Dyes and Pigments 147 (2017) 400e412

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Dyes and Pigments

journal homepage: www.elsevier.com/locate/dyepig

Pyreneamide-based dipodal probes for ultra-sensitive and selective

detection of 3,5-dinitrosalicylic acid in an aqueous solution
Ashwani Kumar*, Pil Seok Chae**
Department of Bionanotechnology, Hanyang University, Ansan, 155-88, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history: Pyrene-appended dipodal probes (probes 1 and 2) with differences in conformational rigidity were
Received 9 August 2017 synthesized for sensitive detection of 3,5-dinitrosalicylic acid (DNSA) in an aqueous solution. Both
Received in revised form dipodal probes had two pyrene units and exhibited quenching caused by aggregation (ACQ) at high water
23 August 2017
content (>60%) in DMSO. As the pyrene dimer (e.g., excimer) was a dominant conformation in 99%
Accepted 23 August 2017
aqueous solution, both probes showed sufficient excimer emission intensity at 486 nm (probe 1) or
Available online 25 August 2017
480 nm (probe 2) upon excitation. The excimer emission of the probes (probes 1 and 2) was sensitively
quenched in the presence of DNSA. Probe 1 selectively responded to DNSA among various carboxylic
Keywords:
Pyreneamide
acids (CAs), while probe 2 was responsive to both DNSA and 5-NSA. Density functional theory (DFT)
Excimer emission calculations suggest that, in the presence of DNSA, the probes undergo structural changes, leading to
DNSA formation of new pep interactions between each pyrene unit of the probe and the benzene ring of the
Fluorescence quenching guest molecule. This new pep interaction enabled energy transfer from pyrene to the non-radiative
guest molecule (DNSA), resulting in fluorescence quenching in the probes. The limit of detection
(LOD) of probe 1 with DNSA was observed to be 1 nM, the lowest value reported so far. This ultra-
sensitivity toward DNSA detection was retained even in the case of a probe 1-coated TLC strip. Probe
3, which had only one pyrene unit, gave produced a monomeric emission spectrum with lmax ¼ 406 nm,
along with aggregation-induced emission enhancement (AIEE) behavior. The interactions of DNSA with
individual probes were examined by UV-visible, fluorescence, and 1H NMR spectroscopies and were
further supported by DFT calculations.
© 2017 Published by Elsevier Ltd.

1. Introduction biological samples.

Aliphatic and aromatic carboxylic acids play significant roles in a
large number of metabolism/biological processes [1] and are
extensively used in various fields and industries. For example,
aliphatic carboxylic acids such as citric, gluconic, lactic, malic, and
tartaric acids are used as additives in the food industry [2]. Aro-
matic carboxylic acids are the main ingredients of paints and wood
preservatives. Perfluorocarboxylic acids are used as components of
fire-retardant foams and fluoroelastomers, in addition to their
widespread applications in process control [3], medical diagnosis
[4], and environmental monitoring [5]. Thus, there is growing in-
terest in sensing these acidic molecules in environmental and
* Corresponding author.
** Corresponding author.
E-mail addresses: ashwanirubal@gmail.com (A. Kumar), pchae@hanyang.ac.kr
(P. Seok Chae).
The medicinal properties of salicylic acid (SA), particularly for
fever reduction, have been known since ancient times. SA also has
anti-inflammatory and analgesic effects. These days, SA and its
derivatives are used as constituents of skin-care products and in
some drugs. For instance, methyl salicylate is a liniment to reduce
muscle and joint pain, while choline salicylate is used topically to
relieve the pain of mouth ulcers and to treat several diseases such
as psoriasis, calluses, corns, acne, seborrheic dermatitis, acanthosis
nigricans, ichthyosis, and warts [6]. SA is an ingredient in shampoos
because it is used for dandruff treatment [7]. Derivatives of SA are
widely used in medical diagnosis, pharmaceuticals, cosmetics, food
technology, and environmental monitoring [8]. SA also plays
important roles in plants including defense regulation against in-
sects and pathogens and in various physiological processes such as
seed germination, fruit yield, protein synthesis, and nutrient uptake
[9]. SA can also cause one of the most prevalent forms of immu-
notoxicity found in humans (i.e., allergic contact dermatitis) [10].

