Selective sensing Ca2+ with a spiropyran-based fluorometric probe

Article abstract of DOI:10.1002/bio.3656

Spiropyran (SP) and its derivatives operate between their ring opening and closing forms as a versatile molecular platform for the fluorescence detection of cations and anions, using a colour change for signalling. A functionalized SP fluorescence probe, L, was prepared and characterized. Probe L can detect Ca²⁺ with a fluorescence ‘turn-on’ response in ethanol solution. It selectively binds Ca²⁺ to form a 1:1 ligand/metal complex, which produced a new emission band centred at 604?nm. The sensing result was clearly observed by the solution colour change from colourless to pink under visible light, and from blue to red under ultraviolet light. The detection limit was calculated to be 4.53?×?10^?8?M for Ca²⁺. The probe provides another possibility that SP-based derivatives could be used for the development and detection of metal ions in environmental and physiological systems.

Full text of DOI:10.1002/bio.3656

Received: 5 January 2019
DOI: 10.1002/bio.3656
Revised: 7 May 2019
Accepted: 10 May 2019
R E S E A R C H A R T I C L E
2
+
Selective sensing Ca with a spiropyran‐based fluorometric
probe
1
2
2
1
1
|
|
|
|
Li Wang
Yuanjun Yao
Jiao Wang
Chuan Dong
Hui Han
1
Institute of Environmental Science, Shanxi
University, Taiyuan 030006, China
Abstract
2
School of Chemistry and Chemical
Spiropyran (SP) and its derivatives operate between their ring opening and closing
forms as a versatile molecular platform for the fluorescence detection of cations
and anions, using a colour change for signalling. A functionalized SP fluorescence
Engineering, Shanxi University, Taiyuan
0
30006, China
Correspondence
2+
probe, L, was prepared and characterized. Probe L can detect Ca with a fluores-
Li Wang, Institute of Environmental Science,
Shanxi University, Taiyuan 030006, China.
Email: wangli@sxu.edu.cn
2
+
cence ‘turn‐on’ response in ethanol solution. It selectively binds Ca to form a 1:1
ligand/metal complex, which produced a new emission band centred at 604 nm.
The sensing result was clearly observed by the solution colour change from colourless
to pink under visible light, and from blue to red under ultraviolet light. The detection
Funding information
Shanxi 131 Leading Talents, Grant/Award
Numbers: 205586801011 and
−8
2+
2
05544901006; Shanxi Provincial Hundreds
of Talents Program, Grant/Award Numbers:
05107004 and 205107001; Shanxi Returned
limit was calculated to be 4.53 × 10 M for Ca . The probe provides another possi-
bility that SP‐based derivatives could be used for the development and detection of
metal ions in environmental and physiological systems.
2
Overseas Scholar Foundation, Grant/Award
Number: 205544901005
[
19,20]
1
|
INTRODUCTION
and signal transduction.
The reversible photochromism of
spiropyran‐based chemicals is related to their chemical structure and
2
+
Calcium ions (Ca ) have several indispensable physiological and bio-
the surrounding micro‐environment, including temperature, pH, and
[21,22]
[1–4]
chemical roles in organisms and cells.
Calcium is required for sev-
solvent polarity.
The ring opening of the closed form of
eral basic functions in the human body such as intracellular signalling,
spiropyran occurs through cleavage of the C (spiro)−O bond to give
[5]
[6–8]
hormone secretion, muscle contraction, nerve function,
vascular
the open form, which absorbs light at a longer wavelength as a result
[
23–25]
contraction and vasodilation, as well as supporting skeletal structure
of the extended conjugated π system.
Exploration of the photo-
[
5]
and function. As an electrolyte, calcium also maintains the regular
chromic properties of spiropyrans has led to the development of
[26]
[
9]
2+
heartbeat. Fluorescent Ca probes have attracted great interest
chemical/biochemical sensors in analytical chemistry
and for
[
20]
due to their capacity for real‐time detection, their simple operation,
molecular switches in material chemistry.
Over the past decades,
[10,11]
and their low detection limit for metal ions.
