A simple Schiff base as dual-responsive fluorescent sensor for bioimaging recognition of Zn2+ and Al3+ in living cells

Article abstract of DOI:10.1039/c8tb01743c

A simple Schiff base fluorescent sensor (BDNOL) was synthesized from the reaction of picolinohydrazide and 4-(diethylamino)salicylaldehyde, and developed for selective detection of Al³⁺ and Zn²⁺. This non-fluorescent sensor displayed obvious fluorescence enhancement after binding to Al³⁺/Zn²⁺ ions with high sensitivity and selectivity, accompanied by obvious fluorescence emission enhancement (504 nm for Al³⁺ and 575 nm for Zn²⁺). The detection limits were found to be 8.30 × 10^-8 M for Al³⁺ and 1.24 × 10^-7 M for Zn²⁺. The binding mechanisms between BDNOL and Al³⁺/Zn²⁺ ions were supported by ¹H NMR and HR-MS analysis, and a density functional theory (DFT) study. The sensing behavior was also studied with molecular logic functions of OR, AND, and NOT gates. Furthermore, the fluorescent sensor was successfully used to recognize Al³⁺ and Zn²⁺ in living cells, suggesting that this simple biosensor has great potential in biological imaging applications.

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Graphical Abstract
The chemosensor BDNOL can selectively recognize Al3+ and Zn2+ with significant
fluorescence enhancement at 504 nm and 575 nm.

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A simple Schiff base as dual-responsive fluorescent sensor for
bioimaging recognition of Zn2+ and Al3+ in living cells
Haiyang Liua,b, Tianqi Liuc, Jia Lia, Youming Zhanga,b, Jiahua Lib, Jun Songa*, Junle Qua*, Wai-Yeung
Wonga,b*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
A simple Schiff base fluorescent sensor (BDNOL) was synthesized from the reaction of picolinohydrazide and 4-
(diethylamino)salicylaldehyde, and developed for selective detection of Al3+ and Zn2+. This non-fluorescent sensor
displayed obvious fluorescence enhancement after binding to Al3+/Zn2+ ions with high sensitivity and selectivity,
accompanied by obvious fluorescence emission enhancement (504 nm emission for Al3+ and 575 nm for Zn2+). The
detection limits were found to be 8.30×10−8 M for Al3+ and 1.24×10−7 M for Zn2+, respectively. The binding mechanisms
between BDNOL and Al3+/Zn2+ ions were supported by 1H NMR, HR-MS analysis, and density functional theory (DFT) study.
The sensing behavior was also studied with molecular logic functions of OR, AND, and NOT gates. Furthermore, the
fluorescent sensor was successfully used to recognize Al3+ and Zn2+ in living cells, suggesting that this simple biosensor has
a great potential in the application of biological imaging.
www.rsc.org/
and nonꢀdestructive nature in the living system.[9ꢀ20] Until
recently, plenty of fluorescent probes have been developed for
monitoring the metal ions of human, such as Fe3+, Zn2+, Cu2+,
Introduction
As an indispensable transition metal for life, Zn2+ plays
significant roles in many physiological processes, and its
metabolism disorder is linked with neurodegenerative diseases,
such as Alzheimer's, Parkinson's and epilepsy disease.[1ꢀ3] It has
been found that Al3+ is not necessary for the human body,
although it ranks as the most prevalent metal element in the
earth's crust.[4,5] People can easily get Al3+ intake via daily diet,
because the aluminum products are widely used in our daily life
[6]. However, excessive intake of Al3+ can also cause serious
damage to the central nervous system for human and increase
the risk of Alzheimer's disease, Parkinson's disease and
amyotrophic lateral sclerosis (ALS).[5,7,8] Therefore, it is of
considerable importance and highly desirable to design and
develop efficient biosensors to monitor Zn2+ and Al3+ in
biological system.
