Novel alkyl chain-based fluorescent probes with large Stokes shifts used for imaging the cell membrane and mitochondria in different living cell lines

Article abstract of DOI:10.1039/c7ra00661f

Fluorescent dyes with large Stokes shifts play a key role in developing multi-purpose fluorescent probes for a wide variety of targets. In this study, we developed two novel alkyl chain-based fluorescent probes (CA-C12 and CA-C2) with large Stokes shifts. The alkyl chain length of the probes affect the membrane permeability, and hence both probes can be successfully applied for sensing the cell membrane and mitochondria in different living cell lines. Furthermore, the probes CA-C12 and CA-C2 exhibited large Stokes shifts and excellent photostability in the different cell lines. The fluorescent dyes with large Stokes shifts were expected to have broader applications for developing various fluorescent probes with excellent optical properties.

Full text of DOI:10.1039/c7ra00661f

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Novel alkyl chain-based fluorescent probes with
large Stokes shifts used for imaging the cell
membrane and mitochondria in different living cell
lines†
Cite this: RSC Adv., 2017, 7, 16087
*
Fangfang Meng, Yong Liu, Jie Niu and Weiying Lin
Fluorescent dyes with large Stokes shifts play a key role in developing multi-purpose fluorescent probes for
a wide variety of targets. In this study, we developed two novel alkyl chain-based fluorescent probes (CA-
C12 and CA-C2) with large Stokes shifts. The alkyl chain length of the probes affect the membrane
permeability, and hence both probes can be successfully applied for sensing the cell membrane and
mitochondria in different living cell lines. Furthermore, the probes CA-C12 and CA-C2 exhibited large
Stokes shifts and excellent photostability in the different cell lines. The fluorescent dyes with large Stokes
shifts were expected to have broader applications for developing various fluorescent probes with
excellent optical properties.
Received 16th January 2017
Accepted 26th February 2017
DOI: 10.1039/c7ra00661f
rsc.li/rsc-advances
Compared to conventional technology, uorescent probes
have become powerful tools for detecting various targets in the
environment, chemistry and medicine.24–26 Recently, a wide
Introduction
The cell membrane is selectively permeable to various biolog-
ical analyses and controls the movement of substances in and
out of cells.1 The cell membrane covers the cytoplasm of living
cells, physically separating the intracellular components from
the extracellular environment.2 The cell membrane can regulate
what enters and exits the cell, facilitating the transport of
materials needed for survival.3 The cell membrane plays a key
role in cell signal transduction and solute transporting.4–6 In
addition, it plays an important role in anchoring the cell cyto-
skeleton and its attachment to the extracellular matrix and
more cells to form tissues.7–9 Consequently, the search for
a method of monitoring the cell membrane has always been
attractive.
Mitochondria is also a membrane organelle and plays a key
role in energy production through respiratory chains,10–12 cell
signalling via the production of reactive oxygen species,13,14 the
regulation of Ca2+ homeostasis,15,16 and triggering cell
death.17–20 Mitochondrial dysfunction is associated with
intrinsic apoptotic pathways and causes a variety of neurode-
generative diseases including Alzheimer,s disease, cancer and
diabetes.21–23 Therefore, it is of importance to image mito-
chondria in biological samples.
variety of uorescent cell membrane and mitochondria probes
have been engineered,27,28 but most of the cell membrane and
mitochondria probes possessed small Stokes shis; however,
a small Stokes shi leads to uorescence imaging errors of the
probes in biological applications.29 Thus, the goal of our study is
to design cell membrane and mitochondria probes with large
Stokes shis for imaging the cell membrane and mitochondria
in different living cell lines.
As we know, carbazole derivatives show a number of attrac-
tive features including large Stokes shis and excellent optical
properties.30,31 In this study, we have developed two novel alkyl
chain-based uorescent probes (CA-C2 and CA-C12) with large
Stokes shis based on a carbazole derivative (Fig. 1). It was
found that both probes containing long and short alkyl chains
can image the cell membrane and mitochondria in different
living cell lines. We envisioned that the alkyl chain length
affects the membrane permeability of the probes and indicated
that the probes can real-time image the cell membrane and
mitochondria in different living cell lines.
Experimental
Measurements equipment and materials
Institute of Fluorescent Probes for Biological Imaging, School of Materials Science and
Engineering, School of Chemistry and Chemical Engineering, University of Jinan,
Shandong 250022, P. R. China. E-mail: weiyinglin2013@163.com
Unless otherwise stated, all reagents were purchased from
commercial suppliers and used without further purication.
