“Turn-on” fluorescent probe detection of Ca2 + ions and applications to bioimaging

Article abstract of DOI:10.1016/j.saa.2017.03.023

Ca^{2 +} is intracellular divalent cation with the largest concentration variations and involved in many biological phenomena and often acted as a second messenger in signaling pathway. Therefore, the development of probes for specific Ca^{2 +} detection is of great importance. Herein, a novel turn-on fluorescent probe for the detection of Ca^{2 +} in MeCN-aqueous medium was designed and synthesized. The probe displayed responses to Ca^{2 +} with a fluorescence enhancement at 525 nm, accompanying with a distinct fluorescence change from nearly colorless to bright yellow-green. Besides, the probe exhibited a rapid signal response time (within 25 s), a good linearity range and a lower detection limit (2.70 × 10^{? 7} M). In addition, the ability of the probe to detect Ca^{2 +} in living cells (HeLa cells) via an enhancement of the fluorescence has also been demonstrated.

Full text of DOI:10.1016/j.saa.2017.03.023

Accepted Manuscript
“Turn-on” fluorescent probe detection of Ca2+ ions and
applications to bioimaging
Huifang Zhang, Caixia Yin, Tao Liu, Yongbin Zhang, Fangjun
Huo
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S1386-1425(17)30183-X
doi: 10.1016/j.saa.2017.03.023
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Spectrochimica Acta Part A: Molecular and Biomolecular
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Please cite this article as: Huifang Zhang, Caixia Yin, Tao Liu, Yongbin Zhang, Fangjun
Huo , “Turn-on” fluorescent probe detection of Ca2+ ions and applications to bioimaging.
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“Turn-on” fluorescent probe detection of Ca2+ ions and applications to
bioimaging
Huifang zhanga,1, Caixia Yina,*, Tao Liua,1, Yongbin Zhangb, Fangjun Huob,**
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.
**Corresponding author: F.J. Huo, Email: huofj@sxu.edu.cn.
*Corresponding author: C.X. Yin, E-mail: yincx@sxu.edu.cn;
1Huifang zhang and Tao Liu contributed equally
Abstract: Ca2+ is intracellular divalent cation with the largest concentration variations
and involved in many biological phenomena and often acted as a second messenger in
signaling pathway. Therefore, the development of probes for specific Ca2+ detection is
of great importance. Herein, a novel turn-on fluorescent probe for the detection of
Ca2+ in MeCN-aqueous medium was designed and synthesized. The probe displayed
responses to Ca2+ with a fluorescence enhancement at 525 nm, accompanying with a
distinct fluorescence change from nearly colorless to bright yellow-green. Besides, the
probe exhibited a rapid signal response time (within 25 s), a good linearity range and
a lower detection limit (2.70×10-7 M). In addition, the ability of the probe to detect
Ca2+ in living cells (HeLa cells) via an enhancement of the fluorescence has also been
demonstrated.
Keywords: Ca2+; Detection; Fluorescent probe; Bioimaging.

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1. Introduction
Ca2+ is the intracellular divalent cation with the largest concentration variations and
involved in many biological phenomena and often acted as a second messenger in
signaling pathway.1-3 For example, changes in the intracellular Ca2+ concentration are
related to physiological responses in obesity, as well as immune responses and
pathological responses in Alzheimer’s disease.4-9
There are a few fluorescent probes for measuring the free intracellular Ca2+
concentration since the first introduction of small-molecule fluorescent indicators by
Tsien and colleagues in the mid-eighties.4,10-17 Draw from the data collection and analysis,
fluorescent Ca2+ probes can fall into two main categories: as genetically encoded
fluorescent proteins for fulfilling the roles of physiological mechanisms including cardiac
function, cell signaling and neuronal network activity18-19 and as fluorescent small organic
molecules for bulk-loading into live cells with no need for transfection and non-disruptive
loading at the same time.20-23
Most currently used small-molecular fluorescent probes for Ca2+ include fluorescein
derivatives, such as Fluo-3, Fluo-4, Calcium Green-1, Oregon Green 488 BAPTA-1 and
rhodamine derivative, such as Rhod-2.23-25 In this contribution, probe (NR) was
successfully synthesized starting from the reported 1,8-naphthalimide-based R126 and
rhodamine derivative as a novel fluorescent probe for Ca2+.
Herein, we have presented the synthesis and spectral properties of probe NR. The
probe exhibited obvious changes in both UV-vis spectra and fluorescence emission
spectra. Moreover, the sensitivity and selectivity of NR for the detection of Ca2+ in the

