Sequential and cellular detection of copper and lactic acid by disaggregation and reaggregation of the fluorescent panchromatic fibres of an acylthiourea based sensor

Article abstract of DOI:10.1039/d1sm00038a

We report, for the first time, the self-assembly of an acyl-thiourea based sensor, N-{(6-methoxy-pyridine-2-yl) carbamothioyl}benzamide (NG1), with panchromatic fluorescent fibres and its dual-sensing properties for the sequential detection of Cu2+ ions and lactic acid. The panchromatic fibres formed by NG1 were disrupted in the presence of Cu2+ ions and this was accompanied by a visible colour change in the solution from colourless to yellow. The addition of lactic acid to the NG1 + Cu2+ solution, on the other hand, induced re-aggregation to fibrillar structures and the colour of the solution again changed to colourless. Hence, it may be surmised that the disaggregation and re-aggregation impart unique dual-sensing properties to NG1 for the sequential detection of Cu2+ ions and lactic acid. The application of NG1 as a selective sensor for Cu2+ ions and lactic acid has been assessed in detail by UV-visible and fluorescence spectroscopy. Furthermore, two structural variants of NG1, namely, NG2 and NG3, were synthesized, which suggest the crucial role of pyridine in imparting panchromatic emission properties and of both pyridine and acyl-thiourea side chain in the binding of Cu2+ ions. The O-methoxy group plays an important part in making NG1 the most sensitive probe of its structural analogs. Finally, the utility of NG1 for the sequential and cellular detection of Cu2+ ions and lactic acid was studied in human RPE cells. The experimental results of the interaction of NG1 with Cu2+ ions and lactic acid have also been validated theoretically by using quantum chemical calculations based on density functional theory (DFT). To the best of our knowledge, this is the first report wherein a dual sensor for Cu2+ ions and lactate ions is synthesized. More importantly, the aggregation properties of the sensor have been studied extensively and an interesting correlation of the photophysical properties of the probe with its self-assembling behavior has been elucidated.

Full text of DOI:10.1039/d1sm00038a

Soft Matter
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PAPER
Sequential and cellular detection of copper and
lactic acid by disaggregation and reaggregation
of the fluorescent panchromatic fibres of an
acylthiourea based sensor†
Cite this: Soft Matter, 2021,
17, 4304
b
Vivekshinh Kshtriya,a Bharti Koshti,a Deepak K. Pandey,
Sumit Kharbanda,c
d
Chandra Kanth P,
Dheeraj K. Singh, *b Dhiraj Bhatia *c and
Nidhi Gour ‡*a
We report, for the first time, the self-assembly of an acyl-thiourea based sensor, N-{(6-methoxy-
pyridine-2-yl) carbamothioyl}benzamide (NG1), with panchromatic fluorescent fibres and its dual-
sensing properties for the sequential detection of Cu2+ ions and lactic acid. The panchromatic fibres
formed by NG1 were disrupted in the presence of Cu2+ ions and this was accompanied by a visible
colour change in the solution from colourless to yellow. The addition of lactic acid to the NG1 + Cu2+
solution, on the other hand, induced re-aggregation to fibrillar structures and the colour of the solution
again changed to colourless. Hence, it may be surmised that the disaggregation and re-aggregation
impart unique dual-sensing properties to NG1 for the sequential detection of Cu2+ ions and lactic acid.
The application of NG1 as a selective sensor for Cu2+ ions and lactic acid has been assessed in detail by
UV-visible and fluorescence spectroscopy. Furthermore, two structural variants of NG1, namely, NG2
and NG3, were synthesized, which suggest the crucial role of pyridine in imparting panchromatic
emission properties and of both pyridine and acyl-thiourea side chain in the binding of Cu2+ ions. The
O-methoxy group plays an important part in making NG1 the most sensitive probe of its structural
analogs. Finally, the utility of NG1 for the sequential and cellular detection of Cu2+ ions and lactic acid
was studied in human RPE cells. The experimental results of the interaction of NG1 with Cu2+ ions and
lactic acid have also been validated theoretically by using quantum chemical calculations based on
density functional theory (DFT). To the best of our knowledge, this is the first report wherein a dual
sensor for Cu2+ ions and lactate ions is synthesized. More importantly, the aggregation properties of the
sensor have been studied extensively and an interesting correlation of the photophysical properties of
the probe with its self-assembling behavior has been elucidated.
Received 10th January 2021,
Accepted 24th February 2021
DOI: 10.1039/d1sm00038a
rsc.li/soft-matter-journal
amplification. Various mechanisms are used to develop novel
sensor molecules which include photo-induced electron transfer
Introduction
There is an ever-increasing need to develop new optical probes (PET),1,2 intramolecular charge transfer (ICT),3,4 fluorescence
that could detect analytes with high sensitivity and good signal resonance energy transfer,5 aggregation-induced emission (AIE),6
aggregation-induced emission enhancement (AIEE),7 metal to
a Department of Chemistry, Indrashil University, Kadi, Mehsana, Gujarat, 382740,
ligand charge transfer,8,9 ligand to metal charge transfer,10 and,
donor–acceptor mechanisms,11 and single electron transfer
processes12 and colourimetric detection.13,14 Another very important
sensing mechanism that can be utilized for the development of
sensitive probes is disaggregation induced emission enhancement
(DIEE).15 The DIEE probes have very low fluorescence or are
non-fluorescent in the aggregated state and the fluorescence is
enhanced on disaggregation.16 Disaggregation of the aggregated
probe leads to a remarkable enhancement of emission and
signal amplification. There are few literature reports that cite
‘‘disaggregation’’ as a mechanism for fluorescence enhancement;
India. E-mail: nidhi.gour@indrashiluniversity.edu.in
b Department of Basic Sciences, Institute of Infrastructure Technology Research and
Management, Ahmedabad, 380026, India
c Biological Engineering Discipline and Center for Biomedical Research,
Indian Institute of Technology Gandhinagar, Palaj 382355, Gandhinagar, India
d Department of Science, School of Technology, Pandit Deendayal Petroleum
University, Gandhinagar, Gujarat, India
† Electronic supplementary information (ESI) available: Synthesis of NG1, NG2
and NG3 and its structural characterizations. It also contains accessory FTIR
analysis, microscopic images and control studies. See DOI: 10.1039/d1sm00038a
‡ Past affiliation: Department of Medicinal Chemistry, Indian Institute of
Advanced Research, Gandhinagar, Gujarat, 382426, India.
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however, such reports are very limited, and not many research the novel probe can also be used for the sequential detection of
groups have explored this phenomenon.17–20 Similarly the Cu2+ and lactate. Lactic acid is also very harmful at concentrations
aggregation behaviour of the probe is seldom discussed, there above a threshold and many diseases like mitochondrial diseases,
are very few reports on the self-assembling properties21,22 and cerebral ischemia and cancer are associated with high lactate
microscopic analysis of the probe.23,24 As a result of the poorly levels in the blood.29 Combined experimental tools (UV-visible
established concept, a proper interpretation of the data or an spectroscopy, fluorescence spectroscopy, Fourier Transform
active application of the disaggregation for novel probe design Infrared (FTIR) Spectroscopy, and Nuclear Magnetic Resonance
has been hampered. Consequently, even if the ‘‘disaggregation’’ (NMR) spectroscopy and quantum chemical calculations based on
may be the real underlying mechanism, researchers remain density functional theory (DFT)) were used for deciphering the
unaware of it due to lack of microscopic analysis. Hence, the sequential identification of copper II (Cu2+) and lactic acid by
self-assembly/aggregation properties of the probe should be fluorescent probe NG1.
adequately discussed while deciphering the sensing mechanism.
