A NIR sensor for cyanide detection and its application in cell imaging

Article abstract of DOI:10.1016/j.saa.2018.03.044

A novel ‘D-π-A’ sensor 1 has been designed and prepared via the condensation reaction of 3?ethyl?2?methyl?1,3?benzothiazol?3?ium iodide and 5?nitro?o?vanillin. Upon treatment with cyanide, sensor 1 exhibited a significant near-infrared (NIR) fluorescence quenching at 663 nm. The MS, IR, ¹H NMR and DFT methods confirmed that the response of 1 to cyanide is due to the nucleophilic addition reaction, which results in the inhibition of the Intramolecular Charge Transfer (ICT) process in the sensor. Furthermore, sensor 1 was used for the determination of CN^? in HeLa cells.

Full text of DOI:10.1016/j.saa.2018.03.044

Accepted Manuscript
A NIR sensor for cyanide detection and its application in cell
imaging
Wei-Na Wu, Hao Wu, Yuan Wang, Xiao-Lei Zhao, Zhou-Qing
Xu, Zhi-Hong Xu, Yun-Chang Fan
PII:
DOI:
Reference:
S1386-1425(18)30230-0
doi:10.1016/j.saa.2018.03.044
SAA 15916
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
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Received date: 20 November 2017
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5 March 2018
12 March 2018
Please cite this article as: Wei-Na Wu, Hao Wu, Yuan Wang, Xiao-Lei Zhao, Zhou-Qing
Xu, Zhi-Hong Xu, Yun-Chang Fan , A NIR sensor for cyanide detection and its application
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ACCEPTED MANUSCRIPT
A NIR sensor for cyanide detection and its application in cell imaging
Wei-Na Wua, Hao Wua, Yuan Wanga,*, Xiao-Lei Zhaoa, Zhou-Qing Xua,*,
Zhi-Hong Xub,*, Yun-Chang Fana
a College of Chemistry and Chemical Engineering, Henan Key Laboratory of Coal Green Conversion,
Henan Polytechnic University, Jiaozuo 454000, P. R. China
b Key Laboratory of Chemo/Biosensing and Detection, School of Chemistry and Chemical Engineering,
Xuchang University, 461000, PR China
*
Corresponding author. Tel.: +86 391 3987818; Fax: +86 391 3987811; e-mail:
wangyuan08@hpu.edu.cn (Y. Wang); zhqxu@hpu.edu.cn (Z.-Q. Xu); xuzhihong1980@yahoo.com
(Z.-H. Xu).
Abstract:
A novel ‘D-π-A’ sensor 1 has been designed and prepared via the condensation reaction of
3-ethyl-2-methyl-1,3-benzothiazol-3-ium iodide and 5-nitro-o-vanillin. Upon treatment with cyanide,
sensor 1 exhibited a significant near-infrared (NIR) fluorescence quenching at 663 nm. The MS, IR, 1H
NMR and DFT methods confirmed that the response of 1 to cyanide is due to the nucleophilic addition
reaction, which results in the inhibition of the Intramolecular Charge Transfer (ICT) process in the sensor.
Furthermore, sensor 1 was used for the determination of CN- in HeLa cells.
Keywords: Cyanide (CN-); fluorescent sensor; Intramolecular Charge Transfer; near-infrared emission;
nucleophilic addition.
1. Introduction
Cyanide is an extremely toxic anion, which can directly lead to the death of organisms [1, 2]. The
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lethal dose of cyanide was 0.7~3.5 mg/kg of body weight [3]. According to the World Health
Organization (WHO), the concentration of cyanide in drinking water should be less than 2.7 M [4].
Nevertheless, cyanide salts are still widely used as industrial materials in plastics production, gold
mining, and other fields [5]. Therefore, the detection of cyanide in environmental and biological
samples is particularly important.
