Development of a new ratiometric probe with near-infrared fluorescence and a large Stokes shift for detection of gasotransmitter CO in living cells

Article abstract of DOI:10.1016/j.saa.2019.117657

A near-infrared (NIR) ratiometric fluorescent probe, NIR-Ratio-CO, was developed for rapid detection of carbon monoxide (CO) in both solution and living cells through the strategy of Pd⁰-mediated Tsuji-Trost reaction. This probe shows a rapid,

Full text of DOI:10.1016/j.saa.2019.117657

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Development of a new ratiometric probe with near-infrared fluorescence and a large
Stokes shift for detection of gasotransmitter CO in living cells
Enbo Zhou, Shengyi Gong, Jiaxin Hong, Guoqiang Feng
PII:
S1386-1425(19)31047-9
https://doi.org/10.1016/j.saa.2019.117657
SAA 117657
DOI:
Reference:
To appear in: Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
Received Date: 12 June 2019
Revised Date: 12 October 2019
Accepted Date: 14 October 2019
Please cite this article as: E. Zhou, S. Gong, J. Hong, G. Feng, Development of a new ratiometric
probe with near-infrared fluorescence and a large Stokes shift for detection of gasotransmitter CO in
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Graphical Abstract

Development of a new ratiometric probe with near-infrared
fluorescence and a large Stokes shift for detection of
gasotransmitter CO in living cells
Enbo Zhou, Shengyi Gong, Jiaxin Hong, and Guoqiang Feng*
Key Laboratory of Pesticide and Chemical Biology of Ministry of Education,
Chemical Biology Center, College of Chemistry, Central China Normal University,
1
*
52 Luoyu Road, Wuhan 430079, P.R. China
Corresponding author. Tel.: +86 2767862163; fax: +86 2767867955
E-mail address: gf256@mail.ccnu.edu.cn (G. Feng)
Abstract: A near-infrared (NIR) ratiometric fluorescent probe, NIR-Ratio-CO, was
developed for rapid detection of carbon monoxide (CO) in both solution and living
0
cells through the strategy of Pd -mediated Tsuji-Trost reaction. This probe shows a
rapid, highly specific and sensitive detection process for CO, accompanied by
colorimetric and distinct ratiometric fluorescence changes at 655 and 592 nm with a
large Stokes shift up to 195 nm. The detection limit for CO was measured to be about
6
1 nM by the fluorescence method. In addition, this probe was successfully applied
for ratiometric imaging of both exogenous and endogenous CO in living cells,
indicating that it can be used as a novel tool for ratiometric fluorescent detection of
CO in living systems.
Keywords: near-infrared; ratiometric; fluorescent probe; carbon monoxide; imaging
1

1
. Introduction
Carbon monoxide (CO), a highly toxic gas molecule, has become recognized as an
important biological gasotransmitter in living systems [1]. CO can be endogenously
generated during the haem catabolism in our human body and other biological
systems by haem oxygenase (HO) enzymes, and plays a significant regulatory role in
various physiological and pathological processes [2]. Abnormal metabolism of CO
has been involved in a numerous of diseases, such as hypertension, inflammation,
Alzheimer’s disease, and heart failure, however, proper amount of CO was found to
be a potential therapeutic agent for therapy of many diseases because of its
cytoprotective effects [3,4]. Despite of these discoveries, many precise cellular
functions of CO remain unclear, which may largely due to the lack of a reliable
method to effectively detect this small gas molecule in living systems [5]. To disclose
more details of the biological CO, the development of convenient and reliable
methods for real-time detection of CO in living systems is therefore important and
urgent [6-8].
Although several methods have been developed to detect CO, such as gas
chromatography [9], chromogenic detection [10,11], and electrochemical analysis
[
12], these methods are limited to detect CO in living systems due to their poor
bio-compatibility. Recently, small molecular fluorescent probes has been proved
particularly useful for detection and imaging in living systems due to their many
advantages such as high sensitivity, great convenience, noninvasive manner, and
2

