Two novel aggregation-induced emission active coumarin-based Schiff bases and their applications in cell imaging

Article abstract of DOI:10.1039/c3nj01557b

Two new coumarin-based Schiff bases, 8,8′-((1E,1′E)-hydrazine- 1,2-diylidenebis(methanylylidene))bis(7-hydroxy-4-methyl-2H-chromen-2-one), (CHC), and 7-hydroxy-8-((E)-((E)-((2-hydroxynaphthalen-1-yl)methylene)hydrazono) methyl)-4-methyl-2H-chromen-2-one, (CHN), with excited-state intramolecular proton-transfer (ESIPT) properties, were synthesized and characterized. Both of the compounds displayed aggregation-induced emission (AIE) characteristics, of which CHC nanoparticles emitted a reddish orange fluorescence, while the CHN nanoparticles exhibited a saffron yellow fluorescence. The appearance of emission peaks in the long wave regions with large Stokes-shifts demonstrated the occurrence of the ESIPT process. Observations of the nanoparticles' morphologies were undertaken through a transmission electron microscope (TEM) method. Furthermore, due to the good biocompatibilities, high stabilities and the large Stokes-shifts, the two compounds were ideal candidates for cell staining. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c3nj01557b

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Two novel aggregation-induced emission active
coumarin-based Schiff bases and their
applications in cell imaging†
Cite this: New J. Chem., 2014,
38, 2386
Hongde Xiao,a Kun Chen,a Dandan Cui,b Nannan Jiang,b Gui Yin,*ac Jie Wanga and
Ruiyong Wang*b
Two new coumarin-based Schiff bases, 8,80-((1E,10E)-hydrazine-1,2-diylidenebis(methanylylidene))bis-
(7-hydroxy-4-methyl-2H-chromen-2-one), (CHC), and 7-hydroxy-8-((E)-((E)-((2-hydroxynaphthalen-1-yl)-
methylene)hydrazono)methyl)-4-methyl-2H-chromen-2-one, (CHN), with excited-state intramolecular
proton-transfer (ESIPT) properties, were synthesized and characterized. Both of the compounds displayed
aggregation-induced emission (AIE) characteristics, of which CHC nanoparticles emitted a reddish orange
fluorescence, while the CHN nanoparticles exhibited a saffron yellow fluorescence. The appearance of
emission peaks in the long wave regions with large Stokes-shifts demonstrated the occurrence of the
ESIPT process. Observations of the nanoparticles’ morphologies were undertaken through a transmission
electron microscope (TEM) method. Furthermore, due to the good biocompatibilities, high stabilities and
the large Stokes-shifts, the two compounds were ideal candidates for cell staining.
Received (in Montpellier, France)
11th December 2013,
Accepted 28th January 2014
DOI: 10.1039/c3nj01557b
www.rsc.org/njc
such as encapsulation by amphiphilic surfactants and blending
with transparent polymers,9 have been undertaken to prevent
Introduction
Over the past few decades, efficient organic fluorescent materials the aggregation but have been met with only limited success.