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presence of other competing species were established. The compatibility and fluorescence
characteristics of NR in physiologically relevant conditions were exploited for the
visualization of living cells exposed to Ca2+ ions.
2. Materials and methods
2.1. Instruments and materials
All solvents and reagents for synthesis and analyses were of analytical grade and
bought from Sigma-Aldrich (St. Louis, MO) without further purification. The
solutions of cation were prepared from their chloride salts. 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 MHz NMR
spectrometer (Bruker, Billerica, MA) respectively and chemical shifts are reported in
ppm relative to the internal standard TMS. ESI-MS was carried out on AB Triple TOF
5600plus System (AB SCIEX, Framingham, USA). The ability of probe reacting to
Ca2+ in the living cells was also evaluated using Leica DMi8 Microsystems.
2.2. Preparation and characterization of the probe NR
2.2.1. Preparation and characterization of compound 2

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The synthesis approach of probe is summarized in Scheme 1. To a solution of
rhodamine B (0.96 g, 2 mmol) in ethanol, N2H4·H2O (80%, 15mmol) was dropwise
added with refluxing at 70°C for 5h. When cooled to room temperature, the mixture
was filtered, washed and recrystallized with ethanol. The pale pink solid thus obtained
1
and dried under vacuum to give compound 2 (0.85 g) in 93% yield. H NMR
(CDCl3-d1, 600 MHz): δ (ppm): 7.96 (d, J = 7.6 Hz, 1H), 7.46 (d, J = 3.9 Hz, 2H),
7.12 (d, J = 6.0 Hz, 1H), 6.52 – 6.38 (m, 4H), 6.31 (d, J = 8.5 Hz, 2H), 3.63 (s, 2H),
3.36 (q, J = 6.9 Hz, 8H), 1.19 (t, J = 6.9 Hz, 12H). 13C NMR (CDCl3-d1, 151 MHz): δ
(ppm): 166.2, 153.9, 151.6, 148.9, 132.5, 130.1, 128.1, 123.9, 123.0, 108.0, 104.6,
98.0, 65.9, 44.44, 12.6 (Fig. S1).
2.2.2. Preparation and characterization of the probe NR
A mixture of R1 (0.28 g, 1 mmol) and compound 2 (0.46 g, 1 mmol) in ethanol (30
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 (0.72 g) in 98%
yield. 1H NMR (CDCl3-d1, 600 MHz) : δ (ppm): 13.55 (s, 1H), 8.72 – 8.63 (m, 2H),
8.58 (d, J = 7.2 Hz, 1H), 8.26 (s, 1H), 8.05 (d, J = 7.4 Hz, 1H), 7.68 (t, J = 7.8 Hz,
1H), 7.55 (d, t, J = 22.8, 7.1 Hz, 2H), 7.18 (d, J = 7.4 Hz, 1H), 6.63 – 6.48 (m, 4H),
6.29 (d, J = 8.3 Hz, 2H), 4.15 (t, J = 7.4 Hz, 2H), 3.36 (d, J = 6.7 Hz, 8H), 1.69 (d, t, J
= 14.8, 7.5 Hz, 2H), 1.43 (d, t, J = 14.3, 7.2 Hz, 2H), 1.19 (t, J = 6.8 Hz, 12H), 0.97 (t,
J = 7.3 Hz, 3H). 13C NMR (CDCl3-d1, 151 MHz): δ (ppm): 164.6, 164.4, 161.7, 153.0,
149.3, 147.8, 134.2, 134.0, 132.5, 130.0, 129.3, 128.6, 127.9, 127.8, 125.9, 124.0,
123.7, 123.3, 122.3, 113.4, 108.3, 104.2, 98.2, 65.8, 44.4, 40.1, 30.2, 20.4, 13.9, 12.6