real-time detection ability with high temporal-spatial resolution [13]. In 2012,
Chang’s group reported a small molecular fluorescent probe based on an organic
palladium complex, which realized CO detection in living cells for the first time [14].
Soon after, many other fluorescent probes for detection of CO in living cells were
0
reported [15-35]. Among them, the Pd -mediated Tsuji–Trost reaction has been
proved to be a simple but very attractive strategy to develop probes for detecting CO
in living systems [22-36]. However, most of the reported CO probes have some
shortcomings. For example, almost all of the reported CO probes are single emissive
(intensity-based), which may be interfered from a variety of factors, such as the
concentration of probe, temperature, pH, and instrument efficiency etc. [26,36]. In
comparison, ratiometric fluorescent probes can effectively overcome these problems
by self-calibration at two emission bands, which greatly increases the reliability and
accuracy of detection [37]. Besides, most of the reported CO probes showed
fluorescence changes in the visible region (<650 nm) and small Stokes shift (<50 nm),
which also limited their biological applications due to the potential strong interference
from the background fluorescence of biomolecules and the scattering light from the
excitation [38,39]. In view of these shortcomings, development of ratiometric
fluorescent CO probes with enhanced near-infrared (NIR) fluorescence and a large
Stokes shift are urgently needed.
Herein, a NIR ratiometric fluorescent probe (NIR-Ratio-CO, Scheme 1) was
designed for detection of CO both in solution and living cells. Probe NIR-Ratio-CO
uses DCI-NH2 as the ratiometric fluorescent signaling unit due to its well-known
3

easy-to-get property with efficient intramolecular charge transfer (ICT) process,
desirable NIR fluorescence properties with a remarkable large Stokes shift (~200 nm),
and excellent biocompatibility to living systems [40,41]. An allyl carbamate moiety
was chosen as the reactive unit because it not only offered an efficient Tsuji–Trost
reaction site [22,26] but also could effectively modulate the electron push-pull system
and alter the photophysical property of DCI-NH2. Due to the electronic-withdrawing
property of allyl carbamate, the probe shows weak ICT and has an emission at 592 nm.
2
+
However, after the addition of CO in the presence of Pd , the allyl carbamate is
cleaved, resulting in the generation of DCI-NH2 with stronger ICT and a red-shifted
emission at 655 nm (Scheme 1). As a result, by self-calibration at two emission bands,
NIR-Ratio-CO could provide an efficient ratiometric NIR fluorescent probe for CO
with desirably enhanced fluorescence in the NIR region. Moreover, we also showed
that this probe exhibited excellent sensing performance for CO and could be used to
detect both exogenous and endogenous in living cells. To the best of our knowledge,
NIR-Ratio-CO is the first ratiometric fluorescent probe reported to date with
enhanced NIR fluorescence and a large Stokes shift for CO detection in living cells
(Table S1 in the supplementary data).
[
Scheme 1]
2
2
. Materials and methods
.1. Materials and instrumentation
Unless otherwise stated, all the chemicals involved in this work were purchased
from commercial suppliers and used without further purification. CORM-3, a
4

