Smart phone assisted quinoline-hemicyanine based fluorescent probe for the selective detection of glutathione and the application in living cells

Article abstract of DOI:10.1016/j.saa.2020.118809

Quinoline appended hemicyanine 6MIM with strong ICT character was successfully synthesized through simple condensation reaction of 6-methoxy-2-chloro-3-formyl quinoline with 2-benzothiazolinium iodide. The photophysical characteristics of synthesized probe revealed that it would selectively detect glutathione (GSH) when it compared with different amino acids including biothiols and the detection limit is found to be 100 nM. The turn off sensor is due to thiol-halogen S_NAr nucleophilic substitution between 6MIM and thiol group in glutathione. More importantly, the biosensor 6MIM was effectively applied in the fluorescence bioimaging of GSH in living cells with low cell toxicity. The colorimetric detectable color change of 6MIM-GSH has been effectively integrated with smartphone assisted RGB color value application with lowest detection value of 120 nM.

Full text of DOI:10.1016/j.saa.2020.118809

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Smart phone assisted Quinoline-hemicyanine based fluorescent
probe for the selective detection of glutathione and the application
in living cells
Kailasam Saravana Mani, Ramar Rajamanikandan, Malaichamy
Ilanchelian, Narenkumar Muralidharan, Mathivanan Jothi,
Subramaniam Parameswaran Rajendran
PII:
S1386-1425(20)30788-5
https://doi.org/10.1016/j.saa.2020.118809
SAA 118809
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date: 25 April 2020
Revised date:
22 July 2020
August 2020
Accepted
date:
2
Please cite this article as: K.S. Mani, R. Rajamanikandan, M. Ilanchelian, et al., Smart
phone assisted Quinoline-hemicyanine based fluorescent probe for the selective detection
of glutathione and the application in living cells, Spectrochimica Acta Part A: Molecular
and Biomolecular Spectroscopy (2020), https://doi.org/10.1016/j.saa.2020.118809
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2020 Published by Elsevier.

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Smart phone assisted Quinoline-hemicyanine based fluorescent probe for the
selective detection of glutathione and the application in living cells
a,b
a
a
Kailasam Saravana Mani* , Ramar Rajamanikandan , Malaichamy Ilanchelian ,
c
c
a,b
Narenkumar Muralidharan , Mathivanan Jothi* , Subramaniam Parameswaran Rajendran*
a
Department of Chemistry, Bharathiar University, Coimbatore, Tamil Nadu, India.
b
Department of Chemistry, Karpagam Academy of Higher Education, Coimbatore, Tamil Nadu,
India.
c
Department of Human Genetics, National Institute of Mental Health and Neurosciences
(
NIMHANS), Bengaluru, India.
Corresponding author: chemsaran.mani@gmail.com, chelian73@yahoo.com,
mjothi@nimhans.ac.in (Biological application)
*
Abstract
Quinoline appended hemicyanine 6MIM with strong ICT character was successfully
synthesized through simple condensation reaction of 6-methoxy-2-chloro-3-formyl quinoline
with 2-benzothiazolinium iodide. The photophysical characteristics of synthesized probe
revealed that it would selectively detect glutathione (GSH) when it compared with different
amino acids including biothiols and the detection limit is found to be 100 nM. The turn off
sensor is due to thiol-halogen S Ar nucleophilic substitution between 6MIM and thiol group in
N
glutathione. More importantly, the biosensor 6MIM was effectively applied in the fluorescence
bioimaging of GSH in living cells with low cell toxicity. The colorimetric detectable color
change of 6MIM-GSH has been effectively integrated with smartphone assisted RGB color value
application with lowest detection value of 120 nM.
