al3+ have
been developed,12 but multi-ion responsive molecular uores-
cence probes which sense Al3+/Mg2+ have rarely been reported.
More problematically, the ionic radius and charge of Al3+ make
it a competitive inhibitor of Mg2+ in the human body. In this
regard, the design of reliable and sensitive multi-target probes
for Mg2+ and Al3+ is highly desirable.
Schiff bases can coordinate with various metal ions and form
stable complexes which are known to be good ligands for metal
ions.13 Beyond this, naphthalene derivatives are excellent uo-
rophores that usually serve as signal reporters in probes
because of their short uorescence life-time, low uorescence
quantum yields, and ability to act as a donor or an acceptor.14
Meanwhile, benzoyl hydrazine derivatives are recognized as
important motifs for use as ligands in coordination chemistry.15
Moreover, based on the special advantage of turn-on uorescent
probes in reducing system errors and background interfer-
ence,16 we aimed to develop a novel turn-on uorescent probe
combining different recognition groups in one molecule, which
was expected to multi-channelly detect Al3+ and Mg2+ with
different turn-on uorescent responses.
1 Introduction
The detection of trace metal ions is becoming an important
concern due to their assignable functions in biological systems
and deleterious effects on public health.1 Among these metals,
Mg2+ is an essential micronutrient element for human life,
participating in gene transcription and neural signal trans-
mission.2 It also plays important roles in enzymes and DNA-
binding proteins.3 However, overloading of magnesium in the
cytosol and subcellular regions exhibits toxicity and is linked
with diseases such as diabetes, hypertension, epilepsy and
Alzheimer's.4 As another highly chemical reactive metal ion,
Al3+ is a non-essential element for living systems and of
signicant environmental concern: an overdose of Al3+ can
damage the central nervous system and produce some neuro-
behavioral and neuro-pathological changes.5 In order to protect
human health, the development of effective tools for the
detection of Al3+ and Mg2+ is of a great importance.
In recent years, molecular uorescence probes with simple
handling, real-time response, high sensitivity and selectivity,
and non-destructive properties have undoubtedly been power-
ful detection tools.6 Compared to several transition metal ions,
such as Hg2+, Cu2+ and Zn2+, only a few uorescent probes have
been developed for the detection of Mg2+ or Al3+ due to their
aLaboratory of Environmental Monitoring, School of Tropical and Laboratory
Medicine, Hainan Medical University, Haikou, Hainan 571101, P. R. China. E-mail:
jun_zh1979@163.com
bWeifang University of Science and Technology, Shouguang, Shandong 262700, P. R.
China
cKey Laboratory of Tropical Translalional Medicine of Ministry of Education, School of
Tropical Medicine and Laboratory Medicine, Hainan Medical University, Haikou,
Hainan 571199, P. R. China
In this work, a uorescent probe (P) based on a Schiff base
combining naphthalene and benzoyl hydrazine was proposed
(Scheme 1). P can be utilized as a multi-ion uorescent probe by
regulating pH: it showed ‘‘off–on’’ uorescent response toward
† Electronic supplementary information (ESI) available. See DOI:
10.1039/d0ra02101f
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solution. For all measurements, excitation and emission slit
widths were 5/5 and 5/10 nm, and excitation wavelengths were
408 and 415 nm for Mg2+ and Al3+, respectively.
2.3 Cell culture
HepG2 cells were purchased from the Committee on Type
Culture Collection of Chinese Academy of Sciences (Shanghai,
China). HepG2 cells were incubated in DMEM (Dulbecco's
Modied Eagle's Medium) supplemented with 10% fetal bovine
serum (FBS).
Scheme 1 Synthesis route of probe P.
Al3+ in ethanol–water solution (v/v, 1 : 9, pH 6.3, 20 mM HEPES)
and Mg2+ in ethanol–water solution (v/v, 1 : 9, pH 9.4, 20 mM
HEPES). More importantly, the probe could be used for cell
imaging of Al3+ and Mg2+ in vivo with negligible cytotoxicity.
2.4 Cell imaging
For imaging experiments, cells were grown in 6-well plates at 70–
80% conuence. Cells were then incubated in DMEM containing
ꢀ
10 mM P for 30 min at 37 C. Cells were then washed with PBS
followed by the addition of 1 mM Al3+ and Mg2+ and incubated for
30 min. Bright eld and uorescence images were captured by
a orescence microscope (Olympus FluoView Fv1000).
