A naphthalimide-based fluorescence ‘‘turn-on'' chemosensor for highly selective detection of carbon monoxide: imaging applications in living cells

Article abstract of DOI:10.1039/c8nj02552e

A naphthalimide-based fluorescence chemosensor, COFP, was designed and synthesized for the detection of carbon monoxide (CO) in HEPES buffer (pH 7.4, 37 °C). The detection process is highly selective towards CO, over a variety of relevant reactive oxygen,

Full text of DOI:10.1039/c8nj02552e

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Naphthalimideꢀbased fluorescence ‘‘turnꢀon’’ chemosensor for
highly selective detection of carbon monoxide: imaging applications
in live cell
Biswajit Das,a Somenath Lohar,b Ayan Patra,b Ejaj Ahmmed,b Sushil Mondal,c
Jatindra Nath Bhakta,c Koushik Dharad,* and Pabitra Chattopadhyayb
a
Department of Chemistry, Sreegopal Banerjee College, Bagati, Magra, Hooghly
712148, West Bengal, India
b Department of Chemistry, The University of Burdwan, Golapbag, Burdwan 713104,
West Bengal, India
c Department of Ecological Studies & International Center for Ecological Engineering
(ICEE), University of Kalyani, Kalyani, Nadia, West Bengal, India
d Department of Chemistry, Sambhu Nath College, Labpur, Birbhum 731303, West
Bengal, India
* Corresponding author’s address: Department of Chemistry, Sambhu Nath College,
Labpur, Birbhum 731303, West Bengal, INDIA; Email: dharachem@gmail.com (Dr.
Koushik Dhara), Mob: +91ꢀ9433125327

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Abstract
A naphthalimideꢀbased fluorescence chemosensor, COFP, was designed and
synthesized for the detection of carbon monoxide (CO) in HEPES buffer (pH 7.4, 37
°C). The detection process is highly selective towards CO, over a variety of relevant
reactive oxygen, nitrogen, and sulfur species. Also, the fluorescence response is not
hampered in the presence of biologically relevant metal ions and/or species. The
detection method was achieved through the transformation of the nitro group of
COFP to the amino functionalized derivative in the presence of CO without using any
heavy metals e.g. Pd, Rh, Ru etc. The chemosensor prompted a ‘turnꢀon’ fluorescence
response to CO with a simultaneous increase of fluorescence intensity by more than
33ꢀtimes. The LOD (limit of detection) was calculated as low as 123 nM using 3σ
method. Moreover, the chemosensor is capable to monitor the changes in intracellular
CO in C6 Glioma cell line.
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Introduction
Carbon monoxide (CO) has been considered as a hazardous gas and
environmental toxicant because of its strong affinity to bind with hemoglobin and
high concentrations of CO (above 70 ppm) can induce both acute and chronic health
hazards.1 Despite its toxic status towards mammals, recent studies have shown that
CO acts as essential gasotransmitter molecule in the body.2,3 CO generally released in
biological system through the catabolism of heme by heme oxygenase (HOꢀ1) and
behaves as a important gas message molecule and attracts attention for its significant
regulatory effect on pathophysiological process.4ꢀ6 CO, a gasotransmitter molecule,
was considered to be an significant versatile signaling bioꢀmolecule protecting from
inflammatory, vascular and cancer diseases.7 The generated CO by heme oxygenase
enzyme, has been shown to prevent both acute and chronic hypertension and also
vasoconstriction through the stimulation of soluble guanylate cyclase.8,9 Endogenous
CO prevents human airway smooth muscle cell proliferation10 protects against
hyperoxia as well as ischemic lung injury11 and inhibits endothelial cell apoptosis.12,13
These aspects have gathered unparalleled attention, deepening the research of CO in
biology. Many biological and chemical aspects of CO remain inexpressible due to the
lack of ways for selective monitoring of this small molecule. Thus the improvement
and development of selective method for direct sensing of CO in living systems is of
great scientific interest.
Although some traditional approaches, such as colorimetric detection14ꢀ17 gas
chromatography18 and electrochemical analysis19 have been developed for CO
detection, these methods are difficult for sensing CO in living systems in a
noninvasive way. In contrast, fluorescence detection techniques using fluorescent
probes are highly attractive due to its high sensitivity and realꢀtime detection
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approach in a nondestructive manner. Recent reports revealed that a genetically
encoded fluorescent platform20 a carbazoleꢀcoumarin fused twoꢀphoton platform,21
and a few fluorescent chemoꢀsensors22ꢀ25 are capable of CO sensing in living systems.