commercially available CO-releasing molecule was used as the safe and
easy-to-handle CO source in all the experiments according to the literature
1
13
[
14,15,23,42]. H NMR and C NMR spectra were recorded on a Varian Mercury 400
or 600 spectrometer. Data of high-resolution mass spectrometry (HR-MS) were
obtained with an LC/Q-TOF MS spectrometer (Agilent). The UV-vis and fluorescent
spectra were recorded on an Agilent Cary-100 UV-vis spectrophotometer and an
Agilent Cary Eclipse fluorescence spectrophotometer, respectively. The pH was
measured by a PB-10 digital pH-meter (Sartorius). Cell imaging was performed on a
Leica TCS SP8 confocal fluorescence microscope.
2
.2. Synthesis
DCI-NH2 was synthesized from isophorone (ISO) as a deep purple solid according
to the literature method [41].
Synthesis of NIR-Ratio-CO. To a solution of DCI-NH2 (289 mg, 1 mmol) was
added allylchloroformate (0.5 ml, 5 mmol) in dry pyridine (8 mL) under the
atmosphere of nitrogen at 0 °C. The resulting mixture was stirred at room temperature
for 4 h. Then, the reaction was quenched with diluted HCl and extracted with ethyl
acetate (20 mL × 3). The combined organic layers were dried, filtered and
concentrated. The crude product was purified by column chromatography (eluent:
petroleum ether/ethyl acetate = 10/1, v/v) to afford NIR-Ratio-CO as a red powder
1
(
112 mg, yield 30 %). M.p.185-186 °C. H NMR (600 MHz, CDCl ) δ 7.47 (d, J = 8.3
3
Hz, 2H), 7.44 (d, J = 8.3 Hz, 2H), 7.01 (d, J = 16.0 Hz, 1H), 6.91 (s, 1H), 6.90 (d, J =
1
5.5 Hz, 1H), 6.81 (s, 2H), 5.97 (ddt, J = 16.6, 11.0, 5.8 Hz, 1H), 5.38 (d, J = 16.9 Hz,
5

1
1
1
6
H), 5.28 (d, J = 10.5 Hz, 1H), 4.69 (d, J = 5.6 Hz, 2H), 2.59 (s, 2H), 2.46 (s, 2H),
1
3
.08 (s, 6H). C NMR (151 MHz, CDCl ) δ 169.24, 154.03, 152.82, 139.11, 136.44,
3
32.10, 130.70, 128.52, 127.81, 123.05, 118.61, 118.45, 113.56, 112.79, 103.81, 78.03,
+
6.02, 42.92, 39.11, 31.95, 27.94. MS (EI): m/z found 373.1 (M ). HR-MS (ESI)
+
+
calcd. for C H N O Na (M + Na ) 396.1682, found 396.1685.
2
3
23
3
2
2
.3. Optical studies
Unless otherwise stated, all the optical tests were performed in PBS buffer (10 mM,
pH 7.4, with 20% DMSO, v/v) at 37 °C with 5 µM of NIR-Ratio-CO in the presence
of 10 µM of PdCl . Standard quartz cuvettes with a 10 mm lightpath were used for the
2
UV-vis and fluorescent spectral measurements. λ = 440 nm and slit width d = d =
ex
ex
em
1
0 nm were set for all fluorescence measurements. The fluorescence quantum yields
were determined in PBS buffer (10 mM, pH 7.4, with 20% DMSO, v/v) using
Rhodamine B (Ф = 0.89 in ethanol) as standard.
2
.4. Imaging of CO in living cells
HeLa cells were cultured in DMEM medium supplemented with FBS (fetal bovine
serum, 10%) at 37 °C in a 5% CO and 95% air incubator. In the cell imaging
2
experiments, a mixture of probe NIR-Ratio-CO (5 µM) and PdCl (10 µM) in PBS
2
buffer with 0.5% of DMSO was used for the imaging. As the control, cells were
imaged after incubation with a mixture of probe NIR-Ratio-CO (5 µM) and PdCl2
(10 µM) at 37 °C for 30 minutes. For imaging of exogenous CO, cells were incubated
with CORM-3 (10, 20 and 30 µM, respectively) for 30 min, and then with a mixture
of probe NIR-Ratio-CO (5 µM) and PdCl (10 µM) for another 30 min. For imaging
2
6