Herein, we report the self-assembly of a new acylthiourea
based probe N-((6-methoxypyridin-2-yl)carbamothioyl)benzamide
(NG1) with panchromatic fluorescent fibres extensively via the
Results and discussion
The structural basis for the design of the NG1 molecule
was the acyl thiourea side chain. There are literature reports
that reveal the exceptional photophysical properties of
acyl thiourea based conjugates and hence they can be
efficiently used as a sensor.30–35 Our group has been actively
studying the self-assembled structures formed by single
amino acids,36 peptides37–41 and heterocycles.42 Our recent
research on pyridothiazole based conjugate revealed its self-
assembly to structures and a subsequent change in the photo-
physical properties due to aggregation. The aggregation-
induced emission enhancement (AIEE) properties of this
conjugate imparted it an exceptional ability to selectively sense
amyloid.42 Hence, we were motivated to design a novel acyl
thiourea conjugate NG1 and assess its aggregation properties to
decipher the underlying mechanism of sensing from an aggregation
perspective. The structure of NG1 was designed with the aim to
make it a more appropriate sensor. The sulphur and oxygen
atoms present in the acyl thiourea side chain of NG1 were
conjugated to O-methoxy pyridine. This conjugation imparted
more electron-donating capacity to the oxygen and sulphur
atoms, through which they can tightly bind with the divalent
metal ions. Due to the presence of an aromatic ring in the
structure, it was also expected that NG1 would tend to aggregate
and self-assemble to fibre like structures via pi–pi stacking as
reported previously.43
microscopic analysis. These self-assemblies are disrupted upon
the addition of Cu2+, and the disaggregation of the aggregated
probe results in the golden yellow colour which could be
visualized by the naked eye. This phenomenon is extensively
studied by microscopic techniques like field emission-scanning
electron microscopy (FE-SEM), atomic force microscopy (AFM),
transmission electron microscopy (TEM) and fluorescence
microscopy (Fig. 1).
The DIEE caused by the addition of Cu2+ ions to NG1 has
been used to design a sensor for the selective and sensitive
detection of Cu2+. Copper is an essential micronutrient; however,
if the concentration of Cu2+ increases beyond a threshold, it
causes deleterious effects on kidney, liver and gastrointestinal
tracts and leads to diseases like Alzheimer’s,25 Parkinson’s,26,27
and Wilson’s disease.59 Chang et al. reported the fluorescent
sensor for the selective detection of Cu2+ ions and citrate
anion.28 Hence, developing a selective and sensitive probe that
could detect copper at a very minute level is of significant
importance. It was further noted that the addition of lactic acid
causes fluorescence quenching of the NG1–Cu2+ complex; hence,
The chemical synthesis of NG1 was carried out in one step
by condensation reaction between benzoyl isothiocyanate
and 2-Amino-6-methoxypyridine as shown in Scheme 1. The
chemical structure of NG1 was characterized by 1H (Fig. S15,
ESI†) and 13C-NMR (Fig. S16 and S17, ESI†), and LCMS,
Fig. 1 A diagrammatic representation of the aggregation properties of
NG1 and its implications in the sequential detection of Cu2+ and Lactate.
(A) Optical microscopy images of NG1: Aggregated state (top) NG1 alone;
disaggregated state (middle) NG1 + Cu2+and re-aggregated state (bottom)
NG1 + Cu2+ + Lactic acid (LA); scale bar: 20 mm. Colourimetric change
in the vial containing NG1 upon the addition of Cu2+ and Cu2+ + LA; (B)
self-assembly of NG1 with panchromatic fibres; (C) sequential cellular
detection of Cu2+ in retinal pigment epithelial (RPE) cells using NG1,
Scale bar: 10 mm.
Scheme 1 Schematic representation of the synthesis of the probe NG1.
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panchromatic properties (Fig. S4, ESI†). Hence, the pyridine group
plays a crucial role in the panchromatic emission.
There are reports in literature wherein acyl thiourea
derivatives have been used as a probe for the detection of Cu2+
ions.33–35 Hence, we were motivated to assess the effect of Cu2+
ions on the aggregation properties of NG1. Interestingly, when
Cu2+ ions were added to these fibres, disruption of the fibril
assembly could be observed. It was also evident that the colourless
solution of NG1 changed to golden yellow on the addition of Cu2+.
When EDTA, a well know metal chelator, was added to the NG1 +
Cu2+ complex, the yellow colour changed to colourless and the
fiber structures were regenerated (Fig. S20, ESI†). This experiment
suggested the crucial role of disaggregation in attributing unique
photophysical properties to NG1. The disaggregation induced
colour production (DICP) was very intriguing to us and we decided
to first assess whether this colour change can be produced only by
Cu2+ or if it can be observed with other metal ions as well.
To assess the specific sensing properties of NG1 for Cu2+,
experiments were performed by co-incubating NG1 with a series
of metal ions commonly found in water. Metal-ion binding
Fig. 2 Microscopy images of self-assembled structures of NG1 (1 mM) in
70% aqueous methanol. (A) SEM at 10 mm scale; (B) SEM at 5 mm scale;
(C) SEM at 1 mm scale; (D) AFM at 100 mm scale; (E) AFM at 50 mm scale bar; studies of NG1 were carried out following the method described
in the experimental section using the metal ions, such as Cu+,
and (F) AFM at 5 mm scale; (G) bright field images of the fibres of NG1,
(H) under the green filter, and (I) under the red filter.
Na+, K+, Ba2+, Ca2+, Mg2+, Cr3+, Mn2+, Co2+, Ni2+, Zn2+, Pb2+, Sr2+,
Fe2+, Fe3+, and Cu2+ ions. As can be surmised from Fig. S6 in the
ESI,† NG1 selectively detected only Cu2+ ions and the colour of the
(Fig. S18, ESI†) and its purity was ascertained through HPLC solution in a vial containing NG1 + Cu2+ changed from colourless
(Fig. S19, ESI†).
to yellow (Fig. S6, ESI†). Furthermore, the microscopic images also
Once the chemical structure of NG1 was characterized revealed that the panchromatic fibres formed by NG1 could only
thoroughly, we studied the self-assembling properties of NG1. be disrupted by Cu2+ and not by any other ions (Fig. S3, ESI†)
For self-assembly studies, NG1 was dissolved at 1 mM concentration suggesting the crucial role of disaggregation in this specific
in 70% aqueous methanol. The scanning electron microscopy sensing. In addition, an interference assay was also performed
(SEM) image of NG1 reveals its assembly to fibre like morphologies. by mixing all the metal ions except Cu2+ with NG1. Notably, there
The diameter of these fibres was 200–400 nm and the length was no visible change in the colour of the solution even in the
varied to several micron ranges (Fig. 2A and C). The AFM presence of all metal ions. However, when Cu2+ ions were added
micrograph studies further confirmed the fibrillar structures to this mixture, the colourless solution changed to yellow.
formed by NG1 (Fig. 2D–F).
As revealed by the colorimetric response, NG1 produced yellow
The fibres formed by the self-assembly of NG1 were also colour only in the presence of Cu2+ and was least affected by the
studied extensively via optical and fluorescence microscopy. It was presence of other types of common ions (250 ppm: Na+, Cu+, Fe3+,
noted that NG1 fibres exhibit fluorescence. The fibres revealed Mn2+, K+, Mg2+, Ca2+, and Cr3+ ions). Hence, it can be inferred that
green emission under the FITC filter (Ex 480/40 Em 527/30) and NG1 shows optimal activity with negligible interference by other
red emission under the rhodamine filter (Ex 546/10 Em 585/40). ions (Fig. S6, ESI†). The microscopic study of fibres formed by
The emission spectra of NG1 also changed in accordance with the NG1 also reveals that only Cu2+ could cause disruption of fibres
excitation wavelength through fluorescence spectroscopy and they remain unaffected by the addition of a mixture of other
(Fig. S14, ESI†). From the emission spectra, it can be interpreted ions (data not shown) confirming a definite correlation of NG1
that NG1 shows maximum fluorescence under blue excitation aggregation properties with its photophysical characteristics.
followed by green and the lowest was red. The fluorescence
The results of the interaction of NG1 with Cu2+ were further
microscopy (Fig. 2G–I, ESI†) and fluorescence spectroscopy validated using UV–visible spectroscopy studies. The UV visible
(Fig. S13, ESI†) clearly suggest that NG1 assembled to spectra of 50 ppm solution of NG1 reveal a broad peak with
panchromatic aggregates which show tunable emission albeit maxima from 290 to 320 nm. When 50 ppm Cu2+ was added to
with low fluorescence intensities. Furthermore, we also this solution, the peak shifted from 310 nm to 339.5 nm. There
synthesized structural analogs of NG1, namely NG2 and NG3, was also a slight enhancement of broad peak in the range of
which does not have the O-methoxy group and the pyridine group, 400–450 nm which corresponded well with the yellow colour
respectively, to decipher the structural basis for the formation of produced in the solution due to interaction between NG1 and
panchromatic self-assemblies. It was noted that NG2 also Cu2+ ions and may be attributed to intramolecular charge
assembles to panchromatic fibres (Fig. 2G–I); however, since transfer (ICT) transition (Fig. 3A). To study the interaction of
NG3 does not have a pyridine group, it does not possess NG1 with Cu2+ ions in more detail, the absorption spectra of
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between NG1 and Cu2+ ions were also evinced via FTIR
presented in the ESI† (Fig. S8). The FTIR spectrum supported
the change in characteristic peaks of probe NG1 in the aromatic
region (1450–1650 cmꢀ1) due to interaction via nitrogen and
sulfur atoms present on NG1 with Cu2+ ions and proved the
formation of the NG1–Cu2+ complex. Thus, the present study
realizes that the probe NG1 can be used for the simultaneous
detection of Cu2+ ions in real water samples as well as it could
potentially be used for sensing Cu2+ ions in mammalian cells
and organisms.
