A new multifunctional Schiff base as a fluorescence sensor for Al 3+ and a colorimetric sensor for CN- in aqueous media: An application to bioimaging

Article abstract of DOI:10.1039/c4dt00361f

A multifunctional fluorescent and colorimetric receptor 1 ((E)-N′-((8-hydroxy-1,2,3,5,6,7-hexahydropyrido[3,2,1-ij]quinolin-9-yl) methylene)benzohydrazide) for the detection of both Al³⁺ and CN ^- in aqueous solution has been developed. Receptor 1 exhibited an excellent selective fluorescence response toward Al³⁺. The sensitivity of the fluorescent based assay (0.193 μM) for Al³⁺ is far below the limit in the World Health Organization (WHO) guidelines for drinking water (7.41 μM). In addition, receptor 1 showed an excellent detection ability in a wide pH range of 4-10 and also in living cells. Moreover, receptor 1 showed a highly selective colorimetric response to CN^- by changing its color from colorless to yellow immediately without any interference from other anions. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4dt00361f

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A new multifunctional Schiff base as a
fluorescence sensor for Al3+ and a colorimetric
sensor for CN− in aqueous media: an application
to bioimaging†
Cite this: Dalton Trans., 2014, 43,
6650
Seul Ah Lee,a Ga Rim You,a Ye Won Choi,a Hyun Yong Jo,a Ah Ram Kim,b
Insup Noh,b Sung-Jin Kim,c Youngmee Kimc and Cheal Kim*a
A multifunctional fluorescent and colorimetric receptor 1 ((E)-N’-((8-hydroxy-1,2,3,5,6,7-hexahydropyrido-
[3,2,1-ij]quinolin-9-yl)methylene)benzohydrazide) for the detection of both Al3+ and CN− in aqueous
solution has been developed. Receptor 1 exhibited an excellent selective fluorescence response toward
Al3+. The sensitivity of the fluorescent based assay (0.193 μM) for Al3+ is far below the limit in the World
Health Organization (WHO) guidelines for drinking water (7.41 μM). In addition, receptor 1 showed an
excellent detection ability in a wide pH range of 4–10 and also in living cells. Moreover, receptor 1
showed a highly selective colorimetric response to CN− by changing its color from colorless to yellow
immediately without any interference from other anions.
Received 4th February 2014,
Accepted 14th February 2014
DOI: 10.1039/c4dt00361f
www.rsc.org/dalton
bind to the iron in cytochrome c oxidase, interfering with elec-
tron transport and resulting in hypoxia. Several researchers
Introduction
Aluminum is the most prevalent (8.3% by weight) metallic also reported that cyanide occasionally plays a significant role
element and the third most abundant of all elements (after in many fire related deaths.8 Despite their toxicity, the use of
oxygen and silicon) in the earth’s crust.1 However, excess cyanides as raw materials for synthetic fibers, resins, herbi-
aluminum can cause damage to certain human tissues and cides, and the gold-extraction process is inevitable.9 Therefore,
cells, resulting in health problems such as Alzheimer’s disease reliable and efficient ways of detecting the presence of cyanide
and Parkinson’s disease.2 Therefore, the World Health Organi- are quite necessary. In recent years, a number of efforts have
zation (WHO) listed excess aluminum as one of the food pol- been devoted to design various chemo-sensors targeting the
lutants and restricted the aluminum concentration to 200 μg L−1 detection of cyanides.10 The most attractive approach focuses
(7.41 μM) in drinking water.3 Additionally, the solubility of on novel colorimetric cyanide receptors, which allow naked-eye
aluminum minerals in water increases under acidic conditions detection by a simple color change without the intervention of
and any such increased amount of Al3+ is fatal to plants and any expensive instruments.11 However, many receptors for
fish.4–6 Hence, it is highly desirable to develop a more sensitive cyanide reported so far have several limitations such as poor
and selective receptor which can detect Al3+ below the limit selectivity over F− or OAc−, or utilization of expensive instru-
that is recommended by WHO in a wide range of pH.7
ments, complicated synthesis, and working only in organic
Cyanide is well known as one of the most rapidly acting media.12,13 Therefore, the search for an effective and selective
and fatal poisons, and its toxicity results from its propensity to cyanide-sensing system in aqueous environments is still a
great challenge.