of endogenous CO, cells were incubated with heme (100 µM) for 1, 4 and 8 h at 37 °C,
respectively, and then with a mixture of probe NIR-Ratio-CO (5 µM) and PdCl (10
2
µM) for another 30 min. Imaging was taken on a confocal fluorescence microscope
after washing the cells three times with PBS buffer solution. The fluorescence of each
channel (red or green) was analyzed with three different cell images, and then the
average fluorescence intensity of each channel was obtained. Finally, the F /Fgreen
red
ratio was calculated.
3
3
. Results and discussion
.1. Probe synthesis
Probe NIR-Ratio-CO was successfully synthesized according to the route as
shown in Scheme 2. DCI-NH2 was prepared from commercially available isophorone
ISO) according to the literature method [41]. Structural identification of
(
1
13
NIR-Ratio-CO was confirmed by H NMR, C NMR and mass spectroscopy, which
can be found in the supplementary data.
[
Scheme 2]
3
.2. Ratiometric fluorescent detection of CO
With NIR-Ratio-CO in hand, we tested its sensing properties for CO in the
presence of PbCl by the absorption and fluorescent spectral changes in PBS buffer
2
(10 mM, pH 7.4, with 20% DMSO, v/v) at 37 °C. As shown in Figure 1, in the
2+
absence of CO, the probe solution (5 µM NIR-Ratio-CO + 10 µM Pd ) showed a
major absorption peak at 418 nm and a marked fluorescence at 592 nm (Ф = 0.02).
Upon addition of CORM-3 (100 µM), the absorption peak at 418 nm decreased
7

gradually and simultaneously a new absorption peak at 460 nm appeared,
accompanied by an apparent color change from light yellow to pink. Meanwhile, a
marked decrease of fluorescence intensity at 592 nm and concurrently a dramatic
enhancement of fluorescence intensity at 655 nm with an obvious red-shift (63 nm) of
emission and a marked emission color change from orange to red can be observed, as
shown in Figure 1b. Furthermore, from the reaction kinetic studies as shown in Figure
S1, upon addition of CORM-3, the fluorescence intensity of the probe solution at 655
nm increased rapidly and reached saturation at about 15 min, indicating it showed a
rapid detection process. In addition, the fluorescence intensity ratio at 655 and 592 nm
(
I /I ) increased from 0.42 to 7.5 after incubation with CORM-3 for 15 min, which
655 592
corresponds to a ∼18 fold enhancement, indicating the probe system shows a fairly
good ratiometric fluorescence response for CO. In contrast, NIR-Ratio-CO shows
negligible response to PbCl and does not respond to CO in the absence of PbCl
2
2
(Figure S2). All these results indicate that NIR-Ratio-CO can be used as a potential
NIR ratiometric fluorescent probe for rapid detection of CO in the presence of PbCl2.
Notably, the fluorescence shows significant enhancement in the far-red to NIR region,
which is highly favorable for the biological applications of NIR-Ratio-CO.
[
Figure 1]
3
.3. The selectivity
To evaluate the selectivity of NIR-Ratio-CO toward CO, we tested its responses
to various biologically relevant analytes. As shown in Figure 2, upon addition of
various analytes including biothiols and amino acids ((cysteine (Cys), homocysteine
8

(
Hcy), glutathione (GSH), hydrogen sulfide (H S, NaHS was used as the source),
2
methionine (Met), threonine (Thr), arginine (Arg), tyrosine (Tyr), alanine (Ala),
aspartic (Asp), lysine (Lys), pyroglutamic acid (Pyr), phenylalanine (Phe), glutamic
acid (Glu), isoleucine (Ile), histidine (His), glycine (Gly), valine (Val), leucine (Leu),
−
−
−
−
−
2−
3
−
−
tryptophan (Trp), serine (Ser)), anions (F , Cl , Br , I , NO , CO , HCO , HSO ,
3
3
4
2−
2−
3
−
2−
7
2−
8
−
−
−
2−
SO , S O , HSO , S O , S O , SCN , Ac , N , and C O , all with sodium
3
2
3
2
2
3
2
4
−
salts), and reactive oxygen/nitrogen species (ROS/RNS) [43] such as ClO , H O ,
2
2
−
−
•
t
•
•−
NO , NO, HNO, ONOO , ROO , BuOO and O to the probe solution (5 µM
2
2
2+
NIR-Ratio-CO + 10 µM Pd ) in PBS buffer (10 mM, pH 7.4, with 20% DMSO, v/v)
at 37 °C, only negligible changes at 592 nm in the fluorescence spectra were observed.
These results are clearly different to the ratiometric fluorescent response of the probe
solution for CO (CORM-3), indicating NIR-Ratio-CO has high selectivity for CO.
[
Figure 2]
3
.4. The sensitivity
Fluorescent titration method was then used to investigate the sensitivity of the
probe for CO. As shown in Figure 3, with the increase of the concentration of
2
+
CORM-3 to the test solution of NIR-Ratio-CO (5 µM with 10 µM of Pd ), the
emission at 592 nm gradually decreased, while the emission at 655 nm increased
dramatically. The ratios of fluorescence intensity at 655 and 592 nm (I /I ) showed
6
55 592
a dose-dependent response to CORM-3 and a saturation was observed after about 50
µM of CORM-3 was added (Figure 3a, inset). Importantly, the ratio I /I showed a
6
55 592
linear increase to the concentration of CORM-3 from 0 to 10 µM (Figure 3b). Since 1
9

