Quinone-based fluorophore: A promising luminescent functional material for effective sensing of serine

Article abstract of DOI:10.1016/j.molstruc.2021.130621

In the recent decade, the redox-active quinone entity has been exhaustively evaluated as a potential drug-like molecule. However, there are only few reports that have demonstrated its applicability as a sensor-like motif against biological analytes. In pu

Full text of DOI:10.1016/j.molstruc.2021.130621

Journal of Molecular Structure 1241 (2021) 130621
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Quinone-based fluorophore: A promising luminescent functional
material for effective sensing of serine
Pooja Chauhana, Aman K.K. Bhasinb, Savita Chaudharya,∗
a Department of Chemistry, Panjab University, Chandigarh, India, 160014
b Department of Chemistry, Amity University Punjab-Mohali, India, 140306
a r t i c l e i n f o
a b s t r a c t
Article history:
In the recent decade, the redox-active quinone entity has been exhaustively evaluated as a potential
drug-like molecule. However, there are only few reports that have demonstrated its applicability as a
sensor-like motif against biological analytes. In pursuit of our curiosity into the sensor-like characteristics
of small molecules, this report investigates into the utility of a sulfur-containing naphthoquinone-based
“ligand” as a selective and sensitive fluorimetric sensor for the amino acid such as serine, in semi-aqueous
medium by distinct mechanisms of fluorescence quenching respectively. The stoichiometry of coordina-
tion of “ligand” with serine was evaluated from B-H plot and found to be 1:1, along with a significantly
low detection limit of 0.28 μM (Stern-Volmer plot), respectively. The efficacy has also been verified in
presence of a select array of competing amino acids, in real water samples.
Received 13 March 2021
Revised 17 April 2021
Accepted 3 May 2021
Available online 8 May 2021
Keywords:
Naphthoquinone
Serine
Aspartic acid
Fluorescence-sensor
Sulphur
© 2021 Elsevier B.V. All rights reserved.
Detection limit
1. Introduction
are some of the state-of-the-art analytical procedures that have
been frequently employed for the detection and characterization of
In addition to playing a significant job in the functioning of
the mammalian fundamental nervous system, abnormal physiolog-
ical concentrations of α-amino acids serve as a critical parameter
in assessing the pathophysiology of various psychiatric disorders
[1,2]. Out of different amino acids, serine is considered as non-
essential α-amino acid. The unbalanced levels of serine in human
plasma have been correlated with the premature onset of several
neurodegenerative diseases like Parkinson’s and Alzheimer’s dis-
ease [3]. It plays a central role in cellular proliferation and fatty-
acid metabolism, apart from serving as a critical neurotransmitter
in the mammalian system [4,5]. However, disturbed levels of serine
in the human serum have been associated with the schizophrenic
disorder [6].
amino acids [8]. In addition, chemical sensor-like molecules (natu-
ral /synthetic) that can analytically sense their chemical interac-
tions with amino acids during alterations in their optical assets
(emission or absorption) have also gained widespread applicabil-
ity in their identification [9]. Amongst a diverse range of highly
fluorescent sensor-like chemical molecules that have been previ-
ously reported in the literature, naphthoquinones are an exciting
category of redox-cycling chemical molecules that are garnering
widespread attention, apparently due to their facile chemical syn-
thesis, appreciable fluorescence and chemical stability in physio-
logical media [10a,11a]. For instance, in a recent report, plumba-
gin (a naturally occurring naphthoquinone) was identified as a sig-
nificant arginine-sensing fluorophore in aqueous medium [11a]. In
another case, a chemically synthesized amino-functionalized naph-
thoquinone was evaluated as a highly selective fluoride-ion sen-
sor in water [10b]. Encouraged by the significant photophysical
properties and chemical robustness of the naphthoquinone mo-
tif, we synthesized a sulfur-containing naphthoquinone derivative
(“ligand”) by a facile one-pot procedure, followed by an investi-
gation into the chemosensors activity of this molecule towards a
gamut of environmentally relevant amino acids (Scheme 1). An es-
sential motive behind hindering the α,β-unsaturated site in “lig-
and” with a relatively inert thiophenyl group was to re-confirm a
repeated pattern of sensitivity towards biothiols like cysteine, as
has been previously reported in the literature (Fig. S1) [9,10]. In
As the respective biogenic amino acid serve as crucial markers
in a series of physiological pathways, their biological detection is
of enormous importance [7]. However, an important road-block in
their analytical identification and quantitation lies in their unfa-
vorable physicochemical properties. This, in turn, can be attributed
to their poor fluorescence and absorption profile, in addition to
their chemical vulnerability under physiological conditions. Chro-
matographic procedures based on GC (gas chromatography), LC/MS
approach, ion exchange, electrochemical and infrared spectroscopy
∗
Corresponding author.
