An endoplasmic reticulum targetable turn-on fluorescence probe for imaging application of carbon monoxide in living cells

Article abstract of DOI:10.1016/j.saa.2020.119150

Carbon monoxide (CO) is a significant mediator in regulating endoplasmic reticulum (ER) stress, and its level may play a potential role in the treatment of vascular diseases combined with ER stress. In-situ visualization of CO in the ER helps to elucidate

Full text of DOI:10.1016/j.saa.2020.119150

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An endoplasmic reticulum targetable turn-on fluorescence probe for imaging
application of carbon monoxide in living cells
Yunyan Zhang, Yonghe Tang, Xiuqi Kong, Weiying Lin
PII:
S1386-1425(20)31129-X
https://doi.org/10.1016/j.saa.2020.119150
SAA 119150
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26 October 2020
Please cite this article as: Y. Zhang, Y. Tang, X. Kong, W. Lin, An endoplasmic reticulum targetable turn-on
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Molecular and Biomolecular Spectroscopy (2020), doi: https://doi.org/10.1016/j.saa.2020.119150
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An endoplasmic reticulum targetable turn-on
fluorescence probe for imaging application of carbon
monoxide in living cells
Yunyan Zhang a, Yonghe Tang b, Xiuqi Kong a, and Weiying Lin a, b*
a
Institute of Fluorescent Probes for Biological Imaging, School of Chemistry and
Chemical Engineering, School of Materials Science and Engineering, University of
Jinan, Jinan, Shandong 250022, P.R. China.
b
Institute of Optical Materials and Chemical Biology, School of Chemistry and
Chemical Engineering, Guangxi University, Nanning, Guangxi, 530004, P. R. China
*Corresponding author. Tel.: +86 53182769108; Fax: +86-531-82769031
E-mail address: weiyinglin2013@163.com.
1

Abstract
Carbon monoxide (CO) is a significant mediator in regulating endoplasmic
reticulum (ER) stress, and its level may play a potential role in the treatment of
vascular diseases combined with ER stress. In-situ visualization of CO in the ER
helps to elucidate its physiological and pathological mechanistic behavior. Herein, a
novel CO fluorescent probe (Na-CM-ER) with ER-targeting characteristics was
structured. Na-CM-ER with naphthalimide as a fluorescent group, under the trigger
of CO, an ICT (Intramolecular Charge Transfer) mechanism was constructed by
converting a nitro group to an amino group and showed dazzling green fluorescence.
Na-CM-ER exhibited satisfactory response speed, selectivity, photo-stability and
sensitivity to CO in vitro. Furthermore, biological imaging experiments demonstrated
that Na-CM-ER could monitor the changes of exogenous/endogenous CO in living
cells and possess an ER-targeting property. To sum up, we hope that Na-CM-ER can
be as a serviceable molecular tool for imaging CO in cellular ER.
Key Words: Carbon monoxide; Fluorescent probe; Endoplasmic Reticulum; Turn-on;
Imaging.
2