mole CORM-3 can release 1 mole CO in aqueous solution [42], the detection limit of
probe NIR-Ratio-CO for CO was thus determined to be about 61 nM based on the
3
σ/k method (S/N = 3). These results indicate that NIR-Ratio-CO can be used as a
ratiometric fluorescent probe for quantitative analysis of CO with high sensitivity.
Figure 3]
[
3
.5. The sensing mechanism
We compared the spectra of the probe solution after addition of CORM-3 with those
of DCI-NH2 and found that they are in good agreement (Figure S3). This suggests
that the formation of DCI-NH2 is responsible for the optical changes. Recently,
several Tsuji–Trost reaction-based fluorescent turn-on probes for CO have been
reported and their sensing mechanism were well studied [22-36]. Based on these
reports, the reaction mechanism of NIR-Ratio-CO for sensing CO in the presence of
2
+
Pd is proposed in Scheme S1. To confirm this sensing mechanism, the reaction
mixture of probe NIR-Ratio-CO system with CORM-3 was analyzed by thin-layer
chromatography (TLC) and high-resolution mass spectrometry. As shown in Figures
S4-5, the red fluorescent product in TLC plate and the peak with a mass of 288.1487
in the mass spectrum clearly showed that DCI-NH2 was formed.
3
.6. The effect of pH
The pH effects of the probe for NIR fluorescent detecting CO was also investigated.
2
+
As presented in Figure S6a, the NIR-Ratio-CO solution in the presence of Pd
shows very weak fluorescence at 655 nm and the fluorescence is not pH sensitive.
However, upon addition of CORM-3, the NIR fluorescence of the probe solution (5
1
0

2
+
µM NIR-Ratio-CO + 10 µM Pd ) showed significant enhancement over a wide
range of pH, and the best pH was found around the physiological pH (~7.0). Similar
results can be obtained from the change of fluorescence intensity ratio (I /I ) under
6
55 592
different pH (Figure S6b). This clearly indicates that NIR-Ratio-CO has great
potential for working in living systems.
3
.7. Bioimaging applications
An evaluation of the ability of probe NIR-Ratio-CO to image CO in living cells
was carried out with a fluorescence confocal microscope. A standard 3-(4,
-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was first
conducted with cells treated with diff erent concentrations of probe NIR-Ratio-CO
10, 20, 30, 40, and 50 µM). As shown in Figure S7, even after incubation with 50 µM
probe, more than 80% of the cells still survive either in the absent or in the presence
5
(
2+
of two equiv. of Pd , indicating that the probe shows low cytotoxicity. In the
bioimaging experiments, the cells were exposed to 0, 10, 20, and 30 µM of CORM-3
for 30 min, washed with PBS, and then stained with a 5 µM probe NIR-Ratio-CO
solution (with 10 µM PdCl ) for 30 min before confocal microscope images were
2
taken (Figure 4). One can see that, the cells incubated with only the probe emitted
fluorescence in the green channel but hardly any fluorescence in the red channel.
However, with exposure to an incremental amount of CORM-3, the fluorescence
emission decreased gradually in the green channel, accompanied by an increase of
fluorescence in the red channel. Correspondingly, the ratiometric images showed that
the F /F
value increased from 0.12 to 4.31. These results indicate that probe
red green
1
1