Keywords
Quinoline derived receptor; hemicyanine; UV-vis and emission spectra; Smartphone integration;
fluorescence bioimaging
Introduction
Thiol group containing amino acids are main factors of numerous peptides that work vital
tasks in sustaining natural redox homeostasis in genetic methods via equilibrium between
oxidized disulfide forms and reduced free thiol group.[1] Significantly, many researchers have
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established that irregular amounts of these thiol containing amino acids are directly connected
with assured infections, including cancer, liver damage, AIDS, Alzheimer’s disease,
osteoporosis, cardiovascular diseases and inflammatory bowel [2-5]. Among the different
biological thiols, glutathione (L-γ-glutamyl-L-cysteinyl-glycine, GSH) is the largely thiol loaded
tri-peptide that serve as eukaryotic and mammalian cellular functions like, xenobiotic
metabolism, maintenance of intracellular redox activity, gene regulation and intracellular signal
transduction. [5,6] GSH is a crucial endogenous antioxidant that acts an essential function in
intercellular defence beside free radicals and toxins.[7] An unusual level of GSH is able to escort
aging, heart problems, cancer and other ailments. [6,8] Therefore, it is crucially imperative to
develop rapid, simple and selective approaches for detection of GSH and checking the changes
of GSH levels under physiological conditions are highly necessary.
Up to now, a lot of tactics have been proposed for the quantification of GSH [9-14]. Even
though, most of the analytical methods are highly expensive, labour-intensive, time consuming,
need sophisticated instrumental facility and which limits their biological applications. In view of
the critical metabolic roles of GSH for intercellular homeostasis, to ascertain the new sensing
tactics are still enviable for selective quantification of cellular GSH level [14,15]. Optical visual
naked eye sensing approaches are compatible for the quantification of thiol containing biothiols
in vitro and in vivo.[15] Generally, the available sensing tactics are based on the operating by
Michael addition,[16] strong nucleophilicity of the thiol group, cleavage of sulfonamide,[17] and
disulfide [18] etc. These reaction based sensing approaches were easily identify the biothiols
from different amino acids. Though, it is static a difficult to distinguish among the molecules
containing thiol group with their analogous reactivities and structures [19,20]. Hence, we have
chosen to progress synthesize an organic molecule based fluorescence probe as GSH specific and
optically accessible as organic dyes but low cost from a synthetic approach. In addition, we have
engaged addicted to account the prospect of utilizing the organic molecules not only for
fluorometric but also for colorimetric detection of GSH, raising the selectivity using
spectrofluorimetric methods.
On continuing our research interest in the detection of various cations with the
application of fluorescence bioimaging in living cells [21, 22], we have decided to synthesize an
organic molecule based probe existing of an intramolecular charge transfer compound whose
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electronic features like absorption and emission spectra. Hemicyanine dye offers such a features
and it is composed of an electron accepter benzothiazolium species and an electron donor that in
this case of methoxy group in quinoline core [23, 24]. In this investigation, we focus to develope
a simple hemicyanine based fluorescent probe 6MIM for the detection of GSH under
physiological conditions, characteristic merits such as simple preparative method, good
photostability, better emission behavior, with super selectivity and sensitivity. It is important to
mention that probe 6MIM shows a high specificity for the quantification of GSH over cysteine
and homo-cysteine and that it can be further applied to examine of GSH in cancer cells. In
addition, the present colorimetric probe was integrated with smart phone detection of GSH.
2
2
. Experimental section
.1. Materials
All the chemicals for the probe synthesis of 6MIM were acquired from commercial
1
13
suppliers. Solvents were distilled according to previously reported procedures. H and C NMR
1
13
spectral studies were recorded in Bruker avance (400 MHz for H and 100 MHz for C) in
CDCl and DMSO-d solvents and chemical shift values were expressed in parts per million
3
6
using TMS as an internal standard. The reaction path was track by TLC on silica gel coated F254
Merck plates. Elemental analyses were examined by using Perkin Elmer 2400 series II Elemental
CHNS analyzer. ESI-MS were recorded on a Thermo Scientific High resolution Magnetic Sector
MS DFS by negative ion electrospray ionization (ESI) method.