have been attracting considerable research interest due to their Fortunately, materials displaying aggregation-induced emission
potential applications, including in organic light-emitting (AIE) characteristics can be used to tackle this problem. Since the
diodes (OLED),1 organic field effect transistors (OFET),2 fluor- first AIE compound, 1-methyl-1,2,3,4,5-pentaphenylsiole, was
escent sensors,3 photovoltaic cells,4 fluorescent biological synthesized by Tang’s group,10 several organic compounds
labels,5 organic lasers,6 etc. Although many traditional organic based on different fluorophores have been reported to display
fluorophores with large delocalized p-conjugated moieties emit AIE characteristics, such as 1-cyano-trans-1,2-bis-(40-methyl-
strongly in their dilute solutions, the emissions become weak at biphenyl)ethylene (CN-MBE),11 diphenyldibenzofulvene (DPDBF)
high concentrations or in aggregated states,7 which is known as derivatives,12 rhodanineacetic acid-pyrene derivative (RAAP),13
concentration quenching or aggregation-caused quenching whose potential applications were further investigated in the
(ACQ). The reason for this is believed to be because the excited fields of bio/chem sensors,14 cell imaging15 and optoelectronic
states of the aggregates often decay via non-radiative pathways devices.16
caused by strong intermolecular vibronic interactions, such as
In recent years, a few interpretations of AIE in terms of the
exciton coupling and excimer formation in the solid state.8 The intra- and intermolecular effects have been reported. Intra-
notorious ACQ effect has greatly limited the efficiency of molecular effects on fluorescence enhancement are simply
organic fluorescent materials because in practice, those mate- explained by the conformational changes from the twisted
rials are commonly used in the solid state. Various approaches, conformations to the planar ones. The planar conformations
are favourable for the radiation process.17 Intermolecular inter-
actions are related with the specific aggregation morphology
(H-type and J-type aggregation). H-aggregates tend to induce
the nonradiative deactivation process, where as J-aggregates
induce a relatively high fluorescence efficiency.18
As far as the specific emission properties are concerned, the
excited-state intramolecular proton-transfer (ESIPT) process
could greatly contribute to the fluorescent emission of the
chromophore in the solid state. The planar conformations are
a State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry
and Chemical Engineering, Nanjing University, Nanjing 210093, People’s Republic
of China. E-mail: yingui@nju.edu.cn
b School of Life Science, Nanjing University, Nanjing, 210093, People’s Republic of
China. E-mail: wangry@nju.edu.cn
c Jiangsu Key Laboratory of Advanced Catalytic Materials and Technology,
Changzhou University, 213164, China
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c3nj01557b
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measured using a Perkin Elmer Lambda 35 spectrophotometer
and Perkin Elmer LS-55 spectrophotometer, respectively. Trans-
mission electron microscopy (TEM) images were recorded on a
JEX-2100 transmission electron microscope. Fluorescence
microscope photographs of the nanoparticles and cell imaging
were captured by n Olympus FV-1000 laser scanning confocal
fluorescence microscope.
Scheme 1 Synthesis of CHC.
Synthesis of compound 2
Compound 2 was synthesized according to our previous
procedure.26
Scheme 2 Synthesis of CHN.
Synthesis of 7-hydroxy-4-methyl-coumarin
4-Methylbenzenesulfonic acid (5 g) was added to a solution of
beneficial to both AIE and ESIPT. The ESIPT dyes can present a resorcinol (11.0 g, 0.1 mol) in toluene (40 mL), and then
large Stokes shift caused by transformation of the excited enol acetoacetic ester (13.0 g) was added drop wise. The reaction
form (E*) to the excited proton-transferred keto form (K*), mixture was refluxed for 3 h and monitored by TLC until the
diminishing the self-absorption to a great extent. This property resorcinol was completely consumed. After cooling, the
enables the ESIPT compounds to have a wide range of applica- solution was poured into ice water (100 mL), a yellow precipi-
tions in systems such as fluorescent probes,19 laser dyes,20 tate was collected by filtration and washed with cold water.
organic electroluminescence optical materials,21 scintillators,22 A pale yellow solid was obtained by recrystallization with 95%
as well as in solar collectors.23
ethanol (11.3 g, yield: 64%). M.p. 185.0–186.0 1C. 1H NMR
In general, an ESIPT mechanism involves a proton transfer from (300 MHz, CD3OD), d = 7.61 (d, 1H, J = 8.9 Hz), 6.83 (d, 1H,
the donor to the acceptor moiety.24 The most common ESIPT donor J = 10 Hz), 6.70 (s, 1H), 6.10 (s, 1H), 2.42 (s, 3H). TOF MS EI
is the phenolic OH, and the basic group is usually a heteroatom, calcd 176.0, found 176.0.