1. Introduction
Carbon monoxide (CO) is known as an odorless and colorless gas. Medical studies
have shown that CO can bind the same site to hemoglobin as oxygen does, and has
higher affinity to hemoglobin (around 210 times) than oxygen (O2) [1]. After entering
the organism, CO is easily combined with hemoglobin to produce carboxyhemoglobin,
which causes hemoglobin to lose its ability and function to carry oxygen, causing
tissue asphyxia [2]. Recent years, CO has been found to be an endogenous gas
signaling molecule in plants and animals, which shoulders a major role in maintaining
the normal operation of animal and plant life [3-5]. In organisms, heme is catalyzed
by heme oxygenase (HO) to produce endogenous CO [6]. In addition, related studies
have found that abnormal endogenous CO concentration is closely related to the
occurrence and deterioration of various diseases, including neurodegenerative
diseases, cardiovascular diseases, lung diseases, inflammation, obesity, sepsis and
cancer [7-9]. Therefore, it is important to design new methods to detect and perceive
the physiological/pathological functions of carbon monoxide selectively and visually
in living systems [10,11].
Endoplasmic reticulum (ER) is the largest organelle with single membrane
structure in eukaryotic cells [12]. The membrane area of ER accounts for at least half
of the total membrane system of eukaryotic cells. ER is involved in important
physiological functions, including synthesizing and modifying protein, stabilizing the
Ca2+ and K+ level [13]. ER participates in life activities with other organelles such as
mitochondria, cell membranes, Golgi apparatus, and lipid droplets in cells [14,15]. It
is noteworthy that the HO which produces endogenous CO is mainly distributed in
ER of the living cells. Accumulation of unfolded proteins, hypoxia and viral
infections could cause ER stress, which leads to ER dysfunction. Exogenous or
endogenous CO could inhibit associated proteins (X-box binding protein 1 and
protein kinase R-like endoplasmic reticulum kinase, PERK) expression, and make
endoplasmic reticulum to restore homeostasis and maintain normal physiological
function [16-18]. However, the detail regulatory mechanisms of CO for ER have not
3

been well defined. Thus, it is in an urgent need for develop effect tools for tracing CO
in ER.
The traditional CO detection methods, including chromogenic detection,
absorption spectroscopy, chromatography, electrochemical assays [19-21], need to
destroy the tested samples and cannot realize the in-situ detection of CO. In contrast,
fluorescence microscopy imaging is a frontier crossover technique for in situ
detection of bioactive molecules [22-25]. Although several CO fluorescent probes
have been developed in recent years [26], to our best knowledge, there is no CO
fluorescent probe targeting ER up to date, thus disclosing the physiological function
of CO in the ER remains very challenging.
Herein, we firstly developed an intelligent ER-specific fluorescent probe
(Na-CM-ER) for imaging of cellular CO. The classic dye naphthalimide dye in
Na-CM-ER was selected as the fluorescence platform, and the labeled methyl
sulfonamide segment as the ER-targeting moiety. Nitro group was employed as a
smart identification site due to the selective "turn-on" response to CO. Na-CM-ER
could selectively detect CO in vitro by reducing nitro group to a highly fluorescent
amino functional derivative (Na-ER-NH). Furthermore, biological imaging
experiments demonstrated that Na-CM-ER could monitor the changes of
exogenous/endogenous CO in living cells and possessed an ER-targeting property.
We expect Na-CM-ER to be a serviceable molecular tool for exploring the
physiologic action of CO in living cellular ER.
2. Experimental section
4

Scheme 1. Synthesis routes for Na-CM-ER.
2.1. Synthesis
Na-CM-ER was synthesized by four steps reaction (Scheme 1). The compound 2
was synthesized according to the previous synthesis method of our research group
[27]. The 1,8-naphthalic anhydride underwent the nitration reaction yielding the
compound 4 [28], which reacted with compound 2 underwent a condensation reaction
1
obtaining the probe Na-CM-ER. The Structural characterization data, including H
NMR, 13C NMR and HR-MS (High Resolution Mass Spectrometry) were provided in
Supporting Information.
2.1.1. Synthesis of the compound Na-CM-ER
Compound 4 (0.216 mmol) and compound 2 (0.216 mmol) were dissolved in 2 mL
ethanol, the mixture was reacted at 80 °C for 2 h with nitrogen protection. Then the
mixture was dissipated to 0 °C and the precipitate was filtered out and washed by
methanol and cyclohexane, and dried to obtain compound Na-CM-ER with a yield of
81 %. 1H NMR (400 MHz, DMSO-d6) δ 9.48 (s, 1H), 8.91 (s, 1H), 8.77 (d, J = 8.0 Hz,
1H), 8.64 (d, J = 4.0 Hz, 1H), 8.05 (t, J = 8.0 Hz, 1H), 7.80 (t, J = 4.0 Hz, 1H), 7.57
5