2
.1.1. Synthesis of probe 6MIM
Probe 6MIM was synthesized through condensation reaction of equimolar solution of
8
-methyl-2-chloro-3-formyl quinoline (1 mmol) and 2,3-dimethylbenzothiazolium iodide
(1 mmol) in refluxing ethanol. After the completion of reaction monitored by TLC (PE:EA) the
obtained precipitate was filtered off and washed with cold ethanol. The purest form of product
will be obtained through recrystallization using chloroform: methanol in 1:1 ratio.
2
-(2-(2-chloro-6-methoxyquinolin-3-yl)vinyl)-3-methylbenzo[d]thiazol-3-ium (6MIM):
-1
1
Orange solid; Yield 92 %; mp 234-236 °C; IR(KBr) υ(cm ): 3407, 2997, 1579, 758; H NMR
400 MHz, DMSO-d ) (ppm) δ: 9.27(s, 1H), 8.49(d, J = 7.6 Hz, 1H), 8.35(d, J = 8.8Hz, 1H),
.27(s, 1H), 7.96-7.93(m, 2H), 7.89(t, J = 8.0 Hz, 2H), 7.61(dd, J = 2.8 Hz, J = 2.8 Hz, 1H),
(
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1
3
7
1
1
.46(d, J = 6.4 Hz, 1H), 4.45(s, 3H), 3.96(s, 3H); C NMR(100 MHz, CDCl ) (ppm)δ: 171.4,
3
48.6, 147.1, 142.6, 141.7, 139.7, 136.3, 133.4, 130.2,129.4, 128.8, 128.7, 127.3, 126.5,
24.9, 118.6, 117.7, 37.3, 36.3; HRMS (ESI-MS) m/z 494.1784; Anal.Calcd. for
C H ClN OS: C, 65.0; H, 4.38; N, 7.62; S, 8.72; Found: C, 65.1; H, 4.37; N, 7.64; S, 8.73.
20
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2
2
2
.2. Methods
.2.1. Absorption and Emission Measurements
UV−vis absorption spectral studies were carried out on a JASCO V-630 UV−visible
spectrophotometer with 1.0 cm quartz cuvettes used to monitor the absorption spectral studies.
Steady-state fluorescence spectral measurements were performed with a JASCO FP-6600
spectrofluorometer attached with a 1 cm quartz cuvette.
2
.2.2. Procedure for GSH sensing
Stock solutions (0.1 M) of GSH were prepared in Tris HCl buffer (pH-7.2). Stock
solutions of the probe 6MIM (0.01 M) were prepared in dimethyl formamide (DMF).
All UV–vis and fluorescence measurements involved use of 10 μM probe 6MIM in Tris
HCl (pH-7.2) and 1 mM GSH. For all fluorescence measurements, the excitation
wavelength was 380 nm for the detection of three biothiols and the excitation and
emission slit widths were 2.5 and 5 nm, respectively.
2
.2.3. Fluorescence bioimaging of living cells
The skeletal myoblasts C2C12 cells were obtained from national center for cell science
(NCCS), Pune, India. These cells were grown in Dulbecco’s Eagles medium (DMEM),
supplemented with 20% Heat inactivated Fetal Bovine Serum (HI-FBS) and 1% Antibiotics and
Antimycoticsat 37 °C in a humidified incubator with 5% CO . Once the cells reached 50-60%
2
confluence we have treated these cells with the probe 6MIM (100 mM) in presence or absence
of GSH (500 mM) for 20 minutes followed by fluorescence imaging with Nuclear counterstain
DAPI(4′,6-diamidino-2-phenylindole) in a BX51 Fluorescence Microscopes (Olymbus).The
fluorescence signal in the cells were quantified using ImageJ Ver1.52a software (NIH, USA) and
represented as the corrected total cell fluorescence (CTCF) using the below mentioned formula.
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CTCF = Integrated Density – (Area of selected cell X Mean fluorescence of background
readings)
3
. Results and Discussion
K. P. Elango et al.[25] reported recently the synthesis of quinoline based hemicyanine
intramolecular charge transfer (ICT) receptor for the selective detection of cyanide ion in
aqueous solution. The synthetic routes for the probe 6MIM were outlined in Scheme-1. To a
solution of 2-methylbenzothiazole (1.49 g, 10 mmol) in CH CN (20 mL) was added methyl
3
iodide (3.548 g, 25 mmol). The mixture was refluxed under an N atmosphere for 24 h. After
2
cooled to room temperature, the solid was filtered off and dried under vacuum to afford a white
solid with 92% yield, which was then used for next step reaction without further purification.