http://dx.doi.org/10.1016/j.dyepig.2017.08.039
0143-7208/© 2017 Published by Elsevier Ltd.
 A. Kumar, P. Seok Chae / Dyes and Pigments 147 (2017) 400e412
One SA derivative, 3,5-dinitrosalicylic acid (DNSA), is used in a
colorimetric biomedical assay for the detection of reducing sugars
[11,12]. Consequently, selective monitoring and quantitative
detection of SA and its derivatives in aqueous solutions are neces-
sary for detailed studies of their effects, functions, and physiological
properties [13].
Among various detection methods, fluorescence-based tech-
niques have many advantages over others. The high sensitivity,
specificity, and real-time monitoring with rapid response and easy
portability make this method irreplaceable and enable in-situ
monitoring of analytes of interest in real time and space.
In the design of fluorescence-based chemical probes, versatile
fluorophores have been connected with distinct binding units such
as a-aminopyridines/a-amidopyridines, ureas, amides/sulfon-
amides, and quaternary ammonium/imidazolium salts to develop
carboxylic acid sensors [13e17]. However, detection systems for a
CA in aqueous solutions are rare because most sensors have been
effective in non-polar or polar organic solvents. Recently, pyrene-1-
sulfonamide-based probes having neutral or ionic character have
been used for the recognition of SA and its derivatives in non-polar
or polar to mixed solvent systems [13]. In continuation of our
previous study of optical molecular probes [18], the aim of this
study is to develop chemical probes with enhanced selectivity to-
ward DNSA and to approach a pure aqueous system, which is an
essential feature for practical applications.
In the current study, we synthesized pyreneamide-based dipo-
dal probes 1 and 2 for sensitive and selective detection of an SA
derivative in pure aqueous solution. Probes 1 and 2 commonly bear
a central 4,40 -methylenediphenylene (MDP) unit flanked by two
glycine residues attached to a pyrene fluorophore. Probe 1 dis-
played high selectivity and sensitivity in DNSA sensing, with a limit
of detection (LOD) of 1 nM, while probe 2 was responsive to both
DNSA and 5-nitrosalicylic acid (5-NSA) with reduced sensitivity. To
the best of our knowledge, this is the lowest LOD (1 nM) observed
for DNSA detection. The ultra-sensitive detection observed for
probe 1 was attributed to strong intermolecular p-p interactions
between pyrene units of the probe and DNSA, allowing energy
transfer from the probe to the guest molecule. The conformational
rigidity of probe 1 appeared to be associated with the high selec-
tivity of the probe toward DNSA over 5-NSA. We discuss in-
teractions of these dipodal probes and their monopod analog
(probe 3) with DNSA in terms of aggregate size and absorption,
fluorescence and nuclear magnetic resonance (NMR) spectros-
copies in addition to DFT calculations frontier molecular orbitals
(FMOs) of the probes and their complexes with the guest molecule.
2. Experimental section
2.1. Details for general procedure and theoretical calculations were
given in ESI
2.1.1. Detailed procedures for probe preparation (synthesis of probes
1e3)
2.1.1.1. Di-tert-butyl (((methylenebis(4,1-phenylene))bis(azanediyl))
bis(2-oxoethane-2,1-diyl))dicarbamate (compound 4). A mixture of
(tert-butoxycarbonyl)glycine (1.93 g, 11 mmol), hydroxybenzo-
triazole (HOBt, 1.62 g, 12 mmol), and 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDC, 2.30 g, 12 mmol) was
dissolved in dichloromethane-dimethylformamide (CH2Cl2: DMF
(1:1), 20 mL) and stirred vigorously at 0  C for 20 min under an
argon atmosphere. Then, 4,40 -methylenedianiline (0.99 g, 5 mmol)
and diisopropylethylamine (DIPEA) (1.55 g, 12 mmol) were added
to the reaction mixture and stirred at room temperature for another
2 h. After completion of the reaction (detected by TLC analysis), the
solvent of the reaction mixture was reduced under vacuum. The
401
crude solid was dissolved in H2O and extracted with CH2Cl2, and
the organic layer was washed with H2O (2  50 mL) and a 5%
aqueous NaCl solution (100 mL). The collected organic layer was
then dried over anhydrous sodium sulfate and filtered. The filtrate
was evaporated under reduced pressure, and the residue was pu-
rified by column chromatography using CH2Cl2 as the eluent to
afford compound 4 as a white solid (2.3 g, 90% yield). 1H NMR
(400 MHz, DMSO-d6): d 1.40 (s, 18H, 6  CH3), 3.69 (d, J ¼ 4.8 Hz, 4H,
2  CH2), 3.83 (s, 2H, CH2), 7.01 (t, J ¼ 4.8 Hz, 2H, 2  NHCOOBut),
7.13 (d, J ¼ 6.8 Hz, 4H, 4  ArH), 7.48 (d, J ¼ 6.8 Hz, 4H, 4  ArH), 9.85
(s, 2H, 2  NHCO).
2.1.1.2. 2,2'-((methylenebis(4,1-phenylene))bis(azanediyl))bis(2-
oxoethan-1-aminium) (compound 5). To a CH2Cl2 (30 mL) solution
of compound 4 (512 mg, 1 mmol), trifluoroacetic acid (TFA, 4 mmol)
was added drop-wise, and the mixture was stirred overnight at
room temperature under an argon atmosphere. After completion of
the reaction (analyzed by TLC), the reaction mixture was evapo-
rated under reduced pressure and precipitated by the addition of
diethyl ether. The suspension was filtered, and the solid was
washed with diethyl ether to afford compound 5 as a white solid
(300 mg, 95% yield). 1H NMR (400 MHz, DMSO-d6): d 3.76 (br s, 4H,
2  CH2), 3.86 (s, 2H, CH2), 7.19 (t, J ¼ 8.4 Hz, 4H, 4  ArH), 7.49 (d,
