Chelation enhanced fluorescence of rhodamine based novel organic nanoparticles for selective detection of mercury ions in aqueous medium and intracellular cell imaging

Article abstract of DOI:10.1016/j.jphotochem.2020.112579

A simple, quick and useful reprecipitation method was used to prepare rhodamine based fluorescent nanoparticles. The characterization techniques such as zeta-particle sizer and scanning electron microscopy were studied to investigate the formation of desired nanoparticles FONs-RSB along with its aqueous stability, surface charge, particle size, and morphological features. The comparative optical properties between parent molecule RSB and FONs-RSB were examined using absorption and fluorescence studies. The sphere morphology of FONs-RSB having 82 nm particle size and negative zeta potential (?33.5 mV) showed selective and sensitive interaction with only Hg²⁺ amongst the series of metal ion studied which was revealed by florescence titration results. The interaction between FONs-RSB and Hg²⁺ leads to enhance the fluorescence of FONs-RSB is because of chelation enhanced fluorescence (CHEF) phenomenon. The interference of foreign metal ions was found to be unaffected to the fluorescence enhancement induced by Hg²⁺. The binding stoichiometry and binding constant were evaluated using Job's and modified Benesi-Hildebrand plot. The mechanism of binding interaction and nature of complexation were analyzed using Infrared (IR), Nuclear Magnetic Resonance (NMR), absorption and fluorescence lifetime titrations in the absence and presence of a variable amount of Hg²⁺ to FONs-RSB. Further, the reaction product of the sensing process in the present investigation was examined using mass analysis. The detection limits calculated using the present method were found to be 1.729 ng/mL (8.619 nM) and 1.68 ng/mL when linear concentration of Hg²⁺ additions were 0?2 μg/mL and 0?100 ng/mL, respectively. The practical application of the present method involves the quantitative determination of Hg²⁺ in environmental samples collected from the local campus and intracellular cell imaging of Hg²⁺ using A375 living cells with non-toxic behavior.

Full text of DOI:10.1016/j.jphotochem.2020.112579

Journal Pre-proof
Chelation enhanced fluorescence of rhodamine based novel organic
nanoparticles for selective detection of mercury ions in aqueous medium
and Intracellular cell imaging
Prasad G. Mahajan, Jin Sik Shin, Nilam C. Dige, Balasaheb D.
Vanjare, Yohan Han, Nam Gyu Choi, Song Ja Kim, Sung Yum Seo,
Ki Hwan Lee
PII:
S1010-6030(20)30378-6
DOI:
https://doi.org/10.1016/j.jphotochem.2020.112579
JPC 112579
Reference:
To appear in:
Journal of Photochemistry & Photobiology, A: Chemistry
Received Date:
Revised Date:
Accepted Date:
7 February 2020
17 April 2020
19 April 2020
Please cite this article as: Mahajan PG, Shin JS, Dige NC, Vanjare BD, Han Y, Choi NG, Kim
SJ, Seo SY, Lee KH, Chelation enhanced fluorescence of rhodamine based novel organic
nanoparticles for selective detection of mercury ions in aqueous medium and Intracellular cell
imaging, Journal of Photochemistry and amp; Photobiology, A: Chemistry (2020),
doi: https://doi.org/10.1016/j.jphotochem.2020.112579

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Chelation enhanced fluorescence of rhodamine based novel organic
nanoparticles for selective detection of mercury ions in aqueous medium
and Intracellular cell imaging
Prasad G. Mahajana*, Jin Sik Shinb, Nilam C. Digec, Balasaheb D. Vanjarea, Yohan Hanc,
Nam Gyu Choia, Song Ja Kimc, Sung Yum Seoc, Ki Hwan Leea*
aDepartment of Chemistry, Kongju National University, Gongju, Chungnam 32588,
Republic of Korea.
bDepartment of Chemistry, Chungnam National University, Daejeon 34134,
Republic of Korea
cDepartment of Biological Sciences, Kongju National University, Gongju,
Chungnam 32588, Republic of Korea.
*Corresponding Email: mahajanprasad2188@gmail.com (P. G. Mahajan);
khlee@kongju.ac.kr (K. H. Lee)
Graphical Abstract
Highlights:
 Fluorescent organic nanoparticles FONs-RSB prepared by reprecipitation
method
 FONs-RSB showed Chelation enhanced fluorescence only in the presence of
Hg2+
 Interference-free detection of Hg2+ ions using a proposed sensing method

 The extremely low detection limit of 1.729 ng/mL for Hg2+ ions in an aqueous
medium
 Feasibilities are analysis of environmental samples and intracellular cell imaging
Abstract
A simple, quick and useful reprecipitation method was used to prepare rhodamine based
fluorescent nanoparticles. The characterization techniques such as zeta-particle sizer and
scanning electron microscopy were studied to investigate the formation of desired
nanoparticles FONs-RSB along with its aqueous stability, surface charge, particle size, and
morphological features. The comparative optical properties between parent molecule RSB
and FONs-RSB were examined using absorption and fluorescence studies. The sphere
morphology of FONs-RSB having 82 nm particle size and negative zeta potential (-33.5
mV) showed selective and sensitive interaction with only Hg2+ amongst the series of metal
ion studied which was revealed by florescence titration results. The interaction between
FONs-RSB and Hg2+ leads to enhance the fluorescence of FONs-RSB is because of
chelation enhanced fluorescence (CHEF) phenomenon. The interference of foreign metal
ions was found to be unaffected to the fluorescence enhancement induced by Hg2+. The
binding stoichiometry and binding constant were evaluated using Job’s and modified
Benesi-Hildebrand plot. The mechanism of binding interaction and nature of complexation
were analyzed using Infrared (IR), Nuclear Magnetic Resonance (NMR), absorption and
fluorescence lifetime titrations in the absence and presence of a variable amount of Hg2+ to
FONs-RSB. Further, the reaction product of the sensing process in the present
investigation was examined using mass analysis. The detection limits calculated using the
present method were found to be 1.729 ng/mL (8.619 nM) and 1.68 ng/mL when linear
concentration of Hg2+ additions were 0-2 µg/mL and 0-100 ng/mL, respectively. The
practical application of the present method involves the quantitative determination of Hg2+

in environmental samples collected from the local campus and intracellular cell imaging of
Hg2+ using A375 living cells with non-toxic behavior.
Keywords: Rhodamine based fluorescent organic nanoparticles; Mercury ions; Chelation
enhanced fluorescence; Environmental samples; Cell imaging.
1. Introduction:
Pollution occurred by contamination of heavy metal ions in the aquatic and
biological systems increased day by day. Concerning about the hazardous effects arises
from heavy metal ions to human being demands acute, rapid, selective and sensitive
method for their qualitative and quantitative determination. The intake of heavy metals
ions is essential for the human body at certain levels to maintain the biological and
therapeutic balance in the function of various processes associated with the human body
[1-3]. One of the toxic and unsafe heavy metal ions is mercury (Hg2+). Mercury originates
in the forms of three groups such as Hg, Hg+ and Hg2+. Among these three groups, mercury
in its divalent oxidation state (Hg2+) is most toxic due to its mobility in the living systems
and ability to cross cell membranes [4-5]. The mining water, power plants, household
bleaches, fungicides, petrochemicals and chemicals including acids are the main sources
through which mercury has potency to release in the environment [6-8]. The severe threats
to human health arise due to the variable concentration of Hg2+ in the body and which can
be enriched through the food chain and cause harmful effects on human health [9-10]. It
affects and damages central nervous system, kidneys, digestive system, lungs and immune
system and further led to increase risk level of hypertension, oxidative stress and anemia
[10-12]. Therefore, continuous research on detection and reduction of Hg2+ pollution in
aquatic and biological systems increases nowadays in science and engineering society.
Numerous analytical techniques have been devoted to examining the Hg2+
concentration in aqueous system. These techniques include atomic emission spectroscopy
(AES) [13], stripping voltammetry [14], atomic fluorescence spectroscopy (AFS) [15],