Fluorometric sensors
several receptors derived from spiropyran have been synthesized
2
+
[26–30]
for Ca detection generally consist of a receptor for interaction with
and used for colorimetric monitoring/detection of metal ions
2+
,
2+
Ca
and a chromophore for signalling the detectable response
but only a few reports have described the detection of Ca using a
after binding or interaction of the analytes with the fluorescent
probe. As a calcium‐specific aminopolycarboxylic acid, 1,2‐bis(O‐
aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA) has been
spiropyran derivative. A series of spiro derivatives (H‐BSP‐n) was syn-
2+
2+
2+
thesized by Yagi and colleagues that responded to Ca , Mg , Sr
2+
[28]
and Ba with no specific selectivity.
2+
[12–15]
reported as
a
Ca
receptor in many previous studies.
This paper reports a simple fluorescent probe L for the colorimetric
2
+
2+
Fluorophores possess exceptional photophysical properties, i.e. qui-
nine, cyanine, rhodamine, indole, boron‐dipyrromethene (BODIPY)
and salicylaldehyde derivatives are usually selected as the chromo-
recognition of Ca (Scheme 1). Upon addition of Ca , a significant
new emission band appeared, centred at 604 nm, along with a solution
colour change from colourless to pink under visible light, and from
blue to red under ultraviolet (UV) light (Figure 3d). The detection limit
[16–18]
phores of choice when developing fluorescent probes.
Spiropyrans or spirobenzopyrans, family of photochromic
2+
−8
a
(DL) of probe L for Ca was 4.53 × 10 M. For practical and conve-
2
+
chemicals, are a fascinating starting point for such specific construc-
tions because of their exceptional binding abilities towards metal ions
nient application, a colorimetric paper strip for sensing Ca was also
developed.
Luminescence. 2019;1–8.
wileyonlinelibrary.com/journal/bio
© 2019 John Wiley & Sons, Ltd.
1

2
WANG ET AL.
SCHEME 1 Synthesis of L
2
2
|
EXPERIMENTAL
triethylamine (0.600 mL) in ethanol (10.0 mL) was refluxed in the dark
for 19 h. Evaporation of the solvent left a purple powdery solid. The
crude product mixture was purified by column chromatography. The
column was packed with silica gel, and a mixed solvent (petroleum
ether:ethyl acetate = 1:3, v/v) was used as the elute solvent. After
.1
|
Materials and instrumentation
All chemical reagents were purchased as analytical grade and were
commercially available from Aladdin Chemicals Co., Ltd and Adamas
Reagent Co., Ltd (Shanghai, China) and Beijing InnoChem Science
and & Technology Co., Ltd. Reagents were used as received without
any further purification except for toluene, which was dried by distil-
evaporation of the solvent, the desired pale‐yellow powdery solid
1
product was obtained at a 10.0% yield. H NMR (CDCl
3
, 600 MHz)
δ (ppm) = 7.74–7.68 (m, 1H), 7.55–7.50 (m, 1H), 7.39–7.29 (m, 2H),
6
2
.81 (d, 1H), 6.72 (s, 2H), 6.50 (d, 1H), 5.78 (d, 1H), 3.52 (d, 2H),
.64 (d, 2H), 1.22 (s, 3H), 1.13 (s, 3H). HRMS (C22 ): calcd
20 2 3
H N O
lation over CaH
2
, and stored over 4 Å molecular sieves. Water for all
+
+
[
L + H] : 361.1507; found value [L + H] : 361.15442 (Figure S3).
experiments was purified using an ELGA PURELAB Ultra water purifi-
−1
cation system with a resistivity higher than 18.2 MΩ•cm . UV–visible
absorption spectra were recorded on a Cary 8454 UV–visible diode
array spectrometer (Agilent Technologies Inc., USA) with 1 nm resolu-
tion. All emission spectra at steady state were recorded on a
QuantaMaster™ 400 fluorescence spectrophotometer in the photon‐
counting mode and corrected for wavelength‐dependent variations
in the emission channels using the correction factors provided by
2.3
|
Sensing of metal ions
The stock solution of L (2.00 mM) was prepared in ethanol (Figure S4),
and then diluted to 40.0 μM for the absorption spectrum and 4.00 μM
for the emission spectrum. Absorption and emission spectra were
2
+
recorded after 30 min upon addition of Ca to the solution of L. All
spectral data were recorded by placing the sample solution in a
10 mm standard quartz cell (Starna cell). The slits for excitation and
1
the manufacturer (Photon Technology International, USA). H NMR
spectra were acquired using a Bruker Avance III HD 600 MHz nuclear
magnetic resonance spectrometer, (Bruker, Billerica, MA, USA).