E-mail address: schaudhary@pu.ac.in (S. Chaudhary).
https://doi.org/10.1016/j.molstruc.2021.130621
0022-2860/© 2021 Elsevier B.V. All rights reserved.

P. Chauhan, A.K.K. Bhasin and S. Chaudhary
Journal of Molecular Structure 1241 (2021) 130621
Scheme 1. Synthetic procedure for chemical synthesis of naphthoquinone-derived molecular “ligand”.
stark contrast, pronounced fluorimetric perturbations in “ligand”
were induced only in the presence of sulfur-less amino acid, ser-
ine, thereby pointing towards the carbonyl motif as a probable
key player in the reported chemical sensing. To sum up, this re-
port highlights a unique amino acid sensing capability of an eas-
ily synthesized naphthoquinone-functionalized molecule in semi-
aqueous medium. Following standard analytical techniques, serine
could be distinctly sniffed by the chemically robust “ligand” via flu-
orescence sequestration respectively, with a considerably low de-
tection limit. An appreciable bio-compatibility and chemoreceptor
efficacy of “ligand” in real water samples has also been identified.
2.2. Chemical synthesis of host ligand (N)
The title ligand was synthesized by a conventional nucleophilic
substitution reaction between 2,3-dichloronaphthalene-1,4–dione
(G) and thiophenol in absolute ethanol under refluxing conditions
(Scheme 1). The structural optimization was confirmed by standard
spectroscopic techniques (Section 2.3) and found to be in accor-
dance with previously published results [11].
2.3. Methods of characterization
Mettler Toledo digital pH meter used to check the stability of
a solution at varying pH (pH 2–14). Perkin Elmer LS55 spectroflu-
orometer was utilized to explore the luminescence properties of
“ligand” in the presence of amino acids. The excitation and emis-
sion slit width of spectrofluorometer was kept static at 10 nm. The
homogeneous solution was prepared with or without amino acid
by utilizing a Sonicator (POWER SONIC 405). B.O.D incubator with
sterilized maintenance was used for the growth of bacterial strains.
The antibacterial activities were performed in laminar air flow. All
the experiments were conducted thrice to ensure the repeatability
of the obtained results.
2. Experimentation scheme
2.1. Materials used
For the synthesis of “ligand”, 2,3-dichloronapthalene-1,4–dione
(G) and thiophenol were employed as starting materials and pro-
cured from Sigma Aldrich. Respective salts of the selected l-amino
acids (aspartic acid, serine, alanine, glycine, leucine, isoleucine, ty-
rosine, tryptophan, histidine, lysine, arginine, glutamic acid, glu-
tamine, proline, valine, methionine, phenylalanine, cysteine and
threonine) were purchased from Sigma Aldrich (HPLC grade; 98%
purity). Concentrated HCl solution (36.5%) and sodium hydroxide
pellets were procured from Fisher Scientific (analytical status; 99%
purity). NaCl and KCl were acquired from Sigma Aldrich (analyt-
ical grade; 99% purity). In order to measure the biocompatibil-
ity, a standard agar-diffusion assay was employed as a qualitative
measure. For the same, Mueller Hinton Agar and Nutrient Broth
were purchased from Hi-media (microbiology grade; 99.9% pu-
rity). The bacterial strains, namely Escherichia coli, Salmonella ty-
phi, Pseudomonas aeruginosa (gram-negative) and Staphylococcus au-
reus (gram-positive) were procured from Department of Microbiol-
ogy (Panjab University, Chandigarh). All anti-bacterial experiments
were carried out under a bench using sterile conditions. During
spiking studies, real water samples were collected from different
resources. Lake water was taken from Sukhna Lake (Chandigarh)
whereas rain water and tap water was taken from Panjab Univer-
sity, Chandigarh. The pH value of all the water samples was acidic
in range 2–8 whereas conductivity was found in range 400–1000
mS/m at 37 °C. All the water samples were used as such without
doing any further purification.