such as carbonyl oxygen or a nitrogen atom on amide. In terms of
the structures of AIE compounds, the molecules have been found to
Synthesis of compound 127
be associated with aromatic groups with rotatable C–C, C–N or N–N
7-Hydroxy-4-methyl-coumarin (5.3 g, 0.03 mol) and hexamine
single bonds.25 Due to the superior light stability, high fluorescent
(9.8 g, 0.07 mol) in glacial acetic acid (50 mL) were refluxed for
quantum yield, as well as the good biocompatibility, coumarin
5.5 h, and then 20% HCl (75 mL) was added and the solution
derivatives have become ideal candidates for fluorescent biological
was heated for 1 h. After cooling, the reaction mixture was
materials. These factors motivated us to design and synthesize two
extracted with ether, and the combined organic layers were
novel coumarin-based ESIPT compounds, 8,80-((1E,1E0)-hydrazine-
evaporated under reduced pressure. The residue was poured
1,2-diylidenebis(methanylylidene))bis(7-hydroxy-4-methyl-2H-
into ice water, and a pale yellow solid of 8-formyl-7-hydroxy-
chromen-2-one), (CHC, Scheme 1), and 7-hydroxy-8-((E)-((E)-
4-methyl-coumarin was obtained. Further purification was carried
((2-hydroxynaphthalen-1-yl)methylene)hydrazono)methyl)-4-methyl-
out by recrystallization with hot ethanol to obtain a light yellow
2H-chromen-2-one, (CHN, Scheme 2). In addition, their nano-
powder (612.1 mg, yield: 10%). M.p. 180–181.0 1C (lit. 120.0–
particles were prepared by a simple reprecipitation method
1
122.0 1C). H NMR (300 MHz, CDCl3): d = 12.22 (s, 1H); 10.62
without using surfactants. What is more, the biocompatibility
(s, 1H); 7.74 (d, 1H, J = 9 Hz); 6.92 (d, 1H, J = 9 Hz); 6.21 (s, 1H);
2.43 (s, 3H). TOF MS EI calcd 204.0, found 204.0.
and the cell uptake behavior of the nanoparticles were investi-
gated to evaluate their potential applications in cell imaging.
Synthesis of CHC
8-Formyl-7-hydroxy-4-methyl-coumarin (408 mg, 2 mmol) was
added to a solution of aqueous hydrazine (85%, 0.1 mL, about
2 mmol) in methanol (20 mL). Then the mixture was refluxed
Experimental
Reagents, materials and equipment
All reagents were of analytical grade unless otherwise stated. for 4 h. After completion of the reaction, the obtained yellow
1H-NMR spectra were recorded on Bruker Ultrashield 300 MHz precipitate was filtered and washed several times with cold
NMR spectrometer. Chemical shifts were expressed in ppm ethanol to give CHC (347 mg) as a pure yellow solid in 86%
(in DMSO-d6 or CDCl3; TMS as an internal standard) and yield. 1H NMR (300 MHz, DMSO-d6): d = 12.78 (s, 1H), 8.40
coupling constants (J) in Hz. Mass spectroscopy was obtained (s, 1H), 7.54 (d, J = 9.0 Hz, 1H), 6.87 (d, J = 9.0 Hz, 1H), 6.19
from a Thermo LCQ Fleet MS spectrometer in negative ion (s, 1H), 2.38 (s, 3H); 13C NMR (75 MHz, DMSO-d6) d 160.69,
mode. Differential scanning calorimetry (DSC) and thermal 160.01, 154.43, 151.21, 135.49, 125.67, 113.33, 112.13, 110.85,
gravimetric analysis (TGA) were investigated on a Netzsch STA 107.22, 18.80. ESI-HRMS: calcd for (C22H16N2O6 ꢀ H)ꢀ m/z
449 C. UV-vis absorption and fluorescence spectra were 403.0930, found (M ꢀ H)ꢀ: 403.0791.
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Synthesis of CHN
fluorescent signals were collected at 420–700 nm for CHC
and 430–650 nm for CHN). The images were captured using a
photomultiplier.