Al3+, Cr3+, Cd2+, Hg2+, etc.[4,6,19ꢀ30] Many of these chemosensors
have excellent performance in the process of sensing one
specific target metal ion, but they can not proceed in oneꢀtoꢀ
multi sensing. Compared to those oneꢀtoꢀone type sensors, to
develop simple, efficient and functionalized fluorescent sensor
with both biological activity and multiꢀanalyte response is
challenging from the viewpoints of economic value and
practical applications.
In recent years, several oneꢀtoꢀmulti type fluorescent
sensors developed for different metal ions have been
reported.[24,30ꢀ37] Kim et al. developed a rhodamineꢀbased and
dualꢀresponsive sensor to selectively recognize Al3+ and Cu2+
by using different signaling channels, UV absorption and
fluorescence.[30] Hu et al. designed a rhodamineꢀphenylurea
conjugate derivative to selectively detect Pb2+ in aceyonitrile,
and Hg2+ in aqueous media, respectively.[31] Emrullahoğlu et al.
Fluorescence detection methods have attracted enormous
attention for qualitative and quantitative detection of relevant
metal ions and anions of organism, owing to their immediate
response, high sensitivity, easy visualization, realꢀtime analysis,
developed
a rhodamineꢀBODIPY derivative for selective
recognition of Hg2+ and Au3+ with different excitation
wavelength.[32] Liu et al. designed and synthesized a quinolineꢀ
based fluorescent sensor, which can be employed to selectively
recognize Cd2+ and Zn2+ via two different binding
mechanisms.[34] However, there are few dualꢀresponsive
chemosensors reported for selective recognition of Al3+ and
Zn2+ in the biosystem, while monitoring these two metals in
organism is of great importance due to their roles to human
health and disease.
a.Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education
and Guangdong Province, College of Optoelectronic Engineering, Shenzhen
University, Shenzhen 518060, PR China Email: songjun@szu.edu.cn;
jlqu@szu.edu.cn
b.Department of Applied Biology and Chemical Technology, The Hong Kong
Polytechnic University, Hung Hom, Hong Kong, PR China Email: wai-
yeung.wong@polyu.edu.hk
c. Department of Chemistry, KTH Royal Institute of Technology, 10044 Stockholm,
Sweden
Salicylaldehyde Schiff base derivatives have received wide
attention in the field of sensing metal ions, because these
chemosensors contain nitrogenꢀoxygenꢀrich coordination
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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circumstance which can coordinate with metal ions, leading to
interesting photophysical property changes.[22,25,28ꢀ30,38] In the
structure of salicylaldehyde Schiff base, the ꢀOH group can
Fluorescence and UVꢀvis studies
form a sixꢀmembered transition state with the adjoining imine Stock solutions of metal chlorides (0.001 M) and sodium
bond, leading to excited state intramolecular proton transfer fluoride (0.001 M) were prepared in 20 mM HEPES buffer (pH
(ESIPT).[38,39] After chelating with metal ions, the ESIPT 7.2), and BDNOL solution was prepared in DMSO (0.001 M).
process is restrained, and meanwhile the ꢀCH=Nꢀ isomerization The concentration of BDNOL for fluorescence and UVꢀvis
is also inhibited, both of which could cause optical signal measurements was diluted to 10 ꢁM in CH3OH/HEPES buffer
changes of the salicylaldehyde Schiff base fluorescent (1/4, v/v, pH 7.2). All spectral data were recorded in 5 min after
sensor.[33,38,39] With this in mind, we synthesized and developed the addition of cations and anions at room temperature.
a simple Schiff base fluorescent sensor (BDNOL), derived from
DFT calculations
the
reaction
of
picolinohydrazide
and
4ꢀ
(diethylamino)salicylaldehyde, which displayed favorable Geometry optimization of BDNOL and [BDNOLꢀ
selectivity for Zn2+ and Al3+ over other competitive metal ions. Zn2+]/[BDNOLꢀAl3+] were performed by the DFT/B3LYP
In the sensing process, the O atom of phenolic hydroxyl group, method using Gaussian 09 software.[41] The 6ꢀ311G basis set
and the N atoms of imine bond and pyridine unit coordinate to was used for C, H, N, O and the LanL2DZ basis set was used
the respective central metal, accompanied by an obvious for the effective potential (ECP) set for Zn2+ and Al3+. The
fluorescence emission enhancement in each case (504 nm geometries of all structures were fully optimized and normal
emission for Al3+ and 575 nm for Zn2+ respectively). The mode analysis was used to confirm the nature of the stationary
density functional theory (DFT) calculations were also points and evaluate the thermochemical properties.