2 Experimental section
All reagents and solvents are commercially available and used
directly. All reagents and solvents are of analytical grade and
used without further purication. The metal ions salts used 2.5 Cell cytotoxicity assay
were NaCl, KCl, CaCl2$2H2O, MgCl2$6H2O, HgCl2, Zn(NO3)2-
To assess the cytotoxicity of P, cytotoxicity was measured by
$6H2O, PbCl2, CdCl2, CrCl3$6H2O, AgNO3, CoCl2$6H2O, NiCl2-
$6H2O, CuCl2$2H2O, FeCl3$6H2O, FeCl2$4H2O and AlCl3$6H2O.
Fluorescence emission spectra were conducted on a Hitachi F-
4600 spectrouorometer. UV-vis spectra were obtained on a Hita-
chi U-2910 spectrophotometer. Nuclear magnetic resonance
(NMR) spectra were measured with a Brucker AV 400 instrument
and chemical shis were given in ppm from tetramethylsilane
(TMS). Mass (MS) spectra were recorded on a Thermo TSQ
Quantum Access Agilent 1100. Fluorescence imaging was per-
formed by confocal uorescence microscopy on an Olympus
FluoView Fv1000 laser scanning microscope (USA). pH values
were measured with a pH-meter PBS-3C (Shanghai, China).
using the methyl thiazolyl tetrazolium (MTT) assay in HepG2
cells. HepG2 cells were seeded into a 96-well cell culture plate at
4000/well, cultured at 37 ꢀC and 5% CO2 for 24 h, and then
different concentrations of P (0, 0.1, 1.0, 10.0 mM) were added to
the wells. The cells were then incubated for 24 h at 37 ꢀC under
5% CO2. Subsequently, 20 mL of MTT (5 mg mLꢁ1) was added to
each well and incubated for an additional 4 h at 37 ꢀC under 5%
CO2. Cells were lysed in triple liquid (10% SDS, 0.012 M HCl, 5%
isopropanol), and the amount of MTT formazan was qualied
by determining the absorbance at 570 nm using a microplate
reader (Tecan, Austria).
The following formula was used to calculate the inhibition of
cell growth: cell viability (%) ¼ (mean of Abs. value of treatment
group/mean Abs. value of control) ꢂ 100%.
2.1 Synthesis of P
2-Hydroxy-1-naphthaldehyde (0.172 g, 1.0 mmol) and sali-
cylhydrazide (0.152 g, 1.0 mmol) were mixed in ethanol (30 mL)
and stirred under reux for 4 h. Aer the reaction was nished,
the mixture was cooled to room temperature and poured into
cold water. The precipitate so obtained was ltered and washed
with ethanol and water in turn, and then dried in a vacuum to
afford P as a yellow solid. Yields: 85.6%. MS m/z: 307.24 [M +
H]+, 329.21 [M + Na]+. 1H NMR (dppm, d6-DMSO): 12.10 (s, 1H),
11.72 (b, 1H), 9.55 (s, 1H), 8.32 (d, 1H, J ¼ 6.00), 7.94 (t, 2H, J ¼
6.00), 7.91 (t, 1H, J ¼ 12.00), 7.61 (s, 1H), 7.48 (s, 1H), 7.42 (s,
1H), 7.24 (d, 1H, J ¼ 6.00), 7.03 (d, 2H, J ¼ 12.00), 7.01 (s, 1H).
13C NMR (dppm, d6-DMSO): 164.90, 159.69, 159.07, 148.61,
134.95, 133.85, 132.62, 129.86, 129.71, 128.75, 128.67, 124.49,
121.91, 120.08, 119.82, 118.23, 116.65, 109.53 (Fig. S1–S3, ESI†).
3 Results and discussion
3.1 Fluorescence study
A pH titration experiment was rst evaluated, as shown in
Fig. S4 (ESI†), from the experimental results, the uorescence
from the free P could be seen to be negligible in the pH range
from 4.0 to 10.0, suggesting that it was not susceptible to the
change in acid–base solution. The uorescence of the P–Al3+
complex displayed a plateau in the pH range from 4.0 to 6.5, and
the maximum response toward Al3+ was obtained under pH 6.3.
