A self-calibrating phosphorescent polymeric probe for measuring pH fluctuations in subcellular organelles and the zebrafish digestive tract

Article abstract of DOI:10.1039/c9tc06285h

The precise regulation of pH in biological systems, including intracellular organelles and tissues, is essential to the normal functions of vital movement. Accurate pH determination will contribute to a better understanding of related physiological processes and the effective monitoring of physiologic derangements. Luminescence imaging and sensing are useful tools for biological study. However, the conventional intensity-based detection method suffers from low signal-to-noise ratio. Herein, we report a water-soluble ratiometric phosphorescent probe (P-pH) for measuring pH fluctuations in biological samples based on ratiometric photoluminescence imaging and photoluminescence lifetime imaging. P-pH consists of a pH-responsive iridium(iii) complex, pH-inert iridium(iii) complex, and hydrophilic poly(N-vinyl-2-pyrrolidone). Owing to its dual emission, P-pH was successfully used to monitor the pH variation in mitochondria and lysosomes based on the ratiometric readout. Using P-pH, the reliable evaluation of three types of pH modifiers in the zebrafish digestive tract was also realized with a distinguishable long emission lifetime. This is the first example of assessing pH modifiers in real animal models using a luminescent approach.

Full text of DOI:10.1039/c9tc06285h

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Nosang V. Myung, Yong-Ho Choa et al.
A
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DOI: 10.1039/C9TC06285H
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Self-calibrating phosphorescent polymeric probe for measuring
pH fluctuation in subcellular organelles and zebrafish digestive
tract†
Received 00th January 20xx,
Accepted 00th January 20xx
Zejing Chen,‡a,b Xiangchun Meng,‡a Mingjuan Xie,a Yuxiang Shi,a Liang Zou,a Song Guo,a Jiayang
Jiang,a Shujuan Liu*a and Qiang Zhao*a
DOI: 10.1039/x0xx00000x
Precise regulation of pH in the biological systems including intracellular organelles and tissues is essential to the normal
functions of vital movement. Accurate pH determination will contribute to better understanding related physiological
processes and effectively monitor physiologic derangements. The luminescence imaging and sensing is a useful tool in
biological study, but the conventional intensity-based detection method often suffers results with low signal-to-noise ratio.
Here we reported a water-soluble ratiometric phosphorescent probe (P-pH) for measuring pH fluctuation in biological
samples via ratiometric photoluminescence imaging and photoluminescence lifetime imaging. P-pH is consisted of a pH-
responsive iridium(III) complex, pH-inert iridium(III) complex and hydrophilic poly(N-vinyl-2-pyrrolidone). Owing to its dual-
emission, P-pH was used successfully for monitoring pH variation in mitochondria and lysosomes by ratiometric readout.
Besides, reliable evaluation of three kinds of pH-modifying in zebrafish digestive tract was realized with the help of P-pH
featuring distinguishable long emission lifetime, which is the first example of pH-modifying assessment in real animal model
through luminescent approach.
released as desired. Therefore, effective pH measurements in
digestive tract of living animals will contribute to the evaluation
of pH-modifying excipients and promote the development of
Introduction
pH is one of the most important physiological indices related to
cellular behaviours and pathological states. It regulates various
physiological processes, such as enzyme reaction, ion transport,
drug excretion, cell proliferation and apoptosis.1-5 It is beneficial
to detect pH variation in living cells, especially in organelles
whose pH values need to be kept in a certain range for specific
process.6 Not only testing intracellular pH, but also monitoring
extracellular pH within living samples is significant to
understand biological events. For example, accurate
evaluations of pH-adjusting effect for pH modifiers towards
digestive juices exert positive effects on drug research and
development.7 This is because there are major challenges in
preparing weakly acidic or basic drugs: they are characteristic
with pH-dependent solubility and release. pH-modifying
excipients are commonly mixed into the drug formulation to
modulate pH of the dissolution media, altering the pH-
dependence of drug dissolution, guaranteeing that drugs can be
pharmaceutical preparations.
