Dual Site-Controlled and Lysosome-Targeted Intramolecular Charge Transfer-Photoinduced Electron Transfer-Fluorescence Resonance Energy Transfer Fluorescent Probe for Monitoring pH Changes in Living Cells

Article abstract of DOI:10.1021/acs.analchem.6b00422

Acidic pH is a critical physiological factor for controlling the activities and functions of lysosome. Herein, we report a novel dual site-controlled and lysosome-targeted intramolecular charge transfer-photoinduced electron transfer-Fluorescence resonance energy transfer (ICT-PET-FRET) fluorescent probe (CN-pH), which was essentially the combination of a turn-on pH probe (CN-1) and a turn-off pH probe (CN-2) by a nonconjugated linker. Coumarin and naphthalimide fluorophores were selected as donor and acceptor to construct the FRET platform. Hydroxyl group and morpholine were simultaneously employed as the two pH sensing sites and controlled the fluorescence of coumarin and naphthalimide units by ICT and PET, respectively. The sensing mechanism of CN-pH to pH was essentially an integration of ICT, PET, and FRET processes. Meanwhile, the morpholine also can serve as a lysosome-targeted group. By combining the two data analysis approaches of the ratios of the two emission intensities (R) and the reverse ratio R′ (R′ = 1/R), the fluorescent ratio of CN-pH can show proportional relationship to pH values in a very broad range from pH 4.0 to 8.0 with high sensitivity. The probe has been successfully applied for the fluorescence imaging of the lysosomal pH values, as well as ratiometrically visualizing chloroquine-stimulated changes of intracellular pH in living cells. These features demonstrate that the probe can afford practical application in biological systems. (Figure Presented).

Full text of DOI:10.1021/acs.analchem.6b00422

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Article
Dual-site Controlled and Lysosome-targeted ICT-PET-FRET
Fluorescent Probe for Monitoring pH Changes in Living Cells
Baoli Dong, Xuezhen Song, Chao Wang, Xiuqi Kong, Yonghe Tang, and Weiying Lin
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00422 • Publication Date (Web): 08 Mar 2016
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Dual-site Controlled and Lysosome-targeted ICT-PET-FRET Fluo-
rescent Probe for Monitoring pH Changes in Living Cells
Baoli Dong, Xuezhen Song, Chao Wang, Xiuqi Kong, Yonghe Tang, and Weiying Lin*
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Institute of Fluorescent Probes for Biological Imaging, School of Chemistry and Chemical Engineering, School of Biological
Science, University of Jinan, Jinan, Shandong 250022, P. R. China. Fax: (+) 86ꢀ531ꢀ82769031, Eꢀmail: weiyingꢀ
lin2013@163.com
ABSTRACT: Acidic pH is a critical physiological factor for controlling the activities and functions of lysosome. Herein, we report
a novel dualꢀsite controlled and lysosomeꢀtargeted ICTꢀPETꢀFRET fluorescent probe (CNꢀpH), which was essentially the combinaꢀ
tion of a turnꢀon pH probe (CNꢀ1) and a turnꢀoff pH probe (CNꢀ2) by a nonꢀconjugated linker. Coumarin and naphthalimide fluoroꢀ
phores were selected as donor and acceptor to construct FRET platform. Hydroxyl group and morpholine were simultaneously emꢀ
ployed as the two pH sensing sites and controlled the fluorescence of coumarin and naphthalimide units by ICT and PET, respecꢀ
tively. The sensing mechanism of CNꢀpH to pH was essentially an integration of ICT, PET and FRET processes. Meanwhile, the
morpholine also can serve as a lysosomeꢀtargeted group. Combing the two data analysis approaches of the ratios of the two emisꢀ
sion intensities (R) and the reverse ratio R’ (R’=1/R), the fluorescent ratio of CNꢀpH can show proportional relationship to pH valꢀ
ues in a very broad range from pH 4.0 to 8.0 with high sensitivity. The probe has been successfully applied for the fluorescence
imaging of the lysosomal pH values, as well as ratiometrically visualizing chloroquineꢀstimulated changes of intracellular pH in
living cells. These features demonstrate the probe can afford practical application in biological systems.
sensitivity and sensing range of pH values, because the fluctuꢀ
ation of the lysosomal pH values is very small (generally less
than 2.0). For example, the variation amplitudes of the ratiꢀ
ometric values for the reported ratiometric pH probes are usuꢀ
ally less than 5 times in the lysosomal lumen pH 4.0ꢀ6.0.