J ¼ 8.4 Hz, 4H, 4  ArH), 8.16 (br s, 6H, 2 x 2NHþ 3 ), 10.45 (s, 2H, 2 
NHCO); 13C NMR (100 MHz, DMSO-d6): d 40.94, 119.33, 129.13,
136.22, 136.87, 164.61.
2.1.1.3. N,N'-(((methylenebis(4,1-phenylene))bis(azanediyl))bis(2-
oxoethane-2,1-diyl))bis(pyrene-1-carboxamide) (probe 1). A
mixture of pyrene-1-carboxylic acid (270 mg, 1.1 mmol), hydrox-
ybenzotriazole (HOBt, 162 mg, 1.2 mmol), and 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDC, 230 mg, 1.2 mmol) was
dissolved in (CH2Cl2: DMF (1:1), 20 mL) solution and stirred
vigorously at 0  C for 20 min under an argon atmosphere. Com-
pound 5 (157 mg, 0.5 mmol) and diisopropylethylamine (DIPEA)
(155 mg, 1.2 mmol) were added to the reaction mixture and sub-
sequently stirred at room temperature for another 8 h. After
completion of the reaction (as detected via TLC analysis), the sol-
vent of the reaction mixture was removed under vacuum. The
crude solid was dissolved in H2O and extracted with CH2Cl2, and
the organic layer was washed with H2O (2  50 mL) and a 5%
aqueous NaCl solution (100 mL). The collected organic extract was
then dried over anhydrous sodium sulfate and filtered. The filtrate
was evaporated under reduced pressure, and the residue was pu-
rified by column chromatography using CH2Cl2:CH3OH (8:2) as the
eluent to afford probe 1 as a white solid (325 mg, 85% yield);
m.p. ¼ 302  C. 1H NMR (400 MHz, DMSO-d6): d 3.91 (s, 2H, CH2),
4.23 (d, J ¼ 6.0 Hz, 4H, 2  CH2), 7.22 (d, J ¼ 8.4 Hz, 4H, 4  ArH),
7.61 (d, J ¼ 8.4 Hz, 4H, 4  ArH), 8.14 (t, J ¼ 8.0 Hz, 2H, 2  ArH),
8.21e8.31 (m, 8H, 8  ArH), 8.36e8.38 (m, 6H, 6  ArH), 8.71 (d,
J ¼ 9.2 Hz, 2H, 2  ArH), 9.03 (t, J ¼ 6.0 Hz, 2H, 2  PyCONH), 10.17
(s, 2H, 2  ArNH); 13C NMR (100 MHz, DMSO-d6): d 43.26, 119.30,
123.62, 123.76, 124.42, 124.94, 125.34, 125.62, 125.79, 126.58,
127.22, 127.92, 128.09, 128.33, 128.98, 130.23, 130.71, 131.59, 131.67,
136.31, 137.03, 167.61, 169.41; HRMS (EI): calc'd. for C51H37N4O4
(M þ Hþ): 769.2809; found: m/z 769.2812.
2.1.1.4. N,N'-(((methylenebis(4,1-phenylene))bis(azanediyl))bis(2-
oxoethane-2,1-diyl))bis(4-(pyren-1-yl)butanamide) (probe 2).
The synthetic procedure for this preparation is similar to that used
for probe 1 using pyrene-1-butyric acid (317 mg, 1.1 mmol) instead
of pyrene-1-carboxylic acid. Probe 2 was obtained as a white solid
(400 mg, 93% yield); m.p. ¼ 246  C. 1H NMR (400 MHz, DMSO-d6):
d 1.99e2.06 (m, 4H, 2  CH2), 2.34 (t, J ¼ 7.2 Hz, 4H, 2  CH2), one
signal of two CH2 groups merged with the signal of H2O, 3.82 (s, 2H,
402 A. Kumar, P. Seok Chae / Dyes and Pigments 147 (2017) 400e412
CH2), 3.88 (d, J ¼ 6.0 Hz, 4H, 2  CH2), 7.12 (d, J ¼ 8.4 Hz, 4H, 4 
ArH), 7.49 (d, J ¼ 8.4 Hz, 4H, 4  ArH), 7.96 (d, J ¼ 8.0 Hz, 2H, 2 
ArH), 8.05 (t, J ¼ 7.6 Hz, 2H, 2  ArH), 8.10e8.26 (m, 14H, 12 
ArH þ 2  PyCONH), 8.41 (d, J ¼ 9.2 Hz, 2H, 2  ArH), 9.94 (s, 2H, 2 
ArNH); 13C NMR (100 MHz, DMSO-d6): d 27.61, 32.20, 34.85, 42.67,
119.25, 123.62, 124.16, 124.24, 124.78, 124.91, 124.96, 126.14, 126.50,
127.20, 127.48, 127.57, 128.18, 128.89, 129.30, 130.44, 130.90, 136.25,
136.68, 136.93, 167.78, 172.58; HRMS (EI): calc'd. for C57H49N4O4
(M þ Hþ): 853.3748; found: m/z 853.3751.
2.1.1.5. Tert-butyl (2-((4-methoxyphenyl)amino)-2-oxoethyl)carba-
mate (compound 6). The synthetic procedure for this preparation is
similar to that used for compound 4 using p-anisidine (370 mg,
3 mmol) instead of 4,40 -methylenedianiline. The procedure affor-
ded compound 6 as a thick liquid (800 mg, 95% yield). 1H NMR
(400 MHz, CDCl3): d 1.47 (s, 9H, 3  CH3), 3.79 (s, 3H, OCH3), 3.92 (d,
J ¼ 6.0 Hz, 2H, CH2), 5.28 (br s, 1H, NHCOOBut), 6.86 (d, J ¼ 10.4 Hz,
2H, 2  ArH), 7.40 (d, J ¼ 10.0 Hz, 2H, 2  ArH), 8.04 (br s, 1H,
NHCO); 13C NMR (100 MHz, CDCl3): d 28.51, 55.68, 77.55, 144.38,
122.04, 130.67, 156.79, 167.77.
2.1.1.6. 2-((4-methoxyphenyl)amino)-2-oxoethan-1-aminium (com-
pound 7). The synthetic procedure for this preparation is similar to
that used for compound 5. The procedure afforded compound 7 as a
brownish-white solid (500 mg, 91% yield). 1H NMR (400 MHz,
DMSO-d6): d 3.73 (s, 3H, OCH3), 3.74 (d, J ¼ 5.6 Hz, 2H, CH2), 6.94 (d,
J ¼ 6.8 Hz, 2H, 2  ArH), 7.50 (d, J ¼ 6.8 Hz, 2H, 2  ArH), 8.10 (br s,
3H, NHþ 3 ), 10.31 (s, 1H, NHCO).
2.1.1.7. N-(2-((4-methoxyphenyl)amino)-2-oxoethyl)pyrene-1-
carboxamide (probe 3). The synthetic procedure for this prepara-
tion is similar to that used for probe 1 using compound 7 (181 mg,
1.0 mmol) instead of compound 5. The procedure afforded probe 3
as a white solid (360 mg, 88% yield); m.p. ¼ 254  C. 1H NMR
(400 MHz, DMSO-d6): d 3.75 (s, 3H, CH3), 4.21 (d, J ¼ 6.0 Hz, 2H,
CH2), 6.94 (d, J ¼ 9.6 Hz, 2H, 2  ArH), 7.60 (d, J ¼ 9.6 Hz, 2H, 2 
ArH), 8.14 (t, J ¼ 8.0 Hz, 1H, ArH), 8.22e8.31 (m, 4H, 4  ArH), 8.37
(t, J ¼ 8.0 Hz, 3H, 3  ArH), 8.71 (d, J ¼ 9.6 Hz, 1H, ArH), 9.03 (t,
J ¼ 6.0 Hz, 1H, PyCONH), 10.07 (s, 2H, ArNH); 13C NMR (100 MHz,
DMSO-d6): d 43.18, 55.17, 113.94, 120.65, 123.63, 123.77, 124.44,
124.97, 125.36, 125.64, 125.81, 126.60, 127.24, 127.93, 128.09, 128.35,
130.25, 130.73, 131.62, 131.67, 132.21, 155.19, 167.28, 169.41; HRMS
(EI): calc'd. for C26H21N2O3 (M þ Hþ): 409.1547; found: m/z
409.1550.
3. Results and discussion
The dipodal probes (1 and 2) share a rigid MDP unit with a bent
