A dual-function fluorescent probe for monitoring the degrees of hypoxia in living cells: Via the imaging of nitroreductase and adenosine triphosphate

Article abstract of DOI:10.1039/c8cc02209g

A new dual-function fluorescent probe is developed for detecting nitroreductase (NTR) and adenosine triphosphate (ATP) with different responses. Imaging application of the probe reveals that intracellular NTR and ATP display an adverse changing trend during a hypoxic process and ATP can serve as a new sign for cell hypoxia.

Full text of DOI:10.1039/c8cc02209g

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A dual-function fluorescent probe for monitoring the
degrees of hypoxia in living cells via the imaging of
nitroreductase and adenosine triphosphate†
Yu Fang,ab Wen Shi,a Yiming Hu,a Xiaohua Lia and Huimin Ma*ab
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
and whether ATP can serve as a hypoxia-sensitive sign, are unclear.
Thus, research on the cell hypoxia by monitoring both intracellular
NTR and ATP would be helpful to better reveal the property of
cancer cells.
A new dual-function fluorescent probe is developed for detecting
nitroreductase (NTR) and adenosine triphosphate (ATP) with
different responses. Imaging application of the probe reveals
that the intracellular NTR and ATP display an adverse changing
trend during a hypoxic process and ATP can serve as a new sign
for cell hypoxia.
Small molecular fluorescence probes have become a powerful tool
for imaging the alteration of intracellular biomolecules in a non-
invasiveness way due to high sensitivity, and great temporal and
spatial resolution.7 In the past several years, many fluorescence
probes with excellent performance have been developed to detect
NTR4 or ATP,8 which promotes the research on the functions of
intracellular NTR and ATP. In spite of these progresses, no single
fluorescent probe is capable of simultaneously detecting both NTR
and ATP to indicate the hypoxic state and understand possible
mutual correlation between NTR and ATP in cells. Perhaps, this
might be realized by using several probes in a cell, but the strategy
would become very complicated due to the effect of different probes
or their cross-talk.9 Therefore, it is necessary to develop a single
fluorescent probe to meet the above requirements.
Herein, we report such a new fluorescence probe (Scheme 1),
which is prepared by using the rhodamine/1,8-naphthalimide hybrid
structure as a platform, nitro and diethylenetriamine as a reacting site
for NTR and ATP, respectively. The probe reacts with NTR, ATP,
and NTR/ATP exhibiting different fluorescence responses.
Significantly, cellular imaging studies with this probe revealed for
the first time that ATP is a hypoxia-sensitive species, and
intracellular NTR and ATP displayed an adverse changing behaviour
during a hypoxic process, i.e., NTR being a roughly exponential
increase but ATP being a decrease, which can be used to effectively
indicate the hypoxic degrees of living cells.
Hypoxia, a general feature in solid tumors, is a result of
inadequate supply of blood flow, and is a critical indicator for cancer,
stroke, ischemia and cardiovascular diseases.1 It is reported that the
mean oxygen content is about only 4% in solid tumors and may be
as low as 0% in some local places.2 There has been much research
on how to reduce the death rate among hypoxic tumor-bearing
patients.3 Therefore, it is of great significance to develop an efficient
method for the accurate monitoring of hypoxia degrees in
biosystems.
Current evidence suggests that hypoxic cells may elevate the level
of nitroreductase (NTR),4 which can reduce nitroaromatic
compounds to the corresponding amines in the presence of reduced
nicotinamide adenine dinucleotide (NADH).5 Thus, NTR is recently
often used as an important sign for different degrees of hypoxia in
living cells. On the other hand, adenosine triphosphate (ATP) can be
found in all forms of life, and provides chemical energy within cells
for a variety of metabolic purposes.6 Nevertheless, there is a
controversy about the main production manner of ATP in the tumor
cells. Some researchers proposed that the ATP production depended
more on glycolysis than aerobic respiration, whereas the others
disagreed with this claim.6d In any event, the decrease of oxygen
may result in the change of ATP in living cells. However, the exact
changing behavior of the intracellular ATP during a hypoxic process,
The design of our probe is based on the following considerations.
(1) Choose suitable spectroscopic units. Naphthalimide and
rhodamine were employed as the fluorochromes, because they have
excellent spectroscopic properties and especially their fluorescence
emissions do not overlap with each other to a large extent.10 (2)
Choose different recognition units. The nitro and diethylenetriamine
groups were utilized, which are specific for NTR and ATP,
respectively. The nitro group in 1,8-naphthalimide may be reduced
to amino by NTR in the presence of NADH,11 possibly resulting in a
a Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of
Chemistry, Chinese Academy of Sciences, Beijing 100190, China. E-mail:
mahm@iccas.ac.cn.
b University of the Chinese Academy of Sciences, Beijing 100049, China.