Luminescence imaging is a popular way to monitor pH in vitro
and in vivo because of its good selectivity, non-invasiveness and
high sensitivity.8-12 Furthermore, possessing super high spatial
resolution, it can be used for real-time detection of analytes at
subcellular levels.13,14 However, it often suffers from
background fluorescence interference due to its technological
characteristics, especially when it is applied to sensing and
imaging in vivo.15 Therefore, in vivo imaging with high signal to
noise ratio (SNR) puts forward higher requirements for
bioprobes. Developing probes with long-lived emission is
gaining popularity with the promotion of imaging technique
which can analyse luminescence signal in the time dimension
and highlight the luminescence of probes even in the complex
environment.15-17
In this work, we reported a ratiometric and long-lived
phosphorescent polymeric probe (P-pH) for pH sensing in vitro
and in vivo via ratiometric photoluminescence imaging (RPIM)
and photoluminescence lifetime imaging (PLIM).18-20 On the one
hand, this approach can avoid the problems caused by
uncertain concentration of probe and fluctuation of excitation
power which frequently occur during the intensity-based
detection, achieving accurate pH determination in subcellular
organelles; on the other hand, it can minimize interferences of
autofluorescence by taking advantage of the long-lived
emission of the phosphorescent probe, thus realizing pH
sensing in zebrafish digestive tract.
a. Key Laboratory for Organic Electronics and Information Displays & Jiangsu Key
Laboratory for Biosensors, Institute of Advanced Materials (IAM), Nanjing
University of Posts & Telecommunications, 9 Wenyuan Road, Nanjing 210023, P.
R. China. E-mail: iamsjliu@njupt.edu.cn, iamqzhao@njupt.edu.cn.
b. Jiangxi Key Laboratory for Nano-Biomaterials, Institute of Advanced Materials
(IAM), East China Jiaotong University, 808 Shuanggang East Main Street,
Nanchang 330013, P. R. China.
† Electronic Supplementary Information (ESI) available: [Experimental information
and procedures, synthesis, characterization, computational details, and additional
tables and figures]. See DOI: 10.1039/x0xx00000x
‡ Chen and Meng contributed equally to this work.
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Scheme 1 (a) Chemical structures of Ir1, Ir2, Ir3 and Ir4. (b) Schematic illustration of polymetric probe P-pH for pH.
(y-intercept) was derived from the plot of pH v.s. log[(Rmax
–
Materials and Methods
R)/(R–Rmin)].
The detailed synthesis and characterizations of intermediate
products, information of experimental devices, theoretical
calculation, cell culture and MTT cytotoxicity, in vitro/vivo
imaging experiments were provided in the ESI.†
Intracellular pH calibration
After incubation with P-pH, the obtained cells were washed
with KRBR buffer containing high K+ at various pH values. Then,
nigericin was added to the medium to allow a rapid exchange of
K+ for H+ which resulted in a rapid equilibration of external and
internal pH. After 5 min, the cell images were obtained using
excitation wavelengths of 405 nm, and collection windows were
set at 460−510 nm (green) and 560−610 nm (red), respectively.
