Aggregation-Induced Emission (AIE) Fluorophore Exhibits a Highly Ratiometric Fluorescent Response to Zn2+ in vitro and in Human Liver Cancer Cells

Article abstract of DOI:10.1002/chem.201701948

Two novel organic fluorophores, containing bis-naphthylamide and quinoline motifs, have been designed and synthesized. One of the fluorophores contains an isobutylene unit and exhibits a significant aggregation-induced emission (AIE) and a remarkable highly selective ratiometric fluorescence response towards Zn²⁺ in solution as well as in human liver cancer cells. The AIE behavior of this fluorophore was fully verified by fluorescence and UV/Vis spectroscopy, quantum yield calculations, and single-crystal X-ray diffraction, which revealed an intricate crystal packing system. Conversely, a fluorophore that lacks the isobutylene moiety did not exhibit any significant fluorescent properties as a result of its more flexible molecular structure that presumably allows free intramolecular rotational processes to occur.

Full text of DOI:10.1002/chem.201701948

A Journal of
Accepted Article
Title: Aggregation-Induced Emission (AIE) Fluorophore Exhibits a
Highly Ratiometric Fluorescent Response to Zn2+ in vitro and in
Human Liver Cancer Cells
Authors: Weitao Gong, Hassan Mehdi, Huimin Guo, Michael
Watkinson, Hua Ma, Ali Wajahat, and Guiling Ning
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To be cited as: Chem. Eur. J. 10.1002/chem.201701948
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Aggregation-Induced Emission (AIE) Fluorophore Exhibits a
Highly Ratiometric Fluorescent Response to Zn2+ in vitro and in
Human Liver Cancer Cells.
Hassan Mehdi, a Weitao Gong, a* Huimin Guob, Michael Watkinsonc*, Hua Mab, Ali Wajahat a and
Guiling Ning a*
Abstract: Two novel organic fluorophores 8AQ and 8WB have been
designed and synthesized. Fluorophore 8AQ, containing an
isobutylene unit, exhibits a significant AIE feature and a remarkable
highly selective ratiometric fluorescence response towards Zn2+ in
solution as well as in human liver cancer cells. The AIE behaviour of
8AQ was fully verified by fluorescence and UV-vis spectroscopies,
quantum yield calculations and through single crystal X-ray
diffraction, which revealed an intricate crystal packing system.
Conversely, fluorophore 8WB, which lacks the isobutylene moiety,
did not exhibit any significant fluorescent properties as a result of its
more flexible molecular structure that presumably allows free
intramolecular rotational processes to occur.
of fundamental academic and industrial exploration.[19-22] The
development of highly selective and sensitive fluorescent
chemosensors for the imaging of ‘mobile’ or ‘free’ Zn2+ also
remains a vibrant area of research due to the pivotal role it plays
in a number of important biological processes, such as brain
activity, gene transcription, immune function, and in reproductive
systems.[23-27] Although a very wide variety of chemosensors
have been reported for the detection and intracellular imaging of
Zn2+, [28-35] most of them exhibit a simple non-ratiometric switch
on fluorescence response. In addition the majority of the
systems that do display a ratiometric response to Zn2+ suffer
from interference with other heavy and transition metal ions like
Fe2+, Co2+, Ni2+, Cu2+, Hg2+ and Cd2+.[36,37] Hence the
development of efficient highly Zn2+ selective ratiometric
chemosensors with rapid response times, accompanied by low
detection limits remains an enduring challenge.[38-41] To address
these issues, researchers have designed several fluorescent
chemosensors for biological imaging of Zn2+ with different
binding motifs.[42-44] Recent reports include molecular systems
based on Schiff bases, peptide derivatives, hydroxyindole-
Bodipy, bipyridine and hydroxynaphthylmethylene, which have
been reported as highly sensitive ratiometric sensors for Zn2+ all
with detection limits in the micromolar range of 1 µM, 0.8 µM,
0.97 µM, 10 µM and 11 µM respectively. [45-49]
Introduction
The design and construction of new organic fluorescent
materials has attracted considerable interest over the past few
decades due to their widespread applications e.g. as sensors, in
electroluminescent devices and in cell imaging.[1-4] Most of these
organic materials exhibit highly emissive properties in dilute
solution but are less emissive at high concentration due to the
phenomenon of aggregation-caused quenching (ACQ),[5-7] which
has proven to be a major obstacle in the advancement of
luminescent materials.[8,9] To overcome these challenges Tang
et al. introduced a novel phenomenon in 2001, aggregation-
induced emission (AIE), which is exactly the reverse process of
ACQ.[10-12] The AIE phenomenon has subsequently attracted
considerable interest,[13-15] and has opened up many
opportunities in the application of novel luminescent materials in
a range of areas including optoelectronic devices, organic light-
emitting diodes OLEDs, organic lasers, photovoltaic cells,
chemo/biosensors and intracellular cell imaging to name a
few.[16-18] As a result, the design and synthesis of fluorescent
materials that can induce AIE continues to be a very active area
Herein we report a new AIE-active fluorophore 8AQ that exhibits
a rapid and highly selective ratiometric response to Zn2+ with a
limit of detection (LOD) up to 2.6 nM which is amongst the most
sensitive reported to date. Furthermore, the fluorophore 8AQ
was successfully applied in ratiometric imaging of intracellular
Zn2+ in human hepatoma cancer cells by fluorescence
microscopy.