Inductively coupled plasma mass spectroscopy (ICP-MS) [16], electrochemical sensors
[17], fluorometric methods [18,19], cold vapor atomic fluorescence (CV-AFS) [20],
polarography [21], colorimetric analyzes [22] and inductively coupled plasma-atomic
emission spectroscopy [23]. Amongst these immense varieties of analytical methods for
the determination of Hg2+ concentration in environmental samples, most are costly, time-
consuming, requires pre-sample treatments, fail to produce extremely low detection limit
and needs sample storage during analysis. These drawbacks encourage to expand sensing
method for qualitative and quantitative determination of Hg2+ in environmental samples
which needs economical, rapid, selective, sensitive and low detection level approach. In an
addition, there were number of probes have been used to monitor Hg2+ such as proteins,
cells, nanomaterials, organic fluorophores, polymers, chromophores and DNA based
molecules [24]. Interestingly, rhodamine based organic fluorophores have been devotedly
used for detection of heavy metal ions due to their spirolactum ring opening and
fluorescence turn-ON characteristics [25-28]. Additionally, considering the design of
nanomaterial-based on rhodamine core will have extra-ordinary properties as compared to
their parent compound in the organic solvent and may use for sensing purpose of heavy
metal ions in aqueous medium at ultra-trace levels, an attempt was made for the preparation
of their fluorescent organic nanoparticles for their sensing application in the present studies.
Currently, one of the swiftly expanding research areas is nanomaterials which offer
remarkable benefits in biological sciences, health sciences and medical engineering [3,4].
There are various many research groups on the development of fluorescent chemosensors
for sensitive detection of metal ions in aqueous medium [1-4]. Traditional fluorescence-
based chemosensors used for the detection of an analyte in aqueous media bears
disadvantages in comparison with the nanomaterials. Such chemosensors are highly
hydrophobic and most of the time were used in organic solvents or mixed organic solvent
systems which may lead to produce interferences from foreign analytes, fail to producing
low detection limits, lack sensitivity, and selectivity. To overcome these shortcomings,
fluorescence-based organic nanoparticles (FONs) become the most capable candidature to
analyze the analyte of interest from the aqueous medium without interferences of other

analytes with an extremely low detection limit [29-31]. The ease of synthesis, real-time
qualitative and quantitative analysis, high surface to volume ratio, extra-ordinary
photophysical properties, sensitivity and selectivity towards analyte of interest, no
interference from other existing or competing analytes and capacity to have an extremely
low detection limit of analyte are the noticeable advantages of fluorescence-based organic
nanoparticles. The increasing industrial and mining activities throughout the whole
population raises the risk of health hazards produced by increasing level of Hg2+
contamination in ecological system. Therefore, to reduce such health risk of Hg2+ pollution,
demands development of selective, sensitive and low cost effective analytical method for
its acute determination in aqueous medium which can be achieved employing design,
preparation and use of fluorescence-based organic nanoparticles (FONs) for heavy metal
ion sensing.
In the present study, we have designed rhodamine Schiff base cored organic
molecule 3',6'-bis(diethylamino)-2-((E)-4-((E)-(pyridin-2-ylimino)methyl)benzylidene-
amino)spiro[isoindoline-1,9'-xanthen]-3-one i.e. RSB which was further used to prepare
fluorescent organic nanoparticles (FONs-RSB) using the reprecipitation method. The
sensing studies reveals FONs-RSB can effectively and selectively interacts with only Hg2+
amongst the series of heavy metal ion. The interaction between FONs-RSB and Hg2+ led
to produce selective chelation enhanced fluorescence in FONs-RSB while other metal ions
produces negligible effects. The present fluorescence-based analytical method provides a
simple, rapid, sensitive and selective quantitative approach for the determination of Hg2+
in environmental samples collected from the local area. The extremely low detection of
Hg2+ concentration at the nano and micro-level can be possible by using the present
analytical approach. Additionally, the prepared nanoparticles showed non-toxic behavior
towards human malignant melanoma A375 cell line. Subsequently, the intracellular
detection of Hg2+ using FONs-RSB is an additional benefit of prepared nanoparticles
which will explore its potent application in the biomedical field.

2. Experimental: Materials and methods:
2.1 Materials:
Rhodamine B, Terephthalaldehyde, hydrazine hydrate, concentrated sulfuric acid and 2-
amino pyridine were procured from Alfa Aesar. The solvents of analytical grade such as
methanol, ethanol and acetone used in the present study were purchased from Samchun
chemicals, Korea and used without further purification. All the metal ion solutions were
prepared by using their salts which were obtained from Alfa Aesar. Double distilled water
used in the series of experiments was collected from Milli-Q system. The 5-
dimethylthiazol-2-yl-2,5-diphenyltetrazoliumbromide (MTT) was obtained from Sigma
Aldrich. The human malignant melanoma A375 cell line and Fetal bovine serum (FBS)
with Dulbecco modified eagle medium (DMEM) were received from Korean Cell Line
Bank (KCLB), Seoul, South Korea and Gibco (Carlsbad, CA, USA), respectively.
2.2 Methods:
2.2.1 Synthesis of 3',6'-bis(diethylamino)-2-((E)-4-((E)-(pyridin-2-ylimino)methyl)-
benzylideneamino)spiro[isoindoline-1,9'-xanthen]-3-one (RSB):
100 mL round bottom flask equipped with rhodamine B (5 mmol, 2.39 g), 50 mL
ethanol and 3 mL concentrated sulfuric acid (H2SO4) was vigorously stirred for 6 h at room
temperature. The formation of rhodamine B ester was monitored on thin layer
chromatography (TLC) progress after specific reaction time. After completion of reaction,
the mixture was allowed to evaporate under reduced pressure to remove excess quantity of
ethanol. To the received crude solid compound (rhodamine B ester), excess quantity of
hydrazine hydrate (1 mL, 20 mmol) and solvent as 10 mL methanol were added. The whole
reaction mass was further stirred for the period of 4 h at room temperature. The progress
of reaction and formation of the compound - rhodamine B hydrazide were monitored on
TLC. Thereafter, reaction mixture was evaporated on rotary using vacuum pressure
technique. Thus, slight pink colored solid received was recrystallized using hot ethanol.
The recrystallized rhodamine B hydrazide was received in appreciable yield of 89% (2.031
g), which was further used for the preparation of compound RSB.

To prepare compound RSB, rhodamine B hydrazide (2 mmol, 0.913 g) was
poured into 25 mL round bottom flask containing 5 mL methanol and allowed to stir at
room temperature. The successive addition of terephthalaldehyde (2 mmol, 0.268 g) was
done and allow the reaction mixture to stir for next 1 h. Afterwards, 2-amino pyridine (2
mmol, 0.188 g) was added to the reaction mixture. The complete reaction mixture was
permitted to stir at room temperature till the completion of reaction and aldehyde became
completely consumed. The consumption of reactants into formation of desired compound
RSB was examined on TLC. After 8 h, the reaction mixture shows completion of reaction
into new product i.e. RSB. The crushed ice was added to the reaction mass and stirred
vigorously for next 30 min. The slight pink colored solid crude compound thus obtained
was filtered and washed with distilled water. The purification of compound RSB was done
using recrystallization process in hot ethanol. The recrystallized product of compound RSB
thus received was in satisfactory yield of 85% (1.103 g). Scheme 1 illustrates schematic
representation of approach for the synthesis of RSB. The recrystallized compound RSB
further used for the spectral characterization such as IR, NMR and Mass analysis followed
by preparation of fluorescent organic nanoparticles of RSB i.e. FONs-RSB.
2.2.1.1 Spectral Characterization of compound RSB:
Melting point (M.P.): Observed: 189-191 0C; IR (Fig. S1): 2923, 2603, 1715 (-C=O), 1637
(-C=N), 1590 (-C=N), 1539, 1455, 1382, 1312, 1267, 1167, 1133, 1116, 1082, 1055, 999,
1
919, 850, 824, 755, 705, 665 cm-1; H-NMR(600 MHz, DMSO-d6) (Fig. S2): δ 8.93 (s,
1H,=CH), 8.79 (s, 1H,=CH), 7.87 – 7.94 (m, 3H, ArH), 7.56 – 7.66 (m, 4H, ArH), 7.36 (s,
1H, ArH), 7.10 – 7.14 (m, 2H, ArH), 6.43 – 6.47 (m, 3H, ArH), 6.41 – 6.43 (t, 1H, ArH),
6.38 – 6.39 (d, 1H, ArH), 6.30 – 6.35 (m, 3H, ArH), 3.28 – 3.31 (q, 8H, -CH2), 1.05 – 1.09
(t, 12H, -CH3,) ppm; 13C-NMR(150 MHz, DMSO-d6) (Fig. S3) : δ 193.08, 164.55, 153.56,
151.60, 149.11, 146.17, 130.74, 128.03, 127.80, 108.31, 105.82, 98.22, 43.93, 12.53 ppm;
LCMS (ESI) (Fig. S4): 649.4 [M+1] m/z.
2.2.2 Preparation of fluorescent organic nanoparticles of RSB (FONs-RSB):