The pH calibration curve was established by the calculated
Igreen/Ired. The details can be seen in the ESI.†
Preparation of P-pH
A mixture of N-vinypyrrolidone (0.55 mL, 5.15 mmol), 2,2-
azobisisobutyronitrile (5.5 mg, 33.5 μmol), complex Ir1 (18 mg,
15.8 μmol) and complex Ir2 (35 mg, 46.6 μmol) was dissolved in
anhydrous tetrahydrofuran. The solution was bubbled with dry
nitrogen for 2 hours to remove dissolved oxygen and then
stirred under nitrogen at 70 °C for 24 h. After cooling, the
reaction mixture was poured into diethyl ether, and the
obtained P-pH was purified by reprecipitation using
dichloromethane/diethyl ether and dialysis. The details can be
seen in the ESI.†
Zebrafish imaging
The zebrafish larvae were purchased from Model Animal
Research Center of Nanjing University. All the zebrafish
experiments were carried out in accordance with the relevant
laws and the guidelines of Institutional Animal Care and Use
Committee. Femtojet controlled by a micromanipulator was
used for microinjection. P-pH (5 mg mL-1) was mixed with the
supersaturated suspension of three acids, respectively, and the
mixture of P-pH and fumaric acid, or P-pH and succinic acid, or
P-pH and adipic acid was microinjected into the zebrafish with
a glass capillary needle. The zebrafish were used for imaging
immediately. After first imaging, the zebrafish were incubated
for another 2 h and used for the second imaging. The zebrafish
Calculation of pKa value
The pKa value for P-pH was determined in
Krebs−Ringer−Britton−Robinson (KRBR) buffer and estimated
from the changes in the phosphorescence intensity ratio with
various pH values by using the relationship, log[(Rmax–R)/(R–
Rmin)]=pH–pKa. R is the ratio of the emission intensity at 470 nm
to the emission intensity at 590 nm. Rmax and Rmin are maximum
and minimum limiting values of R, respectively. The pKa value
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were anesthetized before each imaging. The details can be seen absorption spectra between the protonatedViewfoArrtmicle Oanlninde
in the ESI.†
deprotonated form of Ir3 (ESI†, Fig. DSO1I):.10M.10e3a9n/Cw9hTCil0e6,28th5He
analogous tendency of changes could be observed in the
emission spectra of protonated and deprotonated forms of
these three iridium(III) complexes (Fig. 1a). These results
signified that the protonation of pyridyl group in
cyclometalating C^N ligand had significant influence on
phosphorescence of Ir1.
Results and discussion
Photophysical properties
First,
a pH-responsive phosphorescent complex Ir1 was
Time-dependent density functional theory calculations of
protonated/deprotonated forms of these iridium(III) complexes
were conducted to better understand their excited state
properties (Fig. 1b). The featured emission bands at 565 nm of
these iridium(III) complexes are attributed to the mixed triplet
metal-to-ligand charge-transfer (3MLCT) and ligand-to-ligand
charge-transfer (3LLCT) transitions whether they were
protonated or not (ESI†, Table S2). As for deprotonated and
protonated forms of Ir3, the distributions of lowest unoccupied
molecular orbitals (LUMOs) were nearly the same; both of them
were located at the 1, 10-phenanthroline group of N^N ligands.
The HOMOs of Ir3 were unaffected upon its protonation. For Ir1
and Ir4, the LUMOs were mainly distributed in phenylquinoline
group of C^N ligands in their deprotonated forms, while the
LUMOs transferred to the pyridyl group of C^N ligands in their
protonated forms (Fig. 1b). The protonation of Ir1 and Ir4 nearly
did not affect their HOMOs, but markedly lowered their LUMOs
and band gap, resulting in the bathochromic-shift of
phosphorescence spectra (ESI†, Table S3). This variation of
protonation-induced phosphorescence could be explained by
the intramolecular oxidative quenching.22-24
designed and synthesized. When exposed to strong acid, both
amide in cyclometalating N^N ligand and pyridyl group in
cyclometalating C^N ligand can be protonated. In order to
figure out the protonation effects on phosphorescence of Ir1,
two model iridium(III) complexes (Ir3 and Ir4) displaying similar