Thus, there is a great need in designing ratiometric probes
with high sensitivity and broad sensing range of pH for moniꢀ
toring lysosomal pH values.
Fluorescence resonance energy transfer (FRET) has been
commonly exploited for the design of ratiometric fluorescent
probes relative to other sensing mechanisms including intraꢀ
molecular charge transfer (ICT) and photoꢀinduced electron
transfer (PET), because of its advantages in the high flexibility
of the design of FRETꢀbased probes and the wide choices of
fluorophores. 31ꢀ36 Currently, there are some FRETꢀbased fluoꢀ
rescent probes for monitoring cellular pH values have been
developed.28, 30, 37ꢀ40 However, these probes can be regarded as
■ INTRODUCTION
Intracellular pH is a critical physiological factor that relates
closely with many biological processes including cellular
apoptosis, proliferation and enzymatic activity.1ꢀ3 As an imꢀ
portant organelle for nearly all eukaryotic cells, lysosome
plays important roles in cellular apoptosis, immunologic deꢀ
fence, intracellular digestion and specialized secretory funcꢀ
tions.4ꢀ6 An acidic environment with pH in the range of 4.5ꢀ5.5
could facilitate activating the functions of the acidic hydrolasꢀ
es, cathepsins, nucleases, lipases and other enzymes in lysoꢀ
some.7ꢀ9 By contrast, abnormal lysosomal pH value could give
rise to dysfunctions, which are closely related to many diseasꢀ
es including cancer, shock and rheumatoid arthritis.10ꢀ13 Thus,
to precaution and diagnosis of these diseases, it is of great
important to realꢀtimely and precisely monitor the pH values
of lysosome in living system.
Fluorescence imaging is a great powerful approach for monꢀ
itoring cellular pH because of its numerous advantages, such
as highly sensitivity, imperial spatiotemporal resolution, conꢀ
venient operation, nondestructive test and realꢀtime detecꢀ
tion.14ꢀ19 Fluorescence intensityꢀbased probes are the pretty
common probes, and tend to be disturbed by experimental
conditions including probe environment and excitation intensiꢀ
ty. By comparison, ratiometric fluorescent probe, the other
important class of fluorescent probes, could alleviate aboveꢀ
mentioned problems by outputting ratiometric signal of the
fluorescence intensities at two emissions bands instead of sinꢀ
gle emission intensity.20ꢀ21 That is, the ratiometric probes can
enable the accurate detection of pH values in a quantitative
manner. So far, some ratiometric fluorescent probes that used
for imaging lysosomal pH values have been developed.22ꢀ30
However, these probes usually have obvious limitation in the
Scheme 1. Design strategies for the FRTE-based fluores-
cent probes
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the combination of a turnꢀon pH probe and a dye, and generalꢀ HeLa cells stained with 5 ꢁM CNꢀpH for 20 min at 37 °C were
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ly employ only one pH sensing site to provide the singleꢀsiteꢀ
dependent ratiometric fluorescence signal output by two sensꢀ
ing mechanism such as ICTꢀFRET, PETꢀFRET (Scheme 1a).
washed three times with PBS buffer, and then 100 ꢁM or 200
ꢁM chloroquine was added to stimulate cellular pH change.
Cell images were obtained using a Nikon A1R MP+ confocal
microscope.
Like FRET, ICT and PET are also the conventional sensing
41ꢀ43
mechanisms for developing fluorescent pH probes.
The
fluorescent probes that control fluorescence by multiꢀ
mechanism for generating changes in the emission waveꢀ
lengths can tend to provide diversiform fluorescent signals or
amplify the responding signals, which is beneficial for imꢀ
proving their sensitivity.44ꢀ50 Thus, the development of highly
sensitive fluorescent probe with multiꢀmechanism for monitorꢀ
ing lysosomal pH is still in demand.
