A highly sensitive fluorescent probe for bioimaging zinc ion in living cells and zebrafish models

Article abstract of DOI:10.1039/C8NJ02197J

Zn²⁺ plays fundamental roles in cellular metabolism and apoptosis, as well as neurotransmission. However, how to fast detect Zn²⁺ in biology is still challenging and meaningful. In this study, we rationally synthesized a simple OFF-O

Full text of DOI:10.1039/C8NJ02197J

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ISSN 1144-0546
PAPER
Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
diarylethene-based metal–organic frameworks
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A Highly sensitive fluorescent probe for bioimaging Zinc Ion in
Living Cells and Zebrafish Models
Zhengliang Lu,*, a Wenlong Fan, a Yanan Lu, a Chunhua Fan, *, a Huaiqing Zhao, a Kai Guo, a Wei Chu,
Yizhong Lu*, b
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Zn2+ plays fundamental roles in cellular metabolism and apotosis, as well as neurotransmission. However, it is still
challenging and meaningful how to fast detect Zn2+ in biology. In this report, we rationally synthesized a simple off-on
fluorescent probe for fast detection of Zn2+ based on intramolecular charge transfer and chelation enhanced fluorescence.
Addition of Zn2+ into the probe induced a significant fluorescence enhancement with a bathochromic shift of 55 nm.
Moreover, the probe demonstrated excellent sensitivity (detection limit = 0.072 μM) and outstanding selecitvity toward
Zn2+ and ultra-fast response rate of two seconds. The recognition mechanism of the probe toward Zn2+ was fully confirmed
by HRMS and 1H NMR spectra, as well as TDDFT calculations. Furthermore, the probe could rapidly light-up bioimaging Zn2+
in living cells and zebrafish models with low cytotoxicity and good biocompatibility.
www.rsc.org/
photoinduced electron transfer (PET), intramolecular charge
18
1. Introduction
transfer (ICT),17,
excited-state intramolecular proton,19-22
fluorescence resonance energy transfer,23 or chelation enhanced
It is well known that small-molecular fluorescent probes are now
regarded as an excellent technique that can visualize biological
substances in living cells or organs due to high temporal and spatial
resolution, ease of sample preparation, good sensitivity and
selectivity.1-4 Zn2+, as one of most abundant microelements, has
been attracting more or more interests in biology and pharmacy for
decades due to its important roles in many physiological and
pathological processes including gene transcription and expression,
cell division and proliferation, brain activity.5-7 For example, Zn2+
fluorescence (CHEF).24 In this regard, rational combinations of signal
groups such as rhodamine,25,
peptide,27 diazafluorene,28
26
coumarin,29 4-amino-1,8-naphthalimide,30,
quinoline,
31
32-34
dipyrrometheneboron difluoride,35, 36 fluorescein37 and others,38-42
and recognition sites including di(2-pyridylmethyl)-amine,43-45 and
N,N,-di-(2-picolyl)-ethylenediamine46 have been proved to be an
effective design strategy. It has to be pointed out that many of
them showing remarkable fluorescence response and/or color
change were obtained by complicated synthetic procedures.
Recently, several two-photon (TP) fluorescent probes toward Zn2+
were reported showing fluorescence emission under the long wave
can endogenously neuro-modulate glutamate transmission.8,
9
Being an important structural cofactor and catalytic center of
hundreds of zinc proteins and regulators of enzymes, the
fluctuation of Zn2+ concentration are tightly associated with severe
risks of physical growth retardation and neurological disorders such
as cerebral ischemia, diabetes, Alzheimer’s disease, amyotrophic
lateral sclerosis.10, 11
Since the pioneering work sensing Zn2+ in living cells and organs
from several excellent research groups,12,
numerous Zn2+-specific fluorescent probes reported as a donor‒
acceptor (D‒A) system in recent years.14-16 These probes showed
excellent absorption/fluorescence spectra or color change due to
the effective modulation of electron transfer processes including
light excitation.47 For example, Liu’s group developed
fluorescent probe as quinoline derivative for monitoring
a TP
a
intracellular free Zn2+. Tian’s group reported a TP fluorescent probe
for ratiometric bioimaging Zn2+ in hippocampal tissue and zebrafish
upon excitation at 800 nm.48 With regard to the naphthalene
fluorophore, Kim’s group reported a TP probe for imaging zinc ion
distributed in Golgi upon excitation at 750 nm.49 Meng’s group
synthesized a TP fluorescent probe containing quinoline and DPA
13
there have been
for ratiometric sensing mitochondrial Zn2+ 50
fluorescent probes toward Zn2+ including TP ones still suffered the
interference from Cu2+ Cd2+ or Fe3+ 51-54
Therefore, the
.