intense emission bands with maximum around 565 nm were
prepared (Fig. 1) and their photophysical properties were
summerized in Table S1 (ESI†). Then, the spectral study about
the protonated/deprotonated forms of these iridium(III)
complexes was carried out. The emission and absorption
spectra were shown in the Fig. S1 (ESI†). For Ir1, two intense
absorption bands centred at 300 nm or 380 nm, respectively,
are attributed to 1π–π* transitions of two cyclometalating
ligands; the relatively weak absorption bands at about 415-525
nm are originated from the singlet metal-to-ligand charge-
transfer (1MLCT) and ligand-to-ligand charge-transfer (1LLCT)
transitions.21 The assignments of absorption bands of Ir3 and Ir4
are similar to those of Ir1. It is noteworthy that the same
changes could be observed in the absorption spectral profiles of
Ir1 and Ir4 when their deprotonated forms were transformed
into protonated forms, while no obvious difference occurred in
Fig. 1 (a) Emission spectra of Ir1, Ir3, Ir4 (10 μM) in dichloromethane (black) and in dichloromethane containing 0.1% CF3COOH (red). (b) Calculated LUMOs of deprotonated and
protonated forms of Ir1, Ir3 and Ir4
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Fig. 2 (a) Phosphorescence spectra of P-pH (0.1 mg mL-1) measured in 0.1 M Krebs−Ringer−Britton−Robinson buffer of various pH values. (b) pH dependent ratios of phosphorescence
intensity at 470 nm and 580 nm. (c) pH dependent phosphorescence lifetimes at 470 nm and 580 nm. (d) Luminescence decay curves at 580 nm in different pH buffer. Excitation
wavelength was 405 nm
solutions, respectively, to simulate the complex biological
environments. The emission spectra of P-pH, however, did not
change significantly at the same pH, exhibiting that P-pH is
selectively responsive to pH. P-pH exhibited excellent
reversibility when the pH of solution changed repeatedly
between the two values (ESI†, Fig. S3b). Additionally, P-pH was
stored in solutions at different pH (pH = 7.40, pH = 4.01) for
more than 24 h, and the phosphorescence intensity of P-pH did
not show any fluctuations in the ratios (I470/I580), demonstrating
that P-pH had good chemical stability (ESI†, Fig. S3c). The
toxicity of P-pH towards Hela cells was assessed through MTT
assay. As described in Fig. S3d (ESI†), when the concentrations
of P-pH were higher than 0.1 mg mL-1, the cell survival rates
were still very high, showing its good biocompatibility.
Equipped with good selectivity, stability and low toxicity, P-pH
is a promising pH bioprobe in the complex environments.
Response to pH
Based on the above results, we selected Ir1, pH-inert iridium(III)
complex Ir2 (λmax=470 nm) (ESI†, Fig. S2) and hydrophilic poly(N-
vinyl-2-pyrrolidone) to construct ratiometric phosphorescent
pH probe P-pH with quantum yield of 0.13 (Scheme 1b).25 The
adopted feed ratio was based on the polymerization and
quantum efficiency of Ir1 and Ir2. The content of each
component in P-pH was determined from their absorbance.
First, the photophysical properties of P-pH were investigated in
buffer solutions at different pH. As shown in Fig. 2a, with pH
changing from 7.34 to 3.01, the phosphorescence intensity of
low energy band (550-650 nm) reduced dramatically, while that
of high energy band (450-550 nm) remained unchanged.
Accordingly, the ratio of phosphorescence intensity (I470/I580
)
was increased from 0.36 to 0.87 (Fig. 2b). In addition, the
phosphorescence lifetimes of two emission peaks showed
different variation trends. As demonstrated in Fig. 2c and 2d,
the emission lifetimes of P-pH (λem = 580 nm) decreased from
1073 ns to 530 ns as the pH descended. In contrast, the lifetime
of P-pH (λem = 470 nm) was steadily maintained within 420-450
ns. These results elucidated that P-pH had good responsiveness
to pH. Then, its potential for biological applications was
evaluated. As shown in Fig. S3a (ESI†), various metal cations,
non-metal anions and amino acid were added to the same P-pH
pH detection in living cells
P-pH was further used to monitor pH changes in the living cells.