The development of fluorescent chemosensors towards cyanide ion has been an area of great interest
due to their high selectivity and sensitivity. Nowadays, several chemosensor systems for cyanide
detection have been established [2], including nucleophilic addition [4-22], hydrogen-bonding
interaction [23], cyanide-catalyzed cyclization [24, 25], coordination of copper ions with cyanide [1,
26-28], and so on. Among these strategies, sensors based on nucleophilic addition reaction showed
both specific selectivity and high sensitivity [3]. However, most of the sensors have their maximum
emission wavelengths in the blue or green spectral regions (λ < 600 nm) [20, 23]. Merely a few of them
exhibit the near-infrared (NIR) emission [29]. Indeed, the NIR emission at around 650–900 nm exhibits
special advantages, including minimal interfering absorption and fluorescence from biological samples,
low scattering, and deep penetration into tissues [30]. Furthermore, the reported NIR probes usually
require troublesome and tedious synthetic process, setting a major obstacle for real-world applications
in their industrialization [29].
The receptors with both donor and acceptor units covalently linked through a double bond (D-π-A)
have recently been used for sensing cyanide ions [10, 11, 22]. Particularly, a receptor based on a
conjugated nitrophenol-benzothiazole (I, Scheme 1) system exhibits a red fluorescence with emission
band centered at 612 nm, which will be quenched in the presence of cyanide ions [11]. Furthermore, it
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is believed that introduction of electron-donating groups to receptors in an efficient strategy to red-shift
their emission color [31]. Therefore, we report here a NIR chemosensor (1, Scheme 1) by modification
of the receptor I with a methoxy group to achieve the emission shift (max = 663 nm); thus providing a
simple, cost-efficient method for trace detection of cyanide ion via NIR emission quenching in
bio-chemical systems.
Insert Scheme 1 here.
2. Experimental section
Detailed methods and approaches see S1, ESI.
3. Results and Discussions
3.1 Synthesis
The sensor
1
is synthesized using 3-ethyl-2-methyl-1,3-benzothiazol-3-ium iodide and
2-hydroxy-3-methoxy-5-nitrobenzaldehyde as starting materials according to the literature procedures
[11]. Black rods of 1 suitable for X-ray diffraction analysis were obtained by recrystallization from
DMF solution (Fig. 1).
Insert Fig. 1 here.
3.2 Spectroscopic studies of 1 in the presence of CN-
The selectivity to various anions of sensor 1 in DMSO/H2O (8/2, v/v) solution was investigated. Free
sensor 1 (10 M) features the maximum absorption peak at about 545 nm (Fig. 2) and a broad NIR
fluorescence emission centered at 663 nm (Fig. 3, excited at 540 nm). Upon the addition of 50 eq. of
CN- to the solution of 1, the absorbance peak blue-shifts to 450 nm with color change from pink to
yellow. Simultaneously, the NIR fluorescence emission band was quenched remarkably. In the case of
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−
−
−
−
other anions, such as F−, Cl−, Br−, I−, AcO−, H2PO4 , HSO4 , ClO4 , S2−, N3 and SCN−, no substantial
change in both absorbance and emission spectra of 1 could be observed. To further exploit the utility of
sensor 1 as an anion selection sensor for CN-, competitive experiments were carried out in the presence
of 50 eq. of CN- and various anions (50 eq.). It is worth noting that the competing anions did not lead to
any significant interference with the detection of CN- (Fig. S1 and S2, ESI). This result established the
high selectivity of sensor 1 toward CN- over other anions mentioned above.
Insert Figure 2 here.
Insert Figure 3 here.
To obtain more information of the binding form of 1 with CN-, we performed absorption and
fluorescence spectrum titrations. As shown in Fig. 4, upon the gradual addition of CN- up to 50 eq. into
10 M 1 in DMSO/H2O (8/2, v/v) solution, the absorbance peaked at 545 nm significantly decreased,
accompanying with the absorbance increasement at about 450 nm. Three isosbestic points at 322, 429
and 483 nm were obtained, clearly indicating the reaction between the probe 1 and CN-. The detection
limit calculated on the basis of 3/S is 1.55M for CN- (where  is the standard deviation of blank
measurements and S is the slope of the intensity versus sample concentration), which is lower than the
WHO guideline of 2.7 M cyanide [4], suggesting that sensor 1 is potentially applied to analyze CN-.