†
Electronic supplementary information (ESI) available. See DOI:
10.1039/b000000x/
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the concentration of NTR in the range of 0.25-4.5 μg/mL (Fig. 1E),
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2
DOI: 10.1039/C8CC02209G
with an equation of F = 0.066  [NTR] (μg/mL) + 0.007 (R =
0.99). The detection limit (k = 3) of the probe for NTR was
determined to be 0.12 μg/mL. On the other hand, addition of the
gradually increasing concentration of ATP (0-5 mM) leads to a
strong fluorescence at 580 nm when excited at 540 nm (Fig. 1D),
giving a nearly 110-fold enhancement. However, only a rather small
increase in the fluorescence intensity was produced with excitation
at 420 nm (Fig. 1C). Moreover, as shown in Fig. 1F, there is good
linear dependence between F and the concentration of ATP in the
range of 2-4.5 mM with an equation of F =0.37 × [ATP] (mM) –
0.64 (R2 = 0.99), suggesting that the probe is rather suited for
monitoring the intracellular ATP because its concentration in living
cells is about 3 mM.6c Additionally, a linear relationship also exists
between F and ATP in the low concentration range of 0.5-2 mM
(Fig. S9) with an equation of F = 0.072 × [ATP] (mM) – 0.027 (R2
= 0.86), by which the detection limit (k = 3) of the probe for ATP
was determined to be 0.05 mM.
Scheme 1 Structure of the probe and its reaction with NTR, ATP and
NTR/ATP.
fluorescence off-on response; diethylenetriamine has been proved to
be a good recognition site for ATP8b,10b via multiple hydrogen
bonding.12 Moreover, the π−π stacking interaction between
rhodamine and the adenine part of ATP also contributes to the
spirolactam ring-opening and the fluorescence off-on response (vide
infra; see also Figs. S3 and S4). (3) Easy to prepare. The probe can
be synthesised and purified in three steps with high yields.
The absorption and fluorescence spectra of the probe before and
after reaction with NTR and ATP were studied. As shown in Fig. S3,
reaction of the probe with NTR leads to a small increase of the broad
absorption band at about 420 nm, whereas that with ATP produces a
new absorption peak at 550 nm characteristic of the ring-opening
rhodamine. Notably, the probe itself exhibits
a very weak
fluorescence, due to the quenching effect of the nitro group to 1,8-
naphthalimide and the formation of the spirocyclic rhodamine
structure. However, upon addition of NTR to the probe solution,
significant fluorescence at 520 nm emerges when excited at 420 nm,
resulting from the pull-push conversion from the electron-deficient
nitro to the electron-rich amino and thus the fluorescence recovery of
1,8-naphthalimide; the formation of the corresponding reaction
product was verified by ESI-MS analysis (m/z = 723.2 [M]+; Fig. S5).
Similarly, addition of ATP yields strong fluorescence at 580 nm
when excited at 540 nm (Fig. S3D), which is attributed to the ring-
opening reaction of rhodamine in the presence of ATP.
Then, we optimized the reaction conditions including pH,
temperature, and reaction time. As shown in Fig. S6, the reaction of
the probe with NTR and ATP may be completed in 40 min and 5
min, respectively. The influences of pH and temperature were also
examined (Fig. S7 and S8), showing that the probe can work
Fig. 1 Fluorescence responses of the probe to NTR and ATP. Fluorescence
emission spectra of the probe (10 μM) with different concentrations of NTR
(0-10 μg/mL) in 10 mM Tris buffer (pH 7.4) excited at 420 nm (A) or 540
nm (B); fluorescence emission spectra of the probe (10 μM) with different
concentrations of ATP (0-5 mM) in 10 mM Tris buffer (pH 7.4) excited at
420 nm (C) or 540 nm (D); (E, F) linear relationship between ΔF and the
concentration of NTR (0.25-4.5 μg/mL) and ATP (2-4.5 mM), respectively.
ΔF is the relative fluorescence intensity difference after and before reaction.
o
effectively under the physiological condition (pH 7.4, 37 C). It was
noted that the fluorescence of the probe itself was scarcely affected
by the pH change within 5-10, but its reaction solution with ATP
produced the maximum fluorescence in the range of pH 7.0-7.4 (Fig.