The Hela cells were incubated with nigericin (10 μM) and
potassium ions (20 mM) to homogenize intracellular pH. After
20 minutes, the phosphorescence intensity of red channel
(560−610 nm) decreased obviously as pH descended, while no
emission intensity changes were recorded from green channel
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(460−510 nm) (ESI†, Fig. S4). Meanwhile, phosphorescence lysosome dye was used to recognize locations of lysosomes,
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intensity ratios (I460−510/I560−610) were gradually increased (Fig. while the emission intensity ratios (IGreen/IRed
DOI: 10.1039)/C9aTcCt0e6d285aHs
3a), which was similar to those measured in solutions. Based on parameters to determine pH values in the overlay images (Fig.
the above data, the calibration curve was established and the 3b). Then, ratiometric photoluminescence imaging and λ
pH ranges of different microenvironment can be calculated scanning method were applied in cells treated with NH4Cl.26-29
(ESI†, Fig. S5). Besides, the varied average phosphorescence The results showed that in the NH4Cl-stimulated cells, the
lifetimes of P-pH in cells were recorded. As shown in Fig. 3a, the lysosomal pH (ROI1 and ROI2 in NH4Cl-treated group) was
average phosphorescence lifetimes collected from longpass estimated to be 6.23 ± 0.25 according to the obtained
filter (≥ 550 nm) experienced continuous decline with pH calibration curve, while lysosomal pH (ROI1 and ROI2 in control
decreasing, while that of phosphorescence collected from band group) was 5.02 ± 0.28 in untreated cells. In contrast,
pass filter (482 ± 35 nm) stayed unchanged. The above results cytoplasmic pH (ROI3 and ROI4) in both two groups were within
indicated that P-pH is a reliable ratiometric and lifetime-based 7.42 ± 0.25, showing negligible difference. It is worth
probe to monitor intracellular pH.
mentioning that during the process of locating lysosomes and
sensing pH, P-pH showed good photostability compared with
lysosome dye (ESI†, Fig. S8), which guaranteed pH
determination reliability. Next, similar test in mitochondria was
performed with mitochondria dye (MitoTracker-DeepRed)
(ESI†, Fig. S9). Under double stimulations of both oxidative
stress and starvation,30 the mitochondrial pH in the pretreated
cells decreased dramatically from 7.59 ± 0.35 to 5.17 ± 0.33
(ESI†, Fig. S10). Based on P-pH with self-calibration, the
measurements of subtle pH fluctuation at subcellular levels
were achieved via ratiometric photoluminescence imaging.
pH mapping in lysosomes and mitochondria
Next, P-pH was applied to detecting pH changes at subcellular
levels. As exhibited in Fig. S6 (ESI†), the commercial lysosome
dye (Lyso-Tracker Red) could be excited by 559 nm but not be
excited by 405 nm, which assures that we can only collect the
phosphorescence signals (λGreen = 460−510 nm, λRed = 560−610
nm) from P-pH without interferences from lysosome dye (λPurple
= 570–620 nm) in the co-localization experiments under 405 nm
excitation. In the lysosome-imaging experiment (ESI†, Fig. S7),
Fig. 3 (a) Ratiometric photoluminescence images and PLIM images of Hela cells labeled with P-pH at different pH. The ratiometric photoluminescence images were the green channel
(460 to 510 nm) to red channel (560 to 610 nm), while PLIM images was collected through bandpass filter (482 ± 35 nm) or longpass filter (≥550 nm). Excitation wavelength was
405 nm and the scale bar is 50 μm. (b) Confocal laser scanning microscope images and PLIM images of Hela cells (up) and Hela cells treated with NH4Cl (10 mM) (down) stained with
the P-pH (0.1 mg mL-1) and Lyso-Tracker Red (0.1 μM). The merge channel was the overlay of green channel, red channel and purple channel (570 to 620 nm). The scale bar is 10
μm. (c) Phosphorescence spectra of four regions of interest (ROIs) in Hela cells with internalized P-pH (up) and treated with NH4Cl (10 mM) (down)
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Fig. 4 PLIM images of living zebrafish larva injected with P-pH (a), the mixture of P-pH and FA (b), P-pH and SA (c), P-pH and AA (d) for 0 h and 2 h. (e) The corresponding lifetime
histograms obtained from PLIM images of zebrafish labelled with P-pH. Photoluminescence was collected through bandpass filter (482 ± 32 nm) or longpass filter (≥ 550 nm) and
excitation wavelength was 405 nm. The scale bar is 1 mm.