2.4. Photochemical analysis of host “ligand”
2.4.1. pH studies
In order to check the photochemical stability of the title “lig-
and”, the pH experiments were carried out in acidic and basic
medium. Accordingly, varying dilutions of aqueous solutions of HCl
and NaOH were employed for preparing semi-aqueous solutions of
“ligand” over a relevant pH range (1–14) at room temperature and
their fluorescence profile was recorded respectively under optimal
conditions.
2.4.2. Time studies
The photochemical stability of the title “ligand” in semi-
aqueous medium (ethanol: water; 1:1 v/v) was evaluated with re-
spect to time. In accordance with the observed protocol, the flu-
orescence curve of a 1 ppm solution of “ligand” was recorded at
regular intervals over a 2-day period at different time intervals.
2

P. Chauhan, A.K.K. Bhasin and S. Chaudhary
Journal of Molecular Structure 1241 (2021) 130621
Fig. 1. Antimicrobial activity of host “ligand” against selects bacterial strains at different concentration levels using agar diffusion assay. Ethanol was used as control [13].
2.4.3. Salt effect analysis
solution of serine (10 μM) at room temperature. To the respective
suspension, comparable quantities of the other l-amino acids were
added carefully at room temperature under optimal conditions (at
pH = 8 and temperature 37 °C). Finally, the emission profile of “lig-
and” in the presence of serine (taken as stock solution) was mea-
sured in presence of the other select pool of amino acids at room
temperature.
In order to scrutinize the applicability of the chosen “ligand” in
freshwater samples, the respective effect of salts was investigated.
As per the followed procedure, the fluorescence profile of a semi-
aqueous solution of the native “ligand” was measured upon the ad-
dition of varying concentrations of aqueous solutions of NaCl and
KCl (0–500 mM). The respective analysis was carried out to inves-
tigate the influential behavior of “ligand” in presence of different
ions and electrolytes.
2.6. Antimicrobial activity
2.5. Chemical selectivity of N towards serine: a fluorimetric analysis
2.6.1. Agar-diffusion assay
Fluorescence spectroscopy was exploited to survey the physic-
ochemical properties and amino acid-sensing capacity of “ligand”
in semi-aqueous medium. To perform the respective experiment,
1 ml ethanolic solution of “ligand” (1 ppm) and 1 ml of individ-
ual suspension of selected l-amino acids (analyte; 10 μM) in de-
ionized water were prepared and kept aside for 1 hr. The lumines-
cence profile of the “ligand” (300–800 nm) was recorded in pres-
ence of the selected amino acids at room temperature. The exami-
nation of a distinctive selectivity of “ligand” towards serine was au-
thenticated in parallel by recognizing linear dose-response behav-
ior of the characteristic emission intensity of “ligand” (at 670 nm)
aligned with an enhanced concentration of the target amino acids
(0.01–10 μM for serine respectively). Furthermore, the on-field ap-
plicability of “ligand” as a selective chemical sensor of amino acid
was gauged by scrutinizing a feasible intervention from the various
other amino acids. Accordingly, equivalent volumes of the title “lig-
and” (1 ppm) were taken and consistently mixed with an aqueous
Prior to doing so, a primary overview of the fresh water-
compatibility of the sensor molecule “ligand” was assessed by an
in-vitro agar-diffusion antimicrobial assay. Accordingly, upon auto-
claving the commercially procured Mueller-Hinton agar, the freshly
prepared cooled liquid (40–50 °C) was poured onto sterilized petri
dishes and inoculated with the select bacterial inoculums over the
entire surface [5]. Then, using a sterilized borer, wells (diameter
of 6 mm) dipped in serial ethanolic dilutions of the title “ligand”
(10 ppm, 5 ppm and 1 ppm) were prepared onto the surface of
the inoculated petri dish (Fig. 1). Finally, upon incubation at 37 °C
for 24 h the extent of biocompatibility of title “ligand” was indi-
cated by the absence of bacterial growth directly around the well
(zone of inhibition). Ethanol was used as a negative control in the
assay [12]. The presence of carbon, oxygen and sulfur element in
the “ligand” does not affect the viability of bacterial strains which
suggest a relative non-toxicity of the titular “ligand”. From here, it
can be concluded that, the developed “ligand” acts as biocompati-
3

P. Chauhan, A.K.K. Bhasin and S. Chaudhary
Journal of Molecular Structure 1241 (2021) 130621
Fig. 2. Photochemical stability of host “ligand” in semi-aqueous medium (ethanol: water; 1: 1 v/v) upon varying: (a) pH (2.6, 4.8, 5.8, 7.6, 10.1 and 13.1) (b) Time of
dissolution in medium and (c) Ionic strength of solvent medium (100 mM, 250 mM, 300 mM and 500 mM) in KCl and (d) NaCl.