(d, J = 8.0 Hz, 2H), 7.23 (d, J = 4.0 Hz, 2H), 4.12 (t, J = 8.0 Hz, 2H), 3.11 (q, J =8.0
13
Hz, 2H), 2.25 (s, 3H). C NMR (100 MHz, DMSO-d6) δ 164.1, 163.6, 147.1, 139.0,
137.5, 135.1, 132.1, 130.9, 130.8, 130.7, 130.5, 127.6, 125.4, 124.0, 123.9, 22.1.
HR-MS m/z calculated for C21H17N3O6S [M+H] +: 440.0916; found 440.0914.
2.1.2. Synthesis of the compound Na-ER-NH
The compound Na-CM-ER (88 mg, 0.2 mmol) and SnCl2 (135 mg, 0.6 mmol)
was dispersed in ethanol (10 mL), then added concentrated hydrochloric acid, the
mixture reacted at 80 °C for 3 h under nitrogen protection. After completing the
reaction, the pH was adjusted with 40% NaOH and then extracted with ethyl acetate.
Finally, the mixture was concentrated under vacuum to afford orange solid of the
1
compound Na-ER-NH with a yield of 53 %. H NMR (400 MHz, DMSO-d6) δ 8.04
(s, 1H), 8.03 (d, J = 4.0 Hz, 1H), 7.93 (d, J = 4.0 Hz, 1H), 7.76 (s, 1H), 7.58 (t, J = 6.0
Hz, 3H), 7.28 (d, J = 4.0 Hz, 1H), 7.25 (d, J = 8.0 Hz, 2H), 6.01 (s, 2H), 4.08 (t, J =
8.0 Hz, 2H), 3.05 (s, 2H), 2.28 (s, 3H). HR-MS m/z calculated for C21H19N3O4S
[M+H] +: 410.1163; found 410.1173.
2.2. Optical studies
Unless otherwise specified, all optical tests were conducted in PBS (10 mM,
pH=7.4, 10% DMSO as co-solvent) with Na-CM-ER (10 μM). CORM-2 (A
commercial CO release reagent) was prepared in dimethyl sulfoxide (DMSO), and the
analytes stock solutions were prepared in the ultrapure water at the appropriate
concentration. HITACHI F4600 fluorescence spectrophotometer was used to perform
fluorescence spectrum test. Excitation wavelength was set to 430 nm, and the slit
widths of excitation and emission were set to 5 nm.
2.3. Cell cytotoxic assays
HeLa cells were cultured in Dulbecco's Modified Eagle Medium (DMEM)
provided with 10% FBS in an atmosphere of 5% CO2 at 37 °C. HeLa cells were
planted into aseptic 96-well plates, and then 0-30 μM of Na-CM-ER and Na-ER-NH
6

was added respectively. Subsequently, cells were incubated for 24 h in the same
environment. Finally, the absorbance of the solution was acquired by using the
microplate reader at 570 nm. The cell viability was calculated by the following
formula:
Cell
viability
(%)
=
(OD570(sample)-OD570(blank))/(OD570(control)-OD570(blank))*100%. OD570 sample
denotes the cells incubated with various of concentrations of Na-CM-ER and
Na-ER-NH, OD570 control denotes the cells without the probes, OD570 blank
denotes the wells containing only the culture medium.
3. Results and discussion
3.1. Design of probe Na-CM-ER
Nitro group has electron-withdrawing feature, which could hold electron
distribution as an electron acceptor for fluorescent switch, and easily reduced by the
reducing agents such as CO [29]. By installing a nitro group and an ER targeting
group, the methyl sulphonamide group, on to the 1,8-naphthalimide fluorescent
platform, a ER-targetable turn-on fluorescent probe (Na-CM-ER) was constructed.
Na-CM-ER exhibited non-fluorescent due to the strong electron-withdrawing ability
of the nitro group, the electron structure performed as “A-π-A”, which suppressed the
intramolecular charge transfer (ICT) effect. When CO was introduced, the nitro group
was reduced to amino group due to the reducibility of CO, the electronic structure was
transformed into D-π-A. Then the ICT process was recovered, and the fluorescent
signal was generated (Scheme 2).
7