structure in the central region, making it possible to form a pyrene
excimer in an appropriate environment (see Fig. 1). However, these
two probes differ from each other in terms of conformational ri-
gidity because different pyrene acid derivatives were used for
probe preparation. Probe 2 is less rigid than probe 1 because the
pyrene-1-butyric acid used for probe 2 preparation has an addi-
tional flexible propylene chain compared to pyrene-1-carboxylic
acid used for probe 1 preparation. A short glycine linker and two
rigid amide bonds along with the rigid MDP unit strongly restrict
the conformational flexibility of probe 1. We also prepared probe 3
with a single pyrene unit, which is a monopod analog of probe 1
(see Fig. 1). Probes 1 and 2 were readily synthesized by EDC-
supported coupling of 2,2'-((methylenebis(4,1-phenylene))bis(a-
zanediyl))bis(2-oxoethan-1-aminium) (compound 5) with respec-
tive acid components (Scheme 1). The structures of probes 1 and 2
were supported by 1H and 13C NMR spectroscopies as well as by FAB
mass spectrometry (see ESI). The 1H NMR spectrum of probe 1 in
DMSO-d6 exhibited two aliphatic peaks: one from CH2 groups
(singlet at d 3.91 ppm) and the other from two NCH2 groups of the
glycine units (doublet at d 4.23 ppm). The amide protons of the two
PyCONH and two ArNHCO groups appeared at d 9.03 and
d 10.17 ppm, respectively, which are far downfield. This is pre-
sumably due to intramolecular hydrogen bond formation. In addi-
tion to the 1H NMR spectrum, the 13C NMR spectrum and mass data
are consistent with the chemical structure of probe 1 (see ESI).
Similarly, the 1H NMR spectrum of probe 2 in DMSO-d6 showed
three aliphatic signals arising from the propylene chain. One signal
appeared as a multiplet at d 1.99e2.06 ppm, corresponding to the
central CH2 group of the chain. The signal for one CH2 group of the
chain close to the carbonyl group appeared as a triplet at
d 2.34 ppm, while the signal for the other CH2 group close to pyrene
was obscured by overlapping with a H2O signal. The signals for the
amide protons close to pyrene merged with pyrene signals, giving a
multiplet peak at d 8.10e8.26 ppm, and the peak for the amide
protons of two ArNHCO appeared as a singlet in a far downfield
position (d 9.94 ppm). The 13C NMR spectrum and mass data also
supported the successful formation and isolation of probe 2.
Similarly, the synthesis of probe 3 was also confirmed by 1H and 13C
NMR spectra and mass data.
The UV-visible absorption spectra of the probes (probes 1, 2, and
3) dissolved in DMSO exhibited a typical pyrene signature with
absorption maxima (lmax) at 330, 345, and 372 nm. Due to the
presence of two pyrene units, absorbances of probes 1 and 2 were
found to be roughly two-fold higher than that of probe 3 (Fig. S1).
The optimal excitation wavelength for maximum fluorescence in-
tensity was obtained using a range of wavelengths from 300 to
360 nm with a 5 nm interval. Probes 1 and 2 dissolved in aqueous
solution (99%) produced the maximum fluorescence intensity upon
excitation at 340 nm (Fig. S2-S3). Under these conditions, the
dipodal probes gave rise to green fluorescence with lmax at
480e488 nm, indicative of the formation of a pyrene excimer via
the intramolecular p-p stacking of two pyrene units. An emission
maximum at 383 nm associated with monomeric pyrene units was
also observed. To further investigate the solvent effect on the
fluorescence emission of these probes, 1 mM probe solutions were
prepared in various solvent systems, and their fluorescence spectra
were measured. The solvents used for this experiment include
water (99%), dimethylsulfoxide (DMSO), dimethylformamide
(DMF), ethanol (EtOH), methanol (MeOH), t-butanol (tBuOH),
acetonitrile (ACN), acetone, tetrahydrofuran (THF), ethyl acetate
(EA), dichloromethane (DCM), chloroform (CHCl3), dioxane, and
toluene. Under an illumination of 365 nm, probe 1 showed a high
blue emission in most solvent cases. This emission was from the
pyrene monomer. In contrast, this monomeric fluorescence in-
tensity sharply decreased when water was used as a solvent. This
was accompanied by the emergence of a green excimer emission
with relatively low intensity (Fig. 2). As for probe 2, both excimer
and monomer emissions (designated as I480 and I380, respectively)
appeared in all tested solvent systems, but the relative intensities
(I480/I380) varied significantly depending on the solvent used for the
study (Fig. S4). Of the various solvent systems, 99% water gave the
highest value in this emission ratio (I480/I380), and this was
responsible for the green emission of the probe solution under
illumination at 365 nm (Fig. S4). This behavior was similar to that of
probe 1, although the change in the emission ratio was less dra-
matic than probe 1 (Fig. S5). This result implies that the solvent
plays a crucial role in the formation of intramolecular p-p stacking
of the two pyrene rings present in these dipodal probes. Next, we
measured fluorescence spectra with increasing probe concentra-
tion in DMSO. Probe 1 produced pyrene monomer emission peaks
with little contribution from the pyrene excimer emission (Fig. S6).
Probe 1 has a low tendency to form intramolecular p-p interactions
 A. Kumar, P. Seok Chae / Dyes and Pigments 147 (2017) 400e412 403