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(Fig. S2). ESI–MS m/z: [Probe + H]+ Calcd. for C45H45N5O5 736.34, Found 736.36
(Fig. S3).
Scheme 1 The synthesis of the probe NR.
2.3. Solutions preparation and optical measurements
The optical spectra of the probe NR were measured in a mixture of HEPES:
MeCN = 1:9 (v/v, pH =7.4) solution at room temperature. The stock solutions of
probe was prepared in acetonitrile in the concentration of 0.4 mM, stock solution of
various analytes metal ions, Fe3+, Na+, Zn2+, K+, Pb2+, Al3+, Hg2+, Cd2+, Mn2+, Mg2+,
Cu2+, Ag+, Cr3+, Co2+, Ba2+, Ni2+, and Ca2+ are prepared in deionized water in the
concentration of 20 mM. UV-vis or fluorescence spectra were recorded upon the
addition of various analytes. And any changes in the fluorescence intensity are
monitored using a fluorescence spectrometer (λem = 525 nm, λex = 404 nm, slit: 5 nm/5
nm). Statistical analysis of the data was carried out using Origin 8.6.
2.4. Cell imaging experiments
HeLa cells were cultured in 1× SPP medium (1% proteose peptone, 0.2%
glucose, 0.1% yeast extract, 0.003% EDTA ferricsodium salt) at 37 °C. The cells
were plated on 6-well plates and allowed to adhere for 12 h. Probe dissolved in MeCN
(50 μL, 10 μM) was added to the cell medium (2 mL) at 10 μM final concentration.

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After incubating for 30 min, excess probe was gently washed with phosphate buffered
saline (PBS, pH=7.4) three times. Meanwhile, another portion of HeLa cells
pre-treated with probe was treated with Ca2+ (30 μM) and incubated for further 30
min at 37 °C. Cell imaging was then carried out after washing cells with PBS buffer
three times. Besides, fluorescence images were recorded at blue channel.
3. Results and discussion
3.1. Optical properties of the probe
To investigate the optical properties of the probe, the UV-vis absorption spectra
and fluorescence spectra were both carried out. Fig. 1 (a) showed that the probe (10
μM) exhibited one absorption at 375 nm in the absence of Ca2+, and with the addition
of Ca2+ (0-28 μM), the absorption at 406 nm increased gradually. Accordingly, as
shown in Fig. 1 (b), upon excitation at 404 nm, the probe (10 μM) displays a weak
emission band centered at 537 nm. Addition of Ca2+ (0-28 μM) significantly increases
the fluorescence intensity with a blue-shift centered at 525 nm—a 4.5-fold increase in
fluorescence was observed.
(a)
(b)

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Fig. 1 (a) UV-vis spectra of the probe (10 µM) on the addition of Ca2+ (0-28 µM) to the HEPES:
MeCN = 1:9 (v/v, pH =7.4) solution. (b) Fluorescence spectral change of the probe (10 µM) upon
addition of Ca2+ (0-28 µM) in HEPES: MeCN = 1:9 (v/v, pH =7.4) solution (λex = 404 nm, λem
=525 nm, slit: 5 nm/5 nm).
From the changes in Ca2+-dependent fluorescence intensity, the detection limit is
estimated to be 2.70×10-7 M when the fluorescence emission intensity was plotted as
a function of the Ca2+ concentration (Fig. 2).27-33 The nonlinear curve fitness based on
1:1 complex expression, where F and F0 are the fluorescence intensity of NR in the
presence and absence of Ca2+, Fmax is the final fluorescence intensity in titration
experiment, Ks is the stability constant (Equation (1)). The stability constant Ks is
calculated as 2.05×105 M-1 (R2 = 0.995) for NR-Ca2+ by using a nonlinear
least-square analysis (Fig. 3).34-35 The stability constant K for NR-Ca2+ calculated
from the plots of A0/(A-A0) versus NR-Ca2+ based on the standard Benesi-Hildebrand
method is 1.30×105 M-1 (R2 = 0.976) (Fig. S4), and this value is similar to above Ks
from fluorescence titration experiments.36
Fig. 2 Plot of emission intensity at 525 nm of probe as a function of Ca2+ concentration in HEPES:
MeCN = 1:9 (v/v, pH =7.4) solution.