The probe 6MIM was synthesized through condensation reaction between 6-methoxy-2-chloro-
3
-formyl quinoline (1 mmol) and 2,3-dimethylbenzothiazolium iodide (1 mmol) in refluxing
1
ethanol. The formation of probe 6MIM was attested by spectroscopic techniques. In the H
NMR spectrum of probe 6MIM, the disappearance of aldehyde and appearance of olefinic
protons at δ 8.49 (J = 7.6 Hz) and δ 8.35 (J = 8.8 Hz). The N-methyl (N-CH ) proton appeared at
3
δ 4.44 confirmed the formation of probe 6MIM.
Scheme1. Synthetic route of probe 6MIM
3
.1. Absorption spectroscopic studies
For the importance of GSH detection, we examine its reactivity 6MIM towards GSH
through UV-visible absorption spectral studies. The UV-vis spectra of free 6MIM in DMF/Tris
HCl buffer (pH-7.2) before and after the addition of GSH is displayed Fig. 1. As visualized in
Fig.1, free 6MIM compound showed a characteristic peaks at 306 nm and 382 nm due to the
–* and n–* transitions, respectively. Upon addition of GSH, the initial absorption peak (382
nm) decreased (Fig.1) regularly along with the simultaneous increasing the peak at 306 nm with
slight blue shift, while, color also changes from yellow to colorless.
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Fig. 1. UV–vis absorption spectral changes of 6MIM in DMF/Tris HCl buffer (pH-7.2) with the
increasing concentration of GSH
The decreasing absorption peak at 382 nm and color change is caused by interaction
between the 6MIM and GSH. Furthermore, absorption peak at 306 nm increased with slight blue
shift is owing to the chlorine group of 6MIM is primarily be replaced by the thiol group of GSH
and the succeeding intramolecular rearrangement would quickly lead to the increasing absorption
peak at 306 nm. Similar kinds of results have been earlier made [26, 27] in coumarin systems.
Therefore, in this case we believed that GSH contains the thiol group interacts with chlorine
group of the 6MIM.
3
.2. Emission spectral studies
In addition, we examined the emission behavior of 6MIM in the addition of GSH by
using excitation wavelength at 380 nm and corresponding results were displayed in Fig. 2. As
illustrated in Fig. 2, upon the addition of increasing concentrations of GSH to a solution of
6
MIM in buffer media, the emission peak at 522 nm was decreased steadily and at the same time
a new band at 428 nm increased with a clear isoemissive point at 452 nm, whereas, color also
changes from yellow to colorless. Furthermore, quantum yield (Φ ) of the 6MIM before and
F
after the addition of GSH was measured with compared to the quinine sulphate as a standard.
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The value of Φ also reduced from 15% to 1.3% was noted after the addition of GSH by 6MIM.
F
This observation further supports to the emission quenching experiments. The obtained
isoemissive point solely indicates that reactant and product in equilibrium process. The
calibration plot is relative emission intensity (ΔF=F -F) as a function of the different amounts of
0
GSH is presented in Fig.3. It can be seen from Fig.3, the calibration plot demonstrates good
linear relationship within the experimental concentration range from 30 M to 75M and
yielded detection limit value of 100 nM (S/N=3) [28]. These results are well consistent with the
absorption spectral measurements. The observed spectral and possible sensing mechanism is
represented in Scheme 2.