To assess the role of deprotonation in the NG1–Cu2+
complex formation, we also studied the role of pH in this
complexation. Originally, the pH of the NG1 solution was 7.2.
After the addition of Cu2+, the pH remains unchanged. To assess
the effect of pH on the sensing of Cu2+ ions, NG1 pH was made
acidic at pH 5.0 and also basic at pH 9.0. Also, NG1 was dissolved
in PBS buffer at pH 7.4 and subsequently, Cu2+ was added under
acidic, basic and physiological conditions. In all these vials, NG1
was able to sense Cu2+ and produced yellow colour, indicating
that pH does not affect sensing properties and ruled out the
role of deprotonation in sensing (Fig. S10, ESI†). It further
demonstrates the efficacy of NG1 to be a very good sensor for
the detection of Cu2+ even under acidic and basic conditions and
its utility in real sample analysis of industrial water.
Once the sensitivity and selectivity of the probe NG1 were
ascertained by colorimetry, the fluorescence assay was performed
in the presence of Cu2+ ions since self-assembly of NG1 revealed
the formation of fibres showing panchromatic emission. When
NG1 was excited at 353 nm, the emission spectra were obtained
with the maxima at 450 nm. When Cu2+ ions were added in
increasing concentration there was a significant enhancement in
the fluorescence intensity and the graph exhibited a red-shift
towards 470 nm (Fig. 4A). Interestingly, from UV studies, it can be
assessed that the peak shift of NG1 occurs from 310 to 370 nm on
the addition of Cu2+ ions. Hence, excitation at 353 nm might be
attributed to this absorbance.
Fig. 3 (A) UV-visible spectra of probe NG1 with and without Cu2+ at 50
ppm concentration; (B) UV-visible spectra of probe NG1 after the sequen-
tial addition of Cu2+; (C) LOD of probe NG1 for detecting the presence of
Cu2+ at 339.5 nm, (inset) plot of absorbance vs. Cu2+ at 410 nm; (D) Job’s
plot drawn taking a fixed concentration of probe NG1 and varying
concentrations of Cu2+ ions revealed 1 : 1 stoichiometry between NG1
and Cu2+
.
the probe were studied in the presence of different ppm levels
of Cu2+ ions by UV-visible spectroscopy. As shown in Fig. 3A,
NG1 exhibited a maximum absorption at B310–320 nm. On the
gradual addition of Cu2+ ions, the absorption intensity shifts
from 320 nm to nearly 340–370 nm. There was more and more
red shift as the concentration of Cu2+ ions (0–50 ppm) was
gradually increased and this was attributed to the formation of
the NG1–Cu2+ complex (Fig. 3B). The interaction is replicated
by the visual colour change from colourless to yellow.
To investigate the sensitivity of the synthesized probe NG1
towards Cu2+ ions, a straight-line plot was drawn between the
concentration of Cu2+ and absorbance at 339.5 nm (Fig. 3C).
The plot shows the linear relationship in the concentration
range between 2.5 and 10 ppm. The limit of detection (LOD)
was calculated using the following formula.
3s
LOD ¼
In our body, copper mainly exists in the form of copper citrate.
However, copper also plays an important role in regulating blood
lactate levels and forms copper lactate complex inside the body.35
Slope of calibration curve
where s is the standard deviation.
LOD was found to be 1.5 ppm from the plot of absorbance Hence, we decided to perform titration studies on the interaction
vs. Cu2+ ion concentration for absorbanceat 339.5 nm while it
was calculated to be 8.7 ppm (3.5 mM) for the absorbanceat
410 nm which corresponded to yellow colour (Fig. S6, ESI†).
In conclusion, the convenient detection limit for the newly
synthesized probe NG1 is 1.5 ppm (0.6 mM) for Cu2+ ions, which
is much lower according to the maximal permitted level of Cu2+
ions (15 mM) in water considering the highest safety standards.
The short response time and high selectivity of the probe NG1
in visual inspection could be accounted for its strong and
specific affinity towards Cu2+ ions. (Fig. 3C, inset).
Fig. 4 (A) The curve plotted with the fluorescence intensity of NG1
(250 ppm) at 450 nm versus Cu2+ ion concentration (0.0001 ppm–100 ppm)
showed a steady increase in fluorescence as the concentration of Cu2+
ions was increased; and (B) fluorescence graph shows no enhancement on
the addition of copper lactate to NG1 in increasing amount, on the other
hand, copper citrate showed a steady increase in fluorescence with
increase in its concentration.
Furthermore, Job’s plots were constructed to demonstrate
the complexation between Cu2+ and probe which indicated a
stoichiometry of 1 : 1 complexation between Cu2+ ions and NG1.
The 1 : 1 binding stoichiometry suggests bis-coordination of
Cu2+ ions via nitrogen and sulphur atoms present on the probe
NG1 (Fig. 3D). Moreover, the interaction and binding behaviour
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of NG1 with copper citrate and copper lactate. Interestingly, while
Cu(II) citrate showed a steady increase in fluorescence, the Cu(II)
lactate salt was not able to bind NG1 and no increase in
fluorescence was observed. This study indicated that the Cu(II)
lactate complex was more stable and hence Cu2+ ions could not
form a complex with NG1 and induce a colour change (Fig. 4B).
Hence, motivated by these results, we decided to assess the
application of NG1 for the sequential detection of both Cu2+
ions and lactate ions. The UV visible experiment of the NG1–
Cu2+ complex with lactic acid at high concentrations revealed that
the yellow colour of the NG1 + Cu2+ complex fades to a nearly
colourless solution (Fig. 5A and Fig. S11, ESI†). Interestingly, the
yellow colour of the NG1–Cu2+ complex remains unaffected by
excess addition of other counter anions like oxalate and citrate
and only lactate was able to decolourize the yellow solution
(data not shown). Furthermore, UV-Visible titration of the
NG1–Cu2+ complex with lactic acid was carried out which revealed
Fig. 6 Microscopy image of NG1 (1 mM) in 70% aqueous methanol.
(A) NG1 alone; (B) NG1 + Cu2+(1 : 1); (C) NG1 + Cu2++ lactic acid
(1 : 1 : 5); (D) TEM image of NG1; (E) TEM image of NG1 + Cu2+(1 : 1); and
(F) SAED pattern of NG1 + Cu2+ showing the non crystalline nature.
a
decrease in absorbance on the addition of increasing
concentration of lactic acid to the 50 ppm NG1:Cu2+ yellow
complex from 0 to 500 ppm (Fig. 5B). Notably, the absorbance Cu2+ at a higher magnification. At a higher magnification too,
at both 339.5 nm and 410 nm decreased as can be deciphered NG1 reveals clustered fibres (Fig. 6A). When NG1 + Cu2+ sample
from plots shown in Fig. 5B and C, respectively.
was observed, a disruption in the fiber aggregates could be seen
Furthermore, to assess the role of self-assembly of NG1 in (Fig. 6B). The organic layer appears to be dissolved by Cu2+ and
the sequential detection of Cu2+ and lactate, optical microscopy hence SAED was done to decipher if this organic layer has some
studies of fibres formed by NG1 were performed in the presence crystalline characteristics (Fig. 6C). However, no crystalline
of Cu2+ and lactic acid. It was noted that the fibres which were morphologies, as well as reduced Cu(0) nanoparticles, could
disrupted by the addition of Cu2+ were regenerated on the be observed.
sequential addition of lactic acid. This experiment clearly
To know more about the molecular packing present in NG1
revealed a crucial role of disaggregation in inducing yellow non-assembled, self-assembled, and NG1 + Cu2+ complex we
colour on the addition of Cu2+ and re-aggregation in changing studied its XRD in powder mode (Fig. S12, ESI†). The study
it back to colourless on the addition of lactic acid.
revealed that non-assembled NG1 is more crystalline in nature
Transmission Electron Microscopy was also performed to when compared to the self-assembled material. There were
observe the morphologies of the structures formed by NG1 + more peaks observed in the case of non-assembled material
due to its more uniform lattice arrangement while in the case of
self-assembled NG1, molecules are arranged through weak
interaction force such as hydrogen bonding, and van der Waals
and pi–pi interactions due to which the properties of the
material will change towards more amorphous rather than
crystalline nature.45 The different XRD patterns of the non-
assembled and self-assembled NG1 also indicate their different
morphologies. When Cu2+ is added, the molecular packing
present in NG1 is further disturbed, resulting in obvious
changes in the 2y value. The disappearance of peaks arising
from NG1 on the addition of Cu2+ indicates complex formation
and
a
completely different molecular packing and
morphology.44 These results also further supported the SAED
results obtained using TEM for the NG1 + Cu2+ complex.