From the point of view of sensitivity and speed, in our experi-
ment, pH 6.3 was chosen as the optimum experimental condi-
tion for environmental examples. The host–guest recognition
abilities of P with Al3+ and Mg2+ were investigated via a uo-
rescence spectroscopic method. As shown in Fig. 1a, free P
showed weak uorescence emission at 475 nm when it was
2.2 General spectroscopic methods
Metal ions and P were dissolved in deionized water and DMSO excited at 425 nm in ethanol–water (v/v, 1 : 9, pH 6.3, 20 mM
to obtain 1.0 mM stock solutions. Before spectroscopic HEPES). Only upon the addition of Al3+ did the uorescence
measurements, the solution was freshly prepared by diluting intensity of P show a signicant uorescence enhancement at
the high concentration stock solution to the corresponding 475 nm over other relevant metal ions (Na+, K+, Ca2+, Mg2+, Pb2+,
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Cr3+, Mn2+, Fe2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+ and Hg2+).
The results suggested that probe P, with predominant recog-
nition and selectivity, shows good potential in the detection of
Al3+. More interestingly, when the pH was adjusted to 9.4
(Fig. S5, ESI†), the metal ion switched to Mg2+. The probe did
not give any observable response in the absence or presence of
various other metal ions, and the blue shi of the emission
band at 460 nm was accompanied by a 49-fold uorescence
enhancement for Mg2+, as depicted in Fig. 1b.
Under physiological conditions, the emission of probe P
(10 mM) around 475 nm is very low. However, upon gradual
addition of Al3+ or Mg2+ there was a remarkable enhancement in
uorescence at 475 nm for Al3+ or 460 nm for Mg2+ with an
increasing concentration (0–10 mM) of the metal ion (Fig. 2). The
plots of emission intensity of P as a function of added Al3+ and
Mg2+ are presented in insets to Fig. 2a and b, respectively.
Additionally, from the uorescence titration proles, the
detection limit of P for Al3+/Mg2+ was found to be 0.3 mM/0.2 mM
(based on S/N ¼ 3, inset of Fig. 2), which was sufficiently low to
enable the detection of micromolar concentrations of Al3+/Mg2+
in many chemical and biological systems.
Fig. 1 (a) Fluorescence emission spectra of P (10 mM) in response to
different metal ions (10 mM) in ethanol–water solution (1 : 9, v/v,
20 mM HEPES, pH 6.3); (b) fluorescence emission spectra of P (10 mM)
in response to different metal ions (10 mM) in ethanol–water solution
(1 : 9, v/v, 20 mM HEPES, pH 9.4).
3.2 UV-vis analysis
With the objective of evaluating the potential use of the probe P,
UV-vis analysis upon addition of Al3+/Mg2+ was further evalu-
ated. Firstly, the interaction of P and Al3+ was investigated as
a function of the concentration of Al3+, as shown in Fig. 3.
Fig. 2 (a) Fluorescence response of P (10 mM) with various concen-
trations of Al3+ in ethanol–water solution (1 : 9, v/v, 20 mM HEPES, pH
6.3). Inset: the fluorescence of P (10 mM) as a function of Al3+
concentration (0.5–5.0 mM); (b) fluorescence response of P (10 mM)
with various concentrations of Mg2+ in ethanol–water solution (1 : 9,
v/v, 20 mM HEPES, pH 9.4). Inset: the fluorescence of P (10 mM) as
a function of Mg2+ concentration (1–8.0 mM).
Fig. 3 (a) Absorbance of P (10 mM) with various concentrations of Al3+
in ethanol–water solution (1 : 9, v/v, 20 mM HEPES, pH 6.3); (b)
absorbance of P (10 mM) with Al3+ (100 mM) in ethanol–water solution
(1 : 9, v/v, 20 mM HEPES, pH 6.3).
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The spectrum of free P showed a maximum absorption band a prerequisite in developing a uorescent probe for practical
at 350 nm in ethanol–water solution (1 : 9, v/v, 20 mM HEPES, applications. The reversibility of P was studied by adding Na2-
pH 6.3), which can be assigned to the p–p* transition of the EDTA as a bonding agent (Fig. S8 and S9, ESI†). The addition of
benzoyl hydrazine group.15 With increasing concentration of Na2EDTA to a mixture of P and Al3+/Mg2+ caused a diminution
Al3+, the absorption bands at 325 nm and 375 nm gradually in the uorescence intensity at 475/460 nm, which may produce
decreased, and simultaneously a new band appeared at 425 nm the free probe P. Upon the addition of Al3+/Mg2+, the uores-
with increased intensity. Moreover, a clear isosbestic point at cence intensity of P displayed a signicant uorescence
380 nm was observed, which clearly indicated the presence of enhancement again, which proved the binding between P and
new complex P–Al3+. In contrast, when the UV-vis analysis was Al3+/Mg2+ was reversible.