ble agent in environmental surrounding and can be utilized further
for the environmental remediation process.
of quantum yield for Rhodamine 6 G has been reported in litera-
ture and found to be 0.95. During this study, aqueous solution of
Rhodamine 6 G dye was prepared and its absorption and emission
analysis were carried out. The below mentioned formula was used
for the calculation of quantum yield: -
2.7. Fluorescence studies of spiking samples
ꢂ
ꢃꢂ
ꢃ
In order to evaluate the on-field relevance of the “ligand”, the
sensing efficiency of the same was analyzed in freshwater samples
(collected from rainwater, lake water and tap water). Accordingly,
the real water samples (tap water, lake water and rain water) were
spiked with different concentration of serine in presence of “lig-
and”. The emission behavior was recorded by utilizing fluorescence
spectroscopy. Before carrying out their fluorescence spectrum, the
pH and conductivity values of respective samples have been cal-
culated out. The pH of rain water was found 5.4 (acidic). The pH
of lake and well tap water was found in range 8 to 10 (basic). The
value of conductivity was found in range 400–1000 mS/m. To scru-
tinize the precision of the respective system, all the experimenta-
tion has been carried out in triplicates.
ꢀ
ꢁ
ηX2
ηR2
IX
IR
AR
AX
ϕX = ϕR
(1)
Where subscript X and R refers to “ligand” sample and reference
material, I corresponds to the integrated emission intensity, ɳ cor-
responds to the refractive index of material and A corresponds to
the absorbance values of determined from the absorption spec-
trum. The value of quantum yield was found to be 54% for the de-
veloped “ligand” using the respective dye which suggest the emis-
sion of maximum number of particles during excitation process.
2.8.2. Computation of detection limit & quantitation limit
The value of detection limit (σ) and quantitation limit (β) was
evaluated from Stern-Volmer plot (F/Fo vs concentration) by apply-
ing formula: -
2.8. Evaluation of different parameters from fluorescence data
σ = 3S.D./Slope
β = 10S.D./Slope
(2)
2.8.1. Quantum yield measurement
Rhodamine 6 G dye was utilized as a reference standard while
calculating the quantum yield (ꢀ) of prepared “ligand”. The value
(3)
4

P. Chauhan, A.K.K. Bhasin and S. Chaudhary
Journal of Molecular Structure 1241 (2021) 130621
Fig. 3. Fluorescence emission profile of host “ligand” (λex. = 455 nm; λem. = 670 nm) at 1 ppm concentration) in H2O: ethanol (1:1 v/v) in the presence of a select pool of
amino acids (10 μM) and their selectivity mechanism.
where S.D. refers to the standard deviation of organic “ligand” at
1 ppm concentration level and evaluated by plotting the calibration
curve of emission intensity of “ligand” in presence of amino acids
and at different concentration of amino acids used. The respective
experiment was carried out five times to investigate the repeatabil-
ity of developed system by utilizing fluorescence spectroscopy. The
more details of respective method have been discussed in support-
ing file.