Furthermore, to understand the role of acyl thiourea side
chain present in NG1, the photophysical properties of two other
structural analogs of this probe, namely, NG2 and NG3 were
studied in the presence of Cu2+. Interestingly, both probes NG1
and NG2 show selective sensing for Cu2+ ions. However, probe
NG3 showed very little sensitivity for Cu2+ ions. It also did not
exhibit panchromatic behavior. Hence, the crucial role of
pyridine in sensing as well as in panchromatic emission may
be deciphered. The order of sensitivity of NG1 for the detection
Fig. 5 (A) UV-visible spectra recorded from 370 to 700 nm at high
concentration of NG1 (250 ppm) (black); with Cu2+ (1 : 1) (red); after the
addition of lactic acid (LA) (1 : 1;10) (pink) showing the change of yellow
colour to colourless; (B) the UV spectra of NG1 (50 ppm), NG1–Cu2+
complex (50 ppm : 50 ppm) and varying concentrations of LA to assess
visual colour change; (B) UV-visible spectra of NG1 (50 ppm) in the
presence of Cu2+ ions (50 ppm) and varying concentrations of lactic acid
(0 ppm–500 ppm); (C) the UV spectra of the NG1–Cu2+complex against
varying concentrations of lactic acid from 0 to 500 ppm at wavelength
339.5 nm and (D) the UV spectra of NG1–Cu2+ complex against a varying
concentrations of lactic acid from 0 to 500 ppm at wavelength 410 nm for
detecting decrease in yellow colour.
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of Cu2+ ions was NG1 4 NG2 4 NG3. Furthermore, since NG3
could also sense Cu2+ ions, albeit with low sensitivity, it may be
inferred that the acyl thiourea moiety can also participate in
complex formation. Since NG1 revealed higher sensitivity as
compared to NG2, a crucial role of the –O-methoxy group for
increasing sensitivity could also be surmised. The methoxy
group is known to have electron releasing properties. The lone
pairs of the methoxy groups present in NG1 may be involved in
resonance and could attribute to an increase in the electron
density around the sulphur and oxygen atoms. Hence, sulphur
and oxygen atoms in NG1 can easily donate their lone pairs of
electrons to Cu2+ and cause its effective binding leading to the
yellow colour complex formation.
Cellular detection of Cu2+ and lactate using the NG1–Cu2+
complex
The in vitro sensing capabilities for NG1–Cu2+ were further
explored by testing its capacity to sense Cu2+ and lactate in
mammalian cells.46,47 The application of the NG1–Cu2+
complex was tested in living cells by using MTT cytotoxicity
assay and cellular imaging. Cell viability was measured by
measuring the absorbance of MTT at 570 nm and calculating
the ratio of absorbance of cells treated with respective compounds
to that of control untreated cells. The results as shown in Fig. 7A
indicate that NG1 compounds have minimum cytotoxicity in cells
(B80% cell viability), strongly suggesting that these compounds
can be further exploited for cellular imaging applications. MTT
assays were carried out using a stock solution of 500 : 500 ppm
NG1:Cu2+. The stock was serially diluted and cells were grown in
wells containing 25, 50, 75, 100, 150, 200, and 250 ppm of the
NG1 :Cu2+ complex. To test for cell permeability and fluorescence
properties of NG1 molecule in cells, experiments were carried out
using the retinal pigmented epithelial (RPE1) cells. The cells
were pulsed with NG1 in the absence or presence of different
concentrations of Cu2+ for 15 mins, fixed and imaged using a
confocal microscope. Following in vitro data, imaging of fixed cells
using a confocal microscope displayed that these compounds are
permeable in cells and show bright blue fluorescence in the blue
filter (shown in Fig. 7B). From the fluorescence spectroscopy
analysis, it was evident that the fluorescence of NG1 enhances
with increase in the concentration of Cu2+ added to it. Hence, it
was expected that when the cells are visualized under a blue filter
(Em 450–470 nm) the fluorescence of the dye NG1 would be highly
enhanced in the presence of exogenous Cu2+ (Fig. 7A). Indeed,
similar observations could be noted via cell imaging analysis and
there was a remarkable enhancement of fluorescence on the
addition of Cu2+. This enhancement in the cellular intensity of
dye was rescued to background levels in the presence of lactic
acid, indicating the potential of the dye to sense both Cu2+ and
lactate from cells (Fig. 7C).
Fig. 7 Cytotoxicity measurements and cellular imaging of Cu2+ and lactic acid
in human cells. (A) The MTT assay of RPE1 cell using NG1 molecule. Cells were
treated with different concentrations of NG1 and the cell viability was calculated
using the MTT assay (n = 2 experiments with 10 000 cells under each condition);
(B) retinal epithelial pigmental cells (RPE1) were pulsed with 100 and 250 ppm of
NG1 dye in the absence or presence of Cu2+ in a 1 : 1 ratio of dye : Cu2+
.
Furthermore, the cells were incubated with lactate in presence of dye and Cu2+
for 15 min at 37 1C, fixed with paraformaldehyde, and imaged using a confocal
microscope with a 405 nm excitation laser and images were acquired using
broad width band-pass filters. (C) The images were further quantified using
image-J for the cellular intensity of dye under various conditions and the
normalized intensity plotted in the right figure. Scale bar: 10 mm.
based on density functional theory (DFT). In various fields of
science, DFT-based quantum chemical calculations are used to
elucidate the electronic structures of numerous chemical
species and have become an excellent tool for solving
interesting chemical problems and reaction mechanisms.48–51,58
Here, in the present study, we first conducted a comparison study
on three probes (NG1, NG2, and NG3) and their complex formation
with Cu2+ ions and lactic acid. DFT calculations were performed at
the B3LYP/6-311++G(d,p) level of theory for these probes and their
complexes in the gaseous phase. The vibrational frequencies
were also calculated for all the studied structures, where the
optimization converged successfully to the shallow local minima
on the potential energy surface, which was confirmed by the
absence of negative/imaginary vibrational frequencies. Fig. 8 shows
the ground state optimized geometries of all the three probes and
their association with Cu2+ as well as lactic acid. The binding
energy (BE) of these complexes are elucidated as:
Theoretical studies by DFT calculations
P
BE = Ecomplex
ꢀ
Eindividual
(1)
Furthermore, to perceive a greater insight into the complex
formation by NG1 and its structural analogs NG2 and NG3 with where Ecomplex represents the ground state energy of the
Cu2+ and lactic acid, we resorted to validate theoretically the complex and Eindividual indicates the sum of the ground state
experimental results using quantum chemical calculations energies of all the molecules in a particular complex. The BE of
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Fig. 8 DFT computed optimized geometries of (A) NG1, configuration 1, configuration 2 and configuration 1 + lactic acid, (B) NG2, configuration 1,
configuration 2 and configuration 1 + lactic acid, and (C) NG3, configuration 1, configuration 2 and configuration 1 + lactic acid with their optimized
energy in Hartree.
all the optimized structures is presented in curly braces in Two possible configurations were identified for the study of the
Table 1.
interaction of Cu2+ with these probes. Here, configuration 1
Initially, the pristine probes, i.e., NG1, NG2, and NG3 were indicates when Cu2+ interacts with the sulphur atom of these
optimized using DFT calculations. Based on energetic stability, probes and configuration 2 denotes when Cu2+ interacts near
NG1 was found to be the most stable ligand. To make a better the nitrogen atoms of the probe as can be seen in Fig. 8.
