A fluorescent pH probe for acidic organelles in living cells

Article abstract of DOI:10.1039/c7ob02037f

A water-soluble pH sensor, 2-(6-(4-aminostyryl)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)-N, N-dimethylethanamine (ADA), was synthesized based on the molecular design of photoinduced electron transfer (PET) and intramolecular charge transfer (ICT). The

Full text of DOI:10.1039/c7ob02037f

Organic &
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A fluorescent pH probe for acidic organelles in
living cells†
Jyun-Wei Chen,a Chih-Ming Chen*a and Cheng-Chung Chang
Cite this: Org. Biomol. Chem., 2017,
15, 7936
b
*
A
water-soluble pH sensor, 2-(6-(4-aminostyryl)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)-N,
N-dimethylethanamine (ADA), was synthesized based on the molecular design of photoinduced electron
transfer (PET) and intramolecular charge transfer (ICT). The fluorescence emission response against a pH
value is in the range 3–6, which is suitable for labelling intracellular pH-dependent microenvironments.
After biological evolution, ADA is more than a pH biosensor because it is also an endocytosis pathway
tracking biosensor that labels endosomes, late endosomes, and lysosome pH gradients. From this, the
emissive aggregates of ADA and protonated-ADA in these organs were evaluated to explore how this
probe stresses emission colour change to cause these unique cellular images.
Received 16th August 2017,
Accepted 4th September 2017
DOI: 10.1039/c7ob02037f
rsc.li/obc
which could reveal emission response, colour or intensity with
the association or dissociation of protons. This is because fluo-
Introduction
Acid–base (pH) balance is a key parameter for cell survival.1 rescence detection allows non-invasive measurements with
For example, a higher intracellular pH (≥7.4) and a lower extra- biological objects, parallel monitoring of multiple samples,
cellular pH (∼6.7–7.1) exist in cancer cells compared with and imaging.7 Therefore, ratiometric fluorescence measure-
normal cells (intracellular pH ∼ 7.2; extracellular pH ∼ 7.4) to ment is more desired.8 However, most pH fluorescent probes
form a ‘reversed’ pH gradient2 that promotes proliferation, lack sensitivity or are not suitable for characterizing acidic
cancer cell invasion, and escape from apoptosis.3 Moreover, organelles such as lysosomes, endosomes, and spermatozoa
acid–base dysregulation is a common property of cancer cells acrosomes, because their emissions are weak or disappear in
that may affect the uptake and efficacy of pH-sensitive chemo- concentrated solutions or in acidic environments due to aggre-
therapeutic drugs.4 Thus, the differences in pH between gation-caused fluorescence quenching or changes in intra-
normal and cancer cells may provide a diagnostic tool by using molecular charge transfer (ICT). This is inevitable because the
a pH probe. In addition, tracing pH fluctuations in acidic orga- molecules or probes become encapsulated inside endocytic
nelles is essential to investigate normal cellular functions and vesicles through clustering in the endocytosis pathway. Under
activities in these compartments.
these conditions, a fluorophore which is highly fluorescent in
The endocytosis pathway is a process by which cells inter- the concentrated state, would be a good choice to overcome
nalize biomolecules that are associated with numerous biologi- this challenge by tracing endocytic or lysosome vehicles.