Fig.2. Emission spectral results of 6MIM in DMF/Tris HCl buffer (pH-7.2) with the
increasing concentrations of GSH λ = 380 nm
ex
The plausible mechanism is depicted in Scheme-2 and illustrated as follows.
nd
Chlorine group in the 2 position of quinoline would enable the thiol-halogen S Ar nucleophilic
N
substitution between 6MIM and thiol (GSH). Thus, it was expected that the chloro group of
6
MIM could initially be replaced by thiol group of GSH to produce
thio-quinoline hemicyanine. The –NH group in the terminal position of GSH undergoes Michael
2
addition with two pathways to produce I or II membered ring products thio-quinoline
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hemicyanine I or II. In fact, the hearsay could be supported by a recent article on a large ring
product resulted from the reaction of glutathione with acrylate [14]. Overall, if our mechanism
suggestion is rational, it would be conclude with discrimination of GSH in terms of the different
chemical structures and thus from mass spectrum analysis of 6MIM receptor with GSH, the
molecular ion peak at m/z 637.2118 (Figure S5) corresponds to thiol-quinoline hemicyanine I or
II. (Scheme-3)
Fig.3. Calibration plot for relative emission intensity of 6MIM versus GSH
To know the stoichiometry of the present method, Job’s plot was plotted between the
emission spectral change of at 428 nm and mole fraction of GSH concentration and this plot was
given in Figure S4. As displayed in Figure S4, the maximum intersection point of the absorption
maximum is observed at 0.5, which clearly points out that a 1:1 binding stoichiometry between
GSH and probe 6MIM. This observation was further validated by ESI-MS spectral analysis.
ESI-MS spectrum of organic molecule based probe 6MIM with GSH was displayed in Figure
S5. The observed m/z value of 637.1052 (6MIM with GSH) confirmed the 1:1 binding
stoichiometry between GSH and probe 6MIM.
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Scheme-2. Plausible mechanism of hemicyanine 6MIM with GSH selectivity
Scheme-3. Plausible sensing mechanism of 6MIM with GSH
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3
.3. Quenching mechanism
To find out the emission quenching mechanism, the reduce in fluorescence intensity of
MIM at 552 nm in the incremental addition of GSH were estimated with the assist of
6
predictable Stern-Volmer equation[29, 30]
F0
=
1 + k [Q] … … … (1)
SV
F
where, F and F are fluorescence intensities of with and without quencher, respectively.
0
[
Q] is the concentration of the quencher. kSV is the Stern-Volmer quenching constant which
measures the efficiency of quenching. Fluorescence quenching results of 6MIM in the existence
of GSH were plotted as relative emission intensities at 522 nm vs the different amounts of GSH
and is visualized in Figure S5. Stern-Volmer plot displays that within the experimental GSH
concentrations, it showed an excellent linear association. Generally, a Stern-Volmer linear plot
displays that simply single kind of emission quenching process take places, in either energy
transfer or ground state complex formation pathway [31]. Furthermore, the value of kSV was
calculated from intercept and the slope of the Stern-Volmer plot and it was established as
4
3
.33 × 10 L/ mol (R = 0.9959). The noted k value clearly indicates that the strong binding
SV
interaction involved between the probe 6MIM and GSH.
3
.4. Determination of binding constant and free energy
The binding constant of 6MIM in the presence of GSH was calculated by modified
double logarithmic equation (Eqn. 2)
ꢁꢂ−ꢁ
log ꢀ
ꢃ = log 퐾 + 푛 log [푄] … … … (2)
푏
ꢁ
where, F and F are same as in Eqn. (1), and K is the binding constant. The double logarithmic
0
b
plot of log [F -F]/F vs log [GSH] was shown in Fig. S6. As visualized in Fig. S6, the plot
0
exhibits well fitted straight line within the investigated concentrations. The binding constant Kb
5
value was calculated as 1.80 ×10 L/ mol (R = 0.9914). The collected high binding constant value
demonstrated that the strong binding force occupied between the probe and analyte. The free
energy change (ΔG) of the present case for 6MIM with GSH was computed by the consequent
equation (ΔG = -2.303 RT log K , R shows the universal gas constant, K denotes the binding
b
b
constant, which can be collected from the double logarithmic plot) [32] and the value is
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-29.993 kJ/mol. The observed negative free energy change value revealed that the present
method is highly favorable and spontaneous.