In this work, we present a novel design strategy for developꢀ
ing lysosomeꢀtargeted ICTꢀPETꢀFRET fluorescent pH probe,
which can be regarded as the combination of a turnꢀon pH
probe and a turnꢀoff pH probe by a nonꢀconjugated linker
(Scheme 1b). Two pH sites were employed simultaneously to
control the optical properties of the two fluorophores by ICT
and PET, respectively. Meanwhile, the FRET process exists
between the two fluorophores. The pH probe based on this
novel design strategy possesses highly sensitivity and a very
broad pH detection range, and has been successfully applied
for monitoring lysosomal pH changes in living cells.
■ RESULTS AND DISCUSSION
Design Strategy of the Lysosome-targeted and ICT-
PET-FRET pH Probe. The general design strategy for FRET
probe allows the connection of a dye to a turnꢀon probe by a
nonꢀconjugated linker, and usually employs only one sensing
site (Scheme 1a). Our design strategy features a FRET probe
employing two sensing sites simultaneously to control the
fluorescence of the two fluorophores by ICT and PET, respecꢀ
tively (Scheme 1b). That is, the sensing mechanism of the
probe was essentially an integration of ICT, PET and FRET
processes. More importantly, the novel probe can be regarded
as the combination of a turnꢀon probe and a turnꢀoff probe by
a nonꢀconjugated linker, which is beneficial for enhancing the
range of their fluorescence intensity ratio logically.
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Based on this novel design strategy and our previous studies
on the FRET probes.30, 37 we constructed a novel dualꢀsite conꢀ
trolled and lysosomeꢀtargeted ICTꢀPETꢀFRET probe, CNꢀpH,
which was essentially the combination of the probes CNꢀ1 and
CNꢀ2 by a nonꢀconjugated linker (Figure 1). In the probe,
coumarin and naphthalimide fluorophores were selected to
constructed FRET platform for the two main reasons. Firstly,
the two fluorophores have significant advantages in optical
properties, such as excellent photostability, high molar absorpꢀ
tion coefficient and high fluorescence quantum yield. Secondꢀ
ly, due to the high overlap between the fluorescence of coumaꢀ
rin unit (donor) and the absorption of naphthalimide unit (acꢀ
ceptor), as well as the suitable distance (14.11 Å) between the
two fluorophores calculated by Gaussian 09 software, the
coumarinꢀnaphthalimide platform could possess remarkable
FRET efficiency. Hydroxyl and morpholine were employed as
the two pH sensing sites and combined with coumarin and
naphthalimide units, respectively. With the variation of the pH
from acidity to alkalinity, the probe CNꢀ1 exhibits turnꢀon
fluorescence for the enhancement of the ICT efficiency, while
the probe CNꢀ2 exhibits turnꢀoff fluorescence by the PET proꢀ
cess. In addition, the morpholine can also be served as lysoꢀ
someꢀtargeted group. Figure 1 shows the synthesis routes of
CNꢀpH, as well as CNꢀ1 and CNꢀ2 which can serve as the
building blocks for CNꢀpH chemically. The structures of the
■ EXPERIMENTAL SECTION
Materials and Instruments. Unless otherwise stated, all
reagents were purchased from commercial suppliers and used
without further purification. Twiceꢀdistilled water was used in
all the experiments. TLC analysis was performed on silica gel
plates and column chromatography was conducted over silica
gel (mesh 200ꢀ300, Qingdao Ocean Chemicals). Highꢀ
resolution electronspray mass spectra (HRMS) were obtained
from Bruker APEX IVꢀFTMS 7.0T mass spectrometer. 1H and
13C NMR spectra were recorded on AVANCE III 400 MHz
Digital NMR Spectrometer, using tetramethylsilane (TMS) as
internal reference respectively. The pH measurements were
carried out on a MettlerꢀToledo Delta 320 pH meter. UVꢀVis
absorption spectra were measured on a Shimadzu UVꢀ2600
spectrophotometer. Fluorescence spectra were recorded with a
HITACHI F4600 fluorescence spectrophotometer. PMT Voltꢀ
age was set at 400V or 700V for the fluorescence spectra. Data
were expressed as mean standard deviation (SD) of three sepaꢀ
rate measurements.
Theoretical Calculations. The energy levels of HOMO and
LUMO were determined by the density functional theory
(DFT) and timeꢀdependent density functional theory (TDꢀ
DFT) calculations using the Gaussian 09 software. The exꢀ
changeꢀcorrelation functional of B3LYP with Becke’s three
parameter form was adopted and the basis set of 6ꢀ31G(d) was
used in the calculations.
