Selective “off-on” detection of magnesium (II) ions using a naphthalimide-derived fluorescent probe

Article abstract of DOI:10.1016/j.dyepig.2017.07.033

A novel “off-on” fluorescent probe based on 1, 8-naphthalimide derivative for the detection of Mg²⁺ in ethanol solution was designed and synthesized. The probe displayed responses to Mg²⁺ with a fluorescence enhancement at 523 nm, accompanying with a distinct fluorescence change from nearly colorless to bright yellow-green. Besides, the probe showed a rapid detection process (45 s), high fluorescence enhancement (up to 15-fold), a good binding constant (6.17 × 10⁵ M^?1) and a low detection limit of Mg²⁺ (5.01 × 10^?8 M). Moreover, density functional theory (DFT) calculations were also performed to support the responding mechanism between the probe and the coordination complex. In addition, the cell cytotoxicity experiments suggested that the probe is nearly non-toxic and the fluorescence scanning microscopic experiments demonstrated that the probe was capable of monitoring at the intracellular Mg²⁺ level successfully.

Full text of DOI:10.1016/j.dyepig.2017.07.033

Accepted Manuscript
Selective “off-on” detection of magnesium (II) ions using a naphthalimide-derived
fluorescent probe
Huifang zhang, Caixia Yin, Tao Liu, Jianbin Chao, Yongbin Zhang, Fangjun Huo
PII:
S0143-7208(17)31283-4
10.1016/j.dyepig.2017.07.033
DYPI 6122
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 4 June 2017
Revised Date: 14 July 2017
Accepted Date: 15 July 2017
Please cite this article as: zhang H, Yin C, Liu T, Chao J, Zhang Y, Huo F, Selective “off-on” detection of
magnesium (II) ions using a naphthalimide-derived fluorescent probe, Dyes and Pigments (2017), doi:
10.1016/j.dyepig.2017.07.033.
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Abstract Graphic

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Selective “off-on” detection of Magnesium (II) ions using
naphthalimide-derived fluorescent probe
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Huifang zhanga,1, Caixia Yina,*, Tao Liua,1, Jianbin Chaob, Yongbin Zhangb, Fangjun
Huob,**
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aKey Laboratory of Chemical Biology and Molecular Engineering of Ministry of
Education, Key Laboratory of Materials for Energy Conversion and Storage of
Shanxi Province, Institute of Molecular Science, Shanxi University, Taiyuan, 030006,
China;bResearch Institute of Applied Chemistry, Shanxi University, Taiyuan, 030006,
China.
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*Corresponding author: C.X. Yin, E-mail: yincx@sxu.edu.cn;
**Corresponding author: F.J. Huo, Email: huofj@sxu.edu.cn.
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Abstract: A novel “off-on” fluorescent probe based on 1, 8-naphthalimide derivative
for the detection of Mg2+ in ethanol solution was designed and synthesized. The probe
displayed responses to Mg2+ with a fluorescence enhancement at 523 nm,
accompanying with a distinct fluorescence change from nearly colorless to bright
yellow-green. Besides, the probe showed a rapid detection process (45 s), high
fluorescence enhancement (up to 15-fold), a good binding constant (6.17×105 M-1)
and a low detection limit of Mg2+ (5.01×10-8 M). Moreover, density functional theory
(DFT) calculations were also performed to support the responding mechanism
between the probe and the coordination complex. In addition, the cell cytotoxicity
experiments suggested that the probe is nearly non-toxic and the fluorescence
scanning microscopic experiments demonstrated that the probe was capable of
monitoring at the intracellular Mg2+ level successfully.
Keywords: Fluorescent probe; off-on; Mg2+; DFT calculation; Bioimaging.