performed to investigate the emission difference between Al3+
and Zn2+. Moreover, further application of BDNOL in
Cellular imaging
fluorescence tracing of Zn2+ and Al3+ in living cells was HeLa cells were cultured in RPMIꢀ1640 medium with 10%
studied, suggesting that this Schiff base compound can be used fetal bovine serum (FBS) at 37 oC under a humidified
as a potential biosensor for Zn2+ and Al3+ in living system.
atmosphere of 5% CO2. The cells were incubated with BDNOL
for 6 h and washed with PBS buffer for three times. Then the
cells were incubated with Zn2+ or Al3+ (50 ꢁM) for another 2 h.
Finally, the seeded cells were imaged using a laser scanning
confocal fluorescence microscope.
Experimental section
Synthesis of BDSBL
The cell cytotoxicity of BDNOL was measured by a
standard MTT assay.[42] HeLa cells were seeded in 96ꢀwell
plates with different concentrations of BDNOL (ranged from 2
to 50 ꢁM). After incubation with BDNOL for 12 h, MTT (10
ꢁM) solution was added into each well of a 96ꢀwell plate and
incubated for another 4 h. Then the medium was removed and
replaced with 100 ꢁL DMSO in each well. Finally, the optical
density of each well was measured with a microplate reader at
490 nm. The cell viability was calculated from the optical
density ratio of the treatment sample to the control (The
cytotoxicity measurements were repeated for three times).
Picolinohydrazide was prepared by the reaction of methyl
pyridineꢀ2ꢀcarboxylate and hydrazinium hydrate in the
refluxing methanol via a reported method (1H NMR and 13C
NMR spectra are shown in Fig. S1 and S2).[40] Into a 50 mL
twoꢀnecked flask, picolinohydrazide (0.411 g, 0.003 mol) and
4ꢀ(diethylamino)salicylaldehyde (0.580 g, 0.003 mol) were
added and dissolved in 15 mL anhydrous ethanol. The mixture
solution was stirred at 60 oC for 5 h, and then a yellow
precipitate was obtained. The solid residue was collected by
filtration and washed with cold ethanol (15 mL). After 6 hours
with vacuum drying at 50 oC, a pale yellow powder was
1
obtained, 0.693 g, yield: 74%. H NMR (400 MHz, DMSOꢀd6
)
Results and discussion
d (ppm): 12.22 (s, 1H), 11.59 (s, 1H), 8.71 (d,
8.58 (d, 1H), 8.08 (m, 2H), 7.66 (t, = 3.8 Hz, 1H), 7.13 (d,
8.4Hz, 1H), 6.27 (d, = 8.4 Hz, 1H), 6.13 (s, 1H), 3.36 (q,
7.0 Hz, 4H), 1.11 (t,
DMSOꢀd6) d (ppm): 160.40, 160.15, 151.93, 150.71, 149.98,
149.02, 138.51, 132.41, 127.39, 123.07, 106.94, 104.17, 98.00,
44.31, 13.05. HRMS calcd for C17H21N4O2 [BDNOL+H]+:
313.1665, found: 313.1659.
J = 7.6 Hz, 1H),
J
J
J
=
=
BDNOL was facilely synthesized by the reaction of
picolinohydrazide and 4ꢀ(diethylamino)salicylaldehyde in the
refluxing methanol as shown in Scheme 1, and the structure
was then confirmed by 1H NMR (Fig. S3), 13C NMR (Fig.S4),
and HRMS measurements (Fig. S5).