3
.5. Smartphone integration of 6MIM by the RGB value
On account of innovation in mobile phone aspects in idioms of offering authoritative
integrated-sensors provides important benefit over usual methods in terms of fast investigation,
easy-operation, low-priced, and compacted tool. Therefore, systematic researchers and medicinal
experts have exposed their attention to apply this tool as analytic devices for numerous
uses [33, 34]. Mostly, for embryonic kingdoms they require of such a tool is highly necessary
because of the high priced instrumentation facilities. To achieve the smart phone detection of
GSH, the freely accessible portable RGB Color value APP was downloaded in online the Realme
5
Pro and the alteration in red, blue, green (RGB) values of the glass tubes holding 6MIM
solutions before and after the additions of various amounts of GSH was monitored using back
camera of mobile phone. In general, the model RGB range is characterized by whole number
value for each of the three colours like, red, green and blue (RGB) from 0 to 255. In this RGB
scale, the whole number [255,255,255] matches to accurate white while [0,0,0] to utter black. To
Fig. 4. Smartphone assisted RGB response for the detection of GSH biothiols
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appraise the colour change of 6MIM with the incremental additions of GSH for on-site
quantification[35-37], we have incorporated the present organic molecule based probe with a
smartphone to observe the RGB value changes when the yellow colour of 6MIM changes to
colorless after the mixing of GSH. Smartphone based sensing of GSH by RGB value, the colour
change was easily monitored by tracking the RGB values. The ratio of R/G value was plotted
against the concentrations of GSH and represented in Figure 4. As illustrated in Figure 4, a well
fitted linear relationship range was noted with the concentration range from 20 M to 50 M and
the value of LOD is calculated as 120 nM. This assist of the 6MIM with the smartphone, a low
priced visual naked eye approach was demonstrated for the on-site quantification of GSH.
3
.6. Selectivity
The fluorescence spectral measurements were also monitored to study the reactivity of
probe 6MIM with other amino acids. The emission spectral data of the 6MIM in the presence of
other amino acids (10-fold higher concentrations) counting GSH, Cys, Hcy, Ala, Asn, Trp, Lys,
Leu, Thr, Met, Tyr, Asp, Ser, Gln, Phe, Arg, His, Glu, Pro and Gly were shown in Fig. 5. From
Fig.5, the emission intensity at 522 nm was did not affected even in the presence of 10-fold
higher concentrations of all GSH, Cys, Hcy, Ala, Asn, Trp, Lys, Leu, Thr, Met, Tyr, Asp, Ser,
Gln, Phe, Arg, His, Glu, Pro andGly. Also, the current assay 6MIM is sensitive sufficient to
determine GSH even at very low level (nM range) in same physiological medium.
In addition, GSH can be simply distinguished by the naked eye when introduction of
GSH to the solution of 6MIM results in a color change from yellow to colorless.
These colorimetric changes can be easily visualized by naked eyes (inset of Fig. 4). Therefore,
the colorimetric probe 6MIM is serving as a micromolar level “naked-eye” indicator for GSH.
The observed high selective detection of GSH is due to discrimination of GSH in terms of the
different chemical structures and thus the distinct photophysical properties of the corresponding
thiol-quinoline hemicyanine formation. In the case of other thiol containing amino acids such as,
Cys and Hcy might form stable amino-substituted covalent complexes with 6MIM through a
sequential thiol substitution and rearrangement reaction.
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Fig.5. Relative fluorescence intensity changes (λem = 522 nm) of 6MIM upon addition of 10
folds of various amino acids
3
.7. Fluorescence Bioimaging
GSH is synthesized in mammalian cells and plays a crucial role in cellular protection
from oxidative damage and toxicity [38]. It also important to maintain redox homeostasis in
living cells and any alterations in GSH homeostasis leads to progression of various diseases
including cancer [39]. Thus, detecting the level of GSH in mammalian cells in real-time will be
useful to understand these normal cellular functions [40], disease progression and management.