S8B). This suggests that the probe may be used to detect ATP in
hypoxic cells such as cancer cells that usually have lower
intracellular pH values of pH 7.0-7.2.13
Next, we explored the fluorescence response of the probe in the
presence of both NTR and ATP. In the initial design of our probe,
fluorescence resonance energy transfer (FRET) between
naphthalimide and rhodamine were expected in the presence of both
NTR and ATP. However, such a photophysical process was not
observed, i.e., the fluorescence spectra of the reaction system
showed two emission peaks when excited at 420 nm, but the peak at
520 nm is increased rather than decreased with the increase of NTR
(Fig. S10A). The reason for this phenomenon may be complicated,
but a possible explanation may be that FRET is largely dependent on
the molecular structure and environment. Fortunately, the absence of
FRET in our probe leads to that the peak at 520 nm is only sensitive
to NTR and is scarcely affected by ATP, thus enabling it to indicate
the NTR change. On the other hand, the mixture of NTR and ATP
o
Under the optimized conditions (reaction at pH 7.4 and 37 C for
40 min and 5 min for NTR and ATP, respectively), the fluorescence
responses of the probe to different concentrations of NTR and ATP
were investigated. As shown in Fig. 1, with increasing the NTR
concentration, a gradually enhanced fluorescence emerges at 520 nm
when excited at 420 nm (Fig. 1A), resulting in a nearly 20-fold
increase. In contrast, no significant fluorescence appears when
excited at 540 nm (Fig. 1B), indicating that NTR only turns on the
fluorescence of the 1,8-naphthalimide moiety but not the rhodamine
moiety. Besides, a good linear relationship exists between F and
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exhibits only one emission peak at 580 nm when excited at 540 nm fluorescence was largely decreased (Fig. S15), supporting that the
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DOI: 10.1039/C8CC02209G
(Fig. S10B). This peak at 580 nm is only sensitive to the ATP fluorescence in the red channel is ascribed to ATP. Besides, the
change. The above results manifest that the probe can be used to extremely low fluorescence from the green channel is nearly
detect NTR and ATP respectively and simultaneously by adjusting unaltered (Figs. S14 and S15). It is noted that the electrically neutral
the excitation wavelength.
probe itself does not have good mitochondrial-targeting performance
The specificity of the probe was evaluated for NTR and ATP over (Fig. S16, the Pearson’s and overlap coefficients are 0.61 and 0.85,
other potential interfering species, including inorganic salts, amino respectively), thus the intracellular fluorescence comes from the
acids, proteins, and glucose. The results (Fig. 2) proved that the action of ATP in the whole cells. The above data indicate that the
probe reacted selectively with NTR and ATP instead of the other probe is capable of imaging NTR and ATP in live cells without the
species. Furthermore, we also studied the response of the probe to interference from each other.
various inorganic phosphates or other nucleoside polyphosphates
Having verified the capacity of the probe to image NTR and ATP
(Fig. S11), revealing that, although these phosphates might also form in living cells, we studied the use of the probe to monitor the degrees
hydrogen bonding with the probe, no significant fluorescence is of hypoxia in cells. First, HeLa cells were cultured under normoxic
produced. This phenomenon demonstrates that the specific π−π and different hypoxic (10%, 5% and 1%) conditions at 37 °C for 12
stacking interaction of ATP with the probe (Fig. S3) is an h, and then incubated with 10 μM probe for 40 min under the
indispensable factor to the ring-opening reaction of rhodamine. In respective conditions. As shown in Fig. 3, under the normoxic
addition, the cytotoxicity of the probe toward HeLa cells was condition, no noticeable fluorescence is produced in the green
measured by standard MTT assay (Fig. S12). The cell viability was channel, but strong fluorescence emerges in the red channel, which
still over 80% after incubation even with a high concentration of 25 indicates that ATP instead of NTR is abundant in the cells. However,
μM probe, showing the good biocompatibility of the probe to cells.
with the decrease of the O2 level, the fluorescence in the green
channel becomes more and more strong, whereas that in the red
channel is gradually decreased. A close examination (Figs. 3B and
3C) reveals for the first time an about exponential increase of the
intracellular NTR but a decrease of the intracellular ATP with
decreasing the O2 content from 20% to 1%. The changing trend of
NTR is consistent with the previous finding.4 Although the decrease
of ATP in hypoxic cells was observed before, its exact changing
behaviour of the roughly exponential decrease during a hypoxic
process has not been reported. The mechanism for the decrease of
the intracellular ATP may be rather complicated,6c,14 but a possible
speculation is that hypoxia in tumor cells may lead to a decreased
production of ATP, or more rapid consumption of ATP than its
production. Moreover, the mechanism for the adverse change of
NTR and ATP, and whether there exists an intimate relationship
between NTR and ATP or not, deserve further research.
Nevertheless, the above results clearly indicate that ATP, similar to
NTR, is also sensitive to the intracellular oxygen level and may
serve as a new sign for the cell hypoxia; the probe can be used to
monitor the different degrees of hypoxia by imaging the change of
both intracellular NTR and ATP. Such dual-channel detection is
more powerful than the one-channel imaging.