Cell Culture and Fluorescence Imaging. HeLa cells were
cultured in modified Eagle’s medium supplemented with 10%
calf bovine serum in an atmosphere of 5% CO2 and 95% air at
37 °C. Then cells were seeded into 35 mm glassꢀbottom culꢀ
ture dishes and cultured for 24h. For imaging at various pH,
the cells were incubated with 5 ꢁM CNꢀpH for 10 min at
37 °C, then the media was replaced with PBS buffer at various
pH. The cells were sequentially incubated with the PBS buffer,
10.0 ꢁM nigericin and 5.0 ꢁM monensin for another 30 min,
and then imaged using a Nikon A1R MP+ confocal microꢀ
scope. For imaging pH changes stimulated by chloroquine,
compounds were determined by HRMS, H and 13C NMR
spectra (Supporting Information).
1
pH-dependent Photoproperties of CN-1 and CN-2. Iniꢀ
tially, we determined the pHꢀdependent photoproperties of
CNꢀ1 and CNꢀ2 in BrittonꢀRobinson (BꢀF) buffers. Under
acidic condition (pH = 3.5), CNꢀ1 had an absorption maxiꢀ
mum at 345 nm with molar absorption coefficient (ε) of 2.96 ×
104 molꢀ1.cmꢀ1 (Figure S1). With the pH changing from 3.5 to
9.5, the absorption peaks of CNꢀ1 located in the range of 320ꢀ
370 nm exhibited obvious decrease of absorbance. When the
pH exceeded 5.5, CNꢀ1 showed new absorption bands located
at 400 nm. At pH 3.5ꢀ9.5, CNꢀ2 showed almost same absorbꢀ
ance located at about 430 nm with ε of about 2.02 × 104 molꢀ
1.cmꢀ1 in the visible region. When excited, CNꢀ1 showed emisꢀ
sions at 454 nm and CNꢀ2 showed emissions at 530 nm (Figꢀ
ure S2). As shown in Figure 2a, the overlap between the abꢀ
sorption spectra of CNꢀ2 and the fluorescence spectra of CNꢀ1
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Figure 1. Synthesis routes of CNꢀ1, CNꢀpH and CNꢀ2. (a) tꢀbutyl piperazineꢀ1ꢀcarboxylate, EDCl, HOBt, DIEA, DMF, rt, 12 h; (b)
CF3COOH, CH2Cl2, 2 h; (c) propionic acid, EDCl, HOBt, DIEA, DMF, rt, 12 h; (d) 3ꢀaminopropanoic acid, ethanol, reflux, 3 h; (e) 2ꢀ
morpholinoethanamine, Pd(OAc)2, BINAP, DMSO, Cs2CO3, 80 °C, 8 h; (f) compound 2, EDCl, HOBt, DIEA, DMF, rt, 12 h; (g) propanꢀ
1ꢀamine, ethanol, reflux, 3 h; (h) 2ꢀmorpholinoethanamine, Pd(OAc)2, BINAP, DMSO, Cs2CO3, 80°C, 8 h.
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was significant and beneficial for the FRET from the excitaꢀ
tion state of CNꢀ1 to the ground state of CNꢀ2. More imꢀ
portantly, CNꢀ1 showed turnꢀon fluorescence with the pH
changing from 3.0 to 10.0, while CNꢀ2 showed turnꢀoff fluoꢀ
rescence (Figure 2b). In addition, the pKa values of CNꢀ1 and
CNꢀ2 were calculated to 6.46 and 6.93 using the Hendersonꢀ
Hasselbalch equation. Therefore, the probes CNꢀ1 and CNꢀ2
could probably be combined into a dualꢀsite controlled pH
probe with high sensitivity and broad sensing range of pH.