cal processes.5 In these pathways, every attended organelle
Based on our knowledge, the aggregation induced emission
maintains a unique pH value to provide the appropriate bio- (AIE),9 aggregation induced emission enhancement (AIEE)10
chemical conditions. The pH gradient follows the endocytic and non-resonant exciton system11 are widely used to explain
pathway as ∼6.3 in early endosomes, ∼5.5 in late endosomes, the aggregation-dependent fluorescence enhancement beha-
and ∼4.6 in lysosomes. The apparent decline of proton flow in viours of molecules. The so-called AIE/AIEE dyes are character-
the endocytosis pathway provides a good switch for a fluo- ized by their enhanced emission, usually from non-emissive
rescence biosensor.6 The strategy for measuring the intracellu- molecular states in organic solutions to strongly emitting
lar pH gradient is based on the design of the fluorophore, aggregate states in aqueous media, mostly due to confor-
mational restriction9,10,12 or planarization of “non-flat” mole-
cules.13 Aggregation-dependent non-fluorescence or fluo-
rescence enhancement behaviours of “flat” aromatic dyes
should be discussed in the context of a non-resonant exciton
system because there are no conformational restriction pro-
aDepartment of Chemical Engineering, National Chung Hsing University,
Taichung 402, Taiwan
bGraduate Institute of Biomedical Engineering, National Chung Hsing University,
Taichung 402, Taiwan. E-mail: ccchang555@dragon.nchu.edu.tw
blems in these planar molecules. These flat molecules can
form intermolecular stacks with each other in the concen-
†Electronic supplementary information (ESI) available: Synthesis and character-
isation, Fig. S1–S8, Table S1. See DOI: 10.1039/c7ob02037f
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trated or solid state and lead to an emissive non-resonance
excitonic state of H-aggregates or emissive J-aggregates.11,14
We proposed that these aggregation caused emission mole-
cules, as mentioned above, should brighten the defect and
protonation induced fluorescence quenching once they are
encapsulated by endosomes and become vehicles in the endo-
cytosis pathways. In addition, there is a serious need for the
development of multicolour fluorogens for pH detection,
which usually correlates to discussions about J-aggregate for-
mation, E/Z isomerization, twisted intramolecular charge
transfer (TICT), and excited state intramolecular proton trans-
fer (ESIPT).15,16
In the present study, a pH-response fluorophore, ADA, was
designed and synthesized by incorporating a dimethylamino-
ethylamine group, a proton acceptor, into 1,8-naphthalic anhy-
dride to become a naphthalimide core scaffold, which further
elongated the π-electronic resonance by conjugating a vinyl
aniline functional group. The water-soluble fluorophore ADA
presents red to green emissions when exposed to relatively
neutral to acidic conditions. Its pH dependent AIE behaviour
was also investigated to help explain intracellular molecular
imaging, which was further investigated in live intracellular
images, time-dependent imaging, concentration-dependent
images, and subcellular localization. Finally, we observed that
intracellular luminous behaviour for ADA molecules was
Fig. 1 (a) Chemical structure and push–pull illustration of the com-
pounds ADA, NIM-1 and ANB. Arrows indicate the protonation site.
(b) Solvent effect of compound ADA under neutral conditions, repre-
sented by the extinction coefficient for absorbance and quantum yield
for emission spectra.
Particular optical properties
dependent on pH values in the endocytosis pathway and could Fig. 1b presents broad absorption peaks with a slight solvent
effect, and low quantum yield emission peaks were observed
in high-polarity solvents; brighter emissions with high
quantum yields were obtained in lower polarity solvents.
Compared to the solvent effects of the control compounds
NIM-1 and ANB (Fig. S1†), the basic spectral properties of ADA
are similar to ANB but not NIM-1 with lower quantum yields.
Its absorption spectra showed two main peaks at 310 to
330 nm, assigned to the π–π* transition, and at 435 nm (in
be applied to the distinct intracellular pH gradient of the
endocytosis pathway.