J
J
= 7.0 Hz, 6H). 13C NMR (100 MHz,
Selectivity of BDNOL for Al3+ and Zn2+
The UVꢀvis and fluorescent spectra of BDNOL were tested in
CH3OH/HEPES buffer (1/4, v/v, pH 7.2) in the presence of
various metal ions (Na+, K+, Mg2+, Ca2+, Cu2+, Ni2+, Mn2+, Co2+,
Fe2+, Fe3+, Cr3+, Ag+, Cd2+, Hg2+, Pb2+, Al3+ and Zn2+). As
shown in Fig. S6, broad absorption around 375 nm was
HO
H
N
O
O
N
OHC
N
O
O
NH2NH2
H2O
NH
N
HO
N
EtOH, 5 h, 60 o
C
MeOH, 6 h, Reflux
N
NH2
N
Methyl Picolinate
Picolinohydrazide
BDNOL
Scheme 1. The synthetic route of BDNOL
2 | J. Name., 2012, 00, 1-3
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observed for BDNOL, while there is almost no significant suggesting that the receptor BDNOL has a stronger binding
change in the absorption spectrum within 300ꢀ550 nm among
affinity towards Al3+ than Zn2+. It is an important question to be
solved on how to determine whether Zn2+ exists in the
BDNOLꢀAl3+ solution.
Fig. 1. Fluorescence emission spectra obtained for BDNOL (10 μM) after the
addition of 5.0 equiv. of various metal ions in CH3OH/HEPES buffer (1/4, v/v,
pH 7.2) (λex: 390 nm).
most of the added metal ions (5.0 equiv.) except Al3+ and Zn2+.
Upon the addition of Al3+ and Zn2+, the absorption band
centered at 375 nm exhibited a large redꢀshift to 405 nm and
dramatic absorption enhancement for Al3+, and a larger redꢀ
shift to 434 nm and absorption enhancement for Zn2+,
accompanied by an obvious color change from colorless to light
brown and light yellow respectively. In the fluorescence test,
Fig. 2. (a) The fluorescence intensity at 504 nm of BDNOL (10 μM) with
5.0 equiv. of Al3+, and then added 10.0 equiv. of various metal ions,
free BDNOL exhibited scarcely fluorescence emission. Upon and (b) the fluorescence intensity at 575 nm of BDNOL (10 μM) with
5.0 equiv. of Zn2+, and then added 10.0 equiv. of various metal ions in
addition of the metal ions, only Al3+ and Zn2+ induced apparent
fluorescence changes. On the other hand, other metal ions had
CH3OH/HEPES buffer (1/4, v/v, pH 7.2). 1, Al3+ (a)/ Zn2+ (b);2, Na+; 3, K+; 4,
Mg2+; 5, Ca2+; 6, Mn2+; 7, Cu2+; 8, Co2+; 9, Ni2+; 10, Fe2+; 11, Zn2+; 12, Fe3+; 13,
almost no effects on the fluorescence emission. As shown in
Fig. 1, Al3+ and Zn2+ ions can be easily distinguished from the
different fluorescence emission characteristics, because
significant fluorescence enhancements centered at 504 nm and
575 nm were obtained for Al3+ and Zn2+, respectively.
The BDNOL fluorescence enhancement phenomenon after
encountering Al3+ or Zn2+ can be attributed to the formation of stable
complexes between BDNOL and Al3+/Zn2+, leading to the chelationꢀ
enhanced fluorescence (CHEF) effect via the phenolic hydroxyl O,
the imine bond N, and the pyridine N chelating the central
metal.[25,40,43ꢀ47] More importantly, after BDNOL binds to Al3+/Zn2+,
the ꢀCH=Nꢀ isomerization is inhibited, and meanwhile the
coordination of N,N,O towards Al3+/Zn2+ ions also prevents the
ESIPT process, both of which are conducive to fluorescence
emission enhancement of BDNOL.[38,39,45,48]
Cr3+; 14, Cd2+; 15, Pb2+; 16, Hg2+; 17, Ag+. (λex: 390 nm)
.