Results and Discussion
[a]
[b]
State Key Laboratory of Fine Chemicals, School of Chemical
Engineering Dalian University of Technology, Dalian 610023. PR.
China. Fax: +86 411- 8498-6065; Tel: +86-411-8498-6067 E-mail:
wtong@dlut.edu.cn; ninggl@dlut.edu.cn.
We have designed and synthesized two novel organic
fluorophores 8AQ and 8WB. In fluorophore 8AQ, we rationally
inserted an isobutylene moiety into the structural system in order
to reduce energy loss through free intramolecular rotation, with
the aim of generating an AIE system. Conversely, we expected
fluorophore 8WB, without the isobutylene moiety, would not
display such properties due to its more flexible molecular
structure, should allow free intramolecular rotation and strong π-
π-interactions among its aromatic moieties.
School of Chemistry, Dalian University of Technology, Dalian
610023. PR. China.
[c] The Joseph Priestley Building, School of Biological and Chemical
Sciences, Queen Mary University of London, Mile End Road,
London, E1 4NS, UK. Email: m.watkinson@qmul.ac.uk
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Table 1. Crystallographic data of fluorophores 8AQ and 8WB.
8AQ
8WB.H2O
Molecular formula
C44 H32 N4 O4
680.74
C43 H32 N4 O5
684.73
Formula weight
Crystal system
Triclinic
Monoclinic
P -21/c
Space group
P-1
a/Å
b/Å
c/Å
α/°
β/°
γ/°
10.2139 (4)
13.8506 (5)
14.8388 (6)
63.009
9.811 (3)
23.316 (7)
14.883 (4)
90.00
89.818
98.66
69.710
90.00
Scheme 1. Synthetic route to supramolecular organic fluorophore 8AQ.
V/Å3
Z
1724.38 (12)
3365.8(18)
2
4
The synthetic route towards the two organic fluorophore 8AQ
and 8WB are shown in Scheme 1 and Scheme S1 (see ESI)
respectively. Both organic fluorophores contain bis-
naphthylamide moieties and two quinoline motifs with the only
structural difference between them being the isobutenyl group.
In both cases the syntheses proceeded in good yield.
Dc/(g cm−3
μ/mm−1
F(000)
)
1.311
1.351
0.085
0.090
712
1432
No. of refs measured
No. of unique refs (Rint)
R1[I > 2σ(I)]
12565
21677
7810 [0.0286]
0.0584
7590 (0.0904)
0.0809
Single crystal structure and supramolecular interactions
wR2 (all data)
0.1409
0.2555
The single crystals of fluorophores 8AQ and 8WB were
produced from CH2Cl2: C2H5OH solution via slow solvent
evaporation at room temperature. The structures within the
Goodness of fit
CCDC number
1.021
1.023
1496663
1511124
asymmetric units are displayed in Figure
crystallographic data are listed in Table 1.
1
and the
As shown (Figure 1), the single crystal X-ray structure of
fluorophore 8AQ contains two naphthylamide quinoline moieties
connected to the isobutylene core. The structure contains two
types of intramolecular hydrogen bonds between the H-atoms of
the amide moieties with the O-atoms of the ethers and the N-
atoms of quinoline moieties (Figure 1). This intramolecular
hydrogen bonding network results in one naphthylamide
quinoline moiety being connected to the isobutylene core with a
dihedral angle C(38)-C(25)-C(17)-O(1) of 132.60 (2)o whilst the
other displays a dihedral angle about C(38)-C(25)-C(18)-O(2) of
177.80 (3)o and results in 8AQ exhibiting the classical propeller
conformation in the solid state required in fluorophores
displaying AIE properties. In addition to the restricted molecular
structure within the asymmetric unit, further examination of the
crystal packing system of fluorophore 8AQ clearly reveals
several kinds of intermolecular hydrogen bonding interactions
(Figure 2a).