Results and discussion
Molecular design and synthesis
Naphthalimide is a good electron acceptor to be used to
design spectral pH-response compounds.17 In this study, the toluene) to 477 nm (in DMSO), which was assigned to the
internal charge transfer (ICT) characteristic of the fluorophore
D–π-A type chromophore structure was designed and syn-
with the push–pull effect between the electron donating
thesized by incorporating an electron donor group to naphtha-
limide by means of the π-conjugated linker ethylene. At first, aniline and the electron withdrawing naphthalimide.19
The spectra of ADA were first investigated as a function of
the starting material, 4-bromo-1,8-naphthalic anhydride, was
pH in water. Fig. 2a shows that upon titration with an acid, the
initially reacted with N,N-dimethyl-ethylenediamine (for
NIM-1/ADA) or n-butylamine (for ANB) to form the naphthali- 440 nm peak shifted hypsochromically to 395 nm with an iso-
sbestic point at 415 nm. Meanwhile, upon excitation of the
mide core scaffold. Then, the 4-vinylamine (for ANB/ADA) and
ICT band at 440 nm, a peak appears with λem max at 515 nm,
4-methoxyphenyl (for NM-1) functional groups, or electron-
donating units, were chosen to substitute the 4-positions of which becomes more apparent once excitation was 395 nm,
i.e., the absorption of protonated ADA. This observed emission
change is highly pH dependent and is ‘switched on’ upon acid-
naphthalimide by Heck organic–metal coupling reactions. In
Fig. 1a, the pH-responsive fluorophore ADA is constructed by
introducing a tertiary amine substituent (pKa ∼ 10 of conju- ification with large fluorescence enhancements. The plot of
absorbance at 440 nm against pH showed only minor changes
gate acidic species) and vinylaniline (pKa ∼ 4.73) as a protona-
that occurred above pH 6.2, while the most significant changes
tion site to increase water solubility with the expectation that
ADA will accumulate in acidic organelles in its protonated were observed between pH 3 and 6. From this dynamic region,
form.18 Furthermore, the electron donor dimethylamine acts
a pKa ∼ 5.07 was determined using non-linear regression ana-
lysis. Similar pKa values were also determined by plotting the
as a PET quencher and aniline acts as an ICT responsive
moiety related to naphthalene. Hence, the control compounds emission at 515 nm against pH, and the pKa value at ∼3.64
was unclearly found. The pH-dependent spectral change under
aqueous conditions was not apparent enough to estimate two
pKa values for ADA.
NIM-1 and ANB were prepared according to the relative optical
properties. Compared with these control compounds, we could
easily understand the two-stage acidification process of ADA.
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which is likely due to the PET decrease between dimethyl-
amine and an electron donating group-substituted naphtha-
lene at the 4-position. Meanwhile the pKa2 value (3.64 from
aqueous and 2.68 from DMSO/H2O) corresponds to the proto-
nated form of the aniline moiety of ADA, which is because the
electron donation ability of the NH2 long pair was shared with
naphthalene via a vinyl group. Moreover, when we checked the
viscosity-variation response emission spectra in Fig. S5,† both
emission peaks (∼600 nm and ∼500 nm) presented positive
ICT responses to a high-viscosity environment. From the dis-
cussion above, the emission peak at ∼600 nm was character-
ized as a PET induced ICT change of ADA-H, and the emission
peak at ∼500 nm indicated the ICT change of ADA-H2.
Eventually, the pH response emission changes of ADA cover di-
methylamine, aniline (∼4.87, 4.6), 4-vinyl aniline (∼4.73), and
the fluorescent pH indicator Oregon Green® 488 (∼4.7),21
which demonstrates that this compound is suitable for the
physiological pH range.
Fig. 2 ADA (30 μM) absorption (left) and emission (right) spectral vari-
ations as a function of addition of [H+] in (a) dd-water and (b) DMSO/
H2O = 3/1 in v/v. Inserts show peak intensity plots against pH (excited
wavelength = 400 nm).
pH-Dependent fluorescence mapping
MRC-5 human lung fibroblast normal cell lines, HeLa human
cervix epithelial adenocarcinoma cells and CL1-0 human lung
adenocarcinoma cells were selected for cellular imaging
studies. Fig. 3 shows the cellular imaging of these cell lines
treated with ADA for four hours and then observed by fluo-
rescence microscopy with a colour CCD to obtain the real
emission colour of intracellular ADA. It is interesting that
colourful bright spots were revealed in all three cell lines and that
MRC-5 normal cells exhibited the most abundant fluorescent
colours (Fig. 3b and c). When checking the intracellular mole-
cular imaging of the control compound NIM-1, Fig. S6† shows
similar bright spots, but no apparent fluorescent colour
change as well as fewer bright spots with a unitary fluorescent
colour appeared in the ANB case. It appears that ADA showed
Hence, we investigated the spectral variations of ADA proto-
nation in DMSO/H2O solutions with 0, 1/3, and 1/2 H2O in
volume proportion. Preparations of pH-dependent DMSO/
H3O+ solutions are described in the Experimental section.