comparison, along with NG1, the other two probes (NG2 and After the interaction and geometry relaxation process, it is
NG3) were also interacted with Cu2+ and lactic acid (Fig. 8). noteworthy that among all the six complexes (with Cu2+),
Table 1 Optimized energy (in Hartree) and binding energy (in kcal molꢀ1) of the complexes NG1, NG2, and NG3. The binding energies in kcal molꢀ1 are
provided in curly braces
Configuration 1 (Cu2+ is
interacting near sulphur atom)
Configuration 2 (Cu2+ is
interacting near nitrogen atom)
Configuration 1 With
lactic acid
Ligands
Optimized energy
NG1
NG2
NG3
ꢀ1254.4797
ꢀ1139.9170
ꢀ1123.8654
ꢀ2894.9918 {ꢀ25.07}
ꢀ2780.4292 {ꢀ25.06}
ꢀ2764.3567 {ꢀ11.97}
ꢀ2894.9658 {ꢀ8.71}
ꢀ2780.4032 {ꢀ8.73}
ꢀ2764.3385 {ꢀ0.55}
ꢀ3238.7456 {ꢀ87.05}
ꢀ3124.1970 {ꢀ120.91}
ꢀ3108.1511 {ꢀ124.46}
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Cu2+ tends to strongly bind with the sulphur atom of all the
studied probes (NG1, NG2 and NG3) i.e., in configuration 1 as
shown in the second row in Fig. 8. This trend is also confirmed
from the binding energies which are higher for configuration 1
than configuration 2 in all the studied probes. The BE calculations
for all the complexes (with Cu2+) indicate that configuration 1 of
probe NG1 is the most stable complex. Nevertheless, the BE of
configuration 1 of NG1 is ꢀ25.07 kcal molꢀ1 which is slightly
higher in comparison to configuration 1 of the NG2 complex.
This small energy difference is considerable here because in
experimental observation all the three probes give yellow colour
after incorporating the Cu2+ but the only key difference between
these three probes was their sensitivities. For probe NG1, the
presence of only 1.5 ppm concentration of Cu2+ causes the
occurrence of yellow colour but in the case of other probes
(NG2 and NG3), yellow colour appears only after the incorporation
of high Cu2+ concentration. So, for further studies, we have
selected configuration 1 of NG1 on the basis of BE to validate
our experimental findings.
Here, Cu2+ strongly interacts with the oxygen and sulfur
atoms having a bond length of 1.95 Å and 2.18 Å, respectively
(see second geometry of Fig. 8A). In configuration 2 (all three
probes), Cu2+ is weakly coordinated with nitrogen and oxygen
atoms as their bond length increased on the interaction
(see Fig. 8). We have also optimized the dimer like structure
of NG1 with a single Cu2+ atom to check the assembly of Cu2+
with more NG1 molecules and almost similar structural
features are found as in the single case where Cu2+ interacts
with the S atom of another NG1 (Fig. 9C).
Fig. 9 Optimized structures of (A) lactic acid with Cu2+; (B) Probe NG1
with Cu2+ and lactic acid, and (C) self-associated dimer of NG1 bonded to
single Cu2+ ion.
mapped surfaces were generated to elucidate this behavior in
terms of charge densities.
Electrostatic potential (ESP) mapped electron density surfaces
As experimentally observed, the interaction of Cu2+ with
NG1 yields a yellow-coloured complex but when lactic acid
is incorporated within this complex, the yellow colour is
suppressed. To understand this behavior, we have introduced
a molecule of lactic acid with this stable configuration 1 of NG1
and the optimized geometry can be seen at the bottom of
Fig. 8A (vertically). From Fig. 8A (comparing the structure of
second and fourth geometry from the vertical position), it is
noticeable that Cu2+ is strongly binding with the sulfur and
oxygen atoms of NG1 through 2.18 Å and 1.95 Å, respectively.
The molecular electrostatic potential (MEP) (also called ESP) of
a molecular association is correlated with the corresponding
partial charges, dipole moment, and chemical reactive sites.
The mapping of any molecular system is the way to visualize the
relative polarity of the studied molecules. The expression for
the ESP at any point r in the space near the molecule is given by
ð
X
r
ðr0Þdr0
zA
VðrÞ ¼
ꢀ
(2)
jRA ꢀ rj
jr0 ꢀ rj
However, upon complex formation with lactic acid, both the where ZA is the charge on the nucleus A studied at RA and r(r0)
bond strengths get weakened and the bond lengths are calculated the electron density. For probe NG1 and complex NG1 with
to be 2.26 Å and 1.99 Å, respectively. Also, it can be noticed from Cu2+ and lactic acid, the electron density iso-surface on which
Fig. 9A that Cu2+ forms a strong bond (2.20 Å) with the oxygen the ESP surfaces were calculated and mapped are shown in
atom of –CQO of the carboxylic group in a lactic acid molecule. (Fig. 10). Recently, ESP mapping has played a remarkable role
But after the complex formation of configuration 1 with lactic in the geometrical arrangement, charge density analysis, and
acid, this bond gets stronger with a new bond length of 2.04 Å polarity of different molecular systems.48–51 Generally, MESP
as can be seen in Fig. 9B. Another noticeable feature is the least reflects the relative polarity of the molecule as positive potential
BE of this complex of NG1 (configuration 1 + lactic acid) in indicates the electrophilic sites while adverse potential
comparison to the other complexes. The BE of this indicates the nucleophilic attack sites. To visualize the electro-
complex is ꢀ87.05 kcal molꢀ1 which is much lower than the philic and nucleophilic sites in these complexes, the MESP was
ꢀ95.86 kcal molꢀ1 and ꢀ112.49 kcal molꢀ1 for configuration generated on DFT minimized structures at the B3LYP/
1 + lactic acid of probes NG2 and NG3, respectively. Hence, this 6-311++G(d,p) level of theory.
BE and changes in bond lengths give a clear indication of Cu2+
As shown in the top panel of the mapped optimized structure,
binding with lactic acid and hence altering the properties of the the potential increases in the colour code. The charge distribution
NG1 + Cu2+ complex due to the formation of copper lactate and over pristine NG1 with Cu2+ and lactic acid is visualized in the ESP
resulting decolourization. Furthermore, electrostatic potential map in Fig. 10. Also, in pristine NG1, nitrogen, oxygen, and
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Fig. 11 Molecular orbitals HOMO and LUMO of (A) pristine NG1 and
(B) NG1 with Cu2+. The orbitals were obtained using an iso-surface value
of 0.02 e Åꢀ3
.
Electronic absorption
Time-dependent density functional theory (TD-DFT) calculated
results were used to explain the experimentally measured
absorption bands of the complexes NG1 and NG1 with Cu2+
in the presence of different concentrations of Cu2+. TD-DFT
calculations of NG1 and NG1 with Cu2+ were performed using
DFT calculations in the gas-phase. The frontier molecular
orbitals of pristine NG1 and Cu2+ are shown in Fig. 11. Quantum
chemical calculations show that NG1 reveals three bands at 427,
325, and 311 nm (experimentally observed at 290 nm in 70%
aqueous methanol).
The theoretically calculated absorption at B325 nm red-shifted
when Cu2+ was added and absorption appears at B680 nm, which
shows a similar trend as that of experimentally observed absorption
bands. Energy bands calculated at B325 and B680 nm with
oscillator strengths ( f ) 0.0844 and 0.1445 are due to electronic
excitations from HOMOꢀ1 - LUMO and HOMO - LUMO+1
which are attributed to electron transfer from sulphur atom to
another part of NG1 and from sulphur to Cu2+ atom, respectively.
For both complexes, pristine probe NG1 and NG1 with Cu2+-atom,
the calculated bands are in good agreement with observed
absorption bands and show a similar trend. Overall, a nice
correlation is observed between experimental UV-Vis absorption
and TD-DFT calculated absorption data.