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1. Introduction
The magnesium (Mg2+) ion is an abundant divalent cation in the cells and
environment [1-2]. Intracellular Mg2+ plays important roles in many physiological
processes including the activities of ion channels in membrane proteins and regulation
of hundreds of enzymes such as RNA polymerases, ATPases, protein kinases,
phosphatases, glutathione synthase, and carboxylases and so on [3-18]. Moreover, a
decrease in Mg2+ concentration and abnormal metabolic processing of intracellular
Mg2+ has been implicated in the causes of gastrointestinal, renal losses,
hypocalcaemia and neurological manifestations, especially, a number of chronic
diseases, such as diabetes, osteoporosis, and coronary heart have been associated with
chronic low magnesium [19]. In the environment, high levels of Mg2+ will become
toxic to plants, which can enhance the role of antagonism between ions when crops
absorb the nutrient elements, while magnesium deficiency can affect plant growth and
development, which led to the decrease of productivity and quality in agriculture
[20-21]. Therefore, it is crucial to develop efficient analytical methods for detecting
and monitoring of Mg2+ at the level of cells and in the environment.
Based on that, the fluorescence measurements go into people’s research scope
and small molecules fluorescent probes gradually get an active focus of research in
biological and environment field. Small molecules fluorescent probes are able to bind
the analyte selectively with a change in the sensing system property, such as
absorption, emission and redox potentials [22-24]. Besides, they are usually very
sensitive, low cost, easily performed, capability of real-time detection [25-27]. Until

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now, various fluorescent probes for Mg2+ have been developed. These probes have
receptor groups based upon moieties including diaza-28-crown-6, benzo-15-crown-5,
calix[4]arene, Benzo chromene, Imdiazo-1, 10-phenanthroline and other [1, 7, 17,
28-39]. Most of the reported fluorescent probes for Mg2+ demonstrate poor selectivity
for Mg2+ over Ca2+ and are available only when the Mg2+ ion concentrations are much
high than Ca2+ ion [40].
Enlightened by this, we have driven 1, 8-naphthalimide to react with
2-hydrazinylbenzo[d]thiazole to generate O-hydroxyl Schiff base that possessing O
and N atoms with specific affinity to Mg2+ based on hard and soft acids and bases rule
(HSAB) and we choose O-hydroxyl Schiff base as recognizer to realize “off-on”
fluorescence recognition [41-44]. Herein we demonstrated the recognition
performance of probe for Mg2+ in ethanol solution, and the simple structure molecule
showed the rapid respond ability for Mg2+ with no interference from calcium ion as
we expected. Besides, the probe showed a rapid detection process (45 s), high
fluorescence enhancement (up to 15-fold), a good binding constant (6.17×105 M-1)
and a low detection limit of Mg2+ (5.01×10-8 M). Also the probe could detect the
intracellular Mg2+ in A549 cells by fluorescence bio-imaging under confocal laser
scanning microscopy. All of the above results make probe have a superior potential
for the detection of Mg2+ in complex bio-systems.
2. Experimental section
2.1 Instruments and materials
All chemicals and solvents were of analytical grade and bought from

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Sigma-Aldrich (St. Louis, MO) without further purification. The solutions of cation
were prepared from their chloride (CuCl2, ZnCl2, CrCl3, HgCl2, NiCl2, NaCl, KCl,
BaCl2, CaCl2, FeCl3, MnCl2, CoCl2, PbCl2, CdCl2, AlCl3 and MgCl2) and nitrate
(AgNO3). Fresh double distilled water was used throughout the experiment.
A pH meter (Mettler Toledo, Switzerland) was used to determine the pH.
Ultraviolet-visible (UV-vis) spectra were recorded on a Cary 50 Bio UV-Visible
spectrophotometer and fluorescence spectra were measured on F-7000 FL
fluorescence spectrophotometer. A PO-120 quartz cuvette (10 mm) was purchased
from Shanghai Huamei Experiment Instrument Plants, China. 1H NMR and 13C NMR
spectra were recorded on a Bruker AVANCE III-600 MHz and 150/151 MHz NMR
spectrometer (Bruker, Billerica, MA) respectively, using tetramethylsilane as the
internal standard for chemical shifts. ESI-MS was carried out on AB Triple TOF
5600plus System (AB SCIEX, Framingham, USA). MS was carried out on Thermo
Scientific Q Exactive. The cell viability assay was carried out by CCK-8 and
measured at 450 nm on a microplate reader. The bioimaging was imaged by laser
confocal fluorescence imaging using Airyscan confocal laser scanning microscope.
2.2 Preparation and characterization of the probe
2.2.1 Preparation and characterization of 2
The synthesis approach of probe is summarized in Scheme 1. A mixture of
4-bromo-1, 8-naphthalic anhydride (0.831 g, 3 mmol) and n-butylamine (0.219 g, 3
mmol) in ethanol (30 mL) was refluxed for 5 h. After the reaction was completed, the
reaction mixture was evaporated in vacuum and dried to obtain a solid product. Later