Job’s plots experiment shows that the binding of 1 to CN- is of 1:1 stoichiometry (Fig. S3, ESI). The
association constant (Ka) was determined to be about 2.41×103 M-1, as obtained by fitting the data to
the Benesi-Hildebrand expression (Fig. S4, ESI). Similarly, the fluorescence intensity at 663 nm of
sensor 1 degrades along with the concentration increase of CN- (Fig. 5). The quantum yield of
DMSO/H2O (8/2, v/v) solution of 1 (10 M) with and without CN- (50 eq.) is tested to be 0.016 and
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0.122, respectively, using rhodamine B as standard (ФF = 0.65 in methanol) [32].
Insert Figure 4 here.
Insert Figure 5 here.
The pH dependence of the sensor was detected by UV-vis and fluorescence spectroscopy. The
results indicated that 1 binding with CN- occurred effectively in the range of pH 6-12 (Fig. S5 and S6,
ESI). Furthermore, the fluorescence response of 1 (10 M) with CN- (50 eq.) was adopted in less than
45 min (Fig. S7, ESI), which was a significantly faster analysis time in comparison to the reported
sensor I (about 80 min) [11].
3.3 Sensing mechanism
To explore the reaction mechanism of 1 with CN-, the ESI-MS analysis was employed. As illustrated
in Fig. 6, a peak at m/z 384.0932 corresponding to the additive product [1-CN+H]+ (Cal. 384.10) was
observed upon the addition of CN- to the DMSO solution of 1, confirming the addition reaction
between 1 and CN-. The above conclusion is also supported by the characteristic absorbance band of
CN- group at 2211 cm-1 in the IR spectra of 1+CN- (Fig. S8, ESI).
To further elucidate the binding mode, 1H NMR titration experiment of sensor 1 with
tetrabutylammonium cyanide (TBACN) was conducted in DMSO-d6 (Fig. 7). The peak corresponding
to the vinylic proton Ha showed up field shift from 8.52 ppm to 4.69 ppm in the presence of CN-, while
the peak of proton Hb may be masked by that of TBA. Besides this, the signals corresponding to –N–Et,
i.e. Hc and Hd protons exhibited an up-field shift from 4.80 and 1.45 ppm, respectively, to 3.49 and
1.03 ppm. These observations clearly indicated that the cyanide anion was added to the benzothiazol
C=N group.
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To further clarify the reaction mechanism, we conducted density functional theory (DFT)
calculations with the Becke-3-Lee-Yang-Parr (B3LYP) exchange function (Fig. 8) [33]. The results
show that both the HOMO and LUMO of 1 disperse over the whole molecule, indicating the well
delocalization of the electron cloud density and the active Intramolecular Charge Transfer (ICT)
processes. By contrast, the addition of CN- interrupts the conjugation and blocks the ICT process, since
the HOMO and LUMO of the 1-CN- are centered on the benzothiazol and vanillin moieties, respectively.
Additionally, the energy gap between the HOMO and LUMO of 1-CN- was calculated to be 3.12 eV,
which is much higher than that of 1 (2.50 eV). In other words, compared with that of 1, the absorbance
of 1-CN- should give a blue-shift, and thus 1-CN- is more difficult to excite. As a result, weak
fluorescence emission should occur in the case of 1-CN-. The theoretical results are well in accordance
with the experiment phenomenon. Considering the above evidences, we can roughly conclude that
sensor 1 functions through nucleophilic addition reaction with CN-, as illustrated in Fig. 6, inset.
Insert Figure 6.
Insert Figure 7.
Insert Figure 8.