The development of a chemo-sensor, which is capable of
aDepartment of Fine Chemistry and Department of Interdisciplinary Bio IT Materials,
recognizing a metal ion and an anion simultaneously, is one
Seoul National University of Science and Technology, Seoul 139-743, Korea.
of the most significant tasks because of its important potential
E-mail: chealkim@seoultech.ac.kr; Fax: +82-2-973-9149; Tel: +82-2-970-6693
bDepartment of Chemical Engineering, Seoul National University of Science &
applications in biological industry and environmental pro-
cesses.14 In addition, the detection of multiple targets with a
single receptor would be more efficient and at the same time
less expensive than a one-to-one analysis method, and thereby
would attract more attention.15 Among various approaches for
the detection of both metal ions and anions, especially the
Technology, Seoul 139-743, Korea
cDepartment of Chemistry and Nano Science, Ewha Womans University,
Seoul 120-750, Korea
†Electronic supplementary information (ESI) available. CCDC 972999. For
ESI and crystallographic data in CIF or other electronic format see DOI:
10.1039/c4dt00361f
6650 | Dalton Trans., 2014, 43, 6650–6659
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fluorimetric and colorimetric methods are more popular (m, 4H), 2.62 (m, 4H), 1.86 (m, 4H); 13C NMR (400 MHz
because of their high sensitivities, easy operation, rapid DMSO-d6, ppm): 162.78, 155.37, 151.58, 145.90, 133.83, 132.31,
response rates, and relatively low costs.16
8-Hydroxyjulolidine-9-carboxaldehyde is
129.14, 128.96, 128.15, 113.10, 106.96, 106.45, 50.00, 49.54,
well-known 27.21, 22.20, 21.38, 20.91. ESI-MS m/z (M + H+): calcd, 335.16;
a
chromophore used in fluorescence chemosensors17 and chemo- found, 335.23.
sensors with the julolidine moiety are usually soluble in
aqueous solutions.18 On the other hand, benzhydrazide, con-
taining an amide group, demonstrates an anion recognition
ability by linking the anions with its amide group via a hydro-
gen bond.19 Therefore, we designed to synthesize a structurally
simple chemosensor 1 by integrating the julolidine chromo-
phore and the activated amide functionality for detection of
both cations and anions in aqueous solution, and tested its
sensing properties towards various metal ions and anions.
Herein, we report a chemo-sensor 1 based on the combi-
nation of julolidine and benzhydrazide for a simultaneous
selective detection of Al3+ and CN− in aqueous media. The
chemo-sensor 1 demonstrated the presence of the cation, Al3,+
by fluorescence enhancement and that of the anion, CN−, by
an instant change of color from colorless to yellow. It is
expected that 1 would have potential practical applications,
such as in bio-imaging, through mapping of Al3+ levels in cells.
X-ray data collection and structural determination
A dark color needle-type crystal, approximate dimensions of
0.20 mm × 0.02 mm × 0.01 mm, was used for X-ray crystallo-
graphic analysis. The diffraction data for compound 1 were col-
lected on a Bruker SMART APEX diffractometer equipped with
a monochromator using the Mo Kα (k = 0.71073 Å) incident
beam. The crystal was mounted on a glass fiber. The CCD data
were integrated and scaled using the BRUKER-SAINT software
package, and the structure was solved and refined using
SHEXTL V6.12. All hydrogen atoms except the amide hydrogen
atom were located in the calculated positions. The crystallo-
graphic data are listed in Table 1. The bond lengths and
angles are listed in Table S1.† Structural information was
deposited at the Cambridge Crystallographic Data Center
(CCDC 972999).
