A new fluorescent pH probe for imaging lysosomes in living cells

Article abstract of DOI:10.1016/j.bmcl.2013.12.025

A new rhodamine B-based pH fluorescent probe has been synthesized and characterized. The probe responds to acidic pH with short response time, high selectivity and sensitivity, and exhibits a more than 20-fold increase in fluorescence intensity within the pH range of 7.5-4.1 with the pK_a value of 5.72, which is valuable to study acidic organelles in living cells. Also, it has been successfully applied to HeLa cells, for its low cytotoxicity, brilliant photostability, good membrane permeability and no 'alkalizing effect' on lysosomes. The results demonstrate that this probe is a lysosome-specific probe, which can selectively stain lysosomes and monitor lysosomal pH changes in living cells.

Full text of DOI:10.1016/j.bmcl.2013.12.025

Bioorganic & Medicinal Chemistry Letters 24 (2014) 535–538
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A new fluorescent pH probe for imaging lysosomes in living cells
a,
Hong-Shui Lv a, , Shu-Ya Huang b, , Yu Xu a, Xi Dai a, Jun-Ying Miao b, , Bao-Xiang Zhao
⇑
⇑
a Institute of Organic Chemistry, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, PR China
b Institute of Developmental Biology, School of Life Science, Shandong University, Jinan 250100, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A new rhodamine B-based pH fluorescent probe has been synthesized and characterized. The probe
responds to acidic pH with short response time, high selectivity and sensitivity, and exhibits a more than
20-fold increase in fluorescence intensity within the pH range of 7.5–4.1 with the pKa value of 5.72, which
is valuable to study acidic organelles in living cells. Also, it has been successfully applied to HeLa cells, for
its low cytotoxicity, brilliant photostability, good membrane permeability and no ‘alkalizing effect’ on
lysosomes. The results demonstrate that this probe is a lysosome-specific probe, which can selectively
stain lysosomes and monitor lysosomal pH changes in living cells.
Received 25 September 2013
Revised 2 December 2013
Accepted 6 December 2013
Available online 12 December 2013
Keywords:
Rhodamine B
Fluorescent probe
pH chemodosimeter
Lysosomes imaging
Ó 2013 Elsevier Ltd. All rights reserved.
Intracellular pH plays a critical role in many cellular events,
such as cell growth,1,2 apoptosis,3 autophagy,4 signal transduction5
and proliferation.6 Abnormal intracellular pH values indicate
abnormal cell events and are observed in some diseases including
cancer7 and Alzheimer’s disease.8 The pH value of the cytoplasma
is about 7.2, while some organelles, such as endosomes and
lysosomes, have acidic microenvironment 4.0–6.09,10 which can
facilitate the degradation of proteins in cellular metabolism. Thus
critical information of physiological and pathological processes
can be provided by accurately measuring intracellular pH values.
Fluorescence detection has been widely used because of its high
sensitivity, simple operation and real-time monitoring. The bind-
ing of analytes to probe causes fluorescence enhancement (‘turn
on’) or fluorescence quenching (‘turn off’). Fluorescent quenching
probes may provide incorrect results because quenching can be
caused by other quenchers in real samples.11,12 Therefore, it is
desirable to develop fluorescence-enhanced probes. Some small
molecular fluorescent probe for pH have been reported.13–18 Rho-
damine derivatives with a spirocyclic structure are non-fluorescent
and colorless, while the ring-opening of spirocyclic structure gives
a strong fluorescence emission and a pink color. This makes
rhodamine derivatives serve as excellent ‘turn on’ fluorescent
probes. In recent years, a large number of rhodamine-based fluo-
rescent probes for metal ions and biologically relevant species have
been developed.19,20 The spirocyclic structure is also sensitive to
the pH of the solutions. Under basic conditions, it remains the
spirocyclic form that is non-fluorescent and colorless. While in
acidic solutions, the ring-opened form performs strong fluores-
cence and pink color. Although some rhodamine-based fluorescent
pH probes have been reported, only few are applied for cell imag-
ing.21–23 In order to take advantage of the excellent photophysical
and structural properties of rhodamine derivatives, it is necessary
to design new pH probes which are suitable for cell imaging.
As a continuation of our work on the fluorescent probes for
metal ions and pH,24–27 here, we designed and synthesized a new
rhodamine B-based probe (RB-DFP) for detecting H+. The probe
with a pKa value of 5.72 is suitable for studying acidic organelles
in living cells.
Chemistry: Probe RB-DFP was readily synthesized from rhoda-
mine B hydrazide (4) and 2-(2-formylphenoxy) acetic acid (3) in
EtOH under reflux (Scheme 1). The structure of RB-DFP was con-
firmed by 1H NMR, 13C NMR, IR and HRMS. A solution of RB-DFP
in EtOH/HEPES (pH 7.2) is colorless and non-fluorescent, indicating
⇑
Corresponding authors. Tel.: +86 531 88366425; fax: +86 531 88564464.
E-mail addresses: miaojy@sdu.edu.cn (J.-Y. Miao), bxzhao@sdu.edu.cn (B.-X.
Zhao).
Equal contribution.
Scheme 1. Synthesis of compound RB-DFP.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.12.025