Unfortunately, most
,
.
development of fluorescent probes with excellent selectivity and
sensitivity is still challenging and demanding to rapidly monitor the
homeostasis of biological zinc ion.
Inspired by these nice research results for sensing Zn2+, in this
report, we developed a simple Zn2+ fluorescent probe (Sen-OH)
based on
sensitivity. Sen-OH was synthesized via a one-step condensation of
5-phenylsalicylaldehyde and 2-aminobenzohydrazide. As
a Schiff-base derivative with high selectivity and
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anticipated, addition of Zn2+ rapidly promoted
a
remarkable
The mixture of 5-phenylsalicylaldehyde (0.20 g, 1 mmol) and 2-
fluorescence enhancement with a low detection limit. Moreover, aminobenzohydrazide (0.15 g, 1 mmol) in 20 mL of ethanol was
Sen-OH was low-cytotoxic and cell-permeable and successfully used refluxing overnight under stirring. After the precipitate was formed,
for bioimaging Zn2+ in living cells and zebrafish models.
the solvent was removed by a rotary evaporator. The pure title
compound was obtained by recrystallization from ethanol as a
yellow solid in 60% yield. 1H NMR (400 MHz, Acetone-d6) δ 11.89 (s,
1H), 11.23 (s, 1H), 8.67 (s, 1H), 7.70 – 7.62 (m, 5H), 7.45 (t, J = 7.7
Hz, 2H), 7.37 – 7.30 (m, 1H), 7.25 (ddd, J = 8.4, 7.0, 1.5 Hz, 1H), 7.07
(d, J = 8.4 Hz, 1H), 6.86 (dd, J = 8.4, 1.1 Hz, 1H), 6.61 (ddd, J = 8.1,
7.1, 1.2 Hz, 1H), 6.39 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ
165.04, 157.06, 150.31, 147.42, 139.43, 132.56, 131.38, 129.36,
128.84, 128.29, 127.53, 126.79, 126.13, 119.05, 117.00, 116.50,
114.59, 112.41. HRMS (ESI) calc. for [C20H17N3O2‒H]‒, 330.12425;
found, 330.12476.
Scheme 1. Synthesis of Sen-OH, Ref-OH-NH2, Ref-OH
2.4 Synthesis of Ref-OH-NH2
The reference compound Ref-OH-NH2 was prepared using
salicylaldehyde following the same procedure as Sen-OH in 73%
1
yield. H NMR (400 MHz, Acetone-d6) δ 11.67 (s, 1H), 10.15 (s, 1H),
8.45 (s, 1H), 7.90 (dd, J = 7.8, 1.5 Hz, 1H), 7.37 (dd, J = 7.7, 1.7 Hz,
1H), 7.30 (m, 2H), 7.18 – 7.09 (m, 1H), 7.04 (dd, J = 7.8, 1.6 Hz, 1H),
6.99 (dd, J = 8.1, 1.1 Hz, 1H), 6.94 – 6.85 (m, 1H), 6.83 – 6.77 (m,
1H), 6.71 (td, J = 7.5, 1.1 Hz, 1H). 13C NMR (100 MHz, DMSOꢀd6) δ
160.30, 157.60, 154.74, 149.56, 146.38, 134.27, 131.63, 130.48,
129.91, 128.11, 125.97, 124.13, 119.25, 119.03, 118.34, 117.65,
116.59, 115.84, 114.90, 113.17. HRMS (ESI) calc. for [C14H13N3O2‒
H]‒, 254.0930; found, 254.0932.