A simply operative reprecipitation method was used for the preparation of aqueous
suspension of fluorescent organic nanoparticles of RSB [29-31]. For this purpose, 0.5 mL,
10 × 10−3 M RSB in acetone was swiftly injected using micro syringe into beaker
containing 100 mL of double distilled water. The solution was stirred vigorously on
magnetic stirrer for 45 min followed by 15 min sonication at 30 °C. The colorless aqueous
suspension of nanoparticles FONs-RSB (50 × 10−6 M) thus obtained was further used to
investigate the physical, morphological, optical and sensing properties.
2.2.3 Characterization techniques:
Frontier IR (Perkin Elmer) spectrometer was procured for the purpose of scanning the FT-
IR spectrum. The Bruker Avance 600 MHz spectrometer (Germany) with TMS as an
internal standard was used to perform examination of 1H NMR and 13C NMR spectra. The
LC-MS spectra were analyzed on 2795/ZQ2000 (waters) spectrometer and Bruker
MicroTof-Q spectrometer (Germany). The particle size and charge over the surface of
prepared nanoparticles were measured by Dynamic light scattering (DLS) technique using
Malvern Zeta - Particle size analyzer. The FE-SEM (MIRA3 LMH, TESCAN, USA) was
utilized for investigation of morphology of prepared nanoparticles. The Shimadzu (Japan)
spectrophotometer and FS-2 fluorescence spectrophotometer (Scinco, Korea) were used to
examine the changes observed in UV–visible (absorption) and fluorescence spectra in
corresponding solvents. The fluorescence lifetime decay performance of FONs-RSB in the
presence and absence of Hg2+ ion was evaluated on time correlated single photon counting
(TCSPC) Spectrophotometer (HORIBA-iHR320, Japan) at its excitation wavelength. Thus,
received outputs were analyzed on Data station software equipped with the instrument for
the estimation of fluorescence lifetime values of FONs-RSB in the presence and absence
of Hg2+.
2.2.4 Cell culture and fluorescence imaging:

The A375 cells were grown in DMEM medium using 10% FBS, penicillin (50 units/mL)
and streptomycin (50 µg/mL). The A375 cells were seeded at the density 1 × 105 cells in
35 mm culture plate. The cells were grown until it reaches 60% by following the treatment
of fresh medium every day. Later, A375 cells in respective medium were preserved without
(Control) and with RSB in acetone, aqueous suspension of FONs-RSB and FONs-RSB +
Hg2+ ion at particular concentration of 1 µg/mL and 2 µg/mL for 6 h. The cells were washed
by cold PBS buffer solution for two times. Further, inverted fluorescence microscope
(BX51, Olympus, Tokyo, Japan) under green light was used to examine the treated cells
for fluorescence images. The experiment was carried out three times at optimized
conditions such as constant exposure time, contrast and brightness settings in humidified
atmosphere (95% air and 5% CO2) at 37 °C in CO2 incubator.
2.2.5 Viability assay (MTT):
The A375 cells were plated at a density of 2.5 × 104 cells/well on 96-well plates followed
by incubation overnight. The cells were supplied with fresh medium on each day until they
grown 60%. Thus, such cells were treated without (Control) and with RSB in acetone,
aqueous suspension of FONs-RSB and FONs-RSB + Hg2+ ion at particular concentration.
Finally, warm PBS was used to wash cells for two times followed by addition of 90 µL of
fresh medium and 10 µL of MTT reagent 1 (MTT, 10 mg/mL) to each sample well. Further,
these cells were incubated for next 24 h with the addition of 100 µL of MTT reagent 2
(Solubilisation buffer, 10% SDS with 0.01N HCl and DMSO) at 37 °C in 5% CO2
incubator. Lastly, the cell viability in the form of absorbance was measured, recorded, and
compared with 100% control for each sample on enzyme-linked immunosorbent assay
(ELISA) plate at 595 nm.
3. Results and Discussion:
3.1 Effect of pH on fluorescence of FONs-RSB:
The fluorescence performance of any probe can be variable in the presence of different pH
environment. Therefore, it is essential to examine and optimize the pH effect for the
fluorescence intensity of FONs-RSB. In this study, the fluorescence intensity of FONs-

RSB was tested in the presence of different pH solution from 1 to 12. The fluorescence
intensity at respective pH was plotted and given as Fig. S5. It was noticed that fluorescence
intensity of FONs-RSB found to be maximum at neutral pH = 7. The decrease in the
fluorescence intensity at acidic and basic conditions was attributed to disruption of
structural cavity of nanoparticles followed by aggregation [31-33]. The neutral pH plays
significantly vital role in the biological experiments. Therefore, neutral pH =7 was
optimized for each solution during further experiments.
3.2 Particle size distribution, Zeta potential and Morphology of FONs-RSB:
The particle size distribution curve of FONs-RSB is given as Fig. 1. The analysis of
particle size illustrates narrow distribution of FONs-RSB within the range of 60-150 nm
having average particle size of 82 nm. To investigate the charge over the surface of FONs-
RSB, zeta potential was examined. Fig. 2 represents the zeta potential curve for prepared
FONs-RSB. The value of zeta (ζ) potential -33.5 mV indicates the superior aqueous
stability of FONs-RSB. It is well known that aqueous suspension of nanoparticles having
either high negative or positive zeta potential value is electrically stabilized [34]. In
addition, the negative zeta potential value of FONs-RSB shows negative charge lies over
the surface of nanoparticles. Such negative charge over the prepared nanoparticles FONs-
RSB can be further utilized to examine the sensing ability with oppositely charged metal
ions or analytes in aqueous medium. To investigate the morphological aspects, the
scanning electron microscopy (SEM) was used. Fig. 3 indicates scanning electron
microphotograph of FONs-RSB, which clearly shows the distinct sphere shape
morphology for the prepared nanoparticles. The average particle size estimated through
SEM analysis was found to be 100-120 nm which is in close agreement with the particle
size analyzed using DLS technique (Fig.1). The rationale behind the observation of large
particle size during SEM analysis is aggregation of FONs-RSB while making air -dried
film due to repeatedly injecting aqueous suspension of nanoparticles on glass substrate in
order to have thin film.
3.3 Absorption and fluorescence studies:

It is well known that optical properties such as absorption, fluorescence and fluorescence
lifetime of nanoparticles are much more significantly distinct than those of parent
molecules. Therefore, to recognize the separate identity of nanoparticles than that of
monomer (parent molecule), absorption and fluorescence spectra followed by estimation
of fluorescence lifetime values in respective solvents were performed. Fig. 4 shows
absorption spectrum of RSB in acetone (A) and aqueous suspension of FONs-RSB (B).
The absorption spectrum clearly indicates nanoparticles shown their variable character as
compared with the parent molecule via bathochromically shifting of absorption band. The
distinct absorption band at 420 nm and less pronounced band at 376 nm for RSB in acetone
are assigned to n-π* and π-π* electronic transitions, respectively. In case of nanoparticles
(FONs-RSB), these absorption bands are found to be bathochromically shifted to the
maximum wavelength of 418 nm (π-π*) and 509 nm (n-π*). The structural rearrangement
via head to tail patterns generally known as J- aggregation [35-38] is the reason for shifting
of absorption maxima of parent molecule (RSB) to longer wavelength in FONs-RSB. The
fluorescence spectra of RSB in acetone (A) and aqueous suspension of FONs-RSB (B) is
given as Fig. 5. The excitation wavelength of 420 and 480 nm was used to scan the
fluorescence emission spectrum of RSB in acetone and aqueous suspension of FONs-RSB,
respectively. The broad fluorescence emission peak at 524 nm for RSB in acetone found
to be shifted to the longer wavelength at 558 nm which is assigned to aqueous suspension
of FONs-RSB. Also, 3-fold increase in the fluorescence intensity of FONs-RSB than the
RSB in acetone indicates aggregation induced enhanced emission (AIEE) phenomenon
[36,39]. Such AIEE observed in case of FONs-RSB is because of shifting of zeroth
vibrational level of ground electronic state to higher excited state in RSB molecule during
the formation of J- aggregated nanoparticles i.e. FONs-RSB. The superior photophysical
properties of FONs-RSB was also supported by evaluating the fluorescence quantum yield,
Stokes shift and fluorescence lifetime values. The estimated fluorescence quantum yield
values for FONs-RSB and RSB in acetone were found to be 4.23 and 1.89, respectively.
The calculated fluorescence quantum yields suggest FONs-RSB are more bright and
photophysically active component as compared with RSB in acetone. The estimated Stokes

shift values shows higher value for FONs-RSB (1725.55 cm-1) than that of RSB in acetone
(1189.69 cm-1) which is in support of formation of FONs-RSB. The larger Stokes shift
value of FONs-RSB also supports the origin of bathochromically shifted AIEE of FONs-
RSB due to fluorescence emission from lower lying excited state of J- aggregation of RSB
molecules. The average time spend by any fluorescent molecule in its excited state is
known as fluorescence lifetime of that material. In present case, FONs-RSB (5.63 ns)
illustrates longer fluorescence lifetime value than parent molecule RSB in acetone (2.11
ns). The observation of longer lifetime value for FONs-RSB is attributed to J- aggregates
of nanoparticles, which is responsible to restrict the molecular and vibrational rotations in
RSB molecules [35-38].
3.4 Chelation enhanced fluorescence enhancement of FONs-RSB:
The functionality present in the RSB molecule and negative charge over the surface of
nanoparticles encourage us to examine sensing property of prepared FONs-RSB with
positively charged metal ion solutions. The metal ion solutions (2 μg/mL) of Hg2+, Ag+,
+
Na+, Fe2+, Ni2+, Pb2+, Al3+, Zn2+, Ba2+, Mn2+, Cu2+, NH4 , K+, Ca2+, Mg2+, Cd2+, Cr3+ and
Co2+ was added to the aqueous suspension of FONs-RSB and further examined for their
fluorescence emission response at excitation of 480 nm. Fig. 6 indicates fluorescence
emission spectra of FONs-RSB in the presence and absence of variety of positively
charged metal ions solution having concentration of 2 μg/mL each. The fluorescence
emission of FONs-RSB is remained constant or slight variable when metal ions except
Hg2+ are added to aqueous suspension of FONs-RSB. While, addition of Hg2+ solution
distinctly increases the fluorescence emission of FONs-RSB by nearly 7 times more than
its initial value. Such increase in the fluorescence emission intensity of FONs-RSB by the
addition of Hg2+ is due to the chelation enhanced fluorescence (CHEF) phenomenon
[30,31,41,42]. The opening of spirolactum ring of rhodamine core led to generate more
reactive imine (-C=N) group locate near the spirolactum ring of rhodamine and oxygen (-
O-) of carbonyl group. These functionalities are strongly interacting with the Hg2+ ion and

simultaneously increases the rigidity of molecular assembly through restriction of free
rotations of carbonyl and imine groups which is resulting in CHEF. In comparison, the
sensing ability of parent molecule RSB was also investigated in the presence of a series of
metal ions having a concentration of 2 μg/mL each (Fig. S6). The parent molecule is highly
hydrophobic and hence the sensing experiment was carried out with 10 × 10−3 M RSB in
an acetone solution. Interestingly, the sensing property of RSB also tends towards
interaction with the Hg2+ only but at less extends. After the addition of Hg2+ to the solution
of RSB in acetone failed to produce enhanced fluorescence for a longer time. Such output
received through sensing ability of parent molecule RSB may not able to produce
satisfactory detection limits at extremely low levels. Hence, it was concluded that being of
hydrophilic characteristics, aqueous stability, ability to produce extremely low detection
limits, practical applicability in aqueous systems, selective and sensitive sensing properties
for FONs-RSB would be the preferred choice for the further investigation through present
studies. In addition, the response time is an essential parameter for fluorescent sensors and
which was systematically checked by observing fluorescence intensity as a function of
response time in minutes and given as Fig. S7. The figure illustrates that after the addition
of 2 μg/mL of Hg2+ to the aqueous suspension of FONs-RSB, fluorescence intensity
gradually increases till 5 min and further remains constant for the period of 20 min. This
clearly indicates FONs-RSB is able to sensitize Hg2+ within a short period of time and
remain invariable during the sensing process. Additionally, the change in the fluorescence
intensity was checked after the variation of pH for FONs-RSB when in contact with a
higher concentration of Hg2+ (2 μg/mL). The results are given as supporting information in
Fig. S8. The maximum fluorescence intensity was observed at neutral pH 7 indicates good
stability of formed complexation between FONs-RSB and Hg2+ through sensing process.
This experimental observation clarifies the applicability of FONs-RSB would be possible
to detect Hg2+ in biological samples at an ideal neutral pH range.
3.5 Effect of foreign metal ions:
The selective and sensitive nature for fluorescence enhancement of FONs-RSB by the
addition of Hg2+ was further examined and supported by performing the effect of foreign

metal ion on the performance of fluorescence intensity induced by Hg2+ to FONs-RSB.
The respective solutions of FONs-RSB with Hg2+ (2 μg/mL) and each foreign metal ion (2
μg/mL) were prepared and scanned for the fluorescence measurement. Fig. 7 illustrates the
change in the fluorescence intensity of FONs-RSB in the presence of each metal ion (green
color bars) and in the presence of Hg2+ along with foreign metal ions (brown color bars).
It is observed that only Hg2+ can selectively and sensitively enhances the fluorescence
intensity of FONs-RSB through CHEF process. While, other metal ions failed to produce
significant change in the fluorescence intensity of FONs-RSB. Interestingly, the study on
the effect of foreign metal indicates selective interaction of only Hg2+ ion with FONs-RSB
and could not interfere and affect the detection of Hg2+ by the addition of other analytes.
3.6 Estimation of statistical parameters:
3.6.1 Fluorescence titration and Limit of detection:
Fig. 8 shows fluorescence intensity of FONs-RSB in the absence and presence of variable
concentration of Hg2+ ion from 0 to 2 μg/mL. The results show that progressively change
in intensity of fluorescence emission of FONs-RSB with increase in the concentration of
Hg2+ solution. This increasing pattern of fluorescence intensity was found to be linear in
the concentration range of 0-2 μg/mL of Hg2+ solution. The results were plotted as a change
in the fluorescence intensity from its initial value to the addition of Hg2+ versus the
particular concentration of added Hg2+ in FONs-RSB and used as calibration plot given as
Fig. 9. The results of the fluorescence enhancement of FONs-RSB due to the chelation
process were found to be well fitted into a straight line equation in the linear range. The
limit of detection (LOD) [42] was evaluated using equation 1 given below,
3.3휎
퐿푂퐷 =
(1)
퐾
In the above equation, σ and K represents the standard deviation of blank measurement and
the slope value of the regression line, respectively. The detection limit calculated for Hg2+
using the present analytical approach is found to be 1.729 ng/mL (8.619 nM). To verify the
detection limit of the present investigation at the nano level, the sensing experiment of