Solvents were puried by standard methods prior to use.
Doubly-distilled water was used throughout all the experiments.
Mass spectra were recorded on a 6510 Q-TOF LC/MS or
† Electronic supplementary information (ESI) available: Experimental procedure,
uorescence spectra, tables and characterization data. See DOI:
10.1039/c7ra00661f
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Cell culture and imaging
Different types of cells (A549, 4T-1 and HeLa) were grown in
Dulbecco's modied Eagle's medium, high glucose supple-
mented with penicillin/streptomycin and 10% FBS in a 5% CO2
incubator at 37 ꢀC. Before the imaging experiments, the cells
were cultured overnight on a 35 mm Petri dish in order for their
convenient observation. Cells were incubated with 10 mM CA-C2
and CA-C12 for 10 min at 37 ꢀC, respectively. Aer being washed
twice with PBS, the cells were imaged immediately. In the co-
localization experiment, different cells were incubated with 2
mM commercially available mitochondrial probe MitoTracker
Red (MTR) for 20 min; aer being washed three times with PBS,
10 mM CA-C2 was added to the cells and incubated for 10 min.
Then, the cells were washed three times using PBS before
imaging with a Nikon uorescence microscope.
Fig. 1 (a) The structures of the probes CA-C2 and CA-C12, and (b) the
overall strategy using probes CA-C2 and CA-C12 for imaging the cell
membrane and mitochondria, respectively.
Cell viability evaluated by MTT
We used the colorimetric methyl cytotoxicity assay thiazolyl
tetrazolium (MTT) assay on HeLa cells to measure in vitro
cytotoxicity. About six thousand cells were seeded per well in
a 96-well plate. Aer being incubated for 24 h, different
concentrations (0, 1, 5, 10, 20 and 30 mM) of the probes were
added into each well. Aer 8 h of incubation, the cells were
washed. Subsequently, 100 mL of MTT (5 mg mLꢁ1) was added to
each well and further incubated for 4 h. Aer removing the
medium, 100 mL of DMSO was added to each well to dissolve the
purple crystals. Then, the absorbance at 570 nm was measured
using a microplate reader.
ThermoFisher LCQ FLEET. NMR spectra were recorded on
a Bruker Avance 400 spectrometer using TMS as an internal
standard. The UV-visible-near-IR absorption spectra were ob-
tained on a Labtech UV Power PC spectrometer. Photo-
luminescent spectra were recorded on a HITACHI F-4600
uorescence spectrophotometer with the excitation and emis-
sion slit widths at 5.0 and 5.0 nm, respectively. Fluorescence
imaging of the cells was performed using a Nikon A1MP two-
photon confocal microscope. TLC analysis was performed on
silica gel plates and column chromatography was conducted
over silica gel (mesh 200–300), both of which were purchased
from the Qingdao Ocean Chemicals. HeLa cells, A549 cells, 4T-1
cells and calf serum were obtained from the College of Life
Science, Nankai University (Tianjin, China).
Results and discussion
Preparation of probe
The chemical syntheses of CA-C2 and CA-C12 were accom-
plished in a total of four steps (Scheme 1). N-(2-Ethyl/dodecyl)-4-
Measurement of the uorescence quantum yield
The uorescence quantum yields (F) were calculated using
eqn (1):
ꢀ
ꢁꢀ
ꢁ
2
2
Ar
As
ns
nr
Is
Ir
Fs ¼ Fr
(1)
In this equation, the subscripts s and r refer to the sample
and the reference molecule, respectively. A is the absorbance of
the molecules for both the sample and reference. I represents
the integrated emission area and n is the refractive index of the
solvent. F is the quantum yield.
Preparation of the test solutions
Stock solutions of the probes CA-C2 and CA-C12 were prepared
at 1 mM in DMF. Different solvents were used, including PBS,
H2O, CHCl3, DCM, DMF, DMSO, EtOH, MeOH, MeCN and THF.
Test solutions of the probes CA-C2 and CA-C12 (10 mM) in 5 mL
of the different solvents were prepared. For the uorescence
spectra experiments, the excitation wavelength was 421 nm and
the emission slit widths were 5 nm and 5 nm.
Scheme 1 The synthesis of the probes: (a) 1-bromooctadecane/
bromoethane, KOH, DMF, (b) DMF, POCl3 and CHCl3, (c) 1-iodoun-
decane/iodoethane, EtOH, reflux and (d) EtOH, reflux.