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on, the solid was purified by chromatography on a silica gel column in
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dichloromethane resulted in a pale yellow solid product (0.877 g) in 88% yield. H
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NMR (DMSO-d6, 600 MHz): δ (ppm): 8.53 (d, J = 7.9 Hz, 2H), 8.30 (d, J = 7.6 Hz,
1H), 8.19 (d, J = 7.7 Hz, 1H), 7.98 (t, J = 7.8 Hz, 1H), 4.02 (t, J = 7.3 Hz, 2H), 1.61
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(m, 2H), 1.36 (m, 2H), 0.93 (t, J = 7.4 Hz, 3H). C NMR (DMSO-d6, 150 MHz): δ
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(ppm): 163.2, 132.9, 131.9, 131.7, 131.3, 130.1, 129.5, 129.2, 128.6, 123.1, 122.3,
30.0, 20.3, 14.2 (Fig. S1).
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2.2.2 Preparation and characterization of 3
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To a 100 mL radius flask, compound 2 (0.664 g, 2 mmol) and sodium methoxide
(0.108 g, 2 mmol) in methanol (20 mL) was refluxed for 8 h while CuSO4·5H2O
served as catalyst. Then the crude product was evaporated in vacuum and the residue
was added HCl (1 mol/L, 30 mL) to crystallize out. Subsequently, a solid separated by
filtration was purified by chromatography on a silica gel column in dichloromethane
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to give a white product (0.493 g) in 87% yield. H NMR (DMSO-d6, 600 MHz): δ
(ppm): 8.49 (d, J = 8.3 Hz, 1H), 8.46 (d, J = 7.2 Hz, 1H), 8.42 (d, J = 8.2 Hz, 1H),
7.79 (t, J = 7.8 Hz, 1H), 7.29 (d, J = 8.3 Hz, 1H), 4.12 (s, 3H), 4.01 (t, J = 7.4 Hz, 2H),
1.60 (m, 2H), 1.35 (d, J = 14.8, 7.4 Hz, 2H), 0.93 (t, J = 7.4 Hz, 3H). 13C NMR
(DMSO-d6, 150 MHz): δ (ppm): 164.0, 163.4, 160.7, 133.7, 131.5, 129.0, 128.7,
126.8, 123.2, 122.3, 114.7, 106.7, 57.1, 30.2, 20.3, 14.2 (Fig. S2).
2.2.3 Preparation and characterization of 4
Compound 3 (0.425 g, 1.5 mmol) was added to 55% HI solution with refluxing
at 140°C for 7 h, and then the mixture solution was cooled to room temperature. After

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that, the mixture was slowly poured into ice water, filtered and washed with distilled
water. The pale yellow solid thus obtained was dried under vacuum to give compound
(0.376 g) in 93% yield. 1H NMR (DMSO-d6, 600 MHz): δ (ppm): 11.88 (s, 1H), 8.54
(d, J = 8.3 Hz, 1H), 8.48 (d, J = 7.2 Hz, 1H), 8.36 (d, J = 8.1 Hz, 1H), 7.77 (t, J = 7.7
Hz, 1H), 7.16 (d, J = 8.1 Hz, 1H), 4.02 (t, J = 7.3 Hz, 2H), 1.60 (m, 2H), 1.34 (m, 2H),
0.92 (t, J = 7.3 Hz, 3H). 13C NMR (DMSO-d6, 150 MHz): δ (ppm): 164.1, 163.5,
160.7, 134.0, 131.6, 129.6, 129.3, 126.1, 122.8, 122.3, 113.1, 110.4, 30.2, 20.3, 14.2
(Fig. S3).
2.2.4 Preparation and characterization of 5
To a stirred trifluoroacetic acid (20 mL), compound 4 (0.269 g, 1 mmol) and
hexamethylenetetramine (0.182 g, 1.3 mmol) was added, and then the mixture was
refluxed at 120°C for 10 h. After the remained solution poured into the mixture
solution of CHCl3 (10 mL) and HCl (1 mol/L, 10 mL) and stirred overnight, separate
the organic phase with a separatory funnel and wash the aqueous phase three times
with CHCl3 (10 mL×3). The merged organic layer was dried with sodium sulfate and
evaporated in vacuum to dry to a solid. Then the solid was purified by column
chromatography on a silica gel (ethyl acetate: petroleum ether = 1: 3) to get a pale
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yellow product (0.166 g) in 56% yield. H NMR (DMSO-d6, 600 MHz): δ (ppm):
10.37 (s, 1H), 8.70 (m, 2H), 8.57 (d, J = 7.3 Hz, 1H), 7.87 (t, J = 7.8 Hz, 1H), 4.02 (t,
J = 7.4 Hz, 2H), 1.60 (m, 2H), 1.35 (d, J = 14.8, 7.5 Hz, 2H), 0.93 (t, J = 7.4 Hz, 3H).
13C NMR (DMSO-d6, 150 MHz): δ (ppm): 194.5, 165.1, 163.7, 163.0, 134.0, 133.2,
131.7, 130.5, 127.4, 124.0, 122.8, 117.6, 113.6, 30.1, 20.3, 14.2 (Fig. S4).