FONs-RSB was carried out at the linear concentration range of 0-100 ng/mL Hg2+ solution.
Fig. S9 shows the linear response for change in the fluorescence intensity of FONs-RSB
at the respective Hg2+ concentration. The limit of detection was calculated (1.689 ng/mL)
which was found to be in agreement with experimental set up when the concentration of
Hg2+ with a linear range 0-2 μg/mL. This proves the sensitivity of the proposed analytical
approach for micro as well as the nano level concentration of the analyte of interest. The
present analytical approach based on rhodamine cored fluorescent nanoparticles for the
detection of Hg2+ provides appreciably much lower detection limit in addition to practical
applicability than the already reported sensors and other analytical techniques. Table 1
summarizes comparison syntax of proposed fluorescent nanoprobe with already reported
analytical methods and probes followed by their applicability in the determination of Hg2+.
3.6.2 Evaluation of binding stoichiometry and binding constant for complexation
between FONs-RSB and Hg2+:
The renowned Job’s method [30,43,44] was applied to evaluate the binding stoichiometry
between complex formation of FONs-RSB and Hg2+. The solutions containing varying
mole fractions of Hg2+ from 0.1 to 0.9 were subjected to examine their absorption and
fluorescence spectrum. The total sum of concentration of FONs-RSB and Hg2+ was kept
constant with only changing the mole fraction of Hg2+ from 0.1 to 0.9. Fig. 10 represents
as Job’s plot shows the respective value of absorption and fluorescence intensity for each
of solution holding mole fraction of 0.1 to 0.9 Hg2+ ion. The maximum absorption value
and fluorescence emission intensity noticed only after the mole fraction of Hg2+ achieves
a value of 0.5, which signifies the binding stoichiometry between FONs-RSB and Hg2+
ion could be 1:1. The value of binding constant for complexation between FONs-RSB and
Hg2+ was evaluated using modified Benesi-Hilderbrand (B-H) equation [30,31,45] and
given as equation 2,
1
1
1
1
=
+
(2)
2+ 푛
(
퐼
)
(
퐼
퐵 푀퐴푋
) [
− 퐼 퐻푔
]
퐼−퐼
− 퐼
퐾
0
푀퐴푋
0
0
In equation 2, the maximum fluorescence intensity of FONs-RSB in the presence of Hg2+,
initial fluorescence intensity of FONs-RSB and fluorescence intensity of FONs-RSB at

respective concentration of Hg2+ is represented as Imax, I0 and I, respectively. While, KB and
n stands for the binding constant and number of Hg2+ bound per FONs-RSB i.e. 1,
respectively. The value of binding constant between complexation of FONs-RSB with
Hg2+ was calculated using slope value of modified B-H plot given as Fig. 11. The estimated
binding constant value (KB) for 1:1 complexation between FONs-RSB and Hg2+ in
aqueous medium is found to be 4.149 × 103. The considerably higher binding constant
value supports sensible binding interactions between FONs-RSB and Hg2+.
3.7 Mechanism of chelation enhanced fluorescence of FONs-RSB by Hg2+:
The recent reports were witnessed to recommend the chelation enhanced fluorescence
phenomenon through spirolactum ring opening mechanism in case of rhodamine
derivatives. To investigate the mechanism of chelation enhanced fluorescence of FONs-
RSB by the addition of Hg2+, systematic experiments such as analysis of mass spectrum of
product formation in sensing process, absorption titration, fluorescence lifetime, zeta-
particle size analysis, IR titration and NMR titration were performed in the absence and
presence of variable concentration of Hg2+. The sample used for the IR, NMR and Mass
analysis was the one which showed maximum absorbance and fluorescence response in
experiment of Job’s plot and illustrates 1:1 complexation between FONs-RSB and Hg2+.
The spirolactam ring opening of rhodamine derivative RSB after the addition of Hg2+ leads
to interact metal ion with functionalities namely carbonyl oxygen (O-) and nitrogen of
imine (-C=N) as shown in Scheme 2. The enhanced fluorescence of FONs-RSB by Hg2+
may be assigned to the conformational rigidification imposed on the nano probe upon metal
ion complexation. Such interactions simultaneously restrict the free rotations of carbonyl
and imine groups leading to sensitization and chelation induced fluorescence enhancement
in presence of Hg2+. The titration results of IR and NMR of FONs-RSB in presence of
Hg2+ are given as supporting information (Fig. S10 and Fig. S11). From the IR spectrum,
it seems that shifting of IR stretching frequency of imines from 1590 to 1605 cm-1 and 1637
to 1690 cm-1 along with disappearance of stretching frequency of the carbonyl group (1705
cm-1) clearly indicates the participation of one of imine group which locates near to
spirolactum ring and carbonyl group in the chelation and interaction with Hg2+. These

observations further supported by NMR titration results. The appearance of two singlet
peaks at δ 8.93 and δ 8.79 for the proton belongs to imines (HC=N) in NMR spectrum (Fig.
S2) shows only one singlet peak at δ 9.61 after the addition of Hg2+ (Fig. S11). The singlet
peak observed is assigned to imine group which locates near to pyridine moiety. While,
relevant peak positions (δ values) of other protons in molecules are slightly shifted to either
downfield or upfield. Hence, IR and NMR titration of FONs-RSB with Hg2+ suggests
interaction patterns and supports chelation enhanced fluorescence mechanism. Further, the
product formation of the sensing process was investigated through the Mass analysis. Fig.
S12 given as supporting information shows the mass spectrum of the product of the present
sensing process. The found molecular weight (849.1) of the product of the sensing process
through the complexation between FONs-RSB and Hg2+ was in harmony with the expected
molecular weight (849.28). This observation suggests the complexation between FONs-
RSB and Hg2+ at 1:1 equivalent ratio. Additionally, the complexation between
nanoparticles and Hg2+ was confirmed by examining the zeta-particle size results (Fig.
S13). The zeta potential and particle size were analyzed in the presence of variable
concentration of Hg2+ (0.5, 1 and 2 μg/mL) to the aqueous suspension of FONs-RSB. There
is no change observed in case of zeta potential value but increase in only particle size from
82 nm to 468 nm. This observation suggests the complexation between FONs-RSB with
good aqueous stability. The absorption spectra of FONs-RSB in the absence and presence
of Hg2+ (0 to 2 μg/mL) were examined and results interpret as Fig. S14. The bathochromic
shift of 23 nm in the absorption maximum wavelength and change in the absorption value
after the successful addition of Hg2+ from concentration of 0 to 2 μg/mL ignores the
possibility of ground state complexation and endorses excited state complexation between
FONs-RSB and Hg2+. The excited state complexation of FONs-RSB-Hg2+ was further
supported by analyzing the fluorescence lifetime values in the absence and presence of
different concentration of Hg2+ to the aqueous suspension of FONs-RSB. The rise in the
fluorescence lifetime value from 5.63 ns to 6.12 ns after the addition of Hg2+ solution
having 0 to 2 μg/mL concentration indicates strongly excited state complexation between
FONs-RSB and Hg2+ in an aqueous medium. Table 2 indicates the fluorescence lifetime