2. Experimental
2.1 Instruments and chemicals
All chemical reagents and solvents (analytical grade) were
obtained commercially and used as received without further
purification unless otherwise stated. H NMR and 13C NMR spectra
1
2.5 Cell culture and fluorescence imaging
were performed on a Bruker Advance DRX 400 spectrometer at 400
MHz/100 MHz. Mass spectra (ESI) were recorded on Bruker Waters-
Q-TOF-Premier spectrometer. UV-Vis spectra were recorded on a
TU-1901 spectrometer. Fluorescence spectra were conducted on a
Hitachi F-4600 luminescence spectrophotometer with xenon lamp
(Hitachi High-Technologies C., Japan). The pH value was measured
by a Leici PHS-3C pH meter. The fluorescence images of cell and
HeLa cells were grown in Dulbeco’s Modified Eagle Medium
(DMEM) supplemented with 10% fetal bovine serum, 1% antibiotics
at 37oC in a 95% humidity atmosphere containing 5% CO2. After
washing with Dulbecco’s phosphate-buffered saline (DPBS) twice,
HeLa cells were plated on a flatbottom 96-well plate in 100 μL of
culture medium and incubated in 5% CO2 at 37oC and adhered for
24 h. The cells were washed three times with PBS before further
experiments. The viability was performed by a standard MTT
method after the 24 h incubation of the cells with the probe at
different concentrations. Two groups were set for bioimaging Zn2+
in living cells. In the control group, the cells were imaged after the
cells were treated only with the probe for 30 min. In the
experimental group, the images were taken after the above Sen-
OH-pretreated cells were further incubated with Zn2+ for 30 min
more.
zebrafish were taken using
a Leica confocal laser scanning
microscope (Germany). 2-aminobenzohydrazide and Ref-OH were
synthesized following a revised procedure.55
2.2 Synthesis of 5-phenylsalicylaldehyde (1)
5-phenylsalicylaldehyde was synthesized according to a modified
procedure.56 Dry paraformaldehyde (6.60 g, 220 mmol) was added
to a mixture of 4-phenylphenol (2.72 g, 16 mmol), triethylamine
(8.4 mL, 61 mmol) and anhydrous MgCl2 (2.28 g, 24 mmol) in dry
acetonitrile (50 mL). After refluxing for 6 h, the mixture was cooled
to room temperature, acidified with 1M HCl, and extracted with
ethyl acetate (3 × 20 mL). The combined organic layer was washed
with water and dried over anhydrous MgSO4. After the solvent was
removed under vacuum, the residue was purified by column
chromatography to give 1.77 g of the title compound as a white
solid (yield 65%). 1H NMR (400 MHz, CDCl3) δ 11.01 (s, 1H), 9.98 (d, J
= 0.6 Hz, 1H), 7.79 – 7.75 (m, 2H), 7.58 – 7.54 (m, 2H), 7.46 (dd, J =
8.5, 7.0 Hz, 2H), 7.39 – 7.34 (m, 1H), 7.08 (dd, J = 8.3, 0.7 Hz, 1H).
13C NMR (100 MHz, CDCl3) δ 196.67, 160.97, 139.34, 135.77, 133.34,
131.88, 128.99, 127.40, 126.60, 120.71, 118.14. HRMS (ESI) calc. for
[C13H10O2‒H]‒, 197.0603; found, 197.0601.
2.6 In Vivo Imaging of Zebrafish.
Two-day old zebrafish were obtained from commercial suppliers.
In the control group, the zebrafish were incubated only with the
probe (5 μM) for 30 min and then washed three times with PBS
buffer. In the experimental group, the zebrafish pretreated with
Sen-OH (5 μM) for 30 min were further incubated with Zn2+ (100
μM). The images of zebrafish were taken on a confocal microscope
excited at 405 nm.
3. Results and discussion
3.1 Synthesis
2.3 Synthesis of Sen-OH
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It is well known that many reported probes for Zn2+ were designed
based on its strong affinity. This kind of probe usually holds a
suitable cage to accommodate Zn2+. With this thought, a simple
Schiff-base probe was easily developed by a one-step condensation
of 2-aminobenzohydrazide and 5-phenylsalicylaldehyde in good
yield. In order to elucidate its characteristic, two reference
compounds were synthesized following the similar procedure as
Sen-OH. All compounds were characterized by 1H NMR, 13C NMR
and ESI-MS analysis (SI, Figures S1-S5).