Of particular importance is that fluoride anion (Fꢀ) can be
used as a masking agent towards Al3+ by the formation of stable
aluminum fluoride complexes (AlFx).[49,50] If both of Zn2+ and
Al3+ exist in a solution simultaneously, the introduction of Fꢀ
can cause AlFx species to be generated, releasing free BDNOL
which can subsequently chelate Zn2+ accompanied by a obvious
fluorescence emission redꢀshift to 575 nm from 504 nm (Fig.
3). Through the displacement strategy, it can be used to
determine the existence of Zn2+ in the Al3+ꢀZn2+ solution. The
above results indicate that the simple Schiff base can act as a
potential fluorescent sensor for the selective detection of Al3+
and Zn2+.
The specificity of BDNOL's fluorescence responses
towards Al3+ and Zn2+ ions was examined by competitive
experiments. As shown in Fig. 2a, no significant interference
was observed in BDNOLꢀAl3+ solution in the presence of other
metal ions. Then, BDNOL was treated with Zn2+ in the presence
of other competitive metal ions (10 equiv.). The solution of
BDNOLꢀZn2+ showed almost no fluorescence variation against
other metal ions except Al3+ (Fig. 2b). As seen in Fig. 2b, the
introduction of Al3+ into the system of BDNOLꢀZn2+ can cause
a blueꢀshift of the fluorescence emission from 575 nm to 504
nm (the original fluorescence peak of BDNOLꢀAl3+),
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about 40 seconds. The instant fluorescence response and
fluorescence stability suggests that BDNOL has the potential of
practical application.
Fluorescence titration study
In order to estimate the sensing capability of BDNOL toward
Al3+ and Zn2+, the detail fluorescence titration analysis was
further investigated. As shown in Fig. 5, with gradual addition
of Al3+ and Zn2+ (0ꢀ5.0 equiv.), the fluorescence emission of
BDNOL was significantly enhanced at 504 nm and 575 nm,
respectively. The fluorescence spectra were recorded upon the
addition of different concentrations of Al3+ and Zn2+ from 0.2 to
50 ꢁM (Fig. 5a and Fig. 5b). BDNOL showed nice linear
relationships (R2 = 0.995 for Al3+ and R2 = 0.989 for Zn2+,
respectively) between the fluorescence intensity and Al3+/Zn2+
Fig. 3. Fluorescence emission spectra of 10 μM of BDNOL, BDNOL-Al3+
BDNOL-Al3+, and BDNOL-Al3+&Zn2+ after addition of F- in CH3OH/HEPES
,
buffer (1/4, v/v, pH 7.2) (λex: 390 nm).
Fig. 4. (a) Fluorescence intensity at 504 nm and 575 nm recorded for
BDNOL (10 μM) at various pH values in the absence and presence of 5
equiv. of (a) Al3+, and (b) Zn2+, respectively.
Fig. 5. Fluorescence emission spectra recorded for BDNOL (10 μM) upon
gradual addition 0-50 μM of (a) Al3+ and (b) Zn2+ in CH3OH/HEPES buffer
(1/4, v/v) (λex: 390 nm).
Effect of pH and response time
The pH variation effects on fluorescence intensity of BDNOL
and BDNOLꢀAl3+/BDNOLꢀZn2+ are depicted in Fig. 4. There is
no obvious fluorescence change of BDNOL at a variable pH
range from 3.0 to 12.0. When Al3+ is introduced, a significant
fluorescence intensity enhancement of BDNOLꢀAl3+ is
observed from pH 5.5 to pH 8.5 (Fig. 4a). Little fluorescence
change is found in the pH range from pH 3.0 to pH 5.0 and
from pH 9.0 to pH 12.0. The acidic conditions can induce
dissociation of the BDNOLꢀAl3+ complex because of the
protonation of BDNOL,[51] and the higher pH conditions can
provide sufficient hydroxyl ions to extract metal ion from
BDNOLꢀAl3+ complex to form the hydrolyzed complexes.[50,52]
A similar phenomenon was also found in BDNOLꢀZn2+ system,
and an increase of fluorescence intensity caused by the addition
of Zn2+ was observed in a range of pH 6.0~10.0 (Fig. 4b). The
pH effect study clearly indicated that the best fluorescence
behavior of BDNOL towards both of Al3+ and Zn2+ are under
physiological window at pH=7.4, which is conducive to using
the fluorescent sensors in the biological systems.[53]
The time dependent fluorescence intensity of BDNOL in
presence of Al3+ and Zn2+ ions was also studied, respectively.