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2.2.5 Preparation and characterization of 7
To a solution of 2-chlorobenzo[d]thiazole (0.169 g, 1 mmol) in ethanol, N2H4·H2O
(80%, 5 mmol) was dropwise added with refluxing at 80°C for 5h. When cooled to
room temperature, the mixture was filtered, washed and recrystallized with ethanol.
The white solid thus obtained and dried under vacuum to get compound 7 (0.154 g) in
93% yield. 1H NMR (DMSO-d6, 600 MHz): δ (ppm): 9.01 (s, 1H), 7.68 (d, J = 7.8 Hz,
1H), 7.32 (d, J = 8.0 Hz, 1H), 7.20 (t, J = 7.6 Hz, 1H), 6.98 (t, J = 7.5 Hz, 1H), 5.03 (s,
2H). 13C NMR (DMSO-d6, 151 MHz): δ (ppm): 174.4, 153.9, 130.9, 125.8, 121.4,
120.7, 118.3 (Fig. S5).
2.2.6 Preparation and characterization of the probe
A mixture of 5 (0.149 g, 0.5 mmol) and 7 (0.083 g, 0.5 mmol) in ethanol (15 mL)
was refluxed for 5 h. After the reaction was completed, the reaction mixture was
filtered, washed with ethanol and dried to obtain a yellow solid product (0.218 g) in
98% yield. 1H NMR (CDCl3-d1, 600 MHz): δ (ppm): 8.82 (s, 1H), 8.79 (d, J = 8.3 Hz,
1H), 8.75 (d, J = 7.2 Hz, 1H), 8.69 (s, 1H), 7.87 (t, J = 7.7 Hz, 1H), 7.80 (d, J = 7.9
Hz, 1H), 7.74 (d, J = 8.1 Hz, 1H), 7.63 (t, J = 7.7 Hz, 1H), 7.50 (t, J = 7.6 Hz, 1H),
4.20 (t, J = 7.4 Hz, 3H), 1.84 – 1.63 (m, 3H), 1.46 (dq, J = 15.0, 7.4 Hz, 3H), 1.00 (t,
J = 7.3 Hz, 4H). 13C NMR (CDCl3-d1, 151 MHz): δ (ppm): 167.7, 164.9, 164.2, 160.9,
160.6, 160.3, 160.0, 154.7, 138.1, 135.3, 134.5, 130.6, 130.2, 129.3, 127.3, 126.0,
122.9, 122.6, 122.5, 122.0, 117.4, 115.9, 115.4, 114.5, 111.8, 111.7, 41.0, 30.0, 20.3,
13.7. ESI–MS m/z: [Probe + H]+ Calcd. for C24H21N4O3S 445.13, Found 445.13. IR
(KBr): 3174, 2996, 2959, 2872, 1926, 1886, 1776, 1693, 1649, 1609, 1510, 1470,