3.2 Fluorescence and absorption spectroscopic studies of Sen-OH
toward Zn2+
With the probe in hand, we first determined the feasibility of Sen-
OH sensing Zn2+ by UV-visible and fluorescence spectra in CH3CN
aqueous solution (3:7, v/v, HEPES 20 mM, pH 7.4). As shown in
Figure 1a, the UV-vis spectra of free Sen-OH (10 μM) exhibited an
absorption band at 360 nm, which moved to a broad band at
around 400 nm upon addition of 10 equiv. of Zn2+. Additionally, an
obvious color change from colorless to green was observed under
365 nm UV light (inset of Figure 1a). Free Sen-OH emitted weak
fluorescence at 430 nm excited at 365 nm. Addition of Zn2+ led to
disappearance of this emission and simultaneously formation of
fluorescence at 485 nm (Figure 1b). After these initiation, the
competition experiments of Sen-OH toward other common metal
ions (70 μM) in CH3CN aqueous solution (3:7, v/v, HEPES 20 mM, pH
7.4) were performed (Figure 2). As expected, there were no obvious
fluorescence response even addition of 10 equiv. of competing
metal ions such as Mg2+, Al3+, Ag+, Ba2+, Ca2+, Cr3+, Cu2+, Fe3+, Hg2+,
K+, Li+, Mn2+, Na+, Pb2+, Cd2+ (Figure 2). However, only Zn2+ induced
the distinct color and spectral change. Therefore, these attempts
evidenced the outstanding selectivity of Sen-OH toward Zn2+ over
other metal ions.
Fig. 1 Absorption (a) and fluorescence (b) responses of Sen-OH (10
μM) in the absence and presence of Zn2+ (10 equiv) in CH3CN
aqueous solution (3:7, v/v, HEPES 20 mM, pH 7.4). The
measurements were performed after the addition of Zn2+ for 10
min. Inset: photo graphs of Sen-OH (left) and Sen-OH-Zn2+ (right)
under natural light or an ultraviolet light.
Fig. 2 Fluorescence responses of Sen-OH (10 μM) toward common
competing metal ions (10 equiv.) in CH3CN aqueous solution (3:7,
v/v, HEPES 20 mM, pH 7.4). 1, Blank; 2, Mg2+; 3, Al3+; 4, Ag+; 5, Cd2+
;
6, Ba2+; 7, Ca2+; 8, Cr3+; 9, Cu2+; 10, Fe3+; 11, Hg2+; 12, K+; 13, Li+; 14,
Mn2+ 15, Na+; 16, Pb2+; 17, Zn2+
The measurements were
;
.
performed after addition of metal ions for 10 min. Excitation: 360
nm.
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emission intensities of Sen-OH upon addition of Zn2+ at different
concentrations (20, 60 μM) showed that the response time of Sen-
OH is almost independent of the concentration of Zn2+. Additionally,
photostability was conducted in order to rule out the possibility of
photobleaching interfering with detecting Zn2+. The change of
fluorescence intensity at 485 nm of Sen-OH (10 μM) in CH3CN-H2O
(v/v, 3:7, HEPES 20 mM, pH 7.4) were continuously monitored for
every five minutes in absence and presence of UV light at 365 nm.
As shown in Figure S9, we did not observe any significant change of
fluorescence intensity even under UV irradiation of 365 nm for 30
minutes. These results implied that Sen-OH would rapidly and
reliably sense Zn2+
.
3.3 Effect of pH.
In order to extend biological applications, the pH dependency on
fluorescence intensity of Sen-OH was investigated in the absence
and presence of Zn2+ at various pH value in CH3CN aqueous
solution. As shown in Figure S10, the fluorescence emission of Sen-
OH sharply increased when pH ˃ 8.5, which was aꢀributed to
deprotonation of hydroxyl group at high pH. Another reason could
be that the basic condition accelerated the structural
transformation from amide to imidic acid tautomer, which was also
preferable for an effective conjugate system. However, addition of
Zn2+ did not induce obvious fluorescence change when pH < 2.0
probably because protonation of imine inhibited coordination of
Zn2+ with Sen-OH under strongly acidic conditions. The fluorescence
intensity of Sen-OH with Zn2+ remarkably increased under pH ˃2.0
and reached a plateau with no obvious changes after pH ˃ 4.0,
which was in good agreement with strong affinity toward Zn2+. This
wide pH range of 4-8.5 made Sen-OH suitable for Zn2+ detection
under physiological conditions.