Figure 1. X-ray crystal structures of fluorophores 8AQ and 8WB
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These include hydrogen bonds between the hydrogen bond
acceptors O(4) and O(5) of one fluorophore with hydrogen bond
donors of the naphthalene cores of another fluorophore (H (24)
and H(21) respectively) with distances of 2.798 Å and 2.932 Å
respectively. Similarly, weak intermolecular hydrogen bonding
interactions are also observed between quinoline moieties of
one molecule and adjacent naphthalene groups viz. between
O(4) and H(37) and N(3) and H(28), with bond distances about
2.674 Å and 2.908 Å respectively. Additionally, the packing
arrangement of 8AQ also displays two kinds of CH-π
interactions, C(31)-H(31)-π (Q) and C(30)-H(30)-π (Q) with
distances of 2.926 Å and 2.719Å respectively (Figure 2a).
In addition, three different π-π-interactions are observed
between closely packed molecules in the packing arrangements
of 8WB (Figure 2b) with strong π-π interactions of 3.778 Å (Q-N),
3.825 Å (N-N), and 3.751 Å (Q-Q) which is likely to result in no
AIE in its solid or aggregated form.
Aggregation-induced
fluorophore 8AQ
emission
(AIE)
behaviour
of
Fluorophore 8AQ was readily soluble in common organic
solvents such as C2H5OH, CH3OH, CHCl3, THF, CH3CN and
DMF. In order to investigate the AIE properties of 8AQ we
conducted fluorescence and UV-vis titrations using 10 µM
solution of 8AQ in C2H5OH/H2O mixtures (from 0% to 90 %
water fractions) using 0.05 M Tris-HCl buffer at pH 7.5.
Collectively these interactions generate
a supramolecular
assembly in the solid state which results in the restriction of
intramolecular motion demonstrating that fluorophore 8AQ has
the significant capability of displaying AIE properties in the solid
state. In order to better understand the crucial role of the
isobutylene core in generating the AIE phenomenon, analysis of
the single crystal X-ray structure of 8WB (Figure 1) clearly
shows that the two naphthalene amide quinoline arms are
orthogonal as a result of the increased flexibility of the propyl
spacer.
Figure 2. Packing system in the crystal structure of 8AQ, (a) Intermolecular
hydrogen bonding N-H···O, N-H··N, (N) C-H-π (Q) and (Q) CH-π (Q) stacking
(b) π-π stacking in the crystal structure of 8WB.
Figure 3. (a) Fluorescence spectra of fluorophore 8AQ (10 µM) in pure
C2H5OH with gradual addition of 5% H2O fractions for each spectrum from 0-
90 %.(b) Emission spectra of 8AQ in C2H5OH solution and the solid state
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As shown in Figure 3a fluorophore 8AQ exhibited a weak
fluorescence emission at 418 nm (ex = 366 nm) with a very low
quantum yield (Ф= 0.004). Upon addition of water, the main
emission band at 418 nm is red-shifted to 438 nm with
pH 7.5 (Figure 4a). The fluorescence emission peak of
fluorophore 8AQ occurs at 430 nm upon excitation at 366 nm at
25oC. Addition of one equivalent of Zn2+ into the 8AQ solution
induced a significant ratiometric fluorescence response with a
new emission peak being exhibited with dramatically enhanced
emission intensity at 536 nm with an additional shoulder at 493
nm. This represents a red shift of about 106 nm and is reflected
in the dramatic variation in colour of the solution from blue to
yellow-green upon the addition of Zn2+ observed under a UV-
lamp at 360 nm, as shown in Figure 4. No such changes could
be perceived upon the addition of the other metal ions.
incrementally increasing emission intensity along with
a
concomitant increase in quantum yield (Ф=0.040) (Figure S5).
Similarly an intense fluorescence emission was observed in the
solid state with an enhancement in quantum yield (Ф =0.25)
(Figure 3b). Further the analogous UV-vis experiment reveals
that 8AQ displays an absorption band centred at 333 nm in
ethanol which after addition of 90% water displays
a
bathochromic shift of 16 nm (Figure S1). Hence these
experiments indicate that the weak fluorescence of 8AQ in
ethanolic solution is likely to be due to free intramolecular
rotation, however, upon aggregation or in the solid state, a
significant enhancement in emission is observed, as a result of
bond rotation being prevented and the consequent blocking of
non-radiative decay processes of the photo-excited molecules.