A solution of 1-(hydrazonomethyl)naphthalene-2-ol (2) (186 mg,
1 mmol) and 7-hydroxy-4-methyl-2-oxo-2H-chromene-8-carbaldehyde
(204 mg, 1 mmol) in 20 mL of ethanol was refluxed for 4 h. After
completion of the reaction, the obtained yellow precipitate was
filtered and washed several times with cold ethanol to give
Results and discussion
1
Both CHC and CHN were soluble in common organic solvents,
but insoluble in water. Hence, we employed a simple repreci-
pitation method without surfactants to obtain the CHC and
CHN nanoparticles. The molecularly dissolved dilute DMSO
solutions of CHC and CHN were transparent, emitting very
weak fluorescence, which could hardly be observed (left of the
two inert pictures of Fig. 1). After the addition of water, as the
nanoparticles formed, the reddish orange fluorescence of the
CHC nanoparticles (middle and right of Fig. 1a) and the saffron
yellow fluorescence (middle and right of Fig. 1b) of the CHN
nanoparticles could be observed by the naked-eye. The molar
absorptivities for these two compounds as aggregates were
CHN (346 mg) as a pure yellowish solid in 93% yield. H NMR
(300 MHz, DMSO-d6) d 12.54 (s, 1H), 12.51 (s, 1H), 9.96 (s, 1H),
9.34 (s, 1H), 8.65 (d, J = 8.5 Hz, 1H), 8.02 (d, J = 9.0 Hz, 1H), 7.89
(d, J = 7.8 Hz, 1H), 7.78 (d, J = 9.0 Hz, 1H), 7.60 (t, J = 7.5 Hz, 1H),
7.42 (t, J = 7.5 Hz, 1H), 7.24 (d, J = 9.0 Hz, 1H), 7.01 (d, J = 8.9 Hz,
1H), 6.26 (s, 1H), 2.39 (s, 2H); 13C NMR (126 MHz, DMSO-d6)
d 163.58, 162.61, 160.94, 159.58, 158.06, 154.31, 135.80, 133.49,
132.83, 130.42, 129.41, 128.65, 128.37, 124.39, 122.54, 119.24,
114.03, 112.48, 111.37, 108.88, 105.98, 518.80; ESI-HRMS: calcd
for (C22H16N2O4 ꢀ H)ꢀ m/z 372.1032, found (M ꢀ H)ꢀ:
371.2560.
2.77 ꢁ 104 (CHC, lex = 335 nm) and 2.14 ꢁ 104 (CHN, lex
=
Preparation of the samples
340 nm). The nanoparticle suspensions were transparent without
precipitation and were observed to be stable even after a month.
Preparation of the nanoparticles. CHC or CHN DMSO solu-
tions (10 mL, 1 ꢁ 10ꢀ3 M) were rapidly injected into 2 mL of
DMSO–H2O mixtures of different ratios, respectively, with
vigorous stirring at room temperature. The nanoparticles, with
both having concentrations of 5 mM (represented by the
concentration of monomer, and it means the same herein-
after), were taken for fluorescence emission spectra and
UV-visible absorption respectively.
Preparation of the samples for TEM images. The nano-
particles were deposited on a 300-mesh copper grid coated
with carbon, and observed at an accelerating voltage of 200 kV
at room temperature.
Preparation of the samples for fluorescence microscope
images. A drop of the CHC or CHN nanoparticle suspension
was dropped onto a glass slide, and then covered with a
coverslip. The excitation wavelength was set at 405 nm, with
the choice of suitable filters.
Cell culture
HeLa cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) containing 10% fetal bovine serum and
antibiotics (100 units mLꢀ1 penicillin and 100 mg mLꢀ1 strepto-
mycin), and maintained at 37 1C in a humidified atmosphere of
95% air and 5% CO2.
Cell imaging
A fresh stock of HeLa cells was seeded into a glass bottom dish
with a density of 1 ꢁ 10ꢀ5 cells per dish, and incubated for 24 h.