Fluorescence titration
1 (1.01 mg, 0.003 mmol) was dissolved in methanol (1 mL)
and 10 μL of this solution (3 mM) were diluted with 2.97 mL of
10 mM bis-tris buffer to make the final concentration of
10 μM. Al(NO3)3 (0.10 mmol) was dissolved in methanol
(5 mL) and 1.5–28.5 μL of this Al3+ solution (20 mM) were
transferred to each receptor solution (10 μM) to give 1–19
equiv. After mixing them for a few seconds, fluorescence
spectra were obtained at room temperature.
Experimental section
General information
All the solvents and reagents (analytical and spectroscopic
grade) were purchased from Sigma-Aldrich. 1H and 13C NMR
spectra were recorded on a Varian 400 MHz spectrometer and
chemical shifts are recorded in ppm. Electrospray ionization
mass spectra (ESI-MS) were collected on a Thermo Finnigan
(San Jose, CA, USA) LCQ™ Advantage MAX quadrupole ion
trap instrument by infusing samples directly into the source
using a manual method. The spray voltage was set at 4.2 kV,
and the capillary temperature was set at 80 °C. Absorption
spectra were recorded at room temperature using a Perkin
Elmer model Lambda 2S UV/Vis spectrometer. Emission
spectra were recorded on a Perkin-Elmer LS45 fluorescence
spectrometer. Elemental analysis for carbon, nitrogen, and
hydrogen was carried out using a Flash EA 1112 elemental ana-
lyzer (thermo) at the Organic Chemistry Research Center of
Sogang University, Korea.
UV-vis titrations
For Al3+, 1 (1.01 mg, 0.003 mmol) was dissolved in methanol
(1 mL) and 10 μL (3 mM) of it were diluted to 2.99 mL with
bis-tris buffer–methanol (999/1, v/v) to make
a final
Table 1 Crystal data and structure refinement for receptor 1
Empirical formula
Formula weight
Temperature
Wavelength
Crystal system
Space group
C20H21N3O2
335.40
170(2) K
0.71073 Å
Orthorhombic
Pbca
Synthesis of receptor 1
Unit cell dimensions
a = 12.835(3) Å α = 90.00°
b = 9.3810(19) Å β = 90.00°
c = 28.713(6) Å γ = 90.00°
3457.2(12) Å
An ethanolic solution of 8-hydroxyjulolidine-9-carboxaldehyde
(0.23 g, 1 mmol) was added to benzhydrazide (0.14 g, 1 mmol) Volume
Z
8
in absolute ethanol (3 mL). Two drops of HCl were added to
the reaction solution and it was stirred for 30 min at room
Density (calculated)
Absorption coefficient
temperature. A yellow precipitate was filtered and washed Crystal size
1.289 Mg m−3
0.085 mm−1
0.20 × 0.02 × 0.01 mm3
18 350
Reflections collected
several times with ethanol and dried in a vacuum to obtain the
pure yellowish solid.
Independent reflections
3394 [R(int) = 0.1756]
3394/2/231
0.769
Data/restraints/parameters
Yield 0.17 g (51%); Anal. Calc. for C20H21N3O2: C, 71.62; H, Goodness-of-fit on F2
6.31; N, 12.53; Found: C, 71.45; H, 6.45; N, 12.39; 1H NMR
(400 MHz DMSO-d6, ppm): δ 11.79 (s, 1H), 11.78 (s, 1H), 8.31
Final R indices [I > 2σ(I)]
R indices (all data)
Extinction coefficient
R1 = 0.0607, wR2 = 0.1328
R1 = 0.1587, wR2 = 0.1596
0.0146(11)
(s, 1H), 7.91 (d, 2H), 7.59 (t, 1H), 7.52 (t, 2H), 6.72 (s, 1H), 3.18 Largest diff. peak and hole
0.248 and −0.250 e Å−3
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concentration of 10 μM. Al(NO3)3 (0.10 mmol) was dissolved in hydrochloric acid in bis-tris buffer. After the solution with a
bis-tris buffer (5 mL) and 3–27 μL of the Al3+ ion solution desired pH was achieved, receptor 1 (1.01 mg, 0.003 mmol)
(10 mM) were transferred to the solution of 1 (10 μM) prepared was dissolved in methanol (1 mL), and then 10 μL of the
above. After mixing them for a few seconds, UV-vis spectra methanolic solution of the receptor 1 (3 mM) were diluted
were obtained at room temperature.
with 2.97 mL buffers to make the final concentration of 10 μM.