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Table 1 The photophysical properties of CA-C12/CA-C2 in various
solvents
methylpyridinium iodide (1) and 9-ethyl/octadecyl-9H-carbazole
(2) were prepared via an alkylation reaction. 9-Ethyl/octadecyl-
9H-carbazole-3-carbaldehyde (3) was prepared in one step via
the Vilsmeier reaction of compound (2) with phosphorus oxy-
chloride. The target molecules CA-C2 and CA-C12 were obtained
via a Knoevenagel condensation reaction between compound
(3) and (1). The synthetic details of the products are given in the
ESI.†
CA-C12
CA-C2
laa/lb
SSc
laa/lb
SSc
b
b
Sample
(nm)
(nm)
Fd/%
(nm)
(nm)
Fd (%)
PBS
H2O
410/605
420/573
465/570
473/578
430/580
432/583
445/575
442/578
437/582
437/576
195
153
105
105
150
151
130
136
145
139
3.56
0.73
4.8
15
16
15
18
13
10
18
421/581
421/582
465/564
470/575
433/580
431/580
444/576
437/575
443/574
434/576
160
161
99
105
147
149
132
138
131
142
1.4
1.1
1.6
9.6
11
20
18
11
13
CHCl3
DCM
DMF
DMSO
EtOH
MeOH
MeCN
THF
Optical properties
To demonstrate the potential utility of CA-C2 and CA-C12 as
suitable tracers for live-cell imaging, we evaluated the optical
photophysical properties of both probes. As shown in Fig. 2 and
S1,† we found that the absorbance and emission spectra of CA-
C12 was similar to CA-C2 in various solutions. The maximum
absorption of CA-C12 and CA-C2 was 430 nm (Fig. 2a and S1a†)
and 440 nm (Fig. 2c and S1c†), respectively. CA-C12 and CA-C2
have an emission peak at 570–605 nm in various solutions, but
the uorescence intensities were very high in organic solvents
compared to those in an aqueous solution (Fig. 2b, d, S1b and
d†). The results demonstrate that compounds CA-C12 and CA-
C2 exhibited large Stokes shis in various solutions (Table 1),
which results in an efficient separation of the absorbance and
emission maxima. The quantum yield (F) of the probes CA-C12
and CA-C2 was only 0.01–0.04 in an aqueous solution. In
organic solvents, however, the maximum quantum yield of both
probes reached 0.10–0.20 (Table 1).
14
a
b
Maximum absorption wavelength (nm).
Maximum emission
c
wavelength (nm). Stokes shi (SS). Fd is the uorescence quantum
yield (error limit: 8%) determined using uorescein (F ¼ 0.95) in
aqueous NaOH (pH 13) as the standard.
As we know, mitochondria and the cell membrane are
composed of different organic phases. Thus, we think that
probes CA-C12 and CA-C2 may show strong uorescence in the
mitochondria or cell membrane. In addition, we further inves-
tigated the water solubility of the probes.32 The solubility of CA-
C12 and CA-C2 in water was 1.26 mM and 3.6 mM, respectively.
The results demonstrated that probe CA-C12 exhibited higher
hydrophobicity than CA-C2 (Fig. S2†). Over a wide physiological
pH range from 4.0 to 8.0, the probes were found to be weakly
uorescent (Fig. 3), which demonstrated that CA-C12 and CA-C2
were not affected by pH.
Fig. 3 The intensity of probes CA-C12 and CA-C2 in a buffer solution
at different pH values. Excitation wavelength: 405 nm. [CA-C12] ¼
[CA-C2] ¼ 2 mM.
Cell membrane imaging and photostability
To investigate the imaging of CA-C12 in living cell lines, uo-
rescence imaging experiments were carried out. The cytotoxicity
was evaluated using standard MTT assays, indicating that the
probe CA-C12 showed low toxicity to the cultured cells under the
experimental conditions (Table S1†). Cancer cells (A549, 4T-1
and HeLa cells) were incubated with CA-C12 for 10 min, fol-
lowed by uorescence microscopy. As shown in Fig. 4, the
results of cells imaging demonstrated that the CA-C12 was
located in the cell membrane and the probe was a cell
membrane probe with poor permeability.
The photostability is one of the most important criteria for
constructing novel uorescence probes.33–36 Continuous scan-
ning using a Nikon AMP1 confocal microscope was used to
quantitatively investigate the photostability of CA-C12 (10 mM).