Absorption spectroscopic studies of 8AQ with various
metal ions
To investigate the selective recognition properties of fluorophore
8AQ towards metal ions we measured UV-vis spectra with the
perchlorate salts of a variety of common metal ions (Na+, Ca2+,
K+, Cr3+, Ce3+, Mg2+, Co2+, Ni2+, Cu2+, Pb2+, Mn2+, Ag+, Fe3+, Hg2+,
Cd2+ and Zn2+) in C2H5OH/H2O (1:1) solution in 0.05 M Tris-HCl
buffer solution at pH 7.5 (Figure S2a).
The absorption spectrum of fluorophore 8AQ exhibits
a
characteristic absorption band at 333 nm, which may be
attributed to a π–π* transition and the highly π–conjugated
system existing in the fluorophore. Upon addition of 1 equivalent
of the metal ions into the fluorophore 8AQ, only Zn2+ displayed a
bathochromic shift of ca. 50 nm with a new absorption band
emerging at 382 nm with a simultaneous decrease in the
absorption band at 333 nm with a well-defined isosbestic point at
365 nm. In contrast, the other metals did not induce such
changes (Figure S2a). This result indicates that Zn2+
coordination increases conjugation in fluorophore 8AQ which in
turn leads to a sharp colour variation of the solution from blue to
yellow-green under UV light at 360 nm Figure S2b. This result
clearly demonstrates that fluorophore 8AQ shows highly
selective and sensitive binding capability towards Zn2+.
Fluorescence response of fluorophore 8AQ with various
metal ions
Figure 4. (a) Fluorescence spectra of 8AQ (10 μM, C2H5OH: H2O (1:0.05 to
We also investigated the selective fluorescence recognition
properties of fluorophore 8AQ towards the above mentioned
common metal ions as well as 8WB (see Figure S3) in
C2H5OH/H2O (1:1) solution in 0.05 M Tris-HCl buffer solution at
1:1 ratios) in 0.05 M Tris-HCl buffer (pH=7.5) with 1 equivalent of various
metal ions. (b) Fluorescence titration of 8AQ with increasing (0.05 eq. Zn2+
concentrations of Zn2+
)
.
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These fluorescence emission results indicate that 8AQ exhibits
a highly selective ratiometric response towards the Zn2+ ion over
the other metal ions tested. Furthermore as shown in Figure 4b,
during the fluorescence titration of fluorophore 8AQ with Zn2+, an
increase in fluorescence intensity could be observed with
increasing Zn2+ concentrations until 1 equivalent was added,
thereafter the emission intensity remains the same with further
addition of Zn2+. This fluorescence titration result demonstrates
that fluorophore 8AQ has a highly selective and sensitive
ratiometric response towards Zn2+.
A
Job's plot analysis indicated the expected 1:1 binding
stoichiometry between fluorophore 8AQ and Zn2+ (Figure 5b). To
further verify the high selectivity of fluorophore 8AQ as a
ratiometric responsive sensor for Zn2+, we undertook competitive
experiments by screening the addition of various interfering
metal ions to fluorophore 8AQ in C2H5OH: H2O solution, and
then added 1 equivalent of Zn2+. No significant effect on
fluorescence emission of 8AQ toward Zn2+ was observed. This
result illustrates that fluorophore 8AQ displays robust selectivity
and sensitivity toward Zn2+. In order to further validate the high
selectivity and sensitivity of fluorophore 8AQ towards Zn2+, we
determined the binding constant and detection limit from the
fluorescence titration data. The binding constant of fluorophore
8AQ towards Zn2+ was calculated to be 5 ×107 M−1 with a good
linear relationship value R2= 0.9929 (Figure S6). Moreover the
LOD was calculated to be as low as 2.6 nM by the 3σ method.[42]
As a final investigation into binding selectivity the effect of
counter ions was investigated and Zn(NO3)2, ZnCl2, and ZnSO4
were used the same spectral characteristic were exhibited,
indicating no anion dependence on binding or fluorescence
response (Figure S7).
1H NMR titration of fluorophore 8AQ with Zn2+ to study the
binding mode and composition of metal complex 8AQ-Zn
To gain further insight into the binding mode of Zn2+ with
1
fluorophore 8AQ, we conducted a H NMR titration in CDCl3 at
room temperature (Figure S4). The 1H NMR spectrum of the
fluorophore 8AQ exhibits a singlet peak for the -NH protons of
the amide groups at 11.90 ppm. Additionally, the protons of the
quinoline scaffolds H-14, H-12, H-9, H-13, H-10, appear at 8.83
ppm, 8.58 ppm, 7.49 ppm, 7.47 ppm and 7.41 ppm respectively.