Mass spectra of high resolution were acquired on a Bruker micro
TOF‐Q III mass spectrometer.
emission were opened to
5 nm width. The binding constant
was calculated from the following Benesi−Hildebrand equation:
1
1
1
¼
þ
, where F
0
, F and Fmax
F − F0 KsðFmax − F0Þ½Mþꢀn Fmax − F0
are the fluorescence intensity of molecules in the absence of metal
ions, at a certain concentration of metal ions and at a complete
interaction concentration of metal ions, respectively.
2
.2
|
Synthesis of 3,3‐bimethyl‐6‐cyano‐[(2H)‐1‐
benzopyran‐2,2‐indoline] derivative (L)
L was synthesized in three steps, as shown in Scheme 1. 5‐
2.4
|
Theoretical calculation
Cyanosalicylaldehyde was prepared following the reported proce-
[
31]
1
2+
dure.
A white powdery solid was obtained in 15.0% yield.
, 600 MHz), δ (ppm) = 11.3 (s, 1H), 9.96 (s, 1H), 7.89 (s,
H), 7.78 (d, 1H), 7.13 (d, 1H) (Figure S1). Then, 1‐(2‐carboxyethyl)‐
,3,3‐trimetyl‐3H‐indolium bromide was synthesized following a mod-
H
The electronic properties of L, merocyanine (MC) and the MC–Ca
NMR (CDCl
3
complex were investigated using density functional theory (DFT) cal-
culations. Becke's three‐parameter nonlocal functional exchange along
with the Lee–Yang–Parr nonlocal functional correlation (B3LYP) was
used. The 6–31 + G (d) basis set was assigned for all elements. All elec-
tronic structure calculations were completed with no constraints for
symmetry.
1
2
[
32]
ified published procedure.
An orange solid was obtained with a
, 600 MHz,), δ (ppm) = 8.07–7.90
1
5
9.0% yield. H NMR (DMSO‐d
6
(
m, 1H), 7.89–7.75 (m, 1H), 7.73–7.52 (m, 2H), 4.63 (t, 2H), 2.96 (t,
2
H), 2.84 (s, 3H), 1.51 (s, 6H) (Figure S2).
In the last step, L was prepared from 5‐cyanosalicylaldehyde and
‐(2‐carboxyethyl)‐2,3,3‐trimetyl‐3H‐indolium bromide as previously
2.5
|
Test paper colorimetric experiment
1
[32]
reported, with some modifications.
Briefly, a mixed solution of
L (3.60 mg, 2.00 mmol) was dissolved in 5.00 mL ethanol to obtain a
2
1
‐(2‐carboxyethyl)‐2,3,3‐trimetyl‐3H‐indolium bromide (100 mg,
.320 mmol) and 5‐cyanosalicylaldehyde (40.0 mg, 0.270 mmol), and
2.00 mM solution. The 2 × 1 cm filter paper was soaked in the solu-
0
tion of L for 5 min, and dried at room temperature to get a pale‐yellow

WANG ET AL.
3
strip. Then the test paper was soaked in different concentrations of
calcium chloride solution. After 30 min, the colour changes on the
paper strip were observed after taking out and drying.
photo‐excitation at 290 nm promoted L isomerization from the SP
to MC form, and photo‐excitation at 375 nm did not induce isomeriza-
tion. Therefore, in this study, 375 nm was used as the excitation wave-
length to avoid photoinduced isomerization.