used was: -
Binding constant B.C. = Intercept/Slope
(
)
3. Results and discussion
3.1. Chemical synthesis of host molecule N as a selective amino
acid-sensing system
2.8.3. Estimation of binding constant
In accordance with our recently published article (Scheme 1),
the extensively accepted thiol-mediated Michael addition-
elimination reaction was utilized to assist the chemical synthesis
of the title “ligand” (Scheme 1) (G) [11a]. Accordingly, the one-pot
To estimate the binding efficiency and stoichiometry of “lig-
and”, Benesi-Hilderbrand plot (1/F-Fo vs conc.) was drawn between
emission intensity and reciprocal of concentration. The formula
5

P. Chauhan, A.K.K. Bhasin and S. Chaudhary
Journal of Molecular Structure 1241 (2021) 130621
Fig. 4. (a) Emission profile of serine (10–0.01 μM) in presence of “ligand” (1 ppm), (b) interference study of serine in presence of various other amino acids (10 μM), (c)
Stern-Volmer plot and (d) B-H plot of serine (0–10 μM).
synthetic approach involved a conventional nucleophilic substi-
tution reaction between equimolar amounts of precursor G and
thiophenol under reflux conditions in absolute ethanol, yielding
the title “ligand”. The sulfur-containing “ligand” was obtained in
significant yield upon evaporation of the solvent and followed by
recrystallization in a semi-aqueous solution (Ethyl acetate/hexane;
50.6%). Standard spectroscopic techniques (FTIR, 1H / 13C NMR
and LCMS) were employed to substantiate the analytical purity of
“ligand” [11b] and has been represented in Fig. S2-S5.
dation in its dissolved state over a relevant time interval and has
enhanced the scope in sensing. On a similar pattern, an increase in
the ionic strength of the solvent medium by dissolution of varying
amounts of NaCl or KCl (100–500 nM)) exerted a minimal effect
on the analytical potency of the title “ligand” (Fig. 2(c) and (d)).
On adding the 0.1 M electrolyte to the system, the intensity value
changed. On further increasing the concentration of electrolyte to
0.3 M and 0.5 M, the intensity value has not produced much
change. The ions present in the 0.1 M electrolyte has been com-
pletely adhered over the surface of “ligand” which has produced
influence over the fluorescence intensity of “ligand”. Further incre-
ment in the electrolyte concentration does not affect the physico-
chemical behavior of “ligand” due to the saturation of “ligand” sur-
face, therefore, the emission intensity does not change much [10a].
3.1.1. Photochemical stability of ligand N
The chemical robustness of the title “ligand” was evaluated for
its practical utility at room temperature in presence of different
pH and ionic media [13]. Usually, physiological media (fresh water
samples from lakes, tap or rain water, for instance) exhibit a pH
range of 6.5–8.0, with certain fluctuations depending on weather,
human activity and natural processes [14]. Therefore, an ideal op-
tical sensing system should harbor the potential to exhibit a sig-
nificant efficiency under varying pH conditions. As per our ana-
lytical findings, the title compound exhibited a negligible change
in its spectrochemical behavior within a broad pH range of 3–9
(Fig. 2(a)). The analytical stability of the title “ligand” in semi-
aqueous medium over a 2-day interval was checked by carrying
out the time studies. The intensity of the characteristic fluores-
cence emission peak of the native “ligand” (at 670 nM) was altered
marginally over a 32-h period (Fig. 2(b)) and confirmed the chem-
ical inertness of “ligand” in its corresponding solvent medium. The
fluorophore “ligand” has the ability to resist the chemical degra-
3.2. Serine mediated fluorescence switch-over
In accordance with the widely accepted thiol-ene click chem-
istry, several electrophilic quinone-functionalized molecules have
been successfully investigated for their fluorimetric sensing of bi-
ological thiols (Fig. S1) [9,10]. This has been primarily attributed
to fluorescence alterations in the developed sensing like molecule
upon nucleophilic attack of biological thiol at the α,β-unsaturated
site of the former [15,16]. Due to the presence of a rather in-
ert thiophenyl group at the α,β-unsaturated site in the title com-
pound, the carbonyl motifs apparently serve as the "favored" site
for a chemical interaction with the amino acid analytes. Accord-
ingly, and in stark contrast to previously reported scientific ob-
6

P. Chauhan, A.K.K. Bhasin and S. Chaudhary
Journal of Molecular Structure 1241 (2021) 130621
Fig. 5. (a) Representation of fluorescence emission intensity (λexcitation = 455 nm and λemission = 670 nm) of different water mediums (tap water, rain water, lake water and
freshly distilled water), (b) and (c) Recovery study of serine at different concentration level in different water mediums (deionized water, lake water, rain water and tap
water) and in presence of other 19 amino acids by utilizing “ligand”.