Fig. 1. Chemical structures of pyreneamide-based probes (probe 1, 2, and 3). In dipodal probes 1 and 2, two terminal pyrene fluorophores are connected to a central 4,40 -meth-
ylenediphenylene (MDP) unit through a glycine linker, while probe 3 is the monopod analog of probe 1. Pyrene-1-carboxylic acid and pyrene-1-butyric acid were used for the
preparation of probes 1 and 2, respectively, providing a substantial difference in conformational rigidity between these dipodal probes.

Scheme 1. Synthetic scheme for the preparation of pyreneamide-based molecular probes (probes 1, 2, and 3). (a) HOBt, EDC, DIPEA, 0  C / RT; (b) TFA, RT; (c) pyrene-1-carboxylic
acid, HOBt, EDC, DIPEA, 0  C / RT; (d) pyrene-1-butyric acid, HOBt, EDC, DIPEA, 0  C / RT.

in this solvent system, presumably due to its rigid structure. In
contrast, probe 2, which has a rather flexible architecture, exhibited
both pyrene monomer and excimer emissions under the same
conditions, indicating that the conformational flexibility of this
probe enables pyrene-pyrene interactions. As expected, probe 3,
which has a single pyrene unit, yielded only peaks assigned to
monomeric pyrene emission (Fig. S6c). These peak intensities
tended to increase with increasing probe concentration in all probe
cases, but reached individual maxima at different probe concen-
trations, 4 to 10e12 mM, for probes 1e3, respectively. For probe 2,
the increase in excimer emission intensity (I480) was more promi-
nent than that of the monomer counterpart (I380) (Fig. S6d).
We investigated the solvent composition effect on the photo-
physical properties of probes 1e3 by increasing the concentration
of water from 0 to 99% in DMSO and performing UV-visible (Fig. S1)
and fluorescence spectroscopy (Fig. 3 & Fig. S7-8) measurements.
We also monitored the morphology and size changes for probe
aggregates under the same conditions (Fig. 3). When water content
was increased more than 60%, the UV-visible absorption spectra of
probes 1e3 became abruptly broader, the absorbance significantly
decreased, and a level-off tail was observed in a wavelength region
higher than 360 nm for each probe (Fig. S1). This result suggests the
formation of large probe aggregates at high concentrations of water
in DMSO.
Fluorescence properties of probes 1e3 also significantly varied
with increasing water content in a binary (DMSO-water) solvent
system (Fig. 3). The fluorescence intensity (I384) of probe 1 pro-
duced by monomeric pyrene remained almost constant up to 40%
water content, but decreased drastically with a further increase in
water content from 60 to 100% (Fig. 3A), which suggests aggrega-
tion caused by quenching (ACQ) behavior. Due to this quenching
behavior, an increase in excimer emission intensity (I484) of the
probe with increasing water content was not noticeable in the
spectra (Fig. 3B), but could be observed using the emission intensity
ratio (I484/I384) (Fig. 3C). This result indicates the enhanced ten-
dency to form intramolecular pyrene-pyrene stacking under the
condition tested (i.e., high water content in DMSO). To further
support the ACQ behavior of probe 1, we analyzed probe aggregates
404 A. Kumar, P. Seok Chae / Dyes and Pigments 147 (2017) 400e412

Fig. 2. (A) Solution color image of probe 1 dissolved in an indicated solvent. The image was obtained under UV light illumination at 365 nm, (B) corresponding fluorescence
emission spectra, and (C) normalized fluorescence emission spectra of the probe (1 mM) with lex ¼ 340 nm. In the normalized data, the fluorescence spectrum obtained from the
probe in water is quite different from those in other solvents, as expected from the corresponding solution colors in (A). (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)

via FE-SEM at different water contents (0, 50 and 90%) (Fig. 3D). At
0% water content (DW0), the image showed very few small parti-
cles with a diameter of about 400 nm. At 50% water content
(DW50), the particle size increased to 3e5 mm. When 90% water
content was used, large round plate-shaped aggregates with a
diameter of 30 mm were observed in a free or merged state. Thus,
this FE-SEM study indicates that aggregates formed by probe 1
mainly underwent a morphological change from nano-sized par-
ticles to micro-sized plate-shaped aggregates when water content
was increased from 0 to 50 to 90%. This behavior is likely associated
with a decrease in the monomer emission intensity of this probe at
a high water concentration (i.e., ACQ phenomenon). Unlike probe 1,
the monomer emission intensity (I380) of probe 2 decreased sub-
stantially with increasing water content from 0 to 40%, along with a
concomitant increase in excimer emission (I480) (Fig. S7), indicating
that probe 2 has a greater tendency to form intramolecular pyrene-
pyrene interactions than does probe 1, probably due to its increased
flexibility. When water content was further increased to 60e100%,
probe 2 showed a sharp decrease in both the monomer and excimer
emission intensities (Figs. S7B,C), which was also observed for
probe 1. This result is likely traced to the ACQ effect for both probes.
In this context, probe 3 showed a behavior quite different from
probes 1 and 2. This monopod probe produced a continuous in-
crease in emission intensity with increasing water content up to
80%, and the emission maxima was a little changed from 383 nm to
406 nm (Fig. S8). This result is a typical feature for a probe showing
aggregation-induced emission enhancement (AIEE). We also
analyzed probe 3 aggregates via FE-SEM at 0, 50, and 90% water
content in DMSO (designated as DW0, DW50, and DW90, respec-
tively) (Fig. S8C). At 0% water content (DW0), the image showed a
number of standing random pillars about 100 nm each in size;
while, at 50% water contents (DW50), these pillars moved closely
together to form large aggregates. Finally, at 90% water content, the
FE-SEM image showed large aggregates 20e35 mm in size. Thus,
this FE-SEM study indicates that aggregates formed by probe 3
mainly underwent morphological and size changes from nano-
sized pillars to micro-sized cloud-shaped aggregates. This obser-
vation is consistent with the AIEE behavior of this probe.
In summary, probes 1 and 2, with the dipodal pyrene units,
showed an increased tendency to form intramolecular pyrene-
pyrene stacking with increasing water concentration. They also
showed ACQ phenomena in the high water concentration range of
60e100%. In contrast, probe 3, having a single pyrene unit, dis-
played AIEE behavior with increasing water concentration without
excimer emission. These results are consistent with the previously
reported pyrene sulfonamide-based probes having one or two
pyrene units [18].
The interesting photo-physical properties of probe 1 prompted
us to investigate this probe for DNSA detection in an aqueous so-
lution. For this purpose, a variety of CAs (each 50 mM) was indi-
vidually added to solutions of probe 1 (1 mM). CAs used for this
study includes both aliphatic and aromatic acids (e.g., fumaric acid
(Fum. A), indole-2-carboxylic acid (IND-2-A), maleic acid (Maleic
A), malonic acid (Malonic A), nicotinic acid (NA), picolinic acid (PA),
terephthalic acid (T-PhA), isophthalic acid (Iso-PhA), phthalic acid
(PhA), succinic acid (Succinic A), uric acid (UA), 3,5-dinitrobenzoic
acid (3,5-DNBA), benzoic acid (BA), 2-thiophene carboxylic acid
(2-ThiopheneA), 3-chlorobenzoic acid (3-ClBA), 3-iodobenzoic acid
(3-IBA), 3-nitrobenzoic acid (3-NBA), 4-nitrobenzoic acid (4-NBA),
SA, 2,5-dihydroxybenzoic acid (2,5-DiOH-BA), 5-bromosalicylic
acid (5-BrSA), 5-chlorosalicylic acid (5-ClSA), 5-methylsalicylic
acid (5-MSA), 5-iodosalicylic acid (5-ISA), 5-nitrosalicylic acid (5-
NSA), and 3,5-dinitrosalicylic acid (DNSA)). Among these CAs, the
fluorescence emission of probe 1 was selectively quenched by the
presence of DNSA, presumably due to its highly electron-deficient
nature (Fig. 4 and Fig. S9). The sample containing 5-NSA also
showed fluorescence quenching, but with a much smaller degree
than DNSA. We also carried out an interference study by adding
 A. Kumar, P. Seok Chae / Dyes and Pigments 147 (2017) 400e412 405