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1423, 1388, 1367, 1347, 1315, 1258, 1226, 1183, 1128, 1082, 1067, 1018, 950, 925,
889, 873, 822, 783, 741, 720, 687, 656, 597, 581, 552, 529, 501, 460, 450, 429 (Fig.
S6).
<Inserted Scheme 1>
2.3. Solutions preparation and optical measurements
The stock solutions (0.4 mM) of the probe in DMSO and stock solutions (0.4
mM) of the chlorizated salts of Cu2+, Ag+, Zn2+, Cr3+, Hg2+, Ni2+, Na+, K+, Ba2+, Ca2+,
Fe3+, Mn2+, Co2+, Pb2+, Cd2+, Al3+ and Mg2+ in aqueous solution were prepared
respectively. Test solution was prepared by placing 50µL of the probe stock solution
into a quartz cuvette upon the addition of various analytes and diluting the solution to
2 mL with ethanol at room temperature. UV–Vis and fluorescence spectra were
obtained in ethanol solution. And any changes in the fluorescence intensity are
monitored using a fluorescence spectrometer (λem = 523 nm, λex = 397 nm, slit: 5 nm/5
nm). Statistical analysis of the data was carried out using Origin 9.1.
2.4. Cell experiments
A549 cells were grown in Dulbecco’s Modified Eagle’s medium supplemented
with 12% Fetal Bovine Serum and 1% antibiotics at 37 °C in humidified environment
of 5% CO2 at 37 °C. Cells were plated on 6-well plate and allowed to adhere for 24 h.
Before the experiments, cells were washed with PBS 3 times. Different concentrations
of the probe were loaded into each well, with three duplicates for each concentration
(the final concentration of probe in well was 0, 1, 2, 3, 5, 15, 20 µM, respectivily) for
5 and 10 h. The study of the cellular toxicity of probe was carried out by using the

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Cell Counting Kit-8 (CKK-8) assay.
Probe dissolved in DMSO (50 µL, 0.4 mM) was added to the cell medium
(2mL) at 10 µM final concentration. After incubating for 30 min, excess probe was
gently washed with phosphate buffered saline (PBS, pH=7.4) three times. Meanwhile,
another portion of A549 cells pre-treated with probe was treated with Mg2+ (2 µM)
and incubated for further 30 min at 37 °C and then washed cells with PBS buffer three
times. Cell experiment was imaged with confocal microscopy at green channel.
3. Results and discussion
3.1. Recognition ability toward magnesium ion
3.1.1 Selectivity behavior of probe for Mg2+
The selectivity behavior for one kind of analyte over other substances is one of
the most important characteristics of the fluorescent probe. As shown in Fig. 1, the
fluorescence intensity of probe was weak when excited at 397 nm, no distinct
fluorescence intensity increased with addition of common important metal ions for
human: Cu2+, Ag+, Zn2+, Cr3+, Hg2+, Ni2+, Na+, K+ , Ba2+, Ca2+, Fe3+, Mn2+ , Co2+, Pb2+,
Cd2+, VO2+, and Al3+ up to 2 equiv. compared with Mg2+（Fig. S7 and S8）. We also
carried out the competitive experiments to further study the selectivity behavior and
the results showed that the probe could detect Mg2+ selectively (Fig. S11). The probe
for Mg2+ at about 523 nm displayed an obvious enhancement of fluorescence intensity
in ethanol solution at room temperature immediately, this “off-on” mechanism could
be attributed to the O-hydroxyl Schiff base that possessing O and N atoms with
specific affinity to Mg2+.

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<Inserted Figure 1>
3.1.2 Sensitivity behavior of probe for Mg2+
To evaluate the sensitivity of probe, we had titrated different concentration of
Mg2+ to probe with UV-vis absorption spectra and fluorescence spectra. Fig. 2 (a)
showed that the probe (10 µM) exhibited one absorption at 450 nm in the absence of
Mg2+, and with the addition of Mg2+ (0-2 µM), the absorption at 393 nm increased
gradually, accompanied with a decrease at 480 nm. Accordingly, as shown in Fig. 2
(b), upon excitation at 397 nm, the probe (10 µM) displays a weak emission band
centered at 523 nm. Addition of Mg2+ (0-2 µM) significantly increases the
fluorescence intensity with a 15-fold enhancement in fluorescence observed.
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<Inserted Figure 2>
To investigate the detection limit of probe for Mg2+, probe (10 µM) was treated
with various concentrations of Mg2+ (0-2 µM) and the relative emission intensity at
523 nm in ethanol solution was plotted as a function of the Mg2+ concentration.
Subsequent data analysis revealed an excellent linear relationship (R2 = 0.9975)
between the relative fluorescence intensity at 523 nm and the Mg2+ concentration (Fig.
3). From the linear increase in the Mg2+-dependent fluorescence intensity of probe,
the detection limit (DL) of probe for Mg2+ was found to be 5.01×10-8 M (DL =
K×SD/S, where K= 3; SD is the standard deviation of the blank solution; S is the
slope of the calibration curve), which was enough for practical application in
detecting Mg2+ at the level of cells [45].
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<Inserted Figure 3>