Subsequently, the sensing mechanisms of CNꢀ1 and CNꢀ2
to pH were explored. When pH exceeds 5.5, the hydroxyl
group of CNꢀ1 can ionize a proton to generate negative oxyꢀ
gen ion, consisting with the new absorption located at 400 nm
at pH 5.5ꢀ9.5 (figure S1). Because the negative oxygen ion
possesses stronger electronꢀdonating ability than hydroxyl, the
ionization of hydroxyl is essentially beneficial for enhancing
the ICT efficiency. Thus, CNꢀ1 can show obvious turnꢀon
fluorescence with the increase of pH values. For CNꢀ2, the
Gaussian calculations showed that the HOMO energy of 4ꢀ
ethylmorpholine (ꢀ5.40 eV) is larger than the HOMO energy
of 4ꢀaminonaphthalimide fluorophore (ꢀ5.74 eV), and smaller
than its LUMO energy (ꢀ2.18 eV). It suggests that the reducꢀ
tive PET (aꢀPET) from morpholine to fluorophore occurs
when the fluorophore is excited, and quenches the fluoresꢀ
cence of 4ꢀaminonaphthalimide fluorophore (Figure S3). At
acidic condition, the protonation of amino at morpholine
blocks the aꢀPET process and enables the naphthalimide fluorꢀ
ophore to emit fluorescence. Thus, CNꢀ2 can show obvious
turnꢀoff fluorescence with the increase of pH values. Taken
together, the hydroxyl group and morpholine can be served as
the response sites of CNꢀ1 and CNꢀ2 to pH, and control the
fluorescence of coumarin and naphthalimide fluorophores by
ICT and PET mechanisms, respectively.
Optical Response of CN-pH to pH. UVꢀVis spectra of
CNꢀpH at various pH values are shown in Figure 3a. Comꢀ
pared with the aboveꢀmentioned absorption spectra of CNꢀ1
Figure 2. (a) Normalized absorption spectra (abs.) and fluoresꢀ
cence spectra (flu.) of 5 ꢁM CNꢀ1 and CNꢀ2 at pH 5.0 in BꢀF
buffers (5% MeOH). The gray area represented the overlap beꢀ
tween the fluorescence spectrum of CNꢀ1 and the absorption specꢀ
trum of CNꢀ2. (b) Normalized fluorescence intensity of 5 ꢁM CNꢀ
1 (λem = 454 nm) and CNꢀ2 (λem = 530 nm) at various pH in BꢀF
buffers (5% MeOH). Fluorescence spectra for CNꢀ1, λex = 380 nm;
CNꢀ2, λex = 410 nm.
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Figure 4. Fluorescence intensity ratios I530/I454 (a) and I454/I530 (b)
of 5 ꢁM CNꢀpH in BꢀF buffers (5% MeOH) at various pH. Insets
Figure 3. Absorption spectra (a) and fluorescence spectra (b, λex =
380 nm) of 5 ꢁM CNꢀpH in BꢀF buffers (5% MeOH) at various
pH values.
were the linear plots of fluorescence intensity ratio to pH. λex
380 nm.
=
The fluorescence response mechanism of CNꢀpH to pH was
further investigated on the basic of the pHꢀdependent fluoresꢀ
cence spectra of CNꢀ1, CNꢀ2, as well as the mixture of CNꢀ1,
CNꢀ2 in BꢀF buffers. Obviously, the blue emission located at
454 nm of CNꢀpH arose from coumarin unit and the green
emission located at 530 nm can be ascribed to naphthalimide
unit. Excited at 380 nm, the mixture of CNꢀ1, CNꢀ2 in BꢀF
buffers showed mainly blue emission arose from coumarin
unit because the UV light at 380 nm was primarily absorbed
by coumarin unit instead of naphthalimide unit (Figure S4 and
S5). By contrast, CNꢀpH can show relative strong green emisꢀ
sion arose from naphthalimide unit when excited at 380 nm,
indicating the FRET process from coumarin to naphthalimide
unit substantially occurred (Figure 3b). According to the theoꢀ
retical calculations using MarvinSketch software, three main
forms of CNꢀpH existed at pH 3.0ꢀ10.0 due to the ionization
of hydroxyl and the protonation of amino group at morpholine
(Figure 5 and Figure S6). In form a, the inhibition of the aꢀ
PET process enabled the naphthalimide fluorophore to emit
fluorescence, and the FRET process from coumarin to naphꢀ
thalimide occurred simultaneously. The aꢀPET process can
happen in forms b and c, while the ICT proceeded with the
ionization of hydroxyl in from a. That is, forms a, b and c can
emit strong green fluorescence, weak blue fluorescence and
strong blue fluorescence, which consisted well with the fluoꢀ
rescence photographs of CNꢀpH at pH 4.5, 6.5 and 6.5 (Figure
5). Therefore, the hydroxyl and morpholine can be served as
the two pH response sites of CNꢀpH to synergistically control
its fluorescence properties at various pH, and the response
mechanism of CNꢀpH to pH was essentially an integration of
ICT, PET and FRET processes.