Fig. 2b shows the spectral changes as a function of pH for ADA
compounds in DMSO/H2O (3/1, v/v). When the environment
was changed from neutral to acidic, the λabs was first red-
shifted from 466 to 474 nm and then began to decrease. At the
same time, a new absorption peak at ∼400 nm grew with a
423 nm isosbestic point. Once we fixed the excited wavelength
at 400 nm to track the titration emission spectra, there were
two emission peaks at ∼600 and 495 nm that grew simul-
taneously with different increasing rates. This phenomenon
becomes more obvious on increasing the amount of DMSO,
and the two-step protonation behaviour becomes clearer, as
shown in Fig. 2a, b and Fig. S2.† On the other hand, in Fig. S3
and S4† we checked the pH-titration spectra of the control
compounds NIM-1 and ANB before the characterization of the
possible protonated forms of ADA. At the beginning of the
titration process, a spectral variation of ADA was similar to
NIM-1. Absorption and emission spectra showed only minor
changes with pH variations but were slightly redshifted. For
ideal PET sensors, such shifts do not usually occur. We believe
that this is due to the protonation inhibition of PET from di-
methylamine to naphthalene. This further affects the ICT
between naphthalene and anisole (or aniline). In contrast,
ANB showed apparent changes in its absorption and emission
spectra as a function of pH in DMSO, which is similar to the
spectral change against pH, as ADA showed at a later period of
titration. The marked red-to-green emission colour change in
response to the change in pH should be the cause of protona-
tion of the aniline moiety inducing a push–pull decrease as
well as an ICT change.20
Fig. 3 Fluorescence images of (a) MRC-5 normal, (d) HeLa and (e) CL1-0
cancer cells stained with ADA (5 μM) for 4 hours; (b) magnified version
of (a); (c) confocal λ scanning, 470 to 650 nm with 5 nm integration,
of (b). (f)–(i) Several selected images from the MRC-5 cells pre-loaded
with 5 μM ADA were incubated with nigericin and subsequently imaged
The pKa1 value (5.04 from aqueous and 4.81 from DMSO/
H2O) corresponds to the protonated form of dimethylamine, in pH ∼ 6, 5, 4 and 3 buffers (cube: ex, 390/10 nm; em, 410 nm lp filter).
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special cellular images that combine the intracellular mole-
cular imaging of the control compounds NIM-1 and ANB.
Moreover, these colourful fluorescence spots varied and
showed a different presentation between cancer and normal
cells. These phenomena should be explored based on the
acidic fluorescence-response image consideration. Fig. 3f–i
present the pH dependent cellular imaging of ADA by addition
of a H+/K+ exchanger ionophore nigericin.22 Once the intra-
cellular pH was homogenized by the surrounding medium
clamped at pH 6, 5, 4 or 3, the intracellular pH gradient dis-
appeared, and these colourful fluorescent spots become
unified or faded. Hence, this proves that ADA performed fluo-
rescence cellular imaging against the intracellular pH gradient.
The change in the ratio of red to green emission intensity
showed the ability of ADA to measure a pH dependent cellular
signal over the pH range from 3 to 7. We interpreted ADA to be
suitable for mapping the organelles relevant to the endocytosis
pathway.
Fig. 4 A time-dependent intracellular accumulation illustration of 5 μM
ADA in (a) MRC-5 normal cells, (b) HeLa cancer cells and (c) CL1-0
cancer cells. The UV cube (ex 390/10 nm, 410 long pass filter) was used
to collect the images.
Subcellular localization of ADA
The endocytic pathway is composed of dynamic membrane
processes in an increasingly acidic environment once the sub-
stances pass through the pathway. Endocytic vesicles are
acidic, and the lysosomes can reach pH values of 4.5–4.7.1 To
determine the subcellular localization of ADA, cells were incu-
bated with the compound ADA and then double-stained with
red cellular lysosomal trackers (LysoTracker Red DND-99) and
endosomal trackers (rhodamine DHPE). The rhodamine DHPE
probe can be used to label biological membranes and to probe
phospholipids within membranes, including pathways for
endocytosis.23 Fig. S7† shows that the bright spots of intra-
cellular ADA can partially co-localize with lysosomal markers
(co-localization was analysed by Pearson correlation coefficient
(PCC): 48.2%) and rhodamine DHPE ((PCC): 65.7%). These
results indicated that ADA molecules first pass through cellu-
lar membranes via endocytic vehicles and then accumulate in
lysosomes, resulting in colourful fluorescent spots due to pH
gradient values of the environments that seem to match the
emission variations of the gradually protonated ADA. In the
endocytosis pathway, the participating organelles maintain
unique intravesicular pH values to provide an appropriate
microenvironment for specific biochemical processes. Along
propose that the dark-red spots are the fluorescent presen-
tation of a nearly neutral form of ADA in early endosomes, in
which the environmental acidity is too low to protonate ADA.