Scheme 2. The recognition mechanism of CO by ER-targeted CO fluorescent probe
Na-CM-ER.
3.2. Photophysical properties of Na-CM-ER
With the ER-target CO fluorescent probe Na-CM-ER in hand, we firstly
conducted fluorescence titration experiments with Na-CM-ER (10 μM) and various
concentrations of CORM-2 (the concentration range of CORM-2 was 0-250 μM) in
PBS (10 mM, pH=7.4, 10% DMSO as co-solvent). At first, as expected, the
fluorescent intensity of Na-CM-ER showed no fluorescence because of the strong
electron-withdrawing ability of the nitro group. Satisfactorily, when CO was added,
the fluorescence was generated at 520 nm due to the ICT effect appeared (Fig. 1A),
and the intensity gradually enhanced with the increasing of CO concentrations (Fig.
1B). Fluorescence of probe reached saturation after addition with 210 μM CORM-2,
and the signal enhancement enhanced 143-fold. Besides, fluorescent intensity of
Na-CM-ER at 520 nm enhanced linearly with CORM-2 ranging from 0-100 μM, and
the detection limit of probe for CORM-2 was measured to be 0.42μM (3σ/slope). The
results indicate that the novel probe Na-CM-ER is ultrasensitive to CO with high
signal-to-noise ratio, which is suitable for detecting CO.
8

Fig. 1. (A) Fluorescent curves of Na-CM-ER (10 μM) encountering with CORM-2
(0-250 μM) in 10 mM PBS buffer (pH 7.4, 10 % DMSO v/v). Inset shows the
fluorescent intensity changes of Na-CM-ER (10 μM) at I520 with CORM-2 (0-250
μM). (B) Linear relationship of between intensity and CORM-2 concentration. λex =
430 nm. Error bars represent standard deviation (±S.D.), n =3.
Subsequently, the time-dependent study of Na-CM-ER with CO was conducted.
In the presence of CORM-2 (210 and 40 μM), the fluorescent intensity of Na-CM-ER
at 520 nm increased gradually and reached the equilibrium point after 70 min (Fig. 2
and Fig. S1). Besides, the photo-stability study of Na-CM-ER was also carried out
with or without CORM (210 and 40 μM). After continuous irradiation for 60 min, the
fluorescence intensities of free Na-CM-ER (10 μM) and coexisting CORM-2 (210
and 40 μM) at 520 nm showed little changes (Fig. 3 and Fig. S2). What is more, the
stability of probe Na-CM-ER in serum was also performed. The results in Fig. S3
showed that the fluorescence had satisfactory stability in 10% serum, which was the
concentration of the general cell culture. This offers a well property for further
biological applications. These data indicate that Na-CM-ER has fast recognition
speed for CO and excellent photo-stability.
9

Fig. 2. (A) Time-dependent fluorescence spectra of Na-CM-ER (10 µM) treated with
CORM-2 (210 μM). (B) Fluorescent intensity changes of Na-CM-ER at I520 treated
with CORM-2 (210 μM). Error bars represent standard deviation (±S.D.), n =3. λex =
430 nm..
Fig. 3. Photo-stability profiles of Na-CM-ER (10 μM) in the absence or presence of
CORM-2 (210 μM). Error bars represent standard deviation (±S.D.), n =3. λex = 430
nm.
Then the reactive mechanism was investigated. As above-mentioned, the assumed
reaction between Na-CM-ER and CO could produce Na-ER-NH. Thus, the HRMS
and optical septra were used to test this assumption. Firstly, the HRMS was obtained
after the reaction of Na-CM-ER (10 μM) in the presence of CO. As shown in the Fig.
10

