Novel ratiometric turn-on fluorescent probe for selective sensing of cyanide ions, effect of substitution and bio-imaging studies

Article abstract of DOI:10.1039/c5ra27517b

Novel fluorogenic receptors S1-S4 (imidazo anthraquinone derivatives) were synthesized from o-substituted benzaldehyde and 1,2-diaminoanthraquinone and characterised with various spectroanalytical techniques. In the case of S1, the selectivity towards cya

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Novel Ratiometric Turn-on Fluorescent Probe for Selective Sensing
of Cyanide ions, Effect of substitution and Bio-imaging Studies
Gopal Balamurugana, Parthiban Venkatesanb, Shu Pao Wub, Sivan Velmathia,*
Received 00th January 20xx,
Accepted 00th January 20xx
Novel fluorogenic receptors S1-S4 (imidazo anthraquinone derivatives) were synthesized from o-substituted benzaldehyde
and 1,2-diaminoanthraquinone and characterised well with various spectroanalytical techniques. In case of S1, the
selectivity towards cyanide ions with turn on fluorescence output among other anions due to the inhibition of twisted
intramolecular charge transfer (TICT) mechanism Effect of substitution was successfully compared with sensing studies of
S2-S4. S1 showed good binding constant with 1:1 stoichiometric ratio and micromolar detection limit. The sensing
behaviour was further supported by 1H NMR Titration, TD-DFT calculations and lifetime measurement studies. S1 was
applied for the bio-imaging of the cell line RAW264.7 and successfully sensed the cyanide ions in the physiological
conditions.
DOI: 10.1039/x0xx00000x
www.rsc.org/
Introduction
Cyanide ion is highly toxic and severe health hazard anion that
could be lethal dose for human beings even as low as 1.5 mg/kg of
body weight. Consequently, the utmost tolerant level of cyanide in
drinking water was set at 1.9µM by the World Health Organization
(WHO). The inhibition of cytochrome-c-oxidase in the mitochondria
of the eukaryotic cells has been occurred by cyanide ions due to the
rapid coordination with the iron center led to the inhibition of
oxygen intake by the cells of an organism1. Because of the extensive
usage of cyanide worldwide in industries such as Gold mining
(cyanide leaching process), electroplating, refineries (fluid catalytic
cracking and coking), printed circuit board manufacturing, steel and
chemical industries, the environmental pollution could not be
outmanoeuvre entirely1-4. By considering the above facts, a center
of attention has been created for the articulate design and
synthesis of efficient probes to selectively detect CN- at the
environmental and biological medium. There are surplus of articles
Scheme 1: Design strategy using TICT mechanism.
Molecular Charge Transfer (TICT) mechanism27-30. A clear picture of
the TICT process in case of 2’- substituted imidazole in the ground
state and excited state was shown in scheme 1. (a) Free rotation
could be possible in case of no substitution. The presence of
substitution in 2’ position inhibit the rotation as well in the excited
state (S1),(b) intramolecular charge transfer took place as TICT
process followed by the relaxation to the ground state (S0) (c)But
the deprotonation of the imidazole –NH may liberate the steric
hindrance followed by the inhibition of TICT process resulted in the
enhanced fluorescence. Based on this strategy, we designed the
receptor S1, synthesized in good yield and characterized well with
various spectroscopic techniques, sensing studies towards the
highly toxic anions and biological important anions. Further
supporting information to prove the sensing behaviour and
fluorescence enhancement were obtained using UV-vis,
photoluminescence and 1H NMR titration, fluorescence life
measurement and DFT calculation. Effect of substitution using
electron donating group was also performed by synthesizing S2-S4
to precise the sensing behaviour of S1. Binding constants, detection
limit and quantum yield were thoroughly determined for the
receptors. To reach the ultimate destination of chemosensors, S1
was applied lucratively for real time selective detection of cyanide
ions in domestic waste water. Ultimately, S1 was successfully
applied for bio-imaging of RAW264.7 cell lines.