and CNꢀ2, the absorption bands of CNꢀpH peaked at about
345 nm and 400 nm arose from coumarin moiety, while the
absorption bands located at 257 nm, 280 nm and 430 nm can
be ascribed to naphthalimide unit. That is, the absorption of
CNꢀpH at various pH values was thoroughly superposed by
the absorption of CNꢀ1 and CNꢀ2, indicating that there is no
interaction (mainly πꢀπ interaction) between the coumarin and
naphthalimide fluorophores in CNꢀpH molecule.
We then explored the fluorescence response of CNꢀpH to
pH by determining the fluorescence spectra of CNꢀpH at variꢀ
ous pH in BꢀF buffers. At acidic condition (pH = 3.0), CNꢀpH
showed weak blue emission at 454 nm and strong green emisꢀ
sion at 530 nm (Figure 3b). When the pH changed from 3.0 to
10.0, the blue emission increased and the green emission deꢀ
creased gradually. The ratios of fluorescence intensities R
(I530/I454) showed a significant change (up to 58ꢀfold) from 0.5
at pH 7.4 to 29.3 at pH 4.0, overlapping the lysosomal pH
(4.5ꢀ5.5) and normal physiological pH range (6.2ꢀ7.4) and
indicating CNꢀpH is suitable for assessing acidic media in
lysosome. At pH 4.0ꢀ6.5, the ratio value R (I530/I454) was lineꢀ
arly proportional to pH (Figure 4a). When the data were treatꢀ
ed with the reverse ratio of R’ (R’ = 1/R = I454/I530), R’ can
show an obvious enhancement (up to 15ꢀfold) and linearly
proportional to pH 6.0ꢀ8.0 (Figure 4b). Namely, combing the
two data analysis approaches of R and R’, the fluorescent ratiꢀ
ometric values of CNꢀpH can show proportional relationship
to pH in a very broad range from pH 4.0 to pH 8.0, which was
obviously broader than that of the reported fluorescent pH
probe. Therefore, CNꢀpH can be used for the precise pH
measurement from pH 4.0 to pH 8.0 in a ratiometric manner,
and has potential capability of sensing lysosomal pH changes
in living system.
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Figure 5. Proposed sensing mechanism and the fluorescence photographs of 5 ꢁM CNꢀpH at pH 4.5, 6.5 and 8.5 under 365 nm.
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To evaluate the specificity of CNꢀpH, the fluorescence
spectra of CNꢀpH were determined coexisting with biologicalꢀ
ly relevant species at various pH. We selected a series of ions
and small biomolecules that commonly exist in living systems
as potential interfering substances, including Ca2+, Mg2+, Cu2+,
H2O2, Cys, GSH, H2S, SO2 and NO. Taking into consideration
of the lysosomal pH at 4.5ꢀ5.5 and normal physiological pH at
about 7.4, we chose three test systems at pH 4.5, 5.5 and 7.4.
The ratios (I530/I454) had no significant changes after adding the
various ions or small biomolecules at pH 4.5, 5.5 and 7.4
(Figure S7). Therefore, CNꢀpH can potentially monitor the
lysosomal pH changes in living system.
The timeꢀdependent ratios (I530/I454) of CNꢀpH were also deꢀ
termined at pH 4.5, 5.5 and 7.4. As shown in Figure S8, when
CNꢀpH was added into the solutions with difference pH, the
ratios (I530/I454) reached equilibrium in a short time (less than
1min). In addition, we also studied the reversibility of CNꢀpH
in solutions at pH 5.5 and 7.4. The results suggested that the
process can be reversibly performed for at least six cycles
(Figure S9). Therefore, CNꢀpH was can show realꢀtime and
reversible fluorescence response to pH.
experiments were performed in HeLa cells using CNꢀpH and a
known lysosomeꢀspecific fluorescent probe, LysoTrackꢀ
er Red®, to determine the presumed lysosomeꢀtarget property
of the probe. The HeLa cells were incubated with CNꢀpH (5.0
ꢁM) for 10 min at 37 °C, and then medium was replaced with
fresh medium containing LysoTracker Red® (1.0 ꢁM) and the
cells were incubated for another 10 min. As shown in Figure 6,
the cells showed blue and green fluorescence from CNꢀpH,
and red fluorescence from LysoTracker Red® simultaneously.