S4, the data showed primary signal at 410.1051 (calcd 410.1096), which could be
assigned to [Na-CM-ER+H]+. Secondly, the fluorescence spectra of the reaction
between Na-CM-ER and CO (CORM-2) in PBS (10 mM, pH=7.4, 10% DMSO as
co-solvent) was carried out. After Na-CM-ER interacting with CO, an obvious
fluorescence emission appeared at 520 nm, which was consistence with the maximum
emission peak of the compound Na-ER-NH (Fig. S5). These results directly confirm
our hypothesis that Na-CM-ER can interact with CO through the nitro-reduction
reaction to form Na-ER-NH.
Fig. 4. Fluorescent intensity (I520) changes of Na-CM-ER (10 μM) in the presence of
different biological relevant analytes. The numbers represent: (1) CORM-2，(2) NaHS,
(3) Cys, (4) CaCl2, (5) GSH, (6) NaClO, (7) NaNO2, (8) NaHSO3, (9) KCl, (10)
N-Acetylglycine, (11) free probe, (12) tert-butyl hydroperoxide, (13)
-
N-Acetyl-L-cysteine, (14) ONOO , (15) NO, (16) sodium pyruvate, (17)
formaldehyde, (18) trichloracetic aldehyde, (19) H2O2, (20) acetone. Error bars
represent standard deviation (±S.D.), n =3. λex = 430 nm.
The constructed probe should have exclusive selectivity so that the target molecule
can be detected effectively, thus the selective experiments of Na-CM-ER (10 μM)
11

with different biological relevant analytes were conducted. Other related analytes
including typical representative amino acid (1 mM GSH, 210 μM Cys, 210 μM
N-Acetylglycine, 210 μM N-Acetyl-L-cysteine (NAC)), inorganic salts (210 μM
NaHS, 210 μM NaNO2, 210 μM NaHSO3, 210 μM KCl, 210 μM CaCl2), reactive
oxygen species (210 μM H2O2, 210 μM tert-butyl hydroperoxide (TBHP), 210 μM
-
NaClO, reactive nitrogen species (210 μM NO, 210 μM ONOO ), reactive carbonyl
species (210 μM sodium pyruvate, 210 μM trichloracetic aldehyde, 210 μM acetone,
210 μM formaldehyde (FA)), 210 μM CORM-2 were added into the mixture
containing Na-CM-ER for 70 min. As shown in the Fig. 4, fluorescent signals caused
by other analytes had little changes, and were almost the same as that of the free
probe. However, the signal had distinct enhanced coexisting with CO. Therefore,
Na-CM-ER has well selectivity with CO, which is suitable for detecting exclusive
CO in complex biological environments.
To evaluate whether Na-CM-ER is applicable to detecting CO at physiological
pH, the fluorescence emission spectra of Na-CM-ER in the presence and absence of
CO (210 and 40 μM) in PBS with different pH values (4.5-10.0) were measured.
Fluorescent intensities of Na-CM-ER in different pH (7.0-8.0) buffer reached
maximum, and the fluorescent intensities were consistent with that of concentration
titration results. Thus, Na-CM-ER is favorable for tracing CO in physical
environments (Fig. S6 and Fig. S7).
3.3. Confocal fluorescence imaging
Fluorescent spectra tests show that the probe Na-CM-ER has high sensitivity and
selectivity, well photo-stability, which are benefit for further biological imaging
research. The cellular cytotoxicity of Na-CM-ER was firstly tested. MTT assay
showed Na-CM-ER and Na-ER-NH had no obvious toxic in living HeLa cells at the
different concentrations (0-30 μM), indicating that Na-CM-ER could be used in
biological sample for tracking CO (Fig. S8). Next, we investigated the ability for
detecting exogenous CO in a complex cellular environments. HeLa cells were
12