FA-treated group was lower than those in SA- and AA-treated
groups and FA had the ability to maintain the gastrointestinal
environment at lower pH. This manifested that FA exhibited
best capacities among these three pH-modifiers to regulate the
pH of environment which was consistent with reports.31
pH sensing in zebrafish intestine
Finally, pH imaging in zebrafish intestine with P-pH was
conducted to assess the performance of three pH-modifying
excipients that are often adopted for regulating the pH of the
dissolution media. In this experiment, fumaric acid (FA), succinic
acid (SA) and adipic acid (AA) were introduced into zebrafish,
respectively, to acidize the intestinal juice of zebrafish. The
lower pH the intestinal juice was at and the longer time the
intestinal low pH remained for, the better regulating ability the
pH modifier possessed. As exhibited in Fig. S11 (ESI†), it was not
practical for monitoring pH in zebrafish via ratiometric
photoluminescence imaging due to strong interferences from
background fluorescence. In contrast, photoluminescence
lifetime imaging technology is an efficient way to distinguish
phosphorescence of P-pH from short-lived fluorescence of
living organisms. As shown in Fig. 4a-d, there existed obvious
differences in the averaged phosphorescence lifetimes
collected from longpass band (≥ 550 nm) among four groups. In
the blank group without any stimulation, the averaged
phosphorescence lifetime of P-pH stayed around 600 ns in both
the initial state (0 h) and final state (2 h). In other experimental
groups incubated with FA, SA or AA, the averaged lifetimes were
shorter than that of blank group in the initial state. After 2 h,
their lifetimes showed varying degrees of increase (Fig. 4e). The
average phosphorescence lifetimes in FA-stimulated group
were shorter compared with those in SA- and AA-treated
groups, which meant that the pH of zebrafish intestinal juice in
Conclusions
In conclusion, a water-soluble ratiometric phosphorescent
probe P-pH for measuring pH fluctuation in vitro and in vivo was
developed and the related working mechanism of P-pH was
studied in detail. P-pH displayed good sensitivity,
biocompatibility and stability. With joint use of P-pH and
commercial dyes (Lyso-Tracker Red or MitoTracker-DeepRed),
quantitative detections of pH at subcellular levels (lysosomes
and mitochondria) were achieved via ratiometric
photoluminescence imaging. By using the long-lived
phosphorescence of P-pH, accurate pH sensing in zebrafish with
minimized autofluorescence interference has been obtained
successfully. In particular, this is the first example for the
performance assessment of three kinds of pH-modifying
excipients in real zebrafish digestive tract by luminescent
method.
Conflicts of interest
There are no conflicts to declare.
6 | J. Name., 2012, 00, 1-3
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26 W. Pan, H. Wang, L. Yang, Z. Yu, N. Li and B. TangV,iAewnaArlt.icClehOenmlin.e,
Acknowledgements
2016, 88, 6743.
DOI: 10.1039/C9TC06285H
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This work was supported by the National Funds for
Distinguished Young Scientists (61825503), National Natural
Science Foundation of China (21671108 and 51473078),
National Basic Research Program of China (2012CB933301), the
Priority Academic Program Development of Jiangsu Higher
Education Institutions (PAPD, YX030003), and the Open
Research Fund of State Key Laboratory of Bioelectronics,
Southeast University.
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Journal of Materials Chemistry C
Page 8 of 8
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DOI: 10.1039/C9TC06285H
Self-calibrating phosphorescent polymeric probe for measuring pH
fluctuation in subcellular organelles and zebrafish digestive tract†
Zejing Chen,‡a,b Xiangchun Meng,‡a Mingjuan Xie,a Yuxiang Shi,a Liang Zou,a Song Guo,a
Jiayang Jiang,a Shujuan Liu*a and Qiang Zhao*a
A ratiometric phosphorescent probe P-pH was developed to determine pH in vitro
and in vivo via ratiometric photoluminescence imaging and photoluminescence
lifetime imaging.