carried out in ethanol–water solution (1 : 9, v/v, pH 9.4, 20 mM
HEPES), the metal ion was also changed from Al3+ to Mg2+, as
shown in Fig. 4. The trend in the change in the absorption
3.4 Proposed binding mode of P with Al3+/Mg2+
In order to understand the binding mode of P and Al3+/Mg2+,
spectrum of the latter was similar to the former; the only
the Job's plot of P and Al3+/Mg2+ was conducted (Fig. 5). When
difference was the extent of variation of the absorption at
the molar fraction of P and Al3+/Mg2+ was 0.5, P with Al3+/Mg2+
425 nm. Absorption spectra conrmed the binding ability of P
and Mg2+/Al3+
.
exhibited maximum uorescence emission. The results showed
that P and Al3+/Mg2+ formed 1 : 1 ligand–metal complexes.
Furthermore, the ESI-MS spectra also conrmed this conclu-
sion (Fig. S10 and S11, ESI†), in which the peaks at m/z 331.0,
3.3 Practical applicability of P
366.7, 376.7, 413.2, and 423.2 were assignable to [P + Al3+
2H+]+, [P + Al3+ + Clꢁ ꢁ H+]+, [P + Al3+ + EtOH ꢁ 2H+]+, [P + Al3+
ꢁ
In order to evaluate the practical applicability of P as a selective
multi-analyst uorescent probe for Al3+/Mg2+, competition
experiments were conducted (Fig. S6 and S7, ESI†). It could be
seen that other competitive ions had no obvious interference
with the detection of Al3+/Mg2+ under different pH conditions,
which could be attributed to their inherent magnetic properties.
Meanwhile, reversibility was investigated which is
+
Clꢁ + EtOH ꢁ 2H+]+, and [P + Al3+ + 2EOH ꢁ 2H+]+, respectively.
The peaks at m/z 328.7, 365.1, 347.1, 375.1, and 438.8 were
assignable to [P + Mg2+ ꢁ H+]+, [P + Mg2+ + Clꢁ]+, [P + Mg2+
+
OHꢁ]+, [P + Mg2+ + EtOH ꢁ H+]+, and [P + Mg2+ + 2EtOH + OHꢁ]+,
respectively. As expected, the results indicated that complexes
with a stoichiometry of Al3+/Mg2+ to P of 1 : 1 were formed, and
the results were also supported by the Benesi–Hildebrand
method.15 The Benesi–Hildebrand analysis of the emission data
gives a 1 : 1 stoichiometry for P–Al3+ and P–Mg2+ complexation
Fig. 4 (a) Absorbance of P (10 mM) with various concentrations of
Mg2+ in ethanol–water solution (1 : 9, v/v, 20 mM HEPES, pH 9.4); (b)
absorbance of P (10 mM) with Mg2+ (100 mM) in ethanol–water solution Fig. 5 Job's plot for determining the stoichiometry of P and (a) Al3+
(1 : 9, v/v, 20 mM HEPES, pH 9.4).
and (b) Mg2+. The total concentration was kept at 50 mM.
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Scheme 2 Proposed binding modes of P with Al3+ and Mg2+
.
3.6 Biological application of P with Al3+/Mg2+
Fig. 6 1H NMR titration of P with Mg2+
.
To investigate the potential biological applications of probe P in
living cells, we performed intracellular Al3+ and Mg2+ dual
imaging of HepG2 cells by uorescence microscopy. Aer
incubation with P for 30 min at 37 ꢀC, the cells could not show
any recognizable uorescence, suggesting that auto-
uorescence from the cells can be avoided and no uorescence
signal was detected in cells treated only with P (Fig. 7(a-I) and
(b-I)). However, under these conditions, for the P-loaded HepG2
cells, strong uorescence was detected aer the addition of
exogenous Al3+ and Mg2+ (1 mM each) separately to the cells,
which showed green and orange uorescence (Fig. 7(a-II) and
(b-II)), respectively, demonstrating membrane penetrability by P
and its complexation with Al3+ and Mg2+ inside the cells, clearly
demonstrating that Al3+ and Mg2+ and their interaction with P
are essential for the uorescence turn-on. The uorescence
signal of P in the presence of Al3+ and Mg2+ may be utilized as
a signature for a selective probe response. Hence, these results
indicate that probe P is an efficient candidate for monitoring
changes in the intracellular Al3+ and Mg2+ concentration under
biological conditions. The bright eld images of Fig. 7(a-III) and
(b-III), whose cell shapes indicated that P has low toxicity, reveal
good biocompatibility of P for bioanalysis.