As seen in Fig. S7, the fluorescence intensity of BDNOL (504
nm) basically reached the maximum in one minute after
addition of Al3+ and remained almost unchanged over a period
of 30 min. In the process of sensing Zn2+, a faster recognition
behavior can be observed and the maximum fluorescence
intensity (575 nm) remained almost unchanged (~30 min) after
Fig. 6. Calibration curve obtained for the fluorescence intensity at the
emission peak of BDNOL (10 μM) versus the concentration of (a) Al3+
and (b) Zn2+ (0-50 μM), and the inset shows the linear range (0-10 μM)
of the curve.
concentrations (0.2~10 ꢁM) (Fig. 6a and Fig. 6b). The detection
limits were determined as 8.30 × 10ꢀ8 M for Al3+ and 1.24 ×10ꢀ7
M for Zn2+ on the basis of the IUPAC recommendation of
3σ/S,[54] where σ is the standard deviation of the control sample
and
S is the slope of the calibration curve. The obtained
detection limits are much lower than the enforceable drinking
water standard for Al3+ (7.4 ꢁM) and Zn2+ (45.9 ꢁΜ) developed
by the World Health Organization (WHO).[55]
Considering the potential use of BDNOL for practical
samples, the presence of Al3+/Zn2+ ions in tapꢀwater was tested
to evaluate its practical feasibility. As seen in Fig. S8, the
fluorescence intensity of emission peak increased gradually
upon addition of Al3+ and Zn2+, respectively (2.0ꢀ9.0 ꢁM), and
the total Al3+/Zn2+ concentrations in practical samples were
quantitatively derived from fitted linear calibration curves (each
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practical sample had four replicates). The recovery and relative
standard deviation (RSD) of BDNOL were 97.0ꢀ100.3% and
1.32ꢀ3.82% for Al3+, and 101.5ꢀ102.2% and 0.84ꢀ4.64% for
Zn2+, respectively (Table S1). The fluorescence titration
analysis and tapꢀwater verification test indicate that the Schiff
base sensor can be used to quantitatively detect Al3+/Zn2+ in
practical water samples.
Binding mechanism stud
y
On the basis of the Job's plot analyses, the binding
stoichiometries of the interaction between BDNOL and Al3+,
and BDNOL and Zn2+ were both calculated as 1:1 (Fig. S9).
The mass spectrometry is another direct evidence to investigate
the binding ratio between BDNOL and the central metal. As
shown in Fig. S10, all these ESIꢀmass data clearly indicated the
formation of 1:1 complex between BDNOLꢀAl3+/Zn2+. Based
on the binding stoichiometry analysis and BenesiꢀHildebrand
equation,[56,57] the association constants (Ka) were determined to
be 1.3 × 106 Mꢀ1 for BDNOLꢀAl3+, and 7.9 × 104 Mꢀ1 for
BDNOLꢀZn2+ from the fluorescence titration fitting curves
(Fig. 6). Compared with the BDNOLꢀZn2+ complex, the
generation of more stable aluminum complex is due to the
higher binding affinity towards the Schiff base BDNOL which
provided a hard base environment.[58]
Fig. 7. Frontier molecular orbital profiles of BDNOL, BDNOL–Al3+ and
BDNOL–Zn2+ based on DFT calculations.