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Fig.3 A nonlinear least-square analysis of a 1:1 complex of NR and Ca2+.
It is notable that a 1: 1 complexation stoichiometry for NR-Ca2+ was established
through a Job’s plot analysis at 525 nm, where the products between molar fractions
and the intensity of the fluorescence emission were plotted against molar fractions of
NR under the condition of a constant total concentration. When the molar fraction of
NR/(NR+[Ca2+]) was 0.50, the emission intensity of NR-Ca2+ reached maximum (Fig.
4).37-39
Fig.4 Job’s plot for NR-Ca2+ complex at 525 nm in HEPES: MeCN = 1:9 (v/v, pH =7.4) solution.
The total concentration of NR and Ca2+ is 40 µM.
3.2. The Selective response of probes to Ca2+
The research of NR to the selective recognition of other cations (Fe3+, Na+, Zn2+,
K+, Pb2+, Al3+, Hg2+, Cd2+, Mn2+, Mg2+, Cu2+, Ag+, Cr3+,Co2+, Ba2+,Ni2+ and Ca2+) by

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fluorescent spectra under HEPES: MeCN = 1:9 (v/v, pH = 7.4) was carried out (Fig.
5). The free probe exhibited a weak emission peak at 525 nm (λex= 404 nm, λem =525
nm, slit: 5 nm/5 nm), and upon the addition of 20.0 equiv. of other cations, some
fluorescence emission of the probe did not have obvious increase, while some cations
turn off the fluorescence of the probe, such as (Fe3+, Zn2+, Al3+, Cd2+, Mn2+, Cu2+,
Cr3+, Co2+, Ni2+). Whereas upon the addition of the same amount of Ca2+, the
emission peak at 525 nm enhanced remarkably (the intensity being about 4 times
compared with that of free probe).
Fig. 5 Optical density two-dimensional graph of emission at 525 nm of the probe (10 µM) in the
presence of 20.0 equiv. various metal ions for 5 min in the HEPES: MeCN = 1:9 (v/v, pH =7.4)
solution (λex = 404 nm, λem =525 nm, slit: 5 nm/5 nm) (From left to right: Fe3+, Na+, Zn2+, K+, Pb2+,
Al3+, Hg2+, Cd2+, Mn2+, Mg2+, Cu2+, Ag+, Cr3+,Co2+, Ba2+,Ni2+, Ca2+).
3.3. pH dependent of the probes
The effect of pH on the fluorescence intensity of probe in the absence and
presence of Ca2+ was also investigated as the pH value of system is often considered
as a significant influencing factor on interactions (Figure 6). Under pH range from 4
to 11, the emission intensity of probe showed a weak fluorescence and the addition of
Ca2+ could induce the enhancement of the fluorescence, which showed that the
fluorescence probe to Ca2+ works well during this pH range. The weaker response at

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pH＜4 was presumably attributed to diminished concentration of the probe, because
of the ring-open of the rhodamine. There is no response at pH＞11, which may results
from the hydrolysis of C＝N. Thus, the physiological pH could be selected for the
further research.
Fig. 6 Fluorescence emission at 525 nm of probe at different pH values in the absence or presence
of Ca2+.
3.4. The kinetic study in the detection process of Ca2+
Time-dependent modulation in the fluorescence spectra of probe was monitored
in the presence of 10 equiv. of Ca2+. The kinetic study showed that the fluorescence
emission at 525 nm was immediately initiated and leveled off within 25 s, displaying
a rapid response between probe and Ca2+ under the selected experimental conditions
(Fig. 7).