Because CNꢀpH can emit stronger green fluorescence relative
to blue fluorescence at pH 7.4, the overlap between the green
and red fluorescence images was determined to evaluate the
lysosomeꢀtargeted property of the probe. The Pearson’s correꢀ
lation coefficient between green and red fluorescence images
was calculated to 0. 82, confirming the probe distributed mainꢀ
ly in the lysosomes and possessed lysosomeꢀtargeted property.
In addition, MTT assay indicated that CNꢀpH has no marked
toxicity to HeLa cells below 10ꢁM (Figure S10).
We then utilize CNꢀpH to estimate cellular pH values in a
ratiometric manner. The cells were incubated with 5.0 ꢁM
CNꢀpH for 10 min at 37 °C, then the media was replaced with
buffers at pH 4.0, 5.0, 6.0, 7.0 or 8.0. The cells were sequenꢀ
tially incubated with the buffer, 10.0 ꢁM nigericin and 5.0 ꢁM
monensin for another 30 min.23, 28 As shown in Figure 7a, the
blue fluorescence showed obvious enhancement gradually
with pH varing from 4.0 to 8.0, while the green fluorescence
was was decreased gradually. Meanwhile, when quantified
using Nikon NIS Element software, the blue and green
fluorescence intensity showed turuꢀon and turnꢀoff behaviors,
respectively (Figure 7b). The ratio of R (Igreen/lblue) also
exhibited significant changes from pH 4.0 (R = 3.48 ± 0.21) to
8.0 (R = 0.88 ± 0.11), consisting with the aboveꢀmentioned
response of CNꢀpH to pH in BꢀF buffers. The statistical
Fluorescence Imaging in Living Cells. Encouraged by the
desirable pHꢀdependent spectral properties of CNꢀpH, we then
examined the biological applications of the probe for monitorꢀ
ing pH changes in living cells. Initially, colocalization
significance results showed that the quantified ratio (Igreen/lblue
)
at pH 4.0 exhibited an obvious difference from that at pH 6.0
(R = 2.28 ± 0.20) with p < 0.05, and a significant difference
from that at pH 7.0 (R = 1.17 ± 0.16) and 8.0 with p < 0.01
(Figure 7c). Taken together, CNꢀpH can show desirable sensiꢀ
tivity in monitoring cellular pH values. To the best of our
knowledge, CNꢀpH is the first pH probe based on ICTꢀPETꢀ
FRET mechanism that can be used in the fluorescence imagꢀ
ing of cellualar pH.
Subsequently, CNꢀpH was employed to ratiometrically
visualize the chloroquineꢀstimulated intracellular pH changes
in living HeLa cells. Chloroquine is a lysosomotropic agent
that can inhibit autophagy and protein degradation by raising
the lysosomal pH.51 Before the treatment of chloroquine, the
cells showed fluorescence in both blue and green channels
with the fluorescence intensity ratio (Igreen/lblue) of 2.50 ± 0.13
Figure 6. Colocalization experiments of CNꢀpH and LysoTrackꢀ
er Red® in HeLa cells. (a) blue fluorescence images for CNꢀpH
(425ꢀ475 nm) with excitation at 405 nm; (b) green fluorescence
images for CNꢀpH (500ꢀ550 nm) with excitation at 488 nm; (c)
red fluorescence images for LysoTracker Red® (570ꢀ620 nm) with
excitation at 561 nm; (d) overlay of (a), (b) and (c).
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Figure 7. (a) Fluorescence images for 5 ꢁM CNꢀpH in HeLa cells at various pH. Blue channel, emission at 425ꢀ475 nm with excitation at
405 nm. Green channel, emission at 500ꢀ550 nm with excitation at 488 nm. Ratio images represent the ratio of fluorescence intensity beꢀ
tween green channel and blue channel (Igreen/Iblue). (b) Quantified relative fluorescence intensity at various pH was analyzed using Nikon
NIS Element software and presented as mean ± s.d with, n = 3. (c) Ratio of Igreen/Iblue at various pH obtained from (b). Statistical analyses
were performed with Student’s tꢀtest (n = 3). * P < 0.05, *P < 0.01 and error bars are ± s.d.