In order to understand the binding mechanism of BDNOL
with Al3+/Zn2+, the 1H NMR test of BDNOL has been
conducted by concomitant additon of 1.0 equiv. of Al3+/Zn2+
ions. As seen in Fig. S11 and S12, after addition of these metal
ions, the proton Hf of phenol hydroxyl group at 11.59 ppm
Scheme 2. Proposed binding modes of BDNOL for the recognition of
Al3+ and Zn2+ (SC: Solvent circumstances)
.
The energy band gap ꢂE between the HOMO and LUMO of
BDNOL, BDNOL–Al3+ and BDNOL–Zn2+ were 3.905 eV,
3.502 eV and 2.610 eV, respectively, which indicate that the
interaction of Al3+/Zn2+ and BDNOL resulted in the changes in
electronic and photophysical properties of this Schiff base
sensor. In addition, the theoretical energy gap of BDNOL–Zn2+
(2.610 eV) is narrower than that of BDNOL–Al3+ (3.502 eV),
and the actual fluorescence emission peak of BDNOL–Zn2+
shows a larger redꢀshift to 575 nm compared to the emission
peak of BDNOL–Al3+ at 504 nm, which is also consistent with
the principles of quantum chemistry.[61] According to the Job's
almost disappeared, and the proton Hg of imine group shifted
3+
△
δ was up to 0.
13 ppm for Al and 0.19 ppm for
downfield (
Zn2+, respectively). The protons Hi, Hj, Hk and Hl of pyridine
△
δ was up to 0.22 ppm, 0.22 ppm,
unit also shifted downfield (
0.25 ppm and 0.06 ppm for Al3+ addition, and up to 0.24 ppm,
0.24 ppm, 0.27 ppm and 0.20 ppm for Zn2+ addition,
respectively). The 1H NMR results suggested that the O atom of
hydroxyl unit, the N atoms of imine group and pyridine unit
might coordinate to the central Al(III)/Zn(II). The binding
behavior of BDNOL toward Al3+/Zn2+ was consistent with the
previous reports.[59,60]
1
plot, ESIꢀmass analysis, H NMR study and DFT calculations,
the binding modes of BDNOLꢀAl(III)/Zn(II) are proposed in
Scheme 2.
To obtain a better understanding of the binding mode of
BDNOL and Al3+/Zn2+, and why the introduction of these two
cations could cause different fluorescence emission, the DFT
calculations with B3LYP method were performed. The
calculated minimal structures of BDNOL and BDNOLꢀ
Al3+/Zn2+ complexes with the key structural properties are
shown in Fig. 7. The distances of Al3+ from the pyridine N,
imine N and hydroxyl O are 1.98, 1.93 and 1.77 Å, and Zn2+
from the pyridine N, imine N and hydroxyl O are 2.07, 2.02 and
1.92 Å, respectively. These spatial data indicate that these
optimized structure could provide adequate space to
accommodate Al3+/Zn2+ ions. Timeꢀdependent DFT (TDꢀDFT)
calculations were also performed at the optimized geometries.
Logic gate behavior of BDNOL
The logic gate analysis is an important functionalization and
application of molecular sensor.[62ꢀ64] The fluorimetric sensing
processes of BDNOL towards Al3+ and Zn2+ were also studied
in a molecular logic circuit, in which these two metal ions and
Fꢀ were used as three inputs, and the obtained fluorescence
intensity signals of 504 nm and 575 nm served as two outputs
(Fig. 8a and 8b). As shown in Fig. 8c, in the “Output 504 nm”
logic analysis, either of the inputs “Al3+” and “Al3+ AND Zn2+”
is assigned with “1” (504 nm fluorescence emission “turnꢀon”).
In the “Output 575 nm” logic analysis, each of the inputs
“Zn2+”, “Zn2+ AND Fꢀ” and “Zn2+ AND Al3+ AND Fꢀ” is
assigned with “1” (575 nm fluorescence emission “turnꢀon”).