NIR-Ratio-CO is capable of ratiometric detection of exogenous CO in living cells.
Figure 4]
[
The capability of probe NIR-Ratio-CO to image endogenous CO in living cells
was also investigated. It is known that endogenous CO is generated from the
enzymatic degradation of heme catalyzed by heme oxygenases [2,44]. In this work,
heme was used to stimulate endogenous CO generation [23, 29, 32]. As shown in
Figure 5, when HeLa cells were pre-incubated with 100 µM heme and then indicated
with the probe system, the fluorescence in the green channel decreased gradually over
the incubation time of heme from 0 h to 8 h (images E1, F1 ,G1, and H1), meanwhile,
the fluorescence in the red channel showed an obvious increase over time.
Correspondingly, with the increase of heme-stimulation time, the ratiometric images
showed the F /F
value increased from 0.32 to 2.98, indicating the intracellular
red green
CO level was gradually increased. This result showed that NIR-Ratio-CO can be
used for ratiometric imaging of endogenous CO produced by heme-stimulation in
living cells.
[
Figure 5]
Conclusions
In summary, a NIR ratiometric fluorescent probe NIR-Ratio-CO was successfully
developed for the detection of CO in living cells based on the Pd-triggered Tsuji-Trost
reaction. The probe system shows rapid, colorimetric and distinct ratiometric
fluorescence responses at 655/592 nm for CO in aqueous solution with desirable
properties including high selectivity, good sensitivity and a remarkable large Stokes
1
2

shift. In addition, NIR-Ratio-CO was proved to be capable of ratiometric fluorescent
imaging of both exogenous and endogenous CO in living cells. Taken together, this
work provided a valuable ratiometric NIR fluorescent probe for rapid detection of CO
in living systems.
Acknowledgment
Financial support from the National Natural Science Foundation of China (21672080
and 21472066) and the Fundamental Research Funds for the Central Universities
(CCNU19TS007) is gratefully acknowledged.
Appendix
Structure characterizations for the probe, data for study the sensing mechanism, and
other data, please refer to supplementary data.
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Scheme 1. NIR ratiometric fluorescent probe NIR-Ratio-CO for detection of CO.
1
9

Scheme 2. Synthesis route for NIR-Ratio-CO
Figure 1. (a) UV-vis and (b) fluorescence spectral changes of the probe system (5 µM
NIR-Ratio-CO + 10 µM PdCl ) upon addition of CORM-3 (100 µM) in PBS buffer
2
(
10 mM, pH 7.4, 20% DMSO, v/v) at 37 ff . For fluorescence measurement, λ = 440
ex
nm, slit width: d = d = 10 nm. Inset: color changes and emission color changes
ex
em
under a portable 365 nm light.
(
a) 800
(b)
8
7
6
5
4
3
2
1
0
CO
CO
600
400
200
0
others
others
5
00
600
700
800
0
5
10 15 20 25 30 35 40 45 50
Analytes
Wavelength (nm)
Figure 2. (a) Fluorescent spectra and (b) fluorescent intensity ratio (I /I )
6
55 592
responses of the probe system (5 µM NIR-Ratio-CO + 10 µM PdCl ) for various
2
analytes (100 µM each, except 1 mM of GSH) including: (1) NIR-Ratio-CO itself, (2)
2
+
−
−
−
−
−
2−
−
NIR-Ratio-CO + Pd , (3) F , (4) Cl , (5) Br , (6) I , (7) NO , (8) CO , (9) HCO ,
3
3
3
−
2−
2−
−
2−
2−
−
(
(
(
(
10) HSO , (11) SO , (12) S O , (13) HSO , (14) S O , (15) S O , (16) SCN ,
4
3
2
3
3
2
7
2
8
−
−
2−
4
17) Ac , (18) N , (19) C O , (20) Cys, (21) Hcy, (22) GSH, (23) H S, (24) Met,
3
2
2
25) Thr, (26) Arg, (27) Tyr, (28) Ala, (29) Asp, (30) Lys, (31) Pyr, (32) Phe, (33) Glu,
34) Ile, (35) His, (36) Gly, (37) Val, (38) Leu, (39) Trp, (40) Ser, (41) H O , (42)
2
2
−
t
•
•
•−
ClO , (43) NaNO , (44) BuOO , (45) ROO , (46) HNO, (47) NO, (48) O , (49)
2
2
2
0