The orange fluorescence spots would be the late endosomes.
With prolonged incubation times, these brightly coloured
spots will be redistributed; the dark-red spots will disappear,
and the yellow spots will become dominant, indicating lyso-
somes. Similar fluorescence spot-colour development was also
observed in the HeLa and CL1-0 cancer cell lines, although
there were fewer spot presentations (Fig. 4b and c). However,
the time-dependent cellular images of NIM-1 and ANB showed
no differences between incubation periods (Fig. S8 and S9†).
The former could not present pH-dependent fluorescence
colour changes, while the latter did not display endosomes
because of its lower pKa. That is, compound ADA provides a
full range of colours for the pH scale. These results show that
time-dependent images were associated with the cellular endo-
cytosis pathway.
Formation of intracellular luminescent spots
the endocytic pathway, pH values lowered from ∼6.3 in early Reviewing the optical spectral properties of ADA, we must
endosomes to ∼5.5 in late endosomes, then further down to claim that ADA in most polar solvents showed low fluorescent
∼4.6 in lysosomes.1 Therefore, it is possible that neutral ADA emission. Hence, the fluorescent brightness of intracellular
can pass through the cell membrane and be protonated gradu- molecular imaging (spots) is above what we expected. Through
ally in endosomes to reveal orange coloured fluorescent spots these phenomena, it can be proposed that the compound may
(green < red in Fig. 2b) during the early incubation period. remain in a nonpolar environment within the endosome.
Then, further protonation occurred in lysosomes and yellow However, we observed that the quantum yields of ADA were
spots (green > red in Fig. 2b) were observed.
dramatically reduced in these low polarity solvents during pro-
Thus, to characterize the endocytic pathway-related colour- tonation (EA, THF, toluene etc.) (Table S1†). Hence, we tried to
ful bright spots, we carefully assessed the time-dependent propose the involvement of other possible mechanisms to
intracellular molecular fluorescence images (Fig. 4a). At an explain the intracellular bright spots, and the potential of the
initial ADA incubation period between 30 minutes and 1 hour, emissive aggregates of compound ADA was evaluated. In pre-
the colourful fluorescence spots revealed abundant dark-red vious studies, we applied AIE properties to describe the fluo-
spots, fewer orange spots, and rare bright yellow spots. We rescence behaviour of the solid state and water-soluble fluoro-
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gen and observed the formation of fluorescence organic nano-
particles (FONs).24,25 A typical experimental demonstration is
to show turn-on type fluorescence of a dye as the water content
increases to a certain level where aggregates form.26 Here, we
first explored the emission spectral variations of ADA in a vari-
able ratio of THF/H2O or THF/(H3O+) mixing solutions, and we
found no emission enhancement greater than that in THF
(Fig. S9†). However, it is interesting that red coloured fluo-
rescent spots (FONs) were observed under fluorescence
microscopy by evaporating in a vacuum from 50 μM ADA/THF
solution as shown in Fig. 5a. The TEM image was prepared
from the same solution and clearly showed fine spherical-
shaped nanoparticles with several hundred nanometre dia-
meter FONs (Fig. 5e). Similar sample preparation was also
used to check the FON formation behaviour of the protonated
ADA in acidic solution. As we expected, the orange and yellow
fluorescent bright spots were observed in the preparation from
acid THF/H3O+ mixing solutions at pH ∼ 5.0 and ∼4.0 as
shown in Fig. 5b and c, respectively. Fig. 5d shows the interest-
ing result that the green patterns in the image were observed
in pure acidic aqueous solution (pH ∼ 3.0), in accordance with
the result of Fig. 2. Herein, fluorescence image-related TEM
diagrams are presented in Fig. 5f–h. Combined with the
experimental results of Fig. S10† and Fig. 5, ADA soluble in
organic media is emissive (EA, THF and toluene, as shown in
Fig. 1b). Moreover, the emission was not apparently enhanced
in H2O containing solution. Therefore, ADA should not be
used as an AIE dye but rather as a “non-resonant exciton
system” causing fluorescent aggregation. Usually, such dyes
are emissive in organic solvents as well as in the solid state,
even though they have a pi-stacking pattern. This result pro-
vides evidence that ADA possesses the potential of emissive-
aggregates to form FONs regardless of neutral (ADA) or acidic
Fig. 6 Fluorescence images of MRC-5 normal cells stained with ADA
1 μM (a) and 5 μM for 4 hours (cube: ex, 390/10 nm bp filter; em, 410 nm
lp filter). (c) pH-Dependent fluorescence mapping of the intracellular
endocytosis pathway by ADA.
conditions (ADA-H), which is helpful to infer information from
the unique cellular images of ADA.
Concentration-dependent fluorescence cellular images
further supported this model (Fig. 6). At low concentrations of
ADA, intracellular fluorescent spots were homogeneous and
dispersible. When higher concentrations of ADA were incu-
bated, brighter speckles combined and dense spots were
revealed in the images because the endocytosis pathway
“enclosed” more molecules at a time. The compound ADA was
characterized to present ICT response emission changes as
well as aggregate-caused FONs against pH variation in spectral
studies, which caused the intracellular endocytosis pathway-
related colourful fluorescence spots regardless of whether the
molecule was self-assembled or was enclosed by endocytosis.
The summary is illustrated in Fig. 6c.
Conclusions
We designed and synthesized a pH sensor, ADA, whose emis-
sion response against the pH value was located in a range of
cellular endocytosis pathways. From the results and discus-
sions following the experiments, we initially identified that
ADA penetrated cellular membranes through endocytosis and
then changed their luminous properties by detecting different
concentrations of protons within the endocytic vesicles. The
pH fluctuation related to multi-colour emission changes
within the endocytic pathways arose from the unique
PET/ICT and the properties of emissive aggregates of ADA
and is suitable for labelling intracellular pH dependent
microenvironments.
Experimental
Materials
Fig. 5 FON illustrations for 50 μM of compound ADA. The fluorescence
microscopy images that were prepared from (a) THF solution (cube: ex,
470/20 nm, 515 nm long pass filter); (b) THF/H2O (v/v 1/3, pH ∼ 5);
(c) THF/H2O (v/v 1/3, pH ∼ 4) and (d) H2O (pH ∼ 3) (cube: ex, 390/10 nm
bp filter; em, 410 nm lp filter). The relative TEM images are also shown
in (e)–(h).
General chemicals employed in this study were of the highest
grade available and were obtained from Merck Ltd or Acros/
Aldrich Chemical Co. and used without further purification.
All solvents for spectral measurements were of spectrometric
grade.
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Apparatus
Intracellular pH calibration
Absorption and fluorescence spectra were recorded using a In brief, cells were incubated with the compound for 4 h. The
Thermo Genesys 6 UV–vis spectrophotometer and a HORIBA cover slips were rinsed 2 times with PBS buffer to remove the
Jobin–Yvon Fluoromas-4 spectrofluorometer, respectively. excess compound and then treated with PBS buffer at various
Transmission electron microscopy (TEM) was conducted on a pH values (3–7) in the presence of 5 μM of nigericin. After
Zeiss EM 902A instrument operated at 80 kV. A mixed solution 5 min of incubation, nigericin was removed by gently rinsing
of THF and deionized water containing compound ADA was with PBS (pH 7.4) twice, and then, PBS buffer solutions with
deposited onto a carbon-coated copper grid.
varying pH values (3–7) were added to each cell line and incu-
bated for 1 h at 37 °C. After that, the cells were rinsed twice
again with neutral PBS buffer and transferred to the stage of
the fluorescence microscope.