Fig. 3. (A) Changes in solution color of probe 1 (1 mM) with increasing water concentration in a mixed DMSO:water solvent system under 365 nm illumination, (B) corresponding
spectral changes in fluorescence emission (lex ¼ 340 nm), (C) bar graph of the fluorescence intensity ratio (I488/I388) of the probe, and (D) FE-SEM images for probe aggregates
prepared at 0, 50, and 90% water content in DMSO. D and W represent DMSO and water, respectively, and the numbers 0, 10, 20, 40, …, 100 indicate the percentage of water. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

10 mM of DNSA to a probe 1 solution including individual CAs at
50 mM. No interferences of these CAs were observed in DNSA
detection (Fig. S10).
A fluorescence titration of probe 1 with DNSA was carried out to
understand their binding characteristics. Each additional DNSA
aliquot gradually quenched the emission of probe 1 at 484 nm, with
complete fluorescence quenching observed upon the addition of 2
equivalents of DNSA (Fig. 4BeC). The fitting of this titration data
using a 1:2 binding model gave Ka ¼ 2.92  107 M1 with a small
error (±5%), which indicates a strong association between the
probe and DNSA (Table 1). A binding ratio of 1:2 was deduced from
the titration data and was also confirmed by a Job's plot showing an
inflection point at 0.7 mol fraction of DNSA (Fig. S11). The limit of
detection (LOD) of probe 1 for DNSA was observed to be as low as
1 nM. This value was obtained from the linear relationship between
the fluorescence intensity of probe 1 at 484 nm (I484) and [DNSA]
(inset of Fig. 4C). A similar behavior was observed for probe 2 with
the same guest molecule, but the fluorescence quenching effect of
5-NSA was also pronounced in this case (Fig. S12), indicating the
limited selectivity of this probe in DNSA detection. Probe 2 showed
the same binding stoichiometry (1:2) as probe 1 and bound
strongly to DNSA, although the binding strength of this probe was
approximately two times weaker than that of probe 1
(Ka ¼ 2.92  107 vs. 1.57  107). This probe was calculated to bind to
406 A. Kumar, P. Seok Chae / Dyes and Pigments 147 (2017) 400e412

Fig. 4. (a) Fluorescence quenching effect of various carboxylic acids (CAs) for probe 1 in water. The probe and individual CAs were used at 1 and 50 mM, respectively. Io and I
represent fluorescence intensities of probe 1 at 484 nm in the absence and presence of individual CAs, respectively. The inset shows the color change of probe 1 solution before and
after DNSA addition under illumination at 365 nm. (B) Fluorescence spectral change of probe 1 (1 mM, water) with increasing amount of DNSA ranging from 0 to 4 mM, and (C) a
graphical representation of the change in fluorescence intensity at 484 nm (I484) as a function of DNSA concentration, lex ¼ 340 nm. The inset shows a linear relationship between
I484 and [DNSA]. (D) FE-SEM images for aggregates formed by free probe 1, its DNSA complex, and DNSA alone in a 90% water solution in DMSO. (For interpretation of the references
to colour in this figure legend, the reader is referred to the web version of this article.)

DNSA 100 times stronger than to 5-NSA (Ka ¼ 1.57  107 vs. in detecting DNSA and 5-NSA. The association strengths of the
1.25  105) (Table 1). A control agent, probe 3, displayed a sensing probe with these acid derivatives were only moderately strong or
behavior similar to that of probe 2 in terms of the limited selectivity relatively weak (Fig. 5 and Fig. S13). It is noteworthy that we used
 A. Kumar, P. Seok Chae / Dyes and Pigments 147 (2017) 400e412 407

Table 1 emission intensity at 406 nm instead of 484 nm to calculate the

Association constants (Ka (M1)) of probes 1e3 with DNSA or 5-NSA in water (99%). association constant for the [probe 3 þ DNSA or 5-NSA] system as
Probes 1 2 3 this probe did not show excimer emission.
DNSA 2.92  107 1.57  107 2.42  104
To investigate whether the complexation of probe 1 with DNSA
5-NSA e 1.25  105 9.83  103 affects the aggregate size, we performed FE-SEM analysis on [probe
1 þ DNSA (4 equiv.)] suspended in 99% water solution (Fig. 4D). FE-