published for bio-imaging of cyanide anion (CN-) in the cells based
6
on the strategies such as the formation of cyanide complexes5,
,
nucleophilic addition of CN− to activated double bond or a boron
center and relay recognition7-18. Recently, anthraquinone based
imidazole receptors have been reported for the detection of anions
and metal ions.19-26 In most of the cases, imidazole based receptors
performed as colorimetric sensors and very few of them showed
fluorescence turn off behaviour. Fluorescent off-on imidazo
anthraquinone probes for cyanide may not be prominent in the
picture. From the clear literature review over the structural aspects
like chromophore, fluorophore, electron withdrawing and donating
substitutents in the imidzole based receptors, we could achieve the
fluorescent enhancement for the anions based on Twisted Intra
a.Organic and polymer synthesis Laboratory, Department of Chemistry, National
Institute of Technology, Tiruchirappalli-620015, India.
b.Department of Applied Chemistry, National Chiao Tung University, Hsinchu,
Taiwan
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to room temperature, cold ethanol (5 mL) was added and stirred for
few minutes. The solid obtained was filtered, washed with ethanol
(5 mL x 3), dried. The crude product obtained was again triturated
with hot ethanol (5 mL) to yield pure receptor.
Scheme 2: Syntheses of o-substituted imidazo anthraquinone
derivatives S1-S4.
2-(2-nitrophenyl)-1H-anthra[1,2-d]imidazole-6,11-dione (S1)
Yield: 73%. M.p. 248-250°C. 1H NMR 500 MHz, DMSO-d6: δ 13.85 (s,
1H), 8.28-8.29 (m, 4H), 8.22 (dd, J = 8.50, 32.00 Hz, 2H), 8.01-8.02
(m, 2H), 8.02 (dd, J = 8.00, 23.75 Hz, 2H), 7.92 (t, J = 7.50 Hz, 1H).
13C NMR (75 MHz, DMSO-d6) 120.17, 124.91, 125.46, 125.84, 126.1,
127.27, 128.87, 132.07, 133.30, 133.43, 133.53, 133.74, 134.74,
134.93, 149.01, 155.46, 182.90, 183.50. FTIR ( ν-cm-1) 3427 (N-H
str.), 1666 (C=O str.), 1537 (Nitro asymmetric), 1476 (C=N str.), 1365
(Nitro asymmetric), 715 (N-H bending) HRMS: Calcd. for C21H12N2O4:
m/z 369.075. Found: m/z 370.083(M+1).
2-(2-hydroxyphenyl)-1H-anthra[1,2-d]imidazole-6,11-dione (S2)
Fig. 1 (a) 200ꢀL of anions (1.5 x10-3 M in ACN) was added in 3 mL of
S1 (5 x10-5 M in ACN) (b) 200ꢀL of anions (1.5 x10-3 M in H2O) was
added in 3 mL of S1 taken in 97:3 ACN: water.
Yield: 85%. M.p. 265-267°C. 1H NMR 500 MHz, DMSO-d6: δ 12.65 (s,
1H, -OH), 11.96 (s, 1H, -NH), 8.461-8.269 (m, 2H), 8.177-8.088(m,
2H), 8.044-7.944 (m, 2H), 7.882-7.797 (m, 1H), 7.38 (t, J = 7.30 Hz,
1H), 7.04 (d, J = 8.10 Hz, 1H), 6.98 (t, J = 7.35 Hz, 1H). 13C NMR (75
MHz, DMSO-d6) 117.56, 118.35, 120.29, 121.67, 124.82, 126.70,
127.26, 128.23, 129.55, 133.31, 134.65, 134.96, 156.63, 157.42,
182.58. FTIR ( ν-cm-1) 3548 (O-H str.), 3442 (N-H str.), 1660 (C=O
str.), 1476 (C=N str.), 715 (N-H bending) HRMS: Calcd. for
C21H12N2O4: m/z 340.332. Found: m/z 341.071(M+1).