Determination of quantum yields
The quantum yields of NIM derivatives were determined as
previously described using the following equation:27
Cellular tracker assay
2
2
Φu ¼ Φs ꢀ ½Afu ꢀ AsðλexsÞ ꢀ ηu ꢁ=½Afs ꢀ AuðλexuÞ ꢀ ηs ꢁ
LysoTracker red assay: Cells were seeded onto chamber slides
and incubated with ADA (5 μM) for 4 h at 37 °C. After that, the
cells were gently washed with PBS twice and then incubated
with fresh medium containing 30 nM Hoechst 33342
(ThermoFisher Scientific, Cat. no. H3570) and 100 nM
LysoTracker red DND-99 (ThermoFisher Scientific, Cat. no.
L7528) for 30 min at 37 °C. The fluorescent cellular images were
finally obtained after washing with PBS twice. Rhodamine
DHPE assay: cells were first incubated with ADA (6 μM) for 1 h
at 37 °C. After that, the cells were gently washed with PBS twice
and then incubated with fresh medium containing 30 nM
Hoechst 33342 and 6 μM rhodamine DHPE (ThermoFisher
Scientific, Cat. no. L1392) for 3 h at 37 °C. Cellular images were
finally obtained after washing with PBS twice.
where Φu is the quantum yield of the unknown; Af is the inte-
grated area under the corrected emission spectra; A(λex) is the
absorbance area at the excitation wavelength; η is the refractive
index of the solution; the subscripts u and s refer to the
unknown and the standard, respectively. For the same λex, we
chose 3, 6-bis (1-methyl-4-vinylpyridinium) carbazole diiodide
(BMVC) as the standard, which has a quantum yield of 0.25 in
glycerol and 0.02 in DMSO.24,28
Spectral response of ADA in pH variable solution
ADA was dissolved in DMSO to prepare a stock solution (10−2 M).
The stock solution was diluted with water or DMSO to a concen-
tration of 30 μM. Titration of ADA in DMSO: the acidic ADA/
DMSO solution is achieved by adding very small amounts of
1M–0.1 M HCl solutions (volume <1%) to the 30 μM, 2 mL of
ADA DMSO solution. At the same time, the control titration was
also performed by adding an equal concentration of HCl (equal
volume) to aqueous solution to calculate the pH at regular inter-
vals. Each of these pH values were recorded and analysed by UV
absorption and fluorescence emission. Excitation was at 450 nm
or 400 nm; the emission range was from 420 to 750 nm.
Synthesis
Details of the synthesis of NIM-1,24 ABN29 and ADA are
described in the previous results and ESI† as shown in
Scheme 1.
Cell culture conditions and cellular imaging
MRC-5 human normal embryonic lung fibroblast cells and
HeLa human cervical cancer cells were maintained in modi-
fied Eagle’s medium (MEM) containing nonessential amino
acids, Earle’s salts, L-glutamine, 1 mM sodium bicarbonate,
1 mM sodium pyruvate, 1% penicillin/streptomycin, and 10%
FBS. The CL1-0 human lung adenocarcinoma cancer cell line
was grown in RPMI 1640 (Gibco/Invitrogen, Cat. no. 22400-
089) medium containing 10% foetal bovine serum (FBS). The
cells were cultured at 37 °C under a humidified atmosphere
(95% air and 5% CO2) and were then seeded onto coverslips
and incubated for 24 h before observation by cellular imaging.
On the next day, the cells were incubated with 5 μM of
compounds for 4 h; here the DMSO stock solutions of the
compounds were diluted in serum-free medium before use
(1 : 100 v/v). The fluorescence images were taken under a Leica
AF6000 fluorescence microscope with a DFC310 FX digital
colour camera. The excitation sources were 390/10 nm or
470/20 nm bandpass for compounds. Fluorescence photo-
graphs were taken through related range tubes.
Scheme 1 Reagents and conditions: (i) N,N-Dimethylethane-1,2-diamine
(ADA, NIM-1) or n-butylamine (ANB), EtOH, r.t. 24 h; (ii) Pd(OAc)2/(o-tol)3P,
4-vinylaniline (ADA, ANB), 4-methoxystyrene (NIM-1), Et3N/MeCN, N2,
reflux, 48 h.
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Conflicts of interest
There are no conflicts to declare.
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Acknowledgements
This work was supported financially by the Ministry of Science
and Technology, (MOST 104-2119-M-005-007-MY3) of Taiwan.
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