values of FONs-RSB in the absence and presence of particular concentration of Hg2+
solution. Thus, chelation induced excited state complex of FONs-RSB-Hg2+ remains
longer in the singlet excited state and fluoresces through an increase in the pathways which
results in an enhancement in fluorescence of FONs-RSB when in contact with the
respective concentration of Hg2+ solution. Scheme 2 shows a pictorial representation of a
plausible mechanism for the formation of FONs-RSB and its interaction with Hg2+.
3.8 Application of proposed fluorescence method:
3.8.1 Quantitative determination of Hg2+ in environmental samples using FONs-RSB:
The quantitative determination of Hg2+ from collected environmental samples using
prepared FONs-RSB is the main advantage of proposed analytical method. The samples
under studies were processed for filtration and boiling to remove the solid impurities and
dissolved gases, if any. The solutions were prepared by spiking known quantity of desired
concentration of standard Hg2+ solution followed by dilution with respective environmental
samples. The calibration plot as given in Fig. 9 was used to find out the recovery of spiked
concentration of Hg2+ ion. Pleasingly, observed concentrations of Hg2+ are in harmony with
the spiked concentrations of Hg2+ to the said samples. The obtained data is interpreted in
Table 3. Hence, proposed fluorescence based analytical approach can be successfully
utilized to estimate the Hg2+ quantity in an aqueous medium without interference of foreign
analytes.
3.8.2 Intracellular cell imaging of Hg2+ using FONs-RSB and cell toxicity studies:
The intake of Hg2+ in human body severely affects body organs such as kidney, liver, brain,
etc. Therefore, it is an essential to develop suitable probe which can selectively and
sensitively detects intracellular Hg2+ in cell line. Inspired from the enhanced fluorescence
properties of FONs-RSB when in contact with Hg2+, we performed fluorescence based cell
imaging experiment of proposed sensing method using the human malignant melanoma
A375 cell line. The cultured cells were subjected to treat with the RSB in acetone, aqueous
suspension of FONs-RSB and FONs-RSB + Hg2+ ion at particular concentration. The
fluorescence images for cell imaging studies were scanned at excitation wavelength of 532

nm and given as Fig. 12. It was found that, RSB in acetone (Fig. 12, S2) showed very
fragile fluorescence when in contact with the human malignant melanoma A375 cell line.
While, FONs-RSB (Fig. 12, S3) displayed enhanced fluorescence in cell imaging.
Interestingly, intracellular fluorescence of FONs-RSB was further increased (Fig. 12, S4
and S5) when foreign metal ion Hg2+ of concentration 1 μg/mL (Fig. 12, S4) and 2 μg/mL
(Fig. 12, S5) was externally added to the human malignant melanoma A375 cell line. Fig.
13 illustrates cell viability of A375 cells cultured in complete media with RSB in acetone,
aqueous suspension of FONs-RSB and FONs-RSB + Hg2+ ion at particular concentration.
The MTT assay [46] was performed to check the cytotoxicity effects on A375 cells after
24 h of prepared samples treatments. Satisfyingly, the desired nanoprobe FONs-RSB and
its complex form with Hg2+ were found to be non-toxic with A375 cells. Hence, it was
concluded that prepared rhodamine cored fluorescent organic nanoparticles FONs-RSB
could be the best choice for intracellular detection of Hg2+.
4. Conclusion:
Rhodamine based compound (RSB) was synthesized and characterized at the molecular
scale. The fluorescent organic nanoparticles of RSB (FONs-RSB) were prepared by using
the reprecipitation method. The nano-level particle size of 82 nm and sphere shape
morphology were examined on the particle size analyzer and scanning electron microscope,
respectively. The functionalities present in the molecule and negative charge over the
surface of FONs-RSB suggested examining the fluorescence response in the presence of a
variety of metal ions to evaluate the sensing ability of prepared nanoparticles. Pleasingly,
only Hg2+ ion induced chelation enhanced fluorescence in FONs-RSB, while other metal
ions respond negligibly. Additionally, none of foreign metal ion interferences in the
selective and sensitive detection of Hg2+ in an aqueous medium using FONs-RSB. The
fluorescence enhancement of FONs-RSB was found to be linear in the concentration range
of 0-2 μg/mL of Hg2+. The excited state 1:1 complexation through chelation of Hg2+ with
FONs-RSB was supported by studies on IR, NMR, absorption and fluorescence lifetime
titrations. The formation of complexation i.e. sensing product was confirmed by analysis

of mass spectrum of 1:1 equivalent of FONs-RSB and Hg2+. The limit of detection (LOD)
for Hg2+ is found to be 1.729 ng/mL (8.619 nM) using proposed method and which is
superior than probes and methods reported earlier. The present fluorescence based
analytical method provides simple, rapid, sensitive and selective quantitative approach for
the determination of Hg2+ in environmental samples. The intracellular detection of Hg2+
using FONs-RSB is an additional benefit of prepared nanoparticles which will explore its
potent application in biomedical field.
Conflict of Interest:
Authors declare no conflict of interest.
Author Statement
We are submitting herewith our revised research article entitled, “Chelation
enhanced fluorescence of rhodamine based novel organic nanoparticles for
selective detection of mercury ions in aqueous medium and Intracellular cell
imaging” for consideration and publication in your reputed Journal, “Journal of
Photochemistry and Photobiology A: Chemistry”.
We hereby give the warranty that the manuscript submitted to the
journal for review is original, and has not been published elsewhere; is not currently
being considered for publication by any other journal and will not be submitted for
such review while under review by the Journal “Journal of Photochemistry and
Photobiology A: Chemistry”.
Declaration of Interest Statement
Authors declare no conflict of interest.
Acknowledgements
This research was supported by Basic Science Research Program through the National Research Foundation
of Korea (NRF) funded by the Ministry of Education (NRF- 2019R1I1A3A01059089).

References:
1. S. Sarkar, T. Mondal, S. Roy, R. Saha, A.K. Ghosh, S.S. Panja, A multi-responsive
thiosemicarbazone-based probe for detection and discrimination of group 12 metal
ions and its application in logic gates, New J. Chem. 42 (2018) 15157–15169.
2. (a) P . Miao , L . Liu, Y . Li, G. X. Li, A novel electrochmical method to detect mercury
(II) ions, Electrochem. commun. 11 (2009) 1904–1907.; (b) P. G. Mahajan, N. C. Dige, N.
K. Desai, S. R. Patil, V. V. Kondalkar, S. K. Hong, K. H. Lee, Selective detection of Co2+
by fluorescent nano probe: Diagnostic approach for analysis of environmental samples and
biological activities, Spectrochim. Acta A 198 (2018) 136–144.
3. (a) Y. Ding, Y. Tang, W. Zhu, Y. Xie, Fluorescent and colorimetric ion probes based on
conjugated oligopyrroles, Chem. Soc. Rev. 44 (2015) 1101-1112.; (b) Y. Ding, W. Zhu, Y.
Xie, Development of Ion Chemosensors Based on Porphyrin Analogues, Chem. Rev. 117
(2017) 2203-2256.
4. M. Lufti Firdaus, I. Fitriani, S. Wyantuti, Y. W. Hartati, R. Khaydarov, J. A. Mcalister,
H. Obata, T. Gamo, Colorimetric Detection of Mercury (II) ion in aqueous solution using
silver nanoparticles, Anal. Sciences. 33 (2017) 831-837.
5. M. Harada, Minamata disease: methylmercury poisoning in Japan caused by
environmental pollution, Crit. Rev. Toxicol., 25 (1995) 1-24.
6. A. Gopalakrishna, T. V. Pavan Kumar, Intravenous injection of elemental mercury: A
report of two cases, Indian J. Plast. Surg. 41 (2008) 214–218.
7. N. A. Azmi, S. H. Ahmad, S. C. Low, Detection of mercury ions in water using
a membrane-based colorimetric sensor, RSC Adv. 8 (2018) 251–261.
8. B. V. Tangahu, S. R. Sheikh Abdullah, H. Basri, M. Idris, N. Anuar, M. Mukhlisin, A
Review on Heavy Metals (As, Pb, and Hg) Uptake by Plants through Phytoremediation,
Int. J. Chem. Eng. 2011 (2011) DOI:10.1155/2011/939161.