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H.-S. Lv et al. / Bioorg. Med. Chem. Lett. 24 (2014) 535–538
that RB-DFP has the spirolactam form only. The characteristic peak
of the spiro carbon in the 13C NMR spectrum at 66.6 ppm also
supports this consideration.28
(Fig. S2a). Furthermore, RB-DFP exhibits a good reversibility
between pH 4.2 and pH 7.5 (Fig. S2b). Thus, the probe can monitor
real-time pH changes with very short response time, which is one
of the merits of it.
As Figure 1a shows, RB-DFP is nearly non-fluorescent at pH 7.5.
However, a strong fluorescence enhanced signal appeared at
589 nm and increased drastically caused by the H+-induced ring
opening of spirolactam (Scheme 2) when the pH decreased to 4.1
from 7.5. Meanwhile, the fluorescence quantum yield (UF) of
RB-DFP at pH = 4.2 reached 0.39, using rhodamine B (UF = 0.69
in ethanol) as a standard.29 Moreover, the fluorescence titration
data yielded a pKa of 5.72 (Fig. 1b), indicating that the detection
range of RB-DFP can cover both normal and abnormal lysosome
pH. Also, the trends of absorption spectra depending on pH are
in agreement with emission spectra (Fig. S1).
The time course study showed that the fluorescence intensity of
RB-DFP can reach the maximum in about 1 min in acidic solution
Figure 1. (a) Fluorescence spectra of RB-DFP (10 lM) in aqueous solution (Britton–
Figure 2. Fluorescence microscope images of living HeLa cells with different
concentrations of RB-DFP for 3–12 h at 37 °C.
Robinson buffer–EtOH, 9:1, v/v) with different pH; (b) pH titration curve of RB-DFP,
kex = 563 nm, kem = 589 nm. The inset shows the linear relationship of fluorescence
intensity at 589 nm and varying pH values from 4.6 to 6.5.
Figure 3. Fluorescence microscope images of living HeLa cells co-stained with
1 lM RB-DFP and 0.25 lM LysoSensor green. (a) Red emission from RB-DFP; (b)
green emission from LysoSensor green; (c) overlay of (a) and (b), areas of co-
Scheme 2. Detection mechanism of RB-DFP for pH.
localization appear in yellow.

H.-S. Lv et al. / Bioorg. Med. Chem. Lett. 24 (2014) 535–538
537
Figure 4. (A) Fluorescence microscope images of living HeLa cells co-incubated with 0, 40, 80 and 100 nM bafilomycin A1 (BafA1)and 0.5
lM RB-DFP for 12 h at 37 °C. (B)
Fluorescence intensity quantitation was analyzed using ImageJ image analysis tool. Data are mean SEM. ⁄p < 0.05, ⁄⁄p < 0.01 versus BafA1 0
lM, n = 6.
In order to detect lysosomal pH changes, the pH-responsive
behavior of RB-DFP should not be interfered by complex intracel-
lular environment. As shown in Figure S3, high concentrations of
abundant cations in cells (K+, Na+, Ca2+, Mg2+) and biologically
relevant species (Gly, Phe, Cys, Pro, Ser, vitamin C, H2O2 and glu-
cose) had almost no influence on the fluorescence. The addition
of Cu2+ quenched about 50% of the fluorescence emission, while
Zn2+, Co2+ and Al3+ only caused slight fluorescence quenching. Con-
sidering that the concentrations of these metal ions in intracellular
environment are far lower than those used for the experiment, the
quenching effects of these metal ions can be ignored. All the results
demonstrate that RB-DFP has great potential as a pH fluorescent
probe for real-time monitoring in practical application.
(0.5 lM) for 12 h. With increased concentration of bafilomycin
A1, the fluorescence intensities within the cells gradually weak-
ened (Fig. 4). Thus, RB-DFP can serve as a fluorescent indicator
for lysosomal pH and monitor lysosomal pH changes in living cells.
Cytotoxicity of RB-DFP has also been measured. HeLa cells were
incubated with 5 lM RB-DFP for 12 h. The cell viability was
detected by MTT assays. The results demonstrate that RB-DFP is
non-cytotoxicity to HeLa cells at the experimental conditions
(Fig. S4).
In summary, we have described the synthesis, characterization
and cellular applications of a novel rhodamine B-based probe RB-
DFP for pH. The fluorescence intensity of the probe is sensitive to
acidic pH in very short response time with a pKa value of 5.72. Also,
the probe can be used as a lysosomal pH probe to selectively stain
lysosomes in living cells with low cytotoxicity, excellent photosta-
bility and good cell membrane permeability. Therefore, we believe
this probe is able to be used in reality with potential biological
significance.
Fluorescence imaging in living cells: To investigate the potential
application of RB-DFP for fluorescence imaging in living cells, HeLa
cells were incubated with different concentrations (1, 3 and 5 lM)
of RB-DFP for 3–12 h. As shown in Figure 2, RB-DFP exhibits
excellent cell membrane permeability and staining ability in living
cells. The different fluorescence intensities within single cells dem-
onstrate that intracellular pH is not uniformly distributed. More-
over, the fluorescence intensities within cells increase in
concentration and time dependent manner. To further confirm
the subcellular distribution of RB-DFP, a commercially available
lysosome-specific staining probe LysoSensor Green DND-189 was
used to co-stain HeLa cells with RB-DFP. HeLa cells were first incu-
Acknowledgments
This study was supported by 973 Program (2010CB933504) and
National Natural Science Foundation of China (20972088 and
90813022).
Supplementary data
bated with 1 lM RB-DFP for 3 h, then with LysoSensor Green for
30 min. Confocal fluorescent microscopic images showed that the
distribution of red fluorescence from RB-DFP colocalized with
green fluorescence from LysoSensor Green, suggesting that RB-
DFP selectively stain lysosomes in living cells (Fig. 3). Moreover,
lysosomal pH changes in HeLa cells induced by bafilomycin A1
were detected by the probe. Bafilomycin A1 is a selective inhibitor
of the vacuolar-type H+-ATPase (V-ATPase), which can inhibit
lysosomal acidification to increase lysosomal pH. HeLa cells were
incubated with bafilomycin A1 (40, 80 and 100 nM) and RB-DFP
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.12.025.
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