incubated with culture media containing 10 μM Na-CM-ER for 30 min, then imaged
using a fluorescence confocal microscope. As shown in Fig. 5a-c, cells showed no
fluorescence excited at 405 nm. In stark contrast to phenomenon of free Na-CM-ER
in cells, strong fluorescent signals were observed in HeLa cells further treated with
CORM-2 (40 μM) (Fig. 5d-f). Quantified mean fluorescence intensities were further
confirmed these profiles (Fig. 5g). The cellular imaging results are the same as those
of spectral experiments, which show that the Na-CM-ER has ability for tracking
exogenous CO.
Fig. 5. Fluorescent images of probe Na-CM-ER in HeLa cells for detecting
exogenous CO. (a-c) Cells were treated with free Na-CM-ER (10 μM) for around 30
min; (d-f) Cells were treated with Na-CM-ER (10 μM) for 30 min and further
incubated with CORM-2 (40 μM)
for another 30 min; (g) Quantified mean
fluorescence intensities of cells in the green channel. λex = 405 nm, λem= 500-550 nm,
scale bar = 20 μm.
Next, we tried to test the feasibility of monitoring CO with time-resolved mode
using our probe Na-CM-ER. After addition of CORM-2 (40 μM), imaging was
immediately capatured with confocal microscopy. The results were exhibited in Fig.
S9, observable signals was detected within 2 min at first, demonstrating that probe
can quickly react with CO in cells. With the extension of time, the fluorescence in
cells gradually became much brighter and reached saturation at around 30 min.
13

Moreover, after another 40 min visualization, the fluorescence had little fluctuations,
suggesting that Na- ER-NH, the product of reaction between Na-CM-ER and CO,
has satisfactory photostabilty in living cells. Thus, probe Na-CM-ER can detect CO
with time-solved manner.
After acquiring achievements that Na-CM-ER is capable of tracking exogenous
CO in living cells, we continued to verify the applications of visualizing endogenous
CO in living cells. It is well known endogenous CO is generated by an HO-2
mediated heme, thus, heme is an inducer of endogenous CO [30, 31]. Like the
behaviors of cells treated with free Na-CM-ER (10 μM) in exogenous CO detecting
experiments, cells pretreated with only Na-CM-ER (10 μM) for 30 minutes produced
dull green fluorescence (Fig. 6a-c). However, after the stimulation of 100 μM heme
for 2 h (4 h), cells then loaded with 10 μM Na-CM-ER for further 30 min showed
marked green fluorescence, and the intensities increased with extension of incubation
time of heme (Fig. 6d - i).
After achieving in tracing endogenous CO by stimulation using heme, we next
turned our attention on capturing CO triggered by hypoxia, which is another effective
manner for generation of endogenous CO. During the experiments, the cells were
cultured in hypoxia incubator with 5%. The results were shown as Fig. S10. At 5%
hypoxia incubator, cells firstly cultured for 12 h, then treated with Na-CM-ER (10
μM) for 30 min displayed marked green fluorescence excited at 405 nm, indicating
that Na-CM-ER can detect endogenous CO under hypoxia conditions. Therefore, all
results clearly suggest that Na-CM-ER is capable of tracing endogenous CO in living
cells.
14

Fig. 6. Fluorescent images of probe Na-CM-ER in living HeLa cells for detecting
endogenous CO. (a-c) Cells were treated with Na-CM-ER (10 μM) for 30 min; (d-f)
Cells were firstly treated with heme (100 μM) for 2 h, and then further incubated with
Na-CM-ER (10 μM) for another 30 min; (g-i) Cells were firstly treated with heme
(100 μM) for 4 h, and further incubated with Na-CM-ER (10 μM) for another 30 min.
(j) Quantified mean fluorescence intensities of cells in the green channel. λex = 405 nm,
λem = 500-550 nm. Scale bar = 20 μm.
Inspired by successful results in imaging endogenous CO using Na-CM-ER, we
further performed co-localization experiments to determine the distribution of
Na-CM-ER in cells. Firstly, cells were incubated with 10 μM Na-CM-ER for 30 min,
then CORM-2 (40 μM) was added into cells and further incubated for another 30 min.
After that, ER-Tracker Red (a commercial ER indicator) was put into media for
incubation with 10 min according to protocols. The images in Fig.7 showed that the
fluorescence of ER-Tracker Red in the red channel overlapped well with the
fluorescence of Na-CM-ER in the green channel. Moreover, the Pearson’s correlation
coefficient between them was calculated to be 0.97. Therefore, the results of the
co-localization imaging show that Na-CM-ER has a property for anchoring
endoplasmic reticulum in living cells, and can detect CO in endoplasmic reticulum.
15