9. (a) G. Zeng, C. Zhang, D. Huang, C. Lai, L. Tang, Y. Zhou, P. Xu, H. Wang, L. Qin,
M. Cheng, Practical and regenerable electrochemical aptasensor based on nanoporous gold
and
thymine-Hg2+-thymine
base
pairs
for
Hg2+
detection,
Biosens.
Bioelectron. 90 (2017) 542-548; (b) C. Lai, S. Liu, C. Zhang, G. Zeng, D. Haung, L. Qin,
X. Liu, H. Yi, R. Wang, F. Huang, B. Li, T. Hu, Electrochemical Aptasensor Based on
Sulfur-Nitrogen Codoped Ordered Mesoporous Carbon and Thymine-Hg2+-Thymine
Mismatch Structure for Hg2+ Detection, ACS Sens. 3 (2018) 2566-2573.
10. (a) L. Cherian, V. K. Gupta, A simple field test for the detection of mercury
in polluted water, air and soil samples, Fresenius J. Anal. Chem. 336 (1990) 400-402.; (b)
F. Zahir, S. J. Rizwi, S. K. Haq, R. H. Khan, Low dose mercury toxicity and human
health, Environ. Toxicol. Pharacol. 20 (2005) 351–360.
11. P. Holmes, K. A. James, L.S. Levy, Is low-level environmental mercury exposure of
concern to human health? Sci. Total Environ. 408 (2009) 171-182.
12. E. M. Nolan, S. J. Lippard, Tools and tactics for the optical detection of mercuric ion,
Chem. Rev. 108 (2008) 3443-3480.
13. F. X. Han, W. Dean Patterson, Y. J. Xia, B. B. Maruthi Sridhar, Y. J. Su, Rapid
determination of mercury in plant and soil samples using inductively coupled plasma
atomic emission spectroscopy, a comparative study, Water Air Soil Pollut. 170 (2006) 161-
171.
14. P. Ugo, L.M. Moretto, P. Bertoncello, J. Wang, Determination of trace mercury in
saltwater at screen-printed electrodes modified with sumichelate Q10R, Electroanalysis 10
(1998) 1017–1021.
15. L. P. Yu, X. P. Yan, Flow injection on-line sorption preconcentration coupled with cold
vapor atomic fluorescence spectrometry and on-line oxidative elution for the determination
of trace mercury in water samples, Atomic Spectroscopy 25 (2004) 145–153.

16. J. S. D. Santos, M. D. L. Guárdia, A. Pastor, M.L.P.D. Santos, Determination of
organic and inorganic mercury species in water and sediment samples by HPLC on-line
coupled with ICP-MS, Talanta 80 (2009) 207-211.
17. M. P. Gilad, T. V. Ran, J. Elbaz, I. Willner, Nanoengineered electrically contacted
enzymes on DNA scaffolds: functional assemblies for the selective analysis of Hg2+ ions,
J. Am. Chem.Soc. 132 (2010) 6878-6879.
18. W. Lu, X. Qin, S. Liu, G. Chang, Y. Zhang, Y. Luo, A. M. Asiri, A. O. Al-Youbi,
X. Sun, Economical, green synthesis of fluorescent carbon nanoparticles and their
use as probes for sensitive and selective detection of mercury (II) ions, Anal. Chem. 84,
(2012) 5351-5357.
19. K. D. Bhatt, D. J. Vyas, B. A. Makwana, S. M. Darjee, V. K. Jain, H. Shah, Turn-on
fluorescence probe for selective detection of Hg(II) by calixpyrrole hydrazide reduced
silver nanoparticle: application to real water sample, Chin. Chem. Lett. 27 (2016) 731–737.
20. H. Bagheri, A. Gholami, Determination of very low levels of dissolved mercury(II) and
methylmercury in river waters by continuous flow with on-line UV decomposition and
cold-vapor atomic fluorescence spectrometry after pre-concentration on a silica gel-2-
mercaptobenzimidazol sorbent, Talanta 55 (2001) 1141-1150.
21. L. N. Suvarapu, S. O. Baek, Recent studies on the speciation and determination of
mercury in different environmental matrices using various analytical techniques, Int. J.
Anal. Chem. 2017 (2017) DOI:10.1155/2017/3624015.
22. L. S. Walekar, A. H. Gore, P. V. Anbhule, V. Sudarsan, S. R. Patil, G. B. Kolekar, A
novel colorimetric probe for highly selective recognition of Hg2+ ions in aqueous media
based on inducing the aggregation of CPB-capped AgNPs: accelerating direct detection for
environmental analysis, Anal. Methods 5 (2013) 5501–5507.

23. C. A. Trimble, R. W. Hoenstine, A. B. Highley, J. F. Donoghue, P. C. Ragland, Baseline
sediment trace metals investigation: Steinhatchee river estuary, Florida, Northeast Gulf of
Mexico, Mar. Georesour. Geotechnol. 17 (1999) 187-197.
24. L. H. Jin, C. S. Han, Eco-friendly colorimetric detection of mercury (II) ions using
label-free anisotropic nanogolds in ascorbic acid solution, Sensors and Actuators B 195
(2014) 239-245.
25. H. N. Kim, M. H. Lee, H. J. Kim, J. S. Kim, A new trend in rhodamine-based
chemosensors: application of spirolactam ring-opening to sensing ions Chem. Soc. Rev. 37
(2008) 1465–1472.
26. X. Chen, T. Pradhan, F. Wang, J. S. Kim, J. Yoon, Fluorescent chemosensors based on
spiroring-opening of xanthenes and related derivatives, Chem. Rev. 112 (2012) 1910–1956.
27. M. Saleem, R. Abdullah, A. Ali, B. J. Park, E. H. Choi, I. S. Hong, K. H. Lee, Synthesis,
cytotoxicity and bioimaging of a novel Hg2+ selective fluorogenic chemosensor, Anal.
Methods 6 (2014) 3588-3597.
28. F. Yan, D. Cao, N. Yang, Q. Yu, M. Wang, L. Chen, Sens. Actuators B 162 (2012)
313–320.
29. P. G. Mahajan, N. C. Dige, B. D. Vanjare, S. H. Eo, S. J. Kim, K. H. Lee, A nano sensor
for sensitive and selective detection of Cu2+ based on fluorescein: cell imaging and
drinking water analysis, Spectrochim. Acta A. Mol. Biomol. Spectrosc. 216 (2019)
105–116.
30. P. G. Mahajan, N. C. Dige, B. D. Vanjare, Y. Han, S. J. Kim, S. K. Hong, K. H. Lee,
Intracellular imaging of zinc ion in living cells by fluorescein based organic nanoparticles,
Sens. Actuators B Chem. 267 (2018) 119–128.
31. P. G. Mahajan, D. P. Bhopate, G. B. Kolekar, S. R. Patil, N-methyl isatin nanoparticles
as a novel probe for selective detection of Cd2+ ion in aqueous medium based on chelation

enhanced fluorescence and application to environmental sample, Sens. Actuators B Chem.
220 (2015) 864–872.
32. L. Wang, L. Wang, L. Dong, G. Bian, T. Xia, H. Chen, Direct fluorimetric
determination of γ-globulin in human serum with organic nanoparticle biosensor,
Spectrochim. Acta A 61 (2005) 129–133.
33. P. G. Mahajan, N. K. Desai, D. K. Dalavi, D. P. Bhopate, G. B. Kolekar, S. R. Patil,
Cetyltrimethylammonium bromide capped 9-anthraldehyde nanoparticles for selective
recognition of phosphate anion in aqueous solution based on fluorescence quenching and
application for analysis of chloroquine, J. Fluoresc. 25 (2015) 31–38.
34. J. Lyklema, Fundamentals of Interface and Colloid Science, Elsevier, Wageningen,
(1995)
35. F. Wang, M. Han, M. Yi, Y. Wang, Y. Lai, Aggregation driven growth of size-tunable
organic nanoparticles using electronically altered conjugated polymers, J. Am. Chem. Soc.
127 (2005) 10350–10355.
36. P. G. Mahajan, G. B. Kolekar, S. R. Patil, Recognition of D-penicillamine using Schiff
base centered fluorescent organic nanoparticles and application to medicine analysis, J.
Fluoresc. 27 (2017) 829–839.
37. X. Zhang, X. Zhang, S. Wang, M. Liu, L. Tao, Y. Wei, Surfactant modification of
aggregation-induced emission material as biocompatible nanoparticles: facile preparation
and cell imaging, Nanoscale 5 (2013) 147–150.
38. P. G. Mahajan, D. P. Bhopate, G. B. Kolekar, S. R. Patil, FRET sensor for erythrosine
dye based on organic nanoparticles: application to analysis of food stuff, J. Fluoresc. 26
(2016) 1467–1478.
39. D. K. Dalavi, D. P. Bhopate, A. S. Bagawan, A. H. Gore, N. K. Desai, A. A. Kamble,
P. G. Mahajan, G. B. Kolekar, S. R. Patil, Fluorescence quenching studies of CTAB