Fig. 10 Electrostatic potential surfaces plotted from the total self-
consistent field (SCF) density having iso-value (MO = 0.020000 and
density = 0.000400), calculated at the B3LYP level of theory. The red
colour indicates the most electronegativity, the blue colour shows the
most positive election density and the area of zero potential on the
molecular surface is represented by a green colour.
sulphur are the three electronegative atoms, so a negative charge
distribution can be noticed over both the oxygen as indicated by
the red colour whereas blue colour over the benzene ring and both
the nitrogen atoms near sulphur group indicates low electron
density (Fig. 10A) as these atoms are less electronegative than
oxygen. Shape and charge distribution are found to be considerably
different in NG1 with Cu2+ and lactic acid. Positive charge increases
(blue colour near the Cu2+) as Cu2+ atom attaches with sulphur
in comparison with pristine NG1 and negative charge increases
Experimental
Materials and methods
near the oxygen atom which is attached with the –OCH3 group. All the starting materials for NG1 synthesis were obtained from
On going from NG1–Cu2+ complex to NG1–Cu2+-lactic acid, the commercial suppliers and used as received. 2-Amino-6-
positive charge increases around the Cu2+ as Cu2+ binds with the methoxypyridine and Benzoyl isothiocyanate were purchased
oxygen atom of the carboxylic group of lactic acid. The reason from Combi-Blocks, USA. Acetone, THF, and all-metal ions
may be due to charge transfer between Cu2+ and oxygen atom were purchased from Sisco Research Laboratories (SRL), India.
as it is known that Cu2+ forms copper lactate with lactic acid. Cu(NO3)2ꢁ2.5H2O and CuCl2ꢁ2H2O were purchased from Sigma.
This result indicates that Cu2+ is interacting more with lactic Lactic acid was purchased from Fluka. Moisture sensitive
acid than the probe as yellow colour of NG1–Cu2+ quenches after reactions were performed under an atmosphere of nitrogen.
interacting with lactic acid which is experimentally observed.
All the solvents used for reactions were distilled before use.
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All the microscopy and spectroscopy measurements were SEM samples were prepared on silicon wafers. NG1 (20 ml of a
performed using ultra-pure water obtained from Millipore 1 mM solution) was dispensed and dried at room temperature.
Direct-Qs 5 UV Water Purification System. Rf was recorded The samples were analysed without gold coating under a low
using an Analytical TLC Silica Gel 60F254 instrument purchased vacuum.
from Merck (Germany). The melting point of NG1 and its
Atomic force microscopy (AFM)
derivatives was recorded using a Visual Melting Range Apparatus
1
(MR-VIS) provided by LABINDIA. H NMR (400 MHz) and 13C Neat and coincubated solutions of NG1 were imaged using an
NMR (100 MHz) spectra were recorded using an Avance III 400 atomic force microscope. The samples were placed on freshly
NMR spectrophotometer. Proton chemical shifts were reported cleaved muscovite mica surfaces followed by imaging with an
in parts per million concerning TMS. HPLC was done using AFM (INNOVA, ICON analytical equipment, Bruker, operating
the Waters E2695 instrument. HPLC was performed using under the acoustic AC mode (AAC or tapping mode)), with the
ammonium bicarbonate buffer (ABC) with 27 minutes run time aid of a cantilever (NSC 12(c) from MikroMasch, Silicon Nitride
and water: ACN as the mobile phase in C-18 Column. LC-MS Tip) by NanoDrive version 8 software. The force constant was
was obtained using a Waters 2690 LC-MS instrument using 2.0 N mꢀ1, while the resonant frequency was B276 kHz. All of
ammonium bicarbonate buffer (ABC); 7 minutes run time and the images were taken in the air at rt, with the scan speed of
water: ACN as the mobile phase in C-18 Column. UV-visible 1.5 lines per s. The data analysis was done using the nanoscope
spectra were recorded using a SHIMADZU UV-1900 with a 10 mm analysis software. The sample-loaded substrates were dried in a
quartz cell at 25 1C.
dust-free space under a 40 W lamp for 30 min followed by high-
vacuum drying and subsequently examined under AFM.
Synthesis of probe NG1
Transmission electron microscopy (TEM)
In a three-necked round bottom flask, fitted with a dropping
funnel, 50 mL of dry acetone was filled. 5.0 g (27 mM) of Transmission electron microscopy (TEM) images were taken
2-amino-6-methoxypyridine was placed followed by the dropwise using Zeiss Libra 120. mL of NG1and NG1 with
5
addition of dry acetone under N2 atmosphere during the Cu2+(1 : 1) solutions at a concentration of 1 mM was dropped
constant stirring of the reaction mixture. Next, 7.2 g (44 mM) on copper grids and dried. Finally, they were stained with
of benzoyl isothiocyanate was added and the reaction mixture uranyl acetate and the excess stain was removed by wiping
was then allowed to stir for another 2 h at room temperature. after 1 minute.
The progress of the reaction was monitored through analytical
TLC by using ethyl acetate-hexane (3: 7) as the solvent mixture.
UV visible assay for detection of Cu2+
After the completion of the reaction, the reaction mixture was For colorimetric measurements, stock solutions of 500 ppm
poured carefully with stirring into 500 mL cold water and NG1 and 500 ppm Cu(NO3)2ꢁ2.5 H2O were prepared. The
the resulting yellow precipitate of (N-((6-methoxypyridin-2- sensitivity of probe NG1 for Cu2+ and other metal ions was
yl)carbamothioyl)benzamide) was separated by suction filtration tested by mixing 250 ppm probe NG1 with 100 ppm metals
followed by washing of precipitate with water (3 ꢂ 100 mL). The (100 ppm: Cu2+, Fe3+, Cd2+, Mn2+, Ag+, Hg2+, Ca2+, Cr3+, K+, Mg2+
filtrate was further purified by vacuum distillation which yielded and Na+). The LOD of probe NG1 was detected by making a complex
the desired product (10.0 g, 34 mmol, and yields 86%) as solid of 50 ppm : 50 ppm of NG1 :Cu2+complex. The stoichiometric ratio
light-yellow material. M.P. 136 1C Rf 0.53, (this material was used of the NG1 :Cu2+ complex was determined by adding varying
in the next step without any further purification). The mass of concentrations of Cu2+ (0.5–50 ppm) to 50 ppm NG1.
NG1 was confirmed using EI-MS. The mass spectrum of NG1
Fluorimetric assay for the detection of Cu2+ ions and lactic acid
shows a strong molecular peak at M+ + 1 = 287.86. While the
M+ +1 ion appears for the base peak. Similarly, NG2 and NG3 For fluorimetric measurements, 250 ppm of NG1 stock was
were synthesized using aminopyridine and aniline, respectively, added with varying concentrations of Cu2+ (0.0001–100 ppm).
as the substrate {details of NG2 and NG3 synthesis are provided For lactic acid-mediated quenching, 250 ppm of lactic acid was
in the ESI.† (Scheme S1)}.
added to the 250 ppm : 100 ppm NG1 : Cu2+ complex.
Self-assembling properties of NG1
UV visible assay for lactate detection
The self-assembling properties of NG1 were studied using For sensing of lactate ions, a solution of NG1–Cu2+complex
different microscopic techniques at 1 mM concentration in (50 ppm) was prepared in 70% aqueous methanol. Then
70% methanol aqueous. The disruption studies were carried solutions of lactic acid were prepared (5000 ppm stock solution)
out by incubating NG1 with Cu2+ in a 1 : 1 stoichiometric ratio. in Milli-Qx water. Then 0 to 500 ppm lactic acid was mixed with
Similarly, EDTA was added to the complex in a 1 : 1 : 1 stoichio- of complex solution in a 3 mL vial and the reaction mixtures
metric ratio (Fig. S20, ESI†).
were incubated for 10 minutes. The UV-Vis spectra of all
these solutions were recorded after incubation and those
were compared to the spectrum of Cu2+complex of the same
Field emission-scanning electron microscopy (FE-SEM)
SEM images were taken using a JSM7600F (Jeol) FE-SEM 450 concentration but without any lactic acid to ascertain the
microscope (the accelerating voltage ranging from 1 to 15 kV). selectivity.
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Computational details
The cells were then fixed using 4% paraformaldehyde for
10 min at room temperature. The fixed coverslips were
visualized for dye uptake using Leica SP8 confocal microscope.
The cells were excited using 405 nm with a very low laser power
(B1% laser power) and the images were acquired using a broad
width emission spectrum to capture the maximum of
the emitted photons. 50–60 cells were imaged under every
condition using the same laser power and imaging conditions.
The prediction of certain important molecular properties of the
probes NG1, NG2 and NG3 and their possible complexes with
Cu2+ and lactic acid are calculated using density functional
theory (DFT) calculations. All computational calculations were
carried out using the Gaussian 16 programming package in the
present study.52 GaussView 6 software was used to generate the
input molecular structure and to analyze the output of all
calculated results.53 Geometric optimization calculations were
performed in the gas phase using DFT by employing the
internally stored 6-311++G(d,p)54,55 basis sets and the B3LYP
hybrid functional method.56–58 In general, B3LYP is a promi-
nent method and preferable to the HF and MP2 method as it
includes Becke’s three-parameter exchange with Lee, Yang, and
Parr’s correlational functional as well as an HF exchange term.