servations, the “ligand” employed in this report did not experi-
ence any fluorogenic perturbations in the presence of biothiols like
cysteine. This could be apparently assigned to the "unavailability"
of an unblocked α,β-unsaturated site in “ligand”, thereby hinder-
ing an attack by the thiol-containing nucleophile, cysteine. On the
other hand, serine (hydroxy analogue of cysteine) induced a pro-
nounced dip in the native fluorescence of N in the competing pres-
ence of a diverse pool of select α-amino acids (Fig. 3), thereby
patently highlighting an active role of the hydroxy moiety (of ser-
ine) in contributing to a favorable chemical interaction with the
sensor-like molecule “ligand”. Further, we were curious to explore
the probability of a contribution of the carboxylic entity of ser-
ine in inducing the observed fluorometric perturbations in “lig-
and”. Accordingly, we investigated the fluorogenic effects of serine
and its methyl ester on the sensor-like “ligand”. As per our find-
ings, like serine, its methyl ester also induced a significant drop in
the native fluorescence of “ligand” (Fig. S6). In fact, a marginal dif-
ference between the extent of fluorometric quenching induced by
the select analytes (serine and methyl ester of serine) apparently
pointed towards a mere spectator role played by serine’s carboxyl
group towards modulating the fluorescence of “ligand”. On another
note, in an attempt to identify the critical segment of the fluo-
rophore “ligand” that harbored a potential affinity for serine, we
carried out fluorometric titrations of serine against “ligand” and its
precursor, 1,4-naphthoquinone (L), in parallel. A similar fluoromet-
ric sequestering of “ligand” and its sulfurless precursor (L) by ser-
ine reasonably confirmed a sole active participation of the ketone
motif of “ligand” in mediating a chemical interaction with serine
(Fig. S7).
Therefore, as per our hypothesis, this could most probably in-
volve a condensation reaction between the carbonyl motif of “lig-
and” and the critical functional motifs (-NH2 and -OH) of the target
analyte, serine. Furthermore, in order to assess the real-time effi-
cacy of the proposed sensor-like “ligand” in terms of its response
time towards serine, a time-dependent analysis of “ligand” in pres-
ence of serine identified a quick onset in its fluorescence dip in as
early as 30 s of exposure and reached its fluorescence minimum (~
3-fold) within 120 s (2 min) of serine dosage (Fig. S8).
The phenomenon of quenching fundamentally depends upon
two types of mechanism, namely static and dynamic quenching.
Dynamic quenching results from the electron transfer of fluo-
rophore to the quencher present in ground state due to the pro-
gression of collision whereas in case of serine, static quenching
might take place due to the combination of fluorophore (“ligand”)
and quencher (serine) which results in the formation of very rapid
ground state non-fluorescent complex. This complex formation fur-
ther leads to aggregation of molecules and enhances the internal
charge transfer mechanism (ICT) due to transfer of electrons from
7

P. Chauhan, A.K.K. Bhasin and S. Chaudhary
Journal of Molecular Structure 1241 (2021) 130621
“ligand” (donor) to serine (acceptor). The excellent linear relation-
ship in Stern-Volmer plot also inferred that pure static quenching
exerted by the formed complex. In contrast, the adding up of as-
sorted other selected amino acids (10 μM) wielded a relatively in-
consequential turn down in the fluorescence peak intensity of “lig-
and” at 670 nm, thereby exhibiting efficient "turn-off" fluorescence,
selectively by serine in semi-aqueous medium. In order to investi-
gate the title “ligand” towards serine, the emission intensity of the
characteristic “ligand” at 670 nm was observed to reduce in un-
deviating fraction to an increasing concentration of serine (0.01–
10 μM; Fig. 4(a)) in an aqueous medium. Further, to investigate
the selectivity of serine, interference study was carried out in pres-
ence of other select amino acids at room temperature (Fig. 4(b)). It
was observed that the selectivity of serine amino acid in presence
of “ligand” does not affected when addition of other amino acids
takes place which again suggest the ultimate specificity and selec-
tivity of “ligand” toward serine amino acid. The detection limit was
calculated from the calibration Stern-Volmer plot (F/Fo vs conc. of
serine) and found to be 0.28 μM (Fig. 4(c)) [17,18]. The obtained
value of detection limit suggest that N can detect the serine amino
acid at lower concentration level [19]. Additionally, the value of
detection limit as compared to the reported literature value has
been represented in Table S1. Further, the stoichiometry of ligand:
serine binding system was calculated from the Benesi-Hildebrand
plot to be 1:1 with a binding constant of 3.28 μM (Fig. 4(d)). The
much closer picture of B-H plot has been represented to show
the linear fit behavior of the system at lower concentration level
(Fig. S8).