stabilized perylene nanoparticles for the determination of Cr(VI) from environmental
samples: spectroscopic approach, Anal. Methods 6 (2014) 6948–6955.
40. M. Kumar, A. Kumar, M. Kumar Singh, S.K. Sahu, R.P. John, A novel benzidine based
Schiff base turn-on fluorescent chemosensor for selective recognition of Zn2+, Sens.
Actuators B 241 (2017) 1218–1223.
41. L. N. Wang, W. W. Qin, W. S. Liu, A sensitive Schiff-base fluorescent indicator
for the detection of Zn2+, Inorg. Chem. Commun. 13 (2010) 1122–1125.
42. P. G. Mahajan, N. C. Dige, S. B. Suryawanshi, D. K. Dalavi, A. A. Kamble, D. P.
Bhopate, A. N. Kadam, V. V. Kondalkar, G. B. Kolekar, S. R. Patil, FRET between
Riboflavin and 9-Anthraldehyde based fluorescent organic nanoparticles possessing
Antibacterial activity, J. Fluoresc. 28 (2018) 207–215.
43. A. Samanta, N. Guchhaitb, S. C. Bhattacharya, A simple but highly selective and
sensitive fluorescence reporter for toxic Cd (II) ion via excimer formation, Chem. Phy. Lett.
612 (2014) 251–255.
44. P. Job, Formation and stability of inorganic complexes in solution, Ann. Chim.
9 (1928) 113–203.
45. H. A. Benesi, J. H. Hildebrand, A spectrophotometric investigation of the interaction
of iodine with aromatic hydrocarbons, J. Am. Chem. Soc. 71 (1949) 2703–2707.
46. A. R. Phull, M. Majid, I. U. Haq, M. R. Khan, S. J. Kim, In vitro and in vivo
evaluation of anti-arthritic, antioxidant efficacy of fucoidan from Undaria pinnatifida
(Harvey) Suringar, Int. J. Biol. Macromol. 97 (2017) 468–480.
47. H. E. Kaoutit, P. Estevez, F. Garcia, F. Serna, J. M. Garcia, Sub-ppm quantification of
Hg(II) in aqueous media using both the naked eye and digital information from pictures of
a colorimetric sensory polymer membrane taken with the digital camera of a conventional
mobile phone, Anal. Methods, 5 (2013) 54–58.

48. G. H. Chen, W. Y. Chen, Y. C. Yen, C. W. Wang, H. T. Chang, C. F. Chen, Detection
of Mercury(II) Ions Using Colorimetric Gold Nanoparticles on Paper-Based Analytical
Devices, Anal. Chem. 86 (2014) 6843-6849.
49. Y. Yang, Z. Wang, M. Yang, M. Zhaoyang, W. Guoli, S. R. Yu, Inhibitive
determination of mercury ion using a renewable urea biosensor based on self-assembled
gold nanoparticles, Sensors and Actuators B 114 (2006) 1–8.
50. N. R. Devi, M. Sasidharan, A. K. Sundramoorthy, Gold Nanoparticles-Thiol
Functionalized Reduced Graphene Oxide Coated Electrochemical Sensor System for
Selective Detection of Mercury Ion, Journal of The Electrochemical Society, 165 (2018)
B3046-B3053.
51. A. Samphao, H. Rerkchai, J. Jitcharoen, D. Nacapricha, K. Kalcher, Indirect
Determination of Mercury by Inhibition of Glucose Oxidase Immobilized on a Carbon
Paste Electrode, Int. J. Electrochem. Sci. 7 (2012) 1001–1010.
52. O. O. Soldatkin, I. S. Kucherenko, V. M. Pyeshkova, A. L. Kukla, N. Jaffrezic-Renault,
A. V. Eliskaya, S. V. Dzyadevych, A. P. Soldatkin, Novel conductometric biosensor based
on three-enzyme system for selectivedetermination of heavy metal ions,
Bioelectrochemistry 83 (2012) 25–30.
53. N. Zohora, D. Kumar, M. Yazdani, V. M. Rotello, R. Ramanathan, V. Bansal, Rapid
colorimetric detection of mercury using biosynthesized gold nanoparticles, Colloids and
Surfaces A 532 (2017) 451–457.
54. X. Zeng, J. Liu, Z. Zhang, S. Kong, Sensitive and Selective Detection of Mercury Ions
by Potentiometric Biosensor Based on Urease Immobilized in Chitosan–Poly(Vinyl
Alcohol) Hydrogel Film, Int. J. Electrochem. Sci. 10 (2015) 8344 - 8352
55. E. Coronado, J. R. Galain-Mascarois, C. Marti-Gastaldo, E. Palomares, J. R. Durrant,
R. Vilar, M. Gratzel, M. K. Nazeeruddin, Reversible Colorimetric Probes for Mercury
Sensing, J. Am. Chem. Soc. 127 (2005) 12351-12356.

56. T. K. Krawczyk, M. Moszczyniska, M. Trojanowicz, Inhibitive determination of
mercury and other metal ions bypotentiometric urea biosensor, Biosensors &
Bioelectronics 15 (2000) 681 – 691.
57. J. Do, K. Lin, R. Ohara, Preparation of urease/nano-structured polyaniline-
Nafion1/Au/Al2O3 electrode for inhibitive detection of mercury ion, J. Taiwan Institute of
Chem. Engi. 42 (2011) 662–668.
58. Y. C. Hsieh, J. L. Chir, H. H. Wu, P. S. Chang, A. T. Wu, A sugar-aza-crown ether-
based fluorescent sensor for Hg2+ and Cu+, Carbohydrate Research 344 (2009) 2236–2239.
59. P. G. Mahajan, N. C. Dige, B. D. Vanjare, A. R. Phull, S. Ja Kim, K. H. Lee,
Gallotannin mediated silver colloidal nanoparticles as multifunctional nano platform:
Rapid colorimetric and turn-on fluorescent sensor for Hg2+, catalytic and In vitro anticancer
activities, J. Lumin. 206 (2019) 624–633.
60. D. Hu, Z. Sheng, P. Gong, P. Zhang, L. Cai, Highly selective fluorescent sensors for
Hg2+ based on bovine serum albumin-capped gold nanoclusters, Analyst 135 (2010) 1411–
1416.
61. P. Ncube, R. W. M. Krause, D. T. Ndinteh, B. B Mamba, Fluorescent sensing and
determination of mercury (II) ions in water, Water SA 40 (2014) 175-182.
62. Y. Li-Rong, L. Kai-Bin, Y. Xing, Z. Rong-Bin, H. Zheng, p-Dimethylamino-
benzaldehyde thiosemicarbazone: A simple novelselective and sensitive fluorescent sensor
for mercury(II)in aqueous solution, Talanta 69 (2006) 103–106.
63. C. Huang, Z. Yang, K. Lee, H. Chang, Synthesis of Highly Fluorescent Gold
Nanoparticles for Sensing Mercury(II), Angew. Chem. Int. Ed. 46 (2007) 6824-6828.