We recently described a fluorogenic probe for selective sensing of CO in living cells
based on a Pd(0) mediated reaction.26 In fact, very recently, a few fluorescent probes
for CO detection have been reported to be able to detect CO in living cells, and all
these methods were designed based on transition metal species (e.g., Pd, Rh, Fe, etc.)
mediated reactions.20,24,27ꢀ32 In contrast, we just reported ‘‘targetable’’ fluorogenic
probe for CO detection in living system without using any heavy metals,33 but still,
highly selective and sensitive type ‘‘turnꢀon’’ systems that can perform without using
costly heavy metals are greatly desired. Therefore, there is an urgent need to develop
fluorescent chemosensor that can detect directly the production, distribution and
transportation of CO in biology in order to reveal its mechanistic behavior in
physiological and pathological systems.
In this work, we have designed a new simple fluorescence chemosensor
(COFP) based on 3-nitro naphthalimide developed as a fluorescent platform without
using any heavy metals e.g. Pd, Rh, Ru etc. Here the nitro group in the naphthalimide
system behaves as a CO responsive unit. COFP acts as selective chemosensor for CO
detection in pH 7.4 aqueous HEPES buffer at 37 °C through the transformation of the
nitro group into the highly fluorescent aminoꢀfunctionalized derivative. The detection
process is highly selective towards CO, over a variety of relevant reactive oxygen,
nitrogen, and sulfur species, and thus, it can be useful to detect this gasotransmitter
molecule in an aqueous buffer medium. Also, the fluorescence response is not
hindered in presence biologically and environmentally relevant metal ions and/or
species. The chemosensor prompted a ‘‘turnꢀon’’ fluorescence response to CO with a
4

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simultaneous increase of fluorescence intensity by more than 33ꢀtimes. Moreover, the
chemosensor is capable to monitor the changes in intracellular CO in C6 Glioma
cells.
Results and Discussion
Design and Synthesis of COFP
Synthetic route for the preparation of COFP was depicted in Scheme 1. In this
synthesis, the aniline moiety was attached via amide formation with the reaction of 3-
nitro-1,8-naphthalic anhydride. The chemosensor designing, we choose the 3ꢀnitro
group attached with the fluorophore such that the molecule displays very weak
fluorescence because the electron withdrawing nature of the nitro group can quench
the fluorescence in a good extent. The fluorescence quenching state may be exhibited
due to the photoinduced electron transfer (PET) experience from the naphthalimide
moiety to the nitro group and the weakened the intramolecular charge transfer (ICT)
process.34
Scheme 1. Synthetic route of the chemosensor, COFP.
Fluorescence properties of COFP towards CO detection and proposed
mechanism for sensing
The fluorescence response of 10 ꢁM COFP in HEPES buffer of pH 7.4 at 37
°C displays a very weak emission with a maximum at 522 nm under the excitation of
540 nm. This nonemissive (ΦF = 0.0018) nature COFP is due to the photoinduced
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electron transfer (PET) process operated from the naphthalimide fluorophore to the
nitro group. Then the chemosensor was treated with 100 ꢁM CORMꢀ3 (CORMꢀ3:
carbon monoxide releasing moleculeꢀ3, [Ru(CO)3Cl(glycinate)] 35) in the reaction
°
buffer (10 mM HEPES of pH 7.4, 2% DMSO) at 37 C the fluorescence intensity
remarkably enhanced with the progress of time. Finally, it reaches its maximum
value with a quantum yield, ΦF = 0.0522, after 45 min incubation in reaction buffer at
37 °C (Fig. 1). In this research
Fig. 1 Fluorescence spectrum of COFP (10 ꢁM) upon treatment with CORMꢀ3 (100
ꢁM) with the progress of time in reaction buffer [10 mM HEPES, 2 % DMSO] of pH
7.4 at 37 °C (ex: 440 nm).
work, we have used waterꢀsoluble complex, [Ru(CO)3Cl(glycinate)] (CORMꢀ3) as an
easyꢀtoꢀhandle CO source in buffer medium.35 The chemosensor prompted a ‘turnꢀon’
fluorescence response to CO (CORMꢀ3: 100 ꢁM) with a simultaneous increase of
fluorescence intensity by more than 33ꢀtimes. Next, we studied the concentration
dependent responses of CO with the using of various doses of CORMꢀ3 e.g. 10, 20,
30, 40, 50, 70 and 100 ꢁM with the gradual progress of time under the same
experimental conditions (Fig. 2).