Reagents and Instruments
1,2-Diaminoanthraquinone (DAAQ), 2-nitrobenzaldehyde were
purchased from Sigma Aldrich and utilized without further
purification. Tetrabutylammonium (TBA) salts of anions such as CN-,
-
F-, Cl-, Br-, I-, OAc-, H2PO4 , HSO4- , OH- and analytical grade solvents
2-(2-methoxyphenyl)-1H-anthra[1,2-d]imidazole-6,11-dione (S3)
were purchased commercially and used as such. NMR spectra were
Yield: 78%. M.p. 233-236°C. 1H NMR 500 MHz, DMSO-d6: δ 12.35 (s,
1H), 8.50 (d, J = 6.50 Hz, 1H), 8.22-8.42 (m, 2H), 8.06 (dd, J = 8.00,
32.25 Hz, 2H), 7.74-7.82 (m, 2H), 7.55 (t, 1H), 7.33 (d, J = 7.50 Hz,
1H), 7.16 (d, J = 7.00 Hz, 1H), 4.28 (s, 3H). 13C NMR (75 MHz, DMSO-
d6) 57.11, 113.24, 121.54, 121.99, 125.30, 126.80, 127.42, 130.32,
132.52, 133.54, 134.75, 135.16, 155.46, 158.00, 184.56. FTIR ( ν-
cm-1) 3485 (N-H str.), 1668 (C=O str.), 1472 (C=N str.), 1054 (-OMe),
714 (N-H bending) HRMS: Calcd. for C21H12N2O4: m/z 354.358.
Found: m/z 355.140 (M+1).
obtained using
a
Bruker 500 MHz spectrometer using
Tetramethylsilane (TMS) as an internal standard. Thermo Fisher FT
IR Spectrometer was used to record IR spectra in pellet mode. UV-
vis spectroanalysis were done on Shimadzu UV-2600 UV-vis
spectrophotometer in quartz cell with
1 cm path length.
Photoluminescence spectra were recorded using Shimadzu RF-
5301PC spectrophotometer. 1.5 × 10−3 M aq. solutions of the anions
with 5×10−5 M solutions of S1 in ACN medium had been utilized for
the studies. UV titrations were carried out by the incremental
addition of 0.1 eq. (10 ꢀL) - 2 eq. (200 ꢀL) of guest solutions to 3 mL
of receptor solution in the UV cuvette. 1H NMR titration was carried
out using 0.1 M solution of TBACN in DMSO-d6 and 0.01 M solution
of S1 in DMSO-d6 in Bruker Avance III 500 MHz NMR Spectrometer.
Life time measurement studies were done at Horiba Delta time
instrument coupled with TCSPC. Confocal fluorescence imaging of
cells was performed with a Leica TCS SP5 X AOBS Confocal
2-(2-aminophenyl)-1H-anthra[1,2-d]imidazole-6,11-dione (S4)
Yield: 81%. M.p. 248-250°C. 1H NMR 300 MHz, DMSO-d6: δ 12.43 (s,
1H), 8.13 (s, 2H), 7.99-0.00 (m, 2H), 7.89-7.91 (m, 2H), 7.16-7.24 (m,
2H), 7.21 (d, J = 6.60 Hz, 1H), 6.87 (d, J = 6.00 Hz, 1H), 6.68 (d, J =
6.60 Hz, 1H). 13C NMR (75 MHz, DMSO-d6) 116.66, 118.11, 119.05,
121.38, 124.28, 126.68, 127.11, 128.02, 129.02, 132.18, 133.34,
134.74, 134.70, 149.52, 182.52, 183.49. FTIR ( ν-cm-1) 3432 (N-H
str.), 1661 (C=O str.), 1469(C=N str.), 1102 (C-N str.), 715 (N-H
bending) HRMS: Calcd. for C21H12N2O4: m/z 339.105. Found: m/z
340.086(M+1).
Fluorescence Microscope (Germany), and
a 63x oil-immersion
objective lens was used. The cells were excited with a white light
laser at 456 nm, and emission was collected at 540 ±580nm. DFT
calculation was performed using Gaussian 09 software package and
the optimized structures and MOs were processed from GaussView
5.0 package.