The incremental addition of Zn2+ into the solution of 8AQ showed
variations in the chemical shift values of protons present in the
system. Upon addition of 0.5 to 1 equivalents of Zn2+ into
fluorophore 8AQ, the -NH peak of the amide groups shifts
downfield from 11.90 ppm to 11.97 ppm without an effect on the
peak intensity. Furthermore all quinoline proton peaks shift
downfield on zinc binding and broaden, while the isobutylene
protons H-1 and H-2 are shifted upfield to 5.62 ppm and 5.20
ppm. In contrast minor changes in the naphthalene scaffold are
observed, indicating that, as expected, this moiety is not
involved in binding zinc. This result supports the coordination of
Zn2+ with the N-atoms of quinoline molecules and O-atom of
amide moieties of fluorophore 8AQ, furthermore the upfield shift
of H-1 and H-2 provides solid evidence for the variation in
molecular conformation and the structural rigidity resulting from
Zn-coordination. Moreover no further spectral variation of any
proton signals was recorded upon addition of excess Zn2+ (over
1 equivalent) confirming that metal: ligand binding stoichiometry
is 1:1, consistent with the Job’s plot determinations and the
fluorescence titration experiments.
Figure 5. (a) Job’s plot for determining the stoichiometry of 8AQ with Zn2+. (b)
Fluorescence response of 8AQ-Zn (10 μM) in the presence of 1 equivalent of
various additional metal ions in 0.05 M Tris-HCl buffer solution at pH 7.
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ESI-MS experiment
expected that the quenching from S1 and S2 will result in two
vertical forms of electronic transitions with a more significant
energy difference in the emission spectra. The calculated
vertical emission bands based on TD-DFT optimized excited
state structures are centred at 571 nm and 485 nm, which not
only supports our proposed mechanism but are also consistent
with the experimental findings.
The binding mode between 8AQ and Zn2+ was also investigated
by ESI-MS. In the ESI-MS spectrum molecular ions were
observed at m/z 743.3212 and m/z 843.2919 corresponding to
+
[Zn(8AQ) + H]
and [Zn(8AQ) + ClO4]+ respectively. These
values are in accordance with the expected isotopic distribution
patterns centred as shown in Figure S13 and Fig S14 and also
suggest the binding stoichiometry of 8AQ with Zn2+ is 1:1.
Biological cell-permeability of 8AQ and intracellular
ratiometic fluorescence response towards Zn2+
Signalling mechanism of fluorophore 8AQ with Zn2+
To evaluate the promising applications of fluorophore 8AQ in
biological systems, we examined the effect of pH on the
fluorescence intensity of fluorophore 8AQ and investigated its
recognition properties towards Zn2+ in 0.05 M Tris-HCl buffer
solution (Figure S8). Fluorophore 8AQ continued to display
intense emissions in the presence of Zn2+ in the pH range 6 to 8
indicating that the fluorophore is suitable for biological imaging
applications and monitoring intracellular Zn2+. In order to
demonstrate this, its cell permeability, intracellular ratiometric
fluorescence responsiveness towards Zn2+ and cytotoxicity were
investigated in human hepatoma cancer cells (HepG2). A 1 mM
stock solution of fluorophore 8AQ in C2H5OH/H2O was initially
prepared and then diluted using 0.05 M Tris-HCl buffer solution
of pH 7.5 to 10 μM as the working concentration. We then
investigated bio-imaging applications of HepG2 incubated with
8AQ at 10 μM in 0.05 M Tris-HCl buffer solution of pH 7.5 for 1
hour at 37°C. As shown in Figure 6b, fluorophore 8AQ was well-
distributed and penetrated into live cells and exhibited blue
fluorescence imaging showing that fluorophore 8AQ has
excellent membrane permeability. Consequently HepG2 cells
were incubated with fluorophore 8AQ followed by 10 μM ZnCl2
to explore intracellular Zn-imaging which resulted in an
intracellular ratiometric fluorescence response with the strong
green fluorescence image shown in Figure 6 (c, d). Some
differences in probe localisation were observed upon binding to
zinc, which is perhaps not surprising given the fundamental
differences in structure between the free ligand and its zinc
complex. In addition the morphology of the cells treated with zinc
is somewhat different, however, zinc is known to affect cell
motility and differentiation and it is likely that cells are in different
phases of the cell cycle or differentiation leading to the
heterogeneity observed. [50] In order to evaluate the viability of
HepG2 cells with fluorophore 8AQ and its corresponding
complex 8AQ-Zn, HepG2 cells were treated with 8AQ and its
zinc complex over a range of concentrations for 24 h and 12 h
(Figure 6b) while treated with complex 8AQ-Zn for 24 h (Figure
S19). These data revealed that HepG2 cells exhibit near 100%
viability for 12 h and 24 h treatment with fluorophore 8AQ and
complex 8AQ-Zn. These fluorescence bio-imaging experiments
clearly demonstrate that fluorophore 8AQ has excellent cell
permeability and could be suitable as a ratiometric fluorescence
imaging sensor for intracellular Zn2+ in human liver cancer cells.