Fig. 5. (a) Fluorescence quenching effects of various carboxylic acids (CAs) for probe 3 dissolved in water. The probe and individual CAs were used at 1 mM and 50 mM, respectively. Io
and I represent fluorescence intensities of probe 1 at 406 nm in the absence and presence of individual CAs, respectively. (B) Fluorescence spectral change of probe 3 (1 mM, water)
with increasing amount of DNSA, and (C) a graphical representation of the change in fluorescence intensity at 406 nm (I406) as a function of DNSA concentration, lex ¼ 340 nm. (D)
FE-SEM images for aggregates formed by free probe 3, its DNSA complex, and DNSA alone in a 90% water solution in DMSO.
408 A. Kumar, P. Seok Chae / Dyes and Pigments 147 (2017) 400e412
SEM images showed a decrease in aggregate size from 30 mm to
5 mm upon complexation of the probe with DNSA. The decrease in
aggregate size is likely due to the increased polarity of the probe 1-
DNSA complex relative to that of probe 1 alone. This increase in
polarity was caused by the association with highly water-soluble
DNSA. Such a large polarity increase can induce partial dissocia-
tion of aggregates in an aqueous solution. A similar de-aggregation
behavior was also observed for probe 3 complexed with DNSA
(Fig. 5D).
The fluorescence quenching effect of DNSA for each probe
(probe 1, 2, or 3) was further investigated by plotting the fluores-
cence intensity of the probe as a function of DNSA concentration
(Stern-Volmer plot) (Fig. 6A). Stern-Volmer constants were calcu-
lated using the Stern-Volmer equation (Io/I ¼ 1 þ Ksv [Q]). Stern-
Volmer constants (Ksv (M1)) of probes 1 and 2 with DNSA were
found to be higher (1.19  107 M1 and 7.73  106, respectively)
than that of probe 3 (2.11  105 M1) (Fig. 6B), indicating a more
effective decrease in the fluorescence intensity of these dipodal
probes in the presence of DNSA than probe 3. The relative order of
Stern-Volmer constants (Ksv) for individual probe-DNSA systems
was similar to that of the association constants (Ka) for the same
systems (Table 1). The high Stern-Volmer constants (Ksv) and as-
sociation constants (Ka) observed for the dipodal probes (1 and 2)
strongly suggest that pyrene excimer formation is a main reason for
the enhanced ability of these probes to sensitively detect DNSA
relative to probe 3.
To further address the interactions between the pyrene rings
and DNSA, 1H NMR spectra of probes 1 and 2 were collected in
DMSO-d6 in the absence or presence of DNSA. The addition of 1 or 2
equiv. of DNSA to each probe did not result in any substantial
changes in chemical shifts for the protons of pyrene and benzene
rings or glycine units in DMSO-d6 (Fig. S14-S15). No detectable
change in the chemical shift was observed for the DNSA signals.
When the same NMR experiment was conducted in (DMSO-
d6:D2O) (6:2) instead of DMSO only, we could not still observe any
significant changes in the chemical shifts of the proton signals of
probe 1 in the presence of DNSA (Fig. 7 and Fig. S16). Thus, the
chemical shifts for the protons of both the probes and DNSA were
not significantly affected by the molecular interactions between the
probes and DNSA as observed for DMSO-d6 and (DMSO-d6:D2O)
Fig. 6. (a) Stern-Volmer plot and (b) Stern-Volmer constant (Ksv (M1)) of DNSA for probes 1e3. Emission intensities at 484 nm for probe 1, 480 nm for probe 2, and 406 nm for
probe 3 were measured during the stepwise addition of DNSA (i.e., quencher) to individual probes (1 mM) dissolved in 99% water. Emission intensity ratios (Io/I) were calculated and
plotted against quencher concentration ([Q]), where Io and I are the fluorescence intensities in the absence and presence of DNSA, respectively, and [Q] is DNSA concentration.
(6:2) systems.
Further insight into the conformational changes of probes 1e3
upon complexation with DNSA was obtained by calculating energy-
minimized structures via density functional theory (DFT) at the
B3LYP/6-31G* level. As can be seen in Fig. 8, the optimized struc-
tures for probe 1 or 2 alone showed intramolecular pyrene-pyrene
interactions at a distance of 4.15e4.22 Å, which is comparable to a
previous report [19]. In the presence of DNSA, probes 1 and 2 were
calculated to undergo structural changes from a closed to an open
conformation. Consequently, the pyrene-pyrene interactions of the
probes are converted into newly formed pyrene-DNSA interactions
at a distance of 3.53e3.68 Å (Fig. 8). These new pep interactions
enable energy transfer from the electron-rich pyrene ring to an
electron-deficient DNSA upon excitation at 340 nm, leading to
quenching of fluorescence emission. In Fig. 5, we show that the
fluorescence emission of probe 3 at 406 nm was also quenched in
the presence of DNSA. This result could also be explained by
looking at the optimized structures of the probe-DNSA complex
obtained from the DFT calculation. Like the dipodal probes, the
pyrene ring of this probe forms pep interactions with DNSA at a
distance of 3.58 Å in the presence of the guest molecule (Fig. 8).
The fluorescence emission of the probes and the emission
quenching by their association with DNSA were further analyzed in
terms of electronic transitions from their HOMOs to LUMOs
(Fig. S17). DFT calculations showed that the frontier molecular or-
bitals (i.e., HOMOs and LUMOs) of these probes are mainly localized
on the pyrene rings of the probes. Upon complexation with DNSA,
their locations were changed as follows. For probe 1 or 2-DNSA
complexes, the HOMOs and LUMOs were localized on the pyrene
ring and DNSA, respectively, while for the probe 3-DNSA complex,
these orbitals were localized on p-anisidine and DNSA, respectively.
Thus, it is likely that energy transfer takes place from the pyrene
ring of each probe to DNSA upon excitation. Due to the non-
radiative character of DNSA, this energy transfer results in emis-
sion quenching of the complexes. The HOMO e LUMO energy gaps
(DE) of all tested probes were significantly reduced upon
complexation with DNSA compared to that of the respective probe
alone; this is mainly due to the change in the LUMO location from
the pyrene ring to the DNSA in all probe cases. The HOMO e LUMO
energy gap of the [probe 1(DNSA)2] complex was comparable to
 A. Kumar, P. Seok Chae / Dyes and Pigments 147 (2017) 400e412 409

that of [probe 2(DNSA)2], but substantially larger than that of
[probe 3(DNSA)] (i.e., DE1(DNSA)2 ¼ 1.99 eV > DE2(DNSA)
2 ¼ 1.96 eV > DE3 DNSA ¼ 1.29 eV). The spectral overlap between
the absorption spectrum of DNSA and the fluorescence spectrum of
probe 1 suggests energy transfer from the probe to the guest
molecule as a quenching mechanism (Fig. 9A). This was further
supported by fluorescence lifetime experiments. Fluorescence
emission can be quenched by either static or dynamic process.
Alternatively, both processes could take place together in fluores-
cence quenching. In static quenching, a fluorophore forms a stable
non-fluorescent complex with an analyte in the ground state, while
in dynamic quenching; an excited fluorophore transfers its energy
to an analyte (i.e., a quencher) in close proximity without a need for
stable complex formation. Whether fluorescence quenching occurs
in a static or dynamic process can be determined based on (a) the
Stern-Volmer plot and (b) the excited state lifetime (t) of a sample
of interest. For the current system of probe 1 and DNSA, both
fluorescent intensity and lifetime decreased with increasing
quencher concentration (Figs. 6A and 9B), where the fluorescence
lifetime was reduced from tint ¼ 13.25 to 9.27 ns by the addition of
2.0 equivalents of DNSA. The linear Stern-Volmer plot and the
substantially decreased excited state lifetime in the presence of the
guest molecule suggest that a dynamic process is responsible for
the fluorescence quenching observed for probe 1 with DNSA.
To investigate the potential of the current sensing system as a
convenient and inexpensive sensing tool, silica-coated test strips
were prepared using probe 1. We wrote the letters ‘DNSA’ on a TLC
plate using a probe 1 solution (10 mM, DMSO:H2O (1:9)). We then
dried the plate and then observed the green emission of these
letters under 365 nm irradiation (Fig. 10a). After dipping this TLC
plate into a 1 mM solution of DNSA in tap water, the fluorescence
intensity of the probe was significantly reduced (Fig. 10b). The

Fig. 7. (i) Partial 1H NMR spectra of probe 1 in the absence of DNSA or (ii) in the presence of 1 equiv. of DNSA, (iii) 2 equiv. of DNSA, and (iv) DNSA alone in (DMSO-d6:D2O) (6:2).