DFT calculations using the B3LYP protocol have provided a
meaningful and nice correlation with experimental results.
In order to analyze the charge transfer from Cu to probe NG1,
time-dependent density functional theory (TD-DFT) was also
implemented to compute electronic excitation energies and
molecular frontier orbitals. Besides, the mapping surfaces for
NG1 with electrostatic potential (ESP) were also shown to better
understand the distribution of charges through shape, size and
colour code.
Data analysis
Image analysis and quantification were performed using
Image-J software (nih.gov). 50 cells were randomly selected to
perform the quantification. The value of the total fluorescence
intensity of each cell was obtained from Image-J. The images
were quantified by subtracting the background and measuring
the cellular intensities by measuring the areas of the cells and
the total cellular intensity. The normalized mean value and
standard deviation for each were calculated and plotted in the
graph using Prism 7 software and the statistical significance
was calculated using a one-way Anova testlusions section.
Conclusions
To conclude, we have reported the self-assembling properties of
anacyl thiourea based conjugate NG1 and its implications as a
sensor for the sequential detection of Cu2+ and lactic acid. NG1
self-assembles to fibres which reveal panchromatic emission
properties. These fibres get disrupted only by Cu2+ ions and
their disaggregation led to the formation of yellow colour. The
sensitivity and specificity of NG1 for sensing Cu2+ ions are
tested using various spectroscopic and biophysical assays.
Furthermore, it was found that the sequential addition of lactic
acid suppresses the yellow colour formed by the NG1 + Cu2+
complex and its blue fluorescence is also quenched. Since high
levels of lactate ions and Cu2+ ions are associated with many
pathological disorders, the results presented in the manuscript
could be of immense importance for the designing of a simple
and cost-effective technique for the dual detection of Cu2+ and
lactate. In addition to the experimental study, DFT calculations
were also carried out for assessing the interaction. The comparison
of experimental and theoretical results in combination with cell
studies ensures a good understanding of the mechanism of
the probe along with an immense practical utility for future
applications. To the best of our knowledge, this is the first
report wherein a probe for the sequential detection of copper
and lactate has been designed and the effect of aggregation
properties of the sensor has been studied to decipher its
photophysical characteristics.
Cytotoxic assay
To evaluate the biocompatibility of NG1, we performed a
standard colorimetric MTT assay. Monkey kidney fibroblast
cells were seeded at a density of 5 ꢂ 103 cells per well into
96-well plates and maintained in Dulbecco’s modified Eagle’s
medium (DMEM) containing 10% fetal bovine serum (FBS) at
37 1C in a 5% CO2 incubator. After 24 h, once the cells adhered
to a surface, the culture medium was replaced by a fresh culture
medium containing varying concentrations of NG1 compounds
and again incubated for 24 hrs at 37 1C in a 5% CO2 incubator.
After incubation, the medium was aspirated and replaced by
100 mL per well of MTT prepared in DMEM medium to a final
concentration of 0.5 mg per mL and the plate was incubated
furthermore for 4 h at 37 1C in a 5% CO2 incubator. The formed
formazan crystals were solubilized by adding 100 mL of cell
culture grade DMSO per well and incubated for 2 h at 37 1C in a
5% CO2 incubator. The plate was gently shaken, and then the
absorbance of purple formazan was recorded at 570 nm using a
plate reader.
Cellular sensing of Cu2+ ions
Retinal pigmental epithelial cells (RPE1) were seeded at a
density of 1.5 ꢂ 105 cells per well in Dulbecco’s modified
Eagle’s medium (DMEM) containing 10% fetal bovine serum
(FBS) and incubated at 37 1C in a 5% CO2 incubator for 24 hrs.
The medium was replaced with 20 mM HEPES buffer and
Author contributions
incubated with NG1 ꢃ Cu2+ ꢃ lactic acid in concentrations The manuscript was written by the contribution of all authors.
of 100 ppm and 250 ppm for 15 min at 37 1C in water. NG proposed the application of probe and coordinated the
Post incubation, the cells were washed with ice-cold PBS++ to overall project including drafting of the manuscript and its
block endocytosis and remove the unbound dye and Cu2+ ions. correspondence. VK synthesized the probes NG1, NG2 and
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7 L. Xu, Z. Wang, R. Wang, L. Wang, X. He, H. Jiang, H. Tang,
D. Cao and B. Z. Tang, Angew. Chem., Int. Ed., 2020, 59,
9908–9913.
8 Y. Hong, S. Chen, C. W. T. Leung, J. W. Y. Lam, J. Liu,
N.-W. Tseng, R. T. K. Kwok, Y. Yu, Z. Wang and B. Z. Tang,
ACS Appl. Mater. Interfaces, 2011, 3, 3411–3418.
9 T. J. Meyer, Pure Appl. Chem., 1986, 58, 1193–1206.
10 G. Zhang, G. Kim and W. Choi, Energy Environ. Sci., 2014, 7,
954–966.
NG3. VK, BK and CK did UV. Fluorescence and self-assembly
studies were carried out by the BK and VK jointly. DKS and DKP
performed DFT calculations, structural part was discussed by
DKP and electronic properties of all the probes and their
complexes were explained by DKS. SK and DB devised cell
culture experiments. SK performed the cellular uptake and
quantification of dye uptake in cells. All the authors discussed
the results and contributed to manuscript drafting.
11 X. Y. Shen, Y. J. Wang, E. Zhao, W. Z. Yuan, Y. Liu, P. Lu,
A. Qin, Y. Ma, J. Z. Sun and B. Z. Tang, J. Phys. Chem. C,
2013, 117, 7334–7347.
12 H. Sunahara, Y. Urano, H. Kojima and T. Nagano, J. Am.
Chem. Soc., 2007, 129, 5597–5604.
13 X. Cao, Y. Li, Y. Yu, S. Fu, A. Gao and X. Chang, Nanoscale,
2019, 11, 10911–10920.
14 X. Cao, Y. Li, Q. Han, A. Gao, B. Wang, X. Chang and
J.-T. Hou, J. Mater. Chem. C, 2020, 8, 4058–4064.
15 D. Zhai, W. Xu, L. Zhang and Y.-T. Chang, Chem. Soc. Rev.,
2014, 43, 2402–2411.
Funding source
The work was supported by the DST SERB extramural research
fund (Project No. EMR/2016/003186) received by Dr Nidhi Gour
and DST-SERB Ramanujan Fellowship to Dhiraj Bhatia.
DKS acknowledges financial support from the SERB-DST ECR
project ‘‘ECR/2016/001289’’.
Conflicts of interest
16 M. Deiana, K. Chand, J. Jamroskovic, I. Obi, E. Chorell and
N. Sabouri, Angew. Chem., Int. Ed., 2020, 132, 906–912.
17 J. Yang, C.-C. Dong, X.-L. Chen, X. Sun, J.-Y. Wei, J.-F. Xiang,
J. L. Sessler and H.-Y. Gong, J. Am. Chem. Soc., 2019, 141,
4597–4612.
There is no conflict of interest to declare.
Acknowledgements
18 D. Su, C. L. Teoh, L. Wang, X. Liu and Y.-T. Chang, Chem.
Soc. Rev., 2017, 46, 4833–4844.
19 X. Li, H. Jiang, Z. Luo, Y.-T. Chang and L. Zhang, Chem.
Commun., 2016, 52, 9086–9088.
20 L. K. Kumawat, A. A. Abogunrin, M. Kickham, J. Pardeshi,
O. Fenelon, M. Schroeder and R. B. Elmes, Front. Chem.,
2019, 7, 354.
21 X. Cao, Y. Li, A. Gao, Y. Yu, Q. Zhou, X. Chang and X. Hei,
J. Mater. Chem. C, 2019, 7, 10589–10597.
22 X. Cao, N. Zhao, A. Gao, Q. Ding, Y. Li and X. Chang,
Langmuir, 2018, 34, 7404–7415.
23 M. Reches and E. Gazit, Science, 2003, 300, 625–627.
24 E. Gazit, Prion, 2007, 1, 32–35.
25 R. Scherzer-Attali, R. Pellarin, M. Convertino,
A. Frydman-Marom, N. Egoz-Matia, S. Peled, M. Levy-
Sakin, D. E. Shalev, A. Caflisch and E. Gazit, PLoS One,
2010, 5, e11101.