species, with association constants (Ka) being calculated as 2.9
ꢂ 104 Mꢁ1 and 2.1 ꢂ 105 Mꢁ1 (Fig. S12 and S13, ESI†), corre-
sponding to a stronger binding capability toward Al3+ in
comparison with a naphthalene-based PET chemosensor for
Al3+ (with a K value of 5.1 ꢂ 103 Mꢁ1),16a and a rhodamine spi-
rolactam derivative-based chemosensor for Mg2+ (with a K value
of 2.55 ꢂ 104 Mꢁ1).16b
3.5 Proposed mechanism of P with Al3+/Mg2+
According to reported work,15a,17 probes with only OHa show good
selectivity to Al3+. Based on the so–hard acid–base theory, Al3+
shows affinity to groups with O sites. In order to design probes
with better selectivity and sensitivity to Al3+, P with two OH
groups was proposed in this work. As mentioned above, the
proposed probe still has good selectivity to Al3+ at pH 6.3, and
meanwhile shows good selectivity to Mg2+ at pH 9.4. To evaluate
the binding pattern between P and Mg2+, 1H NMR titration
experiments in DMSO-d6 were carried out (Fig. 6). The phenolic
OH peaks (protons Ha and Hb) and the imine group NH (proton
Hc) of P were observed at d12.10, 11.72 and 9.55, respectively. An
1H NMR titration experiment of P shows that the proton Hb at
d11.7243 ppm disappeared on adding Mg2+, and the proton
signals of the imine group NH (Hc) at d9.5507 ppm and –CH]N
at d7.9390 ppm corresponding to downeld shis to 10.0111 and
7.9473, respectively (Fig. S14, ESI†).
P also was applied to the subcellular locations of Al3+ and
Mg2+ in the HepG2 cells using confocal uorescence micros-
copy. The cells were co-treated with P (10 mM) and Hoechst
33 342 (1 mg mLꢁ1) for 30 min, with the same conditions as
Based on these results, the selective recognition of P for Al3+
/
Mg2+ should be attributed to the interaction of the benzoyl
hydrazine moieties with Al3+/Mg2+, which inhibit the photo-
induced electron transfer (PET) process. As shown in Scheme
2, it seemed that the lone pair of electrons from the nitrogen
atom of the –C]N– group to the benzoyl hydrazine moieties
was responsible for the photoinduced electron-transfer (PET)
process, which quenched the uorescence emission of the
probe. However, upon addition of Al3+/Mg2+, the PET process
was inhibited owing to the chelation of the nitrogen atom of the
–C]N– group with Al3+/Mg2+; as result, the quenched uores-
cence could recur remarkably.
Fig. 7 (a-I) and (b-I) are the field images of P (10 mM) treated HepG2
cells. (a-II) and (b-II) represent fluorescence images treated with P (10
mM), in the presence of Al3+ or Mg2+ (1 mM), respectively; (a-III) and (b-
III) indicate bright field images of cells shown in the panels, respec-
tively. For all imaging, the samples were excited at 375 nm.
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those used in Fig. 8. The results further reveal that P locates
primarily in the cytoplasm of these living HepG2 cells, as shown
in Fig. 8. To evaluate the cytotoxicity of the probe, P was taken as
an example to perform an MTT assay on HepG2 cells with dye
concentrations from 0 mM to 10 mM. The MTT assay results
conrmed that P has no signicant toxicity to cultured HepG2
cells up to 48 h of treatment with 10 mM of P (Fig. S15, ESI†).
Thus, P has promising potential in the dual sensing of Al3+ and
Mg2+ in vitro.
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4 Conclusions
In summary, we developed a single uorescent probe which
displayed a distinct response to Al3+ and Mg2+. The probe
showed ‘‘off–on’’ uorescent responses toward Al3+ at pH 6.3 in
ethanol–water solution. When the pH was changed from 6.3 to
9.4, the detection of the probe could respond to Mg2+. In
addition, the cell imaging for Al3+/Mg2+ was satisfactory.
However, multi-ion responsive molecular probes with multiple
emission modes will be challenging tasks for a long time into
the future.
Conflicts of interest
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There are no conicts to declare.
Acknowledgements
This work was nancially supported by the National Science
Foundation of China (No. 81760387, 81860381, 81660356) and
the Natural Science Foundation of Hainan Province (No.
417149).
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