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Moreover, the Job’s plot analysis using fluorescence intensity changes indicated
a 1:2 complex ratio between Mg2+ and probe, where the products between molar
fractions and the intensity of the fluorescence emission were plotted against molar
fractions of under the condition of a constant total concentration. When the molar
fraction of probe/(probe+[Mg2+]) was 2/3, the emission intensity of probe-Mg2+
reached maximum (Fig. 4 and Fig. S9) [46-47].
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<Inserted Figure 4>
The stability constant for probe-Mg2+ was calculated from the Benesi-Hildebrand
plots based on the standard equation: A0/(A-A0) ={εr/(εr-εc)} (1/K[Mg2+]+1), where A0
is the absorption of probe in the absence of Mg2+, and (A-A0) is the discrepancy of
absorption between the absence and presence of Mg2+; [Mg2+] is the concentration of
Mg2+ (Fig. 5) [48-49]. The stability constant Ks is given by the ratio of intercept/slop,
Ks = 6.17×105 M-1 (R2 = 0.9866) using the linear analysis.
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<Inserted Figure 5>
Time-dependent modulation in the fluorescence spectra of probe was monitored
in the presence of 2 equiv. of Mg2+. The kinetic study showed that the fluorescence
emission at 523 nm was immediately initiated and leveled off within 45 s, displaying
a rapid response between probe and Mg2+ under the selected experimental conditions
(Fig. 6).
<Inserted Figure 6>
3.3 Proposed mechanism between probe and Mg2+

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To gain insight into the sensing mechanism of probe towards Mg2+, a series of
experiments were carried out. Firstly, according to a Job’s plot analysis at 523 nm,
when the molar fraction of probe/( probe+[Mg2+]) was 2/3, the emission intensity of
probe-Mg2+ reached maximum, which further demonstrated a 2: 1 complexation
stoichiometry for [probe-Mg2+] was established (Fig. 4). Besides, the product of
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probe-Mg2+ was also synthesized and characterized, the H NMR, C NMR and MS
were all certified the proposed mechanism Fig. S10. The identification of the
coordination product in the ESI-MS analysis further certified the proposed mechanism:
a peak at m/z = 909.22 was clearly observed, corresponding to [probe+Mg-H]-
perfectly (Fig. 7).
<Inserted Figure 7>
Furthermore, the optimized configuration of probe and probe-Mg2+ was
calculated by Density Functional Theory (DFT) (Fig. 8). The structure for the
probe-Mg2+ complex was calculated, based on the 1:2 stoichiometric between Mg2+
and probe. In view of the participation of possible anions, the optimized structure
(probe-Mg2+) shows that one Mg2+ ion essentially coordinated with two oxygen atoms
of hydroxyl (d(Mg O)= 1.93192 and 1.93185 Å), two nitrogen atom of amino (d(Mg N)
=
…
…
2.12106 and 2.12114 Å), and the bond angle of the geometry are 108.14549º,
101.67700º, 88.44547º and 88.45404º respectively, which means a distorted
tetrahedral coordination geometry. Therefore, the computational results further
confirm the complexation occurs between probe and Mg2+, which are in agreement
with the conclusion above.