Subsequently, the cells were incubated with 5 mM CHC or CHN
nanoparticles in DMEM solution (adding 100 mL 10ꢀ3 M DMSO
concentrated solution CHC or CHN to 1900 mL DMEM) for 4 h
at 37 1C. Afterwards, the cells were washed three times with PBS
to remove any nanoparticles attached to the surface of cells and
then fixed with 4% paraformaldehyde for 10 min at room
temperature. The cells were imaged under a confocal laser
scanning microscope (Olympus, FV-1000; lex = 405 nm;
Fig. 1 Normalized absorption (solid line) and emission (dashed line)
spectra of CHC nanoparticles in the water/DMSO (v/v, 9/1) mixture (a)
and CHN nanoparticles in he water/DMSO (v/v, 7/3) mixture (b). Inert:
photographs of CHC (a) and CHN (b) both with concentrations of 5 ꢁ 10ꢀ5 M
in DMSO dilute solution (left) and in water/DMSO mixtures (middle, v/v, 1/1;
right, v/v, 9/1) under 365 nm irradiation.
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As shown in the normalized absorption and emission spectra of
the CHC nanoparticles and CHN nanoparticles, large stokes
shifts of B270 nm for the CHC nanoparticles and B240 nm
for the CHN nanoparticles could be detected. Moreover, the
quantum yield of the CHC (as aggregates) was 0.827 and the
quantum yield of CHN (as aggregates) was 0.546, (using
quinine as the reference with a known F value of 0.577 in
0.05 M H2SO4), indicating the great properties of CHC and CHN
acting as solid state light-emitting materials.
Thermal stability tests
The thermal stabilities of CHC and CHN were investigated by
differential scanning calorimetry (DSC) and thermal gravi-
metric analysis (TGA). Fig. 2 showed that the decomposition
temperatures of CHC and CHN were both over 300 1C, demon-
strating their thermally stability.
Observation of the CHC and CHN nanoparticles
TEM observation. The transmission electron microscope
(TEM) images were obtained to observe the shape and size of
the nanoparticles. The TEM image in Fig. 3a showed that the
CHC nanoparticles were claviform particles with a mean length
of 500 nm. While, as shown in Fig. 3b, the CHN nanoparticles
were fine spheres with diameters ranging from 60 to 100 nm.
Fluorescence microscope images. The fluorescence micro-
scope images of CHC and CHN are shown in Fig. 4a and b.
Upon excitation at 405 nm, the CHC nanoparticles emitted a
reddish orange fluorescence, and the observed claviform shape
fits with the TEM observation. In addition, for CHN, a large
Fig. 3 Transmission electron microscope (TEM) images of CHC (a) and
CHN (b) nanoparticles obtained from suspensions of the water–DMSO
mixture.
Fig. 4 Fluorescence microscope images of CHC (a) and CHN (b) nano-
particles (water/DMSO = 7/3, v/v).
number of fluorescent dots emitting light could be observed.
Also, the mean length of the CHC fluorescent bodies was 3 mm,
and the diameter of CHC fluorescent bodies was 1 mm. Both
were larger than the sizes observed by TEM which could be
ascribed to the diffusion of light. Accordingly, the mutual
authentication of fluorescent images and the TEM images
confirmed the existence of the CHC and CHN particles further.
Aggregation-induced emission phenomenon
The fluorescence spectra of the CHN nanoparticles in the
water–DMSO mixtures with various ratios (keeping the concen-
tration 5 ꢁ 10ꢀ6 M) upon excitation at 323 nm and 416 nm are
shown in Fig. 5, respectively. As for the excitation at 323 nm,
CHN in DMSO solution without water showed two weak peaks
at 392 and 529 nm. In this situation, the CHN molecules existed
in the isolated state. Because of the free rotations of the C–N
single bonds, the ground state molecules could form a board
range of torsional angles, from planer to twisted. So, in this
Fig. 2 DSC and TG curves of CHC (a) and CHN (b).
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Fig. 5 Fluorescent spectra changes of CHN (5 ꢁ 10ꢀ6 M) depending on the
volume fraction of water in DMSO, lex = 323 nm (a) and lex = 416 nm (b).