Fig. 3 (a) Fluorescence spectra of Sen-OH (10 μM) in the presence
of increasing concentrations of Zn2+ (0, 10, 20, 30, 40, 50, 60, 70, 80,
90 μM) in CH3CN aqueous solution (3:7, v/v, HEPES 20 mM, pH 7.4).
(b) Calibration curve of the fluorescence intensities (I485) versus the
concentration of Zn2+. Excitation: 360 nm. The measurements were
performed after the addition of Zn2+ for 10 min.
Subsequently, the fluorescence titrations of Sen-OH toward Zn2+ in
CH3CN aqueous solution (3:7, v/v, HEPES 20 mM, pH 7.4) were
performed to check the sensitivity of Sen-OH. As shown in Figure
3a, the free probe Sen-OH exhibited weak fluorescence at 430 nm
upon excitation at 365 nm. Addition of Zn2+ reduced a new peak at
386 nm with a large bathochromic shift of 55 nm, which was
assigned to Sen-OH binding Zn2+. The fluorescence at 485 nm
increased with gradually increasing concentration of Zn2+ and
reached a maximum upon addition of 7 equiv. of Zn2+. Furthermore,
as shown in Figure 3b, the fluorescence intensity of Sen-OH at 485
nm was proportional to the gradually increasing concentration of
Zn2+ ranging from 0 to 70 μM with a R2 value of 0.996. The limit of
detection (LOD) was evaluated to be 72 nM using the 3σ/m
equation, which is lower enough than 76 μM of Zn2+ tolerance level
in drinking water WHO recommends. These data suggested that
Sen-OH sensitively responded toward Zn2+
complexation stoichiometry of Sen-OH-Zn2+,
.
To proof the
Job’s plot of
a
fluorescence intensity of Sen-OH against mole fractions of Sen-OH
and Zn2+ was analyzed in CH3CN aqueous (3:7, v/v, HEPES 20 mM,
pH 7.4). As shown in Figure S6, The maximum emission intensity at
a mole fraction of 0.3 indicated this 2:1 stoichiometry between Sen-
OH and Zn2+. Additionally, the reversibility of Sen-OH toward Zn2+
was confirmed by adding EDTA into Sen-OH-Zn2+ in CH3CN-H2O
(Figure S7).
1
Fig. 4 Partial H NMR spectra of (i) Sen-OH in acetone-d6, (ii) Sen-
OH-Zn2+ in acetone-d6, (iii) Ref-OH in acetone-d6 and (iv) Ref-OH-
Zn2+ in acetone-d6.
For on-site practical applications, photostability and response time
are crucial parameters for fluorescent probes toward metal ions.
Thus, we investigated the fluorescence changes of Sen-OH in
presence of Zn2+ toward time. As showed in Figure S8, the
fluorescence emission of Sen-OH sharply reached the maximum
within two seconds after injection of Zn2+ in CH3CN aqueous
solution (3:7, v/v, HEPES 20 mM, pH 7.4), indicating the feasibility
for real-time detection of Zn2+ in living systems with a high temporal
resolution. There was no obvious changes of emission intensity
observed over the subsequent 140 s. Additionally, The fluorescence
3.4 Mechanism Studies
Most fluorescent probes for metal ion detection are designed
based on its strong binding affinity.57-61 Therefore, a suitable cage to
accommodate metal ions is one of the most important structural
characteristics of this kind of probes. The fluorescence intensity of
probes for metal ions usually is reversible upon addition of strongly
chelating reagents such as EDTA. In fact, the selectivity, sensitivity,
or fluorescence intensity and emission wave length could be tuned
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by a suitable substituent or conjugation.62 Thus, we designed Sen- Zn2+, the introduction of 5-phenyl as an hyperchromic group led to a
OH via a one–step condensation of 5-phenylsalicylaldehyde and 2- remarkable emission enhancement at 485 nm by further enlarging
aminobenzohydrazide, which provided
a
three-atom cage the effective conjugated system of Sen-OH. All these data
composed of O, N and O from the hydroxyl, imine and imide, evidenced our speculation that the rational introduction of the
respectively. We imagined that addition of metal ions could phenyl and amino group to Sen-OH can modulate electron transfer
modulate charge/electron processes in Sen-OH which finally process.