For CN−, 1 (1.01 mg, 0.003 mmol) was dissolved in methanol
Al(NO3)3·9H2O (11.3 mg, 0.03 mmol) was dissolved in bis-
(1 mL) and 30 μL (3 mM) of it were diluted to 2.97 mL with bis- tris buffer (1 mL, pH 7.00). 10 μL of the Al3+ solution (30 mM)
tris buffer–methanol (7/3, v/v) to make a final concentration of were transferred to each receptor solution (10 μM) prepared
30 μM. Tetraethylammonium cyanide (TEACN) (0.3 mmol) was above. After mixing them for a few seconds, fluorescence
dissolved in bis-tris buffer (5 mL) and 4.5–90 μL of the CN− spectra were obtained at room temperature.
solution (300 mM) were transferred to the solution of 1 (10 μM)
1H NMR titrations
prepared above. After mixing them for a few seconds, UV-vis
For 1H NMR titrations of receptor 1 with cyanide, five NMR
spectra were obtained at room temperature.
tubes of receptor 1 (3.35 mg, 0.01 mmol) dissolved in DMSO-
d6 (700 μL) were prepared and then five different concen-
trations (0, 0.005, 0.01, 0.02, and 0.05 mmol) of tetraethyl-
Job plot measurements
For Al3+, 1 (1.01 mg, 0.003 mmol) was dissolved in methanol
(1 mL). 100, 90, 80, 70, 60, 50, 40, 30, 20, 10 and 0 μL of the 1
solution were taken and transferred to vials. Each vial was
diluted with bis-tris buffer to make a total volume of 2.9 mL.
Al(NO3)3 (0.003 mmol) was dissolved in bis-tris buffer (1 mL).
ammonium cyanide dissolved in DMSO-d6 were added to each
solution of receptor 1. After shaking them for a minute, 1H
NMR spectra were obtained at room temperature. In order to
check the influence of water on the binding property, the same
0, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 μL of the Al3+ solu-
tion were added to each diluted 1 solution. Each vial had a
total volume of 3 mL. After shaking them for a minute, UV-vis
spectra were obtained at room temperature.
1H NMR titrations of receptor 1 with cyanide were carried out
in a mixture of DMSO-d6–D2O (9/1, v/v).
Methods for cell imaging
The cell imaging test was carried out by the same method as
our previous study.20 Human dermal fibroblast cells in low
passage were cultured in FGM-2 medium (Lonza, Switzerland)
supplemented with 10% fetal bovine serum, 1% penicillin/
streptomycin in an in vitro incubator with 5% CO2 at 37 °C.
Cells were seeded onto a 12 well plate (SPL Lifesciences,
Korea) at a density of 2 × 105 cells per well and then incubated
at 37 °C for 4 h after the addition of various concentrations
(0–100 μM) of Al(NO3)3. After washing with phosphate buffered
saline (PBS) two times to remove the remaining Al(NO3)3, the
cells were incubated with 1 (20 μM) for 30 min at room temp-
erature. The cells were observed using a microscope (Olympus,
Japan). The fluorescent images of the cells were obtained
using a fluorescence microscope (Leica DMLB, Germany) at
the excitation wavelength of 515–560 nm.
Competition with other metal ions or anions
For Al3+, 1 (1.01 mg, 0.003 mmol) was dissolved in methanol
(1 mL) and 10 μL of this solution (3 mM) were diluted with
2.99 mL of 10 mM bis-tris buffer to make the final concen-
tration of 10 μM. MNO3 (M = Na, K, 0.20 mmol) or M(NO3)2 (M
= Mn, Co, Ni, Cu, Zn, Cd, Mg, Ca, Pb, 0.20 mmol) or M(NO3)3
(M = Fe, Cr, Ga, In, 0.20 mmol) or M(ClO4)2 (M = Fe,
0.20 mmol) was dissolved in methanol (5 mL). 27 μL of each
metal solution (20 mM) were taken and added to 3 mL of the
solution of receptor 1 (10 μM) to give 19 equiv. of metal ions.