Fluorophore 8AQ exhibited very weak fluorescence in solution
due to the free intramolecular rotation among rotatable moieties
and the consequential loss of excited state energy via non-
radiative relaxation.
In designing fluorophore 8AQ, amide groups were inserted into
the molecular system to link the naphthalene moieties along with
two quinoline groups. In fluorophore 8AQ, naphthalene scaffolds
are used as the signalling part and the peripheral quinoline
group served as a Zn2+ chelator. Upon addition of Zn2+ into the
fluorophore a remarkable enhancement in emission intensity
associated with a highly ratiometric red shifted response is
observed. The ratiometric fluorescence behaviour of fluorophore
8AQ after coordinating with Zn2+ may be attributed to binary
signalling mechanisms involving
a
change in molecular
conformation and intermolecular charge transfer ICT
mechanisms. As illustrated in the partial 1H NMR titration (vide
infra), upon addition of Zn2+ into 8AQ, Zn2+ strongly coordinates
with two O-atoms of the amide groups along with two N-atoms of
quinoline moieties. This result is fully consistent with
theoretically optimized structures. The DFT optimized structure
of complex 8AQ-Zn also shows that the O-atom of the
naphthylamide and the N-atom of the quinoline moieties are
strongly coordinated with Zn2+ which displays a tetrahedral
geometry. Furthermore according to the optimised structure of
8AQ-Zn, one quinoline moiety is perpendicularly situated to the
naphthylamide moiety Figure S16. These DFT calculations
demonstrate that fluorophore 8AQ is strongly coordinated with
Zn2+ which induces an enhancement in fluorescent emission
behaviour as shown in Figure S16. Additionally due to the strong
coordination of Zn2+ with the quinoline N-atoms intramolecular
charge transfer to Zn2+ is promoted, resulting in the remarkable
ratiometric response displayed. The calculations revealed that
the HOMO-LUMO energy gap of the 8AQ-Zn complex was also
lower (3.86 eV) compared to the fluorophore 8AQ (4.42 eV). In
order to explain the appearance of two prominent emission
peaks after binding with Zn2+, the TD-TDFT calculation
demonstrated that the structural distortion induced in 8AQ by
coordination with Zn2+ results in an approximately 20 nm
difference in absorption wavelength of S1 and S2. It can thus be
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naphthalene-amide groups connected to the quinoline moieties
which act as the metal chelating sites for Zn2+. In fluorophore
8AQ, the isobutylene group is introduced into the system in
order to produce
a certain level of rigidity to reduce
intramolecular molecular rotations. The AIE behaviour of 8AQ
was clearly demonstrated by fluorescence spectroscopy,
quantum yield calculations and in the solid state through single
crystal X-ray diffraction, which revealed a crystal packing system
with extensive intermolecular interactions. Furthermore
fluorophore 8AQ displayed a highly ratiometric fluorescence
response towards Zn2+. In contrast 8WB has a much more
flexible molecular structure due to the lack of the isobutylene
group and exhibits poor selectivity towards metal ions. The 8AQ-
Zn complex stoichiometry ratio was confirmed to be 1:1 by Job’s
plots analysis and was also supported by 1H NMR titrations, DFT
calculations and ESI-MS analysis. A binding constant of 5 ×107
M−1 and LOD of 2.6 nM were calculated by using linear fittings
and standard deviation methods. Importantly fluorophore 8AQ
also exhibited
a highly ratiometric fluorescence response
towards Zn2+ in human hepatoma cancer cells (HepG2).
Investigations into the development of more sophisticated AIE
active fluorophores, biosensors and chemosensors with
comparable selectivity and sensitivity are in progress in our
laboratories.
Experimental Section
Materials and Instrumentation: All commercially available chemicals
were purchased from commercial suppliers except when specified.