Fig. 6 Variation in the fluorescence intensity of different emission peaks
for CHN with the increasing volume fraction of water in DMSO,
lex = 323 nm (a) and lex = 416 nm (b).
state we expected to have no emission due to the nonradiative
decay processes brought about through the molecular torsions conformation was favored in the aggregation state. Hence the
and rotations.17 However, different from the hypothetical con- nonradiative decay could be prevented. Therefore, the confor-
ditions, in the actual excited state, the existence of hydrogen mational changes relating to the rotation of the N–N moiety
bonds to some extent hinders the free rotations. What is more, played an important role on the AIE.
the relatively high viscosity of the DMSO solution also attri-
In addition, it was probable that the aggregation-induced
butes to the restriction of intramolecular rotation (RIR).28 As a planarizations inevitably induced the intermolecular inter-
result, a weak and broad emission is formed due to the actions. It is widely acknowledged that the formation of an
restriction of intramolecular rotation, the enhancement of excimer complex, which can be ascribed to the strong inter-
planarity, and the improvement of conjugation.
molecular interactions of planar p-conjugated in the solid state,
As the water fraction increased, a new emission peak located leads to fluorescence quenching.8 However, in CHN the bulky
at 447 nm appeared. The fluorescence intensity enhanced and polar hydroxy group restricts the parallel face-to-face
gradually namely the AIE characteristic of CHN, indicating intermolecular interactions in the aggregated state, namely
the formation of aggregated nanoparticles. This enhancement the H-aggregate formation which exhibits blue-shifted absorp-
was depicted in Fig. 6a. In the long wavelength region, when tion bands compared to the isolated state.17 The prevention of
the fraction of water increased to 60%, an emission peak H-aggregation forces the head-to-tail aggregation, the J-aggregation,
located at 534 nm appeared, with a shoulder emission peak which induces a great enhancement in the fluorescence. The
at 565 nm. The fluorescent intensity values of the three peaks absorption spectra of CHN in the isolated state and aggre-
all reached a maximum (FF = 0.546) in the presence of gated state, shown in Fig. S1 (ESI†), were also in accordance
70% water.
with the bathochromic shift in the absorption maximum of
The enhanced fluorescence emission could be explained in J-aggregation.
terms of intra- and intermolecular effects. The CHN molecule
Therefore, most probably, the fluorescent enhancement of
consists of an N–N moiety as a bridging group. Rather than CHN could be ascribed to the combined effects of aggregation-
there being free rotation in the isolated state, a more planar induced planarization and J-aggregate formation.
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Fig. 8 Mechanism of ESIPT process (a). Excited-state tautomers for CHC
(b) and CHN (c).
could form intermolecular H-bonds with protic or polar
solvents.29 Thus, this phenomenon indicated the existence of
the ESIPT process to some extent.
The basic process of the ESIPT has been illustrated in the
literature,30 and a brief introduction of ESIPT is shown in
Fig. 8a. In the ground state, the enol form (E) was more stable
than the keto form (K). Upon photoexcitation, ESIPT occurred
on an fs-timescale, transforming the excited enol form (E*) to
the excited proton-transferred keto form (K*), which was pre-
ferred in the excited state. Then, the excited keto form (K*)
relaxed radiatively to the ground-state keto form (K) releasing a
large Stokes-shifted fluorescence. Finally, the ground-state keto
form (K) transformed to the original enol form (E) in the
ground-state.
It is widely acknowledged that ESIPT only occurs through a
planar structure because of an increasing ESIPT barrier corre-
lating with the twist angle.31 On the basis of molecular struc-
ture, it had a twisted structure at the energetic minimum of the
ground state; while on the other hand, a planar structure was
more stable in the excited state. Hence, the transition energy
gap of the planar molecules was lower. Therefore, the long
wavelength photons, 416 nm for CHN and 390 nm for CHC in
our experiment, exclusively excite planar or near-planar mole-
cules, and the proton transfer took place right after excitation
leading to no short wavelength region emission being observed.
At a short wavelength, 323 nm in our experiment, all molecules
were excited. For the planar molecules, the ESIPT process
could occur directly. However, for the twisted molecules, the
increased barrier enforced the molecules to planarize first
before proton-transfer. Therefore, the fluorescence spectra are
closely related to the molecular population and reaction rate
Fig. 7 Fluorescent spectra changes of CHC (5 ꢁ 10ꢀ6 M) depending on the
volume fraction of water in DMSO, lex = 323 nm (a) and lex = 390 nm (b).