Synthesis of o- substituted anthra(1,2-d)imidazole-6,11-dione (S1-
S4):
o-substituted
benzaldehyde
(0.5
mmol)
and
1,2-
diaminoanthraquinone (0.5 mmol) were taken in DMSO (1 mL) and
heated to 120°C for 6 h. (Scheme 2) The completion of the reaction
was monitored by thin layer chromatography (TLC) using hexane:
ethylacetate (6:4) as an eluent. After cooling the reaction mixture
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Results and Discussion
Naked eye sensing
Initially, 200 µL of anions, CN-, F-, Cl-, Br-, I-, OAc-, H2PO4 , HSO4 &
OH- (1.5x10-3 M in ACN) were added to 3 mL of S1-S4 (5x10-5 M in
ACN) to examine the sensitivity visually. S1 (Fig. 1a), S2 and S4 were
shown strong color change for CN- ions whereas F- and OAc- ions
showed slight colour change. But S3 did not show any color change,
this may be furnished as the electron donating –OMe in S3
decrease the acidity of the imidazole –NH. (Supporting information
fig. A1) By taking into account the compatibility of the receptor
with water to analyze the environmental measures, anions were
taken in H2O for further analysis. Interestingly, S1 showed the
intense yellow color change selectively for cyanide among anions.
To increase the ecological factor, the percentage of water in ACN
for S1 had been altered and it showed intense yellow color change
only for cyanide ions in 3% Aq. ACN medium(Fig. 1b). No sensing
was observed above 3% aq. ACN solution of S1. In the same way, S2
and S4 were displayed the same result alike S1. (Supporting
Information fig. A1)
-
-
Fig. 2 (a) UV-vis spectral studies of S1 (5 x10-5 M in ACN) with 200ꢀL
of anions (1.5 x10-3 M in ACN) (b) UV-vis spectral studies of S1 (5
x10-5 M in 97:3 ACN: water) with 200ꢀL of anions (1.5 x10-3 M in
H2O)
UV-vis spectral studies
By analyzing the sensing behaviour using UV-vis spectroscopy,
under 100% ACN medium, S1 showed an absorbance maxima at
384 nm and the addition of cyanide ion into S1 suppress the
corresponding band and formed a new band at 460 nm whereas
fluoride and acetate ions interact with S1 weakly compared to
cyanide. (fig. 2a)This proves the higher nucleophilicity of cyanide
played a vital role in the sensing behaviour. While using aq. solution
of anions to S1 in ACN, due to the effect of solvation by water
molecules, cyanide ions only showed the bathochromic shift to 450
nm. (fig. 2b) As we saw in the naked eye experiment, beyond 10%
of water content in ACN, no shift was observed. The incremental
addition of cyanide ions (20-200 µL) to S1 clearly exposes the
gradual decrease in the band at 384 nm and increase in the band at
455 nm. (fig. 3a) The ratiometric behaviour of S1 towards cyanide
ions could be helpful to quantitatively analyze the same in the real
sample by using Beer-Lambert’s law.(fig. 3b) Interference of other
anions towards cyanide sensing of S1 was demonstrated
successfully that shows there is no effective interference in the
absorbance maxima of R1-CN- complex. (fig. 3c) In case of S2 and S4
band at 410 nm and 448 nm were red-shifted to 480 nm and 500
nm respectively. (Supporting Information fig. A2, A3). Using
Benesi-Hildebrand plot, from the incremental addition of CN- ions
to S1, S2 and S4, S1 displayed a very good association contant
(1.5x10-4) whereas S2 and S4 showed slightly lesser binding
constants. By using Job’s plot, the stoichiometric ratio of the guest-
host complexes were calculated and listed in table 1. Except S2,
others possess 1:1 stoichiometry. Both the binding sites in S2 (-OH
and imidazole –NH) were deprotonated to give 1:2 host:guest
stoichiometric ratio. In addition, detection limit and limit of
quantification were also calculated for the receptors. All the
experiments were reiterated to assure the repeatability and
confirmed with the low error percentage.
Fig. 3 (a) Incremental addition of CN- to S1 (b) Support for the
ratiometric detection of CN- by S1 with 1:1 stoichiometric ratio (c)
Interference of other anions in the sensing behaviour of S1 with
cyanide ions.
Fig. 4 (a) 2D contour image of Photoluminescence S1 (b) S1-
Fig. 5 (a) Photoluminescence studies of S1 (5 x10-5 M in 97:3 ACN:
water) with 200ꢀL of Anions (1.5 x10-x10-3
M in H2O) (b)
Photoluminescence studies of the incremental addition of CN- to S1.
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Table 1: Binding constant, detection limit and stoichiometric ratio of the host-guest complexes formed.