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<Inserted Figure 8>
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All of above presumed that the “off-on” fluorescence could be attributed to the
coordination of hydroxyl group and N atom in C＝N to Mg2+ with 2:1 stoichiometry.
The proposed detection mechanism is shown in Scheme 2.
<Inserted Scheme 2>
3.4 Cell experiments
For the better practical application of the cellular probe, we studied the
concentration employed which could disturb living systems minimally. The cytotoxity
of probe was determined using the Cell Counting Kit-8 (CCK-8) by incubation with
A549 cells. The experiment showed that there is no significant cytotoxic responses
(cell viability ≥ 80%) for all of the tested cells after incubation for 5 h and 10 h with
different concentration of the probe (the final concentration of probe in well was 0, 1,
2, 3, 5, 15, 20 µM) (Fig. 9).
<Inserted Figure 9>
In order to evaluate the reaction of probe to intracellular Mg2+, cellular imaging
studies were also carried out (Fig. 10). As shown in Fig. 10b, A549 cells incubated
with 10 µM of probe for 30 min at 37 ℃ showed nearly no fluorescence. In a further
experiment it was found that A549 cells displayed bright green fluorescence when the
cells were first incubated with 10µM of probe for 30 min and then incubated with 2
µM Mg2+ for 30 min at 37 ℃ (Fig. 10e). These imaging experiments indicated that
the probe can be used to detect Mg2+ in imaging modality.
<Inserted Figure 10>

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4. Conclusions
In summary, we have developed a novel “off-on” fluorescent probe based on 1,
8-naphthalimide derivative for the detection of Mg2+ with highly selectivity and
sensitivity in ethanol solution. Moreover, the probe showed a rapid detection process
(45 s), high fluorescence enhancement (up to 15-fold), a good binding constant
(6.17×105 M-1) and a low detection limit of Mg2+ (5.01×10-8 M). Optical properties,
1H NMR, ESI-MS and DFT calculations also showed response mechanism towards
Mg2+ as a 1: 2 binding mode at ethanol media. The cell cytotoxicity experiments
fluorescence scanning microscopic experiments demonstrated that the probe can be
used to detect intracellular Mg2+ in living A549 cells.
Acknowledgments
The work was supported by the National Natural Science Foundation of China (No.
21472118, 21672131), Talents Support Program of Shanxi Province (2014401),
Shanxi province Outstanding Youth Fund (2014021002) and Scientific Instrument
Center of Shanxi University.
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Figure captions
Scheme 1 The synthesis route of the probe.
Fig. 1 (a) Optical density two-dimensional graph of the probe (10 µmol/L) in ethanol
solution in the presence of 2 equiv. various metal ions. (b) Photographs of the visual
color and the fluorescence under illumination at 365 nm. From left to right: Probe,
Cu2+, Ag+, Zn2+, Cr3+, Hg2+, Ni2+, Na+, K+ , Ba2+, Ca2+, Fe3+, Mn2+ , Co2+, Pb2+, Cd2+,
Al3+, and Mg2+.
Fig. 2 (a) UV-vis spectra of the probe (10 µM) with the addition of Mg2+ (0-2 µM) in
ethanol solution. (b) Fluorescence spectral change of the probe (10 µM) upon addition
of Mg2+ (0-2 µM) in ethanol solution (λex = 397 nm, λem =523 nm, slit: 5 nm/5 nm).
Insert: photographs of probe before (left) and after (right) addition of Mg2+.
Fig. 3 Plot of emission intensity at 523 nm of probe as a function of Mg2+
concentration in ethanol solution.
Fig. 4 Job’s plot for probe-Mg2+ complex at 523 nm in ethanol solution and the total
concentration of probe and Mg2+ is 15 µM.
Fig. 5 Benesi-Hildebrand plots of probe-Mg2+ complex in ethanol solution.
Fig. 6 Reaction time profiles of probe in the presence of Mg2+ in ethanol solution.
Fig. 7 The MS of probe-Mg2+ in the DMSO.
Fig. 8 Calculated structure for the probe (a) and the complex [probe-Mg2+] (b) by
Gaussian05.
Scheme 2 Proposed sensing process of the probe for Mg2+
Fig. 9 CCK-8 assay of A549 cells treated with probe at different concentrations for 5

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h and 10 h.
Fig. 10 Confocal fluorescence images in A549 cells. Fluorescence image of A549
cells incubated with probe (10 µM) for 30 min at 37 °C (a-c); Cells pretreated with
probe and then incubated with 2 µM Mg2+ for 30 min at 37 °C (d-e).

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Scheme 1
O
O
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O
N
3
O
O
N
O
NH2
CH3ONa
CH3OH/CuSO4
Br
OCH3
Br
2
1
O
N
O
O
N
O
O
N
O
HI
HMTA/TFA
HCI/H2O
S
N
N
CHO
N
H
OH
OH
OH
N1
5
4
S
N
NH2NH2
NH2
NH
S
N
Cl
6
7
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Fig. 1 (a)
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