Within 360 s, the uorescence signals of CA-C12 decreased
slightly in living HeLa cells (Fig. 5) and other living cells lines
(Fig. S3†). The results proved that the probe CA-C12 was capable
of real-time imaging cells membrane in different cell lines.
Fig. 2 The absorption and fluorescence spectra of CA-C12 (a and b)
and CA-C2 (c and d) in various solvents. CHCl3 (-), DMF (C), EtOH
(:), H2O (;) and PBS (A). [CA-C12] ¼ [CA-C2] ¼ 10 mM.
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Fig. 4 (a–i) Confocal images of the probe CA-C12 (10 mM) in different
cells lines. (a, d and g) Bright-field images, (b, e and h) fluorescence
images of CA-C12 to the cell membrane (lex ¼ 405 nm; lem ¼ 570–
620 nm) and (c, f and i) merged images. Scale bar ¼ 20 mm.
Fig. 6 (a–i) Confocal images of the different cells incubated with 10
mM probe CA-C2. (a, d and g) Bright-field images, (b, e and h) fluo-
rescence images of CA-C2 (lex ¼ 405 nm; lem ¼ 570–620 nm) and (c, f
and i) the merged images. Scale bar ¼ 20 mm.
Fig. 5 (a) Fluorescence images (the red channel) of Hela cells incu-
bated with CA-C12 acquired at different times under successive
excitation. (b) The mean intensities of the cells incubated with CA-C12
(10 mM) in the red channel under successive excitation at different
times. Excitation wavelength: 405 nm.
Mitochondria imaging and photostability
Contrary to the probe CA-C12 with a long alkyl chain, CA-C2
should possess superior permeability. Subsequently, we inves-
tigated whether CA-C2 can enter cells and image the mito-
chondria. Similarly, the cytotoxicity of CA-C2 was evaluated by
MTT assays (Table S1†) and the data proved that CA-C2 had low
toxicity towards living cells. As shown in Fig. 6, the probe CA-C2
possessed superior permeability to living cells and exhibited
strong uorescence in different living cells (A549, 4T-1 and
HeLa cells).
Furthermore, to prove that the probe can image the mito-
chondria in different living cell lines, co-localization experi-
ments were carried out. To determine the intracellular
location of CA-C2 inside the cells, probe CA-C2 and the
commercial mitochondrial tracker MTR were co-incubated in
different cell lines (Fig. 7a–l). The intensity prole of the linear
regions of interest across a HeLa cell in the green and red
channels also varies in close synchrony (Fig. 7j–m). The
Mander's overlap coefficient and Pearson's co-localization co-
efficient were determined as 0.95 and 0.90, respectively
(Fig. 7n). The imaging results demonstrate that probe CA-C2
was capable of imaging the mitochondria in different living
cells lines. Similar to CA-C12, probe CA-C2 showed a high
Fig. 7 The confocal images of cells treated with MTR (2 mM) for
20 min and probe CA-C2 (10 mM) for 10 min. (a, e and i) Bright-field
images, (b, f and j) fluorescence images of probe CA-C2 collected
between 500 and 550 nm upon excitation at 405 nm, (c, g and k)
fluorescence images of MTR collected between 570 and 620 nm
upon excitation at 561 nm and, (d, h and l) the merged images.
(m) The intensity profile of the linear region of interest (white arrow)
across the HeLa cells co-incubated with MTR and CA-C2. (n)
The correlation plot of MTR and CA-C2 intensities. Scale bar ¼
20 mm.
uorescence intensity within 360 s and this feature is prom-
ising as the probe can real-time image the mitochondria over
a long period of time (Fig. 8). In addition, we further proved
that probe CA-C2 possessed high photostability in living A549
and 4T-1 cells (Fig. S4†).
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In summary, we described two novel alkyl chain-based uo-
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alkyl chain length of the probes affects the membrane perme-
ability and allows both probes to be successfully applied for
sensing the cell membrane and mitochondria in different living
cell lines. Furthermore, probes CA-C12 and CA-C2 exhibited
excellent photostability in different cell lines. This nding may
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Acknowledgements
This study was nancially supported by the NSFC (21472067,
21672083 and 51503077), the Taishan Scholar Foundation (TS
201511041) and the startup fund of the University of Jinan (309-
10004) as well as the Doctor start up fund of University of Jinan
(160082102) and NSFSP (ZR2015PE001).
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