Solvents such as CH2Cl2, CHCl3, CH3OH, C2H5OH, and n-hexane and
petroleum ether were used as obtained. Anhydrous solvents such as
DMF, THF were also purchased from suppliers. All intermediates and
target molecules were satisfactorily characterised by 1H and 13C NMR
spectroscopy, ESI-MS and in some cases single crystal structures. NMR
spectra were recorded on Bruker AVANCE-400 NMR spectrometer at
400 MHz (1H NMR) and 100 MHz (13C NMR). High resolution mass
spectra were recorded on an Agilent 6310 MS spectrometer and a Q-
TOF MS spectrometer. UV-vis spectra were recorded using a HITACHI
U-4100 spectrophotometer. Fluorescence spectroscopic measurements
and fluorescence quantum yields were measured on a JASCO FP-8500
spectrofluorimeter. Single-crystal X-ray diffraction experiments were
Figure 6. Fluorescence microscopy images of HepG2 cells. Bright-field
transmission images of cells after incubation with (a) 10 μM 8AQ (c) 10 μM
8AQ and 10 μM Zn2+ for 2 hours at 37°C (b, d) Fluorescence transmission
images of 8AQ, 8AQ-Zn (above) and cell viability values (%) estimated by
MTT assay versus incubation concentrations of 8AQ (below).
performed with
a Bruker SMART AXS CCD diffractometer. The
fluorescent bioimaging assays were performed with a laboratory use
luminescence microscopy.
UV-Vis and fluorescence measurements
Conclusions
For absorption and fluorescence measurements, 1 mM stock solutions of
fluorophores 8AQ and 8WB were prepared in 50 ml graduated volumetric
flasks. These stock solutions were diluted to 10 μM with CH3CH2OH/H2O
(1:1 v/v) solution. Meanwhile, solutions of perchlorate salts of all tested
metal ions were also prepared in water. The recognition properties of
In conclusion, we have designed and synthesized two novel
organic fluorophores 8AQ and 8WB with and without an
isobutylene group that display AIE and non-AIE characteristics
respectively. Both fluorophores 8AQ and 8WB contain two
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fluorophores 8AQ and 8WB towards metal ions were investigated
through UV-vis and fluorescence spectroscopies in 0.05 M Tris-HCl
buffer solution of CH3CH2OH/H2O. The association constant and
detection limit of fluorophore 8AQ towards Zn2+ was determined from the
fluorescence emission intensity data. [51]
incubator. The supernatants were removed, cells were suspended in 1%
DMSO, and then the absorbance was measured at 490 nm.
Synthesis of fluorophore 8AQ
Theoretical Methods
Intermediate compound L-2 was synthesised according to the literature
[60]
and used as a starting material for construction of fluorophore 8AQ.
L-2 (0.23 g, 0.50 mmol) was dissolved in dried THF (10-15 mL) and
We performed extensive Density Functional Theory/Time-dependent
Functional Theory (DFT/ TD-DFT) based calculations to investigate the
correlation between structure of 8AQ and its sensing behaviour towards
added dropwise into 8-aminoquinoline (0.3 g,
1
mmol) containing
oC.
triethylamine (0.2 g, mmol) with continuous stirring at
2
0
Subsequently the reaction mixture was stirred for 12 hours at room
temperature. The THF was evaporated under reduced pressure.
Purification was performed by column chromatography using
CH2Cl2/ethyl acetate (3:2) as the eluant giving pure 8AQ (75% yield).
Single crystals of 8AQ were obtained CH2Cl2 /CH3CH2OH via slow
evaporation within one week. Crystal Data are summarized in Table 1.
ESI-MS m/z C44H32N4O4 : 730.23 [M+ Na]+, 1H NMR (400 MHz, CDCl3) δ
11.86 (s, 2H, -NH), 8.82 (d, J = 4.0 Hz, 2H, Q-H14), 8.67 (s, 2H, N-H8),
8.58 (d, J = 4.0 Hz, 2H, Q-H12), 7.98 (d, J = 8.0 Hz, 2H, N-H7), 7.84 (d, J
= 8.0 Hz, 2H, N-H4), 7.46-740 (m, 4H, Q-H9, H10), 7.38-736 (m, 4H, Q-
H11, H13), 7.18 (s, 2H, N-H3), 7.22-7.16 (m, 4 H, N-H6, H5), 5.67 (s, 2H,
H-1), 5.24 (s, 4H, H-2). 13C NMR (100 MHz, CDCl3) δ 163. 5, 153.3,
147.8, 139.2, 138.8, 136.1, 135.8, 135.6, 135.2, 134.0, 129.0, 128.4,
128.2, 127.9, 127.0, 126.4, 124.7, 123.9, 121.7, 118.2, 117.6, 108.2,
70.1 ppm.