3.4 Fluorescence imaging experiments of 1 in living cells
To exhibit the cytotoxicity of the ensemble, HeLa cells were treated with different concentration of 1
for 24h (Fig. 9). The observation clearly indicates that the 1 have almost no cytotoxicity (even up to 20
M). Encouraged by the biocompatibility of 1, we examined the ability of the fluorescence
chemosensor 1 to detect CN- in HeLa cells. Cells were supplemented with 1 (10 μM) in Dulbecco’s
modified Eagle’s medium (DMEM) contained with 10% fetal bovine serum for 30 mins at 37℃,
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leading to significant intracellular fluorescence as determined by laser scanning confocal microscopy
(Fig. 10a). Removing free 1 by rinsing the cells with DMEM, then 50 μM CN- in DMEM
supplemented with 10% fetal bovine serum was added and the cells were incubated for another 30 mins
at 37℃ under same imaging condition, whereupon an obvious decrease in the fluorescence from the
intracellular area was observed (Fig. 10d). The results indicate that sensor 1 can be used to image
intracellular CN- in living cells.
Insert Figure 9.
Insert Figure 10.
4. Conclusion
In summary, a ‘D-π-A’ sensor 1 was synthesized and fully characterized, which exhibits remarkable
fluorescence emission in the NIR region. Based on this NIR signal, 1 has been presented as a
fluorescent on-off sensor for CN-. The mechanism is proved to be a nucleophilic addition reaction via
MS, IR, 1H NMR and DFT methods. The detection limit of the sensor towards CN- was tested at 1.55
M. Furthermore, the sensor 1 was used to imaging CN- in living cells.
Acknowledgements
This work was supported in part by the National Natural Science Foundation of China (21001040),
the Joint Program for Fostering Talents of National Natural Science Foundation of China and Henan
Province (U1304202 and U1604124), the Science and Technology Department of Henan Province
(162300410011, 152102210343 and 152102210314), the Education Department of Henan Province
(15B150016, 2014GGJS-045 and 15HASTIT002).
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Figure captions
Scheme 1. Synthesis route of sensor 1.
Fig. 1. Crystal structure of 1.
Fig. 2. UV–vis spectra of 10 M sensor 1 in DMSO/H2O (8/2, v/v) solution with 50 eq. of anions: F-,
-
-
-
-
Cl-, Br-, I-, AcO-, H2PO4 , HSO4 ,ClO4 , S2-, N3 , SCN-, CN- and blank. The insert shows the solution
color of 1 with CN-.
Fig. 3. Fluorescence emission spectra of 10 M sensor 1 in DMSO/H2O (8/2, v/v) solution with 50 eq.
-
-
-
-
of anions: F-, Cl-, Br-, I-, AcO-, H2PO4 , HSO4 ,ClO4 , S2-, N3 , SCN-, CN- and blank. Excitation
wavelength was 540 nm. The insert shows the solution color of 1 with CN- under 365 nm UV lamp.
Fig. 4. UV–vis spectra of 10 M sensor 1 upon the addition of CN- (0–50 eq.) in DMSO/H2O (8/2, v/v)
solution.
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Fig. 5. Fluorescence emission spectra of 10 M sensor 1 upon the addition of CN- (0–50 eq.) in
DMSO/H2O (8/2, v/v) solution. Excitation wavelength was 540 nm.
Fig. 6. MS spectral changes with the addition of CN- to 1. The inset shows the proposed reaction
mechanism of 1 with CN-.
Fig. 7. 1H NMR spectral changes with the addition of CN- to 1.
Fig. 8. Energy-minimized structures and HOMO/LUMO of 1 and 1-CN- by DFT calculation.
Fig. 9. Cytotoxicity of 1 in HeLa cells. Cells were treated with different concentrations of 1 for 24 h
and cell viability assay were determined by MTT assay.
Fig. 10. Confocal fluorescence images of Hela cells: Confocal fluorescence (a), brightfield (b), and
overlay (c) images of Hela cells incubated with 10 M of 1 for 30 mins at 37℃; Confocal fluorescence
(d), brightfield (e), and overlay (f) images of Hela cells incubated with 10 M of 1 for 30 mins at 37℃
and then incubated with 50 M CN- for another 30 mins at 37℃.
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Highlights
A quite simple turn-off fluorescent probe for CN- has been developed.
The probe displays NIR emission at 663 nm.
Response of probe to CN- is a nucleophilic addition reaction.
The probe has been used for imaging of CN- in living cells.
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