3
3
|
RESULTS AND DISCUSSION
Isomerization of compounds
3
.2
|
Selectivity behaviour of L
.1
|
Binding selectivity towards different metal ions was examined by
monitoring the fluorescence changes of a mixed solution of L
(4.00 μM) and various metal ions (80.0 μM). As shown in Figure 3a,
L produced no emission after 550 nm upon excitation at 375 nm. After
Spiropyran derivatives, as a class of photochromic compounds,
undergo reversible isomerization between their colourless cyclic
spiropyran (SP) form and the coloured ring‐open MC form upon light
[
33,34]
excitation (Figure 1).
The isomerization equilibrium between SP
2
addition of CaCl into the the solution of L, an intense emission band
and MC is heavily dependent on the energy of the spiro C–O bond
appeared that was centred at 604 nm. However, upon addition of
[20]
+
+
+
+
2+
2+
2+
2+
cleavage of the SP form.
Substitution of the electron‐withdrawing
other metal ions such as Li , Na , K , Ag , Ba , Cd , Hg , Mg ,
2
+
2+
3+
3+
3+
group, i.e. cyano, weakens the C–O bond and facilitates isomerization.
Shiraishi and colleagues used a Gaussian calculation to study the ring
opening/closing reactions of spiropyan derivatives from the potential
Mn , Zn , Al , Fe and Cr , there was almost no change in the
fluorescence of the mixed solution. The observed new emission band
2
+
might arise from the MC–Ca complex, which was generated follow-
[33]
energy surface, and reached the following conclusion:
photo‐
ing isomerization and L ring opening and resulted from interaction
2
+
2+
isomerization from SP to MC is a two‐step reaction, including (i) spiro
C–O bond cleavage; and (ii) isomerization from cis to trans.
with Ca . The specificity of L for Ca detection was also examined
in the presence of other metal ions (Figure 3b). The emission intensity
2
+
Photoinduced isomerization properties of L (4 μM) were investi-
gated and the spectra are shown in Figure 2. The absorption spectrum
of L in ethanol displayed almost no change with photo‐excitation at
of MC–Ca complex was not markedly affected by coexisting metal
3+
3+
ions except for Fe and Cr , for which fluorescence quenching
occurred (Figure S5). Upon addition of 4.00 μL triethanolamine
3+
3+
3
75 nm. This result suggested that L exists in solution as the SP form,
(0.100 mol/L) to the Fe and Cr solutions, fluorescence of the solu-
3
+
3+
and 375 nm excitation did not promote isomerization. Figure 2 also
displays the spectral changes of absorption and fluorescence for L
with photo‐excitation at 290 nm. A new absorption band appeared
centred at 554 nm, and a new emission band also appeared at about
tions was restored (Figure 3b). The interference of Fe and Cr with
2+
the MC–Ca complex could be avoided by adding 0.100 mol/L
triethanolamine (TEA) (4.00 μL) as a masking agent, and the excess
2+
of TEA had hardly any effect on fluorescence of the MC–Ca com-
plex. As expected, when the absorption spectrum was recorded, the
new absorption band centred at 526 nm did not appear, except fol-
6
45 nm; these were both attributed to the MC form of L. Therefore,
2+
lowing addition of Ca to the solution (Figure 3c). Consistent with
the spectral change, solution L (100 μM) changed from colourless to
pink under natural light (Figure S6), and from blue to red under UV
2+
light (Figure 3d). Therefore, the presence of Ca can be readily differ-
entiated from other metal ions by the naked eye.
2+
The response time for the binding process of Ca to probe L was
2+
studied (inset in Figure 3a). Following the addition of 80.0 μM Ca to
.00 μM probe L, the fluorescence intensity of L decreased rapidly,
4
FIGURE 1 Isomerization of spiropyran derivatives between the SP
and MC forms upon light stimulus
reaching a stable value within 30 min.
(
a)
(b)
FIGURE 2 Spectral change of absorption (a) and fluorescence (b) for L (4.00 μM) and L after the fluorescence was recorded with λex at 375 nm
and 290 nm in ethanol

4
WANG ET AL.