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While all the other inputs, includig “Fꢀ ” and “Al3+ AND Fꢀ”,
are assigned with “0” (no fluorescence emission “turnꢀon”).
This logic gate study showed that this single fluorescence
sensor could perform arithmetic operations, which is like a
signal transmission computer.
Cytotoxicity and cell imaging
The cytotoxicity of BDNOL was also performed by a MTT
assay before its bioimaging application (Fig. S13). The
following MTT study shows that BDNOL (20 ꢁM) has low
cytotoxicity towards cells, indicating that this simple
fluorescent chemosensor has a good potential for biological
applications. In the experiment of cell imaging, the HeLa cells
were incubated with BDNOL (20 ꢁM) for 4 h and then treated
with metal ions (50 ꢁM of Al3+ or Zn2+ ions) for another 2 h.
The cells incubated with the chemosensor only exhibited
negligible fluorescence (Fig. 9a), while an obvious green
fluorescence image was observed after the addition of Al3+ ions
(Fig. 9b), and a new orange fluorescence image could be
observed for Zn2+ ions addition (Fig. 9c), indicating that
BDNOL has a very good performance for Al3+ and Zn2+ ions
imaging in Hela cells. The cell imaging results suggest that this
Schiff base sensor can be employed for fluorescence imaging of
Al3+ and Zn2+ ions selectively in living cells.
Fig. 8. The corresponding visualization figure, (b) the fluorescence emission
output bar diagram, and (c) the molecular logic circuit with three inputs
(Zn2+, Al3+ and F-) and two outputs (fluorescence emission at 504 nm and 575
nm.
(b) BDNOL+Al3+
(c) BDNOL+Zn+
(a) BDNOL
B
r
i
g
h
t
ꢀ
f
i
e
l
d
40 µm
D
a
r
k
ꢀ
f
i
e
l
d
40 µm
O
v
e
r
l
a
y
40 µm
Fig. 9. Fluorescence microscopy images of HeLa cells. (a) Cells were
incubated with 20 μM BDNOL for 4 h, (b) Cells were incubated with 10
μM BDNOL for 4 h, and further incubated with 50 μM Al3+ for 2 h, (c)
Cells were incubated with 10 μM BDNOL for 4 h, and further incubated
with 50 μM Zn2+ for 2 h
.
Conclusions
In summary, a simple Schiff base fluorescent sensor BDNOL
was developed for the selective recognition of Al3+ and Zn2+
ions. This nonꢀfluorescent sensor can be used to sensitively
detect Al3+ and Zn2+, leading to obvious fluorescence
enhancement at 504 nm and 575 nm, respectively. The
fluorescence enhancement mechanism of sensing Al3+ and Zn2+
is based on the CHEF effect, the ꢀCH=Nꢀ isomerization, and the
inhibition of ESIPT process. The recognition mechanism has
also been investigated by 1H NMR, ESIꢀmass analysis and DFT
calculations in details. In addition, BDNOL was used for the
6 | J. Name., 2012, 00, 1-3
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ARTICLE
fluorescence imaging of Al3+ and Zn2+ selectively in living 16 X. Kong, F. Su, L. Zhang, J. Yaron, F. Lee, Z. Shi, Y. Tian, D. R.
cells, indicating that this simple biosensor can be potentially
applied for the recognition of Al3+/Zn2+ in biological systems.
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This work was supported by the National Basic Research
Program of China (2015CB352005); the National Natural
Science Foundation of China (61775145, 61605124, 31771584,
61525503, 61620106016, 81727804, 61605130, 51602201);
Guangdong Natural Science Foundation Innovation Team
(2014A030312008); and Shenzhen Basic Research Project
(JCYJ20170412110212234,
JCYJ20150930104948169,
JCYJ20160308093035903,
JCYJ20160328144746940,
GJHZ20160226202139185), the Hong Kong Polytechnic
University (1ꢀZE1C). T.L. is supported by the China
Scholarship Council and Ms. Clarea Au (847S for the Endowed
Professorship).
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