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Fig. 7 Reaction time profiles of probe in the presence of Ca2+ in HEPES: MeCN = 1:9 (v/v, pH
=7.4) solution.
3.5. Proposed mechanism
The reaction mechanism of the system was also studied. It was presumed that the
turn-on fluorescence could be attributed to the coordination of probe with Ca2+. The
measured intensity F at 525 nm varied as a function of ln[Ca2+] in a nonlinear
relationship (R2 =0.995), which indicated that the formation of 1:1 stoichiometry
between Ca2+ and probe and the association constant of probe with Ca2+ was
calculated to be 2.05×105 M-1 (Fig. 3). Moreover, according to a Job’s plot analysis at
525 nm, when the molar fraction of NR/(NR+[Ca2+]) was 0.50, the emission intensity
of NR-Ca2+ reached maximum, which further demonstrated a 1: 1 complexation
stoichiometry for NR-Ca2+ was established (Fig. 4). In addition, the identification of
the coordination product in the ESI-MS analysis made it possible to propose the
signaling mechanism: a peak at m/z = 863.24, corresponding to [probe-Ca + H2O +
1
Cl], is clearly observed (Fig. S5). H NMR titration were carried out, results were
showed in fig. S6. The addition of Ca2+ into probe will induce some protons to shift,
such as Hb, Hc.40-43 The proposed detection mechanism of Ca2+ was shown in Scheme
2.
Scheme 2 Proposed sensing process of the probe for Ca2+
3.6. Imaging of living cell

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It is known that cell imaging experiments could prove further application for
fluorescent probes.44-45 In order to evaluate the cell permeability and reaction of probe
NR to Ca2+, cellular imaging studies were also carried out (Fig. 8). As shown in Fig.
8b, HeLa cells incubated with 10 μM of NR for 30 min at 37 ℃ showed nearly no
fluorescence. In a further experiment it was found that Hela cells displayed bright
yellow-green fluorescence when the cells were first incubated with 10μM of NR for
30 min and then incubated with 30 μM Ca2+ for 30 min at 37 ℃ (Fig. 8e). These
imaging experiments indicated that probe can be used to detect Ca2+ in imaging
modality.
Fig. 8 Fluorescence images in HeLa cells. Fluorescence image of HeLa cells incubated with probe
(10 µM) for 30 min at 37 °C (a-c); Cells pretreated with probe and then incubated with 30 µM
Ca2+ for 30 min at 37 °C (d-f).
4. Conclusions
In this study, a novel fluorescent probe based on 1, 8-naphthalimide and
rhodamine derivative for the detection of Ca2+ has been developed, which displayed
highly selective and sensitive fluorescence enhancement and color change upon the
addition of Ca2+ over other metal ions. The experiment also showed response

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mechanism towards Ca2+ as a 1: 1 binding mode at MeCN-aqueous media. Moreover,
the probe showed a rapid detection process (within 25 s), a good linearity range and a
low detection limit of Ca2+ (2.70×10-7 M). The fluorescence scanning microscopic
experiments demonstrated that the probe can be used to detect intracellular Ca2+ in
living HeLa cells.
Acknowledgments
The work was supported by the National Natural Science Foundation of China (No.
21672131), Talents Support Pro-gram of Shanxi Province (2014401), Shanxi
Province Outstanding Youth Fund (No. 2014021002).

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Abstract Graphic
The Ttitle:
“Turn-on” fluorescent probe detection of Ca2+ ions and applications to
bioimaging
List of Authors:
Huifang zhanga,1, Caixia Yina,*, Tao Liua,1, Yongbin Zhangb, Fangjun Huob,**
The statement:
Based on the coordination reaction between probe and Ca2+ under mild conditions, we
herein report a turn-on fluorescent probe which features a rapid signal response time
(within 25 s), a good linearity range and a low detection limit (2.70×10-7 M). The
application of this fluorescent probe was demonstrated by fluorescent imaging in HeLa
cells.

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Highlight
a) The compound is a novel fluorescent probe for the detection of Ca2+.
b) The probe exhibited rapid detection time (within 25 s) and low
detection limit: 2.70×10-7 M.
c) The ability of the probe to detect Ca2+ in living cells (HeLa cells) was
also illustrated.