(a)
(b)
(
c)
(d)
FIGURE 3 (a) Fluorescence spectra changes of L (4.00 μM) in the presence of various metal ions (80.0 μM) in ethanol. Inset: Response time of L
2
+
2+
(
4.00 μM) to Ca (80.0 μM) in ethanol. (b) Fluorescence intensities of L (4.00 μM) at 604 nm (I604nm) upon addition of Ca in the presence of
3+
3+
interfering metal ions (80.0 μM). Addition of 4.00 μL triethanolamine (0.100 mol/L) of Fe and Cr solution. λex = 375 nm. (c) Absorption spectra
of L (40.0 μM) with various ions in ethanol solution (the concentration of metal ions was 4.00 mM). (d) Colour changes of L (100 μM) and the MC–
Ca complex in visible light (left) and ultraviolet light (right)
2+
2
+
3
.3
|
Absorption studies on the binding of Ca
recorded at an emission maximum of 604 nm, on increasing addition
2+
of a Ca solution and excitation at 375 nm. As shown in Figure 4b,
2
+
The absorption and emission of L (40 μM) in ethanol solution were
investigated. As shown in Figure 4a, the absorption spectrum of L
displayed several bands at 236 nm, 287 nm, 357 nm, 414 nm, but
the lack of absorption around 526 nm suggested that L existed in
fluorescence intensity increased upon addition of Ca . The dynamic
range of response was from 8.00 μM to 80.0 μM, observed from the
2
+
fluorescence titration data. The DL of L for Ca was found as
−
8
2
4.53 × 10 M (R = 0.997) (inset in Figure 4b) (DL = 3σ/k, in which
2
+
the SP form. When Ca was added to the solution of L, a new absorp-
σ is the standard deviation of the blank solution; and k is the slope
[35]
2+
tion band appeared at 526 nm. With the incremental addition of Ca
4.00 mM), the new absorption band centred at 526 nm increased
of the calibration curve).
Sensor performance was compared with
2+
[36–38]
(
other previously published data for Ca sensors.
As listed in
together with the absorption band at 357 nm, and the absorption
band at 414 nm gradually decreased (inset in Figure 4a). In addition,
two isosbestic points emerged at 396 nm and 444 nm, indicating that
two or more chemical species coexisted in the solution. This result
Table 1, the DL values and linear range from this study approximated
to or were better than other reported data.
3
.5
|
Binding mode study
2+
suggested that Ca induced ring opening of L and converted it into
2
+
the MC form. In the MC form, phenolic oxygen and carbonyl in MC
Binding stoichiometry of L with Ca was determined using Job's plot,
known as the method of continuous variation. Determination of
stoichiometry was based on the measurement of physical properties
associated with complex formation at various mole fractions of the
2+
bound to Ca via a coordination bond. This chelation resulted in con-
formational rigidity, suggesting that intramolecular charge transfer
(
ICT) occurred from the phenolate oxygen to the electron‐deficient
2+
[30]
reactants with Ca
. In this case, the measurable parameters
quaternary nitrogen centre.
were fluorescence changes. Job's plot as shown in Figure 5a
2+
revealed that Ca formed a 1:1 complex with L. The association con-
2+
[39]
stant of L with Ca was obtained from the Benesi–Hildebrand plot
2
+
3
.4
|
Fluorescence studies on the binding of Ca
ꢀ
ꢁ
1
1
1
¼
þ
and observed to be
2+
Binding of L (4.00 μM) with Ca was also assessed by fluorescence
titration. Concentration‐dependent fluorescence changes were
F − 3:48E − 6 1:30E6½Ca2þꢀ 3:82E5
3 −1
2.47 × 10
M
(Figure 5b).

WANG ET AL.
5
(
a)
(b)
2+
FIGURE 4 (a) Absorption spectra of L (40.0 μM) in the presence of different concentrations of Ca (4.00 mM). (b) Fluorescence spectra of L
2
+
2+
(
4.00 μM) in the presence of different concentrations of Ca (80.0 μM). Inset: Fluorescence intensity of L as a function of Ca concentration.