Thus, we have evaluated the potential of probe 6MIM to GSH in biological system. We have
used this 6MIM in cell culture studies to determine its biological relevance. First, we treated
skeletal muscle cells C2C12 with or without 6MIM for 20 minutes and subjected to fluorescence
imaging. The results showed that, the probe is capable to cross the membrane and enter to the
live cells and fluorescence signal present predominantly in cytoplasm not in the nucleus. We
have observed quenching of 6MIM fluorescence signal when we incubate the cells with GSH.
We have also noticed the change in cell morphology when we treated with the 6MIM (Fig. 6A).
Then we have analyzed the potential of 6MIM to detect endogenous GSH. It is known that
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oxidative cellular insult leads to increased productions endogenous antioxidant GSH to protect
the cells from damage [38]. We have tested the ability of 6MIM to detect oxidative insult
induced endogenous GSH. For this we have treated cells with H O a strong oxidizer and probed
2
2
endogenous GSH induction with 6MIM. Here we have observed 6MIM fluorescence quenching
in H O treated cells compared to untreated control (Fig. 7). Hence these results suggest that
2
2
probe 6MIM can act as a GSH biothiol sensor in mammalian live cells.
Fig.6. GSH sensing in mammalian cells. Fluorescence microscope images of C2C12 cells
treated with 6MIM (100 μM) in presence or absence GSH (500 μM) and the cells were also
counter stained with nuclear stain DAPI (A). Cell images were obtained for 6MIM and DAPI
using an excitation filter BP545-580 and BP330-385 respectively. Magnification 10X.
Quantification of fluorescence signal in these cells was shown in a graph (B). Error bar
indicates +/- Standard Error Mean (SEM)
Conclusion
Quinoline bearing hemicyanine fluorescence probe 6MIM with strong ICT character was
synthesized and investigated. The synthesized probe will selectively detect glutathione over
cysteine / homocysteine and the detection limit was found to be nanomolar range. The initial
probe 6MIM having yellow in color in solution, with the addition of GSH led to quenching of
yellow color to colorless. Moreover, the cell studies further illustrated that 6MIM could serve as
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Fig.7. Endogenous GSH sensing in mammalian cells. Fluorescence microscope images of
C2C12 cells treated with 6MIM (100 μM) in presence or absence H O (150 μM) and the cells
2
2
were stained with nuclear stain DAPI (A). Cell images were obtained as indicated in Fig 6.
Magnification 20X. The quantified fluorescence signal in these cells was shown in B. Error bar
indicates +/- SEM.
a potential fluorescent probe for the detection of GSH in living cells. Finally, probe has been
successfully integrated with smartphone RGB color value application for the real-time online
analysis of GSH with the detection limit of 120 nM.
Declaration of competing interests
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
The author KSM would like to thank Department of Chemistry, Gandhigram Rural
Institute for NMR studies. Further, I would thank Dr. Balasubramanaian Murugesapandian,
Department of Chemistry, Bharathiar University for our constant research support. M.J.
acknowledges DST-SERB, New Delhi, India for financial support (EEQ/2016/000322;
ECR/2017/000265).
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Appendix A. Supplementary data
The NMR spectra, mass spectra, UV absorbance and fluorescence spectra and additional
data (.doc) file will be available as Supporting Information.
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Smart phone assisted Quinoline-hemicyanine based fluorescent probe for the
selective detection of glutathione and the application in living cells
a,b
a
a
Kailasam Saravana Mani* , Ramar Rajamanikandan , Malaichamy Ilanchelian ,
c
c
*a,b
Narenkumar Muralidharan , Mathivanan Jothi* , Subramaniam Parameswaran Rajendran
a
Department of Chemistry, Bharathiar University, Coimbatore, Tamil Nadu, India.
b
Department of Chemistry, Karpagam Academy of Higher Education, Coimbatore, Tamil Nadu,
India.
c
Department of Human Genetics, National Institute of Mental Health and Neurosciences
(NIMHANS), Bengaluru, India.