Then, 29 μL of Al3+ solution (20 mM) were added to the mixed
solution of each metal ion and 1 to make 19 equiv. After
mixing them for a few seconds, fluorescence spectra were
obtained at room temperature.
For CN−, 1 (1.01 mg, 0.003 mmol) was dissolved in metha-
nol (1 mL) and 30 μL of this solution (3 mM) were diluted with
2.88 mL of bis-tris buffer–methanol (7/3, v/v) to make the final
concentration of 30 μM. Tetraethylammonium salts of F−, Cl−,
Results and discussion
Synthesis and X-ray crystal structure of 1
−
Br−, and I−, tetrabutylammonium salts of H2PO4 and OAc−,
and sodium salts of SO42− and N3− (0.20 mmol) were dissolved
in bis-tris buffer (1 mL). 90 μL of each anion solution
(200 mM) were taken and added to 2.91 mL of the solution of
receptor 1 (10 μM) to give 200 equiv. of anions. Then, 90 μL of
tetraethylammonium cyanide solution (200 mM) were added
to the mixed solution of each anion and 1 to make 200 equiv.
After mixing them for a few seconds, UV-vis spectra were
obtained at room temperature.
Receptor 1 was obtained by the coupling reaction of 8-hydroxy-
julolidine-9-carboxaldehyde and benzhydrazide with 51% yield
1
in ethanol (Scheme 1), and characterized by H NMR and 13C
NMR, elemental analysis, ESI-mass spectrometry analysis, and
pH effect test
A series of buffers with pH values ranging from 2 to 12 was
prepared by mixing sodium hydroxide solution and Scheme 1 Synthetic procedure of 1.
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Scheme 2 Fluorescent enhancement mechanism of the 1–Al3+
complex.
Fig. 1 Crystal structure of receptor 1. Displacement ellipsoids are
shown at the 50% probability level.
excitation at 410 nm. Upon the addition of 20 equiv. of the
afore-mentioned metal ions to the solution of 1, only Al3+
caused a remarkable enhancement of the fluorescence inten-
sity with a high quantum yield (Φ = 0.502). This fluorescence
enhancement could be explained by an excited state intramole-
cular proton transfer (ESIPT) mechanism, CvN isomerization,
and chelation enhanced fluorescence (CHEF) (Scheme 2).
When 1 exists as an unbound form, the excited state intra-
molecular proton transfer21 and CvN isomerization of the imine
double bond in receptor 1 are responsible for nonradiative de-
activation, showing a very low fluorescence intensity.22 By con-
trast, upon addition of Al3+ to 1, a stable chelation of 1 with
Al3+ prevents both the CvN isomerization and ESIPT in 1,
resulting in a strong fluorescence intensity. In conjunction,
stable chelate complexation of Al3+ with 1 possibly induces
rigidity in the resulting complex, thereby generating efficient
chelation enhanced fluorescence.23 The selectivity of receptor
1 for Al3+ has been plotted as a bar graph in Fig. 2b. On the
other hand, Cr3+ and Fe3+, considered as strong Lewis acids,
did not show any fluorescence enhancements. This might be
due to their paramagnetic properties which promote dissipa-
tion of the excited state energy in a non-radiative process as a
result of spin–orbital coupling.24
X-ray crystallography. Crystals of 1 were obtained by slow evap-
oration in methanol and its structure is shown in Fig. 1.
Fluorescence and absorption studies of 1 toward different
metal cations
The selectivity of receptor 1 toward various metal cations, viz.,
Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Na+, K+, Mg2+, Ca2+,
Cr3+, Hg2+, Pb2+, Ga3+, In3+, Pb2+ and Fe3+, was primarily
studied by fluorescence in bis-tris buffer–methanol (999/1, v/v).