target amino acid has been observed to be facilitated by distinctly
mechanisms of fluorescence quenching, respectively. As per our hy-
pothesis, a facile chemical interaction between the carbonyl mo-
tif of the title “ligand” and the carboxyl or hydroxy–functionalized
target amino acid (serine, respectively) could be responsible for
rendering certain selectivity in “ligand”. The resulting fluorimetric
perturbations observed in “ligand” remained rather unaffected in
the presence of a competing pool of other amino acids. A similar
pattern of high selectivity and sensitivity for the target amino acid,
serine, was also evident in natural water samples. The utility of the
naphthoquinone motif as a biologically dynamic fragment, in accu-
mulation to its prospective convenience in selectively sensing spe-
cific intracellular amino acid such as serine may render this partic-
ular motif as a potential candidate for comprehending the under-
lying pathology of amino acid-based disorders.
Declaration of Competing Interest
There are no conflicts to declare.
CRediT authorship contribution statement
Pooja Chauhan: Conceptualization, Data curation, Formal anal-
ysis, Investigation, Software, Writing - original draft. Aman K.K.
Bhasin: Conceptualization, Funding acquisition, Methodology, Visu-
alization, Writing - original draft. Savita Chaudhary: Investigation,
Project administration, Resources, Supervision, Validation, Visual-
ization, Writing - review & editing.
Acknowledgement
3.3. Fluorometric spiking activity
Savita Chaudhary is thankful to DST Inspire Faculty award
[IFACH- 17], Haryana State Council for Science and Technology
[HSCSIT/R&D/2020/476] and DST Purse grants II for financial as-
sistance. The authors are thankful to the Sophisticated Analytical
Instrumentation Facility (SAIF, CIL), Panjab University, Chandigarh.
In order to assess the realistic efficacy of the utilized “ligand”
as a sensing system against serine in semi-aqueous standard, com-
petitive binding experiments were carried out in presence of dif-
ferent real water samples [20,21]. Firstly, fluorescence spectra of a
solution of the host ligand in select water samples such as lake
water, tap water and rain water were recorded (Fig. 5(a)). After-
wards, the emission profile of a real water solutions of the title
“ligand” in the vicinity of serine was recorded (Fig. 5(b)). In ac-
cordance with our findings, the competing amino acids exerted an
inconsequential interference in the serine–induced quenching of
the native fluorescence profile of “ligand”. Furthermore, to validate
the on-field relevance of this sensor-like “ligand” in varying phys-
iological media, a spiking study was conducted at different con-
centration level of aspartic acid and serine (2, 4, 6, 8 and 10 μM)
[21]. Further, recovery study of serine in different water mediums
(deionized water, lake water, rain water and tap water) in pres-
ence of 19 amino acids (alanine, valine, leucine, isoleucine, lysine,
phenylalanine, glutamic acid, aspartic acid, glutamine, proline, ty-
rosine, threonine, arginine, glycine, histidine, methionine, cysteine,
asparagine and tryptophan) has also been calculated (Fig. 5(c)). Ac-
cording to the results obtained, the reported “ligand” demonstrated
a closely significant sensitivity towards serine in varying fresh wa-
ters samples (Fig. 5), thereby also corroborating the minimal effect
of dissolved salts and varying pH (a characteristic of natural wa-
ters) on the fluorimetric sensitivity of the former towards serine.
To conclude, the title “ligand” demonstrates a highly perceptive
and discerning detection of serine, by distinctly opposing routes of
fluorescence quenching respectively.
Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.molstruc.2021.130621.
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