Fig. 2 Fluorescence responses of the probe (10 μM) to other species. (A): (0)
blank; (1) KCl (150 mM); (2) CaCl2 (2 mM); (3) MgCl2 (1 mM); (4) ZnCl2
(100 μM); (5) CuCl2 (100 μM); (6) glucose (1 mM ); (7) cysteine (1 mM); (8)
glutathione (1 mM ); (9) serine (1 mM); (10) tyrosine (1 mM); (11) alanine (1
mM); (12) HOCl (100 μM); (13) H2O2 (100 μM); (14) OONO– (100 μM); (15)
NaHS (100 μM); (16) vitamin C (1 mM); (17) 1 μg/mL DT-diaphorase; (18)
4 μg/mL NTR. λex/em = 420/520 nm. (B): (0) blank; (1) KCl (150 mM); (2)
CaCl2 (2 mM); (3) MgCl2 (1 mM); (4) ZnCl2 (100 μM); (5) CuCl2 (100 μM);
(6) glucose (1 mM); (7) cysteine (1 mM); (8) glutathione (1 mM); (9) serine
(1 mM); (10) tyrosine (1 mM); (11) alanine (1 mM); (12) HOCl (100 μM);
(13) H2O2 (100 μM); (14) OONO– (100 μM); (15) NaHS (100 μM); (16) ATP
(2.5 mM). λex/em = 540/580 nm.
The potential ability of the probe to image NTR and ATP in live
cells was evaluated by using different lasers with the excitation
wavelength of 405 nm and 559 nm, respectively. As shown in the
image b of Fig. 3A, the cells incubated with the probe exhibit no
significant fluorescence in the green channel (λex = 405 nm);
however, if the cells were grown under the hypoxic condition of 1%
O2 for 12 h and then incubated with the probe, apparent fluorescence
is produced (image e), consistent with the known observation that
hypoxia may lead to the increase of intracellular NTR.4 Moreover,
when the cells were pretreated with 0.1 mM dicoumarin (inhibitor of
NTR) for 30 min, and then incubated with the probe, the
fluorescence largely decreases (Fig. S13), further confirming that the
fluorescence in the green channel is attributed to NTR. The change
of NTR hardly affects the intracellular fluorescence intensity from
the red channel (Fig. S13). On the other hand, as is seen from Fig.
S14, HeLa cells incubated with the probe for 30 min show a
significant fluorescence in the red channel (λex = 559 nm) when
compared to the cells themselves, and the fluorescence keeps nearly
unchanged at least for 1 h. Furthermore, if the cells were pretreated
with 0.5 U/mL apyrase for 30 min (apyrase can transform ATP into
adenosine 5’-monophosphate or organic phosphate), the
Fig. 3 Confocal fluorescence images of HeLa cells under noxmoxic (20% O2)
and the different degrees of hypoxic conditions (10%, 5% and 1%). (A)
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Control (HeLa cells themselves); the cells were grown and then incubated
with 10 μM probe for 40 min at different O2 concentrations. The fluorescence
L. Yuan, X. Zeng, J. J. Peng, Y. Ni, J. C. Er, W. Xu, B. K. Agrawalla, D.
D. Su, B. Kim and Y. T. Chang, Angew. Chem. Int. Ed., V2i0ew16A,rt5ic5le, O17n7lin3e-
DOI: 10.1039/C8CC02209G
from the green (ex= 405 nm, em= 430-530 nm) and red (ex= 559 nm, em
=
1776; (d) Z. C. Xu, N. J. Singh, J. Lim, J. Pan, H. N. Kim, S. Park, K. S.
Kim and J. Yoon, J. Am. Chem. Soc., 2009, 131, 15528-15533; (e) D.
Maity, M. Li, M. Ehlers and C. Schmuck, Chem. Commun., 2017, 53, 208-
211.
570-670 nm) channels was detected, respectively. The differential
interference contrast (DIC) images of the corresponding cells are given in the
bottom. Scale bar: 10 μm. (B) Relative pixel intensity (n = 3) of the
fluorescence images from the green channel in panel A (the pixel intensity
from image e is defined as 1.0). (C) Relative pixel intensity (n = 3) of the
fluorescence images from the red channel in panel A (the pixel intensity from
image g is defined as 1.0).
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In summary, we have reported the design, preparation and
application of a novel dual-function fluorescence probe, which
shows different fluorescence off-on responses to NTR, ATP and
NTR/ATP simultaneously. This permits the probe to be capable of
detecting the two hypoxia-sensitive species of NTR and ATP.
Notably, such a detection has been demonstrated with living HeLa
cells, which firstly uncovers that the intracellular NTR and ATP
exhibit an adverse changing trend during a hypoxic process, and
ATP is indeed a hypoxia-sensitive species. This may enable ATP to
serve as a new sign combined with NTR for the accurate monitoring
of the hypoxic situation in cells.
We thank the financial support from the 973 Program (No.
2015CB932001), the NSF of China (Nos. 21535009, 21435007,
21621062, 21675159 and 91732104), and the Chinese Academy of
Sciences (XDB14030102).
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