As shown in Fig. 2a, receptor 1 exhibits a little emission
with a low fluorescence quantum yield (Φ = 0.035) upon
A quantitative investigation of the binding affinity of 1 with
Al3+ was performed by a fluorescence titration (Fig. 3). The
fluorescence intensity increased up to 19 equiv. of Al3+, and no
Fig. 2 (a) Fluorescence spectra of 1 (10 μM) before and after addition of
Fig. 3 Fluorescence spectra of 1 (10 μM, λex = 410 nm) after addition of
increasing amounts of Al3+ (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19 and 20 equiv.) in bis-tris buffer–methanol (999/1, v/v) at
room temperature. Inset: the intensity at 488 nm versus the number of
equiv. of Al3+ added.
various metal ions (200 μM) of Mn2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+
,
Hg2+, Na+, K+, Mg2+, Ca2+, Al3+, Pb2+ Cr3+, Ga3+, In3+ and Fe2+ in bis-tris
buffer–methanol (999/1, v/v). (b) Bar graph shows the relative emission
intensity of 1 at 483 nm upon treatment.
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complex was determined to be 0.193 μM on the basis of 3σ/K
(Fig. S4†).28 Importantly, the detection limit of 1 is much lower
than the WHO limit (vide supra),3 and suggests that 1 could be
an effective sensor for the detection of aluminum in drinking
water.
To further check the practical applicability of receptor 1 as
an Al3+-selective fluorescence receptor, we carried out a com-
petitive experiment with different metal ions in an aqueous
solution (Fig. 6). It was found that most of the metal ions did
not interfere with the detection of Al3+ by 1. However, Fe2+,
Fe3+, Cu2+ and Cr3+ quenched about 39, 37, 58 and 38% of the
fluorescence obtained with Al3+ alone, respectively. Neverthe-
less, 1 still had sufficient “turn-on” ratios for the detection of
Al3+ in the presence of Fe2+, Fe3+, Cu2+ and Cr3+. These results
indicate that 1 could be a good sensor for Al3+ over competing
metal ions.
Fig. 4 Absorption spectra changes of 1 (10 μM) after addition of the
gradual amounts of Al3+ (1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 equiv.) in bis-tris
buffer–methanol (999/1, v/v) at room temperature. Inset: absorption at
475 nm versus the number of equiv. of Al3+ added.
For biological applications, the pH dependence of the
1–Al3+ complex was examined. Over the pH range tested, the
fluorescence intensity of 1–Al3+ displayed strong pH dependence
(Fig. 7). An intense and stable fluorescence of 1–Al3+ found in
further change was observed. A UV-visible spectrometric titra-
tion of 1 with Al3+ solution revealed that the absorption band
at 380 nm decreased and two new bands at 290 nm and
450 nm emerged and increased gradually to maxima at 19
equiv. of Al3+ (Fig. 4). In addition, an isosbestic point was
observed at 326 nm, indicating that only one product was gen-
erated from 1 upon binding to Al3+.
The Job plot analysis showed a 1 : 1 stoichiometry for the
1–Al3+ complex (Fig. S1†), which was further confirmed by
positive-ion ESI-mass spectrometry (Fig. 5). The mass spec-
trum showed a peak at m/z 423.60 that corresponds to the
complex, [1 + Al + NO3]+, and demonstrates a 1 : 1 binding of
the Al3+ to 1.
Based on the Job plot, ESI-mass spectrometry analysis, and
the crystal structures reported in the literature,25 we propose
the structure of a 1 : 1 complex of 1 and Al3+ as shown in
Scheme 2.
Fig. 6 Relative fluorescence of 1 and its complexation with Al3+ in the
presence of various metal ions. Response of 1 was included as controls.
1 alone, 1 + Al3+, 1 + Al3+ + Mn2+, 1 + Al3+ + Fe2+, etc. (left to right). Con-
The binding constant Ka of 1 with Al3+ has been determined
as 1.8 × 103 M−1 on the basis of Li analysis (Fig. S2†).25a,26 This
value is within those (103–107) previously reported for Al3+-
binding chemosensors.27 The detection limit for the 1–Al3+
ditions: 1, 10 μM; Al3+, 19 equiv.; other metal ions, 19 equiv. (λex
=
410 nm).