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Species
S1-CN-
S2-CN-
S4-CN-
Binding Constant
15466
Detection Limit LOD
3.960 x 10-6
Limit of Quantificaiton LOQ
1.31 x 10-5M
Stoichiometric Ratio
M
1:1
1:2
1:1
13862
2.96 x 10-6
7.25 x 10-7
M
9.87x10-6
M
M
7103
M
2.41 x 10-6
fluorescent enhancement was observed. (Supporting Information
fig. A3) It can be inferred from the above the observation that the
introducing bulkier electron withdrawing group in the 2’ position of
phenyl ring will induce the fluorescent enhancement through TICT
process.
Quantum yield
Quantum Yield of S1 and depronated form of S1(S1-) have been
calculated by using the following method with Rhodamine-B as a
reference(R).
2
Φꢀ
Φꢁ
Iꢀ ꢃ1 ꢄ 10ꢅAꢆꢇ nꢀ
ꢂ
x
x
2
ꢃ1 ꢄ 10ꢅAꢈꢇ
Iꢁ
nꢁ
(Equation 1)
Φs – Quantum Yield of the sample; ΦR – Quantum yield of the
reference (Quantum yield of Rhodamine B is 0.68 in EtOH); I –
Fluorescence intensities of the sample (S) and reference(R); A – Uv-
vis absorbance of the sample (S) and reference(R); n – Refractive
Index of the solvent used (For EtOH - 1.36 and for DMSO – 1.47); As
quantum yield is temperature dependent, in both the cases
temperature was maintained same. By applying the experimental
value in the equation 1, quantum yields of S1 and S1+CN- were
found to be 0.0033 and 0.033 in DMSO. 10-fold enhancement
clearly tells the efficient usage of the receptor S1 as a selective
fluorescent probe.
1H NMR Titration
Fig. 6 1H NMR titration of S1 (0.01 M in DMSO-d6) with cyanide ion
The recognition of cyanide by S1 can further be supported by 1H
NMR titration (fig 6). 0.01 M solution of S1 and 0.3-2.0 eq of TBACN
(0.1 M in DMSO-d6) were used for the 1H NMR titrations. Highly
acidic in nature and the ability to interact through hydrogen
bonding of the –OH and imidazole NH with cyanide drives the
proton to appear at down field. Due to the addition of cyanide ion
with the receptor, the peak at 13.85 ppm (a) corresponding for
imidazole –NH, was disappeared. On increasing the concentration
of cyanide, the shifting of the peaks at 7.8-8.0 ppm (c-f protons)
were shifted to 7.4-7.8 ppm belonged to the anthraquinone protons
and the protons (b and g) in the nitrophenyl ring were remained
same. As the deprotonation occurred during the addition of cyanide
ions, the electron density around anthraquinone moiety might be
increased and resulted in the local diamagnetic shielding of the
corresponding protons. But the electron density around nitrophenyl
ring may not be altered much by the deprotonation process
therefore no change was observed in the corresponding protons of
the same.
(0-2.0 eq. of 0.1 M in DMSO-d6)
Fluorescence spectral studies
3D fluorescence spectral study was carried out to prop up the
effective fluorogenic sensing of cyanide by S1. The fluorescence
property of the receptor S1 was investigated by using 3D scan
recording emission spectra from 300 to 600 nm while the excitation
was subsequently tuned from 300 to 600 nm. The contour image of
the spectra of the receptor showed very weak fluorescence
whereas after the addition of cyanide to the receptor, there was a
new band formed around λem 570 - 625 nm range in the excitation
wavelength of (λex) 425 - 475nm (Fig 4a, b). The photolumiscence
spectral studies were carried out with the excitation at 455 nm and
the slit width of 5 nm. S1 showed very weak fluorescent and an
emission peak at 554 nm. Among various anions, only for the
addition of cyanide ion, S1 displayed 10-fold fluorescent
enhancement with a bathochromic shift from 554 nm to 570 nm
(Fig 5a, b). It could be inferred from the results that the coplanarity
of the system was retained by the deprotonation of 1-H of imidzole
moiety of S1 followed by the fluorescence enhancement via the
inhibition of twisted intramolecular charge transfer (TICT)
mechanism. S2 was excited with 500 nm and emission was
observed at 558 nm by adding cyanide ions into S2, fluorescence
quenching took place whereas in case of S4, comparatively
Life Time Measurement
Further, time dependent photoluminescence decay process had
been carried out for the life time measurement of S1 and S1-CN ion
pair complex. A pinch of milk powder in water had been used to
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prompt the instrument. The proposed mechanism was also well
propped up by the fluorescence lifetime data. In the fluorescence
Fig. 9: The proposed mechanism for the sensing behaviour of S1
lifetime decay experiment (λem
= 554 nm), the average
luminescence lifetime of S1 was found to be 3.5 ns at which 455 nm
nanoLED source was used. After the addition of 10ꢀM of CN- ions to
the solution of S1, the average luminescence lifetime (λem = 570
nm) of S1-CN system increased to 4.7 ns. In both the cases, the
fluorescence decay curve was fitted to double-exponential decay
theoretical calculation. From the obtained results, there is a delay
(1.5 ns) in the decay process when cyanide ion interacts with the
receptor; hence it is proved that there is a platform for the
enhanced fluorescence in the case S1-CN- interaction. (Fig. 7a, b)
DFT theoretical calculations
Fig.7 Lifetime measurement studies of S1 (a) and S1- (b)
DFT theoretical calculations were demonstrated for S1 and S1- for
supporting the deprotonation occurred during the addition of
cyanide ion. The structural optimization and the computational
calculations were carried out using Gaussian09 quantum chemistry
package and the results were viewed with GaussView5 GUI.31
Geometry optimizations were conducted by DFT using Becke’s
three parameter exchange functional (B3)32 and includes a mixture
of HF with DFT exchange terms associated with the gradient
corrected correlation functional of Lee, Yang, and Parr (LYP)29, 33and
the 6-31G basis set. Highest occupied molecular orbital (HOMO)
and lowest unoccupied molecular orbital (LUMO) for S1 and its
deprotonated form (S1-) have been generated from optimized
structures of the same (fig. 8). From the calculated results, the
energy gap between HOMO and LUMO of S1 had decreased after
deprotonation of –NH of imidazole by cyanide ions. The calculated
absorbance maxima of S1 and S1- from the energy gap (ꢁES1 and
-
ꢁES1 ) were 364 nm and 454 nm respectively and well coincided
with the experimental values. From the TD-DFT studies of the
receptors S1, S2 and S4 and their corresponding deprotonated form
S1- , S2- and S4- were carried out with TD-SCF CAM-B3LYP with the
basis set 6-31G (keyword- td cam-b3lyp/6-31g geom=connectivity).
From the results, it could be concluded that the experimental λmax
of the receptors and their respective deprotonated forms were well
agreed with those of calculated λmax which further supports the
sensing behaviour of the receptor. (Table 2)
Fig.8 DFT Theoretical studies using Gaussian 09 software with B3LYP
6-31G basis set of S1 and S1-.
Table 2: The comparison of experimental and theoretical λmax of S1
and S1- using TD-DFT Studies.
Species
Calculated λmax
(nm)
Experimental λmax
(nm)
Mechanism
S1 and S1-
S2 and S2-
S4 and S4-
382.12/455.33
380.76/495.22
380.97/521.00
384/455
From 1H NMR spectral titration, DFT Calculation and life time
measurement studies, S1 was deprotonated at –NH imidazole by
cyanide ions and the electron density over benzimidazo
anthraquinone (chromophore) moiety was increased that driven
the color change. The phenomenon was simply illuminated by the
following statements; the bulkier nitro group in the o-position and
1-H of imidazole tend to disturb the co-planarity of the molecule
whereas the co-planarity may be achieved in the excited state on
deprotonation of the same followed by the fluorescence
410/480
443/500
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Table 3: Real sample analysis of S1 using waste water sample.
10% fetal bovine serum (FBS) at 37 °C under an atmosphere of 5%
CO2. Cells were plated on 18 mm glass coverslips and allowed to
adhere for 24 h. Experiments to assess CN- ions uptake were
performed in PBS with 10 μΜ (CH3CH2CH2CH2)4N(CN). Treated the
cells with 2 μL of 10 mM CN- ions (final concentration: 10 μM)
dissolved in sterilized PBS (pH 7.4) and incubated for 30 min at
37°C. The treated cells was washed PBS (3×2 mL) to remove
remaining (CH3CH2CH2CH2)4N(CN) ions. Culture media (2 mL) was
added to the cell culture, which was treated with a 10 mM solution
of S1 (2μL; final concentration: 10 μM) dissolved in DMSO. The
samples were incubated at 37°C for 30 min. The culture media was
removed, and the treated cells were washed with PBS (3×2 mL)
before observation. (Fig. 10) From the images, it could be inferred
that S1 has very good permeability through the cell membrane and
has very weak fluorescent whereas after the addition of cyanide
ions, S1 sensed the guest selectively with enhanced yellowish green
fluorescent.
Sample (ppm)
Average Recovery
% Recovery
(ppm)
0
0
5
Nil
4.3
86%
92%
96%
10
15
9.2
14.4
Conclusion
Novel TICT based fluorescent turn on probe S1 was synthesized
from 2-nitrobenzaldehyde and 1,2-diaminoanthraquinone and
characterized well with various spectroanalytical techniques.
Cyanide ions were selectively sensed by S1 through the
deprotonation of 1-H imidazole followed by the inhibition of TICT
occurred led to 10-fold fluorescent enhancement. Effect of
substitution was discussed using S2-S4. In case of S2, fluorescence
quenching was observed due to the deprotonation of both the sites
(-OH and imidazole –NH). But S4 showed sufficient bulkiness to give
slight fluorescent enhancement. S1 showed relatively very good
association constant with 1:1 stoichiometric ratio and micromolar
detection limit. 1H NMR titration clearly supported the
deprotonation of 1-H imidazole and the sensing behaviour was
further supported by DFT as well as TD-DFT studies. Moreover,
lifetime measurement showed that there is a delay (1.5 ns) in
double exponential decay of S1 and S1- which was essential
information for the fluorescence enhancement behaviour. S1 was
successfully applied for the real time analysis of waste water and
the bio-imaging of the cell line RAW264.7 and sensed the cyanide
ions with yellowish green fluorescence output under physiological
conditions.
Fig.10 Fluorescence images of macrophage (RAW 264.7) cells
treated with S1 (a) and CN- (b) ions. (Left) Bright field image;
(Middle) fluorescence image; and (Right) merged image. (Ex 456nm,
Em.540-580nm.)
enhancement; In other words, the higher electron withdrawing
nature of nitro group makes the phenyl ring as an acceptor and
there will be a Twisted Intramolecular Charge transfer (TICT) from
electron rich imidazoanthraquinone moiety to nitrophenyl ring in S1
whereas the deprotonation inhibits the TICT followed by the
fluorescent enhancement. (Fig. 9)
Real Sample Analysis
Role of a chemosensor could be accomplished only when it might
be useful in practical applications. Sensing cyanide ions in waste
water collected in our premises was demonstrated with S1. The
samples were collected every one hour and mixed with together.
The resulting turbid sample as a whole was filtered to remove the
suspended particle led to a light grey solution (pH 8.0). On testing
the filtered sample with S1, no color change was observed. 5, 10
and 15 ppm cyanide solutions prepared from the filtered water
samples were added with S1. The color change as well as
bathochromic shift in the UV-vis spectra were observed. From the
absorbance at 455 nm of the three solutions, using Beer-Lambert’s
Law, the corresponding concentrations were calculated for all the
solutions. Overall 90% recovery was observed it could be concluded
that S1 acts as a highly sensitive and selective chemoreceptor for
CN- ion (Table 3).
Acknowledgement
Author GB thanks UGC (University Grant Commission, India) for the
financial support through Junior Research Fellowship scheme (F.2-
8/2011(SA-1) dated 29/11/2013).
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DOI: 10.1039/C5RA27517B
Novel Ratiometric Turn-on Fluorescent Probe for Selective Sensing of
Cyanide ions, Effect of substitution and Bio-imaging Studies
Gopal Balamurugana, Parthiban Venkatesanb, Shu Pao Wub, Sivan Velmathia,*
aOrganic and polymer synthesis Laboratory, Department of Chemistry, National Institute of
Technology, Tiruchirappalli-620015, India.
bDepartment of Applied Chemistry, National Chiao Tung University, Hsinchu, Taiwan
*Corresponding Author velmathis@nitt.edu, svelmathi@hotmail.com
Graphical Abstract