ex = 375 nm
λ
2+
TABLE 1 Comparison of sensors for Ca detection
Structure of sensors
Detection limit (μM)
Linearity range (μM)
References
[
36]
CdTe/MA‐EGTA QDs
0.740
_
1.00–15.0
10.0–70.0
[
37]
[
9]
0
.270
1.00–4.00
[
38]
0
.0700
0.0500–1.00
0
.0453
8.00–80.0
This work
2
+
To understand the mechanism of the responses of L and Ca , the
assigned to aliphatic protons of the carboxylate chain, had two chem-
ical shifts, which also merged into a single chemical shift as indicated
by b′ and c′, with a shift to downfield. Signals of the vinylic (d and e)
as doublets at 5.93, 5.95 and 6.99, 7.00 ppm disappeared, along with
two new singlets that appeared in the range 8.40–8.60 ppm. Protons
from the aromatic rings also shifted to the lower field. Complex forma-
1
change in the H NMR spectrum of L (40.0 μM) in MeOH‐d
4
was
2+
investigated by adding Ca (40.0 μM) into the solution. As shown in
Figure 6, chemical shifts of H at a position assigned to methyl protons
on the N‐heterocycle took the form of two different chemical shifts
2
+
that merged into a single shift after the binding of L with Ca . This
2+
was resulted from the breaking of ether linkage in L and the symmetry
tion involving carboxy and phenolate with Ca would deshield the
protons at the vinylic (d and e) and the aliphatic protons adjacent to
[40]
of the quasi‐planar merocyanine.
Protons at positions b and c,

6
WANG ET AL.
(
a)
(b)
2
+
2+
FIGURE 5 (a) Job's plot for MC–Ca in ethanol. Total concentration of L and Ca is 40.0 μM. (b) Benesi–Hildebrand plot from fluorescence
2+
titration data of L with Ca . λex = 375 nm
2+
1
FIGURE 6 Top: Probable mode of binding of sensor L (40.0 μM) with Ca . Bottom: H NMR spectra of L in MeOH‐d
4
and L with 10.0
equivalent CaCl
2
2+
FIGURE 7 Diagrams of HOMO and LUMO levels of L, MC and the MC–Ca complex

WANG ET AL.
7
5
|
CONCLUSIONS
In conclusion, a spiropyran fluorescence probe L was prepared for col-
2+
orimetric detection of Ca by fluorescence ‘turn‐on’ response in eth-
2+
anol solution. Probe L selectively bound Ca to form a 1:1 ligand:
metal complex, which had a new emission band centred at 604 nm.
The sensing result was easily observed by a solution colour change
from colourless to pink under visible light, and from blue to red under
FIGURE 8 Photographs of test strips of probe L with various
concentrations of Ca (from left to right 0 mol/L, 5.00 mM, 0.0100
M, 0.0500 M, 0.100 M)
2+
2+
−8
UV light. The DL of probe L for Ca was found as 4.53 × 10 M.
Therefore, L could be potentially used in chemical, environmental
and physiological systems.
[
41]
the acid group (b, c).
The observed chemical shifts to downfield
for protons at positions b, c and d, e, as well as aromatic protons,
2
+
ACKNOWLEDGEMENTS
confirmed MC–Ca complex formation.
As shown in Figure 6, L reacted with Ca in two steps: (i) Ca
induced ring opening of L and isomerization from SP to MC; and (ii)
2+
2+
Authors thank Shanxi Returned Overseas Scholar Foundation
(205544901005), Shanxi 131 Leading Talents (205544901006,
205586801011) and Shanxi Provincial Hundreds of Talents Program
(205107001, 205107004) for funding support.
2+
2+
coordination of the MC ligand with Ca to form a 1:1 MC–Ca com-
plex from the phenolic oxygen and carbonyl oxygen of MC.
ORCID
Li Wang https://orcid.org/0000-0002-0412-2617
3
.6
|
Density functional theory studies
REFERENCES
2
+
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calculation was conducted using DFT at the B3LYP/6–31 + G(d) level
in ethanol. The energy minimized structure and highest occupied
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1
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2+
2+
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(
(
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4
|
APPLICATION
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2
+
For convenience of Ca sensing, 3.60 mg (2 mmol) L was dissolved in
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[
12] M. Kamiya, K. Johnsson, Anal. Chem. 2010, 82, 6472.
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2
5
.00 mL ethanol to obtain a 2.00 mM solution. A 2 × 1 cm filter paper
was soaked in the solution of L for 5 min, and dried at room temper-
2
+
2+
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