Zn2+ [52,53]
All the calculations were performed with B3LYP functional in
.
combination with 6-31G(d) and Lanl2dz basis sets as implemented in
Gaussian 09.[54-56] The molecular structures of ground states and low-
lying excited states of both 8AQ and the corresponding zinc complex
8AQ-Zn were fully optimized and verified with frequency calculations.
The CPCM polarizable conductor model was used to handle the impact
solvent environment on geometric and electronic structures. [57]
Fluorescence imaging in living Cells
For fluorescence imaging experiments, all cultured human liver cancer
cells HepG2 were obtained from Dalian Medical University. HepG2 cells
cultured on a 35 mm glass-bottom culture dish (φ 20 mm) in RPMI-1640
medium containing 10% fetal bovine serum, 1% penicillin, and 1%
streptomycin, cells were washed three times with an isotonic saline
solution (140 mM NaCl, 10 mM glucose and 3.5 mM KCl). After that
prepared 10 μM solutions of the fluorophore 8AQ in the isotonic saline
solution was added to the culture medium and cells were incubated for 2
hours at 37 oC in a 5% CO2/95% air incubator. For intracellular Zn2+
imaging experiments the fluorophore 8AQ loaded cells were washed
three times with isotonic saline solution after 1 h incubation then
incubated again in the presence of isotonic saline solution containing 10
μM Zn2+ and 0.5 mg/ml cremophor C040 for 2 h under identical conditions.
The cells were washed five times with isotonic saline solution and then
subjected to the fluorescence imaging using a fluorescent microscopy
with an excitation wavelength of 360 nm.
Synthesis of compound 8WB
The synthetic procedure for compound 8WB is the same as fluorophore
8AQ. Single crystal of compound 8WB were produced from solution of
CH2Cl2 /CH3CH2OH via slow evaporation within one week. Crystal Data
are summarized in Table 1. ESI-MS m/z C43H32N4O4 : 691.23 [M+ Na]+,
1H NMR (400 MHz, CDCl3) δ 11.98 (s, 2H, -NH), 9.10 (d, J = 8.0 Hz, 2H,
Q-H14), 8.82 (s, 2H, N-H8), 8.76 (d, J = 4.0 Hz, 2H, Q-H12), 7.93-7.88
(m, 4H, N-H7,H4), 7.54 (t, J = 8.0 Hz, 4H, Q-H13), 7.46 (t, J = 7.5 Hz, 2H,
N-H5), 7.41-7.34 (m, 4H, Q-H11, H10), 7.20-7.17 (m, 2H, N-H6), 7.11 (s,
2H, N-H3), 4.67 (t, J = 6.0 Hz, 4H, H-2), 3.21 (Quintet, J = 4 Hz, 2H). 13
C
NMR (100 MHz, CDCl3) δ 163.7, 153.6, 147.8, 138.9, 136.1, 135.8,
135.5, 134.2, 129.1, 128.3, 128.0, 127.4, 126.2, 124.6, 123.4, 121.8,
121.4, 117.7, 107.3, 66.0, 44.9, 28.8 ppm.
MTT assay
Acknowledgements
The cytotoxicity of 8AQ and 8AQ-Zn to human liver cancer cells (HepG2)
was tested by using a previously reported method of the MTT assay.[58,59]
HepG2 cells cultured in DMEM medium, containing 10% FBS (fetal
bovine serum) and antibiotics (100 units/ ml penicillin and 100 mg ml
streptomycin), were washed with an isotonic saline solution (140 mM
NaCl, 10 mM glucose and 3.5 mM KCl), and then incubated with various
concentrations of 8AQ (5-15 μM) for 12 hours and 24 hours, in case of
complex 8AQ-Zn (5-30 μM) for 24 hours at 37 oC in a 5% CO2/ 95% air
incubator. After the culture medium was removed, the cells were further
incubated with the PBS buffer containing 0.5 mg ml of MTT for 4 h in the
The authors are grateful for financial support from the National
Natural Science Foundation of China (NO.21206016) and the
Fundamental Research Funds for the Central Universities
(DUT17LK07). MW would like to acknowledge the contribution of
the COST Action TD1304 Zinc-Net.
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Keywords
Fluorophore• Ratiometric • Bioimaging • Sensing • AIE •
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