After the water fraction increased to 70%, the fluorescence
intensities decreased with further increases of the water frac-
tion. This emission decrease could be explained by the change
in the shapes of the aggregates. When the water fraction was
lower than 70%, the molecules accumulate slowly in an ordered
way to form more emissive nanoparticles.12 Whereas in a
system with a higher water fraction, the CHN molecules
agglomerated quickly in a random way to form amorphous
nanoparticles that were less emissive. Concerning the emission
spectra upon excitation at 416 nm shown in Fig. 5b, the
conditions were similar to excitation at 323 nm for the long
wavelength region. However, no emission peak in the short
wavelength region could be observed. As for the CHC nano-
particles, the conditions were similar except for the specific
water fractions, and they are depicted in Fig. 7.
Investigation of the contribution of ESIPT to fluorescent
emission features
In order to verify whether proton-transfer exists and how it of ESIPT.
impacts the excited state of CHC and CHN, we measured the
Concerning our fluorescence spectra of CHN upon excita-
FL spectra in solvents with different polarities. At the same tion at 323 nm, as the water fraction increased, the ratio of
concentration, we noticed that the emission intensities planar molecules increased as the nanoparticles form. After the
decreased with the increase of the polarity of the solvent, as water fraction increases to 60%, the ESIPT emission peak could
shown in Fig. S2 (ESI†), because these two ESIPT compounds be observed. Based on the discussion above, nanoparticles of
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CHN (namely the planar molecules) were expected to emit three CHC and CHN have good photophysical properties and could
maximum peaks coming from the E*E*, E*K* and K*E* tauto- be good candidates for cell staining, avoiding the impacts of
mers, respectively. The emission maximum peak at 447 nm, excitation light.
with a small Stokes shift and mirror-image shape, could be
ascribed to the radiative decay from the E*E*. As for the two
emission maximum peaks in the long wavelength region, the
Conclusions
large Stokes shifts indicated they are originated from the E*K*
In summary, we have designed and synthesized two coumarin-
and K*E* tautomers separately. The ESIPT emission peak of the
based fluorescent molecules, CHC and CHN. The two com-
phenolic OH on 2-hydroxy-1-naphthaldehyde to nitrogen atom
pounds are stable and show high thermal stabilities. In addition,
had been reported to be located at 531 nm.32 In addition, the
the ESIPT fluorescence could be utilized as a new strategy to
fluorescence spectra of the CHC nanoparticles which only had a
develop other AIE-active materials. The success of the coumarin-
single ESIPT emission peak coming from the E*K* tautomers
based series is expected to accelerate organic photo-material
are shown in Fig. 7, revealed the ESIPT emission peak of
discovery and lead to further technological applications in the
phenolic OH on 8-formyl-7-hydroxy-4-methyl-coumarin to the
fields of nanobiotechnology and optoelectronics, and potential
nitrogen atom appeared at 570 nm. Accordingly, emission peak
uses in cell imaging.
at 534 nm could be assigned to E*K* species and the other
emission peak at 565 nm probably came from K*E* species.
Acknowledgements
Cell staining
This work was supported by the National Basic Research Program
Cell permeability enabled the compounds to enter living cells,
hence further intracellular investigations became possible. To
test whether CHC and CHN could enter cells and serve as
biological fluorescence probes, HeLa cells were incubated with
the CHC and CHN nanoparticles and then examined by fluores-
cence confocal microscopy. As shown in Fig. 9, the HeLa cells
incubated with the nanoparticle solution and excited by
405 nm, emitted reddish orange fluorescence due to staining
with CHC, and a saffron yellow fluorescence due to staining
CHN. The merged image revealed that fluorescence could only
be detected inside the HeLa cells instead of in random positions.
Therefore, CHC and CHN could be assimilated by HeLa cells and
aggregates in their cytoplasm. Moreover, cell morphology
remained in good condition after intake of the nanoparticles,
indicating the great cytocompatibility of CHC and CHN. The
biological imaging tests on the HeLa cells have shown that both
of China (No. 2014CB846004) and Jiangsu Key Laboratory of
Advanced Catalytic Materials and Technology (No. BM2012110).
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