Fig. 7 Fluorescence intensity of 1–Al3+ (1, 10 μM) after addition of 19
equiv. of Al3+ at various ranges of pH in bis-tris buffer–methanol (999/1,
v/v) at room temperature. Inset: intensity at 483 nm.
Fig. 5 Positive-ion electrospray ionization mass spectrum of 1 upon
addition of 19 equiv. of Al3+ in methanol.
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Fig. 8 Fluorescence images of fibroblasts cultured with Al3+ and 1.
Cells were exposed to 0 (A and H), 10 (B and I), 20 (C and J), 40 (D and
K), 70 (E and L), 90 (F and M) and 100 μM (G and N) Al(NO3)3 for 4 hours
and then later with 1 (20 μM) for 30 min. The top images (A–G) were
observed with the light microscope and the bottom images were taken
with a fluorescence microscope. The scale bar is 50 μm.
the pH range of 4.0–10.0 warrants its application under physio-
logical conditions, without any change in detection results.
Based on the pH dependence experiment, further experi-
ments were conducted to test whether intracellular Al3+ could
be monitored by fluorimetry. Adult human dermal fibroblasts
were first incubated with various concentrations of Al3+ (0, 10,
20, 40, 70 90 and 100 μM) for 4 h and then exposed to 1
(20 μM) for 40 min before imaging, so that 1 could permeate
easily through the living cells without any harm. The fibro-
blasts that were cultured with both Al3+ and 1 exhibited fluo-
rescence (Fig. 8), whereas the cells cultured without Al3+ or 1
did not exhibit fluorescence. The intensity and region of the
fluorescence within the cell containing 1 gradually increased
with an increase in the Al3+ concentration from 10 to 100 μM.
In addition, the fluorescence intensity of the cells persisted even
after 5 h from exposure to 1 at 20 μM. These results show that
the new receptor 1 is efficient to image Al3+ in cells, and could be
useful in the determination of the exposure level of cells to Al3+.
Fig. 9 (a) Absorption spectral changes of 1 (30 μM) in the presence of
200 equiv. of different anions. (b) The color changes of 1 (30 μM) upon
addition of various anions (200 equiv.) in bis-tris buffer–methanol
(7/3, v/v).
to 40 equiv. of CN− shows that the absorption band at 478 nm
gradually decreased and two new bands at 350 nm and
400 nm appeared with two clear isosbestic points at 337 and
408 nm (Fig. 10b). The second step up to 200 equiv. of CN−
shows that the absorption peak at 370 nm decreased obviously,
whereas two new prominent bands at 290 nm and 450 nm
were developed with well-defined isosbestic points at 324 and
403 nm (Fig. 10c). Based on this two-step UV-vis process, we
propose that the first and second steps are the deprotonation
of phenolic OH and NH of amide, respectively. Moreover, the
bathochromic shift of the absorption bands led us to propose
the transition of the intramolecular charge transfer (ICT) band
through the deprotonation of the chemosensor 1 by the CN−,
Colorimetric and spectral response of 1 toward CN−
Receptor 1 was treated with a variety of anions to investigate based on Bhattacharya’s proposal.29,30 To identify the ICT
the selectivity in a bis-tris buffer–methanol mixture (7/3, v/v). property of 1, we have checked the change of its absorption
As shown in Fig. 9, the addition of CN− to 1 caused a signifi- spectra in both polar and non-polar solvents such as bis-tris
cant bathochromic shift in absorption spectra (Fig. 9a) and buffer, dimethylsulfoxide, methanol, and toluene, because it
showed a color change from colorless to yellow instantly has been reported that the solvent dipole can relax the ICT
(Fig. 9b).