Figure 8. (a) Fluorescence imaging of lysosomal pH changes in HeLa cells stimulated with 100 ꢁM and 200 ꢁM chloroquine. Blue channel,
emission at 425ꢀ475 nm with excitation at 405 nm. Green channel, emission at 500ꢀ550 nm with excitation at 488 nm. (b) Quantified relaꢀ
tive fluorescence ratio of Igreen/Iblue of images analyzed using Nikon NIS Element software and presented as mean ± s.d, n = 3.
(Figure 8a and 8b). According to the aboveꢀmentioned ratios
(Igreen/lblue) of CNꢀpH in living HeLa cells (Figure 7c), the lysoꢀ
somal pH value of the cells untreated with chloroquine was
calculated to be about 5.5, which is in the normal lysosomal
pH range of 4.5 ꢀ 5.5. After the treatment with 100 ꢁM chloroꢀ
quine for 20 min, the cells exhibited slightly increased blue
and decreased green fluorescence with the fluorescence intenꢀ
sity ratio (Igreen/lblue) of 1.78 ± 0.15 corresponding to the pH of
about 6.4. Under the same conditions, when the cells were
treated with higher concentration of chloroquine (200 ꢁM), the
cells can show relative obvious decreased green fluorescence
with the fluorescence intensity ratio (Igreen/lblue) of 1.50 ± 0.17
corresponding to the pH of about 6.6. These results indicate
that CNꢀpH can be applied for monitoring the chloroquineꢀ
induced lysosomal pH changes within a small range.
■ CONCLUSIONS
In summary, we have developed a novel dualꢀsite controlled
and lysosomeꢀtargeted ICTꢀPETꢀFRET fluorescent probe
(CNꢀpH) for monitoring lysosomal pH values in living cells.
The probe adopted coumarin and naphthalimide fluorophores
as donor and acceptor to construct FRET platform, as well as
employed hydroxyl and morpholine simultaneously as the two
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Analytical Chemistry
(12) Zhang, R. G.; Kelen, S. G.; Lamanna, J. C. J. Appl. Phys.1990,
pH sensing sites to control the fluorescence of coumarin and
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8
68, 1101ꢀ1106.
naphthalimide by ICT and PET, respectively. With the pH
varying from 3.0 to 10.0, the coumarin unit showed blue turnꢀ
on fluorescence at 454 nm while the naphthalimide unit exhibꢀ
ited green turnꢀoff fluorescence at 530 nm. Unꢀ
der the synergistic effects of ICT, PET and FRET, CNꢀpH can
show excellent response to pH with high sensitivity. Combing
the two data analysis approaches of R and the reverse ratio R’,
the fluorescent ratios of CNꢀpH can show proportional relaꢀ
tionship to pH values in a broad range from pH 4.0 to pH 8.0,
which obviously exceeded that of the developed FRETꢀbased
pH probe. CNꢀpH had no marked toxicity to living cells and
possessed desirable lysosomeꢀtargeted property. CNꢀpH has
been successfully applied for the fluorescence imaging of the
lysosomal pH values, as well as ratiometrically visualizing
chloroquineꢀstimulated changes of intracellular pH in living
cells. These features demonstrate the probe can afford practiꢀ
cal application for monitoring pH changes in pathogenic cells.
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ASSOCIATED CONTENT
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Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
Synthesis of the probes, absorption and fluorescence spectra, theꢀ
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1
oretical calculations, H and 13 C NMR spectra. (PDF)
AUTHOR INFORMATION
Corresponding Author
Fax: (+) 86ꢀ531ꢀ82769031, Eꢀmail: weiyinglin2013@163.com
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This work was financially supported by NSFC (21172063,
21472067), Doctoral Fund of University of Jinan (160082104),
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Shandong
Provincial
Natural
Science
Foundation
(ZR2014BP002), and the startup fund of University of Jinan. We
also thank the Special Foundation for Taishan Scholar Professorꢀ
ship of Shandong Province (TS 201511041).
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