emitted fluorescence. A feasible mechanism of Sen-OH detecting
TDDFT calculations using Gaussian 09 program at the B3LYP/6-31G
Zn2+ was demonstrated in Scheme S1. Sen-OH emitted weak level were performed to illustrate the above feasible mechanism of
fluorescence probably because of the PET process on. As Sen-OH sensing Zn2+. The optimized structures of Sen-OH and the
anticipated, addition of Zn2+ into Sen-OH induced a remarkable complex Sen-OH-Zn2+ are shown in Figure 5. The HOMO mainly
fluorescence intensity enhancement. Concomitantly, the large distributed in amino benzene moiety. However, the LUMO located
bathochromic shift was also observed. Reasonably, these changes on amide, imine and hydroxyl benzene groups other than the 5-
were attributed to the combination of ICT and CHEF induced by Zn2+ phenyl group. The energy gap between HOMO and LUMO of free
binding. The three-atom cage holding Zn2+ limited the free rotation probe was 2.56 eV. In the probe-Zn2+ (2:1) complex, the HOMO
and twist of Sen-OH, which led to the good rigidity and high mainly distributed in the entire 5-phenylsalicylaldehyde moiety due
coplanarity of the molecular assembly. The similar cage was to the presence of Zn2+. The LUMO still located on amino benzene
confirmed to be much effective for Zn2+ selectivity by two or three- moiety from the other probe molecule of the complex. The lower
input logic gate designed by Yin or Glass’s group.63, 64 Moreover, the energy gap of 1.61 eV in the complex corresponded to the red shift
structural transformation from amide to imidic acid tautomer of fluorescence. These computational calculations supported our
happened in the presence of Zn2+, being propitious to form an design concept, in which the substituent was allowed to endow
extended conjugate system. In order to more clearly elucidate the Sen-OH with the promising properties.
proposed mechanism, HRMS of Sen-OH were conducted in the
absence and presence of Zn2+. Besides a peak at m/z = 332.12476
assigned to the free probe ([M+H+]: calcd 332.13941, Figure S11),
Figure S11 demonstrated a new peak at m/z = 725.18491, which
corresponded to Sen-OH-Zn2+ ([2M+Zn2+-H+]: calcd 725.18548).
This complexation was fully consistent with the 2:1 stoichiometric
ratio from the Job’s plot.
Moreover, 1H NMR titrations of Sen-OH without or with Zn2+ in
acetone-d6 were carried out to evidence its strong binding ability to
Zn2+ (Figure 4). Without Zn2+, the probe Sen-OH in acetone-d6
showed an amide proton peak at 11.89 ppm and a hydroxyl proton
peak at 11.23 ppm (Figure 4i). As expected, both chemical shifts
completely disappeared in the presence of Zn2+, which indicated
that the three-atom cage chelated Zn2+ and amide NH–C(=O) –)
transformed its imidic acid tautomer (–(N=C(OH) –) (Figure 4ii). This
structural transformation enlarged the conjugate system, in which
Zn2+-binding could inhibit PET and concomitantly enable both ICT
and CHEF. The complexation of Sen-OH and Zn2+ was further
evidenced by the fact that the chemical shift of imine proton at 8.67
Fig. 5 Optimized structures and frontier molecular orbitals of (a)
ppm downfield shifted to 9.05 ppm companied by considerable
Sen-OH, (b) Sen-OH:Zn2+ (2:1). Calculations were performed at
downfield shifts of aromatic protons.
B3LYP/6-31G level.