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Fig. 2 The chemosensor, COFP, displayed timeꢀcourse fluorescence response to CO
in the reaction buffer [10 mM HEPES, 2 % DMSO] of pH 7.4 at 37 °C to various
concentration of CORMꢀ3 under the following experimental conditions: (1) 10 ꢁM
COFP, (2) 100 ꢁM CORMꢀ3, (3) 10 ꢁM COFP+10 ꢁM CORMꢀ3, (4) 10 ꢁM
COFP+20 ꢁM CORMꢀ3, (5) 10 ꢁM COFP+30 ꢁM CORMꢀ3, (6) 10 ꢁM COFP+40
ꢁM CORMꢀ3, (7) 10 ꢁM COFP+50 ꢁM CORMꢀ3, (8) 10 ꢁM COFP+70 ꢁM CORMꢀ
3 and (9) 10 ꢁM COFP+100 ꢁM CORMꢀ3 (ex: 440 nm).
The proposed mechanistic route for the detection is shown in Scheme 2. Due
to the possible transformation of the 3ꢀnitro moiety of the chemosensor into a possible
highly fluorescent aminoꢀfunctionalized 1,8ꢀnaphthalimide moiety, the enhanced
fluorescence was encountered in the reaction medium. This transformation was
triggered by the analyte, CO, and that can be oxidized into carbon dioxide. In this
way, the 3ꢀnitro group functioned as the CO reaction site, which can be easily
transformed in to the possible amino compound.33 The formation of the aminoꢀ
derivative was confirmed by the ESIꢀMS study. The peak found at m/z = 311.0887
(calculated m/z = 311.0796 for [C18H12N2O2+Na+]+, Fig. S1, Supplementary data)
demonstrates the presence of the amino functionalized product. Finally after the
conversion of the nitro group into the aminoꢀfunctionalized moiety, the possible
intramolecular charge transfer
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Scheme 2. Possible mechanistic path of COFP for CO detection through the
transformation of nitro group into the aminoꢀfunctionalized derivative.
(ICT) process improves due to the disruption of PET process, and thus, a fluorescence
‘‘turnꢀon’’ signal can be observed. The LOD (limit of detection) was calculated as
123 nM from the calibration curve (Fig. S2, Supplementary data) using 3σ
method.36,37 A variable pH dependency study of fluorescence response showed that
the maximum enhancement was observed in the range of pH 7.0 to 7.8 (Fig. S3,
Supplementary data).
Selectivity study
The selectivity of the chemosensor toward CO sensing was investigated with a variety
of biologically relevant metal ions and molecules. To investigate this study,
selectivity assays were performed while keeping the other experimental condition
°
unchanged in the reaction buffer at 37 C. Enhanced fluorescence of COFP (10 ꢁM)
was not encountered upon the addition of a large excess 100 equiv (1.0 mM) of
biologically relevant metal ions, i.e., Na+, K+, Ca2+, Mg2+, Mn2+, Fe2+, Fe3+, Cu2+,
Zn2+, Cd2+, Hg2+, Pd2+, creatinine and urea). So, in presence large excess of
biologically significant ions and other molecules, almost no effect on fluorescence
intensity was observed (Fig. 3). Furthermore, the selectivity of the chemosensor
towards CO detection was examined with a variety of biologically relevant reactive
species that may hamper the detection behavior. The experimental finding shows that
the enhanced fluorescence was succeeded only when it can be prompted by CO, and
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no important changes were found when various biologically significant reactive
oxygen, nitrogen and sulfur species, e.g., H2O2, tertꢀbutyl hydroperoxide (tBuOOH),
superoxide (O2 −), NaOCl, NO and H2S were introduced (Fig. 4). Moreover, the
ꢀ
fluorescence enhancement due to CO response was almost unperturbed in presence of
these competitive species in addition to CORMꢀ3 (Fig. S4, Supplementary data).
Thus, the selectivity study clearly demonstrates that the chemosensor, COFP, should
have potential applications to detect CO over a variety of biologically relevant metal
ions, molecules and reactive species.
Fig. 3 The fluorescence intensity change profile of COFP (10.0 ꢁM) in the presence
of various metal ions in the reaction buffer [10 mM HEPES, 2 % DMSO] of pH 7.4 at
37 °C under the following experimental conditions (1) only chemosensor COFP, (2)
100 ꢁM CORMꢀ3, (3) Na+, (4) K+, (5) Ca2+, (6) Mg2+, (7) Mn2+, (8) Fe2+, (9) Fe3+,
(10) Cu2+, (11) Zn2+, (12) Cd2+, (13) Pb2+, (14) Hg2+, (15) creatinine and (16) urea
(ex: 440 nm).