Fig. 7. Co-localization imaging between probe Na-CM-ER and ER-Tracker Red. a)
Fluorescence imaging of cells incubated with Na-CM-ER (10 μM) for 30 min, then
further loaded with CORM-2 (40 μM) for another 30 min in green channel; b)
Fluorescence imaging of cells treated with ER-tracker Red in red channel; c) The
merged imaging of a and b; d) Intensity scatter plot of the green and red channels; e)
Intensity profile changes of red channel of ER-Tracker Red and green channel of
probe. The green channel: λex = 405 nm, λem= 500-550 nm; the red channel: λex = 561
nm, λem= 570-620 nm. Scale bar: 20 μm.
4. Conclusions
In conclusion, we have rationally designed and synthesized the first ER-targeted
turn-on CO fluorescent probe, named as Na-CM-ER. Based on the nitro reduction,
the probe Na-CM-ER emits a strong fluorescence after interacting with CO.
Na-CM-ER possesses high sensitivity and selectivity to CO during the spectrum
testing. Furthermore, Na-CM-ER has the ability to detect exogenous and endogenous
CO in living cells. Importantly, Na-CM-ER can effectively accumulate in cellular ER
verified by the co-localization imaging experiments. Taken together, Na-CM-ER is a
powerful molecule tracking reagent for imaging CO, especially CO in ER, and will
play vital roles in further exploring the physiological and pathological functions
related to ER CO.
16

Acknowledgements
This work was financially supported by NSFC (21672083, 21877048, 21807039,
22077048), Taishan Scholar Foundation (TS 201511041) and the startup fund of
University of Jinan (309-10004, 160100137)
Declaration of competing interest
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.
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Biographies
Yunyan Zhang obtained her B.S. degree at University of Jinan in 2018. Currently,
she is a graduate student in the Institute of Fluorescent Probes for Biological Imaging,
University of Jinan, and her supervisor is Professor Weiying Lin. Her research
interests focus on the design and synthesis of novel functional fluorescent probes and
their biological applications.
Yonghe Tang got his doctor’s degree at University of Jinan in 2019. Now, he is a
lecturer in the Institute of Optical Materials and Chemical Biology, School of
Chemistry and Chemical Engineering, Guangxi University. His research interests
mainly include the design and synthesis of fluorescent probes.
Xiuqi Kong got her doctor’s degree at Nankai University in 2015. Now, she is a
lecturer in the Institute of Fluorescent Probes for Biological Imaging, University of
Jinan. Her research interests mainly including the design, synthesis and biological
applications of fluorescent probes.
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Professor Weiying Lin received his Ph.D. from the University of Kansas in 2000.
After completing postdoctoral research at Massachusetts Institute of Technology, in
2005, he joined the faculty at Hunan University. Subsequently, he moved to the
University of Jinan as the Dean and the Distinguished Professor of the Institute of
Fluorescent Probes for Biological Imaging. His research interests cover the
interdisciplinary areas of molecular recognition, photochemistry, analytical chemistry,
and chemical biology.
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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:
23

Graphic Abstract
The first endoplasmic reticulum-targeted fluorescent probe (Na-CM-ER) was
designed and synthesized for monitoring carbon monoxide (CO) in endoplasmic
reticulum of the living cells.
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Highlights
1) The probe Na-CM-ER is the first endoplasmic reticulum-targeted
fluorescent probe for CO.
2) The probe Na-CM-ER shows high selectivity and sensitivity to CO.
3) The probe Na-CM-ER is applied for sensing exogenous/endogenous
CO in the living cells.
4) The probe Na-CM-ER is successfully employed to monitor CO in
subcellular endoplasmic reticulum.
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