In order to validate the significant contribution of the 5-phenyl
and aniline group of Sen-OH, two reference compounds Ref-OH and
Ref-OH-NH2 were synthesized using salicylaldehyde and amino
salicylaldehyde. As shown in Figure 4iii and iv, the vanishing of
chemical shifts of amide proton at 11.67 ppm and phenolic proton
at 10.15 ppm in 1H NMR spectrum of Ref-OH in the presence of Zn2+
confirmed the strong coordination ability of the three-atom cage to
Zn2+. A series of fluorescence spectra of Ref-OH and Ref-OH-NH2
with or without Zn2+ were performed in CH3CN aqueous solution
(3:7, v/v, HEPES 20 mM, pH 7.4). As shown in Figure S12, Ref-OH
showed
a slight fluorescence enhancement. But Ref-OH-NH2
demonstrated only a small red shift from 450 to 485 nm, which
indicated that the amino group could act as an auxochrome. Such a
group containing lone pair electrons can shift the maximum
emission peak to the long wave range. Probably, in the presence of
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Fig. 6 Confocal fluorescence images of living HeLa cells treated with Fig. 7 Confocal fluorescence images of living zebrafish treated with
Sen-OH in the absence and presence of Zn2+. (a) Bright field image Sen-OH with or without Zn2+. (a) Bright field image of zebrafish. (b)
of HeLa cells with Sen-OH. (b) Image of (a). (c) Overlay of (a) and Green channel image of (a). (c) Overlay of (a) and (b). (d) Bright field
(b). (d) Bright field image of HeLa cells with Sen-OH and Zn2+. (e) image of zebrafish with Sen-OH and then Zn2+. (e) Green channel
Image of (d). (f) Overlay of (d) and (e). The excitation wavelength is image of (d). (f) Overlay of (d) and (e). The excitation wavelength is
405 nm and the emission was collected at 500-550 nm.
405 nm and the emission was collected at 500-550 nm.
3.5 Imaging of Zn2+ in Living Cells and zebrafish models.
4. Conclusions
Before we pursued more potential applications in biology, first,
the cell cytotoxicity and biocompatibility of the probe were been
In summary, we have reported a simple off-on fluorescent probe
for Zn2+ detection under physiological conditions. Addition of Zn2+
into the probe led to the strong green fluorescence emission in
CH3CN aqueous solution with a large red shift. This probe showed
high selectivity and sensitivity to Zn2+ over other competing metal
ions. The response time of less than 2 seconds made the probe
suitable for further on-site applications. The Zn2+ recognition
examined
by
standard
3-(4,5-dimethlthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assays. HeLa cells were
incubated with different concentration probe Sen-OH (0, 5, 10, 15,
20 μM) for 24 h. As shown in Figure S13, we observed that the cell
maintained its high viability of 90% even the concentration of the
probe was up to 15 μM. Thus, the probe Sen-OH demonstrated the
low cytotoxicity and good biocompatibility to living HeLa cell
samples.
1
mechanism was fully supported by HRMS, H NMR titrations, as
well as theoretical calculations. Furthermore, the probe was
successfully applied for bioimaging Zn2+ in living HeLa cells and
zebrafish models with excellent biocompatibility and low cell
cytotoxicity.
Inspired by the aforementioned sensing performance including
high specificity, quantitative detection, high temporal resolution,
low cytotoxicity and good biocompatibility, we next tested the
feasibility of Sen-OH for bioimaging Zn2+ in living HeLa cells with a
confocal laser scanning microscope (Leica, Germany). After HeLa
cells were incubated with Sen-OH (5 μM) for 10 min and washed
three times with PBS buffer, as shown in Figure 6a-c, no obvious
fluorescence no matter under bright field or green channel was
observed upon excitation at 405 nm. As anticipated, when the cells
pretreated with the probe (5 μM) for 10 min were further
incubated with Zn2+ for 10 min more, they emitted the remarkable
green fluorescence (Figure 4e-f). These results indicated that Sen-
OH could fast visualize intracellular Zn2+ in living cells with a good
cell-penetrating ability.
Next, we tested the feasible application on bioimaging Zn2+ in
living Zebrafish models. As show in Figure 7 a-c, first, 2-day-old
zebrafish treated only with Sen-OH (5 μM) for 10 min did not emit
any fluorescence. However, there was the strong fluorescence
when zebrafish was pretreated with the probe and then Zn2+ (Figure
7e-f). These results suggested that Sen-OH could be used for
bioimaging Zn2+ in vivo.
Acknowledgments
We thank for the supporting from Natural Science Foundation of
China (grant No. 21101074), Shandong Provincial Natural Science
Foundation of China (grant No. ZR2013BQ009 and ZR2016BL17),
the Doctor’s Foundation of University of Jinan (Grant No.
XBS1320).
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A simple off-on fluorescent probe was prepared and successfully applied for bioimaging
Zn2+ in living systems.