Corresponding author: chemsaran.mani@gmail.com, chelian73@yahoo.com,
mjothi@nimhans.ac.in
Author statement
Kailasam Saravana Mani: Supervision, Visualization, Writing - original draft,
Writing - review & editing, Project administration, Funding acquisition. Ramar
Rajamanikandan: Formal analysis, Data curation, Validation, Writing - original draft.
2
0

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Malaichamy Ilanchelian: Formal analysis, Data curation, Validation, Writing - original draft.
Narenkumar Muralidharan: Formal analysis, Data curation, Validation, Writing - original
draft. Mathivanan Jothi: Formal analysis, Data curation, Validation, Writing - original draft.
Subramaniam Parameswaran Rajendran: Supervision, Visualization, Writing - original draft,
Writing - review & editing, Project administration, Funding acquisition.
2
1

Journal Pre-proof
Smart phone assisted Quinoline-hemicyanine based fluorescent probe for the
selective detection of glutathione and the application in living cells
a,b
a
a
Kailasam Saravana Mani* , Ramar Rajamanikandan , Malaichamy Ilanchelian ,
c
c
a,b
Narenkumar Muralidharan , Mathivanan Jothi* , Subramaniam Parameswaran Rajendran*
a
Department of Chemistry, Bharathiar University, Coimbatore, Tamil Nadu, India.
b
Department of Chemistry, Karpagam Academy of Higher Education, Coimbatore, Tamil Nadu,
India.
c
Department of Human Genetics, National Institute of Mental Health and Neurosciences
(NIMHANS), Bengaluru, India.
Corresponding author: chemsaran.mani@gmail.com, chelian73@yahoo.com,
mjothi@nimhans.ac.in
Conflict of Interest Statement
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
2
2

Journal Pre-proof
Smart phone assisted Quinoline-hemicyanine based fluorescent probe for the
selective detection of glutathione and the application in living cells
a,b
a
a
Kailasam Saravana Mani* , Ramar Rajamanikandan , Malaichamy Ilanchelian ,
c
c
a,b
Narenkumar Muralidharan , Mathivanan Jothi* , Subramaniam Parameswaran Rajendran*
a
Department of Chemistry, Bharathiar University, Coimbatore, Tamil Nadu, India.
b
Department of Chemistry, Karpagam Academy of Higher Education, Coimbatore, Tamil Nadu,
India.
c
Department of Human Genetics, National Institute of Mental Health and Neurosciences
(NIMHANS), Bengaluru, India.
Corresponding author: chemsaran.mani@gmail.com, chelian73@yahoo.com,
mjothi@nimhans.ac.in
Graphical Abstract
2
3

Journal Pre-proof
Smart phone assisted Quinoline-hemicyanine based fluorescent probe for the
selective detection of glutathione and the application in living cells
a,b
a
a
Kailasam Saravana Mani* , Ramar Rajamanikandan , Malaichamy Ilanchelian ,
c
c
a,b
Narenkumar Muralidharan , Mathivanan Jothi* , Subramaniam Parameswaran Rajendran*
a
Department of Chemistry, Bharathiar University, Coimbatore, Tamil Nadu, India.
b
Department of Chemistry, Karpagam Academy of Higher Education, Coimbatore, Tamil Nadu,
India.
c
Department of Human Genetics, National Institute of Mental Health and Neurosciences
(NIMHANS), Bengaluru, India.
Corresponding author: chemsaran.mani@gmail.com, chelian73@yahoo.com,
mjothi@nimhans.ac.in
Research Highlights
1
2
3
4
. Quinoline appended hemicyanine fluorescence probe 6MIM was synthesized and
attested through a range of spectroscopic techniques
. The synthesized probe 6MIM detects glutathione (GSH) selectively over cysteine and
homocysteine through fluorescence quenching.
. The quenching will be visualized through naked eye from yellow color to colorless and
limit of detection will be 100 nM.
. The colorimetric detectable color change of 6MIM-GSH has been effectively integrated
with smartphone assisted RGB color value application with lowest detection value of 120
nM.
5
. The biosensor 6MIM was effectively applied in the fluorescence bioimaging of GSH in
living cells with low cell toxicity
2
4