Fig. 8. Energy-minimized structures of probes 1e3 in the absence (left) or presence of DNSA (right), pep interactions between two pyrene rings or between a pyrene ring and DNSA
were indicated by yellow dotted lines, along with inter-ring distances. The structures were obtained from DFT calculations at the B3LYP/6-31G* level. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article.)
410 A. Kumar, P. Seok Chae / Dyes and Pigments 147 (2017) 400e412

Fig. 9. (A) Spectral overlap between the normalized absorption spectrum of DNSA and
normalized fluorescence spectrum of probe 1 in water. (B) Fluorescence lifetime decay
profile of probe 1 (1 mM) alone or in the presence of 2.0 equiv. of DNSA in water. The
inset shows a change in the ratio of excited state lifetime (t0/t) of probe 1 induced by
the presence of 2.0 equiv. of DNSA. t0 and t represent excited state lifetimes of the
probe in the absence and presence of the guest molecule, respectively.
detection limit of this system was investigated by preparing a TLC
plate with four spots using a probe 1 solution (10 mM, DMSO:H2O
(1:9)) (Fig. 10c). When these spots were individually in contact with
water and solutions of 1, 10 and 100 nM DNSA, a notable quenching
of the green emission was observed even in DNSA concentrations as
low as 1 nM. This demonstrates the potential of these probes in real
time sensing applications (Fig. 10d).
Considering that probe 1 was highly sensitive to DNSA, a highly
electron-deficient compound, it is interesting to investigate the
behavior of the same probe with other highly electron-deficient
compounds such as 2,4,6-trinitrotoluene (TNT) and 2,4,6-
trinitrophenol (TNP). For this purpose, we tested various phenol
derivatives including TNP and TNT with probe 1 under the same
conditions tested above. Among the various phenol derivatives and
TNT, only TNP exhibited the selective fluorescence quenching of
probe 1 (Fig. S18). However, the binding strength of probe 1 for TNP
was 100 times smaller than that of the probe with DNSA
(Ka ¼ 1.80  105 vs. 2.92  107 M1). Furthermore, an observed LOD
for TNP sensing was 500 nM, significantly larger than that (1 nM)
for DNSA sensing. Thus, probe 1 is an optimal chemical probe for
selective and sensitive detection of DNSA. DNSA, TNT and TNP
commonly have a highly electron-deficient ring system due to the
presence of multiple electron-withdrawing substituents on the
benzene ring. However, probe 1 was not responsive to TNT at all,
indicating that electron deficiency of a guest molecule is not suf-
ficient for the fluorescence quenching of the probe. DNSA and TNP
respectively contain a carboxylic acid and hydroxyl group with the
ability to bind to hydrogen-bond acceptor (e.g., carbonyl group)
while TNT doesn't have such a functional group. Based on the
selectivity on DNSA and TNP over TNT, we conceive that the pres-
ence of hydrogen-bond donor in a guest molecule (e.g., carboxylic
or and hydroxyl group) may play a key role in the sensing system of
probe 1 although this hydrogen-bond was not evident in the mo-
lecular structure of probe 1-DNSA complex obtained from the DFT
calculations.
4. Conclusions
In summary, the dipodal pyreneamide-based probes (probes 1
and 2) displayed ACQ behaviors, while the monopod counterpart
(probe 3) showed AIEE character at a high water content (>60%) in
DMSO. In addition, with increasing concentration of water in

Fig. 10. Fluorescence quenching behaviors of DNSA for (a,b) written letters or (c,d) dropped spots on a TLC plate prepared using a probe 1 solution (10 mM, DMSO:H2O (1:9)).
Fluorescence intensity of the letters ‘DNSA’ was detected under 365 nm irradiation, (a) before or (b) after dipping the TLC plate into a 1 mM DNSA solution. Fluorescence intensities
of the spots on a TLC plate were measured under 365-nm irradiation (c) before and (d) after exposure to a drop of H2O or different concentrations of a DNSA solution: 1, 10, or
100 nM. 5 mL of water or DNSA solution of indicated concentration was added to the TLC plate.
 A. Kumar, P. Seok Chae / Dyes and Pigments 147 (2017) 400e412
DMSO, the dipodal probes gave strong fluorescence emission at 480
or 484 nm upon excitation as a result of the high tendency to form
the pyrene dimer (i.e., excimer). This excimer emission was
completely quenched in the presence of 2 equiv. of DNSA. A
conformational change of the probes from a closed to an open
structure and the resulting energy transfer from the probe to the
guest molecule upon excitation are mainly responsible for the high
sensitivity of these probes toward DNSA detection. The enhanced
selectivity of probe 1 toward DNSA detection compared to probe 2
is, at least in part, attributed to its conformational rigidity caused by
a short linker between the central MDP unit and pendant pyrene
rings. In the case of probe 3 (with no excimer emission), DNSA
association also resulted in fluorescence quenching, but with less
sensitivity and selectivity than probe 1. Among the three probes,
probe 1 exhibited the highest selectivity and sensitivity toward
DNSA detection, reaching an LOD of 1 nM in an aqueous solution.
This ultra-sensitivity was well retained in an application of a probe
1-coated TLC strip for DNSA detection. DFT calculations supported
that quenching the fluorescence emission of the probes by DNSA
was mostly caused by energy transfer from electron-rich pyrene to
electron-deficient DNSA in an excited state. The current result is a
step toward the development of new sensing molecules for DNSA
detection.
Acknowledgements
This work was supported by the National Research Foundation
of Korea funded by the Korean government (MSIP) (grant number
2008-0061891 to P.S.C. and A.K.).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.dyepig.2017.08.039.
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