−
ONOO , (50) CO (CORM-3). All spectra were obtained 15 min after mixing in PBS
buffer (10 mM, pH 7.4, 20 % DMSO, v/v) at 37 ff , λ = 440 nm, d = d = 10 nm.
ex
ex
em
(
a) 800
8
6
4
2
0
(b) 3.5
.0
0
-100 µM
3
600
400
200
0
2.5
2.0
0
20
40
60
80
100
CORM-3 (µM)
1.5
1.0
0.5
y = 0.625 + 0.256*[CO]
2
R
= 0.9977
5
00
600
700
Wavelength (nm)
800
0
2
4
6
M)
8
10
[CO] (
µ
Figure 3. (a) Fluorescent spectral changes of the probe system (5 µM NIR-Ratio-CO
10 µM PdCl ) upon addition of different concentrations of CORM-3. Each spectrum
+
2
was obtained 15 min after mixing. Inset: the saturation curve of the fluorescence
2
intensity ratio changes. (b) Linear relationship (I = 0.625 + 0.256 × [CORM-3], R =
0
.9977) of the fluorescence intensity ratios (I /I ) to CO concentrations from 0 to
655 592
1
0 µM. All data were obtained in PBS buffer (10 mM, pH = 7.4, with 20% DMSO,
v/v) at 37 ff . λ = 440 nm, d = d = 10 nm.
ex
ex
em
Figure 4. Confocal fluorescent imaging of CO in HeLa cells by the probe system (5
µM NIR-Ratio-CO + 10 µM PdCl ). A, A1, A2 and A3: the cells were incubated with
2
2
1

the probe system for 30 min. B, B1, B2 and B3, C, C1, C2 and C3, and D, D1, D2 and
D3: the cells were pre-incubated with 10, 20 and 30 µM of CORM-3 for 30 min, then
with the probe system for 30 min, respectively. Scale bar = 10 µm. Green channel: λex
=
458 nm, λem was collected at 549-620 nm. Red channel: λ = 458 nm, λem was
ex
collected at 620-721 nm. A histogram on the right shows the quantitative information
of fluorescence ratio of the red channel and the green channel (F /Fgreen) upon
red
incubation of different concentrations of CO.
Figure 5. Confocal imaging of endogenous CO via heme stimulation in HeLa cells
with the probe NIR-Ratio-CO system (5 µM probe + 10 µM PdCl ). E-H: Bright
2
field images. E1-H1: Green channel (λ = 458 nm, λem = 549-620 nm). E2-H2: Red
ex
channel (λ = 458 nm, λ = 620-721 nm). E3-H3: Merged images of green and red
ex
em
channel. The cells were pre-incubated with 100 µM heme for 1 h (F, F1, F2 and F3), 4
h (G, G1, G2 and G3), and 8 h (H, H1, H2 and H3), and then incubated with the probe
system for 30 min, respectively. Scale bar = 10 µm. A histogram on the right shows
the quantitative information of fluorescence ratio of the red channel and the green
channel (F /Fgreen) upon incubation of heme at different times.
red
2
2

Highlights
1
2
3
4
. A NIR ratiometric fluorescent probe for detection of CO was reported.
. This probe shows enhanced fluorescence in the NIR region with a large Stokes shift.
. This probe shows high selectivity and sensitivity for CO with a rapid response.
. Imaging both exogenous and endogenous CO in living cells was successfully applied.
1

Declaration of interests
☒
The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐
The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: