Highly reactive (<1 min) ratiometric "naked eye" detection of hypochlorite with real application in tap water

Article abstract of DOI:10.1039/c3dt51238j

The de-diaminomaleonitrile reaction in a simple carbazole diaminomaleonitrile hybrid (CDH) was promoted by OCl^- which disrupted the ICT mechanism by breaking the donor and acceptor linkage. This system utilized an irreversible OCl^- promoted oxidation reaction and it responded instantaneously at room temperature. The chemosensor also showed excellent performance in tap water and the "dip-stick" method.

Full text of DOI:10.1039/c3dt51238j

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Highly reactive (<1 min) ratiometric “naked eye”
detection of hypochlorite with real application in tap
Cite this: Dalton Trans., 2013, 42, 10097
Received 10th May 2013,
Accepted 13th May 2013
water†
DOI: 10.1039/c3dt51238j
Shyamaprosad Goswami,* Sima Paul and Abhishek Manna
www.rsc.org/dalton
The de-diaminomaleonitrile reaction in
a
simple carbazole
In recent years, a number of analytical methods, such as
diaminomaleonitrile hybrid (CDH) was promoted by OCl− which colorimetric, luminescent, electrochemical and chromato-
disrupted the ICT mechanism by breaking the donor and acceptor graphic methods9 have been proposed for the detection of
linkage. This system utilized an irreversible OCl− promoted ClO−. Fluorescent probes play an important role in this
oxidation reaction and it responded instantaneously at room respect, due to their great temporal and spatial resolution
temperature. The chemosensor also showed excellent perform- capability, as well as high sensitivity, simplicity of implemen-
ance in tap water and the “dip-stick” method.
tation, fast response times and offering application methods
for not only in vitro assays but also in vivo imaging studies.10
Till now, only a few fluorescent probes for ClO− have been
reported.11 Fluorescent probes are sensitive, but the signal
read-out still needs the aid of bulk instruments. In compari-
son, colorimetric probes, in particular naked eye detection
methods, have obvious advantages. Moreover, spectral studies
are significantly influenced by some factors including probe
concentration, sample environment and light scattering. By
contrast, these factors may be potentially avoided by employ-
ing ratiometric probes, as they allow the measurement of
absorbance and intensities at two wavelengths.12 Thus, we
have designed a ratiometric “naked eye” as well as fluorescent
probe for the detection of ClO−.
Generally, when a fluorophore contains an electron-donat-
ing group conjugated to an electron-withdrawing group, it
undergoes intramolecular charge transfer (ICT) from the
donor to the acceptor upon excitation by light. The conse-
quence of the ICT is a red shifted emission and the compound
becomes non-fluorescent. Many fluoroionophores have been
designed by employing the interaction of cations with the elec-
tron-donating group.13 After being bound to a cation, the elec-
tron-donating group loses its donating ability, so the ICT
ceases, a blue shift appears and the compound becomes
strongly fluorescent, which is often used for the fluorescent
ratiometric determination of cations (Scheme 1).14 Instead of
the suppression of ICT through metal co-ordination with a
receptor, it is also reasonable to deduce that the fluorescence
intensity can be enhanced by breaking the linkage of the elec-
tron-withdrawing and electron-donating groups in the sensor
molecule through a chemical reaction promoted by special
analytes. However, there is no report concerning this thought,
to suppress the ICT mechanism.
The development of selective and sensitive signaling systems
for the determination and visualization of anions with biologi-
cal interest is a very attractive research topic in the chemosen-
sing and molecular imaging fields.1 Reactive oxygen species
(ROS) are produced mainly through an electron transfer reac-
tion from oxygen, and they play a crucial role in many physio-
logical processes and are also implicated in many human
diseases, including cancer, neurodegenerative disorders, and
inflammatory processes.2 Hypochlorite (OCl−)/hypochlorous
acid (HOCl) is one of the biologically important reactive
oxygen species (ROS) and is produced in organisms by peroxi-
dation of the chloride ions catalyzed by the heme enzyme
myeloperoxidase (MPO).3 It is implicated in a variety of patho-
logical diseases including atherosclerosis, neuron degenera-
tion, cystic fibrosis, arthritis, and cancers.4,5 Hypochlorite also
plays a critical role in hosting innate immunity during
microbial invasion.6 Hypochlorite is also widely used in our
daily life, for example, in household bleach, disinfection of
drinking water and cool-water treatment, with a concentration
in the range of 10−5–10−2 M.7 Such highly concentrated hypo-
chlorite solutions are a potential health hazard to humans and
animals.8 Because of the biological and environmental impor-
tance of HOCl and its anion, the development of highly sensi-
tive and selective probes for HOCl has become an important
issue.
Bengal Engineering and Science University, Shibpur, Howrah 711103, India.
E-mail: spgoswamical@yahoo.com; Fax: +91-3326682916
†Electronic supplementary information (ESI) available: Synthetic procedure with
characterisation and spectral data are available. See DOI: 10.1039/c3dt51238j
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Fig. 1 (a) UV-vis absorption titration spectra of the CDH (c = 2.0 × 10−5 M) in
the presence of OCl− (c = 2.0 × 10−4 M) at pH 7.4 in CH3CN–H2O (4 : 6, v/v). The
naked eye colour change of the CDH on addition of OCl− (inset). (b) Time-
dependent absorption intensity of the CDH at 283 nm (in red) and 400 nm (in
black) in the presence of OCl− (2 equiv.).
Scheme 1 Schematic approach for the detection of ClO−.
The plausible mechanism of sensing can be ascribed to the
rapid de-imination of the aldehyde group by hypochlorite
under mild conditions (i.e. ambient temperature). By utilizing
the virtue of this specific deprotection reaction promoted by
hypochlorite, recently W. Lin et al. reported a highly selective
probe for hypochlorite.15 In our receptor, we protected the
aldehyde group with diaminomaleonitrile (donor), which
becomes non-fluorescent due to intramolecular charge trans-
fer. Taking advantage of the high oxidizing properties of OCl−,
our receptor, carbazole diaminomaleonitrile hybrid (CDH),
behaved as a fluorescent chemosensor for OCl−. The caged
fluorescence moiety of the chemosensor was oxidatively
removed by OCl− (scheme presentation of the reaction in
Fig. 4). We expected the protective group, diaminomaleonitrile,
to be removed and the fluorescent carbazole aldehyde to be
generated, accompanied by the “off–on” signal. If this was the
case, the design approach in this work should open up a new
avenue for the development of fluorescence turn-on chemo-
dosimeters for OCl−, by suppressing the ICT mechanism
through breaking the linkage of the electron-donating and
electron-withdrawing groups (Scheme 1).
The sensing properties of the CDH in response to OCl−
were investigated by both UV-visible and fluorescence titration
in a CH3CN–H2O solution (4 : 6, v/v, 10 mM HEPES, pH 7.4).
The CDH absorbed strongly around 283 nm and 400 nm,
which was attributed to the ICT transition in CDH. However,
an increasing absorption around 283 nm emerged gradually
and the absorption around 400 nm decreased simultaneously
with a clear isosbestic point at 316 nm, when OCl− was added
to the CDH (Fig. 1a). The bathochromic shift was apparently
attributed to the ICT.18 This phenomenon was likely due to the
oxidation of the CDH and led to the formation of the carbazo-
ledialdehyde. In fact, in the presence of 2 equiv. of OCl−, an 82
fold enhancement in the ratiometric value of A283/A316 (0.056
to 4.565) was achieved with respect to the hypochlorite free
solution (Fig. S3b†). Under the present conditions the reaction
of the CDH with OCl− was rather fast and it reached equili-
brium within 1 min after the addition of OCl− (Fig. 1b).
In agreement with the absorption spectral change for the
CDH after introducing OCl−, the orange colored solution of
the CDH became colorless on addition of only 2 equiv. of OCl−
(see the inset of Fig. 1a). Thus, this detection behavior could
be easily seen with the naked eye under a normal UV lamp.
The absorption spectra of the CDH probe in the presence of
different anions and oxidants were investigated. As shown in
Fig. S3a,† the representative species including Cu2+, Co2+,
Hg2+, Mg2+, Fe3+, F−, I−, NO2−, NO3−, SO42−, SO32−, PO4−, OH−,
H2O2, O2−, CH3COOOH, showed nearly no changes in the
absorption spectra observed upon the addition of those
species at a concentration of 40 mM, indicating that our probe
showed a selective response towards OCl− over other anions
and oxidants. In the time dependent absorption spectra, it is
seen that the reaction is completed within 1 min, with a rate
constant of 10.4 × 10−2 s−1, which strongly supports the high
reactivity of the probe (Fig. S4 and S5†).
The target compound was synthesized through the series of
reactions detailed in Scheme 2. The intermediate compounds
(A)16 and (B)17 were synthesized according to the reported
procedures. The structure of the receptor was confirmed by
1H NMR, 13C NMR and ESI MS spectra (ESI†).
The fluorescence signaling behavior of the CDH towards
OCl− and the representative anions and oxidants was surveyed
upon excitation at 316 nm. In the case of the fluorescent titra-
tion the free probe displayed two emission peaks with a
Scheme 2 Synthetic route of the receptor (CDH). Reagents and conditions: (a)
butylbromide, K2CO3, KI, CH3CN, heated to 80 °C, 4 h; (b) POCl3, DMF, 100 °C,
20 h; (c) diaminomaleonitrile, ethanol, reflux, 8 h.
10098 | Dalton Trans., 2013, 42, 10097–10101
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Fig. 3 (a) Comparison of the emission intensity ratio (F456/F376) of the CDH
probe at pH 7.4 in CH3CN–H2O (4 : 6, v/v), in the presence of various anions and
oxidants. (b) Competitive fluorescent signaling of OCl− (2 equiv.) by the CDH in
the presence of various anions and oxidants (10 equiv.), λex = 316 nm.
Fig. 2 (a) Fluorescence emission spectra of the CDH (c = 2.0 × 10−5 M) with
OCl− (c = 2.0 × 10−4 M) at pH 7.4 in CH3CN–H2O (4 : 6, v/v). The naked eye fluo-
rescence change of the CDH on addition of OCl− (inset). (b) Plot of the fluo-
rescence intensity vs. the whole range of hypochlorite concentration tested at
456 nm.
maximum intensity at around 376 nm and 456 nm (Φ = 0.01),
which are characteristic of the carbazole moiety. By contrast,
upon addition of OCl−, the intensity of the emission peak at
376 nm gradually decreased with the simultaneous increased
intensity at 456 nm (Fig. 2a), with a clear isosbestic point at
398 nm (Φ = 0.65). The fluorescence maximum was red-shifted
by about 8 nm. The non-fluorescent solution exhibited blue
fluorescence, when OCl− was gradually added to the solution
of the CDH (Fig. 2a, inset). A linear calibration graph of the
response to OCl− concentrations from 0.99 to 23 μM was
obtained (Fig. 2b), indicating that the CDH probe can be
potentially employed to quantitatively detect OCl− concen-
trations. The detection limit was found to be 1.07 μM based on
K × Sb1/S, where Sb1 is the standard deviation of blank
Fig. 4 Partial 1H-NMR spectra of CDH and CDH + OCl− in CDCl3.
measurements and S is the slope of the calibration curve NMR spectra of the CDH and the resultant compound of CDH
(ESI†).
and OCl− are shown in Fig. 4. The resonance signal at δ =
To test the selectivity, the CDH probe was incubated with 2.050 ppm in the CDH was attributed to the primary amine
the different anions and oxidants at pH 7.4 in CH3CN–H2O protons. Upon addition of 2 equiv. of OCl− a new signal at δ =
(4 : 6, v/v). As shown in Fig. 3a, OCl− elicited a large ratiometric 9.810 ppm appeared corresponding to the aldehyde protons
signal with F456/F376 = 12.14. By contrast, Fe3+, I− and H2O2 and the resonance signal at δ = 2.050 ppm completely dis-
induced a very modest ratiometric response with F456/F376
<
appeared. Meanwhile, carbazole dialdehyde was generated due
0.45, and the remaining species caused only negligible to the de-diaminomaleonitrile reaction in CDH which occurred
changes in the emission ratio (F456/F376 < 0.3). Thus, these in presence of OCl−. In the 13C NMR spectra of CDH and CDH
data indicated that the probe has high selectivity for OCl−. + OCl−, we found that the four peaks at 110.01, 114.70, 115.25
Practical applicability of the CDH as a OCl− signaling device and 125.07, corresponding to the diaminomaleonitrilic
was verified by competitive signaling experiments with carbon, disappeared and the peak at 164.35 was shifted to
different anions and oxidants (Fig. 3b). The CDH probe exhibi- 179.35 (generation of aldehydic carbon). In addition, the mass
ted unaltered signaling for OCl− in the presence of commonly spectrometry analysis after addition of OCl− showed that the
encountered anions and oxidants (except Fe3+, I− and H2O2, peak at m/z 459.47 disappeared and a new peak appeared at
which interfere slightly).
m/z 279.32 corresponding to carbazole dialdehyde.
The ratiometric responses of the CDH probe on addition of
Many sensors for OCl− detection can only be performed in
OCl− at different pH values were also investigated. Fig. S1† solution, which limits their application under special circum-
depicts the effect of the pH, which reveals that the detection of stances, such as on-site detection in situ. To demonstrate the
hypochlorite by the CDH is pH-dependent.
practical application of our sensor, we prepared TLC plates of
To confirm the ratiometric response of the CDH towards the CDH to determine the suitability of a “dip-stick” method
OCl−, the reaction of the CDH with 2 equiv. OCl− was carried for the detection of OCl− (Fig. 5(i)).
out in CH3CN–H2O (4 : 6, v/v). After stirring at ambient temp-
Hypochlorite is used in many industrial processes and also
erature for 4 h, the major product was purified by chromato- in daily life. Therefore, the detection of hypochlorite in water
1
graphy on a silica gel column and was subjected to H NMR, is of interest. We analyzed OCl− in tap water and distilled
13C NMR and ESI TOF mass analysis (ESI†). The partial 1H water. The experimental results showed that this sensing
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S. Goswami, A. Manna, S. Paul, K. Aich, A. K. Das and
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OCl− (×10−3 M) (a) 0, (b) 5, (c) 9, (d) 11. (ii) Competition fluorescence emission
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system gave no fluorescence response to the deionized water
(Fig. S7†). Thus, deionized water samples had no interference
in the performance of this sensing system. Nevertheless, the
addition of tap water (100 μL) led to a significant increase in
the fluorescence intensity of the sensing system, and a bigger
change was observed when increasing the amount of tap water
(Fig. 5(ii)), which further revealed that such a sensing system
was sensitive towards hypochlorite, even in real water samples.
The concentration of hypochlorite in tap water was determined
to be 0.256 mol L−1 from comparing the fluorescence intensity
of the CDH in the presence of tap water with the titration data.
In conclusion, a new carbazole based “naked eye” and ratio-
metric fluorescence probe, CDH, was developed for selective
and rapid detection of OCl− at physiological pH of 7.4 in
aqueous media. The chemical reaction was fast at room temp-
erature and irreversible. In this reaction, the addition of OCl−
to the CDH disrupted the ICT mechanism by opening a new
avenue for the development of a fluorescence turn-on chemo-
dosimeter for OCl−. This chemodosimeter is also a good can-
didate with great potential for the selective detection of OCl−
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The authors thank the DST and CSIR (Government of India)
for the financial support. S. P. and A. M. acknowledge the UGC
and CSIR respectively for providing fellowships.
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