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Fig. 4 Fluorescence responses of 10 ꢁM COFP to CO in the reaction buffer [10 mM
HEPES, 2 % DMSO] of pH 7.4 at 37 °C with the progress of time in the presence of
t
.−
various species: (1) none, (2) CORMꢀ3 (100 ꢁM), (3) H2O2, (4) BuOOH, (5) O2
(source: KO2), (6) NaOCl, (7) NO (source: NOCl3) and (8) H2S (source: NaHS). [3ꢀ8:
Con. 100 ꢁM each]
The responses of COFP towards CO and biologically relevant metal ions
and/or species were also examined using UVꢀvis spectroscopic study (Fig. 5A & B).
The absorption spectrum of the probe showed maximum at 435 nm that is attributed
to the charge transfer transition of COFP. Upon addition of the 100 ꢁM CORMꢀ3 to
the probe solution, the absorbance at 435 nm was gradually increased with the
progress of time. On the other hand, the absorbance at 435 nm was remain unchanged
in the presence of biologically relevant metal ions and/or species (Fig. 5B) under the
same experimental condition. Thus, the selectivity study clearly reveals that the probe
should have potential applications to detect CO over a variety of competitive metal
ions and relevant species.
Fig. 5 (A) UVꢀvis absorption spectra of COFP (10 ꢁM) and the progress of reaction in
the presence of CORMꢀ3 and (B) Selectivity study of COFP (10 ꢁM) in presence of
various metal ions and relevant species (100 ꢁM each) in the reaction buffer [10 mM
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HEPES, 2 % DMSO] of pH 7.4 at 37 °C.
Cellular imaging study
Based on the result found from the spectral response toward carbon monoxide in an
aqueous buffered medium, we have introduced the chemosensor, COFP, to
C6 Glioma cells, to check the applicability in biological systems. Control experiments
were studied by treating cells with only 10 ꢁM COFP and 100 ꢁM CORMꢀ3
incubated for 45 min and the images were shown in Fig. 6A and 6B respectively. The
images displayed almost no fluorescence. Then, cells were incubated with 10 ꢁM
COFP for 20 min and then coincubated with CORMꢀ3 (10, 50 and 100 ꢁM) for
another 45 min. The addition of various doses of CORMꢀ3 may increase the
intracellular CO concentration. After being washed several times with PBS, cells
exhibited strong fluorescence images with high contrast (Figure 6 C, D and E). These
results demonstrate that the newly designed chemosensor, COFP, could display a
brilliant and high contrast images for the CO detection in the living cells. In addition
to that, 10 ꢁM of COFP did not show any significant cytotoxic effect (Fig. 7) on the
C6 Glioma cell line for at least up to 4 h of its treatment. However, significant
cytotoxicity was observed for the incubation at higher doses after 4 h. These results
implied that the chemosensor could be served as an efficient candidate for monitoring
changes in intracellular CO.
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Fig. 6 Fluorescence microscopic images of C6 Glioma cells for CO detection using
COFP in DMEM (2% DMSO) of pH 7.4 incubated at 37 °C. Control experiment with
A) 10 ꢁM COFP and B) 100 ꢁM CORMꢀ3; imaging experiment: C) 10 ꢁM COFP +
10 ꢁM CORMꢀ3, D) 10 ꢁM COFP + 50 ꢁM CORMꢀ3, and E) 10 ꢁM COFP + 100
ꢁM CORMꢀ3. The upper, middle, and bottom row indicate the brightꢀfield,
fluorescence, and merged images (ex ~440 nm).
Fig. 7 Cytotoxic effect of COFP (1: control, 2: 10 ꢁM, 3: 40 ꢁM, 4: 70 ꢁM and 5: 100
ꢁM) in C6 Glioma cells incubated for 3 h.
Experimental section
Materials and methods
All reagents and chemicals were purchased from Sigma and used without
further purification. Solvents used for spectroscopic experiments were purified and
dried by standard procedures. Fluorescence and absorption spectra were performed
using a HITACHI Fꢀ4600 Fluorescence Spectrophotometer and Shimadzu 2450 UV
Spectrophotometer attached with temperature controlling unit respectively. NMR
spectra of organic compounds were obtained on a Bruker Advance DPX 400. Electron
spray ionization (ESI) mass spectra were recorded on a Qtof Micro YA263 mass
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spectrometer.
General method of fluorescence studies
COFP was dissolved in DMSO to make the stock solutions. For fluorometric
analyses, desired amount of DMSO stock was taken and diluted in 10 mM HEPES
°
buffer (2 % DMSO) of pH 7.4 at 37 C to get the final concentration (10 ꢁM) of the
chemosensor. All the fluorescence spectra of the chemosensor were recorded at an
excitation wavelength of 440 nm.