However, other species such as F−, Cl−, Br−, I−, OAc−, SO4
excited by polar solvents.31 As shown in Fig. S4† and summar-
ized in Table 2, the absorption spectra of 1 featured a marginal
and N3 demonstrated almost no change in the UV-visible red-shift of absorption maxima (Δλabs = 8 nm), indicating an
spectra under identical conditions.
apparent solvent dependence of the absorption band. There-
2−
−
The binding properties of 1 with CN− were further studied fore, this solvatochromic behavior demonstrates the occur-
by UV-visible titration experiments (Fig. 10). If one looks care- rence of the ICT transition in receptor 1.14,31 In order to
fully into the UV-visible titration and inset in Fig. 10a, there is confirm whether the color change originated from the
the two-step change of the absorption bands. The first step up transition of ICT through a deprotonation mechanism, the
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Table 2 Absorption properties of 1 in various solvents
Solvent
λabs [nm] (log ε)
Buffera
DMSO
MeOH
Toluene
386 (3.93)
384 (4.18)
382 (4.10)
378 (4.12)
a 10 mM bis-tris, pH 7.0.
deprotonation mechanism between 1 and CN− (Fig. S5†).
Based on the UV-vis titrations, the binding constant of each
step of receptor 1 and CN− was determined using Li’s
equation. As shown in the insets of Fig. 10b and c, K1 and K2
were determined to be 6.6 × 102 (0–40 equiv.) and 4.2 × 102
(50–200 equiv.), respectively.
The preferential selectivity of 1 as a colorimetric chemosen-
sor for the detection of CN− was studied in the presence of
various competing anions. For competition tests, receptor 1
was treated with 200 equiv. of CN− in the presence of 200
equiv. of other anions. No interference was observed in the
detection of CN− in the presence of other anions (Fig. 11). This
Fig. 10 (a) Absorption spectral changes of 1 (30 μM) after addition of
increasing amounts of CN− (10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110,
120, 130, 140, 150, 160, 170, 180, 190 and 200 equiv.) in 10 mM bis-tris
buffer–methanol (7/3, v/v) at room temperature. Inset: absorption at
450 nm versus the number of equiv. of CN− added. (b) Absorption spec-
tral changes of 1 in the range of 0–40 equiv. of CN− in (a). (c) Absorption
spectral changes of 1 in the range of 50–200 equiv. of CN− in (a).
Fig. 11 (a) Absorption spectra of 1 and its complexation with CN− in
the presence of various anions. Response of 1 was included as controls.
1 alone, 1–CN−, 1–F−, 1–Cl−, 1–Br−, 1–I−, 1–OAc−, 1–SO4 2−, and 1–
N3−. (b) The color changes of 1 and its complexation with CN− in the
presence of various anions in bis-tris buffer–methanol (7/3, v/v). Con-
interaction between 1 and OH− was conducted. UV-visible
spectral change of 1 upon addition of OH− was almost identi-
cal to that of 1 upon addition of CN−, demonstrating the ditions: 1, 30 μM; CN−, 200 equiv.; other metal ions, 200 equiv.
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of Education, Science and Technology (2012001725 and
2012008875) is gratefully acknowledged.
Notes and references
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Fig. 12 1H NMR titration of 1 with CN− in DMSO-d6.
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cule.11 To check the influence of water on the binding property,
1H NMR titration experiments of 1 with CN− were also carried
out in a mixture of DMSO-d6–D2O (9 : 1, v/v) (Fig. S6†). Almost
identical results were observed, indicating that water did not
affect the binding interaction of receptor 1 with CN−.
Conclusion
We have successfully designed and synthesized a simple, fluo-
rescent and colorimetric chemosensor 1, capable of recogniz-
ing both cations and anions in aqueous solution. 1 exhibited
an excellent selectivity and sensitivity towards Al3+ by fluo-
rescent intensity enhancement and towards CN− by inducing a
rapid color change from colorless to yellow. The detection
limit of 1 for Al3+ (0.193 μM) was much lower than that men-
tioned in guidelines of the WHO (7.41 μM). Moreover, 1 could
operate in a wide range of pH and can be successfully applied
to living cells for detecting Al3+. On the basis of the results, we
believe that receptor 1 will offer an important guidance to the
development of single receptors for recognizing both cations
and anions both in vivo and in vitro.
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Acknowledgements
The Basic Science Research Program through the National
Research Foundation of Korea (NRF) funded by the Ministry
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