NG thanks Indrashil University and Institute of Advanced
research Gandhinagar for the infrastructure support. NG, VK,
and CKP greatly acknowledge support from DST and SERB
research grant (EMR/2016/003186) for funding and fellowships.
BK thanks Indrashil university for funding and infrastructure
support. DKP is grateful to DST, India for providing financial
support under the INSPIRE Fellowship No. IF170625. DS thanks
GSBTM for the fellowship. DB thanks Central Imaging Facility,
(CIF) at IITGN for confocal microscopy, XRD and the initial
seed funding from IITGN to DB. Mr. Ashadul Haque (IITGn) is
thanked for helping in XRD analysis. Ramesh Singh Chilwal
and Dr K. B. Joshi (Sagar University) are thanked for their help
in AFM imaging. NG also gratefully acknowledges Prof
Ehud Gazit (Tel Aviv University, Israel) and his PhD student
DorZaguri for useful discussion and experimental support.
26 A. Paul, B.-D. Zhang, S. Mohapatra, G. Li, Y.-M. Li, E. Gazit
and D. Segal, Front. Mol. Biosci., 2019, 6, 16.
27 W. Zhang, T. Wang, Z. Pei, D. S. Miller, X. Wu, M. L. Block,
B. Wilson, W. Zhang, Y. Zhou and J.-S. Hong, FASEB J., 2005,
19, 533–542.
28 Y. Chang, J. Fu, K. Yao, B. Li, K. Xu and X. Pang, Dyes Pigm.,
2019, 161, 331–340.
29 K. Aou, S. Kang and S. L. Hsu, Macromolecules, 2005, 38,
7730–7735.
Notes and references
1 Y. Jiao, J.-F. Xu, Z. Wang and X. Zhang, ACS Appl. Mater.
Interfaces, 2017, 9, 22635–22640.
2 N. S. Sariciftci, L. Smilowitz, A. J. Heeger and F. Wudl,
Science, 1992, 258, 1474–1476.
3 E. Wang, J. W. Lam, R. Hu, C. Zhang, Y. S. Zhao and
B. Z. Tang, J. Mater. Chem. C, 2014, 2, 1801–1807.
4 N. Lu, X. Gao, M. Pan, B. Zhao and J. Deng, Macromolecules,
2020, 53, 8041–8049.
¨
30 A. Saeed, R. Qamar, T. A. Fattah, U. Florke and M. F. Erben,
Res. Chem. Intermed., 2017, 43, 3053–3093.
31 G. Singh and S. Rani, Eur. J. Inorg. Chem., 2016, 3000–3011.
32 S. Suganya and S. Velmathi, Sens. Actuators, B, 2015, 221,
1104–1113.
5 B. Zhang, H. Wu, Z. Wang, A. Qin and B. Z. Tang, J. Mater.
Chem. C, 2020, 8, 4754–4762.
6 Y. Hong, J. W. Lam and B. Z. Tang, Chem. Soc. Rev., 2011, 40,
5361–5388.
This journal is © The Royal Society of Chemistry 2021
Soft Matter, 2021, 17, 4304–4316
| 4315

View Article Online
Soft Matter
Paper
33 W. M. Khairul, M. F. A. Hasan, A. I. Daud, H. M. Zuki and 50 P. Sanchora, D. K. Pandey, D. Rana, A. Materny and
M. A. Kadir, Malaysian Journal of Analytical Sciences, 2015, 20(1),
73–84.
34 Y. R. Sadaphal and S. S. Gholap, Sens. Actuators, B, 2017,
253, 173–179.
D. K. Singh, J. Phys. Chem. A, 2019, 123, 4948–4963.
51 D. K. Pandey, H. L. Kagdada, A. Materny and D. K. Singh,
J. Mol. Liq., 2021, 322, 114538.
52 M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria,
M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone,
G. A. Petersson, H. Nakatsuji, X. Li, M. Caricato,
A. V. Marenich, J. Bloino, B. G. Janesko, R. Gomperts,
B. Mennucci, H. P. Hratchian, J. V. Ortiz, A. F. Izmaylov,
J. L. Sonnenberg, D. Williams-Young, F. Ding, F. Lipparini,
F. Egidi, J. Goings, B. Peng, A. Petrone, T. Henderson,
D. Ranasinghe, V. G. Zakrzewski, J. Gao, N. Rega,
G. Zheng, W. Liang, M. Hada, M. Ehara, K. Toyota,
R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima,
Y. Honda, O. Kitao, H. Nakai, T. Vreven, K. Throssell,
J. A. Montgomery Jr., J. E. Peralta, F. Ogliaro,
M. J. Bearpark, J. J. Heyd, E. N. Brothers, K. N. Kudin,
V. N. Staroverov, T. A. Keith, R. Kobayashi, J. Normand,
K. Raghavachari, A. P. Rendell, J. C. Burant, S. S. Iyengar,
J. Tomasi, M. Cossi, J. M. Millam, M. Klene, C. Adamo,
R. Cammi, J. W. Ochterski, R. L. Martin, K. Morokuma,
O. Farkas, J. B. Foresman and D. J. Fox, 2016.
35 S. Liu, J. Kang, X. Cao and X. Yue, Spectrochim. Acta, Part A,
2016, 153, 471–477.
36 N. Gour, C. Kanth P, B. Koshti, V. Kshtriya, D. Shah, S. Patel,
R. Agrawal-Rajput and M. K. Pandey, ACS Chem. Neurosci.,
2018, 10, 1230–1239.
37 N. Gour and S. Verma, Soft Matter, 2009, 5, 1789–1791.
38 N. Gour, D. Kedracki, I. Safir, K. X. Ngo and C. Vebert-
Nardin, Chem. Commun., 2012, 48, 5440–5442.
39 N. Gour, J. N. Abraham, M. Chami, A. Castillo, S. Verma and
C. Vebert-Nardin, Chem. Commun., 2014, 50, 6863–6865.
40 J. N. Abraham, N. Gour, S. Bolisetty, R. Mezzenga and
C. Nardin, Eur. Polym. J., 2015, 65, 268–275.
41 N. Gour, S. Mondal and S. Verma, J. Pept. Sci., 2011, 17, 148–153.
42 N. Gour, V. Kshtriya, S. Gupta, B. Koshti, R. Singh, D. Patel
and K. B. Joshi, ACS Appl. Bio. Mater., 2019, 2, 4442–4455.
43 E. Gazit, FASEB J., 2002, 16, 77–83.
´
44 J. M. Aguirre, A. Gutierrez and O. Giraldo, J. Braz. Chem.
Soc., 2011, 22, 546–551.
45 P. Singh, S. K. Brar, M. Bajaj, N. Narang, V. S. Mithu,
53 T. Keith and J. M. Millam, GaussView, Semichem Inc.,
Shawnee Mission, KS, 2016.
O. P. Katare, N. Wangoo and R. K. Sharma, Mater. Sci. 54 M. A. McAllister, THEOCHEM, 1998, 427, 39–53.
Eng., C, 2017, 72, 590–600.
46 D. Bhatia, S. Surana, S. Chakraborty, S. P. Koushika and
Y. Krishnan, Nat. Commun., 2011, 2, 1–8.
55 D. K. Singh, S. Cha, D. Nam, H. Cheong, S. W. Joo and
D. Kim, ChemPhysChem, 2016, 17, 3040–3046.
56 A. D. Becke, Phys. Rev. A: At., Mol., Opt. Phys., 1988, 38, 3098.
47 D. Bhatia, S. Mehtab, R. Krishnan, S. S. Indi, A. Basu and 57 A. D. Becke, J. Chem. Phys., 1992, 96, 2155–2160.
Y. Krishnan, Angew. Chem., Int. Ed., 2009, 48, 4134–4137.
48 A. Bharti, P. Bharati, R. Dulare, M. Bharty, D. K. Singh and
N. Singh, Polyhedron, 2013, 65, 170–180.
49 D. Majumdar, S. Dey, D. Das, D. K. Singh, S. Das, K. Bankura
and D. Mishra, J. Mol. Struct., 2019, 1185, 112–120.
58 D. K. Pandey, H. L. Kagdada, A. Materny and D. K. Singh,
J. Phys. Chem. A, 2021, DOI: 10.1021/acs.jpca.0c10912.
59 A. Ala, A. P. Walker, K. Ashkan, J. S. Dooley, and
M. L. Schilsky, Wilson’s disease, The Lancet, 2007,
369(9559), 397–408, DOI: 10.1016/S0140-6736(07)60196-2.
This journal is © The Royal Society of Chemistry 2021
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