Cell culture and imaging
C6 Glioma cell line, was purchased from National Center for Cell Science
(NCCS), Pune, India and was used throughout the study. Cell were cultured in Dulbecco’s
modified Eagle’s medium (DMEM, Gibco BRL) supplemented with 1% antibiotic mixture
containing penicillin, 10% FBS (Gibco BRL), streptomycin and neomycin (PSN, Gibco
BRL), at 37 °C in a humidified incubator with 5% CO . Cells were grown to 80-90 %
2
confluence, harvested with 0.025 % trypsin (Gibco BRL) and 0.52 mM EDTA (Gibco
BRL) in PBS (phosphate-buffered saline, Sigma Diagnostics) and plated at desire cell
concentration for the experiment. Cells were incubated with COFP in DMEM (2%
DMSO) of pH 7.4 at 37 °C for 15 minutes to allow the chemosensor entering into
cell. After that, cells were washed thrice with DMEM medium and were again
incubated with 10, 50 and 100 ꢁM CORMꢀ3 for 45 min at 37 °C. After every
incubation, cells were washed with DMEM three times. Brightꢀfield and fluorescence
images of C6 Glioma cells were taken by a fluorescence microscope (ZEISS
Axioskop 2 plus).
Synthesis of COFP (Scheme 1)
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A ethanolic (20 mL) solution of aniline (0.112 g, 1.2 mmol) was added
dropwise to a 40 mL ethanolic solution of 3ꢀnitroꢀ1,8ꢀnaphthalic anhydride (0.243 g,
1 mmol) under stirring condition. Then the solution mixture was refluxed for 4 h. The
progress of the reaction was checked by TLC. After the completion of the reaction,
the solution was cooled to room temperature and filtered. The filtrate was taken for
next few days at room temperature under slow evaporation. Crystalline solid
compounds were obtained after few days. The crud product was purified by column
chromatography on silica gel using CH2Cl2/MeOH (6/1) to afford the pure product,
COFP (calculated yield, 84%). 1H NMR (400 MHz, d6ꢀDMSO):
δ 7.47ꢀ7.57 (m, 5H),
8.58ꢀ8.64 (m, 1H), 8.67ꢀ8.69 (d, 1H, J=7.2), 8.79ꢀ8.81 (d, 1H, J=8.4), 8.94 (s, 1H),
9.49 (s, 1H). 13C (100 MHz, d6ꢀDMSO)
δ
124.7, 125.1, 128.9, 129.3, 129.5, 129.7,
130.2, 130.4, 130.6, 131.4, 132.2, 134.4, 136.0, 136.9 and 146.3. HRMS (m/z) found
319.0799, 341.0654 and 659.1408; Calculated for [M+H+]+ 319.0719, [M+Na+]+
341.0538, and [2M+Na+]+ 659.1179 respectively where M formulated as C18H10N2O4,
COFP (Fig. S5ꢀS7, Supplementary data)
Conclusions
In summary, we designed and synthesized a naphthalimideꢀbased fluorescence
chemosensor, COFP, and presented a new approach for CO detection by a
fluorescence method in aqueous buffer medium without using any heavy metals e.g.
Pd, Rh, Ru etc. The detection method is based on the transformation of a nitro group
of COFP molecule into an aminoꢀfunctionalized system in the presence of CO. The
chemosensor exhibits “turnꢀon” fluorescence response with more than 33ꢀfold
enhancement on fluorescence intensity. The response is selective over a variety of
relevant reactive oxygen, nitrogen and sulfur species. Also, the fluorescence response
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is not hampered in presence biologically relevant metal ions and /or species.
Moreover, the chemosensor is capable to monitor the changes in intracellular CO in
C6 Glioma cells.
Acknowledgements
K.D. acknowledges the Department of Science and Technology (DST), Fast
track research grand, New Delhi (vide project no. SB/FT/CSꢀ142/2012). S.L. is
thankful to UGC, New Delhi, India for offering the fellowship as SRF. Financial
assistance from Department of Science and Technology, Govt. of West Bengal (DST,
GoWB, vide project no. 698 (Sanc.)/ST/P/S & T/15ꢀG/2015) is gratefully
acknowledged.
Conflicts of interest
There are no conflicts to declare.
†Electronic Supplementary Information (ESI) available: Supplementary data (Fig.
S1ꢀS7) associated with this article can be found, in the online version, at
https://doi.org/xxxxxxxxxx
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